ProfilerPro Glycan Profiling Assay User Guide
Contents
1. Peaks by Co migration with Known Glycan It is sometimes interesting to compare the migration time of peaks observed in your samples to the migration time of known glycan standards to help identify the peaks in your samples When running glycan standards it is important that the buffer the standards are presented in is closely matched to the buffer your samples are presented in If the buffers are not well matched the migration time of the standards may not correspond to the migration time of your sample A simple method to achieve matched buffers is to label your standards using the reagents from the Glycan Release and Labeling kit Prepare the standards the same way as you prepared your test samples Any dilutions of the standards prior to addition in the Denaturing Plate should be done with water Milli Q or equivalent PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Analyzing Data 23 Glycan Standard Mixture Man G0 GUE G1F G2 G2F D4 196D 4500 4000 3500 g 3000 L S 2500 D S 2000 1500 1000 500 Time sec Figure 15 Example showing a mix of glycan standards compared to an IgG sample run in well matched buffers Blue IgG Red Mix of six glycan standards Man5 GO GOf Gif G2 G2f The standards were run at about 50 ng L concentration PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Results 24 Results In the Glycan Ladder the Lower Ma2. C 8 UL sample m gt Transfer OOO Transfer Reconstitute mAb Bij M 11 pLof A 8 uL of e M and run ecccccce denature x digest escccece all ecccecce eeccccce Denaturing Plate PNGase F Plate Dye Plate LabChip GXII Touch Figure 2 Sample preparation procedures Spin down All plates must be spun down before removing the seal to collect any reagent that may be attached to the plate seal Plates have to be kept frozen at all times Remove plate from freezer only prior to use Cutting the plates The 96 well Denaturing PNGase F and Dye Plates can be cut vertically into sections of 24 48 or 72 wells if running fewer than 72 samples To cut a plate first stack it on top of a 96 well skirted PCR plate for support Using a sharp blade carefully score and cut the top and sides of the plate along the segmented groove then carefully break off the section After cutting inspect the plate wells along the cut edge for cracks Do not use wells with cracks Storage If not using a whole 96 well Denaturing PNGase F or Dye Plate after cutting the plate freeze the remainder immediately Partial Plates When working with partial or cut plates it s helpful to stack the plate on top of a 96 well skirted PCR plate for support especially when spinning Plate Seals Use plate seals with a minimum thermal range of 20 C to 80 C For all plate sealing use of a rubber roller is recommended ProfilerPro Glycan Profiling Assay
3. 1 Place the chip into the plastic storage container The sipper should be submerged in the fluid reservoir Remove the reagents from each well of the chip using vacuum Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and aspirated twice with water Milli Q or equivalent Add 120 uL water Milli Q or equivalent to the active wells Cover the wells with Parafilm to prevent evaporation and store the chip at room temperature until next use The chip must be used to its lifetime to the total number of 400 samples within 30 days of analyzing the first plate of samples Chip Cartridge Cleaning 1 2 PN CLS140161 Rev C Daily a Inspect the inside of the chip cartridge and O rings for debris b Use the provided lint free swab dampened with water Milli Q or equivalent to clean the O rings using a circular motion If the O rings stick to the chip or a pressure leak is detected perform the more extensive monthly cleaning procedure Monthly a To reduce pressure leaks at the chip interface clean the O rings frequently Remove the O rings from the top plate of the chip interface on the LabChip GXII Touch instrument Soak O rings in water Milli Q or equivalent for a few minutes Clean the O ring faces by rubbing between two fingers Wear gloves b To reduce the occurrence of current leaks clean the chip interface frequently Clean the top plate of the chip interface using the provided lint fr
4. Assay Performance 1 PN CLS140161 Rev C Can concentrations lower than 1 25 mg mL be tested using this assay Yes concentrations as low as 0 5 mg mL can be tested however the percent area of peaks for samples lt 1 25 mg mL are less reproducible CV gt 10 than samples tested at gt 1 25 mg mL What other glycoproteins in addition to monoclonal antibodies can be tested using this assay Other N linked glycoproteins can be tested using this assay However this assay was optimized to provide a glycosylation pattern for 5 major glycans Man5 GO GOf G1f and G2F Other glycans or oligosaccharides may not be detected according to the assay performance specifications If am using this kit for the first time how do confirm that the kit is working properly Deglycosylation can be confirmed by testing unlabeled digested samples running Protein Express Kit for reduced conditions Sample labeling can be confirmed by labeling glycan standards see Analyzing Data on page 21 Can charged N glycans be detected using this assay Some charged glycans will migrate faster than the lower marker If this is the case and the peak is within the baseline window then they can be manually included in the analysis For instruction on how to perform this see Troubleshooting on page 26 How does this assay compare to HPLC and CGE LIF The Glycan assay is a high throughput assay in which 96 samples from sample preparatio
5. Run Operator N C Read Plate Barcode Data Path C Program Files x86 PerkinElmer LabChip GX Touch Data kan ran kan raen rowse Defaul Iv Copy to C Program Files x86 PerkinElmer LabChip GX Touch DataCopy kan rnn v Create Daily Sub Directory I Auto Export Data File Name Labchip_ 2014 06 Ors 07 55 10 gxd Default File Prefix Project name Labchip mj Computer Name m Barcode Iv Date lv Time Advanced Settings S Sample Names File C Expected Peak File C Excluded Peak File Figure 11 Run setup screen 4 Touch Start to begin the run LabChip GX H Touch HT a Run Select Wells Setup Run Start Run Chip Status SR Type HT High Resolution AG oe OC TL N TT OOOO Chip Reagents N A pid en Chip Expiration Jan 1 2020 B 16 20 OC OOOO Chip Life 400 Samples Left C L E E L 22 L sess OC p E B Run Parameters tae aaa OOO Operator Irina Assay HT Glycan r Oc O OC QO OO OC Q Data Path C Program Files x86 PerkinElmer 2 6 D ox D Q O C7 O O OC CT LabChip GX Touch Data Plate Name BioRad 7 HSP 96xx Sip 4 mm H D COL D L T L T OOOOC L O Barcode N A Plate Cycles 1 Auto Export No Sample Saver No Random Selection No Figure 12 Starting a run PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 20 Storing the Chip After use the chip must be cleaned and stored in the chip container
6. do 1 Doone or all of the following Replace the 18 megohm 0 22 um filtered water Milli Q or equivalent water used for chip preparation e Replace the buffer used for sample and reagent preparation e Use a0 22 micron filter for all water and buffers used for chip sample and reagent preparation e Spin down sample plate to pellet any particulates PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 33 Symptom Humps in several electropherograms which do not correspond to sample data 1250 1000 750 500 Fluorescence 250 y 0 d ES T SAT a Pe C L 1 20 25 30 35 40 45 50 55 60 Time seconds Figure 26 Humps in several electropherograms Possible causes 1 Electrode 7 is dirty and has contaminated the Gel mixture in well 7 2 High concentrations of detergent in the sample buffer can sometimes cause humps in the electropherogram What to do 1 Before restarting the run clean electrode 7 Remove the chip and follow the electrode cleaning procedure We recommend using the provided swab and isopropanol to manually clean electrode 7 2 Lower the detergent concentration in the sample Symptom Peaks migrating much faster or slower than expected Note Some migration time variance between chips or within a plate is considered normal chip performance All chips are QC tested at PerkinElmer prior to shipment Possible causes
7. mg mL 10 60 ug of MAb total Reproducibility of Area CV lt 10 for a peak gt 2 5 of total glycan Limit of Detection 1 ng of GOf standard smallest amount of labeled GOf standard that can be detected Deglycosylation gt 95 of all N linked glycans will be released from MAb Usable Size Range Appropriate for neutral glycans found on MAbs some charged glycans may run outside of our usable range Sizing Reproducibility CV lt 2 5 Sample Prep Chip Prep and Analysis Time lt 8 hours for a 96 well plate Number of Samples per Chip Prep Up to 192 samples before chip needs to be reloaded with fresh gel Kit Contents The ProfilerPro Glycan Profiling system consists of Table 2 Glycan Release and Labeling Kit Part Number 760523 Item Plate Quantity Denaturing Plate Glycoprotein denaturing plate One 96 well plate PNGase F Plate Oligosaccharides releasing One 96 well plate plate Dye Plate Oligosaccharides labeling plate One 96 well plate Note Store plates at 20 C PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Table 3 Glycan LabChip Reagent Kit Specifications 6 Part Number 760525 Reagent Vial Quantity Glycan Gel Matrix Red 2 vials 1 8 mL each Glycan Marker dry Green 4vials Glycan Marker Diluent White l1 vial 0 65 mL Glycan Ladder dry Yellow 4 v
8. prior section for guidance Peaks that are found to the left of the marker are labeled with by the software Hover over the peak you wish to include with the mouse right click and select Include peak Once included into the analysis all analysis features can be applied to the peak Symptom Marker is miscalled in the ladder If the ladder peaks are compressed and or broad the software may be miscalling the marker peak There are two ways to correct this 1 Manually select the proper marker peak by hovering over the peak with the mouse right click and select Force Lower Marker It is often helpful to click Turn Off Analysis in order to identify the proper peak 2 If the marker peak height is lower than the ladder peaks the software often miscalls the marker peak In this case remove the chip from the instrument clean out well 4 and replace with fresh marker Glycan Ladder Ladders Ladders 22 Aligned Time sec Figure 22 Compressed and broadened ladder peaks due to miscalled marker PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 29 Glycan Ladder Ladders Ladders Figure 23 Example of a degraded marker peak with analysis turned off Symptom No ladder or sample peaks but marker peaks detected Note The lower marker peak height will most likely be greater than normal height Possible causes 1 Air bubble in sipper introd
9. the unsealed plate for 2 hours at 55 C or until sample is completely dry using a PCR machine with lid open or heat block Caution The dye contains acetic acid Be wary of fumes Incubating under a fume hood is recommended Sample must be completely dry to ensure sufficient labeling ProfilerPro Glycan Profiling Assay User Guide Sample Preparation Procedures 12 Reconstitution 1 Add 100 uL of water Milli Q or equivalent to dried samples 2 Seal plate carefully with an adhesive plate seal Note Ensure the plate is completely sealed to prevent spilling during mixing 3 Mix samples on a plate shaker at maximum speed for at least 1 minute Check to be sure the pellet has completely dissolved 4 Spin plate at 1200 g for 1 minute Note After spinning there may be a white pellet at the bottom of the wells Be careful not to disturb the pellet as it may cause a chip sipper clog 5 Carefully remove the plate seal The plate is ready to run on LabChip GXII Touch PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 13 Chip Preparation Procedures Note Equilibrate the chip and all components of the Glycan Reagent kit to room temperature for 20 30 minutes before use Preparing the Buffer Tube 1 Add 750 uL of water Milli Q or equivalent to the 0 75 mL Buffer Tube Ensure there are no air bubbles in the Buffer Tube 2 Insert the Buffer Tube into the buffer slot on the LabChip G
10. 0161 Rev C Can samples be digested overnight at 37 C using this assay No overnight digestion of samples may lead to poor percent area reproducibility and sample resolution Can samples be labeled overnight at 55 C using this assay No samples should be labeled until dry Extended incubation of dry labeled samples at 55 C may lead to glycan degradation Can an evaporator be used to label the glycans instead of incubating at 55 C for 2 hours No the labeling efficiency using an evaporator is much lower and will result in low sample signal Can labeled samples be reconstituted in less or more than 100 uL of water No reconstituting samples in 100 uL results in a buffer composition optimal for glycan migration in this assay Reconstituting in less or more than 100 uL will change this buffer composition and potentially alter migration and data resolution After labeling standards using this assay can they be frozen and used later Yes labeled standards may be frozen for several weeks and tested again at a later time Thawed labeled standards should be kept on ice when not in use ProfilerPro Glycan Profiling Assay User Guide Frequently Asked Questions 36 6 Can samples be prepared using this assay and then analyzed using HPLC or CE LIF e HPLC No the reagent chemistry of the Glycan assay is not compatible with the HPLC method e CE LIF Yes provided the optics system is compatible or adjusted accordingly
11. 1 Particulates from the samples may be clogging the separation channel this will slow down migration 2 Gel was not primed properly into the chip PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 34 What to do 1 PN CLS140161 Rev C If fast or slow migration is observed repeatedly on a new chip contact technical support to arrange return of the chip to PerkinElmer Please send a data file showing the failure along with the return request Minimize the loading of particulates in the sample by performing a centrifuge spin of the sample plate e g 3000 rcf for 5 minutes and or ensuring the Sip 4 mm plate type is selected in the Select Wells screen before starting a new run The debris may be flushed out of the chip by washing and re priming the chip See LabChip Kit Essential Practices on page 38 for instructions on how to wash and reprime the chip Check the O rings on the top surface of the chip interface and clean if necessary ProfilerPro Glycan Profiling Assay User Guide Frequently Asked Questions 35 Frequently Asked Questions Kit Specification 1 2 What is in the Lower Marker The Lower Marker consists of dye labeled maltohexaose What is in the Ladder The ladder is a dye labeled glucose homopolymer standard consisting of a 1 6 linked glucose oligosaccharides with variable number of monomeric glucose units 1 23 or more Assay Preparation 1 PN CLS14
12. Express Kit for reduced conditions Note It may be necessary to dilute the digested sample depending on the starting concentration A starting concentration of 2 mg mL or below is recommended for the Protein Express assay Under reduced conditions you will see a shift from the heavy chain HC to the non glycosylated heavy chain NGHC for the digested sample Figure 19 Blue control undigested Red Digested MAb Symptom Digestion is incomplete Possible causes 1 The samples were incompletely denatured 2 There was an insufficient amount of PNGase F in the reaction What to do 1 Repeat the sample preparation being careful to spin down the Denaturing Plate Small amounts of denaturing buffer attached to the plate seal may significantly decrease the amount or concentration of reagents needed to sufficiently denature samples Additionally verify that the samples are being denatured at 70 C for 10 minutes PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 27 2 Ifthe sample tested was on the high end of the Glycan assay concentration range then dilute the sample down to 5 mg mL and repeat sample preparation Some MAbs may be more difficult to deglycosylate than others In this event a greater amount of PNGase F to MAb and longer incubation time may be needed to completely deglycosylate samples 3 Repeat the sample preparation being careful to spin down the PNGase F Plate Small amo
13. User Guide Procedure Digestion Hints Procedure PN CLS140161 Rev C 4 Sample Preparation Procedures 10 Thaw and spin Denaturing Plate at 1200 g for 1 minute Carefully remove the plate seal Add 8 UL of sample monoclonal antibody with concentration range of 1 25 7 5 mg mL 10 ug to 60 pg total protein to Denaturing Plate Mix by pipetting up and down or with a plate shaker a Samples should be at least one step purified Crude sample media or buffers that contain sugars may interfere with the labeling efficiency of the desired glycans b For a control blank sample add 8 uL of 18 Megohm 0 22 um filtered water Milli Q or equivalent instead of sample and follow all digestion and labeling steps Seal plate carefully with an adhesive plate seal Note Ensure the plate is completely sealed to prevent evaporation 5 6 Spin Denaturing Plate at 1200 g for 1 minute Incubate Denaturing Plate for 10 minutes at 70 C using a PCR machine with lid temperature at 70 C heat block or incubator Break point If needed after digestion samples may be frozen at 20 C and labeled the next day Overnight digestion is not recommended Incubators Use of incubators or ovens for incubation is not recommended If using an incubator for the labeling step be careful of acetic acid fumes when opening the incubator door Thaw PNGase F Plate Spin both PNGase F Plate and Denaturing Plate at 1200 g for 1 minute Caref
14. XII Touch instrument Buffer Tube Ladder Tube AKEREKE Sample LAA AAA Well A1 vuevvevevvevvvwv LS DAI IAIIYI4YA IY IY AAAI IAI IAI IY YY Ladder Tube Buffer Tube Figure 3 Locations of the Buffer Tube and Ladder Tube in the GXII Touch instrument Preparing the Ladder Tube 1 Add 145 uL of Glycan Ladder Diluent purple cap to one of the Ladder tubes 2 Vortex at highest speed for about 30 seconds and spin down Make sure the ladder is completely reconstituted 3 Transfer 120 uL of prepared ladder to the 0 2 mL Ladder Tube Ensure there are no air bubbles in the Ladder Tube 4 Insert the Ladder Tube into the ladder slot on the LabChip GXII Touch instrument PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 14 Preparing the Marker Tube Notes Prepare marker solution just before loading the chip into the LabChip GXII Touch and starting the assay Do not prepare marker solution in advance as the marker signal degrades over time 1 Prepare marker solution by adding 125 uL of Marker Diluent white cap to one of the Marker tubes 2 Vortex at the highest speed for about 30 seconds and spin down Make sure marker is completely reconstituted Preparing the Chip 1 Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells see Figure 4 For more details on how to set up a vacuum line see page 44 2 Each active ch
15. an Ka Run Test Ladder after Prime Status Buffer Figure 8 Chip priming screen 1 Touch the Run button see Figure 8 2 Select the appropriate assay type see Figure 9 plate name well pattern and whether to read wells in columns or rows Select number of times each well is sampled under Adv Settings Figure 10 Touch the green arrow Assay HT Glycan HT Glycan HT Protein Express Low MW Figure 9 The Assay Choices menu PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 18 T R Run Select Wells Setup Run Start Run Change Assay Folder Assay Folder C Program Files x86 PerkinElmer LabChip GX Touch Assay Select Assay Select Plate Type IR R 1 Fil HT Glycan w BioRad 96 HSP 96xx Sip 4 mm eee Rant ER 23 45 67 8 9 1011 12 ieee Size 4 11100000000 A Status 3 4 Column Wise Selection Sip Order Adv Settings 24 of 96 selected Figure 10 Selecting wells 3 Inthe Setup Run tab select the operator name the option to read barcode the destination of the file the inclusion of sample names expected peaks and excluded peaks and the filename convention Select Auto Export to export results tables automatically Figure 11 Touch the green arrow PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 19 LabChip GX H Touch HT SRI ZT Run Select Wells Setup Run Start
16. an be run as needed throughout the day PerkinElmer recommends the chip be re prepared after it has been idle for 8 hours but the chip can be used continually over an 8 hour work day as long as the maximum recommended idle time of 8 hours and total chip lifetime of 400 samples are not exceeded ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 43 Samples e Prepared sample plates should be free of gas bubbles and particulate debris both of which may inhibit sipper flow e Sample plates containing gas bubbles and or particulate debris should be spun down at 3000 rpm 1250 rcf prior to analysis Up to four 96 well plates 400 samples can be run with a single chip preparation when running the GXII Touch HT instrument Up to 48 samples can be run with a single chip preparation when running the GXII Touch 24 instrument PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Well Aspiration Using a Vacuum 44 Chip Well Aspiration Using a Vacuum Aspirating with a pipette can leave used reagents in the chip wells For this reason PerkinElmer recommends vacuuming the wells instead This can be accomplished by attaching a permanent pipette tip to a house vacuum line with trap Figure 30 To avoid contamination use a new disposable pipette tip over the permanent tip for each chip aspirated Figure 31 Figure 30 A Permanent pipette tip attached to a house vacuum line B vacuum line
17. and 10 should be rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry e Add 120 uL of water Milli Q or equivalent to each active well 1 2 3 4 7 8 9 and 10 e Touch the Unload Chip button on the Home screen and place the chip into the instrument e Close the chip door securely Transfer 750 uL of water Milli Q or equivalent into the Buffer Tube Install into the instrument e Touch the Wash button Figure 28 PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 41 Chip Status Type HT High Resolution Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Figure 28 Wash screen After completion of the wash cycle open the chip cartridge and return the chip to the chip container ensuring the sipper is immersed in fluid e Thoroughly aspirate all fluid from the chip wells using a vacuum line e Replace fluid in the wells with freshly made reagents as described in Preparing the Chip on page 14 Do not let wells remain dry Transfer 750 uL of water Milli Q or equivalent into a clean Buffer Tube Install into the instrument Install the Ladder Tube sample plate and chip into the instrument and run the assay If air bubbles are not dislodged after a reprime apply a vacuum to the sipper Perform this by filling all active wells with 100
18. ckman Coulter Cat 538619 or Axygen Cat PCR AS 200 Centrifuge with rotor that accepts a 96 well PCR plate Optional Plate shaker for example a VarioMag Magnetic Shaker Cat 51110 2000 rpm amplitude 2 mm ProfilerPro Glycan Profiling Assay User Guide Safety Warnings and Precautions 8 Safety Warnings and Precautions WARNING A For Research Use Only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals CAUTION We recommend that this product and components be handled only by those who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In case of contact with skin or eyes wash immediately with water WARNING AA Denaturing Buffer Ladder and Gel Matrix contain SDS Avoid inhalation and contact with skin and eyes PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Sample Preparation Procedures 9 Sample Preparation Procedures Denature Hints PN CLS140161 Rev C 1 25 7 5 mg mL Denature Digest for Label for 10 min at 70 C 1 hr at 37 C 2 hrs at 55 C C fore
19. ee swab dampened with water Milli Q or equivalent c Allow the O rings and chip interface to air dry Reinsert the O rings into the chip cartridge ProfilerPro Glycan Profiling Assay User Guide Analyzing Data 21 Analyzing Data Assignment of Size and Identification of Peaks in the Electropherogram The LabChip GX Touch software analyzes the electropherograms assigning a size among other things to each observed peak It may not be intuitively obvious what this size means given that glycan molecules with the same mass can sometimes have very different apparent electrophorectic mobility During the labeling process the glycans are derivatized with a charged fluorophore The electrophorectic mobility of each glycan in our specific separation medium is dependent on its charge mass and structure where structural elements such as monosaccharide composition mannose glucose galactose glycosidic linkage position C2 C3 C4 C6 and anomercity a vs B have been shown to affect electrophorectic mobility Considering these complexities the size that the software assigns does not have a direct physical meaning like the size of a DNA molecule in base pairs or the size of a protein in Daltons Rather the sizes assigned to glycan peaks by the LabChip GX Touch software relate the migration time of each peak to the migration time of the glucose peaks in the glycan ladder It is a way of normalizing the migration time of each peak so
20. he first stop 4 Withdraw the pipette from the well Chips Repriming Chips e Touch the Unload Chip button on the Home screen to open the instrument door Place the chip into the instrument e Close the chip door securely and choose the corresponding assay e Touch the Prime button on the Home screen to reprime the chip PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 40 Washing Chips Important Note Wash chips only with water Milli Q or equivalent Use of any other reagents including Wash Buffer is likely to cause even more artifacts in subsequent data Notes Some protein samples may have components which produce data with extra peaks spikes or other artifacts When these artifacts are present washing chips on the LabChip GXII Touch immediately before the next use can often restore data quality Chips should only be washed on the LabChip GXII Touch immediately before they are prepared with fresh reagents and primed on the instrument Chips should not be washed and left with water in the chip channels for any extended period of time For most protein samples the only chip cleaning protocol that is required is to rinse and aspirate the active wells twice with water Milli Q or equivalent and store the chip with 120 uL of water in each active well e Thoroughly aspirate all fluid from the chip wells using a vacuum line e Each active well 1 2 3 4 7 8 9
21. ials Glycan Ladder Diluent Purple 1 vial 0 75 mL Note Store reagents at 4 TC Table 4 Consumable Items Item Supplier and Catalog Number Quantity Ladder Tubes Genemate Cat C 3258 1 10 0 2 mL Detection Window VWR Cat 21912 046 1 Cleaning Cloth Swab ITW Texwipe Cat TX758B 3 Buffer Tubes E amp K Scientific Cat 697075 10 0 75 mL NC Table 5 High Resolution LabChips Item Catalog Number Touch HT High Resolution Chip for use with GXII Cat 760524 Touch 24 High Resolution Chip for use with GXII Cat CLS138951 PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Specifications 7 Additional Items Required GX Plate Adapter for Deep Well PCR Plates Cat 126209 Note The PerkinElmer Dye Plate can be read directly in the GXII Touch instrument using the Plate Adapter Alternatively after reconstituting with water Milli Q or equivalent labeled samples can be transfered to a standard 96 well PCR plate prior to placing them into the instrument PN CLS140161 Rev C PerkinElmer Hard Shell thin wall 96 well skirted PCR plate blue Cat 6008870 recommended Heating device any PCR thermocycler is best A heat block that accepts a 96 well PCR plate will also work An incubator may work but is less desirable Adhesive plate seals PCR type with a minimum thermal range of 20 C to 80 C For example a Be
22. ion and analysis using the LabChip GXII Touch instrument Fast separation time less than 45 seconds per sample makes this method valuable for use in high throughput screening of antibody glycosylation patterns in early stage development The microchip based CE analysis gives resolution sufficient to determine the relative abundance of the major N glycan types found on antibodies which contain primarily neutral N linked glycans consisting of complex hybrids and high mannose type oligosaccharides MAb Glycosylation Pattern Demo D6 MAb14 cence N 2 S u 16 18 20 22 Total Area 6914 43 Aligned Time sec Figure 1 Glycosylation profile of a therapeutic MAb Relative abundance of the major glycan types can be reliably quantified PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Introduction 4 Features e Fast separation time less than 45 seconds per sample about 1 5 hours for 96 samples e Software determines the relative amounts of each glycan present All included reagent set provides predictable reproducible results Reagents provided in 96 well plate format for convenience and ease of automation e Automatic sampling from a 96 well plate PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Specifications Assay Specifications Specifications Table 1 Assay Specifications Amount of Sample Required 8 UL with concentration range of 1 25 7 5
23. ip well 1 2 3 4 7 8 9 and 10 should be rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry 3 If any water spilled onto the top or bottom of the chip surfaces during rinsing aspirate using a vacuum line DO NOT run the tip over the central region of the detection window Use the provided Detection Window Cleaning Cloth to clean the chip detection window Figure 4 Using a vacuum to aspirate the chip wells is more effective than using a pipette See page 44 for more details 4 Using a reverse pipetting technique add 75 uL of Gel Matrix red cap to chip wells 2 3 7 8 and 9 and 120 uL to well 10 Figure 6 PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 15 Glycan Gs KT S je MOOG Marker Gel Figure 5 Reagent placement Note Be sure to periodically clean the O rings on the top plate of the chip interface on the LabChip GXII Touch instrument Use the provided lint free swab dampened with water Milli Q or equivalent or 70 isopropanol solution in DI water to clean the O rings using a circular motion Allow the O rings to dry before inserting a chip Inserting a Chip into the LabChip GXII Touch Instrument 1 Check that the sample plate Buffer Tube and Ladder Tube are placed on the instrument properly 2 Remove the chip from the chip storage container and inspect the chi
24. j gt ProfilerPro Glycan Profiling Assay User Guide For LabChip GXII Touch d Copyright 2014 PerkinElmer Inc All rights reserved PerkinElmer is a registered trademark of PerkinElmer Inc All other trademarks are the property of their respective owners PN CLS140161 Rev C Contents Introductio M saiia earan aeaa des ae aa dig dads aaa en decent aiana Eiai 3 Applications 5 vices e aa eaae Ee Aaaa a E E tases AA AAS cant E E es 3 Specifications ardir aaa eet nian oe 5 ASSAY Specification S emeena enn a E TE a E EEn EEE 5 PARo a lac E ened E E E E L E 5 Additional Items ReqUire s asrarrarersennrnii a iiaa cer E mess dee 7 Safety Warnings and PrecautiOns ccccccceeeeeeeeeeeeeeneeeeeeeeeeeeeeeeeeeeeeeneeeeseeeeeneees 8 Sample Preparation Procedures ccccceeeeseeeeeeeeeeneeeeeeeeeeeeeeeseeeeeeeeeeeaeeaeeneeeeseeeeenes 9 Denature onena nir r a a E geet E a E EE daai 9 PISS WON aces E TTT T 10 Labeling xes endo Sea yao SOR 22 atin TS asin eine T ee ee CRT aU 11 FROGOMS TMU Le fice tadect cakes TE 12 Chip Preparation Procedures cccecceeeeeseeeeeceeeseeeeeeeeeeeeeeeseeeeeeneeeeeeaeeenseeesesens 13 Preparing the Buffer Tube sss sese sees sees eee ereenn 12 Preparing the Ladder Tube esse eee eee 12 Preparing the Marker Tube sss es sese e sees eee seene renee 14 Preparing the Chip see eee eee ee e eee ee 14 Inserting a Chip into the LabChip GXII Touch Instrument esse eee ee e ee e e 15 Running LG e U 17 Sto
25. n to data analysis can be completed in approximately 8 hours Using HPLC or CGE LIF a few samples from sample preparation to data analysis are completed on the order of days ProfilerPro Glycan Profiling Assay User Guide 6 PN CLS140161 Rev C Frequently Asked Questions 37 Why do need a plate adapter The 96 well Glycan plates do not fit in the LabChip GXII Touch plate tray The plate adapter provides a support base so a Glycan plate can be positioned in the plate tray Is it possible to run the assay without the plate adapter Yes after sample reconstitution the samples can be transferred to another 96 well plate that fits the LabChip GXII Touch plate tray ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 38 LabChip Kit Essential Practices General PN CLS140161 Rev C To ensure proper assay performance please follow the important handling practices described below Failure to observe these guidelines may void the LabChip Kit product warranty Note It is important to keep particulates out of the chip wells channels and capillary Many of the following guidelines are designed to keep the chips particulate free For assay and instrument troubleshooting refer to the LabChip GX Touch software Help file or call PerkinElmer Technical Support at 1 800 762 4000 Allow the chip sample plate and all reagents to equilibrate to room temperature before use approximately 20 to 30 minu
26. o the chip container ensuring the sipper is immersed in fluid e Thoroughly aspirate all fluid from chip well 4 using a vacuum line e Ensure that chip well 4 is rinsed and completely aspirated twice with water Milli Q or equivalent e Add Marker Solution green cap to chip well 4 e Reinsert the chip back into the instrument e Restart the run 2 Perform a marker channel unclogging procedure by repriming the chip See LabChip Kit Essential Practices on page 38 for instructions on how to reprime the chip Symptom Ladder traces show up in the lanes following the ladders delayed sip Y Marad Time sec Figure 24 Small ladder peaks in sample well caused by delayed sip Possible causes 1 Separation channel overloaded with sample 2 Partial clog in the separation channel PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 32 What to do 1 Lower the starting sample concentration 2 Reprime the chip See LabChip Kit Essential Practices on page 38 for instructions on how to reprime the chip Symptom Unexpected sharp peaks 600 500 Lower Upper marker marker Peak caused by particulates 400 300 Fluorescence 200 100 innne 20 25 30 35 50 55 40 45 Aligned Time sec Figure 25 Unexpected sharp peak Possible causes Dust or other particulates introduced through sample or reagents What to
27. or any other purposes contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com Rights of Use The chip and reagents supplied with this kit are sold with limited rights of use The chip may only be used with the specific quantity of reagents supplied with this kit The purchaser has no right or license to refurbish reuse remanufacture or otherwise use the chip with any other reagents than those specifically supplied in this kit For more information on the terms and conditions of use of these chips and reagents please read your LabChip GX Touch User Guide and refer to the applicable label license The reagent kit contains materials that are provided under a license from Life Technologies Corporation for research use only PerkinElmer LabChip and the LabChip logo are registered trademarks of PerkinElmer Inc and or its parent affiliates and or subsidiary companies collectively PerkinElmer The PerkinElmer logo is a registered trademark of PerkinElmer Inc All other trademarks and registered trademarks are the property of their respective holders 2014 PerkinElmer Inc http www perkinelmer com PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide j gt PerkinE PerkinElmer Inc 68 Elm Street Hopkinton Massachusetts 01748 U S A TEL 508 435 9500 FAX 508 435 3439 http www perkinelmer com
28. p window Clean BOTH sides of the chip window with the PerkinElmer supplied clean room cloth dampened with a 70 isopropanol solution in DI water 3 Touch the Unload Chip button on the Home screen PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 16 R Chip Status Type HT High Resolution j Chip Reagents N A Prime Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Unload Plate Figure 6 Home screen 4 Insert the chip into the LabChip GXII Touch instrument Figure 7 and close the chip door securely Figure 7 Chip in the LabChip GXII Touch instrument B Touch the Load Plate button on the Home screen Figure 6 to retract the sample plate and send the sipper to the Buffer Tube Note Do not keep the chip door open for any length of time Dye is sensitive to light and can be photobleached PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Chip Preparation Procedures 17 Running the Assay Note Chips can be primed independently from running assays Select the assay of choice from the insert see Figure 9 Touch the Prime button on the Home screen Make sure the Buffer Tube is placed on the instrument TT R Chip Status Assay Folder Change Assay Folder Type HT High Resolution C Program Files x86 PerkinElmer LabChip GX Touch Assay Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Assay uT Glyc
29. ring the MMP T 20 Chip Cartridge Cleaning sese eee ee eee e 20 Analy Zing Dave TT 21 Assignment of Size and Identification of Peaks in the Electropherogram 21 Identifying Peaks by Co migration with Known Glycan Standards 0 22 FRG SUES TTT 24 Troubleshooting TTT 26 Frequently Asked Questions ss sssss secs ecss ese s sees ereenn ennenen ennenen 35 LabChip Kit Essential Practices ccccesseseeeeeeeeeeeeeeeeeeeeeeeeeeeeneenseeeseeeeeseeeeseeees 38 SIGMA als odio are ik he Aca a EE L l te 38 GINDS aer r E e E eagle hneduaginne Gauss puns a eas a a a 39 STe oE A E E E AAT SE A EET 43 Chip Well Aspiration Using a Vacuum cssseeeeessseeeeeess sees sss ee eree eers seene rees 44 Customer Technical Support sss seeses eser ess eee eee seer essere rennes 45 Licenses and Rights of Use sssssssesescessseeessssesssreeereree resse nnee nenen ennenen 46 PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Introduction 3 Introduction Applications The ProfilerPro Glycan Profiling system provides a high throughput method for analysis of the glycosylation patterns of monoclonal antibodies MAb and other glycosylated proteins The system provides methods for 1 deglycosylation where N linked glycans are released enzymatically using PNGase F 2 fluorescent labeling of the released glycans in presence of released proteins 3 separation by microchip based capillary electrophoresis CE and 4 detect
30. rker should be the tallest peak Following the Lower Marker peak are 7 ladder peaks with Relative Glucose Units RGUs of 7 to 13 Marker and Ladder Ladder1 Ladder Fluorescence nm w A n o o o Q o o o oO Oo o oOo G t t 12 00 1200 S SS SS SS Es 20 22 Aligned Time sec Figure 16 Glycan Ladder Lower Marker LM and ladder peaks L annotated with RGU units For monoclonal antibody samples the Lower Marker should migrate before the major N glycan peaks Man5 GO GOf Gif G1f and G2f MAb Demo E6 MAb14 S 1000 D S T 20 22 Aligned Time sec Figure 17 MAb14 Major N glycan peaks are annotated with percent area PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Results 25 MAb Demo D9 MAb1 Gor n t a o 0 o 2 5 DC S L 20 Aligned Time sec Figure 18 MAb17 Major N glycan peaks are annotated with percent area PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 26 Troubleshooting Note Some of the data examples shown in this section were generated with assays other than the assay described in this user guide How to Test for Complete Digestion PerkinElmer recommends testing for complete deglycosylation by PNGase F using the Protein Express assay Compare the digested sample unlabeled to the undigested sample Follow the protocol for the Protein
31. tes Clean the O rings in the chip interface weekly and the electrodes daily Refer to the Instrument Users Guide Maintenance and Service section for procedures Avoid use of powdered gloves Use only non powdered gloves when handling chips reagents sample plates and when cleaning the instrument electrodes and electrode block Calibrate laboratory pipettes regularly to ensure proper reagent dispensing Only the PerkinEImer supplied clean room cloth can be used on the chip to clean the detection window Water used for chip preparation procedures must be 18 megohm 0 22 um filtered water Milli Q or equivalent Using the Reverse Pipetting Technique described next will help avoid introducing bubbles into the chip when pipetting the gel PerkinElmer Inc warrants that the LabChip Kit meets specification at the time of shipment and is free from defects in material and workmanship LabChip Kits are warranted for 60 days from the date of shipment All claims under this warranty must be made within thirty days of the discovery of the defect ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 39 Reverse Pipetting Technique A STEP 1 STEP 2 STEP 3 STEP 4 Figure 27 Reverse pipetting 1 Depress the pipette plunger to the second stop 2 Aspirate the selected volume plus an excess amount from the tube 3 Dispense the selected volume into the corner of the well by depressing plunger to t
32. that results can be more easily compared across different runs different instruments different reagent lots etc _ Ladder1 Ladder ce ceni N S E u 20 22 Aligned Time sec Figure 13 The Ladder is a mixture of a 1 6 linked glucose oligosaccharides with a various number of monomeric glucose units Lower Marker LM and ladder peaks used for sizing L annotated with RGU units The Lower Marker is maltohexaose which runs with a size of 6 6 RGU PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Identifying Standards Analyzing Data 22 As an example if a peak has a migration time half way between the 9 mer and 10 mer a 1 6 linked glucose oligosaccharides in the ladder that peak will be assigned a size of 9 5 Relative Glucose Units RGU These units are specific to the particular gel separation matrix and the fluorophore that are provided in the ProfilerPro Glycan Profiling kits They may not correspond to Glucose Units assigned by other analysis methods MM16RL10 test_2010 01 21_04 28 22 Point to Point Fit Size RGU Figure 14 Example of two standard curves that relate migration time to size in Relative Glucose Units RGU The LabChip GX Touch software allows the user to assign names to peaks based on their size If you observe that the GOf peak in your sample has a size of 9 3 RGU you can name the peak OUT using the Analysis Settings dialog Expected Glycan tab
33. u suspect there may be debris in your samples spin the sample plate down in a centrifuge e g 3000 rcf for 5 minutes Unclog the sipper by repriming the chip See LabChip Kit Essential Practices on page 38 for instructions on how to reprime the chip 6 Make sure the correct plate type is selected If using the Plate Type 96 Deep Well PCR with Adapter verify the sample volume is at least 100 UL 7 To ensure complete drying repeat the sample preparation procedures allowing the drying reaction to extend beyond 2 hours until the samples are completely dry Symptom No ladder peaks but sample peaks and marker peaks are present Possible causes 1 Low orno ladder volume in the Ladder Tube What to do 1 Add more ladder to the Ladder Tube and restart the run Recommended standard ladder volume is 120 uL minimum volume is 100 uL Symptom No marker peaks but sample peaks are present Possible causes 1 No marker added to chip well 4 PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 31 2 If there is marker solution in chip well 4 the problem may be due to a marker channel clog What to do 1 This may be due to not filling marker well or chip remaining idle on instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure e Touch the Unload Chip button on the Home screen to open the chip door e Return the chip t
34. uL water Milli Q or equivalent Then suction the sipper with a vacuum line as shown in Figure 29 until droplets of fluid flow out from the sipper When suctioning the sipper be careful not to bend or break the sipper To facilitate this cut the end of the pipette tip attached to the vacuum line to widen the mouth PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide LabChip Kit Essential Practices 42 E A a J ATT Figure 29 Removing an air bubble or clog by suctioning the sipper with a vacuum line Other Considerations PN CLS140161 Rev C New chips should be stored refrigerated prior to first use After running the first set of samples chips must be stored at room temperature and used within 30 days Do not allow the liquid in the chip container to freeze as this may lead to poor chip performance Do not submerge the chip in any solution The entire chip surface must be thoroughly dry before use The sipper must be kept immersed in fluid at all times and should not be exposed to an open environment for long periods of time Use care in chip handling to prevent sipper damage Damage to the sipper can result in inconsistent sampling Avoid exposing the chips to dust by keeping them in a closed environment such as in the chip container or in the instrument before and after chip preparation Chips can be prepared and left in the instrument for extended periods of time so that samples c
35. uced during chip priming What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 38 for instructions on how to reprime the chip Symptom Missing sample ladder and marker peaks Possible causes 1 Clog in sipper or marker channel of chip What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 38 for instructions on how to reprime the chip Symptom Ladder detected but no sample peaks Possible causes 1 The sipper is not reaching the sample due to low sample volume in the well of the plate 2 Ifthe missing sample peaks occurred only in a few wells of the plate check those wells for air bubbles 3 The sipper is not reaching the sample due to an incorrect capillary height setting or incorrect plate definition PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 30 4 Ifthe plate has been uncovered for some time sample evaporation might have occurred 5 Debris from the sample or sample prep is clogging the sipper 6 The samples did not get labeled completely due to incomplete drying What to do 1 Add more sample to the well 2 Manually insert a larger volume pipette tip 100 uL into the sample well and dislodge the bubble Rerun these sample wells 3 Check the plate definitions 4 Check the sample wells especially around the edge of the plate where evaporation is fastest and make a fresh plate if volumes are low 5 If yo
36. ully remove plate seals Transfer all 11 uL denatured sample to PNGase F Plate Mix by pipetting up and down or with a plate shaker ProfilerPro Glycan Profiling Assay User Guide Labeling Hints Procedure PN CLS140161 Rev C bk O N o Sample Preparation Procedures 11 Seal plate carefully with an adhesive plate seal Note Ensure the plate is completely sealed to prevent evaporation Spin PNGase F Plate at 1200 g for 1 minute Incubate for 1 hour at 37 C using a PCR machine with lid temperature at 37 C heat block or incubator Can more than 8 uL of digested sample be labeled Sample buffer conditions resulting in optimal glycan peak intensity was observed when labeling 8 uL of the digested sample Labeling more or less will result in lower signal intensity Break point If needed after labeling dried samples may be sealed and stored frozen at 20 C and reconstituted the next day Overnight labeling is not recommended Mixing It is best to use a plate shaker with max speed Incubators Carefully inspect samples to ensure they are completely dry It s likely that using an incubator or oven will increase the drying time to more than 2 hours Thaw Dye Plate Spin both Dye Plate and PNGase F Plate at 1200 g for 1 minute Carefully remove plate seals Transfer 8 uL of digested sample to the Dye Plate Mix by pipetting up and down or with a plate shaker Spin Dye Plate at 1200 g for 1 minute Incubate
37. unts of digestion buffer attached to the plate seal may significantly decrease the available amount of PNGase F Verify that the digestion plate is incubated at 37 C for 1 hour or longer Symptom Peaks ahead of the marker so marker is miscalled If the sample contains peaks that run faster than the marker the software may identify them as the marker incorrectly This can be corrected by manually selecting the proper marker peak Hover over the peak with the mouse right click and select Force Lower Marker To identify the correct peak as the marker click Turn Off Analysis in the Analysis menu and overlay the sample with a ladder Look for the peak in the sample that aligns with the marker in the ladder should be the tallest peak Turn the analysis back on and select the marker peak GtycanDemo D4 MAb12 22 Aligned Time sec Figure 20 Miscalled marker GlycanDemo IM kasser Lasoen Figure 21 Sample and ladder overlayed with analysis turned off PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Troubleshooting 28 How to include peaks ahead of the marker in the analysis By default the software will only include peaks that migrate after the marker into the analysis If there are peaks before the marker that you would like to include in the analysis you can do so by manually including them First be sure that the marker is assigned to the proper peak see
38. with trap Figure 31 Replacing the disposable pipette tip PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Customer Technical Support 45 Customer Technical Support PerkinElmer Inc 68 Elm Street Hopkinton MA 01748 1668 PerkinElmer Technical Support Phone USA Toll Free 800 762 4000 Phone Worldwide 1 203 925 4602 Fax 1 203 925 4602 Email global techsupport perkinelmer com Internet www perkinelmer com For additional assay and instrument troubleshooting refer to the LabChip GX Touch Software Help file PN CLS140161 Rev C ProfilerPro Glycan Profiling Assay User Guide Licenses and Rights of Use 46 Licenses and Rights of Use Label Licenses This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the data for internal research and training purposes solely in combination with PerkinElmer Inc instrumentation and not to generate revenue which may include but is not limited to use of the product 1 in manufacturing or in quality assurance or quality control of a commercial product 2 to provide a service information or data for a fee or other consideration 3 for therapeutic or prophylactic purposes 4 for diagnostic use and 5 for resale whether or not such items are resold for use in research For information on purchasing a license to this product f
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