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1. SSS 5 After a number of dialogs depending on selections a Summary dialog is shown e A list of calculated volumes of required solutions is displayed Note To prepare a run with solutions we recommend printing this list using the Print button This list will also be displayed in Method Notes in the Run Setup dialog Method Wizard for System AKTAcrossflow1 x Summary Basic Setings System AKTAcrossflowl ry Preproduct Steps E Summary Inlet number Definition Volume max 1 Sample 2 Conditioning butter 360 ml 3 Diafiltration buffer 1 See note 4 Diafiltration buffer 2 See note 5 Water 1850 ml 6 CIP solution 1 630 ml 7 CIP solution 2 0 ml 8 Storage Solution 0 ml Retentate out Definition 1 Flush 2 Waste 3 Product Petmeate out Definition Waste 2 Cone 3 DF Note 1 Please note that this info will be stored i Method notes after Finish is executed Note 2 The DF volumes are not estimated here ext Help Set Default 6 Click Finish in the Summary dialog Leesa 58 AKTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow 7 The Run Setup window that appears consists of a number of tab pages On the variable page chosen running conditions are displayed e Toshow detail variables check Show details box Detail variables are indicated by a D in the column immediately to the left of the Variable column e To show unused variables check Show unused variables box Unused variables are indicated
2. AKTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters 12 5 2 Regulation of the Permeate pump example The TMP_PI_PermeatePump instruction was used when the system was run manually TMP Control was set to 1 Bar and was monitored at different PI values in the instruction PI settings Curve example TMP 1 Settings The P parameter was set to 1 The parameter was set to 100 Result Pressure peaks were observed both in the beginning of the regulation and also when TMP was reached 2 Settings The P parameter was decreased to 0 2 The parameter was decreased to 50 Result Smooth regulation up to set TMP and no pressure peaks were observed when TMP was reached AKTAcrossflow User Manual 11 0012 32 Edition AB 167 Feedback tuning and PID parameters 168 AKTAcrossflow User Manual 11 0012 32 Edition AB 13 Strategy instructions Strategy instructions 13 13 1 System settings and instruction boxes 13 1 1 System setting The order of instructions within each group Some instructions are only shown when chosen as components Alarms Specials Warnings UV RetentateHoldUpVol pH PressureOffset Cond RPCVoffset Feed_Press RPCVhysteresis Hires PPCV_Setp TMP Total_Membrane_Area Flux Lumen_Diameter Shear Total_Number_of_Fibers pNFF AuxOut1 Valves AuxOut2 Flowpath AuxOut3 AirSensor AuxOut4 Zerotevel FeedPressure_Pl DeltaP_PI TMP_PID_Retentat
3. Result No result Add unique identifier to result name Directory Home Scouting subdirectory Name Batch ID Cancel AKTAcrossflow User Manual 11 0012 32 Edition AB 141 9 Running KTAcrossflow methods 9 4 During the run The progress of the method being used can be viewed in detail in UNICORN The System Control module displays the current status of AKTAcrossflow and displays up to four view panes for monitoring different aspects of the run File View Manual System Help Run Pause Continue End En Er EAA Ef Cond TMP Flux ConcFactor bar 555 04 454 40 35 3 04 0 0 prrTT I I rere T T T T T T T T T T T T T T T T T T T T T T T T min 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 4 4 4 amp 6 4 amp 8 5 5 4 5 5 60 33 25 min Hold_Until Condition_Occurred TransVol Greater_Than 500 0000 Infinite ml Logbook 33 25 min Constant_Retentate_Volume Disabled 33 25 min Watch_RetVol Less_Than 50 0000 END_BLOCK 33 25 min Block PURGE_TRANSFER_LINE For Help press F1 Run INITIAL_CONCENTRATION Active watch Controlled By alisa Astarte lt gt lt buntcoAN manager Method Editor Fulleroces 4 System Control 1 Sy ConcTestFo IHAL DPS 3 44PM sil To customize the view panes Run data Curves Flow scheme and Logbook right click in the respective view pane and select
4. Feed Control Retentate Flow DeltaP Retentate flow Feed Flow Retentate Flow Enter the value for the selected constant parameter for example Retentate Flow 25 ml min Enter the desired TMP Note When using TMP in a method a value close to the TMP limit set in the Basic Settings dialog may lead to the run being paused Feed Control Retentate Flow v Retentate flow 2 mimin 0 800 ml min TMP 25 bar 0 0 5 2 bar KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard Endpoint There are two kind of endpoints Watch for Endpoint or Retentate Volume Concentration Factor The Watch for options are OFF UV Greater than and UV Less than Endpoint Watch for Endpoint UV Less than x Level UY Greater than UV Less than Enter a Level value for a selected Watch for Endpoint e Level The signal falls outside a specified value If the first endpoint is selected a Maximum endpoint has to be entered Retentate Volume or Concentration factor Select and enter a value Endpoint Watch for Endpoint UV Less than vi Level 200 mau 7000 7000 mau Max Endpoint Retentate Volume 24 ml 24 319 ml Concentration Factor CAUTION Do not use a volume below 50 ml in the Large reservoir as this carries a risk of getting air in the retentate If the Watch for option is set to
5. Filter List XM 3KD 0 50 mm v lt D 0 50 mm e Select the type of process step Steps C Preproduct V Product _ Postproduct e The Hollow Fibre specification per filter area displays the recommended default values for the selected filter Accept or edit the values Hollow Fibre specification per filter Lumen Diameter 05 mm 0 10 10 00 mm Number of Fibes 12 11000 Surface Area 50 cm 2 40 150 cm 2 Lumen Hold Up Vol 117 mi 0 0 25 0 mi Feed Pressure Limit 4 3 bar 0 5 2 bar TMP Limit 3 0 bar 0 5 2 bar If Other filter has been selected enter Lumen diameter Number of fibers Surface area Lumen hold up volume Feed pressure limit and TMP limit for the hollow fiber Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit 114 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard E e If several filters are assembled together in parallel select in the System setup section the Number of filters 1 3 If only one filter is used keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System setup 3 Number of filters i B Extra Tubing Volume 0 mil 0 0 25 0
6. Parameter 3 name Setpoint default underlined LowAlarm 0s 0 20000 Instruction Help text Alarm Help text HighAlarm cause The flow has exceeded the HighAlarm limit LowAlarm cause The flow has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue Sets the alarm limits for Shear When reached the system is set to Pause Alarm Shear _HighAlarm Shear __LowAlarm AKTAcrossflow User Manual 11 0012 32 Edition AB 193 Strategy instructions _ 13 5 14 All Valves V1 V4 Instruction name Group Alarm_Valves System Settings Alarms ethod Manual Alarms amp Monitors Parameter name Position name default underlined T_VB1 Disabled Enabled Parameter name Position name default underlined T_VB2 Disabled Enabled Parameter name Position name default underlined R_VB Disabled Enabled Parameter name Position name default underlined P_VB Disabled Enabled nstruction Help text Enable Disable the valve alarms If a valve is not responding an alarm is raised and the system is set to Pause After at least 5 sec press Continue button If the alarm persists try to restart the system If the alarm still persists check if the valve is damaged permanently For details see AKTA Instrument Handbook Alarm Help text Valve error valve not responding After at least 5 sec press Continue button If the ala
7. Instruction name Lumen_Diameter Group Method Manual Recirc System Settings Specials Parameter 1 name Diameter Position name default underlined 1 00 mm 0 1 10 0 mm Instruction Help text Together with Total_Number_Of_Lumen Shear can be calculated using Shear 4 Qf No of Fibers n fiberradius 3 AKTAcrossflow User Manual 11 0012 32 Edition AB 181 Strategy instructions _ 13 3 12 Total_Number_of_Fibres Instruction name Group Total_Number_Of_Fibers Method Manual Recirc System Settings Specials Parameter 1 name Position name default underlined Quantity 1 1 1000 Instruction Help text Together with Lumen_Diameter Shear can be calculated using Shear 4 Qf No of Fibers n fiberradius 3 13 3 13 PPCV_Setp Instruction name Group PPCV_Setp System Settings Specials Parameter name Set point default underlined Pressure 700 0 999 Instruction Help text Sets offset on PermeateControlValve used by system during TMP_Control Flux_Contro and Permeate_Unrestricted_Flow 13 3 14 TMP_PI_RetentateControlValve nstruction name Group TMP_PI_RetentateControlValve System Settings Specials Method Manual Permeate Parameter 1 name Setpoint name default underlined P 0 100 0 000 10000 000 Parameter 2 name Setpoint name default underlined 20 000 sec 0 000 10000 000 Parameter 3 name Setpoint name default
8. P Ue Recucla Retentate UB Air Trans fer UB1 Permeate UB F Ue Recucla E U Transfer PurgeValve Transfer UB2 Ene Mune The reservoir is filled with 5 ml buffer from the Transfer valve block Which inlet is dependent of the previous type of process concentration diafiltration 1 or diafiltration2 Air Trans fer UB1 Permeate UB F UB Recuclel P UB OueT Trans fer UB2 En Mune Retentate UB Cartridge permeate The product between reservoir and R VB Out3 is chased with buffer and is emptied through R VB Out3 Air Trans fer UB1 Permeate UB F UB Recucte EMN Muna Trans fer UB2 Cartridoe permeate AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard lt a Ce uM Recovery with buffer flushes In this example the retentate volume will be recovered with two buffer flushes of 16 9 ml each 1 The reservoir is emptied through R VB Out3 Air Trans fer VB1 Permeate UB Mun UB Recucle T UB Tnz Trans fer VB2 M Y8 1In7 A1 628 2 paaa Cortridoe permeate 2 The reservoir is filled with 16 9 ml buffer according to the last prod
9. Note The value infinity is set as 9999 in UNICORN D In certain cases the D parameter can reduce the oscillations introduced by a Pl regulator D can have values between 0 and infinity where larger values have a greater effect and a value of 0 has no effect Note Most often a simple Pl regulator is preferable for control of pressure and AKTAcrossflow is therefore configured by default with the D parameter set to zero Table 12 3 PID parameters 12 4 Setting up feedback tuning UNICORN uses so called PI feedback tuning where P and are parameters that determine the tuning characteristics There are two ways to apply the feedback tuning instructions e In the Method Editor module e In the System Control module reached with the commands Manual or System Settings AKTAcrossflow User Manual 11 0012 32 Edition AB 161 Feedback tuning and PID parameters aE 12 4 1 Instruction groups The instructions are found in the following Instruction groups see Table 12 4 Instruction name Instruction Group FeedPressure_PI System Settings Specials ethod Manual Recirculation TMP_PID_RetentateControlValve System Settings Specials ethod Manual Permeate TMP_PID_PermeatePump System Settings Specials ethod Manual Permeate Flux_Pl_RetentateControlValve System Settings Specials ethod Manual Permeate PUF_PI_RetentateControlValve System S
10. e TMP control mode e ATMP value for the water flux test is entered Default for hollow fibers with cut off smaller than 0 1 um is e TMP control mode Default for hollow fibers with cut off 0 1 um and larger is e NFF Normal Flow Filtration control e Feed flow or Feed pressure is selected as Feed control and a value is entered Note When comparing status of a filter as a function of time and number of experiments it is important to have performed the same type of method ie same TMP NFF feed control value The principle of the test method is 1 If control mode is TMP DeltaP is set NFF control starts the feed pump it self 2 When the TMP NFF has been reached and the flux is stabilized for 3 minutes a normalized water flux is measured and an instruction Set_Eval_ Mark with the parameter Normalized_Water_Flux is set E KTAcrossflow User Manual 11 0012 32 Edition AB 71 Creating Preproduct methods using the Method Wizard The data is analyzed by the Evaluation module of UNICORN For example normalized water flux is plotted against number of performed runs with the filter Temperature effect due to viscosity is compensated for using a correction table Results are compared to previous tests and provide information about the quality status of the filter Normalized Water Flux 20 mmia RetFlow and 0 65 bar TMP a 8 E 3 as a a SS i en ms ce 8 LLMH ban
11. FeedPressure 00 5 20 bar e Enter a value for the selected Feed control Water Flush Water Flux Test O TMP NFF FeedFlow Feed Control for NFF 30 ml min 0 200 ml min 132 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Postproduct methods using the Method Wizard 8 ss Filter Storage Solution e The liquid in the filter is exchanged to an appropriate storage solution For information on recommended storage solution see enclosed filter instruction from manufacturer The procedure is the same as for Buffer conditioning see Section 5 2 5 and Section 5 4 7 e To include the step in the method check the Filter Storage Solution box Filter Storage Solution Note A recommended storage solution for the system is 20 Ethanol To exchange solution in the system perform the Sanitization method and use 20 Ethanol as liquid It is important to use a three way connector instead of a filter See Section 10 1 AKTAcrossflow User Manual 11 0012 32 Edition AB 133 8 Creating Postproduct methods using the Method Wizard 134 KTAcrossflow User Manual 11 0012 32 Edition AB Running AKTAcrossflow methods 9 9 Running KTAcrossflow methods 9 1 Final preparation This section describes the final preparations that should be done before starting a run The Summary page created by the Method Wizard will list calculated required volumes and corresponding inlet positions 9 1 1 Solutions 1 Immerse
12. Parameter 2 name Setpoint default underlined Delta_Base 0s 0 20000 Instruction Help text For Watch on Shear reported from feed pump The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 36 WatchPar_pNFF Instruction name Group WatchPar_pNFF System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 bar 0 00 5 20 Parameter 2 name Setpoint default underlined Delta_Base 0 00 bar 0 00 5 20 Instruction Help text For Watch on pNFF The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this con dition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval speci
13. Transfer_Purge_Valve Alarm _UV Watch_Cond Shear NFFConstanthlow Set_TrfVol_Totalizer Alarm _pH Watch_FeedPress NFF_ConstantPressure 2 Retentate_Valve_Block Start_Eval_Window2 DF_ExchangeFactor Alarm _Cond Watch_RetenPress EmptyReservoir Stop_Eval_Window2 Method_Base Alarm_FeedPress Watch_PermPress EmptyReservoirAbort Set_Eval_Mark2 ConstRVol_P Alarm_TrfPress Watch_FeedFlow Concentration_Factor Set_PermVol_Totalizer Alarm _DeltaP Watch_Reten_Flow MixerSpeed Flux_Drop_Calc Alarm _TMP Watch_PermFlow Set_ResVol_Totalizer Total_Membrane_Surface_Area Alarm _Flux Watch_TrfFlow ManSample a Alarm _pNFF Watch_RetVol FeedPressure_PIl oe P PI RetentatControlValve Alarm _Shear Watch_ResVol DeltaP_PI TMP_PI_Permeate Pump2 Alarm_Valves Watch_ PermVol EmptyResFeed_PI Flux_PI_RetentatControlvalve2 Alarm_Flowpath Watch_TransfVol RetentateHoldupVol PUF_PI_RetentatControlValve Alarm_AirSensor Watch_DeltaP PUF_PI_PermeatePump2 Alarm_ZeroLevel Watch_TMP PNFF_PI WatchPar_UV Watch_Flux Fractionation WatchPar_pH Watch_Shear FractionationStop WatchPar_Cond Watch_pNFF Reset ubeNumber WatchPar_FeedPress Watch_ConcFact FeedTube WatchPar_RetenPress WatchPar_ PermPress WatchPar _FeedFlow WatchPar _Reten_Flow WatchPar _PermFlow WatchPar _TrfFlow WatchPar_ ResVol WatchPar_ RetVol WatchPar_ PermVol WatchPar_ TransfVol WatchPar_ DeltaP WatchPar_ TMP WatchPar_ Flux WatchPar_Shear WatchPar_ pNFF WatchPar_ConcFactortor WatchPar_DF_X_Fct WatchPar _ Flux_Drop
14. o amp 8 l 1 2 3 4 s 6 Data point Fig 5 1 Example of plotted Normalized Water Flux Note Some Hollow fibers might not wet out very well with water If problems occur for example a failed water flux test it can be necessary to flush the filter manually with 10 Isopropyl alcohol For more information refer to the Hollow fiber operating guide 5 2 5 Buffer conditioning The purpose of the method is to replace the water in the system with a buffer which is suitable for the protein cell in the planned Product filtration run The created method will e Fill the system with the selected buffer e Condition the filter with buffer e The first block in the method is Preparing the system Note Ifa CIP has been performed we recommend performing a buffer conditioning after the water flush to ensure that the pH in the system is OK before starting a product method 72 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea SSS 5 3 Preproduct methods Method Wizard dialogs Note To set default values click the Set Default button in the first dialog To get help instructions for a dialog click Help or press F1 5 3 1 Basic settings Flat sheets Note To get the Method Wizard for flat sheets Flat sheet has to be selected as component in System Setup See Section 2 4 Method Wizard for Syste
15. see also Table 12 1 e PID settings for process steps where the membrane is not exposed to proteins thus avoiding the risk for membrane fouling or gel layer formation For these steps a high permeate flow rate and a rather linear pressure flow behaviour of the filter is typical AKTAcrossflow User Manual 11 0012 32 Edition AB 157 Feedback tuning and PID parameters nl e PID settings for process steps with risk of membrane fouling or gel layer formation like product steps Under these conditions a slower but more robust control accounts for low permeate flow rates and a non linear pressure flow behaviour These slow settings are selected as default values in the strategy to provide maximum robustness of the control Control PID settings P D element Feed pump Hollow Fibers control FeedPressure_PI 0 05 50 A DeltaP_Pl 0 05 20 A EmptyResFeed_Pl 0 05 20 A Flat Sheet Cassettes FeedPressure_Pl 0 05 150 A DeltaP_PI 0 05 150 A EmptyResFeed_Pl 0 05 150 A TMP control TMP_PID_RetentateControlValve 0 1 20 TMP_PID_PermeatePump membrane not exposed to proteins 0 1 20 TMP_PID_PermeatePump membrane exposed to proteins 0 03 300 TMP_PID_PermeatePump hollow fibers 0 05 75 PUF control PUF_PI_RetentateControlValve 0 1 20 A PUF_PI_PermeatePump 0 001 200 A Flux control Flux_Pl_RetentateControlValve 0 1 20 A NFF control pNFF_PI 0 2 20 A 0 05 150 Level control Const_RVol_P 50 NA A
16. 73 0 l l i hg N i my 7 W d ul 2 5 72 0 l p s 40 1 1 3 y zoh f 20 p s 0 0 0 50 100 150 200 min ee AKTAcrossflow User Manual 11 0012 32 Edition AB 149 After run procedures 3 To change the layout of the Chromatogram window right click in the window and select Properties Chromatogram Layout 10 Curve Style and Color Edit Texts Layout Library Header CuveNames YArs XAsis Cuve Peak Table Select curves to display 02 03 M04 TMP method deltaP scouting003 10_Cond M05 TMP method deltaP scouting003 10_Cond TMP method deltaP scouting003 10_pH V07 TMP method deltaP scouting003 10_F_Flow 08 TMP method deltaP scouting003 10_T_Flow v09 TMP method deltaP scouting003 10_P_Flow V10 TMP method deltaP scouting003 10_R_Flow Clear Select all Apply to all chromatograms Help 4 Inthe Curve tab page uncheck the check boxes to remove the curves you do not want to display 5 Click OK Refer to the UNICORN user manuals Online Help and the User Reference Manual UNICORN 5 1 Evaluation for Cross Flow Filtration for more information about viewing the result and customizing the layout 150 AKTAcrossflow User Manual 11 0012 32 Edition AB After run procedures 10 2 2 Printing the result To print the chromatograms 1 2 7 8 Open all chromatograms you want to print in the Evaluation module Select the File Print command In the Print Chromatograms di
17. Eil Contents 13 5 16 15 517 13 5 18 13 5 19 13 5 20 13 5 21 13 5 22 13 5 23 13 5 24 13 5 25 13 5 26 13 5 27 13 5 28 13 5 29 13 5 30 13 5 31 13 5 32 13 5 33 13 5 34 155 35 13 5 56 13 5 37 13 5 38 13 5 59 13 6 13 7 13 7 1 13 7 2 13 7 3 13 8 13 9 13 9 1 13 9 2 13 9 3 Alarm AISES Of niaaa a aa a aiaa 195 Alarmszerokev lrenenennin n o A A AED 195 Wath POR U a n A ARARE 195 WatchPar_pH 196 WatchPar_Cond 196 WatchPar_FeedPress 197 WOtGH RGF RETOMPEOSS esces E E A E eia 197 WatchPar PermPreSS ssseidsccecusssesseciccesisntcedossisbaelebesvessestateadssssesseliiedeees 198 WatchPar_FeedFlow a WatchPGr RETFIOW seen n a hentia scents 199 WatehPar Pern FOWL deccsccsstccsscaserscessenssesstanttave comsausconcagenviendavcaneucte 199 WatehPar THEI OW sssccetcedehscecds co bestalucsasssotcscheats a Ra WatehPar ROEV OM cc zsieecseccosssoicuassceosatenceeiianctiteseitiama tical weit bois WatchPar_ResVol WatchPar_PermVol WGLCHR GT TRONSV Ol iiinis a aaa 202 WatchPar_DeltaP 202 WatchPar_TMP 203 WatchPar_Flux 203 WOLCH POR She GPSi ain ice lae salads ted ight eae 204 WatehPa ONFE Fronin A noecceice EER 204 WatchPar_ _FLUX_Drop 205 WatchPar_ConcFacto 205 WatchPar_DF_X_Fact 206 Fraction Collector iNStrUCtiONS cccecccccsssesessssssssseesessssssesseessssseeesssssseee 206 PCE E EE AAA E A esse 207 UV Monitor 20
18. Feed Control e Select to perform Feed control with constant DeltaP constant Feed Flow constant Retentate Flow or constant Shear Feed Control Feed Flow v Feed flow KTAcrossflow User Manual 11 0012 32 Edition AB 117 Creating Product methods using the Method Wizard e Enter a value for the selected constant parameter for example Feedflow 500 ml min Feed Control Feed Flow v Feed flow 500 ml min 0 800 ml min Control Mode e Select to have Flux or TMP as Control mode Control Mode Flux be Flux ape e Enter a value for the selected control mode for example Flux 45 LMH Control Mode Flux he Flux 5 LMH 0 4800 LMH Endpoint e There are two kind of endpoints Watch for Endpoint or Retentate Volume Concentration Factor e The Watch for options are OFF UV Greater than and UV Less than Endpoint ee Watch for Endpoint UV Less than x Level UY Greater than Max Volume UV Less than e Enter a Level value for a selected Watch for Endpoint e Level The signal falls outside a specified value e Ifthe first endpoint is selected a Max endpoint has to be entered Retentate Volume or Concentration factor Select and enter a value Endpoint Watch for Endpoint UV Greater than b Level 10 mAU 7000 7000 mAU Max Endpoint Retentate Volume od ml 30 1021 ml Concentration Factor e ifthe Watch for option is set
19. Parameter 1 name Mode Note Position name default underlined Shear Control Mode Direct Only for HF 0s 0 20000 Instruction Help text Starts the flow on the feed pump Shear should stabilize at 98 of set value before new instruction is executed Instruc tion resets FeedFlow FeedPressure DeltaP and RetFlow To allow new instruction after stabilized Shear Watch_Stable_Baseline DeltaP should be programmed in method 13 2 6 Retentate_Valve_Block Instruction name Group Retentate_Valve_Block Method Manual Recirc Parameter name Position name default underlined Macro R VB Recycle R VB Out1 R VB Out2 R VB Out3 Description Selects the position for R VB Recycle R VB Out1 Port located prior to filter devise R VB Out2 R VB Out3 When Feed_ flow gt 0 or Constant_Retentate_Volume is active no action is allowed A warning will be raised Instruction Help text Selects the position for R VB Recycle R VB Out1 Port located prior to filter devise R VB Out2 R VB Out3 When Feed_ Flow gt 0 or Constant_Retentate_Volume is active no action is allowed A warning will be raised Alarm help text 1 Retentate_Valve_Block instruction not allowed during Constant_Retentate_Volume Alarm help text 2 Retentate_Valve_Block instruction not allowed when Feed setpoint gt 0 Alarm help text 1 Retentate_Valve_Block instruction not allowed during EmptyReservoir Alarm help text 2 Retentate_Valve_Block instru
20. Pp Pp Feed retentate and permeate pressure respectively Bar e The filtration effect in cross flow filtration is a result of the applied transmembrane pressure TMP Pe PR Mis Pp e DeltaP ap is the pressure drop between feed and retentate AP Pe Pp e Cross flow is proportional to DeltaP if permeate flow is zero Pr PR Permeate Line Fig 1 3 A cross flow system simple illustration KTAcrossflow User Manual 11 0012 32 Edition AB Introduction a z 1 5 AKTAcrossflow applications 1 5 1 Ultrafiltration Typical cut offs 1 kD to 1000 kD Ultrafiltration is a pressure driven convective process using semi permeable membranes to separate species by molecular size or shape Removing solvent from solution results in solute concentration or enrichment In ultrafiltration species smaller than the membranes pores pass through the membrane while larger species are retained UF membranes may also be used for diafiltration to remove salts or other microspecies from a solution via continuous dilution and re concentration Protein concentration The volume of sample in the reservoir is reduced The product is retained at the retentate side of the membrane If the sample volume is larger than the reservoir volume the reservoir can be continuously fed with sample solution Fed Batch Ultra filtration Retentate Proteins Membrane Permeate Small peptides and salts Cut off 1 kD to 10
21. Table 12 1 Recommended settings for PID control strategy default settings in bold style CC 158 AKTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters 12 1 3 Tuning and troubleshooting of PID control If tuning of the PID control is required it is most likely that control settings for feed pump or permeate pump need to be adjusted The settings for the Retentate Control Valve given in Table 12 1 should not be modified at all Common methods for PID optimization e g Ziegler Nichols method can be applied In most situations good results are obtained when using the following rules of thumb 1 Slower control Increase parameter decrease P parameter Faster control Decrease parameter increase P parameter 2 No drastic changes should be applied an appropriate measure is to change the parameter by factor or 2 respectively while initially keeping the P parameter constant 12 1 4 PI parameters for larger filter areas When using larger filter areas e g 100 cm it is necessary to optimize the parameters When optimizing the parameters to obtain a faster regulation a recommended start may be to e Decrease parameter by dividing with 2 e Increase P parameter by multiplying with 1 2 e An example for the parameters of TMP_PID_RetentateControlValve is to e Decrease parameter from 20 to 10 e Increase P parameter from 0 1 to 0 12 When the regulation is too fast e g noisy curves
22. nstruction Help text Sets the alarm limits for Flux When reached the system is set to Pause Alarm Flux_HighAlarm Flux_LowAlarm Alarm Help text HighAlarm cause The flow has exceeded the HighAlarm limit LowAlarm cause The flow has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue 192 KTAcrossflow User Manual 11 0012 32 Edition AB fe 13 5 12 Alarm_pNFF Strategy instructions Instruction name Group Alarm_pNFF System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined Mode Disabled Enabled Setpoint default underlined 5 20 bar 0 00 5 20 Parameter 2 name HighAlarm Parameter 3 name Setpoint default underlined 0 00 bar 0 5 20 LowAlarm Instruction Help text Alarm Help text HighAlarm cause The pressure has exceeded the HighAlarm limit LowAlarm cause The pressure has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue Sets the alarm limits for pNFF When reached the system is set to Pause Alarm pNFF_HighAlarm pNFF_LowAlarm 13 5 13 Alarm_Shear Instruction name Group System Settings Alarms Method Manual Alarms amp Monitors Alarm_Shear Parameter 1 name Position name default underlined Mode Disabled Enabled Parameter 2 name Setpoint default underlined HighAlarm 20000 st 0 20000
23. select Time as base and click Change peeve oes ee O Chen Tree O00 en oh Seo ro a lt oe z one The principle of inserting an instruction in a method is 1 Enter the 2 Select 3 Select 4 Select the 5 Click Insert to Breakpoint in Instruction group Instruction command in the add the minutes for the Macro list instruction to the method instruction Parameters Macro TransferFlow OP 7 Var Closed ermeate _Purge_Valve Set_TrfVol_Totalizer Transfer DF_Exchange_Factor MethodBase OAlaimstMion ConsthiVol_P A i eplace OWatch ConstRVolLag Oore Fig 3 2 Principle of the instruction editor The instruction is inserted in the method E Main 0 00 Base Time 0 00 Transfer_Yalve_Blocks Closed 5 Continue with the next instruction Tropan Trason Prrameters an ne va ow D 5 erwstant_Retentate Volume 5 2 is Tesrites Valve Blocks vs J Orp OWate OPemeate Turies Page Valve Cv Ser Tivol Torsizer Tunde DF Exchange _F acio A Meri jane OAlomatMen Consfiva_ P Fi ComatVell OWatch tag O Other 6 Repeat the procedure until all instructions are inserted in the method AKTAcrossflow User Manual 11 0012 32 Edition AB 53 Handling methods in AKTAcrossflow as 7 To end the method select Instruction group Other and the instruction End_Method Breakpoint Instruc
24. 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 500 kD 0 5 30 12 50 24 200 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 0 1 um 1 0 30 6 50 70 560 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 0 2 um 1 0 30 6 50 70 560 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 0 45 um 1 0 30 6 50 70 560 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 0 65 um 0 75 30 8 50 40 320 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Table 1 2 Hollow fiber cartridges with UNF fittings For assembling instructions and ordering information see AKTAcrossflow Instrument Handbook Ea 28 AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction 1 8 Associated documentation The following documentation is included with AKTAcrossflow AKTAcrossflow manuals are delivered as spiral bound books or binders as indicated in the margin Screen versions of the documentation are available from UNICORN software under Help a 1 8 1 AKTAcrossflow Installation Guide This guide provides technical information and basic operating instructions for the unpacking and installation of AKTAcrossflow 1 8 2 AKTAcrossflow Instrument Handbook This handbook provides technical information and basic operating instructions for the AKTAcrossflow In addition maintenance schedules instructions for troubleshooting and user maintenance are included 1 8 3 AKTAcrossflow Safety Handbook Provides the safety instructions that must be strictly followed for the safe use of the AKTAcro
25. 15 1 1 5 2 1 6 1 6 1 1 6 2 1 6 3 1 7 1 7 1 1 7 2 1 8 1 8 1 1 8 2 1 8 5 1 8 4 1 8 5 The User Man a sseinccsscnitisastsiccccectevosuswid E RRE 13 Mtr rodU CON iasaiten e E AANEEN E 13 GIOSSGWORLEIIS sscscsiacisticecaseccscossessssbesccdaausussecebelasosiessuusesccsssssssousevecbats 14 PRUAGI PIS sscisacinsccasusssccecsossovsnshacvsstatecccceosssoon nee E EEE AA 15 Normal flow filtration versus cross flow FiItFAtiONn uu 15 SVSTOM OWS ireen a AAAA OE NR ce tees Filtration effectS s s011000 KTAcrossflow applications CHET OM sssscsiczcarsdet seals cessscheusshaesscbsuceotestcisseluachdeissvelesl a np aa MICE OTNERQUION Mss ea a terse ater ete i iad His aaa The AKTACrOSSIIOW waacssssccssssccssssccssseccssscessescesssccesseccesececeescecreseecrecceseessereeeeeeee Description a COMTO O CS aii a A E ate eae R a hades S nsorsand VAIVES renna prae les ulate hesel cased aa aasia FIMO cartridges e aaae aa a Kvick Start flat sheet COSSCtLOS ceeeecssssessscsssssesssssssssesssssssessssssseeessssssees 27 Hollow fibers H Associated documentation ss ssssessssssisssssresssstrsssstersssterrssrerresseerresseerrrse 29 KTAcrossflow Installation Guide sscccssssssscssssssssssecssssssesssseesssssesssssseesen 29 KTAcrossflow Instrument HONGDOOK vvcssssssssssecesssssssesseesssssssessseeesen 29 AKTAcrosstow Safety HONABOOK ssecsscccsscsessssssssesessisssssusessennesssnsseee KTAcrossflow Method Handbook a UNICORN CGOCU
26. 5 Inthe UF Process Optimization dialog e Select to perform buffer conditioning before experiment or not e Enter Sample Volume Note This will be the total volume in the reservoir retentate holdup volume Note The created optimization method will include a chasing block which will fill the system with sample without any dilution e Select number of crossflows e Select Feed Parameter Retentate Flow Note The other options are DeltaP and Feed flow e Enter the retentate flows of the experiment plan e Enter the TMP settings of the plan e There is an option to select Retentate recovery after the run The retentate loop will be emptied according to the procedure Recovery with no flushes see Section 7 1 6 UF Process Optimization Buffer Conditioning before Start Fill step will use 26 ml extra volume i e if you write 100 ml when the system will use 126 ml Sample Volume 100 38 350 ml Note Sample volume is total retantate volume Number of crossflows 3 Mi Feed Parameter Retentate Flow x Crossflow 1 24 ml min 0 600 ml min Crossflow 2 16 ml min 0 600 ml min Crossflow 3 8 ml min 0 600 ml min TMP Test Points Number of Points 5 TMP Paint 1 1 0 bar 0 0 5 2 bar TMP Point 2 1 6 bar 0 0 5 2 bar TMP Point 3 22 bar 0 0 5 2 bar TMP Point 4 28 bar 0 0 5 2 bar TMP Point 5 34 bar 0 0 5 2 bar Retentate Recovery AKTAcrossflow User Manual 11 0012 32 Edition
27. AB 93 6 Process optimization in Ultrafiltration Sry 6 Click Next When Next is clicked a Summary of required solutions will be displayed Note An extra volume of sample is needed which is specified in the Summary page 7 To display the list of variables click Finish To edit a variable s value click in the cell and change the value 8 To edit a variable name click Edit Variable 9 Select a variable and edit Note favariable s value is changed it will affect the created method A changed variable may also affect the calculated required solution volumes in the previous Summary dialog Note We do recommend to not edit any variables especially not the detail variables 10 To view the method click the Text Editor icon The method is displayed as blocks 11 To view and expand the method Use the keyboard arrows 12 To save this first method step select File Save As in the UNICORN Method Editor 13 Browse for a folder enter a method name and click OK 6 4 Continuing the experiment Two protein concentrations were included in the experiment plan So the procedure has to be repeated for the other protein concentration 1 Run the method with the first protein concentration 2 Clean the filter 3 Run the method with the second protein concentration Note It may also be possible to perform this kind of experiment using the Scouting function in UNICORN See UNICORN documentation 94 KTAcrossflow User Manual 11 0012
28. Air Trans fer VB1 Permeate UB Pegal aee T UB Ine Trans fer VB2 T UB Tn 5 Torinse the distance between Retentate valve block and reservoir the Retentate VB is set to recirculation 25 ml min 15 ml Air Trans fer VB1 Permeate UB F UE Recucte T UB Tnd Trans fer VB2 M Y8 1In7 AED a Cartridoe permeate UB 0ut 1 6 5mlis emptied and the reservoir is filled with 5 ml 7 At Constant Retentate Volume a new flush is performed out to R VB Out2 Air Trans fer VB1 Permeate UB T VB Ini UB Recucle Trans fer VB2 T VB Tn Soe a paad Cartridoe permeate 8 The reservoir is filled with a volume defined by the used filter area Air Trans fer VB1 Permeate UB MPSS Sie a T VB Inz Trans fer VB2 3 Retentate UB T UB Tn 5 Gi i o paaa Cartridee permeate EA KTAcrossflow User Manual 11 0012 32 Edition AB 81 Creating Preproduct methods using the Method Wizard 82 9 Recirculation at the retentate side for one minute with FeedPressure control Air Trans fer UB1 Permeate UB F UB Recuclel P UB OueT Muna Transfer UB2 Mune Ue Ine 10 Then the reservoir is emptied through R VB Out2 Air Transfer UB1 Mueni Permeate UB F UB Recuclel E E Mena Transfer VB2 AED a Cartridge permeate 5 4 2 Visualized procedure of Preparing the system Depending on previous step some of methods begin
29. Feed Flow Retentate Flow Shear AKTAcrossflow User Manual 11 0012 32 Edition AB 119 Creating Product methods using the Method Wizard Sey e Enter a value for the selected constant parameter for example Feedflow 500 ml min Feed Control Feed Flow x Feed flow 500 ml min 0 600 ml min Control Mode e Select to have Flux or TMP as Control mode Control Mode Flux be Flux HE e Enter a value for the selected control mode for example Flux 45 LMH Control Mode Fus x Flux 45 LMH 0 4800 LMH Endpoint e There are two kind of endpoints Watch for Endpoint and Permeate Volume Diafiltration DF Exchange Factor Endpoint Watch for Endpoint OFF f x Permeate Volume 0 ml 0 3999 ml DF Exchange Factor e The Watch for options are shown in the screen below Endpoint Watch for Endpoint UV Less than 3 Cond Greater than and Stable Cond Level Cond Less than and Stable Cond OFF racm PH Greater than and Stable pH Permeate Volume pH Less than and Stable pH O DF Exchange Factor ween ell e Enter parameters for the selected Watch for Endpoint e Level The signal falls below a specified value e Time Stable Delta Base The signal is stable within the limits of the Delta Base value for the period specified by the minutes parameter Time Stable Endpoint Watch for Endpoint Cond Less than and Stable Cond v Level 2 mS cm 0 999 mS cm Time Stable 1 min 0 9999 min Delta B
30. Normal flow filtration NFF is used as control mode Feed flow is not necessary GED 2 If Water flux test is a part of Water flush or Rinse the method will be ended as these methods If the test is performed as a single method the reservoir will be emptied using the function EmptyReservoir 3 The rest of the reservoir liquid is emptied through R VB Out2 At level O ml in the reservoir the test is complete conse SSS 88 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea Se 5 4 7 Visualized procedure of the Buffer conditioning method 1 The system is prepared according to the procedure Preparing the system described in Section 5 4 1 The Transfer Inlet used T VB In2 is connected to buffer 2 The reservoir is filled with a volume defined by used filter area Air Trans fer VB1 Permeate UB M Y8 Ini P UB Recucle T UB Tn2 T UB Tn T UB Ind Trans fer VB2 Retentate UB T UB Tn R VB Out3 5 A1 su 2 Cartridge permeate 3 Permeate recycle to waste is opened The permeate pump is started using PUF control Flat sheets or TMP Flux control Hollow fibers 30 ml is emptied through Transfer Purge Valve Waste Then the valve is set to reservoir Air Trans fer VB1 Permeate UB
31. Properties An alternative is to select View Properties For more information about customizing the view panes see the UNICORN user manuals To stop the run before it is finished click the End button above the Run data view pane Note Ifthe run is in Hold and the run pauses it is necessary to click Hold to continue AKTAcrossflow User Manual 11 0012 32 Edition AB 9 5 for the sample volume 1 4 5 Running KTAcrossflow methods ex Manual sampling during run Itis possible to perform sampling from the reservoir during a run and compensate In UNICORN select Manual Recirc and select the instruction ManSample CrossFlow Wizard 06T Recirc Instructions Instructions Recire Permeate O Transfer O Alarms amp Mon Other Open the lid of the reservoir Parameters Volume 0 00 100 00 2 Sml Auto update If this is checked the parameter fields will be updated during method run Enter the planned sample volume Take a sample with an appropriate pipette device Immediately click Execute in the ManSample dialog Note f not clicked immediately the system will start to compensate the lost volume before ManSample instruction is used 6 Close the lid Note To get an Eval mark in the result file it may be convenient to set an Eval_Mark using the instruction Set_Eval_Mark with the parameter ExtData_vs_Capacity AKTAcrossflow User Manual 11 0012 32 Edition AB 143 9
32. System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 bar 0 00 5 20 bar Parameter 2 name Setpoint default underlined Delta_Base 0 00 bar 0 00 5 20 bar nstruction Help text atch on DeltaP reported The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 202 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions m 13 5 33 WatchPar_TMP Instruction name Group WatchPar_TMP System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 bar 0 00 5 20 bar Parameter 2 name Setpoint default underlined Delta_Base 0 00 bar 0 00 5 20 bar Instruction Help text Watch on TMP reported The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected pe
33. User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard uM Concentration 1 Concentration starts with TMP control which activates the permeate pump and permeate valve block P VB Out2 is opened lt gt 2 Theconcentration continues still with P VB Out2 open until the end point has been reached specified Concentration factor Retentate volume or Watch condition T UB Tn2 Perwecte mae P Diafiltration The volume in the reservoir is the volume specified as concentration end point for example 50 ml 1 At Constant Retentate Volume 50 ml and TMP control the permeate and transfer pumps are activated and P VB Out3 and T VB In3 are opened 2 Liquid leaving the system through P VB Out3 is replaced with diafiltration buffer through T VB In3 until the end point has been reached DF Exchange factor Permeate volume or Watch for Endpoint pH conductivity or UV If two diafiltration steps are performed the second step will use buffer from T VB In4 je Panata Pump a AKTAcrossflow User Manual 11 0012 32 Edition AB 109 Creating Product methods using the Method Wizard 110 Product recovery with no flushes Option to Recirculate before emptying the reservoir Flowrates are the same as for the previous step Air Transfer UB1 Permeate UB
34. W 0 00 Block Step1_Conc W 0 00 Block Restore CONCENTRATION 0 00 End_Block W 0 00 Block RECOVERY To save the method select File Save Select location Enter method name select System and Technique Click OK Note Any must be selected as Technique Save As Name System Size Type Prev Folder BufferConditi AkTAcrossflow 185KB Method File AkTAcrossflow 185KB Method File AkTAcrossflow 184KB Method File AkTAcrossflow 189KB Method File AkTAcrossflow 185KB Method File Tei AKTAcrossflow vis 187KB Method File Ayo AkTAcrossflow 187KB Method File Anion_Exchange AkKTAcrossflow 187KB Method File Cation_Exchange ak AkTAcrossflow 187KB Method File RPC HIC Size_Exclusion Affinity Method name Tesi Chromatofocusing OK Cancel Help KTAcrossflow User Manual 11 0012 32 Edition AB 51 Handling methods in AKTAcrossflow 52 3 4 Example of creating an AKTAcrossflow method This is an example of a simple method which checks the function of the valves and pumps of AKTAcrossflow system m 0 00 0 00 0 00 0 00 0 50 0 50 0 50 1 00 1 00 1 00 1 00 1 50 1 50 1 50 2 00 2 00 2 00 2 00 2 00 2 50 3 00 3 00 3 00 3 00 3 00 3 50 3 50 3 50 4 00 4 50 Base Time Transfer_ alve_Blocks Closed Transfer_Purge_ alve To_Reservoir Permeate_ alve_Block Closed Transfer_ alve_Blocks T B In1 Permeate_ alve_Block P B Recycle
35. by a U in the column immediately to the left of the Variable column e To edit a variable s value click in the cell and change the value Note Ifa variable s value is changed it will affect the created method A changed variable may also affect the calculated required solution volumes in the previous Summary dialog Note We do recommend to not edit any variables especially not the detail variables Note Do not delete variables with the Edit Variable Delete command Start Protocol Questions Result Name Variables Scouting Notes Gradient Columns Reference Curves Evaluation Procedures Method Information Block Variable Value Range Start_Settings_System TMP_Alarm bar 4 00 0 00 5 20 Retentate_Holdup ml 19 00 1000 00 1000 00 jAlarm_FeedPress Feed_Press_Alarm bar 4 00 0 00 5 20 Empty_Reservoir_Completely FeedPress_EmptyRes bar 3 00 0 00 5 20 MaxFlow_EmptyRes ml min 600 0 0 0 600 0 Start_Settings_FS Filter_Area cr 50 0 1 0 1200 0 Rinse_RVB_Vol Rinse_Ret_Vol ml 50 00 0 00 999999 00 Rinse_Vol Rinse_Volume ml 300 00 0 00 999999 00 CIP_Flux CIP_Max_Flux LMH 30 0 0 0 4800 0 TMP_Limit_CIP bar 4 00 0 01 5 20 Time_PreCIP1 Pre_CIP1_Time min 60 00 0 00 999999 00 Flush_Vol Flush_Volume ml 300 00 0 00 999999 00 TMP_PreWaterFluxT est TMP_PreWaterFlux bar 1 00 0 00 5 20 BufferCond_Fill Vol ButferCond_Fill
36. constant Set point 20 of Feed R PCV Closed Pressure fulfilled ONFF flow P PCV Opened Normal Flow Filtration is used in Water flux test for hollow fibers with cut off 0 1 um and larger AKTAcrossflow User Manual 11 0012 32 Edition AB 25 Introduction 1 6 3 Sensors and valves The system consists of several valves and sensors as shown in Fig 1 6 There are sensors for e Pressure e Conductivity e UV absorbance DH e Temperature e Reservoir level and e Air The sensors and valves are described in KTAcrossflow Instrument Handbook Transfer Flow Pressure Restrictor Sensor Transfer Transfer Pump Valve Block 1 Module A Paa Transfer line Transfer Purge Valve Air Sensor In pd wastet In3 In4 Level amp Retentate Transfer Temperature Pressure Control Valve Block 2 Sensor R Valve Stirrer n5 ae Permeate Reservoir R PCV Retentate Valve Block In6 Valve Block iha Recycle n ns Out2 Out1 Out 3 Out2 Out 3 Recirculation line Retentate Permeate line Pressure Sensor Feed Permeate Permeate Pressure Pressure Pressure Control RA Sensor Sensor Valve gt GD GA MANNA P Pe Cond UV pH gt S eX LZ P PCV Feed Pump out ui Cartridge Permeate Pump Module B Fig 1 6 Detailed flow path The output from the sensors are monitored during the process run with UNICORN 26 KTAcrossflow User Manual 11
37. default underlined Delta_Base 0 0 ml min 0 200 nstruction Help text atch on flow reported from transfer pump The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 28 WatchPar_RetVol Instruction name Group WatchPar_RetVol System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 ml 0 00 80000 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 ml 0 00 80000 00 nstruction Help text atch on retentate volume The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condi tion For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_basel
38. ea e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 350 mi 1100 mi Tubing kit Small ID 1 7 mm O Large ID 2 9 mm 5 3 3 Preproduct setup Method Wizard for System AKTAcrossflow2 x Preproduct setup Basic Settings Preproduct Steps Rinsing Summary Fiter CIP mcr Length of Time marz Length of Time 60 min 0 9999 min Z Water Flush between CIP 60 min 0 9999 min Z Water Flush H Water Flux Test TMP i bart052baq ONFF Z Buffer Conditioning Frish Tarcal Set Dota Rinsing e To include a Rinsing in the method check the Rinsing box Rinsing AKTAcrossflow User Manual 11 0012 32 Edition AB 77 Creating Preproduct methods using the Method Wizard Filter CIP e To include a Filter CIP check the Filter CIP box e There is an option to select two CIP with a optional Water flush between e The circulation time of CIP solution is entered in the Length of Time boxes Filter CIP CIP 1 Length of Time 60 CIP 2 Length of Time 60 Water Flush between CIP min 0 9999 min min 0 9999 min e Ifthe large reservoir is used Volume to Fill has to be entered Note The minimum volume is 200 ml Volume to Fil 200 nl 200 1100 mi Length of Time 60 min 0 3999 min Oop Water flush e To include a Water flush in the method check the Water Flush box Note After a Filter CIP we reco
39. for the period specified by the minutes parameter Time Stable Endpoint Watch for Endpoint Cond Less than and Stable Cond Level 2 mS cm 0 999 mS cm Time Stable 1 min 0 9999 min Delta Base 2 mS cm 0 999 mS cm e Ifthe first endpoint is selected a Max endpoint has to be entered Permeate Volume or Diafiltration DF Exchange Factor Select and enter a value Max Endpoint Permeate Volume DF Exchange Factor ho 0 50 AKTAcrossflow User Manual 11 0012 32 Edition AB 105 Creating Product methods using the Method Wizard e Ifthe Watch for option is set to OFF the selected Permeate Volume or Diafiltration DF Exchange Factor will be the only endpoint e For two endpoints the endpoint of the process will be when one of the two endpoint conditions is met 7 1 6 Recovery dialog Method Wizard for System AKTAcrossflow1 Recovery Basic Settings Recovery Option Product Steps Step 1 Initial Recovery collected through RVB 3 includes empty reservoir Step 2 and retentate tubing product displacement Recovery Summary V Recirculation before initial Recovery with no backpressure V Buffer Flushes collected through RVB 1 Number of Buffer Flushes 1 O2 Volume Flush 1 163 mol 16 9 350 m V Recirculation before Flush Recovery e Inthe Recovery dialog there are two alternatives e No Recovery An option to select if the retentate volume will be drained
40. is corrosive and therefore dangerous to health Avoid spillage and wear safety glasses safety gloves and protective lab coat CAUTION Always make sure that the system components are compatible with sodium hydroxide at the concentration time and temperature used 1 Prepare a sanitization solution which is compatible with the AKTAcrossflow system for example 1 M NaOH Reservoir 350 ml 5000 ml Reservoir 1100 ml 7000 ml 2 Fill the Rinsing system bottles with 1 M NaOH 2 x 200 ml 3 Replace the filter device with a three way connector 4 Replace the pH electrode with a dummy electrode 5 Remove the float from the reservoir 6 Ifanairfilter is connected to the reservoir remove the filter Connect a tubing to waste The tubing is included in the Reservoir cleaning kit 11 0033 86 WARNING The reservoir is over filled during the sanitization It is important to have tubing to waste NaOH is corrosive and therefore dangerous to health Avoid spillage and wear safety glasses safety gloves and protective lab coat Note To avoid siphoning back into the reservoir keep tubing free from solution in the waste bottle 7 Place all inlet tubing in the prepared sanitization solution 8 Place all outlet tubing in a waste container AKTAcrossflow User Manual 11 0012 32 Edition AB 147 After run procedures 9 Run the method Note Itis also possible to run System sanitization as an Instant Run see Sectio
41. method run 6 Start Flux Control under Permeate set flux to 800 LMH and pump for three minutes AKTAcrossflow System 1 Permeate Instructions Instructions Parameters i TMP Control a z O Recic Flux Control A Fu a any Permeate Permeate_Valve_Block eod LMH Permeate_Unrestricted_Flow zeoe O Transter Stort Eval Window TMPlimit 0 01 5 20 top_E val window T O AlamstMon Set_Eval_Mark 0 01 bar Off O Other Set_PemiVol_Totalizer Auto update If this is checked the parameter fields will be updated during method run es 37 System preparation SSS 7 After three minutes set Flux to O LMH and stop feed pump 8 Select EmptyReservoir under Recirc select R VB Out1 as RetValveOutlet set MaxFeedPressure to 0 4 bar and click Execute AkKTAcrossflow System 1 Recirc Instructions x Instructions Parameters Recic Feed RetValveQutlet inset O Petmeate Recdrteesias RVBOuD Jelete Transfer Retr Valve Block MaxFeedPress 0 00 5 20 O Alarms amp Mon ee 0 4 T bar O 0e ee m Kakkar 10 600 e 800 0 a ml min Auto update If this is checked the parameter fields will be updated during method run System will stop when reservoir is empty 9 Immerge rinsing system tubing to rinsing solution bottles and repeat from the beginning Step 3 ss 38 AKTAcrossflow User Manual 11 0012 32 Edition AB System preparation 2 4 Selecting filter component for the system strategy 1 InUNICOR
42. methods using the Method Wizard ea 2 The reservoir is completely filled to 350 ml 350 ml small reservoir large reservoir Entered Desired volume with CIP1 solution Constant Retentate Volume is activated The permeate valve is set to P VB Out1 A retentate flow is started The permeate pump is started by Flux control The first 30 ml passes out through P VB Out1 Air Trans fer UB1 Permeate UB Transfer PurgeValve P UB Recucie EMN MEn Muna Trans fer UB2 UB In MEn Mune 3 After 30 ml the permeate valve is set to P VB Recycle and the liquid is recycled to the reservoir Air Trans fer UB1 Permeate UB P UB Recucie Muna Mena Trans fer UB2 Ls Cartridge Permeate R UB OutT 4 After the specified CIP 1 circulation time for example 30 min ConstRetVol is disabled and the reservoir is emptied through R VB Out2 Air Permeate UB F Ue Recucls Ap y Cartridoe permeate fF UB OutT AKTAcrossflow User Manual 11 0012 32 Edition AB 85 Creating Preproduct methods using the Method Wizard 5 If Water Flush between CIP has been selected the reservoir is filled with 100 ml water and 50 ml leaves via Permeate recycle to Transfer Purge Valve Waste Air Trans fer UB1 uen Permeate UB F Ue Recucla F UB Oubt
43. ml min 0 0 600 0 Parameter 2 name Delta_Base Setpoint default underlined 0 0 ml min 0 0 600 0 nstruction Help text atch on flow reported from feed pump The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 198 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions a 13 5 25 WatchPar_RetFlow Instruction name Group WatchPar_RetFlow System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 ml min 0 00 600 ml min Parameter 2 name Setpoint default underlined Delta_Base 0 00 ml min 0 00 600 ml min Instruction Help text Watch on flow reported for retentate flow The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the St
44. process fluid that passes through a membrane 14 AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction 1 4 Principles 1 4 1 Normal flow filtration versus cross flow filtration GE Healthcare AKTAcrossflow separation cartridges flat sheet cassettes and hollow fibres are designed for cross flow tangential flow TFF operation Unlike normal flow filtration or dead ended filtration cross flow methodology continuously sweeps the membrane surface by recirculating the feed flow across it Doing so minimizes blinding of the membrane and promotes consistent long term productivity It also allows units to be cleaned stored and re used as needed Feed Flow Cross Flow i ot i ae a Filtrate Flow Permeate Flow Fig 1 1 Normal flow and tangential flow filtration 1 4 2 System flows As the feed flow is pumped through the cartridge the retentate the material excluded by the membrane pores continues through the recirculation loop The permeate including solvent and solutes is transported through the membrane pores and is collected separately Permeate au E Retentate i Permeate Individual Membrane Lumen Fig 1 2 Principle of cross flow filtration AKTAcrossflow User Manual 11 0012 32 Edition AB 15 Introduction 16 1 4 3 Filtration effects Term Definition Or Qr Op Feed retentate and permeate flow respectively ml min Pe
45. specification per filter area displays the recommended default values for the selected filter type Accept or edit the values Flat Sheet specification per filter Surface Area 50 cm 2 40 150 cm 2 Filter Hold Up Volume 1 4 mil 0 0 25 0 mi Feed Pressure Limit 4 0 bar 0 5 2 bar TMP Limit 4 0 bar 0 5 2 bar If Other filter type has been selected enter Surface Area Hold Up Volume Feed pressure and TMP limits for the flat sheet Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit e if several filters are assembled together in parallel select in the System setup section the Number of filters 1 3 If only one filter is used keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System setup Number of filters 1 a Extra Tubing Volume 0 ml 0 0 25 0 mi e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 350 ml 1100 mI Tubing kit Small ID 1 7 mm O Large ID 2 9 mm Ge 74 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ee 5 3 2 Basic settings Hollow fibers Note To get the Metho
46. the concentration This is possible when concentration is the first step The initial volume in reservoir has to be defined on the next page Step 1 Concentration Reservoir fill volume ea ml 5 350 ml to start concentrate AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard A Minimum working volume is displayed The minimum working volume is the system holdup volume including filter with an addition of a small volume in the reservoir This volume depends on the reservoir size tubing kit and filter volume This is the lowest working volume that is recommended Note Minimum working volume will vary with reservoir size and tubing kit used and is not the same as system holdup volume To ensure process performance a smaller volume is added to the system holdup volume in the calculation of Minimum working volume For information on system holdup volume see AKTAcrossflow Instrument Handbook CAUTION Do not use a volume below 50 ml in the Large reservoir as this carries a risk of getting air in the retentate 7 2 4 Step 1 Concentration dialog Method Wizard for System AKTAcrossflow System 2 Step 1 Concentration Basic Settings Product Steps Feed Control ete ae Step 1 Retentate flow tol min 0 600 ml min Step 2 Recovery Flux LMH 0 4800 LMH Summary Endpoint Watch for Endpoint OFF Retentate Volume 27 ml 30 1021 mil O Concentration Factor coe J m J F
47. to OFF the selected Retentate Volume or Concentration factor will be the only endpoint CAUTION Do not use a volume below 50 ml in the Large reservoir as this carries a risk of getting air in the retentate 118 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard e For two endpoints the endpoint of the process will be when one of the two endpoint conditions is met bd Concentration factor Max Endpoint O Retentate Volume Concentration Factor 0 50 Note A Concentration factor between 0 50 can be entered A value of 1 means no concentration A value lt 1means dilution Values between 0 1 are used by the software to compensate for dilutions for example due to the hold up volume on the retentate side This feature can be used when performing diafiltration of small volumes as a planned single step See Section 7 1 1 Diafiltration 7 2 5 Step 2 Diafiltration dialog Method Wizard for System AKTAcrossflow System 2 Step 2 Diafiltration Basic Settings Product Steps Feed Control L Step 1 Retentate flow O mimin 0 600 ml min Step 2 A Recovery Flux LMH 0 4800 LMH Summary Endpoint Watch for Endpoint Permeate Volume D o ml 20 9999 ml O DF Exchange Factor Fns Feed Control e Select to perform Feed control with constant DeltaP constant Feed Flow constant Retentate Flow or constant Shear Feed Control Feed Flow v Feed flow
48. underlined D 1 000 sec 0 000 10000 000 nstruction Help text These parameters are used to tune the feedback control when TMP_Control is active For details see Feedback tuning chapter in User Instrument Manual 182 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions _ 13 3 15 TMP_PI_PermeatePump Instruction name Group TMP_PI_PermeatePump System Settings Specials Method Manual Permeate Parameter 1 name Setpoint name default underlined P 0 030 0 000 10000 000 Parameter 2 name Setpoint name default underlined 300 000 sec 0 000 10000 000 Parameter 3 name Setpoint name default underlined D 0 000 sec 0 000 10000 000 Instruction Help text These parameters are used to tune the feedback control when TMP_Control is active For details see Feedback tuning chapter in User Instrument Manual 13 3 16 Flux_Pl_RetentateControlValve Instruction name Group Flux_Pl_RetentateControlValve System Settings Specials Method Manual Permeate Parameter 1 name Setpoint name default underlined P 0 100 0 000 10000 000 Parameter 2 name Setpoint name default underlined 20 000 sec 0 000 10000 000 Instruction Help text These parameters are used to tune the feedback control when Flux_Control is active For details see Feedback tuning chapter in User Instrument Manual AKTAcrossflow User Manual 1
49. variables Variables Variable Value Range Fiter_Area or 40 0 1 0 1000 0 P_Parameter_FeedPress _Parameter_FeedPress P_Parameter_DeltaP 0 005 0 001 1000 000 150 000 0 000 10000 000 0 100 0 001 1000 000 Parameter_DeltaP 10 000 0 000 10000 000 arameter_EmptyFeedPress 0 005 0 001 1000 000 _Parameter_EmptyFeedPress P_Parameter_PUF_Perm 150 000 _ 0 000 10000 000 0 001 0 001 1000 000 _Parameter_PUF_Perm P_Parameter_Flux_Ret 60 000 0 000 10000 000 0 200 0 001 1000 000 _Parameter_Flux_Ret P_Parameter_TMP_Perm 50 000 0 000 10000 000 0 001 0 001 1000 000 _Parameter_TMP_Perm 25 000 0 000 10000 000 Empty_Reservoir_Completely Ret Vor tmh 17 00 0 00 5000 00 RetFlow_Step1 Ret_Flow_Step1 ml min 0 0 0 0 600 0 TMP_Step1 TMP Step fba 0 00 0 00 5 20 Flush_Vol Flush_Volume ml 400 00 0 00 999999 00 ow Show details Show unused variables Display tooltip for extended variable cells Cancel 6 Click the Next button to proceed through several pages AKTAcrossflow User Manual 11 0012 32 Edition AB Running AKTAcrossflow methods ex S 7 Select Evaluation procedure if appropriate 8 Click the Start button on the last page The Run starts Result Name Run info Date 2005 User default Method CN WizardGeneratedm03
50. 0 150 cm 2 Fiter Hold Up Volume 1 4 ral 0 0 25 0 mi Feed Pressure Limit 40 bar 0 5 2 bar TMP Limit eo bar 0 5 2 bar System setup Number of filters Extra Tubing Volume mi 0 0 25 0 ml Reservoir Size 350 ml 1100 mi Tubing kit Small ID 1 7 mm Large ID 2 9 mm Set Default 3 Gothrough all selections on the Method Wizard pages Click the Next button to proceed through several pages C t AKTAcrossflow User Manual 11 0012 32 Edition AB 139 Running AKTAcrossflow methods 4 Click the Run button on the last page Method Wizard for System AKTAcrossflow System 1 Summary Basic Settings System Preproduct Steps Inlet number AKTAcrossflow System 1 Definition Volume max Product Steps Step 1 Recovery Postproduct Steps Summary Retentate out Sample Conditioning buffer Diafiltration buffer 1 Diafiltration buffer 2 Water CIP solution 1 CIP solution 2 Storage Solution Definition 40 ml 1220 ml See note See note 2830 ml 1040 ml 0 ml 0 ml 1 2 3 Peimeate out Flush Waste Product Definition 1 2 3 Note 1 Note 2 5 Verify the method on the Variables page Waste Cone DF Please note that this info will be lost after Finish is executed Print this page in order to keep this information The DF volumes ate not estimated here Print Set Default Note We do recommend to not edit any variables especially not the detail
51. 00 kD M KTAcrossflow User Manual 11 0012 32 Edition AB 17 Introduction Diafiltration Diafiltration is a unit operation that incorporates ultrafiltration membranes to remove salts or other microsolutes from a solution Small molecules are separated from a solution while retaining larger molecules in the retentate Buffer exchange is a filtration process used for the removal of smaller ionic solutes whereby the feed solution is continuously or repeatedly filled up with a buffer One buffer is removed and replaced with an alternative buffer A buffer exchange is typically run after an UF concentrating step using the same cassette hollow fiber as for the UF step Again the product is retained at the retentate side in the reservoir Typical cut offs are 1 kD to 1000 kD Ultra filtration Retentate Proteins in new buffer Membrane Permeate Small peptides and salts old buffer Cut off 1 kD to 1000 kD 18 AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction eC ____ 1 5 2 Microfiltration Microfiltration is a pressure driven convective process intended to separate larger unsoluble particles sub micron size species resulting in solution concentration or clarification It is usually an upstream recovery process where cells and cell debris are separated from the other components in the solution The product can be the cells in the retentate or the clarified protein solution in the permeate Typica
52. 000 _Parameter_FeedPress 150 000 0 000 10000 000 P_Parameter_DeltaP 0 100 0 001 1000 000 l_Parameter_DeltaP 10 000 0 000 10000 000 P_Parameter_EmptyFeedPress 0 005 0 001 1000 000 _Parameter_EmptyFeedPress 150 000 0 000 10000 000 P_Parameter_PUF_Perm 0 001 0 001 1000 000 _Parameter_PUF_Perm 60 000 0 000 10000 000 P_Parameter_Flux_Ret 0 200 0 001 1000 000 _Parameter_Flux_Ret 50 000 0 000 10000 000 P_Parameter_TMP_Perm 0 001 0 001 1000 000 l_Parameter_TMP_Perm 25 000 0 000 10000 000 Empty_Reservoir_Completely Ret Vol ml 0 00 5000 00 Start_Settings_FS Filter_Area cr 40 0 1 0 1000 0 RetFlow_Step1 Ret_Flow_Step1 ml min 25 0 0 0 600 0 TMP_Step1 TMP_Step_1 bar 2 50 0 00 5 20 Step1_Conc_G Conc_Factor_Step1Cont 0 00 50 00 Show details Show unused variables Display tooltip for extended variable cells 4 Click Next or Back to go through the dialog boxes and add the information that is required as well as your own comments Note This list will also be displayed in Method Notes in the Run Setup dialog Note Incorrect information is shown in the Method Duration tab in the Method Information dialog Instead use the information shown in the Summary dialog in the Method Wizard The Summary information is also displayed in the Method Notes tab in the Start protocol SSS AKTAcrossflow User Manual 11 0012 32 Edition AB 137 9 Running KTAcrossflow methods 5 Click the START button in the Result Name dialog box T
53. 0012 32 Edition AB Introduction ey 1 7 Filtration cartridges 1 7 1 Kvick Start flat sheet cassettes The cassettes are available in 50 cm for ultrafiltration process development with minimum working volume O PERM 1 O FEED Fig 1 7 Ultrafiltration cassette Name Kvick Start Cut offs 5kD 10kD Select 10kD 30kD 50kD and 100kD Nominal area 50 cm Feed flow rate 300 480 LMH 25 40 ml min Flux range low 15 LMH 1 2 ml min Flux range high 150 LMH 12 0 ml min 30 200 L m vol challenge 135 900 ml The ranges relate to NMWC and are typical not absolute values Table 1 1 Flat sheet cartridges with UNF fittings For ordering information see AKTAcrossflow Instrument Handbook 1 7 2 Hollow fibers GE Healthcare manufactures a complete selection of cross flow ultrafiltration and microfiltration membranes with a surface area of 50 cm specially dedicated for the AKTAcrossflow instrument These designs are configured to both afford convenient linear scaling and to optimize any candidate application around reproducible and predictable fluid mechanics The tube like structure is made from a membrane and sealed inside a cross flow cartridge When in use the feed stream flows into the inner diameter of one end of the hollow fiber and the retentate the material that does not permeate through the walls of the hollow fiber flows out the other end The
54. 1 AKTAcrossflow User Manual 11 0012 32 Edition AB 125 ED Creating Postproduct methods using the Method Wizard A 8 2 Postproduct methods Method Wizard dialogs 8 2 1 Basic settings Flat sheets Note To get the Method Wizard for flat sheets Flat sheet has to be selected as component in System Setup See Section 2 4 Method Wizard for System AKTAcrossflow1 Basic Settings Basic Settings Method 3 Preproduct Steps Filter List Kvick Start 5KO s Product Steps Concentration Diafitration Recovery Postproduct Steps Steps Summary V Preproduct v Product Postproduct Flat Sheet specification per filter Surface Area i cm 2 40 150 cm 2 Filter Hold Up Volume 1 4 ml 0 0 25 0 ml Feed Pressure Limit 40 bar 0 5 2 bar TMP Limit bar 0 5 2 bar System setup Number of filters Estra Tubing Volume mil 0 0 25 0 ml Reservoir Size 360 mi 1100 mi Tubing kit Small ID 1 7 mm Large ID 2 9 mm t Set Default e inthe Method list select Ultrafiltration Basic Settings Method Ultrafiltration v 2 7 Calibrate level sensor Filter List Installation Test System Sanitization Ultrafiltration e inthe Filter List available GE Healthcare filters are displayed Select the used filter or select Other Method Ultrafiltration v Filter List Kvick Start 5KD v 10KD 10 KD Select i 30KD Ti Kuck Stat 100 RD Preprodul Other gt A 126 KTAcrossfl
55. 1 0012 32 Edition AB 183 Strategy instructions Es 13 3 17 PUF_PI_RetentateControlValve Instruction name Group PUF_PI_RetentateControlValve System Settings Specials Method Manual Permeate Parameter 1 name Setpoint name default underlined P 0 100 0 000 10000 000 Parameter 2 name Setpoint name default underlined 20 000 sec 0 000 10000 000 Instruction Help text These parameters are used to tune the feedback control when Permeate_Unrestricted_ Flow is active For details see Feedback tuning chapter in User Instrument Manual 13 3 18 PUF_PI_PermeatePump Instruction name PUF_PI_PermeatePump Group System Settings Specials Method Manual Permeate Parameter 1 name P Setpoint name default underlined 0 001 0 000 10000 000 Parameter 2 name Setpoint name default underlined 200 000 sec 0 000 10000 000 Instruction Help text These parameters are used to tune the feedback control when Permeate_Unrestricted_Flow is active For details see Feedback tuning chapter in User Instrument Manual 13 3 19 pNFF_PI Instruction name pNFF_PI Group System Settings Specials Method Manual Permeate Parameter 1 name P Setpoint name default underlined 0 050 0 000 10000 000 Parameter 2 name Setpoint name default underlined 150 000 sec 0 000 10000 000 Instruction Help text These p
56. 16 FeedPressure_Pl nstruction name Group FeedPressure_Pl System Settings Specials Method Manual Recirc Parameter 1 name Position name default underlined P _0 050 0 000 10000 000 Parameter 2 name Position name default underlined 150 000 sec 0 000 10000 000 nstruction Help text These parameters are used to tune the feedback control when FeedPressurecontrol is active For details see Feedback tuning chapter in User Instrument Manual 13 2 17 DeltaP_PIl nstruction name Group DeltaP_PI System Settings Specials Method Manual Recirc Parameter 1 name Position name default underlined P 0 100 0 000 10000 000 Parameter 2 name Position name default underlined 10 000 sec 0 000 10000 000 nstruction Help text These parameters are only used to tune the feedback control during DeltaP For details see Feedback tuning chapter in User Instrument Manual 176 AKTAcrossflow User Manual 11 0012 32 Edition AB 13 2 18 EmptyResFeed_Pl Strategy instructions Instruction name EmptyResFeed_PI Group System Settings Specials Method Manual Recirc Parameter 1 name FPC_P Position name default underlined 0 05 0 000 10000 000 Parameter 2 name FPC_ Position name default underlined 150 000 sec 0 00 10000 000 Instruction Help text These parameters are only used to tune the feedback control during EmptyReserv
57. 2 32 Edition AB Strategy instructions fe 13 3 4 Permeate_Unrestricted_Flow Instruction name Formula Group Permeate_Unrestricted_Flow Pp Method Manual Per meate Parameter name Mode Note Position name default underlined Pressure Control Mode PID Pp lt r Disabled Enabled Instruction Help text Starts the flow on the permeate pump at Offset permeate pressure If RetenPress gt PermPress RetenPress value is used as Offset RetentateControlValve lifts RetenPress to Offset if RetenPress lt Offset Resets TMP_Control and Flux_Control 13 3 5 Normal_Flow_Filtration NFF_ConstantFlow Instruction name Formula Group NFF_ConstantFlow Of Method Manual Permeate Parameter name Mode Position name default underlined Flow Control Mode Direct Off 0 0 ml min 0 600 Instruction Help text Starts set feed flow and starts permeate pump at 20 of set feed flow Retentate PressureControlValve close retentate loop Permeate PressureControlValve is open Instruction ignored if feed flow gt 0 ml min NFF_ConstantPressure Instruction name Formula Group NFF_ConstantPressure Pp Method Manual Permeate Parameter name Mode Note Position name default underlined Pressure Control Mode PID Pp Pr Off 0 0 bar 0 00 5 20 bar Instruction Help text Starts the flow on the permeate pump at Offset permeate pressure If RetenPress gt PermPress RetenPress value is used as Offset Re
58. 2 32 Edition AB 185 Strategy instructions 13 4 Transfer instructions 13 4 1 Transfer Flow Instruction name Group Transfer Flow Method Manual Transfer Parameter name Position name default underlined FlowRate 0 0 ml min 0 200 0 Instruction Help text Starts the flow on the Transfer pump at the set flow rate 13 4 2 Constant_Retentate_Volume Instruction name Mode Note Group Constant_Retentate_Volume Control Qt Qp Method Manual Transfer Mode Parameter name Posistion name Default underlined Mode CRV_Off CRV_On Instruction Help text CRV_On liquid level in the reservoir is kept constant at the level detected when instruction activated Instruction adjusts Transfer Flow to compensate for detected changes in level CRV_Off immediate inactivation of instruction If retentate valve is open when executing Constant_Retentate_Volume a warning will be raised Instruction ignored If P VB recycle is open when executing Constant_Retentate_Volume CRV_On a warning will be raised Instruction ignored Warning Help text 1 Constant_Retentate_Volume is not allowed with Retentate_Valve_Block open Instruction ignored Warning Help text 2 Constant_Retentate_Volume is not allowed with P VB recycle position open Instruction ignored 186 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions __________ _ ___ 13 4 3 Transfer_Valve_Blocks Instructi
59. 3 2 12 Concentration _Factor es 32413 MIXCISPCCd ione E R ae he ie casas a oe T5224 SCRIROSV Ol POLAIIZON escccsestsces science atari can cts caitse datentiasuntctaa ts 13 2 15 ManSample 13 2 16 FeedPressure_PI E viii KTAcrossflow User Manual 11 0012 32 Edition AB AKTAcrossflow User Manual 11 0012 32 Edition AB Contents Ew 13 217 DOP Pl sigs cascsatscsblstu dst seealehiabee teleseed AA AOAN 13 2 18 gt EmptyResF ed Plaane ipini e iananacinmiananeaianiee 13 2 19 RetentateHoldupVol oieeeecccccsccsssssssssscssescescsssssssssssssnssssunususssssseecsesseseeeeeeeees 13 3 Permeate instructions 13 3 1 TMP_Control 13 3 2 Flux_Control oe 13 3 3 Permeate VAIVE_BIOGCKS sessi uinean a 13 3 4 Permeate Unrestricted FIOW cceeccssssssscsssssessssssssesscsssssesssssssseessssssseessssssee 13 3 5 Normal_Flow_Filtration 13 3 6 PressureOffset 13 3 7 Evaluation Instructions 13 3 8 Set_PermVol_Totalizer ie c ceeeeccssssessssssssesssssssssesssssseessssssesssssssssesssesseeesessse 133 9 YER Drop CALCULATION scccssccccccescssscs csssssacesasnsnsctantiastobesasaa shasieciannscsettines 13 3 10 Total_Membrane_Surface_Area 13 311 L men Diameter desann ann DNS 13 3 12 Total_Number_of_ Fibres ooo ecccccecsscssssesscsssssesssssssssscsssseeesssssssseessssseessssssee 15 5 132 PPCV StD aiian eonenn A E an 13 3 14 TMP_PI_RetentateControlValve
60. 32 Edition AB Process optimization in Ultrafiltration 6 6 5 Evaluating results Evaluate the results with the UNICORN Evaluation module see User Reference Manual UNICORN 5 1 Evaluation for Cross Flow Filtration Example of results A Flux vs TMP 5 mgml 1X conc Ca Cx Smm rece Faron __ Rethow E L mer Rethow Se ee T Ee 10 15 20 25 30 35 40 B Flux vs TMP 50 mg m 10X cone Ce Ca 24 mumin Rethow 16 mimin Rethiow amp mimin RetFiow Fig 6 1 Evaluation of TMP excursions runs with A 5 mg ml B 50 mg ml Antibody samples Note To be able to show several cross flows in a graph chromatograms have to be bundled See Section 11 3 2 AKTAcrossflow User Manual 11 0012 32 Edition AB 95 6 Process optimization in Ultrafiltration 96 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard 7 Creating Product methods using the Method Wizard 7 1 Ultrafiltration 7 1 1 Introduction Concentration The volume of sample in the reservoir is reduced The product is retained at the retentate side of the membrane If the sample volume is larger than the reservoir volume the reservoir can be continuously fed with sample solution Fed Batch Diafiltration Diafiltration is a filtration process used for the removal of smaller ionic solutes whereby the feed solution is continuously filled up with a buffer One buffer is removed and replaced with an alternativ
61. 5 4 3 Visualized procedure of the rinsing method y 5 4 4 Visualized procedure of the Filter CIP method 2 91191121121212 84 5 4 5 Visualized procedure of the Water flush MethO eecssssssssssssssse 86 5 4 6 Visualized procedure of the Water flux tt 88 5 4 7 Visualized procedure of the Buffer conditioning method 89 Process optimization in Ultrafiltration GLa sIMMOAU COM xc excel oe teen ce tamed Meade cdtl tha Mattes 91 6 2 GERMS TIT SMEG aama a ANE e 91 GS SCLECUING a Method ennan a e a n en 91 6 4 Continuing the experiment sssssssssisssssisssisrisssisssrsrrssrissssssrssrrenrseenens 94 6 5 GE VAUGEIMOSSUIS nnna aa a a aa 95 KTAcrossflow User Manual 11 0012 32 Edition AB Contents ee 7 Creating Product methods using the Method Wizard 7 A Ultrafiltration eisenai 97 7 1 1 Introduction 97 7 1 2 Basic settings dialog 98 7 1 3 Product steps dialog 100 7 1 4 Step 1 Concentration dialog ssssessssssssssssssssssssssssscsesssseeseecesesesssssssssssssssises 102 7 1 5 Step 2 Diafiltration dialog essssssssssssssssssssssssssssssssessscsessesssseeseesessssssssssssssses 104 7 1 6 Recovery dialog 7 1 7 Visualized procedure of an Ultrafiltration method ues 108 1 2 Microfiti ation iis cezecscccsscscseasiassssseeticcccssessneasdaaaceieanecssscstiontanuaactttehecccoesends 112 7 2 1 TR OGUCTION insae r E E EET 112 7 2 2 BOSIC set
62. 5000 Ret_vol_Step1Cont ml 0 00 80000 00 RetFlow_ProdRec Ret_Flow_ProdRec ml min 0 0 600 0 Ret_Flow 0 0 600 0 Show details Show unused variables Z Display tooltip for extended variable cells 3 2 Creating a new method To create a new method there are two alternatives e In the Method Wizard customized methods for most purposes are made by setting appropriate values for the method variables e Inthe Text Instructions editor in the Method Editor module more advanced editing facilities are available aa KTAcrossflow User Manual 11 0012 32 Edition AB 47 Handling methods in AKTAcrossflow 48 3 3 Creating cross flow methods using the Text instructions The Text instruction editor in the Method Editor can be used to build methods step by step The editor can also be used to modify instructions in methods created by the Method Wizard Note All Strategy instructions for AKTAcrossflow are listed in Chapter 13 3 3 1 Opening the Text Instruction Editor 1 Select the Method Editor module in UNICORN If a method is opened in the Method Editor click the Text Instruction icon Method Editor Test1 To create a new method click the New Method icon in UNICORN Manager y UNICORN Manager File view Administration Tools Window Help jee RE Ge amp IE Methods or click the New icon in the Method Editor eR guyo aN AKTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAc
63. 61 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 e Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 815 65 555 Fax 815 65 666 e Portugal Tel 21 417 7035 Fax 21 417 3184 Russia amp other C I S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 e Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 e USA Tel 800 526 3593 Fax 877 295 8102 imagination at work User Manual 11 0012 32 AB
64. 7 pH 207 Cond 207 WITE E aaa AT a A aa aN ke a N e iia N 208 CGI CTO eee ee anae Ea ae AANE nt ae NA 209 CETOL S oree EAA AR E A A NAIN ES 209 LevelsensorZeroCalibration ccsssssssesssscssssssssssssssssssssssssssssssscssssssssesssseeesees 209 UPC aea nr E E TA N Sin Ooh AA 209 KTAcrossflow User Manual 11 0012 32 Edition AB List of figures Fig 1 1 Fig 1 2 Fig 1 3 Fig 1 4 Fig 1 5 Fig 1 6 Fig 1 7 Fig 1 8 Fig 2 1 Fig 2 2 Fig3 1 Fig 3 2 Fg 5 1 Fg 6 1 Fig 11 1 Fig 11 2 Normal flow and tangential flow filtration eee 15 Principle Of Cross flow FIItFOtiON esses A cross flow system simple illustration 3 Flow pathi OvervieW erini E NEEE A EAE T Detailed flow PUM a a cscesssesssezveassussscesstesscesssassoccaaechedevesstinssSuceiceesdcadssnsdss ised E Ultrafiltration COSSCttE oe esessssssssssssssssssssssssssessssssssssssssssstseeststtestsssteetesstteetteeeeeeeeeeeeees Hollow fiber Piston rinsing system Location of the bottles Ri Example of a simple KTAcrossflow MethOG cessssssssscssssssssssssssesseenssssseeeensse 52 Principle of the instruction CCTM ceessssssssssccsssssssssssssccessccscsssssssssssssssesssssssssseneeesee 53 Example of plotted Normalized Water FlUX sssessssccccssssssssessscsesssssssnneeseessees 72 Evaluation of TMP excursions runs with A 5 mg ml B 50 mg ml Antibody samples Example Of Process OPtiMIZAtiON sssecssssccccsssss
65. Blocks E Transfer SA TNA Taker O Alarms amp Mon ae O Other Auto update If this is checked the parameter fields will be updated during method run 6 Select parameter for example Transfer_Flow 7 Enter a parameter value for example 50 ml min AKTAcrossflow System 1 Transfer Instructions Instructions Parameters O Recic Constant_Retentate_Volume revise Wet O Permeate Transfer_Valve_Blocks 50 ml min Transter_Purge_Valve e Transfer Set_TifVvol_Totalizer O Alarms amp Mon npara O Other Transfer_Yalve_Blocks T VB In1 Auto update If this is checked the parameter fields will be updated during method run 8 Click Insert 9 When all commands are set for the desired operation click Execute 10 To stop the operation click the End or Pause button in the System Control window Note Reset ResVol_Totalizer under Recir to avoid values below zero Eas 64 AKTAcrossflow User Manual 11 0012 32 Edition AB Performing cross flow runs manually 4 2 1 WARNINGS and CAUTIONS CIP method When running a method using a CIP Clean in place solution containing sodium hydroxide NaOH WARNING NaOH is corrosive and therefore dangerous to health Avoid spillage and wear safety glasses safety gloves and protective lab coat CAUTION Always make sure that the filters and system components are compatible with sodium hydroxide at the concentration time and temperature used General WARNING Do not operate the AKTAcrossflow s
66. Evaluating AKTAcrossflow results 154 11 3 Main functions 11 3 1 Any vs any Used to generate a plot of any combination of process data collected during a AKTAcrossflow run The process data could be for example detector signals flows flux and pressures 11 3 2 Process optimization Used to get a graphical analysis of process opimization run s For example a process optimization to find an optimal flux as a function of TMP Flux vs TMP zm Gd ao 24 mumin RetRow 16 mimin Rethiow E mumn Rethow 110 7 os E C 7 60 so LMH 10 15 20 25 30 35 40 bar Fig 11 1 Example of process optimization AKTAcrossflow User Manual 11 0012 32 Edition AB Data Selection Evaluating AKTAcrossflow results Feature in the Evaluation module The example is a result from a run with up to three different cross flows in the same run 1 When a process result has been selected Found chromatograms are displayed 2 Check the box Bundle chromatograms and chromatogram groups will be displayed to the right 3 Check the box for the first chromatogram e g crossflow 1 4 Click the New data group button 5 Check the box for the second chromatogram crossflow 2 and click New data group 6 Repeat the procedure until all chromatograms have been selected as groups 7 Click Next to continue the evaluation ES Search filter setup
67. Folder bc Asa Wizard 30 T Result lid 27 Process Opt 3retflow Packet holder001 Chromatogram k 7 Curves Ys Found chromatograms i ECTS I Select Fle Chrom Curves New data oroup 27 Process Opt 3 retflow Packet holderd id 27 Process Opt 3reti 11 TMP Flux SPP Group 2 id 27 Process Opt 3 retflow Packet holder i group EiGop3 P a es oe ee id 27 Process Opt 3 retflow Packet holder0 Search Clear Bundle chromatograms AKTAcrossflow User Manual 11 0012 32 Edition AB 155 Evaluating AKTAcrossflow results 156 11 3 3 Normalized Water flux Is used to get normalized water flux values calculated The results can be used to maintain long term analysis of the filter recovery Normalized Water Flux 20 mimin RetFlow and 0 68 ber TMP 200 175 wo SS SS So E 128 LLMH bai o 8 l 1 2 3 4 s 6 Data point Fig 11 2 Example of plotted Normalized Water Flux 11 3 4 Capacity plots Is used to get a graphical analysis of the filter capacity during a filtration KTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters 12 Feedback tuning and PID parameters 12 1 PID control Whenever an automated process step requires the control of pressure feed retentate and permeate pressure the UNICORN control software of KTAcrossflow employs PID type controllers to control the pressure to its setpoint Recommende
68. GE Healthcare AKTAcrossflow User Manual O KTA Important user information All users must read this entire manual to fully understand the safe use of AKTAcrossflow WARNING The WARNING sign highlights instructions that must be followed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood CAUTION Note The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood The Note sign is used to indicate information important for trouble free and optimal use of the product Declaration of conformity This product meets the requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a laboratory device It is not intended for clinical or in vitro use or for diagnostic purposes used asa stand alone unit or connected to other CE marked GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual Contents 1 Introduction 1 1 1 2 1 3 1 4 1 4 1 1 4 2 1 4 5 1 5
69. LowAlarm cause The pressure has fallen below the LowAlarm limit Pressure limits are set by AlarmFeed_Press Press Acknowledge on the error message window 190 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions s 13 5 8 Alarm_TrfPress Instruction name Group Alarm_ TrfPress System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined Mode Disabled Enabled Parameter 2 name Setpoint default underlined HighAlarm 5 20 bar 0 00 5 20 bar Parameter 3 name Setpoint default underlined LowAlarm 0 00 bar 0 00 5 20bar Instruction Help text Sets the alarm limits for the pressure from Transfer Pump When reached the system is set to Pause Alarm TrfPress HighAlarm TrfPress LowAlarm Alarm Help text HighAlarm cause The pressure has exceeded the HighAlarm limit LowAlarm cause The pressure has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue 13 5 9 Alarm_DeltaP Instruction name Group Alarm_DeltaP System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined Mode Disabled Enabled Parameter 2 name Setpoint default underlined HighAlarm 5 20 bar 0 00 5 20 bar Parameter 3 name Setpoint default underlined LowAlarm 0 00 bar 0 00 5 20bar Instruction Help text Sets the alarm limits for DeltaP When reached th
70. M Y8 Ini P UB Recucle T UB Tn2 T VB Tns T UB Ind Trans fer VB2 4 Recirculation for 5 minutes Air Trans fer VB1 Permeate UB UbRecucle T VB Tn3 T UB Tns 5 The rest of the reservoir liquid is emptied through R VB Outz Air Trans fer VB1 Permeate UB T VB Ini UB Recucle T UB Ins T VB In T UB Tns A re ERC paad Cartridoe permeate SSS AKTAcrossflow User Manual 11 0012 32 Edition AB 89 Creating Preproduct methods using the Method Wizard 90 AKTAcrossflow User Manual 11 0012 32 Edition AB Process optimization in Ultrafiltration 6 6 Process optimization in Ultrafiltration 6 1 Introduction A TMP excursion method can be created to determine the optimal cross flow and TMP settings for an ultrafiltration process of a specific protein 6 2 Experiment plan The goal of an optimization can for example be to find the optimal conditions for a 10x concentration of a 5 mg ml antibody protein solution to 50 mg ml The experiment plan includes therefore different Retentate flows Qp and TMP settings for the two antibody concentrations Antibody concentration Set1 Set2 Set3 mg ml 5 Qr ml min T bar 0 Ltrs 6 5 x 2 8 a 4 50 Qg ml min a 6 ee Se bar 1 0 1 6 2 2 2 8 3 4 6 3 Creating a method 1 Open the Method Wizard by clicking the Wizard icon in the Method Editor in ANI UNICORN 2 Select a system and click OK Method Wizard Select a system AK TAcrossflow System 1 AkTActossflow Sys
71. MENTOTION u cesecsssssessssssssessssssssesssssssessssssssesssssseesessssseeesssens 2 System preparation 2 1 2 2 2 3 2 3 1 2 3 2 2 3 3 2 4 2 5 2 6 Siro ANE BINE VSE a AEE E A EET Motors S 31 Aae E ai E E E E E E N E 33 The PUMP piston rinsing SyYStem oac eeesesssssssssessesssscccssssssssseseeseseesecsssseees 34 Introduction Ta Using the pump piston FINSING SYSTEM sssssssssssssssssssssssssssccccessecseeeeeeees 35 Cleaning the PUMP piston rinsing System cceccccssssscssssssccscccceccssesssssessene 36 Selecting filter component for the system Strategy s s s 39 Selecting type of Retentate Valve block o cccecssesssssssssssecsssssssssuseeees 41 Calibrating the level Sensor o cecssssssssssssessssssssscsssssssssesesssssecseesssssesssesees 43 3 Handling methods in AKTAcrossflow 3 1 3 1 1 3 1 2 3 1 3 3 1 4 3 1 5 A UNICORN method 45 BloGK S mina 45 BOS EE PERENE ENSE EEEE A 45 COS EEEIEE O AEAEE TA A 46 Watch and Hold_Until 46 Block PONG Hick naar tawuattaduy stead cnet E E R ERG 46 AKTAcrossflow User Manual 11 0012 32 Edition AB Vv Contents vi 3 1 6 Run Setup ekra ea N 47 32 Creating a MEW MELA OG rarman tuen ERER 47 3 3 Creating cross flow methods using the TEXt INSTRUCTIONS ioeie enini a e E 48 3 3 1 Opening the Text Instruction Editor ccecccccscccsssssscsessssssssssssssstssssssssssneeee 48 3 4 Example of creating an KTAcrossflow Method oss 52 3 5 Creating cross flow
72. Mena Transfer UB2 Permeate UB F UB Recuclel Cartridge permeate RUE 7 Then if a CIP 2 has been selected the reservoir is filled with 100 ml CIP2 solution and the procedure described above is repeated except for that less volumes are used 5 4 5 Visualized procedure of the Water flush method If the previous method was Filter CIP or if the method is a post product method the system and reservoir are prepared according to the procedure Preparing the system and reservoir described in Section 5 4 1 Otherwise the system will be prepared according to the procedure Preparing the system 1 The reservoir is prepared according to the procedure described in Section 5 4 1 The Transfer Inlet used is T VB In5 water 86 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea 2 The reservoir is filled with 100 ml water At Permeate_Unrestricted_ Flow FeedPressure Control and Constant_Retentate_Volume a volume defined by used filter is passed through the filter out to P VB Out1 For hollow fibers 750 kD and smaller TMP control is used For 0 1 um and larger Flux control is used Air Trans fer UB1 e n Permeate UB P UB Recucie EMN Ena Trans fer UB2 UB Ine Ls Cartridge permeate R UB OutT 3 Permeate Unrestricted Flow is disa
73. Method Manual Permeate Parameter 1 name Mode Position name default underlined Q ProcessOptimisation 1 ExtData_vs_Capacity 2 NormalisedWaterflux Instruction Help text Sets a mark for data collection Several Mark instructions can be given during a run For more information see UNICORN manual Evaluation Appendix 180 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions 13 3 8 Set_PermVol_Totalizer Instruction name Set_PermVol_Totalizer Group Method Manual Permeate Parameter name Volume Set point default underlined 0 00 ml 0 00 5000 00 Instruction Help text Sets a desired volume for totalizer Permeate pump flow will increase value 13 3 9 Flux_Drop_Calculation Instruction name Flux_Drop_Calculation Group Method Manual Permeate Parameter 1 name Mode Position name default underlined Off On Instruction Help text Latches reference flux and calculates drop with current flux Increase in flux values not relevant 13 3 10 Total_Membrane_Surface_Area Instruction name Total_Membrane_Surface_Area Method Manual Permeate Group System Settings Specials Parameter 1 name Area Position name default underlined 50 cm 1 1200 cm Instruction Help text Used for calculation of flux from the equation Flux Qp I h Alm 13 3 11 Lumen_Diameter
74. N Manager select Administrator System Setup Tools Window He User Setup EH Audit Trail fi System Setup Create System Report Generate File List Controller Settings Create Restore Backup Change User Attributes Options 2 Select a system and click Edit System Setup Systems EE SEUPPD 02610 AKTActossflow System 1 EE Ak TActosstlow System 2 aa KTAcrossflow User Manual 11 0012 32 Edition AB 39 System preparation SSS 3 Inthe Edit System dialog click Component Edit System System name KTAcre System type Membrane Strategy Computer name SEUPPD 02610 Control unit number 1 4 1 Auto save interval 5 minutes 4 Inthe Component dialog select filter type e Hollow fiber or e Flat Sheet Component Select components owl Fraction Collector Filter Type Hollow Fibre Flat Sheet xiliary Equipment etentate Valve Block Old Type New Type FRO a Of 5 Click OK to close the Component dialog 6 Click OK to close the Edit System dialog 7 Click Close to close the System Setup dialog 40 AKTAcrossflow User Manual 11 0012 32 Edition AB System preparation lt a 2 5 Selecting type of Retentate valve block 1 InUNICORN Manager select Administrator S ystem Setup Tools Window He User Setup EH Audit Trail fi System Setup Create System Report Generate File List Cont
75. OFF the selected Retentate Volume or Concentration factor will be the only endpoint For two endpoints the endpoint of the process will be when one of the two endpoint conditions is met Concentration factor Max Endpoint O Retentate Volume Concentration Factor 0 50 Note A Concentration factor between 0 50 can be entered A value of 1 means no concentration A value lt 1means dilution Values between 0 1 are used by the software to compensate for dilutions for example due to the hold up volume on the retentate side This feature can be used when performing diafiltration of small volumes as a planned single step See Section 7 1 1 Diafiltration AKTAcrossflow User Manual 11 0012 32 Edition AB 103 Creating Product methods using the Method Wizard es 7 1 5 Step 2 Diafiltration dialog Note If diafiltration of a small volume is planned we recommend performing a concentration as an initial step This is to avoid dilution of the sample due to the hold up volume on the retentate side For information see Section 7 1 1 Diafiltration and Section 7 1 4 Endpoint Method Wizard for System AKTAcrossflow System 1 Step 2 Diafiltration Basic Settings Product Steps Feed Control L Step 1 Retentate flow L ml min 0 600 ml min Step 2 Recovery TMP i bar 0 0 5 2 bar Summary Endpoint Watch for Endpoint Permeate Volume ml 20 9999 ml DF Exchange Factor Feed Control e Selec
76. Parameters nstruction name Group FracParameters System Settings Specials Parameter 1 name Setpoint default underlined DelayVol 0 000mlI 0 000 10 000 Parameter 2 name Setpoint default underlined TubeChange Tube DropSync WasteBetweenTubes nstruction Help text Parameter settings for fraction collector Delay volume is defined as the volume from the UV cell to the end of the tubing for the fraction collector Collection of the flow during tube change can be handled in different ways Tube No synchro nisation of collection DropSync Tube change synchronised to drop release Should only be used at flow rates generat ing drops WasteBetweenTubes The flow will be diverted to waste when moving between tubes FracNumberingMode Instruction name Group FracNumberingMode System_Settings Specials Parameter name Position name default underlined Mode Reset Continue Instruction Help text Determines whether fraction number is reset at the end of a method or not Reset sets Tube No to 1 after method end Continue continues numbering from the last tube in the previous method 206 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions Ct 13 7 UPC 13 7 1 UV Monitor UVLampOFF Instruction name Group UVlampOFF Method Manual Alarms amp Mon Instruction Help text Sets the UV lamp permanently to OFF To start UV lamp system and computer has to be re booted 13 7 2 pH pHTem
77. Retentate_ Yalve_Block R B Out1 Transfer_ alve_Blocks T B In2 Transfer_Purge_Yalve Waste Permeate_ alye_Block P B Out1 Retentate_ alve_Block R B Out2 Transfer_ alve_Blocks T B In3 Permeate_ alye_Block P B Out2 Retentate_ alye_Block R B Out3 Transfer_ alve_Blocks T B In4 Permeate_ alve_Block P B Out3 Retentate_Yalve_Block R B Out3 Transfer_ alve_Blocks T B In5 TransferFlow 0 1 ml min Permeate_ alve_Block Closed TransferFlow 0 0 ml min Transfer_ alve_Blocks T B In6 FeedFlow 5 0 ml min Permeate_Unrestricted_Flow Enabled Permeate_ alve_Block P B Out2 FeedFlow 0 0 ml min Permeate_ alyve_Block Closed Transfer_ alve_Blocks T B In Transfer_ alve_Blocks T B In8 End_Method Fig 3 1 Example of a simple AKTAcrossflow method 1 Inthe Method Editor of UNICORN select File New 2 Select system select to use Method Editor and click OK New Method For system AKTActossflow System 1 Template selection Technique Template For column Use O Wizard lat Method Editor Method notes AKTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow Ea E 3 First the base of the method is selected As default in UNICORN column volumes CV is set as base This is not useful in AKTAcrossflow methods E Main 4 inthis case Time minutes is more appropriate Click the instruction row above select Instruction group Other select Base
78. Running KTAcrossflow methods 144 KTAcrossflow User Manual 11 0012 32 Edition AB After run procedures 10 After run procedures 10 1 System sanitization If you want to be sure that the system is clean for example when a new filter is used or a process is changed it is recommended to perform a sanitization of the system CAUTION Instead of a filter use a three way connector 18 1170 59 Filters are not compatible with the used flows and pressures in the System sanitization method Replace the pH electrode with a dummy electrode Remove the reservoir float If an air filter is connected to the reservoir remove the filter Connect the tubing Reservoir cleaning kit 11 0033 86 at the reservoir top Place the tubing in waste WARNING The reservoir is over filled during the sanitization It is important to have tubing 11 0033 86 to waste NaOH is corrosive and therefore dangerous to health Avoid spillage and wear safety glasses safety gloves and protective lab coat Note The stirrer should be present in the reservoir during sanitization However it should be replaced with a new aseptic one after the sanitization Note The System sanitization method can also be used to pH neutralize the system solutions after sanitization and to exchange the solution in the system to an appropriate storage solution for example 20 Ethanol 10 1 1 Sanitization of the pump piston rinsing system When perfo
79. UVlampOFF LevelSensorCalibration Watch_DF_X_Fact Watch_Flux_Drop Watch_AirSensor Watch_ ZeroLevel Watch_Auxin1 Watch_Auxin2 Watch_Aux In3 Watch_Auxin4 Watch_Off 1 Only in Method Editor 2 Only in Method Editor and System control when running a method 3 Only in System Control when not running a method 4 Only in System control 5 Note Some instructions are only shown when chosen as components e g Frac 6 Only for Hollow fibers 170 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions 13 2 Recirculation instructions 13 2 1 FeedFlow Instruction name Formula Group FeedFlow Qf Method Manual Recirc Parameter 1 name Mode Position name default under lined FlowRate Control Mode Direct 0 0 ml min 0 600 Instruction Help text Starts the flow on the feed pump Feed Flow should stabilize at 98 of set value before a new instruction is executed To allow new instruction after stabilized DeltaP Watch_Stable_Baseline DeltaP should be programmed in methods Instruction resets RetFlow FeedPressure DeltaP and Shear 13 2 2 RetFlow Instruction name Formula Group RetFlow Qr Qf Qp Method Manual Recirc Parameter 1 name Mode Position name default underlined FlowRate Control Mode Direct 0 0 ml min 0 0 600 0 Instruction Help text Starts the flow on the feed pump RetFlow should stabilize at 98 of set value before new instruction is executed In
80. _ Vol mi 130 00 0 00 999999 00 Show details Show unused variables 7 Display tooltip for extended variable cells In the Run Setup dialog there are tabs to select to for example make notes Edit Variable enter method information and make selections of start protocol Start Protocol Variables Scouting Notes Gradient Columns Questions Reference Curves Evaluation Procedures Result Name To display the text instructions of the created method click the Text Instruction icon KTAcrossflow User Manual 11 0012 32 Edition AB Method Information Handling methods in AKTAcrossflow 10 To view and expand the method click the plus An alternative is to use the keyboard arrow keys Main Ml 0 00 Block Info_Inlets_and_Outlets 0 00 Block INITIAL_SETUP W 0 00 Block PREPRODUCT_STEP_WATER_FLUSH S W 0 00 Block PREPRODUCT_STEP_WATERFLUX_TEST PREPRODUCT_STEP_WATERFLUX_TEST 0 00 Base SameAsMain 0 00 Set_Mark Preproduct Step Water Flux Test E 0 00 Block Water_Inlet Water _Inlet 0 00 Base Time 0 00 Transfer_ Yalve_Blocks T B In5S W 0 00 Block DelayOp05min 0 00 End_Block W 0 00 Block Prime_line W 0 00 Block Prepare_Reservoir_Full W 0 00 Block Fill_Yolume_100ml W 0 00 Block Default_Ret_Flow W 0 00 Block Flowpath_PermRecycle 0 00 Block TMP_PreWaterFluxTest 0 00 Block Empty_Reservoir_Recycle W 0 00 Block St
81. _Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 197 Strategy instructions 13 5 23 WatchPar_PermPress Instruction name WatchPar_PermPress Group System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name DeltaPeak Setpoint default underlined 0 00 bar 0 00 5 20 bar Parameter 2 name Delta_Base Setpoint default underlined 0 00 bar 0 00 5 20 bar nstruction Help text atch on pressure at permeate filter port The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 24 WatchPar_FeedFlow Instruction name Group WatchPar_FeedFlow System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name DeltaPeak Setpoint default underlined 0 0
82. able_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 26 WatchPar_PermFlow Instruction name Group WatchPar_ PermFlow System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 0 ml min 0 0 200 0 ml min Parameter 2 name Setpoint default underlined Delta_Base 0 0 ml min 0 0 200 0 ml min Instruction Help text Watch on flow reported from permeate pump The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 199 Strategy instructions _ 13 5 27 WatchPar_TrfFlow Instruction name Group WatchPar_ TrfFlow System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 0 ml min 0 200 Parameter 2 name Setpoint
83. ak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 34 WatchPar_Flux Instruction name Group WatchPar_Flux System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 0 LMH 0 0 120000 0 LMH Parameter 2 name Setpoint default underlined Delta_Base 0 0 LMH 0 0 120000 0 LMH Instruction Help text For Watch on flux reported from permeate pump The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 203 Strategy instructions _ 13 5 35 WatchPar_Shear Instruction name Group WatchPar_Shear System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak O s 0 20000
84. ally 4 4 Performing cross flow runs manually 4 1 Introduction The most convenient way to perform cross flow runs is of course to use Method Wizard created methods However it is always possible to perform process steps using the manual mode in UNICORN Note All Strategy text instructions and parameters are listed in Chapter 13 4 2 Executing text instructions 1 Inthe System Control window in UNICORN select the desired instruction group from the menubar under Manual System Help 1 Recirc Ctrl M 2 Permeate 3 Transfer 4 larms amp Mon 5 Other Run Shift R An instruction box is displayed KTAcrossflow System 1 Transfer Instructions Instructions Parameters ir al 4 FlowR ate 0 0 200 0 O Recic Constant_Retentate_Volume ee l l O Permeate Transfer_Valve_Blocks 0 0 ml min Transfer_Purge_Valve Transfer Set_TrfVol_Totalizer Execute DF Exchange Factor OTE ConstRVol_P Close Other Help Auto update If this is checked the parameter fields will be updated during method run AKTAcrossflow User Manual 11 0012 32 Edition AB 63 Performing cross flow runs manually Ss 2 Select instruction group for example Transfer 3 Select parameter for example Transfer_Valve_Blocks 4 Select an inlet valve 5 Click Insert AKTAcrossflow System 1 Transfer Instructions Instructions Parameters ORecite Cet riai Volume eee Orereste Transfer Valve
85. alog select print formats and layout options Print Chromatograms Print Format L B OBE Chromatograms in each column 1 Chromatograms in each row 1 Use thick lines Landscape OK Cancel Help Click Preview In the Customise Report window verify that the layout is correct Click Edit Mode to make changes Click Preview to return to the preview mode Click Exit to return to the Print Chromatograms dialog Click OK Refer to the UNICORN user manuals for more information about printing results AKTAcrossflow User Manual 11 0012 32 Edition AB 151 After run procedures 152 AKTAcrossflow User Manual 11 0012 32 Edition AB Evaluating AKTAcrossflow results 11 Evaluating AKTAcrossflow results 11 1 Associated documentation The use of the Crossflow Evaluation module in UNICORN is described in detail in the User Reference Manual UNICORN 5 1 Evaluation for Cross Flow Filtration 11 2 Opening the Evaluation module In UNICORN Evaluation click the Membrane System Evaluation icon Evaluation File View Help Oela Gaoakere ay ay TE f kaj Lu mm Operation dialog is displayed Operation Select operation Process optimization O Normalized Water Flux Diatiltration time optimization Capacity plots O Any vs any Open Previous Analysis Next gt Cancel Help KTAcrossflow User Manual 11 0012 32 Edition AB 153
86. ant and will lead to incorrect methods AKTAcrossflow User Manual 11 0012 32 Edition AB 45 Handling methods in AKTAcrossflow 3 1 3 Calls To execute the instructions contained within a block in a method the block must be called by the program When a block is called the instructions in the block are executed in the order that they are written until the block is finished or the End_Block instruction is executed There are two types of calls e Unconditional calls which are made with a Block instruction e Conditional calls which are made with a Watch instruction This makes it possible to call a specified block or instruction when a particular monitor signal meets a given condition 3 1 4 Watch and Hold_Until The breakpoint when the Watch instruction is issued determines when the watch begins A watch remains active until the condition is met or a new Watch instruction is issued for the same monitor The watch is cancelled automatically when the condition is met A watch can also be turned off with the Watch_off instruction The Hold_Unitil instruction is a special kind of Watch instruction The method is put on hold until a specific condition is met signal test or value or the time out is reached Thereafter the remaining instructions in the method are executed 3 1 5 Block pane The organization of blocks in the method is shown graphically in the Block pane of the Method Editor Each block is represented by a gray bar with the
87. arameters are used to tune the feedback control when Normal_Flow_Filtration is active For details see Feedback tuning chapter in User Instrument Manual 184 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions ___________ _ ___ 13 3 20 Fractionation Instruction name Group Fractionation Method Manual Permeate Parameter name Position name default underlined FracSize 0 000 ml 0 50 Instruction Help text Starts fraction collection if the fraction size specified by the FracSize parameter is gt 0 ml Tube change is delayed with respect to the set delay volume Fractionation is stopped with the instruction FractionationStop FeedTube Instruction name Group FeedTube Method Manual Permeate Instruction Help text During Fractionation FeedTube moves the tube rack forward one tube after the set delay volume has been collected and a fraction mark is given When Fractionation is not used FeedTube moves the rack instantly and no fraction mark is given FractionationStop Instruction name Group FractionationStop Method Manual Permeate Instruction Help text Stops fraction collection after the set delay volume has been collected ResetFracNumber Instruction name Group ResetFracNumber Method Manual Permeate Warning Help text ResetFracNumber is not allowed during fractionation Instruction ignored AKTAcrossflow User Manual 11 001
88. ase 2 mS cm 0 999 mS cm Ge 120 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard lt a E e Ifthe first endpoint is selected a Max endpoint has to be entered Permeate Volume or Diafiltration DF Exchange Factor Select and enter a value Endpoint Watch for Endpoint Cond Less than and Stable Cond v Level 20 mS fem 0 399 mS cm Time Stable 1 min 0 9999 min Delta Base d mS cm 0 999 mS cm Max Endpoint O Petmeate Volume DF Exchange Factor 5 0 50 e Ifthe Watch for option is set to OFF the selected Permeate Volume or Diafiltration DF Exchange Factor will be the only endpoint e For two endpoints the endpoint of the process will be when one of the two endpoint conditions is met 7 2 6 Recovery dialog Method Wizard for System AKTAcrossflow2 Recovery Basic Settings Recovery Option Recovery Preproduct Steps Product Steps Retentate Dutlet Step1 Product Flush Waste Recovery Postpraduct Steps The system will flush the retentate to waste RVB 2 Summary M Recirculation before initial Recovery with no backpressure SSS AKTAcrossflow User Manual 11 0012 32 Edition AB 121 Creating Product methods using the Method Wizard SS ey Recovery of permeate e For Lysate clarification the target product is passing the filter to the permeate During the concentration step through P VB Out2 and during the diafiltrat
89. bar default If Retentate pressure gt Offset the Retentate pressure value is used as a new Offset If Retentate pressures lt Offset the Retentate control valve lifts Retentate pressure to Offset Permeate unrestricted flow resets TMP control and Flux control The mode is used for example in the Method Wizard created preproduct method Rinsing see Section 5 2 1 AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction Normal Flow Filtration In the Normal Flow Filtration mode the flow and pressure over the filter are controlled by the feed pump only In the permeate line the permeate control valve P PCV is fully opened As a consequence liquid can be pushed through the permeate pump as the check valves of the permeate pump are not active In order to allow for a homogeneous flushing of the pump heads of the permeate pump during Normal Flow Filtration the permeate pump is running idle at a low flow rate 20 of Feed flow while liquid is transfered by the feed pump The pressure over the NFF filter DNFF is determined as the difference between feed pressure and permeate pressure in order to compensate for a minor pressure loss in the components of the permeate line pNFF feed pressure permeate pressure Control Mode Control Element Normal flow filtration Feed Pump Permeate PCV Pump NFF control with constant 100 20 of Feed R PCV Closed Feed flow flow P PCV Opened NFF control with
90. bled 50 ml is emptied through the filter P VB is set to recirculation and the liquid leaves the system through Transfer Purge Valve Waste Air Trans fer UB1 Permeate UB P UB Recucie EMN Transfer PurgeValve Ena Muna Trans fer UB2 UB In 4 The rest of the reservoir liquid is emptied through R VB Out2 At level O ml in the reservoir the water flush is complete Trans fer UB1 Permeate UB P UB Recucie Transfer PurgeValve MEn Muna Trans fer UB2 Mune KTAcrossflow User Manual 11 0012 32 Edition AB 87 Creating Preproduct methods using the Method Wizard ay 5 4 6 Visualized procedure of the Water flux test The filling of reservoir procedure is included in the previous method which should be Rinsing or Water flush So the Water flux test is just a continuation of these methods If the Waterflux test is used as a single method Prepare system will be the first step Additional 50 ml will be used for the test 1 The permeate valve block is set to recirculation and the transfer purge valve is set to reservoir A permeate flow is started by TMP control When a stable flux has been achieved a value of a normalized water flux is collected by setting a Set_Eval_Mark with the parameter Normalized_Water_Flux If
91. block name and the length of the block The line is shifted down to indicate calls to other blocks estore CONCE 0 00 min Cond Buffer Inl 15 00 ml 999999 00 ml Stop Perm Pun DelayOp05min DelayOp05min 0 05 min 0 05 min Watch_ConeFactor Greater_Tha Watch RetVol Less Than 13 m 0 00 min 0 05 min 0 05 min 0 00 min Hold_Until Trans ol Greater_Than 0 00 ml INFINITE base Hold_Until TransVol Greater_Than 5 ml INFINITE base Hold_Until DeltaP Stable_Baseline 1 00 Minutes 5 00 base Hold_Until Trans_Flow Less_Than 0 1 ml min 5 00 base 46 AKTAcrossflow User Manual 11 0012 32 Edition AB 3 1 6 Run Setup Handling methods in AKTAcrossflow Ea Run Setup in the Method Editor is a dialog box with a number of tabs that define the method properties Start Protocol Questions Result Name Variables Scouting Notes Gradient Columns Reference Curves Evaluation Procedures Method Information Block la Variable Value Range Start_Settings_System Ret_Vol ml 13 00 0 00 5000 00 Feed_Press_Alarm bar 5 20 0 00 5 20 TMP_Alarm bar 0 00 5 20 Start_Settings_FS Filter_Area crr 1 0 1000 0 Sample_volume Sample_Vol_High ml 0 00 80000 00 Sample_Volume ml 0 00 80000 00 RetFlow_Step1 Ret_Flow_Step1 ml min 0 0 600 0 Hold_Until_DeltaP DeltaP bar 0 00 5 20 TMP_Step1 TMP_Step_1 bar 0 00 5 20 Sieni Cone 000
92. buffer 1 See Help Calibrate pH electrode for buffer 2 See Help Parameter 1 name Reference value 1 Input default underlined 0 00 pH 0 00 14 00 Parameter 2 name Reference value 2 Input default underlined 0 00 pH 0 00 14 00 Result Value 1 Calibrated electrode slope 0 0 Result Value 2 Asymmetry potential at pH 7 0 0 mV Help text The pH Monitor is calibrated using standard buffer solutions in a two point calibration Difference between the buffers should be at least 1 pH unit A new electrode has a slope of typically 95 102 and an asymmetry potential within 30mvV As a rule when an electrode has an asymmetry potential outside of 60mV and a slope lower than 80 and no improvements can be achieved by cleaning it should be replaced AKTAcrossflow User Manual 11 0012 32 Edition AB 209 Strategy instructions oe Cond_Calib Monitor Function Cond_Calib Calibration of cell constant Text 1 Determination of cell constant for Cond cell See Help Parameter name Input default underlined Reference value 1 1 000 mS cm 1 0 999 9 Result Value Cell constant 1 cm 0 1 300 0 Help text Calibration of Cond conductivity cell Normally it is not necessary to adjust the cell constant as the flow cell is pre cali brated on delivery Set CondTempComp to 0 in System Settings prior calibration of cell constant The temperature sen sor must be calibrated before adj
93. calibration Text 1 Sets the pressure reading to zero See Help Help text Calibrate pressure offset according to step 1 Calibrate pressure offset for Transfer pump Click the Start calibrate button For details see AKTA Instrument Handbook AKTAcrossflow User Manual 11 0012 32 Edition AB 211 Strategy instructions 212 AKTAcrossflow User Manual 11 0012 32 Edition AB Index Index A after run AKTAcrossflow AKTAcrossflow run alarms applications assembling filters B basic settings Io LSE MTE E ES A EEEE N E AE EEE AA hollow fibers Block pane blocks buffer conditioning buffer exchange 18 Cc CONDORONT tsi ate eee Ate ose beech sted bee otto boa 0 cel lhe tated A 136 20 calls ee cartridges CAUTION cell harvesting cell washing control modes Cross flOW oe CLOSS TOW MONUGIIV FUAS cc eee se tae tase ah a ete dee se et aaseel 63 N D DeltaP ow diafiltration diafiltration method documentation E CRNA OUAE E EA AEAEE PENE A E EI A E ENE EE A 103 105 EXthO TUBING Volume wait a a a a aaa 74 F feed flow filter CIP filter component flat sheet cassette flow path flux flux control mode H ONO R T ai i EES EAEE TAEAE EAEAN EN NAAA EAEAN AA DANAEA ASEE TAE A E TE 46 MOMOW FIBERS lt scgiscteee etescsveenc ceca ce sccncetiel dl saaysaueesinctedseescobepdetitaecsctbacutetdatas
94. ck Start 5KD he Product Steps Concentration Diafitration x Recovery Postproduct Steps Steps Summary 7 Preproduct X Product Postproduct Flat Sheet specification per filter Surface Area a cm 2 40 150 cm 2 Filter Hold Up Volume 14 ml 0 0 25 0 m Feed Pressure Limit bar 0 5 2 bar TMP Limit j bar 0 5 2 bar System setup Number of filters Extra Tubing Volume ml 0 0 25 0 ml Reservoir Size 360 mi 1100 mi Tubing kit Small ID 1 7 mm Large ID 2 9 mm Finish Cancel Help Set Default e inthe Method list select Ultrafiltration Basic Settings Method Ultrafiltration v gt 5 Calibrate level sensor Filter List Installation Test System Sanitization Ultrafiltration e inthe Filter List available GE Healthcare filters are displayed Select the used filter or select Other Method Ultrafiltration v Filter List Kvick Stat 5KD v 10KD 10 KD Select i OKD piens Keck Stat 100 KD Preprodul Other A 98 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard e Inthe next list select Concentration Diafiltration Basic Settings Method Ultrafiltration v Filter List Kvick Stat 5KD 50 cm 2 Concentration Diafiltration v UF Process Optimization e Inthe next area select the type of process step Steps C Preproduct V Product _ Postproduct e The Flat Sheet specification per filter area displays the recommended default values for the sel
95. ction not allowed when during NFF 172 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions fe 13 2 7 RPCVoffset Instruction name Group RPCVoffset System Settings Specials Parameter name Set point default underlined Pressure 350 0 999 Instruction Help text Sets offset on RetentateControlValve used by system during TMP_Control Flux_Contro l and Permeate_Unrestricted_Flow 13 2 8 RPCVhysteresis Instruction name Group RPCVhysteresis System Settings Specials Parameter name Set point default underlined Setpoint 50 0 100 Description Instruction Help text 13 2 9 Reservoir_Size Instruction name Group Reservoir_Size System Settings Specials Parameter 1 name Position name default underlined Size 350 ml 350 1100 ml Instruction Help text Sets reservoir size 350 or 1100 ml AKTAcrossflow User Manual 11 0012 32 Edition AB 173 Strategy instructions _ 13 2 10 EmptyReservoir nstruction name Group EmptyReservoir ethod Manual Recirc Parameter 1 name Position name default underlined RetValveOutlet R VB Out1 R VB Out2 R VB Out3 Parameter 2 name Position name default underlined axFeedPressure 0 00 bar 0 00 5 20 bar Parameter 2 name Position name default underlined axFeedFlow 600 0 ml min 0 0 600 0 ml min nstruction Help text Instr
96. d The program can also be set up so that you can log on without a password See UNICORN manuals and Online Help Or login as default user with password default UNICORN logon Users 5 Inthe System Control module select System Connect to connect UNICORN to the instrument unit System Help F Rgonect Ctrl N 32 AKTAcrossflow User Manual 11 0012 32 Edition AB System preparation ss 6 Select the appropriate system name and click OK System Connect W AKTActosstlow System 1 When the communication between UNICORN and the instrument unit is established e There is a constant light on the Power indicator on the instrument unit e The green Run indicator in the status bar in UNICORN is lit e The Connection box shows Yes The Instrument box shows Ready se 4 g Run a ai ET AF Run Data ia a 4 aa Run Status Acc Volume Block Volume Acc Time Block Time 7 wa z3 2 2 Assembling Filters For preparing and connecting Flat sheet membrane cartridges or Hollow fiber membrane cartridges see KTAcrossflow Instrument Handbook peN KTAcrossflow User Manual 11 0012 32 Edition AB 33 System preparation Sess 2 3 The pump piston rinsing system 2 3 1 Introduction Leakage between the pump chamber and the drive mechanism is prevented by a seal The seal is continuously lubricated by the presence of eluent To prevent any deposition of salts from aqueous buff
97. d Editor of UNICORN displays the method as a list of text instructions The instructions are usually organized in blocks denoted by blue square symbols for a specific functional use for example to load a sample to concentrate a sample etc A block may contain other blocks or individual instructions The blocks can be expanded to show the instructions within the block E Main W 0 00 Block Info_Inlets_and_Outlets W 0 00 Block INITIAL_SETUP W 0 00 Block FILL_SAMPLE S W 0 00 Block CONCENTRATION_1 CONCENTRATION_1 0 00 Base SameAsMain 0 00 Set_Mark Concentration 1 W 0 00 Block Flowpath_PermConc Flowpath_PermConc 0 00 Base Time 0 00 Permeate_ alye_Block P B Out2 W 0 00 Block DelayOp05min 0 00 End_Block W 0 00 Block RetFlow_Step1 W 0 00 Block TMP_Stepi W 0 00 Block Chase_Inlet_to_Reservoir Mi 0 00 Block Stepi_Conc W 0 00 Block Restore_CONCENTRATION 0 00 End_Block Mi 0 00 Block RECOVERY 3 1 2 Base Every method block must start with a Base instruction defining the base for calculating breakpoints Different blocks can use different bases In AKTAcrossflow the base can be one of the following e Volume the unit depends on the scale defined in the system strategy e Time minutes e SameAsMain all blocks apart from the main block which means that the block will inherit the base defined in the main block Note Do not use Column Volume CV as base in AKTAcrossflow methods as it is not relev
98. d Wizard for hollow fibers Hollow fiber has to be selected as component in System Setup See Section 2 4 Method Wizard for System AKTAcrossflow2 Basic Settings Basic Settings Method Preproduct Steps Filter Type Product Steps Filter List AXM 3 KD 0 50 mm Recovery Postproduct Steps Steps Summary Preproduct v Product Postproduct Hollow Fibre specification per filter Lumen Diameter 25 mm 0 10 10 00 mm Number of Fibers 12 11 1000 Surface Area 50 cm 2 40 150 cm 2 Lumen Hold Up Vol mil 0 0 25 0 ml Feed Pressure Limit 43 bar 0 5 2 bar TMP Limit 30 bar 0 5 2 bar System setup Number of filters B Extra Tubing Volume ml 0 0 25 0 ml Reservoir Size 350m 1100 m Tubing kit Small ID 1 7 mm Large ID 2 9 mm Cancel Help SetDefaut e Inthe Method list select Ultrafiltration Cell Processing Basic Settings Method Ultrafiltration Cell Processing v Calibrate level sensor Filter Type Installation Test iF System Sanitization Filter List Ultrafiltration Cell Processing e Select Filter Type AXM or AXH AXM hollow fibers have a straight linear shape AXH hollow fibers are curved into a loop aa KTAcrossflow User Manual 11 0012 32 Edition AB 75 Creating Preproduct methods using the Method Wizard e Inthe Filter List available GE Healthcare filters are displayed Select the used filter or select Other Basic Settings Method Ultrafiltration Cell Processing x Filter T
99. d default settings for the controllers P PI or PID type are listed in Table 12 1 These settings provide fast response and robust control for most operating situations However the following controllers may require adjustment depending on the type and behaviour of the filter 12 1 1 Feed pump control FeedPressure_PI DeltaP_PI and EmptyResFeed_PI These three controllers ramp the flow rate of the feed pump to achieve a desired pressure The default settings are appropriate for Flat Sheet Cassettes that give higher back pressure than Hollow Fiber cartridges at a given flow rate When using Hollow Fiber cartridges the action of the Integral controller I parameter is recommended to be increased by reducing the figure for the parameter to yield a faster pump response and shorter ramp time respectively For the alternative settings see Table 12 1 When creating methods with the Method Wizard the alternative settings in Table 12 1 are automatically installed for the different process steps when appropriate 12 1 2 TMP control TMP_PID_PermeatePump TMP_PID_RetentateControlValve During TMP control the permeate pump controls the permeate pressure by adjusting the flow rate of the permeate pump Depending on membrane area filter cut off and process conditions the magnitude of the permeate flow rate may vary in a wide range from approx 1 ml min to 50 ml min and higher Two settings are recommended to provide fast and robust control
100. d is T VB In5 water The reservoir is filled with 100 ml water At Constant Retentate Volume and TMP regulation a volume defined by filter type and area is passed through the filter out to P VB Out1 Waste Air Trans fer UB1 Permeate UB UB Recucls AENA Cartridge permeate N Constant Retentate Volume is disabled 50 ml of the reservoir volume is emptied through the filter P VB is set to recirculation and the liquid leaves the system through Transfer Purge Valve Waste Air Transfer VB1 Permeate UB F UB Recuctel E E Transfer PurgeValve Transfer UB2 Ey Cartridge permeate Rue The rest of the reservoir volume is emptied through R VB Out2 At level 0 ml in the reservoir the rinsing is complete Air Transfer UB1 UB Int Permeate UB P Ue Recucla AENA MEn Trans fer UB2 Hee Cartridge permeate Rue 5 4 4 Visualized procedure of the Filter CIP method If the method is a post product method the system and reservoir are prepared according to the procedure Preparing the system and reservoir described in Section 5 4 1 The Transfer Inlet used is T VB In6 CIP solution If the method is a pre product method Preparing the system will be used 1 84 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct
101. ds using the Method Wizard e Circulate at set time e Empty the system of CIP solution Note The system will not be sanitized For sanitization of the system see Section 10 1 5 2 3 Water flush A used filter can be rinsed with the Water flush method It is also the recommended method to be performed after a Filter CIP method where NaOH was used The method should also be used if a filter has not been rinsed with water before a water flux test is performed The method will rinse e The water inlet tubing in the transfer section e Retentate loop e Reservoir Note Ifthe previous method is Filter CIP or the Water flush method is a post product step system and reservoir will be washed Otherwise only system will be washed e Retentate and permeate side of filter 70 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea a a 5 2 4 Water Flux test Note Water flush or Rinsing has to performed before Water Flux test This is a method to have control of the quality status of the membrane cartridge By comparing obtained water fluxes as a function of time of usage it is possible to see when it is necessary to extensively clean or discard the filter We recommend performing the test e before a product step and e after a product step and filter cleaning The way to perform the test by used control mode is dependent on the filter type used Default for flat sheets is
102. e UV signal Averaging time is the time interval used for calculating the moving average of the absorbance signal A long averaging time will smooth out noise efficiently but will also distort the peaks 188 AKTAcrossflow User Manual 11 0012 32 Edition AB es 13 5 3 Pressure_Filter_Factor Strategy instructions Pressure_Filter_Factor Instruction name Group System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined Mode 30 NoFilter 2 100 Instruction Help text i e all control modes When active all pressure signals is filtered to avoid overshooting and hysteresis during pressure regulating operations 13 5 4 Alarm _UV Instruction name Group Alarm _UV System Settings Alarms Method Manual Alarms amp Mon Parameter 1 name Position name default underlined Mode Disabled Enabled Parameter 2 name Setpoint default underlined High Alarm 6000 000 mAU 6000 6000 Parameter 3 name Setpoint default underlined Low Alarm 6000 000 mAU 6000 6000 Instruction Help text Sets the Alarm limits for the UV signal 13 5 5 Alarm _pH Instruction name Group Alarm _pH System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined Mode Disabled Enabled Parameter 2 name Setpoint default underlined High Alarm 14 00 pH 0 14 Parame
103. e Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condi tion For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 201 Strategy instructions _ 13 5 31 WatchPar_TransVol Instruction name Group WatchPar_TransVol System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 ml 0 00 80000 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 ml 0 00 80000 00 nstruction Help text atch on transfer volume The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 32 WatchPar_DeltaP Instruction name Group WatchPar_DeltaP
104. e buffer buffer exchange The product is retained at the retentate side A buffer exchange is typically run after an concentrating step using the same filter as for the concentration step So the methods can be combined to one method If diafiltration is the single step and the sample volume is low the hold up volume on the retentate side will dilute the sample Therefore in this situation we recommend to perform an initial concentration and enter a Concentration factor of 1 as endpoint See Section 7 1 4 Endpoint Product recovery after Concentration Diafiltration There are two alternatives to recover the product in the retentate e No Recovery An option to select if the retentate volume will be drained manually e Recovery The reservoir is first emptied through RVB Out3 A defined number of flushes can then be selected and the retaining product will be emptied through RVB Out1 For descriptions of the Recovery procedures see Section 7 1 7 Recovery section AKTAcrossflow User Manual 11 0012 32 Edition AB 97 Creating Product methods using the Method Wizard es 7 1 2 Basic settings dialog Note To get the Method Wizard for flat sheets Flat sheet has to be selected as component in System Setup See Section 2 4 Method Wizard for System AKTAcrossflow1 Basic Settings Basic Settings Method Preproduct Steps Filter List Kvi
105. e measured conductivity values will be converted CondRefTemp is active when a factor of CondTempComp is selected 13 8 Watch These signals should have a Watch instruction defined by UNICORN in the following order and also be possible to select Watch_Off for Hold_Until UV pH Cond Feed_Press Reten_Press FeedFlow R_Flow Perm_Flow Trans_Flow RetVol PermVol TransVol DeltaP TMP Flux Shear ConcFactoror DF_X_Fact FluxDrop Airsensor ZeroLevel Auxin1 Auxin2 Auxin3 AuxIn4 These signals should be possible to select Hold_Until for defined by UNICORN in the following order UV pH Cond Feed_Press Reten_Press FeedFlow R_Flow Perm_Flow Trans_Flow RetVol PermVol TransVol DeltaP TMP Flux Shear ConcFactoror DF_X_Fact FluxDrop Airsensor ZeroLevel Auxin1 Auxin2 Auxin3 AuxiIn4 208 AKTAcrossflow User Manual 11 0012 32 Edition AB 13 9 Calibration 13 9 1 ZeroLS Strategy instructions Monitor Function ZeroLS 0 calibration of level sensor Text 1 0 calibrate the level sensor See Help 13 9 2 LevelsensorZeroCalibration Instruction name LevelsensorZeroCalibration Group System Settings Monitors Instruction Help text Give user access to Calibration instruction in order to make it possible to calibrate Level sensor from a method 13 9 3 UPC pH Monitor Function pH Calibration of pH electrode Text 1 and 2 Calibrate pH electrode for
106. e parameters are used to tune the feedback control when DeltaP is active ConstRVol_P This parameter affects the responsiveness of the CRV algorithm It controls how fast the transfer pump will compensate changes of the reservoir volume when using ConstantRetentateVolume EmptyResFeed_PI This instruction allows manipulation of the P and parameters of the regulator algorithm used to keep the feed pressure at the requested level during the EmptyReservoir procedure Table 12 5 Descriptions of PI instructions AKTAcrossflow User Manual 11 0012 32 Edition AB 163 Feedback tuning and PID parameters 164 12 5 Optimizing the PI parameters The objective to optimize the PI parameters is to obtain a e Smooth regulation e without over pressure peaks but e fast enough Too weak PI values may lead to a smooth but slow ramping Too aggressive PI values may lead to a fast but instable ramping 12 5 1 Regulation of the Feed pump example The Feedpump_PI instruction was used when the system was run manually The Feed pump was run using different PI parameter values to obtain a set Feed pressure PI settings Curve example Feed pump flow 1 Settings The parameter was set to a high value 1000 which means it has minimal effect of the regulation The P parameter was set to a high value 1 0 Result Totally uncontrolled regulation with high fluctuations 2 Settings The paramet
107. e passed and finally the method is saved e When an Instant Run is performed In this case the Method Wizard dialogs are passed before the run is performed The method is not saved in this procedure See Section 9 3 To create a new method using the Method Wizard in the Method Editor Note Depending on used filter type in the method Hollow fiber or Flat Sheet remember to select correct component according to instruction in Section 2 4 S 1 Inthe Method Editor of UNICORN click the Method Wizard icon 2 Select System Method Wizard Select a system KTAcrosstlow System 1 AKTAcrossflow System 2 KTAcrossflow User Manual 11 0012 32 Edition AB 55 Handling methods in AKTAcrossflow 56 3 Adialog of Basic Settings will first be displayed Note This example is from when Flat Sheet is selected as component To obtain default values in the Method Wizard click Set Default This is only possible in the first dialog In the Method list the type of process is selected The Filter List section will display available GE Healthcare filters The used filter type is selected here Below the Filter List the type of method and included steps are selected The Flat Sheet specification per filter area displays the recommended default values for the selected filter type These can be edited by the user In the System setup section it is possible to select number of filters and extra tubing volume This is necessa
108. e system is set to Pause Alarm DeltaP _HighAlarm DeltaP _LowAlarm Alarm Help text HighAlarm cause The pressure has exceeded the HighAlarm limit LowAlarm cause The pressure has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue AKTAcrossflow User Manual 11 0012 32 Edition AB 191 Strategy instructions 13 5 10 Alarm_TMP Instruction name Group Alarm_TMP System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined ode Disabled Enabled Parameter 2 name Setpoint default underlined HighAlarm 5 20 bar 0 00 5 20 bar Parameter 3 name Setpoint default underlined LowAlarm 0 00 bar 0 00 5 20bar nstruction Help text Sets the alarm limits for TMP When reached the system is set to Pause Alarm TMP_HighAlarm TMP_LowAlarm Alarm Help text HighAlarm cause The pressure has exceeded the HighAlarm limit LowAlarm cause The pressure has fallen below the LowAlarm limit Press Acknowledge on the error message window Press Continue 13 5 11 Alarm_Flux Instruction name Group Alarm_Flux System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined ode Disabled Enabled Parameter 2 name Setpoint default underlined HighAlarm 400 LMH 0 400 Parameter 3 name Setpoint default underlined LowAlarm O LMH 0 400
109. eControlValve TMP_PID_PermeatePump Flux_ Pl_RetentateControlValve PUF_PI_RetentateControlValve PUF_PI_PermeatePump pNFF_PI EmptyResFeed_PI ConstRVol_P FracParameters Frac_Numbering_Mode Reservoir_Size fC AKTAcrossflow User Manual 11 0012 32 Edition AB Monitors AveragingTimeUV Pressure_Filter_Factor CondTempComp CondReffemp pHTempComp AirSensor WatchPar_UV WatchPar_pH WatchPar_Cond WatchPar_Feed_Press WatchPar_Reten_Press WatchPar_ PermPress WatchPar_FeedFlow WatchPar_Ret_Flow WatchPar_ PermFlow WatchPar_TrfFlow WatchPar_ RetVol WatchPar_ ResVol WatchPar_ PermVol WatchPar_ TransVol WatchPar_ DeltaP WatchPar_ TMP WatchPar_ Flux WatchPar_ Shear WatchPar_ pNFF WatchPar_ConcFact WatchPar_DF_X_Fact WatchPar_ Flux_Drop Curves UV Cond FeedFlow RetFlow PermFlow TrfFlow Feed_Press RetenPress PermPress pH Temp DeltaP TMP Flux Shear ConcFact DF_X_Fct RetVol ResVol PermVol TransVol ConvRatio pNFF Auxin1 AuxOut1 169 13 Strategy instructions 13 1 2 Instruction box The order of instructions within each group Note Only groups defined by the strategy are described Recirc Permeate Transfer Alarms Watch Warnings and Monitors FeedFlow TMP_Control Trans_Flow AutoZeroUV Hold_Until RetFlow2 Flux_Contro Constant_Ret_Volume AveragingTimeUV Watch_UV FeedPressure Permeate_Valve_Block Transfer_Valve_Blocks Pressure_Filter_Factor Watch_pH DeltaP2 Permeate_Unrestricted_Flow
110. ected filter type Accept or edit the values Flat Sheet specification per filter Surface Area 50 cm 2 40 150 cm 2 Filter Hold Up Volume 1 4 ml 0 0 25 0 ml Feed Pressure Limit 4 0 bar 0 5 2 bar TMP Limit 40 bar 0 5 2 bar If Other filter type has been selected enter Surface Area Hold Up Volume Feed pressure and TMP limits for the flat sheet Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit e If several filters are assembled together in parallel select in the System setup section the Number of filters 1 3 If one filter is used only keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System setup Number of filters 1 ad Extra Tubing Volume oo ml 0 0 25 0 ml e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 350 mi 1100 mI Tubing kit Small ID 1 7 mm O Large ID 2 9 mm AKTAcrossflow User Manual 11 0012 32 Edition AB 99 Creating Product methods using the Method Wizard 100 7 1 3 Product steps dialog Method Wizard for System AKTAcrossflow1 Product Steps v Basic Settings Number of Steps l Product Steps Step1 Step1 Concentration Diaf
111. edures 10 1 System Sanitization aaa a i 145 10 1 1 Sanitization of the pump piston rinsing System woeeeeccssscscscsscsceseeseeeee 145 10 1 2 Sanitization of reservoir FOC eesssssssseeesssessssseessseeceesesestssseentenanenee 145 10 1 3 Creating a method isos a TE a EE dissents 146 10 1 4 Running the System sanitization MethO ssssssssssssssssssssssscccecseseceeees 147 10 2 Viewing and printing the result 148 102 1 Viewing the Tesultiscciissidcahet As A was R UR 149 102 2 PRINTING the FeSUl tas esccastecssctcnaitaaiasaaununnaraaantoadiiatarncne 151 E KTAcrossflow User Manual 11 0012 32 Edition AB vii Contents Sra 11 Evaluating AKTAcrossflow results 11 1 Associated documentation oeceeccceeeccssssssssssssssssssssssessseseesecsenceceeceecceneeceee 153 11 2 Opening the Evaluation module oo cccccssssssessscscssccssssesssseseesseescessssees 153 TLS Mai fUNCtON S eneiniad an iN 154 11 3 1 Anyvs any w 11 3 27 Process Optimizat N sren innean ae 154 11 3 3 Normalized Water flUX eccccccccccsssssssscssssscssssessssssssssssssssssnussessssssseceeesesseeeeeeees 156 11 34 CGPACIY plotr nnna A AERAN 156 12 Feedback tuning and PID parameters 12 L A PID CONTO ian r oa a A N aa 157 12 1 1 Feed pump control FeedPressure_PI DeltaP_PI and EmptyResFeed PI sesssssssssses 157 12 1 2 TMP control TMP_PID_PermeatePump TMP_PID_RetentateControlValve 157 12 1 3 Tuning and troubleshooti
112. eed Instruction name Group MixerSpeed System Settings Specials Manual Method Parameter 1 name Position name default underlined Speed Auto 0 600 rpm Instruction Help text Sets Mixer speed Auto adjusts mixer speed to reservoir size SystemSettings and actual ResVol in a linear relation Min Speed 80 rpm Max Speed for 350ml 200 rpm and Max Speed for 1100m 300 rpm 13 2 14 Set_ResVol_Totalizer Instruction name Group Set_ResVol_Totalizer Method Manual Recirc Parameter name Set point default underlined Volume 0 0 ml 0 00 5000 00 Instruction Help text Sets a desired volume for totalizer Transfer pump flow will increase value if it is directed to reservoir Permeate flow if permeate outlet valve is opened will decrease volume but not during position Recycle of the Permeate_Valve_Block and if Transfer_Purge_Valve is in position reservoir Feed flow with retentate valve open will also decrease volume AKTAcrossflow User Manual 11 0012 32 Edition AB 175 Strategy instructions O 13 2 15 ManSample Instruction name Group ManSample Manual Recirc Parameter 1 name Position name default underlined Volume 0 00 ml 0 00 100 00 Instruction Help text Reduce totalizer manually with the amount of removed sample at that very moment See equation under Concentration_Factor in User Manual It is very important that a correct value is inserted ASAP 13 2
113. el An alarm will set the system to Pause if zero level is indicated by the level sensor 13 5 18 WatchPar_UV Instruction name WatchPar_UV Group System Settings Monitors Method Manual Alarms amp Mon Parameter 1 name DeltaPeak Setpoint default underlined 0 00 mAU 0 6000 Parameter 2 name Delta_Base Setpoint default underlined 0 00 mAU 0 6000 Instruction Help text Watch on UV signal The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN User Manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this con dition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 195 Strategy instructions 13 5 19 WatchPar_pH Instruction name Group WatchPar_pH System Settings Monitors Method Manual Alarms amp Monitors Parameter name Setpoint default underlined DeltaPeak 0 00 pH 0 14 Parameter name Setpoint default underlined Delta_Base 0 00 pH 0 14 nstruction Help text atch on pH signal The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using
114. en reservoir and RVB Out3 will be emptied through RVB Out3 e There is also an option of performing recirculation between the flushes Recovery Recovery Option Initial Recovery collected through R VB 3 includes empty reservoir and retentate tubing product displacement Recirculation before initial Recovery with no backpressure Buffer Flushes collected through R VB 1 Number of Buffer Flushes 1 O2 Volume Flush 1 16 3 mi 16 9 350 ml M Recirculation before Flush Recovery Note The product will leave the system through R VB Out3 and the flushed volumes through R VB Out1 AKTAcrossflow User Manual 11 0012 32 Edition AB 107 Creating Product methods using the Method Wizard ee 7 1 7 Visualized procedure of an Ultrafiltration method Note For information on the specific instructions for example Constant Retentate Volume see Chapter 13 Sample filling Example for a 100 ml sample volume 1 To prime the sample inlet tubing Transfer pump fills the tubing with 6 ml sample from T VB In1 sample 2 To prime the distance to Transfer Purge Valve Waste additional 10 ml is filled iS Vanna 7 Pump 3 Reservoir is filled with 90 ml sample 4 The last 10 ml of the sample is chased with buffer to the reservoir 5 Recirculation on the retentate side oa SSS 108 AKTAcrossflow
115. ength of Time 60 min 0 9999 min CIP 2 Length of Time 60 min 0 3999 min M Water Flush between CIP e Ifthe large reservoir is used Volume to Fill has to be entered CIP 1 Volume to Fill 200 ml 0 1100 mi Lenath of Time 60 min 0 9999 min CIP 2 Volume to Fill 200 ml 0 1100 mi Length of Time 60 min 0 3999 min M Water Flush between CIP Water flush e To include a Water flush in the method check the Water Flush box Note After a Filter CIP we recommend running a water flush Note Ifa Water Flux test will be performed Water Flush should be performed before the test M Water Flush pN KTAcrossflow User Manual 11 0012 32 Edition AB 131 ED Creating Postproduct methods using the Method Wizard aes Water Flux test e To include a Water Flux test in the method check the Water Flux Test box e Select to use TMP or NFF Normal Flow Filtration as control mode NFF is default for hollow fibers with cut off 0 1 um and larger e For TMP as control mode e Enter a TMP value for the test Note When comparing status of a filter as a function of time and number of experiments it is important to have performed the same type of method i e same TMP value Water Flush Water Flux Test TMP 1 bar 0 5 2 bar ONFF e For NFF as control mode e Select to perform Feed control for NFF with FeedFlow or FeedPressure Water Flush Water Flux Test OTMP ike FeedPressure x Feed Control for NFF FeedFlow
116. entate valve retentate valve block type rinsing RUN indicator in UNICORN Run Setup S SAMPING assan A E E T R 143 scouting shear starting a run starting AKTAcrossflow system storage solution filter system strategy instructions system preparation system sanitization T tangential flow filtration text instructions TMP TMP control mode transfer instructions transmembr ne presul sanss A a a an AA ai 16 U ultrafiltration ultrafiltration method UNICORN methodi tannanna aa a a a cae dhe vine ea Baal 45 starcand OJON errr nea ret i a E 31 WwW WARNING warnings Watch water flush water flux test a673 71 WOPKING VOIUIMES wctscncsticisiteanitadaslsscctdttearancaguanlahaustem S TAA 101 117 AKTAcrossflow User Manual 11 0012 32 Edition AB 215 216 AKTAcrossflow User Manual 11 0012 32 Edition AB www gehealthcare com GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden AKTA Drop Design AKTAcrossflow UNICORN and Kvick Start are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric Company Milli Q is a trademark of Millipore Corp Microsoft and Windows XP are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries All goods and services are sold subject to the terms and conditi
117. er was kept to 1000 The P parameter was set to 0 5 Result The size of the first step is too large AKTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters PI settings Curve example Feed pump flow 3 Settings The parameter was kept The P parameter was set to 0 2 Result The size of the first step is still too large 4 Settings The parameter was kept The P parameter was set to 0 1 Result The size of the first step is still too large 4b Settings The parameter was kept The P parameter was set to 0 05 Result The size of the step seems to be correct 5 Settings The P parameter is kept to 0 05 The optimization of the parameter starts with the settings 500 and continues with 1 200 Result The slope of the curve is increasing with decreased values AKTAcrossflow User Manual 11 0012 32 Edition AB 165 Feedback tuning and PID parameters 166 PI settings Curve example Feed pump flow 6 Settings The parameter is further decreased 200 100 Result Increased slope is obtained 7 Settings The P parameter was kept at 0 05 and the parameter was set to 25 Result The curve indicates a too high flow before it stabilizes The parameter should be increased 9 Settings The parameter is increased to 50 Result The PI parameters are optimized and a smooth and fast regulation is obtained
118. ers on the pistons and to prolong the life of the seals the low pressure chamber behind the piston can be flushed continuously with a low flow of 10 mM NaOH in 20 ethanol prepared in Milli Q water or equivalent The piston rinsing system tubing is connected to the rearmost holes on the pump heads The following flow diagram and table show the tubing configuration of the piston rinsing system Feed pump P 984 Optional path j Waste H Rinsing solution Transfer amp Permeate pump P 982 Optional pati S4 S5 I I Waste w Rinsing solution Fig 2 1 Piston rinsing system E 34 KTAcrossflow User Manual 11 0012 32 Edition AB System preparation To eliminate the risk of re introducing proteins cells into subsequent batch runs always use a separate waste container Do not recirculate the same rinsing solution If rinsing solution is recirculated the solution should be replaced with fresh solution every day or between cross flow runs To estimate the required volume of rinsing solution the flow rate of rinsing solution is approx 2 3 of the set flow of the pump In connection to System sanitization the rinsing bottles should be cleaned 2 3 2 Using the pump piston rinsing system To use the piston rinsing system 1 Fillthe rinsing system bottles with 10 mM NaOH in 20 ethanol see Fig 2 2 2 Insert the rinsing inlet and outlet tubing ends into the rinsing solution see Fig 2 1 Note To eliminate the risk of re in
119. ettings Specials ethod Manual Permeate PUF_PI_PermeatePump System Settings Specials ethod Manual Permeate pNFF_PI System Settings Specials ethod Manual Permeate DeltaP_PI System Settings Specials ethod Manual Recirculation ConstRVol_P System Settings Specials ethod Manual Transfer EmptyResFeed_Pl System Settings Specials ethod Manual Recirculation Table 12 4 PI instruction groups ee 162 AKTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters 12 4 2 Description of the PID instructions In Table 12 5 the PID instructions are described Instruction name Description FeedPressure_Pl These parameters are used to tune the feedback control when Feed_Pressure_Control is active TMP_PID_RetentateControlValve These parameters are used to tune the feedback control when TMP_Control is active TMP_PID_PermeatePump These parameters are used to tune the feedback control when TMP_Control is active Flux_Pl_RetentateControlValve These parameters are used to tune the feedback control when Flux_Control is active PUF_PI_RetentateControlValve These parameters are used to tune the feedback control when Permeate_Unrestricted_Flow is active PUF_PI_PermeatePump These parameters are used to tune the feedback control when Permeate_Unrestricted_Flow is active pNFF_PI These parameters are used to tune the feedback control when Normal_Flow_Filtration is active DeltaP_Pl Thes
120. etup section the Number of filters 1 3 If only one filter is used keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System setup Number of filters Extra Tubing Volume 00 ml 0 0 25 0 ml 1 v KTAcrossflow User Manual 11 0012 32 Edition AB 129 ED Creating Postproduct methods using the Method Wizard e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 350 ml 1100 ml Tubing kit Small ID 1 7 mm O Large ID 2 9 mm 8 2 3 Postproduct setup Method Wizard for System AKTAcrossflow1 Postproduct setup Basic Settings Postproduct Steps M Flush Summary Conditioning Buffer O Water M Fitter CIP moar Length of Time Ocr2 Z Water Flush Z Water Flux Test T P bar 0 5 2 bar ONFF Filter Storage Solution Flush e To include a Flush in the method check the Flush box and select to perform the flush with conditioning buffer or water Flush Conditioning Buffer Water 130 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Postproduct methods using the Method Wizard 8 ss Filter CIP e To include a Filter CIP check the Filter CIP box e There is an option to select two CIP with a optional Water flush between e The circulation time of CIP solution is entered in the Length of Time boxes Filter CIP CIP 1 L
121. ever a new filter does not need to be washed with a CIP method The method starts with Preparing the system block The method will rinse e The water inlet tubing in the transfer section e Retentate loop e Reservoir e Retentate and permeate side of filter Hollow fibers for microfiltration are dry at delivery However they need to be wetted with the Rinsing method before use Note Some Hollow fibers might not wet out very well with water If problems occur for example a failed water flux test it can be necessary to flush the filter manually with 10 Isopropyl alcohol For more information refer to the Hollow fiber operating guide 5 2 2 Filter CIP A filter which has been used in a process of a protein should be cleaned with a Filter CIP method The Filter CIP method includes an option to perform two Filter CIP procedures with an optional water flush between The created method will e Wash the transfer line e Wash the reservoir Note Ifthe Filter CIP method is a postproduct step the system and the reservoir will be cleaned Preparing system and reservoir see Section 5 4 1 If the method is a preproduct step the system will be cleaned Preparing system see Section 5 4 2 e Wash the filter both on retentate and permeate side with CIP solution e f large reservoir is used Volume to fill has to be entered E KTAcrossflow User Manual 11 0012 32 Edition AB 69 5 Creating Preproduct metho
122. fied in the Stable_baseline condition in the Watch instruction 204 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions a 13 5 37 WatchPar_ _FLUX_Drop Instruction name Group WatchPar_ FluxDrop System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 0 0 0 100 0 Parameter 2 name Setpoint default underlined Delta_Base 0 0 0 0 100 0 Instruction Help text Watch on _Flux_Drop reported The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 38 WatchPar_ConcFactor Instruction name Group WatchPar_ConcFactor System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 0 00 50 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 0 00 50 00 Instruction Help text Watch on concentration factor reported The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_c
123. g the Method Wizard 8 1 Introduction The following method steps in this order are recommended to run after a cross flow filtration 1 Buffer or Water Flush Flushes out remains of protein cells before the system is treated with NaOH The procedure is the same as for Water flush The first block is Preparing the system and reservoir See Section 5 4 1 Section 5 2 3 and Section 5 4 5 Filter CIP Clean in place For a description see Section 5 2 2 and Section 5 4 4 The first block is Preparing the system and reservoir See Section 5 4 1 Water Flush For a description see Section 5 2 3 and Section 5 4 5 The first block is Preparing the system and reservoir See Section 5 4 1 Water Flux Test For a description see Section 5 2 4 and Section 5 4 6 Filter Storage Solution The liquid in the filter is exchanged to an appropriate storage solution For information on recommended storage solution see enclosed filter instruction from manufacturer The procedure is the same as for Buffer conditioning see Section 5 2 5 and Section 5 4 7 Note A recommended storage solution for the system is 20 Ethanol To exchange solution in the system perform the Sanitization method and use 20 Ethanol as liquid It is important to use a three way connector instead of a filter See Section 10 1 Note Ifthe system needs more intensive cleaning it may also be necessary to perform a System sanitization See Section 10
124. gin with Table 5 1 Table of Cross references KTAcrossflow User Manual 11 0012 32 Edition AB 67 Creating Preproduct methods using the Method Wizard _ Depending on how the system and or filter was treated the day before different combinations of the methods above may be used If the system has not been cleaned for a while it may be necessary to start with a System sanitization See Section 10 1 To avoid contaminations when a different protein is introduced it may also be necessary to perform a System sanitization Condition Rinsing Filter Water Filter Water Water Buffer CIP 1 Flush CIP2 Flush Flux Conditioning Test New filter x x X After Recommended X postproduct procedure and same filter After Recommended x x X postproduct procedure but other filter Table 5 2 Preproduct steps Condition Flush Filter Water Filter Water Water Filter Storage Water CIP1 Flush CIP2 Flush Flux Solution Buffer Test Recommended x x X x x x x postproduct procedure Table 5 3 Postproduct steps 68 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea a 5 2 Preproduct methods Description 5 2 1 Rinsing A new filter for ultrafiltration is stored in NaOH and Glycerol which means it has to be rinsed with an increased volume to get rid of the glycerol Use the Rinsing method How
125. harvested cells Lysate clarification After cell harvesting mechanical disruption of the cells releases the product of interest from the cells and creates a lysate e Ina concentration step cells cell debris or other insoluble matter are retained by the filter and the target product is passing the filter to the permeate Note In this step the product leaves the system through P VB Out2 e Adiafiltration step is then performed to flush the rest of the product of interest through the membrane Note In this step the product leaves the system through P VB Out3 112 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard ey Product recovery after Concentration Diafiltration e For Lysate clarification the target product is passing the filter to the permeate The retentate can be directed to waste e Ifthe product is in the retentate there are two alternatives to recover the product e No Recovery An option to select if the retentate volume will be drained manually Note Ifa retentate valve block of the old type is used by the system To avoid siphoning do not place the recovery vials too low e Recovery The reservoir is first emptied through RVB Out3 A defined number of flushes can then be selected and the retaining product will be emptied through RVB Out1 For descriptions of the Recovery procedures see Section 7 1 7 Recover
126. his will initiate the method run Result Name Run info Date 2004 10 15 10 52 15 User default Method c Default UFConcDiafiltrO6T m04 Result No result Add unique identifier to result name Directory Home Browse Scouting subdirectory Name UFConcDiafiltrO6T 001 Batch ID 9 3 Instant Run Help This instruction describes the easiest way to create a method run the system and generate results The procedure is based on an Instant run The use of Instant Run can be recommended when methods do not need to be saved for example when running an Installation test or System sanitization e 1 Click the Instant run icon in the UNICORN Manager module or select File Instant Run 2 Inthe Instant Run dialog e select to use Wizard e Select a System 138 AKTAcrossflow User Manual 11 0012 32 Edition AB Running AKTAcrossflow methods ex ey e Click the Run button InstantRun For system l A Template For column The Method Wizard is opened in the System Control module Method Wizard for System AKTAcrossflow1 Basic Settings Basic Settings Method Mi Preproduct Steps Filter List Kvick Start_5KD Product Steps Concenttation Diafitration Recovery Postproduct Steps Steps Summary V Preproduct V Product Postproduct Flat Sheet specification per filter Surface Area 50 cm 2 4
127. iltration Recovery Summary Sample loading Use airsensor to terminate sample fill ja Sample Volume i ml 0 80000 ml Note If sample volume is larger than the size of the reservoir specify the reservoir fill volume to start concentrate on the next page Note Recommended Min Working Volume 24 ml In the Product Steps dialog select e Number of steps 0 3 e Select the type of ultrafiltration for the selected steps Product Steps Number of Steps 2 Step 1 Concentration Diatiltration Step 2 O Concentration Diafiltration e Enter Sample Volume Sample loading C Use airsensor to terminate sample fill Sample Volume 300 mi 0 80000 ml e If Use airsensor to terminate sample fill is selected the air sensor connected to Transfer_Inlet 1 in the transfer block will stop sample loading when the sample container is empty AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard Note A Maximum Sample Volume has to be entered Sample loading Use airsensor to terminate sample fill Maximum Sample Vol 300 ml 0 80000 mi e Fed Batch A volume larger than the reservoir can be selected In this case the Fed Batch function continuously fills the reservoir meanwhile permeate leaves the system during the concentration This is possible when concentration is the first step The initial volume in reservoir has to be defined on the next page Step 1 Co
128. ine condition in the Watch instruction 200 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions m 13 5 29 WatchPar_ResVol Instruction name Group WatchPar_ResVol System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 ml 0 00 80000 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 ml 0 00 80000 00 Instruction Help text Watch on reservoir volume The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condi tion For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 30 WatchPar_PermVol Instruction name Group WatchPar_PermVol System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 ml 0 00 80000 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 ml 0 00 80000 00 Instruction Help text Watch on permeate volume The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using th
129. ion step through P VB Out3 The retentate can be directed to waste R VB Out 2 by selecting Waste Recovery Recovery Option Recovery Ms Retentate Outlet Product Flush Waste The system will flush the retentate to waste R VB 2 Recirculation before initial Recovery with no backpressure Note To clean the retentate side it may be recommended to select Recirculation before initial Recovery Recovery of retentate If the product is in the retentate as for Cell harvesting Cell washing there are two alternatives to recover the product e No Recovery An option to select if the retentate volume will be drained manually Note Ifa retentate valve block of the old type is used by the system To avoid siphoning do not place the recovery vials too low e Recovery The reservoir is first emptied through RVB Out3 A defined number of flushes can then be selected and the retaining product will be emptied through RVB Out1 For descriptions of the Recovery procedures see Section 7 1 7 Recovery section No Recovery e When this alternative is selected the system will hold after the product steps No postproduct steps are allowed in the method Recovery Recovery Option No Recovery x Note No Postproduct Steps are allowed when No Recovery is selected The system will hold when product steps have finished Note Ifa retentate valve block of the old type is used by the system To avoid siphoning do not place the recovery vials
130. ist Ultrafiltration Cell Processing e Select Filter Type AXM or AXH AXM hollow fibers have a straight linear shape AXH hollow fibers are curved into a loop A 128 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Postproduct methods using the Method Wizard 8 e inthe Filter List available GE Healthcare filters are displayed Select the used filter or select Other Basic Settings Method Ultrafiltration Cell Processing w Filter Type AXM v Filter List KD 0 50 mm e Select the type of process step Steps C Preproduct 7 Product Postproduct e The Hollow Fibre specification per filter area displays the recommended default values for the selected filter Accept or edit the values Hollow Fibre specification per filter mm 0 10 10 00 mm 1 1000 cm 2 40 150 cm 2 ml 0 0 25 0 ml bar 0 5 2 bar bar 0 5 2 bar Lumen Diameter Number of Fibers Surface Area Lumen Hold Up Vol Feed Pressure Limit TMP Limit If Other filter has been selected enter Lumen diameter Number of fibers Surface area Lumen hold up volume Feed pressure limit and TMP limit for the hollow fiber Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit e If several filters are assembled together in parallel select in the System s
131. it may be necessary to optimize the parameters in the opposite way e Increase parameter and decrease P parameter E KTAcrossflow User Manual 11 0012 32 Edition AB 159 Feedback tuning and PID parameters 12 2 Hardware components using PI and PID parameters PI and PID parameters are used for the following Hardware components and Instructions See Table 12 2 Hardware component Instruction Feed Pump Feedpump_PI DeltaP_PI Retentate Control Valve TMP_PID_RetentateControlValve Flux_Pl_RetentateControlValve PUF_PI_RetentateControlValve Permeate Pump TMP_PID_PermeatePump PUF_PI_PermeatePump pNFF_PI Reservoir ConstRVol_P EmptyResFeed_PI Table 12 2 Hardware components using PI parameters A 160 KTAcrossflow User Manual 11 0012 32 Edition AB Feedback tuning and PID parameters 12 3 Description of the PI parameters and regulators The table below describes the three PID parameters used Parameter Description P The P parameter reduces the effect of an error but does not completely eliminate it A simple P regulator results in a stable stationary error between actual and requested flow or pressure The I parameter eliminates the stationary error but results in a slight instability leading to oscillations in the actual flow or pressure The parameter can have values between 0 and infinity Smaller values have a greater effect and a value of infinity has no effect
132. l cut offs are 100 nm to 10 um Micro filtration Retentate Intact cells cell debris Membrane Permeate Colloidal material viruses proteins and salts Cutoff 100 nm to 10 um Cell harvesting This may be the process of concentrating dewatering the cell mass after fermentation With cell harvesting the cells are the target material and are recovered as product in the retentate Cell washing The cells may also be washed to prepare them e g transfer them into a specific buffer for further processing such as freezing or lysing Together with cell harvesting a cell washing step can be performed Lysate clarification Cells cell debris or other insoluble matter are retained by the membrane and the target product is passing through the membrane into the permeate Together with lysate clarification a diafiltration step can be performed to maximize target product recovery from the lysate AKTAcrossflow User Manual 11 0012 32 Edition AB 19 Introduction 20 1 6 The AKTAcrossflow 1 6 1 Description In AKTAcrossflow the sample solution is loaded into the Reservoir with the Transfer pump The Feed pump transports the solution to the filtration device The flow through the membrane is regulated by adjusting the pressure using the Retentate pressure control valve R PCV The remaining retentate flow cross the membrane is recirculated back to the Reservoir In diafiltration the Transfer pump is transporting solution t
133. lab coat CAUTION Always make sure that the filters and system components are compatible with sodium hydroxide at the concentration time and temperature used General WARNING Do not operate the AKTAcrossflow system at pressures above the specified maximum pressure 5 2 bar 9 2 Starting a run 1 Inthe System Control module select File Run 2 Select the method to start Click OK System j Type Modified CrossFlow Wiza Method File 2004 10 06 16 37 CrossFlow Wiza Method File 2004 10 06 16 38 CrossFlow Wiza Method File 2004 10 06 16 39 MPexcursion CrossFlow Wiza Method File 2004 09 23 08 53 CrossFlow Wiza Method File 2004 10 05 10 32 UFConcentration06T CrossFlow Wiza Method File 2004 10 05 10 24 UFDiafiltration06T CrossFlow Wiza Method File 2004 10 05 10 22 WaterFlushO6T CrossFlaw Wiza Method File 2004 09 28 13 35 y gt OK Cancel Help A Start Protocol appears consisting of a number of dialog boxes KTAcrossflow User Manual 11 0012 32 Edition AB Running AKTAcrossflow methods ex SSS 3 On the Variables page it is possible to fine tune the method before proceeding Note We do recommend to not edit any variables especially not the detail variables Variables Block Variable Value Range PID_and_filter_block P_Parameter_FeedPress 0 005 0 001 1000
134. leyOp0Sene 278 min Base Tene min 283 min End Block 2 83 min Watch_RetVol Greater Than 100 0000 Stop_increasing_volume 2 83 min End Block Fax Heb press FL fet Push val ative watch Coriroled Oy Asa 66 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard 5 Creating Preproduct methods using the Method Wizard 5 1 Preproduct methods Introduction There are different methods which are used before the actual cross flow run is performed e Rinsing e Filter CIP Clean in place e Water Flush e Water Flux test and e Buffer conditioning Preproduct Method Description Method Wizard dia Visualized Procedure logs Basic Settings Section 5 3 1 p 73 Flat Sheets Basic Settings Hollow Section 5 3 2 p 75 Fibers Rinsing Section 5 2 1 p 69 Section 5 3 3 p 77 Section 5 4 3 p 84 Filter CIP Section 5 2 2 p 69 Section 5 3 3 p 78 Section 5 4 4 p 84 Water Flush Section 5 2 3 p 71 Section 5 3 3 p 78 Section 5 4 5 p 86 Water Flux test Section 5 2 4 p 71 Section 5 3 3 p 78 Section 5 4 6 p 88 Buffer conditioning Section 5 2 5 p 72 Section 5 3 3 p 79 Section 5 4 7 p 89 Preparing system and Dependent on previous Section 5 4 1 p 80 reservoir procedure method a procedure which some of the methods begin with Preparing system Dependent on previous Section 5 4 2 p 82 procedure method a faster procedure which some of the methods be
135. m AKTAcrossflow1 Basic Settings Basic Settings Method Preproduct Steps Filter List Kvick Start_ 5 KD Product Steps Concentration Diafitration Recovery Postproduct Steps Steps Summary V Preproduct v Product Postproduct Flat Sheet specification per filter Suttace Area 50 em 2 40 150 em 2 Filter Hold Up Volume 1 4 mil 0 0 25 0 mi Feed Pressure Limit bar 0 5 2 bar TMP Limit bar 0 5 2 bar System setup Number of filters Extra Tubing Volume _ ml 0 0 25 0 mi Reservoir Size 350 mi O1100 m Tubing kit Small ID 1 7 mm O Large ID 2 9 mm ni Cancel Hep Set Defaut e Inthe Method list select Ultrafiltration Basic Settings Method Ultrafiltration v m n Calibrate level sensor Filter List Installation Test System Sanitization Ultrafiltration e In the Filter List available GE Healthcare filters are displayed Select the used filter or select Other Method Ultrafiltration v Filter List Kvick Stat 5KD v Kyick Start ae Kvick Start 100 KD Preprodul Other a SSS AKTAcrossflow User Manual 11 0012 32 Edition AB 73 Creating Preproduct methods using the Method Wizard Sey e Inthe next list select Concentration Diafiltration Basic Settings Method Ultrafiltration S Fiker List Kvick Start 5KD 50cm 2 x Concentration Diafiltration v Concentration ation e inthe next area select the type of process step Steps V Preproduct _ Product _ Postproduct e The Flat Sheet
136. manually Note Ifa retentate valve block of the old type is used by the system To avoid siphoning do not place the recovery vials too low e Recovery The reservoir is first emptied through RVB Out3 A defined number of flushes can then be selected and the retaining product will be emptied through RVB Out1 For descriptions of the Recovery procedures see Section 7 1 7 Recovery section 106 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard ee No Recovery e When this alternative is selected the system will hold after the product steps No postproduct steps are allowed in the method Recovery Recovery Option No Recovery b Note No Postproduct Steps are allowed when No Recovery is selected The system will hold when product steps have finished Note Ifa retentate valve block of the old type is used by the system To avoid siphoning do not place the recovery vials too low Recovery e Select whether or not to perform Recirculation before initial recovery The recirculation without pressure on the filter will wash out product e g in the gel layer from the filter e f Buffer flushes are selected enter number of flushes and flush volumes Default volumes are dependent on filter hold up volumes e If buffer flushes are not selected e the reservoir will first be emptied through R VB Out3 e The reservoir will be filled with 5 ml buffer and the product betwe
137. material that passes through the membrane walls of the hollow fiber is called the permeate S J Fig 1 8 Hollow fiber a AKTAcrossflow User Manual 11 0012 32 Edition AB 27 1 Introduction For AKTAcrossflow following hollow fibers are recommended Name Cuttoff Inner Length fibers Nominal Area Feed flow rate Flux range Flux range 30 2001 m2 diameter cm cm 2000 16000 s low 10 LMH high 80 LMH vol challenge mm Start AXH 3kD 0 5 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXH 10 kD 0 5 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXH 30 kD 0 5 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXH 100 kD 0 5 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXH 300 kD 0 5 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXH 500 kD 05 60 4 40 10 85 ml min 0 7 ml min 5 6 ml min 0 13 0 84 L Start AXM 500 kD 1 0 30 6 50 70 560 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 750 kD 1 0 30 6 50 70 560 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 3kD 0 5 30 12 50 24 200 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 10 kD 0 5 30 12 50 24 200 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 30 kD 0 5 30 12 50 24 200 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 100 kD 0 5 30 12 50 24 200 ml min 0 8 ml min 6 4 ml min 0 15 1 0 L Start AXM 300 kD 0 5 30 12 50 24 200 ml min
138. methods using thie MethOd Wizard s2 citadsnecceutin ceed a 55 Performing cross flow runs manually AL MALPOPU COM sseni a 63 4 2 Executing text iINStrUCtIONS o ceecccsesssssseescsssmeeccsseecsssnsecssessecsessseeccesseecesniee 63 4 2 1 WARNINGS and CAUTIONS ccssssssssssssssssssssssssssssssssssssssssscscecsessssessceeeeseessees 65 AS MOMITOMNAG THETA cla Sis cinta in AAR ER 65 Creating Preproduct methods using the Method Wizard 5 1 Preproduct Methods Introduction ssesssssesesssescccssssssssusseessesseesecsssssees 67 5 2 Preproduct Methods Description eecsseessssssssccsssssssssusessessssssssesssesssees 69 5 2 1 RIPSING si neea aticcn tere n btal bag 8 E Lost URANO oe 5 2 2 Filter CIP aE ERRE EE toed it ARTO canons 5 2 3 WWGLOD USI cies ausesasstatssaseccosonseceornscizshstaaisstsaessdesarsocdterneeceasocu sanutaattacssenstesoete 5 2 4 Water Flux test 5 2 5 Buffer conditioning 5 3 Preproduct methods Method Wizard dialogs ssessecscssccsssesssseenees 73 5 3 1 Basic settings FIGtSNCCES 2os cicadas iene a ease 73 5 3 2 Basic Settings Hollow FIDCLS ssssssssssssssssssssssssescscssescesesseeceseeessesssssssssssssses 75 5 3 3 Preproduct setup a 5 4 Preproduct methods Visualized procedures essessssessesssssccssssesssees 80 5 4 1 Visualized procedure of Preparing the system and theTreserv il sensura nanao n EnA A A AN 80 5 4 2 Visualized procedure of Preparing the SYStOM ccccccsssssssssssssssss 82
139. mi e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 350 ml 1100 mi Tubing kit Small ID 1 7 mm O Large ID 2 9 mm 7 2 3 Product steps dialog Method Wizard for System AKTAcrossflow System 2 Product Steps Basic Settings Number of Steps 1 Product Steps Step 1 Step1 Concentration Diafitration Recovery Summary CI Load all Sample Sample Volume oo mi 0 80000 ml Note Recommended Min Working Volume 30 ml SSS AKTAcrossflow User Manual 11 0012 32 Edition AB 115 Creating Product methods using the Method Wizard 116 In the Product Steps dialog select Number of steps 0 3 Select the type of ultrafiltration for the selected steps Product Steps Number of Steps al Step 1 Concentration Diafiltration Step 2 O Concentration Diatiltration Enter Sample Volume Sample loading C Use airsensor to terminate sample fill Sample Volume 300 ml 0 80000 mi If Use airsensor to terminate sample fill is selected the air sensor connected to Transfer_Inlet 1 in the transfer block will stop sample loading when the sample container is empty Note A Maximum Sample Volume has to be entered Sample loading Use airsensor to terminate sample fill Maximum Sample Vol 309 ml 0 80000 mi Fed Batch A volume larger than the reservoir can be selected In this case the Fed Batch function continuously fills the reservoir meanwhile permeate leaves the system during
140. mmend running a water flush Note Ifa Water Flux test will be performed Rinsing or Water Flush should be performed before the test Water Flush Water Flux test e To include a Water Flux test in the method check the Water Flux Test box e Select TMP or NFF Normal Flow Filtration as control mode NFF is default for hollow fibers with cut off 0 1 um and larger e For TMP as control mode e Enter a TMP value for the test Note When comparing status of a filter as a function of time and number of experiments it is important to have performed the same type of method i e same TMP value M Water Flush M Water Flux Test TMP f ber 05 2be0 ONFF 78 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ey e For NFF as control mode e Select to perform Feed control for NFF with FeedFlow or FeedPressure M Water Flush M Water Flux Test O TMP NFF FeedPressure x Feed Control for NFF 2 5 e Enter a value for the selected Feed control M Water Flush M Water Flux Test OTMP ONFF FeedFlow Y Feed Control for NFF 0 69 nin 0 200 min Buffer conditioning e To include a Buffer conditioning in the method check the Buffer Conditioning box Note We recommend performing a Buffer conditioning before a Product filtration run especially when a Filter CIP has previously been performed Buffer Conditioning EE AKTAcrossflow User Manual 11 0012 32 Edi
141. n 9 3 10 Empty the system rinsing bottles and fill them with ordinary rinsing solution 20 ethanol in 10 mM NaOH 11 After the System sanitization method repeat the method using Milli Q water or buffer instead of sanitization solution 12 Empty the system rinsing bottles and fill them with rinsing solution 20 ethanol in 10 mM NaOH 13 Replace the stirrer with a new aseptic one 10 2 Viewing and printing the result This section describes the basics of how to view and print the result in the Evaluation module For evaluating AKTAcrossflow results see Chapter 11 and User Reference Manual UNICORN 5 1 Evaluation for Cross Flow Filtration 148 AKTAcrossflow User Manual 11 0012 32 Edition AB After run procedures 10 10 2 1 Viewing the result To view the result 1 Locate the result file in the Results pane in the Main Menu module 2 Double click the file The result file opens in a Chromatogram window in the Evaluation module See below Evaluation TMP method deltaP scouting003 10 gt UNICORN Local Fil Maria Result strat ver O33 TMPrun040224 TMP method deltaP scouting001 TMP method delt eae File Edit View Integrate Operations Procedures Window Help Oege JE ONH C TMP methad deltaP lt enutinanina AN IAF TMP mathad daltaP sanatinannadn mand TMP mathad deltaP snantinaniadn hand TMP mathed deltaP lt nantinaniniaan nH Cond FeedPress TMP Flux LMH bar 3 5 k 7 N l
142. nadc diets c sign ie ieee 14 27 AKTAcrossflow User Manual 11 0012 32 Edition AB 213 214 instant run instructions fraction collector permeate recirc transfer K Kvick Si rt flatsheet Cassettes iota ieee ee ee ted See la 27 L level se nsor Calibration EE E EEEE E eee ED E AE SO EEAO SE 43 lysate ClO HIC CIC yea na a a aa n ane ra lai 19 M mains power switch manual sampling method new run save Method Wizard basic settings default values Help microfiltration Open post product methods preproduct methods product methods Summary dialog microfiltration minimum working volume monitoring a run monitors N NaOH normal flow filtration P PDGF COLO cuenino annan a n ia inann O a n aA steeds E RTE permeate instructions permeate unrestricted flow control mode aise Noea LENE E E EE E T N ET N EEE E TEA PID troubleshooting PID CONtFOL sssi piston rinsing system postproduct methods POWER indicator preproduct methods process optimization he product MENGIS airen e e eR E A AAEREN KTAcrossflow User Manual 11 0012 32 Edition AB Index product recovery protein concentration protein concentration method R FECIFCUIOTION IMSTFUGHIONS cece eeaerls acct E ce necaea Greg einen at Bole 171 BASON EIN E E cea anche cian T Ae ET ae oe oll bbl i edad result evaluate print oe view retentate ret
143. ncentration Reservoir fill volume m ml 5 350 ml to start concentrate A Minimum working volume is displayed The minimum working volume is the system holdup volume including filter with an addition of a small volume in the reservoir This volume depends on the reservoir size tubing kit and filter volume This is the lowest working volume that is recommended Note Minimum working volume will vary with reservoir size and tubing kit used and is not the same as system holdup volume To ensure process performance a smaller volume is added to the system holdup volume in the calculation of Minimum working volume For information on system holdup volume see KTAcrossflow Instrument Handbook CAUTION Do not use a volume below 50 ml in the Large reservoir as this carries a risk of getting air in the retentate KTAcrossflow User Manual 11 0012 32 Edition AB 101 Creating Product methods using the Method Wizard 7 1 4 Step 1 Concentration dialog Method Wizard for System AKTAcrossflow System 1 Step 1 Concentration Basic Settings Product Steps Feed Control Retentate Flow Step 1 Retentate flow fo ml min 0 600 ml min Step 2 2 Recovery TMP 0 0 bar 0 05 2 bar Summary Endpoint Watch for Endpoint OFF Retentate Volume 26 ml 26 320 ml Concentration Factor Cancel Help Feed Control 102 Select to perform TMP control with constant DeltaP constant Feed Flow or constant Retentate Flow
144. ng of PID control ee 12 1 4 PI parameters for larger filter GPCOS ceessssssssssssssssssssssssssscsccssssssessesseee 12 2 Hardware components using PI and RID OOROMelE NS ss avasiracstentanatucianmmamii ha tanntna tit 160 12 3 Description of the Pl parameters and regulators wees 161 12 4 Setting UP feedback tUNING reececssccccsssssssssssuemesssssscsssssssstusesssessesseesses 161 12 4 1 Instruction QrOUPS ieeeeseesssssseesseseseeeeee 162 12 4 2 Description of the PID instructions 163 12 5 Optimizing the Pl parameters 164 12 5 1 Regulation of the Feed PUMP CXAMPIE ssseseccsssssscscssssssscsceseseeee 164 12 5 2 Regulation of the Permeate PUMP CXGMPe ceccccscsccsscsssscscceesessseee 167 13 Strategy instructions 13 1 System settings and instruction boxes Loeeecccssccssssssssteseessessssssseens 169 13 1 1 System setting The order of instructions within each group 169 13 1 2 Instruction box The order of instructions within each group 170 13 2 Recirculation INSEFUCTIONS isione 171 1321A SRCCURIOW serorea anae O E A R EEA 171 13 2 2 RetFlow 13 2 3 FeedPressure P3224 D ltgP poisike nune a NE Iaz5g Sheaks ere EA O ATOR 13 2 6 Retentate_Valve_BloCk sss ssssissesissssesseresserrssseresserressrrsssrreserrssrresseress 172 13 2 7 RPCVoffset aya T3258 RRCVAYVSLCLOSIS snina ai 1329 Reservoir SIZE aE EnaA E O OAA ER RA 1322 10 EMIPtVRESCIVOME neea E O EE 13211 EmMptyReSeTvVoirA DO ietan a E E 1
145. nsor as Method Basic Settings Method Ultrafiltration x Calibrate Filter List Installation Test System Sanitization Ultrafiltration AKTAcrossflow User Manual 11 0012 32 Edition AB 43 System preparation Ss Method Wizard for System Obelix Basic Settings Basic Settings Method Calibrate level sensor vj Summary The purpose with the calibrate level sensor method is to increase the accuracy when emptying the reservoir Function The method will empty the reservoir to R VB out1 and make sure that there is air in the retentate tubing before calibration When the calibration is finished the method will deair the system with water T VB in5 Note The reservoir lid must be open during level sensor calibration to ensure a good result 4 Click Finish 5 Torun the method there are two alternatives e Save the method and run it see Section 9 2 e Run the method as an Instant run see Section 9 3 Note Ifthe level sensor is too far off the correct value e g the system has not been used for some days this method will not run properly It is then necessary to manually make sure that the reservoir is empty and to perform a manual level sensor calibration in System Control See AKTAcrossflow Instrument Handbook Ss 44 AKTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow 3 Handling methods in AKTAcrossflow 3 1 AUNICORN method 3 1 1 Blocks The Text pane in the Metho
146. o the reservoir during the process in order to maintain a constant retentate volume Transfer Line Transfer Pump Retentate Pressure Control Valve Reservoir Recirculation Line Permeate Line Permeate Pump Feed Pump Cartridge Fig 1 4 Flow path Overview Fig 1 5 AkTAcrossflow system AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction Es 1 6 2 Control modes Term definitions Term Definition QF Qr Op Feed retentate and permeate flow respectively ml min Pe Pp Pp Feed retentate and permeate pressure respectively bar TMP Transmembrane pressurelbar Pe PR TMP Pp 2 DeltaP Pressure drop bar AP P PR Flux I h m2 Permeate flowrate per membrane area I h m2 Qp Permeate flow l h A Membrane area m Flux Qo Shear rate sect Qr Feed flow ml min n Number of fibers r Fiber radius mm Shear 169 76527 x Qr nx r CC Ty AKTAcrossflow User Manual 11 0012 32 Edition AB 21 Introduction SSS TMP Control mode Control Mode Control Element TMP Control Feed Pump Permeate R PCV Pump TMP control with constant Q gt 0 Offset TMP Feed flowrate TMP control with constant Qr gt O Offset TMP Retentate flowrate TMP control with constant Pe Pp gt O Offset TMP DeltaP TMP control is usually used in ultrafiltration where the system has to push the retentate th
147. oir For details see Feedback tuning chapter in User Instrument Manual 13 2 19 RetentateHoldupVol Instruction name RetentateHoldupVol Group System Settings Specials Method Manual Recirc Parameter 1 name Volume Position name default underlined 18 20 ml 0 00 100 000 Instruction Help text Sets the calculated or meassured value for Holdup Volume 13 3 Permeate instructions 13 3 1 TMP_Control Instruction name TMP_Control Formula TMP Pf Pr 2 Pp Group Method Manual Permeate Parameter name Pressure Mode Control_Mode PID Note point No overshooting max 10 of set Position name default under lined 0 00 bar 0 0 5 20 bar Instruction Help text Adjusts Retentate_Control_Valve and permeate pump to maintain set TMP Prior to activation of TMP control make sure that DeltaP is stable Resets Flux_Control and Permeate_Unrestricted_Flow AKTAcrossflow User Manual 11 0012 32 Edition AB 177 Strategy instructions 13 3 2 Flux_Control nstruction name Formula Group Flux_Control Flux Qpll h A m2 Method Manual Permeate Parameter name1 Mode Note Position name default underlined Flux ControlMode Direct Pp lt r 0 0 LMH 0 0 4800 0 Parameter name2 Position name default underlined TMPLimit Off 0 01 5 20 bar nstruction Help text Starts the flow on the pe
148. on name Group Transfer_Valve_Blocks Method Manual Transfer Parameter name Position name default underlined Macro Closed T VB In1 T VB In2 T VB In3 T VB In4 T VB In5 T VB In6 T VB In7 T VB In8 Instruction Help text Selects the position for Transfer_Valve_Blocks T VB In1 T VB In2 T VB In3 T VB In4 T VB In5 T VB In6 T VB In7 T VB In8 The inlet valves will not be set to their default position closed at End until the flow on the pump has gone down to 0 mi min 13 4 4 Transfer_Purge_Valve Instruction name Group Transfer_Purge_Valve Method Manual Transfer Parameter name Position name default underlined Instruction To Reservoir Waste Instruction Help text Selects transfer flow direction to Reservoir or Waste Waste position is useful when cleaning the transfer line 13 4 5 Set_TrfVol_Totalizer Instruction name Group Set_TrfVol_Totalizer Method Manual Transfer Parameter name Set point default underlined Volume 0 00 ml 0 00 5000 00 Instruction Help text Sets a desired volume for totalizer Transfer pump flow will increase value 13 4 6 DF_Exchange_Factor Instruction name Group DF_Exchange_Factor Method Manual Transfer Parameter 1 name Position name default underlined Mode Off On Instruction Help text DiaFiltration Exchange Factor calculates the relation between total buffer volume introduced during diafiltration step and the re
149. onditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction AKTAcrossflow User Manual 11 0012 32 Edition AB 205 Strategy instructions _ 13 5 39 WatchPar_DF_X_ Fact Instruction name Group WatchPar_DF_X_Fact System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 0 00 50 00 Parameter 2 name Setpoint default underlined Delta_Base 0 00 0 00 50 00 nstruction Help text atch on DF_X_aFct reported The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condi tion For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 6 Fraction collector instructions Frac
150. ons of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information 2005 General Electric Company All rights reserved GE Healthcare Bio Sciences AB a General Electric company GE Healthcare Bio Sciences AB Bjorkgatan 30 SE 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Paci c Tel 852 2811 8693 Fax 852 2811 5251 e Australia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 57606 1619 Fax 01 57606 1627 e Belgium Tel 0800 73 888 Fax 03 272 1637 e Canada Tel 800 463 5800 Fax 800 567 1008 e Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 490 Fax 07
151. op_Pumps 0 00 End_Block Block PREPRODUCT_STEP_BUFFER_COND Block FILL_SAMPLE W 0 00 Block CONCENTRATION_1 W 0 00 Block RECOVERY W 0 00 Block POSTPRODUCT_STEP_FLUSH1 E 0 00 Block POSTPRODUCT_STEP_CIP E 0 00 Block POSTPRODUCT_STEP_WATER_FLUSH E 0 00 Block POSTPRODUCT_STEP_WATERFLUXTEST 2 2 2 oo o o D H EE pop 2p 66 D DD H H 11 The method can be edited in the Text editor as described in Section 3 3 12 To save the method select File Save 60 KTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow Ea as 13 Select location 14 Enter method name select System and Technique Click OK Note Any must be selected as Technique c Default For System KT Actos stem 1 sheds ee AKTActossflow System 2 Prev Folder BufferConditi AKTAcrossflow Method File CIPO6T AkTAcrossflow Method File LysateClarific AKTAcrossflow Method File PostProduct AKTAcrossflow Method File RinsingO6T AKTAcrossflow Method File Techn TMP Excursion AKTAcrossflow Method File AkTAcrossflow Method File Anion_Exchange AKTAcrossflow Method File Cation_Exchange AkTAcrossflow Method File ne Size_Exclusion Affinity Method name Method 1l Chromatofocusing OK Cancel Help aa KTAcrossflow User Manual 11 0012 32 Edition AB 61 Handling methods in AKTAcrossflow 62 AKTAcrossflow User Manual 11 0012 32 Edition AB Performing cross flow runs manu
152. ow User Manual 11 0012 32 Edition AB Creating Postproduct methods using the Method Wizard 8 e inthe next list select Concentration Diafiltration Basic Settings Method Ultrafiltration v Filter List Kvick Stat 5KD 50cm 2 Concentration Diafiltration v Concentration Diafiltration UF Process Optimization e Inthe next area select the type of process step Steps Preproduct 7 Product V Postproduct e The Flat Sheet specification per filter area displays the recommended default values for the selected filter type Accept or edit the values Flat Sheet specification per filter Surface Area 50 cm 2 40 150 cm 2 Filter Hold Up Volume 1 4 ml 0 0 25 0 ml Feed Pressure Limit 4 0 bar 0 5 2 bar TMP Limit 40 bar 0 5 2 bar If Other filter type has been selected enter Surface Area Hold Up Volume Feed pressure and TMP limits for the flat sheet Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit e If several filters are assembled together in parallel select in the System setup section the Number of filters 1 3 If only one filter is used keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System
153. pComp Instruction name Group pHTempComp System Settings Monitors Parameter name Position name default underlined pHTempComp OFF ON Instruction Help text Sets the temperature compensation on or off For more accurate measurements during temperature changes the pH measurement can be temperature compensated When using pHTempComp it is important that the temperature of the pH electrode is the same as that of the conductivity flow cell since that is where the temperature is measured 13 7 3 Cond CondTempComp Instruction name Group CondTempComp System Settings Monitors Parameter name Setpoint default underlined CompFactor 2 0 0 0 9 9 Instruction Help text Relates conductivity to temperature The compensation consists of a compensation factor together with a reference temperature CondRefTemp All conductivity values will then automatically be converted to the set reference tempera ture The factor is expressed in percentage increase of conductivity per C increase in temperature If the CompFactor is unknown a general approximate value of 2 can be set for many common salt buffers 0 off AKTAcrossflow User Manual 11 0012 32 Edition AB 207 Strategy instructions ee CondRefTemp Instruction name Group CondReffemp System Settings Monitors Parameter name Setpoint default underlined Reffemp 25 0 C 0 0 99 9 Instruction Help text Sets the reference temperature to which th
154. r Felentata Valva Black 3 7 Enter a parameter value Pennee pey 0 0 600 0 len O Pi Var 50 ja ml min 8 Usethe Insert Change Replace or Delete buttons All text entries are shown in the Text pane Applicable variables can be edited for each selection e Click Insert to place the instruction in the method e Tochange a value in an instruction select the instruction row enter a new value and click Change SS 50 AKTAcrossflow User Manual 11 0012 32 Edition AB ss 9 10 11 E Handling methods in KTAcrossflow Ea e To replace an instruction select the row edit the instruction and click Replace e To delete an instruction row select the row and click Delete Individual text instructions can be grouped in blocks of instructions marked by blue square symbols for a specific functional use for example to set a flow path to rinse a filter etc A block may contain other blocks or individual instructions This is an example of text instructions in the Text pane Main W 0 00 Block Info_Inlets_and_Outlets W 0 00 Block INITIAL_SETUP W 0 00 Block FILL_SAMPLE W 0 00 Block CONCENTRATION_1 CONCENTRATION_1 0 00 Base SameAsMain 0 00 Set_Mark Concentration 1 0 00 Block Flowpath_PermConc Flowpath_PermConc 0 00 Base Time 0 00 Permeate_ alye_Block P B Out2 W 0 00 Block DelayOp05min 0 00 End_Block Mi 0 00 Block RetFlow_Step1 W 0 00 Block TMP_Stepi W 0 00 Block Chase_Inlet_to_Reservoir
155. rm persists try to restart the system If the alarm still persists check if the valve is damaged permanently For details see AKTA Instrument Handbook 13 5 15 Alarm_FlowPath Instruction name Group Alarm_FlowPath System Settings Alarms Method Manual Alarms amp Mon Parameter name Position name default underlined Mode Enabled disabled Instruction Help text Enable Disable the Alarm_Flow Path The Alarm_FlowPath checks for closed flow paths if all Inlets closed all Outlets closed when Flow gt 0 An alarm will be raised and the System is set to Pause Alarm1 Help text TrfFlow gt 0 and inlets are closed Alarm2 Help text PermFlow gt 0 and outlets is closed 194 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions 13 5 16 Alarm_Airsensor Instruction name Alarm_AirSensor Group System Settings Alarms Method Manual Alarms amp Monitors Parameter name Mode Position name default underlined Disabled Enabled Instruction Help text Sets the alarm for air sensor An alarm will set the system to Pause if air is detected in air sensor flow cell 13 5 17 Alarm_ZeroLevel Instruction name Alarm_ZeroLevel Group System Settings Alarms Method Manual Alarms amp Monitors Parameter name Mode Position name default underlined Disabled Enabled Instruction Help text Sets the alarm for ZeroLev
156. rmeate pump as flux If permeate pressure lt 0 2 bar R PCV will lift permeate pressure to Offset If system reaches TMPLimit when active system will Pause Resets TMP_Control and Permeate_Unrestricted_Flow RPCV will lift permeate pressure to 95 of Offset before Flux will be ramped up during 30 sec 13 3 3 Permeate_Valve_Blocks Instruction name Group Permeate_Valve_Block Method Manual Permeate Parameter name Position name default underlined Macro Closed P VB Recycle P VB Out1 P VB Out2 P VB Out3 Instruction Help text Selects the position for the Permeate_Valve_Block to P VB Recycle P VB Out1 P VB Out2 P VB Out3 No valve block action allowed when P VB recycle is used and a permeate control mode is active A warning will be raised During Constant_Retentate_Volume P VB recycle position is not allowed A warning will be raised It is possible to change position when permeate flow is greater than 0 although volume will not be accurate When opening a new valve outlet the old outlet will be open for about one second Permeate_Valve_Block shall remain in position at Pause and the flow on the pump s goes down to 0 ml min Warning help text 1 Instruction ignored No valve block action allowed when P VB recycle is used and permeate contro mode is active Warning help text 2 Instruction ignored P VB recycle is not allowed during Constant retentate volume 178 AKTAcrossflow User Manual 11 001
157. rming system sanitization the pump piston rinsing system should be sanitized by using 1 M NaOH as liquid in the rinsing system bottles 10 1 2 Sanitization of reservoir float The reservoir float has to be sanitized separately Remove it from the reservoir before system sanitization The float can be autoclaved or equivalent AKTAcrossflow User Manual 11 0012 32 Edition AB 145 After run procedures 146 10 1 3 Creating a method 1 2 In the Method Editor of UNICORN open the Method Wizard Select the System In the Basic settings dialog select System Sanitization as method Basic Settings Method System Sanitization v Enter Recirculation time A minimum recirculation time of 30 min is recommended Recirculation Time 30 min 0 999999 min Select the Reservoir used System setup Reservoir Size 350ml 1100 mi Note Information on important preparations and notes before the method is run is displayed in the Basic Settings dialog All In and Outlet connections will be used Click Finish In the Summary dialog click Print to print the summary Click Finish to end the Method Wizard Note Do not edit any variables or method instructions 8 Save the method AKTAcrossflow User Manual 11 0012 32 Edition AB After run procedures 10 1 4 Running the System sanitization method When running a method using a sanitization solution containing sodium hydroxide NaOH WARNING NaOH
158. roller Settings Create Restore Backup Change User Attributes Options 2 Select a system and click Edit System Setup Systems EE SEUPPD 02610 ow System 1 EE Ak TActosstlow System 2 aa KTAcrossflow User Manual 11 0012 32 Edition AB 41 System preparation SSS 3 Inthe Edit System dialog click Component Edit System System name Systemtype Membrane Strategy Computer name SEUPPD 02610 Control unit number 1 4 1 Auto save interval 5 minutes 4 Inthe Component dialog select type of Retentate valve block used e Old type or e New type Component Select components owl Fraction Collector Filter Type Hollow Fibre Flat Sheet xiliary Equipment etentate Valve Block Old Type New Type rRO a Of 5 Click OK to close the Component dialog 6 Click OK to close the Edit System dialog 7 Click Close to close the System Setup dialog 42 AKTAcrossflow User Manual 11 0012 32 Edition AB System preparation 2 6 Calibrating the level sensor There is a method for automatic calibration of the level sensor available for the system The calibration should be performed before every run e 1 Inthe Method Editor of UNICORN click the Method Wizard icon 2 Select System Method Wizard Select a system A tem 1 AKTActossflow System 2 OK Cancel Help 3 Inthe Basic Settings dialog select Calibrate level se
159. rossflow Sy 2 Select System and specify to use Method Editor Click OK New Method For system Use AKTActossflow System 1 O Wizard e Method Editor Template selection Technique Method notes Template For column 3 Click the Customise Panes icon O sA SQnD an E Main 0 00 Base CY 0 10 ml Any 4 Select Text and Instruction Box and click OK Customize Panes Flow scheme Instruction box Block Gradient The Instruction box is displayed in the lower part of the Method Editor oe gt C O Pease mhd Base O Tunta tnd Me Tee Comas O MermatMon Owah ionnd Message O Othe Nen Checenanoansen a Replace AKTAcrossflow User Manual 11 0012 32 Edition AB 49 Handling methods in AKTAcrossflow See 5 First select group of instructions for example Recirc Breakpoint Instructions A oo min Recire RetFlaw x DeltaP va O Permeate FeedPressure Var Shear O Transfer FeedPump_PID TMP_Control O Alaims amp Mon TMP_PID_RetentateControlValve TMP_PID_PermeatePump O Watch Man_PCV_Retentate O Other aaa a 6 Select instruction within the group Breakpoint Instructions p a oo min Recirc pan Je DeltaP O Permeate FeedPressure Shear O Transfer FeedPump_PID TMP_Control O Alatms amp Mon TMP_PID_RetentateControlalve TMP_PID_PermeatePump O Watch Man_PCV_Retentate O Othe
160. rossflow is intended for laboratory use only not for clinical or in vitro diagnostic purposes AKTAcrossflow User Manual 11 0012 32 Edition AB 13 Introduction 1 3 Glossary of terms Term Explanation Cartridge Also Module or Cassette The unit encapsulating the membrane The membrane can have different format Flat sheet cassette Example of the unit encapsulating the membrane A cassette has at least one inlet feed and two outlets retentate and permeate Hollow fiber The tube like structure made from a membrane and sealed inside a cross flow cartridge Cross flow Also called tangential flow iltration In cross flow filtration he feed solution flows parallel to he surface of the membrane Driven by pressure some of the eed solution passes through the membrane filter Most of the solution is circulated back to the eed tank The movement of the eed solution across the membrane surface helps to remove the buildup of foulants on he surface Cut off MWCO Molecular weight cutoff The size designation in Daltons D for ultrafiltration membranes The molecular weight of the globular protein that is 90 retained by the membrane No industry standard exists hence the MWCO ratings of different manufacturers are not always comparable Retentate The portion of the feed solution that does not pass through a cross flow membrane filter Permeate Also called filtrate The portion of a
161. rough the relatively small pores of the membrane The control mode is used at a e Constant Feed flow e Constant Retentate flow or e Constant DeltaP The TMP is mainly controlled by the Retentate valve R PCV The TMP control mode adjust the Retentate control valve and permeate pump to maintain set TMP Before activation of TMP control DeltaP has to be stable Offset is 0 2 bar as default and is used to avoid low or negative pressure on the permeate side Too low pressures will affect the permeate pump s function as a flow meter If TMP lt AP 2 the Permeate pump Offset will automatically be changed The TMP control mode resets Flux control and Permeate Unrestricted flow Note The TMP has to be optimized for different applications filter and sample See Chapter 6 22 AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction Flux Control mode Control Mode Control Element Flux Control Feed Pump Permeate R PCV Pump Flux control with constant Or gt 0 Flux gt 0 Offset Feed flowrate Unrestricted for Pp gt Offset Flux control with constant Qk gt 0 Flux gt O Offset Retentate flowrate Unrestricted for Pp gt Offset Flux control with constant shear rate gt 0O Flux gt 0 Offset Shear rate Unrestricted for Pp gt Offset Flux control with constant Pe PR gt O Flux gt 0 Offset DeltaP Unrestricted for Pp gt Offset Flux control is usually used in microfiltration filtra
162. ry when using several filters assembled together in parallel The Extra tubing volume has to be calculated manually if other tubing than standard are used Reservoir size and tubing kit to be used in the recycling loop is selected To get help instructions for each Method Wizard dialog click Help or press the F1 key To go back to the previous dialog click Back To stop the Method Wizard click Cancel AKTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow Ea e To proceed with the next dialog click Next Method Wizard for System AKTAcrossflow1 Basic Settings Basic Settings Method x Preproduct Steps Filter List Kvick Start 5KD e Product Steps Concentration Diafitration x Recovery Postproduct Steps Steps Summary V Preproduct V Product Postproduct Flat Sheet specification per filter Surface Area w cm 2 40 150 cm 2 Filter Hold Up Volume 1 4 ml 0 0 25 0 ml Feed Pressure Limit bar 0 5 2 bar TMP Limit bar 0 5 2 bar System setup Number of filters Extra Tubing Volume 2 ml 0 0 25 0 mi Reservoir Size 350 mi 1100 mi Tubing kit Small ID 1 7 mm Large ID 2 9 mm Newt gt Finish Cancel Help Set Default 4 In each dialog select the appropriate parameter values and click Next to continue o KTAcrossflow User Manual 11 0012 32 Edition AB 57 Handling methods in AKTAcrossflow
163. setup Number of filters L Extra Tubing Volume 00 ml 0 0 25 0 ml e Select Reservoir size and Tubing kit used in the recycle loop Reservoir Size 30 mi 1100 mi Tubing kit Small ID 1 7 mm O Large ID 2 9 mm AKTAcrossflow User Manual 11 0012 32 Edition AB 127 ED Creating Postproduct methods using the Method Wizard E 8 2 2 Basic settings Hollow fibers Note Togetthe Method Wizard for hollow fibers Hollow fiber has to be selected as component in System Setup See Section 2 4 Method Wizard for System AKTAcrossflow2 Basic Settings Basic Settings Method Preproduct Steps Filter Type AXM Product Steps Fiter List AXM_3KD 0 50 mm _ Recovery Postproduct Steps Steps Summary V Preproduct v Product Postproduct Hollow Fibre specification per filter Lumen Diameter 05 rmm 0 10 10 00 mm Number of Fibers 12 1 1000 Surface Area 50 cm 2 40 150 cm 2 Lumen Hold Up Vol mil 0 0 25 0 ml Feed Pressure Limit 43 bar 0 5 2 bar TMP Limit 30 bar 0 5 2 bar System setup Number of filters Extra Tubing Volume ml 0 0 25 0 mi Reservoir Size 350 ml 1100 mi Tubing kit Small 1D 1 7 mm Large ID 2 9 mm Cancel Help _ Set Default e inthe Method list select Ultrafiltration Cell Processing Basic Settings Method Ultrafiltration Cell Processing v Calibrate level sensor Fiter Type Installation Test Jarli System Sanitization Fiter L
164. ssflow 18 4 AKTAcrossflow Method Handbook Provides more detailed information on applications 1 8 5 UNICORN documentation UNICORN control system includes three manuals e Getting Started e User Reference Manual 2 pcs e Administration and Technical Manual Documentation of the AKTAcrossflow specific Evaluation module includes X User Reference Manual UNICORN 5 1 Evaluation for Cross Flow Filtration e Specific sections in Online Help AKTAcrossflow User Manual 11 0012 32 Edition AB 29 Introduction 30 AKTAcrossflow User Manual 11 0012 32 Edition AB System preparation 2 System preparation 2 1 Starting the system To start AKTAcrossflow system 1 Switch on the instrument at the mains power switch located on the rear panel Mains power switch The Power indicator on the front panel flashes slowly until the internal communication with the CU Control Unit is established Power indicator 2 Switch on power to the PC and the monitor 3 Start and log on to UNICORN by double clicking on the icon on the Microsoft Windows xP desktop AKTAcrossflow User Manual 11 0012 32 Edition AB 31 System preparation es 4 Enter User name and Password and click OK Ab Y7 UNICORN logon User name O Password l Normally the system administrator defines the users and creates your first passwor
165. sss1ssseissssissseresenresssrresserreseresreess 13 3 15 TMP_PI_PermeatePump 13 3 16 Flux_Pl_RetentateControlValve ssseisessssesseiseereeersssrrrsereesses 13 3 17 PUF_Pl_RetentateControlValve s ssssissesissseisssereseerrsessresserrsserresseeess 13 3 18 PUF_Pl_PermeatePump 13 3 19 pNFF_PI 13 3 20 Fractionation 13 4 Transfer inStrUctiOns aineinaan aiii 1341 rIransterFloWranrorirn oaan E E E 13 4 2 Constant_Retentate_Volume 13 4 3 Transfer_Valve_Blocks 13 4 4 Transfer_Purge_Valve 13 45 Set NOl TOZET eonan a e a Re 13 46 DF Exch nge Factor ssrscryer ir e E 187 13 47 Method BOSC irc i i a NNE REENA 188 i RSE S e EE S09E11 n1740 L EIAI E EASO T 188 13 5 Alarms Warnings and Monitors 188 D551 AUtOZ OL OV cos penrian AE R sess diss E tbetes 188 155227 HAVCFAGING TANI Os 2Ne i trans tetuthin atroita cs a settee tha celeb ee tite 188 135 3 Pressure Filter FOCtOP arannana iiaeiai 189 BSS AICI Uorn sass ctpezes A anata ada aa Rea co 189 13 5 5 ALRITE pH ppan A 189 135 6 Alarm Cond e aeaa a E N Ea RRA 190 13 5 7 Alarm FEedPr S Sinaine na aan i 190 13 5 8 Alarm_TrfPress 13 5 9 Alarm Delta srainnsear nnn 13510 AlE TMP iE AN E E EAA E HBG Seog I AlN FUR sinne EA NA EA 135 1 Alara PNE F era oie E E acti iad clan dee AN ere adi 13 5 13 Alarm_Shear 13 5 TA AI VGIVOS VIVE en a EE E 194 13 5 15 Alar FOWP AN insana E T it 194
166. sssescscecssssssssuunssessesssssssssnneeesee 154 Example of plotted Normalized Water Flux csescscccsssssssseeessssssscsssssssnnseesssee 156 AKTAcrossflow User Manual 11 0012 32 Edition AB xi List of figures xii AKTAcrossflow User Manual 11 0012 32 Edition AB Introduction 1 Introduction 1 1 The User Manual The AKTAcrossflow User Manual provides instructions for performing cross flow filtration also called tangential flow filtration using the automatic AKTAcrossflow instrument and UNICORN software 1 2 Introduction The purification of biological therapeutics requires processes which usually employ two technologies used in combination membrane separations and chromatography Chromatography offers greater selectivity than membrane separations and can purify much greater quantities of solution in one operation However membrane separations technologies offer a number of other advantages including the fact that they are a fast and robust process step AKTAcrossflow covers three main filtration techniques e Ultrafiltration of proteins e Diafiltration of proteins e Microfiltration of cell and lysate solutions The system supports both hollow fibers and flat sheet cassettes with sizes suitable for flow rates up to 300 ml min for flat sheet cassettes and up to 600 ml min for hollow fibers The main application is method development and scale up in process development laboratories WARNING AKTAc
167. struction resets FeedFlow FeedPressure DeltaP and Shear To allow new instruction after stabilized DeltaP Watch_Stable_Baseline DeltaP should be programmed in methods 13 2 3 FeedPressure Instruction name Formula Group FeedPressure Qp Method Manual Recirc Parameter 1 name Mode Position name default under lined Pressure Control Mode Direct 0 00 bar 0 00 5 20 Instruction Help text Starts the flow on the feed pump Feed Pressure should stabilize at 98 of set value before new instruction is executed Instruction resets FeedFlow RetFlow DeltaP and Shear To allow new instruction after stabilized DeltaP Watch_Stable_Baseline DeltaP should be programmed in methods AKTAcrossflow User Manual 11 0012 32 Edition AB 171 Strategy instructions _ 13 2 4 DeltaP Instruction name Formula Group DeltaP DeltaP Pf Pr Method Manual Recirc Parameter 1 name Mode Position name default underlined Pressure Control Mode PID 0 0 bar 0 5 2 Instruction Help text Starts the flow on the feed pump DeltaP should stabilize at 98 of set value before new instruction is executed Instruction resets FeedFlow Shear and FeedPressure To allow new instruction after stabilized DeltaP Watch_Stable_Baseline DeltaP should be programmed in method 13 2 5 Shear Instruction name Formula Group Shear Shear 4 Qf No of Fibers x fiberradius Method Manual Recirc
168. t to perform TMP control with constant DeltaP constant Feed Flow or constant Retentate Flow Feed Control Retentate Flow Delta P Retentate flow Feed Flow Retentate Flow e Enter the value for the selected constant parameter for example Retentate Flow 25 ml min 104 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard lt a e Enter the desired TMP Note When using TMP in a method a value close to the TMP limit set in the Basic Settings dialog may lead to the run being paused Feed Control Retentate Flow z Retentate flow 2 ml min 0 600 ml min TMP 25 bar 0 0 5 2 bar Endpoint e There are two kind of endpoints Watch for Endpoint and Permeate Volume DF Exchange Factor Endpoint Watch for Endpoint OFF E E Permeate Volume fo ml 0 9999 ml O DF Exchange Factor e The Watch for options are shown in the screen below Endpoint Watch for Endpoint UV Less than M Cond Greater than and Stable Cond Level Cond Less than and Stable Cond OFF ee ene PH Greater than and Stable pH Permeate Volume pH Less than and Stable pH UVG h O DF Exchange Factor e Enter parameters for the selected Watch for Endpoint e Level The signal falls below a specified Less than value or is above a Greater than value e Time Stable Delta Base The signal is stable within the limits of the Delta Base value
169. tem 2 OK Cancel Help AKTAcrossflow User Manual 11 0012 32 Edition AB 91 6 Process optimization in Ultrafiltration 92 3 Inthe Basic Settings dialog e Select Ultrafiltration as method e Select the used filter from the list of available GE Healthcare filters or select Other e Select UF Process Optimization e Inthe Flat Sheet specification per filter section recommended default values are displayed Accept these values or edit them e If Other has been selected as filter enter parameters for the used flat sheet cassette Check information from filter manufacturer e If several filters assembled together in parallel are used enter number of filters If other tubing than standard is used calculate the extra volume and enter the value e Select reservoir size and tubing kit used in the recycle loop Basic Settings Method Ultrafiltration Filter List Kvick Start 5KD s Optimization Flat Sheet specification per filter Surface Area 50 cm 2 40 150 cm 2 Filter Hold Up Volume 14 ml 0 0 25 0 mi Feed Pressure Limit 4 0 bar 0 5 2 bar TMP Limit 14 9 bar 0 5 2 bar System setup Number of filters u Mi Entra Tubing Volume 20 ml 0 0 25 0 m Reservoir Size 350 ml 1100 mi Tubing kit Small ID 1 7 mm O Large ID 2 9 mm 4 Click Next AKTAcrossflow User Manual 11 0012 32 Edition AB Process optimization in Ultrafiltration 6
170. tentate volume when diafiltration was started E KTAcrossflow User Manual 11 0012 32 Edition AB 187 Strategy instructions 13 4 7 MethodBase Instruction name Group MethodBase Method Manual Transfer Parameter 1 name Position name default underlined Base FeedPump TransferPump and PermeatePump Instruction Help text Sets the base for method volume calculation to chosen pump 13 4 8 ConstRVol_P Instruction name Group ConstRVol_P System Settings Specials Method Manual Permeate Parameter 1 name Setpoint default underlined P 50 000 mbar 0 000 10000 000 Instruction Help text Parameter affects respons of CRV algorithm A higher value causes faster transfer flow indrease to compensate for sinking level and vice versa Parameter also affects how fast startlevel is restored after CRV instruction is turned off Note A higher value amplifies noice in the Level Sensor signal and instability may occur 13 5 Alarms Warnings and Monitors 13 5 1 AutoZeroUV Instruction name Group AutoZeroUV Method Manual Alarms amp Mon Instruction Help text Sets the relative AU to zero 13 5 2 AveragingTime Instruction name Group AveragingTimeUV System Settings Monitors Method Manual Alarms amp Mon Parameter name Setpoint default underlined AvTimeUV 0 02 0 04 0 08 0 16 0 32 0 64 1 30 2 60 5 10 and 10 Instruction Help text Filters the noise in th
171. tentateControlValve lifts RetenPress to Offset if RetenPress lt Offset Resets TMP_Control and Flux_Control AKTAcrossflow User Manual 11 0012 32 Edition AB 179 Strategy instructions se 13 3 6 PressureOffset Instruction name PressureOffset Group System Settings Specials Parameter name Pressure Set point default underlined 0 2 bar 0 2 1 0 bar Instruction Help text Sets offset pressure used by system during TMP_Control Flux_Control and Permeate_Unrestricted_Flow 13 3 7 Evaluation Instructions Start_Eval_Window Instruction name Start_Eval_Window Group Method Manual Permeate Parameter 1 name Mode Position name default underlined O Any vs any 1 Capacity 2 DFTimeOpt Instruction Help text Starts collection of chosen sets of data for evaluation Several Window instructions can be given during a run For more information see UNICORN manual Evaluation Appendix Stop_Eval_Window Instruction name Stop_Eval_Window Group Method Manual Permeate Parameter 1 name Mode Position name default underlined Q Any vs any 1 Capacity 2 DFTimeOpt Instruction Help text For more information see UNICORN Stops collection of chosen sets of data for evaluation Several Window instructions can be given during a run manual Evaluation Appendix Set_Eval_Mark Instruction name Set_Eval_Mark Group
172. ter 3 name Setpoint default underlined Low Alarm 0 00 pH 0 14 Instruction Help text Sets the Alarm limits for the pH signal AKTAcrossflow User Manual 11 0012 32 Edition AB 189 Strategy instructions 13 5 6 Alarm _Cond Instruction name Alarm _Cond Group System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name ode Disabled Enabled Position name default underlined Parameter 2 name High Alarm Setpoint default underlined 999 99 mS cm 0 00 1000 00 Parameter 3 name Low Alarm Setpoint default underlined 0 00 mS cm 0 00 1000 00 nstruction Help text Sets the Alarm limits for the conductivity signal 13 5 7 Alarm_FeedPress Instruction name Alarm_FeedPress Group System Settings Alarms Method Manual Alarms amp Monitors Parameter 1 name ode Disabled Enabled Position name default underlined Parameter 2 name HighAlarm Setpoint default underlined 5 20 bar 0 00 5 20 bar Parameter 3 name LowAlarm Setpoint default underlined 0 00 bar 0 00 5 20 bar nstruction Help text Sets the alarm limits for pressure from FeedPump When reached the system is set to Pause An alarm FeedPressHi ghAlarm FeedPressLowAlarm will set the system to Pause Alarm Help text Press Continue HighAlarm cause The pressure has exceeded the HighAlarm limit
173. the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this con dition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 20 WatchPar_Cond Instruction name Group WatchPar_Cond System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 mS cm 0 999 9 Parameter 2 name Setpoint default underlined Delta_Base 0 00 mS cm 0 999 9 nstruction Help text atch on conductivity signal The DeltaPeak setting affects only the Less_than_or_valley and Peak_max conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condi tion For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition for the Watch instruction 196 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions 13 5 21 WatchPar_FeedPress Instruction name Group WatchPar_FeedPress S
174. the ends of the solution inlet tubing in the appropriate solution containers 2 Check that there are sufficient solution volumes available 3 Set ResVolTotalizer to zero in UNICORN System Control Note Use Milli Q water or equivalent when preparing solutions and buffers 9 1 2 Sample 1 Put the ends of the sample inlet tubing in the appropriate sample containers 2 Check that there is sufficient sample available 9 1 3 Waste 1 Check that following outlet tubing are placed in waste containers e Transfer Purge Valve Waste e R VB Out2 Retentate Waste valve e P VB Out1 Permeate Waste valve 2 Check that the waste containers are not full and will accept the volume diverted to it during the run 9 1 4 Filter 1 Check that the correct filter is assembled in a correct way 2 Ensure the filter is clean and has acceptable quality Appropriate test is Water flux test AKTAcrossflow User Manual 11 0012 32 Edition AB 135 9 Running KTAcrossflow methods 136 9 1 5 Calibration Calibrate the pH electrode conductivity meter and pressure sensors if required Refer to AKTAcrossflow Instrument Handbook The Level sensor should be calibrated before every run See Section 2 6 9 1 6 WARNINGS and CAUTIONS CIP method When running a method using a CIP solution containing sodium hydroxide NaOH WARNING NaOH is corrosive and therefore dangerous to health Avoid spillage and wear safety glasses safety gloves and protective
175. tings didog inisieer cs nai a E R 113 7 2 3 Product steps didog in a ie e E E aaakte a 115 7 2 4 Step 1 Concentration CIGOG sssssssssssssesssssssssssssscsesssceeseeeeeesssssssssssssssssiass 117 7 2 5 Step 2 DiPiltration didlo eesssssssssssssssssssssssssssssssessessesssssssesesesessssssesssssssees 119 7 2 6 RECOVEFV CIGOG esses scessesstaecesdicectaacssessicclinnchlstacttoletabadahielduscaaens 121 8 Creating Postproduct methods using the Method Wizard BEL SITFODW COME asses cca aaenestert osetia E E A 8 2 Postproduct methods Method Wizard dialogs 8 2 1 BASIC settings Flat SHCCES eececcsssssssssssssssssssssssssssessssssssssecessessesseeeeeeeeeseeees 8 2 2 Basic settings Hollow FIDO eeecccssssssssssssssssssssssssssssssssssssssssssessceceeseeeeeeees 8 2 3 Postproduct Setup woeeescssssssssssssssssssecsescssessssssssssssssnssssnsusssssseceecceseeeeeseeeeeeeees 9 Running KTAcrossflow methods 9 1 Final preparation 135 9 1 1 SOMOS tsi enia EAER 135 9 1 2 SOMDl E peginan ri EN E E A O O EE 135 9 1 3 WASTE y iei E OE OTE E EOE 135 9 1 4 FUROR S aieiaa A E A EN nS 135 9 1 5 Calibra ON ziers o a 136 9 1 6 WARNINGS and CAUTIONS wccccsssssssssssssssssccccsssssessssssssssssssnssnnnsnussssessecseceees 136 9 2 Starting A TUN oine aE N ONNEA 136 9 3 PPUSTOUATARDM enparen easa e aE ARa aait 138 QA PUAN AVS ae EAN N EREN 142 9 5 Manual sampling during FUN ssssssssssssssssssssrssssssssssisssrsssisssrsnrssronrrsnrssnrne 143 10 After run proc
176. tion AB 79 Creating Preproduct methods using the Method Wizard 5 4 Preproduct methods Visualized procedures Note For information on the specific instructions for example Constant Retentate Volume see Chapter 13 5 4 1 Visualized procedure of Preparing the system and the reservoir Depending on the previous step some of the methods begin with a preparation of the system and reservoir However different transfer inlet tubing is used depending on the preferred liquid 1 Transfer pump fills the tubing from Transfer Inlet to Transfer Purge Valve Waste Air Permeate UB Trans fe Purgeli F Ue Recucla er alve Retentate UB R UB dutS rae a Cartridge permeate EN Transfer VB1 Permeate UB F Ue Recucla E U Transfer PurgeValve Transfer UB2 EnS Retentate UB Cartridge Permeate Pump Air Transfer UB1 Permeate UB F UB Recuclel Mene 80 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ea E 4 At Constant Retentate Volume reservoir volume 5 ml transfer flow feed flow 50 ml is pumped at the retentate side out to R VB Out2
177. tion where the system has to hold back the permeate from the relatively large pores of the membrane The control mode is used at a e Constant Feed flow or e Constant Retentate flow or e Constant Shear rate or e Constant DeltaP In this mode the TMP value is a function of the permeate flux If the permeate pressure is lt 0 2 the R PCV has to increase the Retentate pressure When permeate pressure is above 0 2 the permeate pump can start Aconstant ramping during 60 sec from flux 0 to the set flux is performed During the ramping the function for R PCV is to maintain the permeate pressure above 0 2 Note Ifthe system reaches TMP limit when active the system will Pause Flux control resets TMP control and Permeate Unrestricted flow Note The flux has to be optimized for different applications filter and sample E KTAcrossflow User Manual 11 0012 32 Edition AB 23 Introduction 24 Permeate Unrestricted flow Control Mode Permeate Unrestricted Control Element flow Feed Pump Permeate R PCV Pump With constant Feed flow rate Q gt 0 PR Pp PR Pp gt Offset With constant Retentate Qr gt O PR Pp PR Pp flow rate gt Offset With constant Shear rate Shear rate gt 0 PR Pp PR Pp gt Offset With constant DeltaP Pe Pp gt O PR Pp PR Pp gt Offset Permeate unrestricted flow starts the flow on the permeate pump at Offset permeate pressure 0 2
178. tions Base a 450 2 min O Recirc Block Continue O Permeate End Block Var End Method O Transfer Evaluate Flow_Warni O Alarms amp Mon Hold one Loop O Watch Loop_End Message Other New Chromatogram M4 8 To save the method select File Save Select System and Technique Any Enter a method name and click Save c Default For System ee Name System Size Type SEUPPD 02610 c Prev Folder AkTAcrossflow System 2 jufferConditi AKTAcrossflow 185KB Method File IPO6T AKTAcrossflow 185KB Method File oncDiaMeth AKTAcrossflow 192KB Method File oncentration AKTAcrossflow 182KB Method File ysateClarific KTAcrossflow 184KB Method File ostProduct AKTAcrossflow 189KB Method File i insingO6T AkTAcrossflow 185KB Method File Anion_Exchange MP Excursion AKTAcrossflow 187KB Method File Cation_Exchange MP1 kTAcrossflow 187KB MethodFie RPE gt Size Enclusion Method name TestMethod Chromatofocusing Lo Cancel Help A 54 KTAcrossflow User Manual 11 0012 32 Edition AB Handling methods in AKTAcrossflow 3 5 Creating cross flow methods using the Method Wizard AKTAcrossflow methods are complicated and include many blocks and instructions So it may be convenient to use the Method Wizard to build methods for different applications The Method Wizard can be used in two different ways e When a method is created in the Method Editor of UNICORN The Method Wizard dialogs ar
179. too low 122 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Product methods using the Method Wizard es Recovery e Select to perform Recirculation before initial recovery or not The recirculation will wash out product e g in the gel layer from the filter e f Buffer flushes are selected enter number of flushes and flush volumes Default volumes are dependent on filter s hold up volumes e If buffer flushes are not selected e the reservoir will first be emptied through R VB Out3 e The reservoir will be filled with 5 ml buffer and the product between reservoir and RVB Out3 will be emptied through RVB Out3 e There is also an option to perform recirculation between the flushes Recovery Recovery Option Recovery b Initial Recovery collected through R VB 3 includes empty reservoir and retentate tubing product displacement Recirculation before initial Recovery with no backpressure Buffer Flushes collected through RVB 1 Number of Buffer Flushes 1 O2 Volume Flush 1 16 3 mi 16 9 350 ml Recirculation before Flush Recovery Note The product will leave the system through R VB Out3 and the flushed volumes through R VB Out1 AKTAcrossflow User Manual 11 0012 32 Edition AB 123 Creating Product methods using the Method Wizard 124 AKTAcrossflow User Manual 11 0012 32 Edition AB Creating Postproduct methods using the Method Wizard 8 8 Creating Postproduct methods usin
180. troducing bacteria etc use the optional path with a separate waste bottle 3 Fill the tubing with solution using a syringe connected to the outlet tubing end 4 Repeat the procedure for all pumps AKTAcrossflow User Manual 11 0012 32 Edition AB 35 System preparation es 2 3 3 Cleaning the pump piston rinsing system 1 Remove the rinsing solution bottles and empty them Place the tubing in waste containers 2 Fill the bottles with freshly prepared rinsing solution 20 ethanol in 10 mM NaOH However do not connect the rinsing system tubing yet Rinsing system bottle Transfer amp Permeate pump Permeate p bottles Buffer bottles Rinsing system bottle Feed pump Retentate bottle Fig 2 2 Location of the bottles 3 Connect one Transfer Inlet port to distilled water In Manual select this port under Transfer Transfer_Valve_Block click Execute AKTAcrossflow System 1 Transfer Instructions Instructions Parameters OFeae TransferFlow Macro ee TVB n1 Constant Retentate Volume lock OPermeate z ah ranster_t 2_ Valve Transfer Set_TiVol_Totalizer DF_Exchange_Factor O AlarmstMon ConstRVol_P Other ConstRVolLag Auto update If this is checked the parameter fields will be updated during method run 36 AKTAcrossflow User Manual 11 0012 32 Edition AB AKTAcrossflow User Manual 11 0012 32 Edition AB S
181. uct step Air Trans fer VB1 Permeate UB M Y8 In1 P UB Recucle T UB Tn2 T UB Ind Trans fer VB2 Retentate UB T UB Tn R VB Out3 5 ci A1 EEN 2 paaa Cartridee permeate 3 Approx 12 ml is emptied through R VB Out3 The product is chased with buffer through R VB Out3 Air CE Transfer namari UB Recucte EDA PurgeValve T UB Ind Trans fer VB2 Retentate UB T VB In 4 Recirculation for five minutes Air Trans fer VB1 Permeate UB Ub Recucle Trans fer UB2 IB Tnt M Y8 1In7 5 5 mlthrough R VB Out1 is emptied a AKTAcrossflow User Manual 11 0012 32 Edition AB 111 Creating Product methods using the Method Wizard Sarg 6 The reservoir is filled with 16 9 ml buffer The product is chased with approx 10 ml buffer to R VB Out1 Recirculation for 5 minutes Reservoir is emptied to R VB Out1 7 The reservoir is filled with 5 ml 8 At ConstantRetentateVolume the retaining product between reservoir and R VB Out1 is chased with buffer and is emptied through R VB Out1 9 The rest of the system volume is emptied through waste 7 2 Microfiltration 7 2 1 Introduction Depending on the application the product of interest will e stay in the retentate or e pass through the filter to the permeate side Cell harvesting washing In cell harvesting and cell washing the product of interest the cells will stay in the retentate e Aconcentration reduces the volume of the cell solution e Adiafiltration is run to wash the
182. uction uses R VB Out to open one position FeedPump to empty reservoir in a controlled manner In order to per form this FeedFlow is reduced when reservoir level is below a certain value and R VB Out creates a back pressure to avoid siphoning about 2 bar When O level is reached defined as just below reservoir bottom surface Feedflow is immediately stopped 13 2 11 EmptyReservoirAbort nstruction name Group EmptyReservoirAbort Method Manual Recirc Parameter 1 name Position name default underlined RetValvePort R VB Recycle Parameter 2 name Position name default underlined FeedFlow 0 0 ml min 0 0 600 0 ml min nstruction Help text EmptyReservoirAbort aborts the sequence initiated by the EmptyReservoir instruction Instruction immediately stops Feedflow Sets R VB Out to Recycle position Eventually Watch EmptyResFinished signal is generated Ooo 174 KTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions n 13 2 12 Concentration_Factor Instruction name Group Concentration_Factor Method Manual Alarms amp Monitors Parameter 1 name Position name default underlined Mode Off On Parameter 2 name Position name default underlined Type FedBatch TankBatch Instruction Help text Starts the Concentration factor calculation Use watch command in method to set the endpoint FedBatch cannot be operated without a Set_ResVol_Totalizer volume entered Detailed information in User Manual 13 2 13 MixerSp
183. usting the cell constant Fill the flow cell with calibration solution of 1 00 M NaCl Wait until the temperature is constant in the range 20 30 C Enter the theoretical conductivity value according to graph in Instrument Handbook Basic operation Calibration Cond_Cell Monitor Function Cond_Cell Enter a new cell constant Text 1 Add cell constant value of a new Cond cell See Help Parameter name Input default underlined Reference value 40 0 1 cm 0 1 300 0 Help text To be performed when you are replacing the current cell with a new conductivity cell The cell constant is shown on the packaging In case you have thrown the outer package perform Cond_Calib 210 AKTAcrossflow User Manual 11 0012 32 Edition AB Strategy instructions Cond_Temp Monitor Function Cond_Temp Temperature sensor Text 1 Calibrate the Cond temperature sensor See Help Parameter name Input default underlined Reference value 0 0 C 5 0 60 0 Help text Calibration of the temperature sensor in the conductivity flow cell is only necessary if the monitor is used in high accu racy measurements Place the Cond flow cell together with a precision thermometer inside a box or empty beaker to ensure that they are not exposed to draft Leave them for 15 min to let the temperature stabilise Read the temperature on the thermometer and enter this as Reference value TrfPress Monitor Function TrfPress Pressure
184. with a Preparing the system This method is faster than Preparing the system and reservoir and is used when the reservoir does not need to be treated However different transfer inlet tubing is used dependent of preferred liquid 1 Transfer pump fills the tubing from Transfer Inlet to Transfer Purge Valve Waste Air Trans fer UB1 Permeate UB Trans fe Purge F Ue Recucls er alve Muna Transfer UB2 Mene rae w Cartridge permeate KTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard ey 2 Reservoir is filled to a volume defined by used filter area Air Trans fer VB1 Permeate UB J P rans ter P UB Recucte R VB Out3 5 A1 LENS Cartridge permeate 3 25 ml of the reservoir volume is emptied 4 Recirculation at the retentate side for one minute with FeedPressure control Air Permeate UB Eto She SUES 5 The reservoir is emptied through R VB Out2 Waste Permeate UB prilive aara 285 Cartridee permeate EE AKTAcrossflow User Manual 11 0012 32 Edition AB 83 Creating Preproduct methods using the Method Wizard ay 5 4 3 Visualized procedure of the rinsing method The system is prepared according to the procedure Preparing the system 1 described in Section 5 4 1 The Transfer Inlet use
185. y section 7 2 2 Basic settings dialog Note To get the Method Wizard for hollow fibers Hollow fiber has to be selected as component in System Setup See Section 2 4 Method Wizard for System AKTAcrossflow2 Basic Settings Basic Settings Method Ultrafitration Preproduct Steps Filter Type es Product Steps Filter List AXM 3 KD 0 50 mm Recovery Postproduct Steps Steps Summary v Preproduct v Product Postproduct Hollow Fibre specification per filter Lumen Diameter mm 0 10 10 00 mm Number of Fibers 1 1000 Surface Area cm 2 40 150 cm 2 Lumen Hold Up Vol mil 0 0 25 0 ml Feed Pressure Limit bar 0 5 2 bar TMP Limit bar 0 5 2 bar System setup Number of filters Extra Tubing Volume ml 0 0 25 0 ml Reservoir Size 350 ml 1100 mi Tubing kit Small ID 1 7 mm Large ID 2 9 mm F Cancel Set Default a AKTAcrossflow User Manual 11 0012 32 Edition AB 113 Creating Product methods using the Method Wizard e Inthe Method list select Ultrafiltration Cell Processing Basic Settings Ultrafiltration Cell Processing Calibrate level sensor Method Filter Type Filter List e Select Filter Type AXM or AXH AXM hollow fibers have a straight linear shape AXH hollow fibers are curved into a loop e inthe Filter List available GE Healthcare filters are displayed Select the used filter or select Other Basic Settings Method Ultrafiltration Cell Processing v AXM v Filter Type
186. ype AxM Filter List 3 KD 0 50 mm e Select the type of process step Steps M Preproduct _ Product _ Postproduct e The Hollow Fibre specification per filter area displays the recommended default values for the selected filter Accept or edit the values Hollow Fibre specification per filter Lumen Diameter 05 mm 0 10 10 00 mm Number of Fibers 12 1 1000 Surface Area 50 cm 2 40 150 cm 2 Lumen Hold UpVol 117 ml 0 0 25 0 ml Feed Pressure Limit 43 bar 0 5 2 bar TMP Limit 3 0 bar 0 5 2 bar If Other filter has been selected enter Lumen diameter Number of fibers Surface area Lumen hold up volume Feed pressure limit and TMP limit for the hollow fiber Information is usually available from the manufacturer Note When using TMP in a method a value close to TMP limit may lead to the run being paused Always choose a lower working TMP than the TMP limit e If several filters are assembled together in parallel select in the System setup section the Number of filters 1 3 If one filter is used only keep the default value 1 e If other tubing than standard is used calculate the extra tubing volume and enter the value in the Extra Tubing Volume box System setup Number of filters i Extra Tubing Volume 9 4 ml 0 0 25 0 ml 76 KTAcrossflow User Manual 11 0012 32 Edition AB Creating Preproduct methods using the Method Wizard
187. ystem Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 bar 0 00 5 20 bar Parameter 2 name Setpoint default underlined Delta_Base 0 00 bar 0 00 5 20 bar Instruction Help text Watch on pressure reported at filter feed port The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta_Base setting affects only the Stable_baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_baseline condition in the Watch instruction 13 5 22 WatchPar_RetenPress Instruction name Group WatchPar_RetenPress System Settings Monitors Method Manual Alarms amp Monitors Parameter 1 name Setpoint default underlined DeltaPeak 0 00 bar 0 00 5 20 bar Parameter 2 name Setpoint default underlined Delta_Base 0 00 bar 0 00 5 20 bar Instruction Help text Watch on pressure at filter retentate port The DeltaPeak setting affects only the Less_than_or_valley and Peak_max_conditions when using the Watch instruction see UNICORN user manual As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height Delta
188. ystem at pressures above the specified maximum pressure 5 2 bar 4 3 Monitoring the run 1 In System Control in UNICORN select View Panes 2 Check the desired boxes and click OK Customize Panes MRundata MLogbook Cancel Help 3 To edit the displayed panes right mouse click in the window and select Properties AKTAcrossflow User Manual 11 0012 32 Edition AB 65 4 Performing cross flow runs manually as 4 Select appropriate tab and choose how to display data Properties ee ee OEE Run Data Groups Run Data Color Curves XAxis Y Axis Run Data Instruments Connection Run Status Scouting No Acc Volume Block Volume Acc Time Block Time TrfFlow pH RetFlow PermFlow FeedPress RetenPress ResVol v Example of displayed information 2 System Control 1 SEUPPDIO386 Method x Chriss Water Flush Test m02 Result x MsalWater Flush Test001 res Run data Flow scheme 1 48 min End Block Ty 1 48 min Block Woter_Flush_Pet_Flow 1 48 min Base SemeAeMain 148mm Reflow 500 ml min 1 48 min End Block Log book i 6 rin Block 0 Fush_vot 1 48 min Base Volume onl 1 48 min MathodBase FeedPumnp 2 78 min Condhion_Occured Re VolLess_Than 40 0000 Increase_volume 278 min Base SemeAsMem 2 78min Trenstor Flow 80 0 mlmin 2 78 min Block Oa
189. ystem preparation SS 4 Start transfer pump at 200 ml min and pump 200 ml distilled water to the reservoir At the same time as the transfer flow is started start a Feedflow at 400 ml min AKTAcrossflow System 1 Transfer Instructions bg Instructions Parameters Bree Flowfiate 00 2000 Constant_Retentate_Volume Permeate Transfer_Valve_Blocks 200 ml min D Transfer_Purge_Valve So Transfer Set_TrVol_Totalizer DF_Exchange_Factor ONerpeton ConstfVol_P O Other ConstRVolLag Auto update If this is checked the parameter fields will be updated during method run AKTAcrossflow System 1 Recirc Instructions Instructions Parameters 1 t i a j neen Recite cog FeedFlow f 10 0 600 0 Permeate FeedPressure 400 mimin Jelete DeltaP z O Transfer Retentate_Valve_Block Execute OAlaimstMon EmpyReservoi Concentration_Factor Ch Other MixerSpeed v re Auto update If this is checked the parameter fields will be updated during method run 5 Open permeate valve block Out 2 When 200 ml water is in the reservoir stop transfer pump AKTAcrossflow System 1 Permeate Instructions Instructions Parameters P TMP_Control a O Recic Flux Contil pe Permeate A Permeate_Unrestricted_Flow F O Transfer Start_Eval_ Window Stop_Eval_ Window Dorao Set Eval Mark O Other Set_PermVol_Totalizer Auto update If this is checked the parameter fields will be updated during

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