ProfilerPro Glycan Profiling User Guide - PerkinElmer

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ProfilerPro Glycan Profiling Kit, Ver 2, User GuideIntroductionApplicationsThe ProfilerPro Glycan Profiling system provides a high throughput method for analysis of the glycosylationpatterns 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 ofthe released glycans in presence of released proteins; 3) separation by microchip based capillary electrophoresis(CE); and 4) detection and analysis using the LabChip GXII instrument. Fast separation time–less than 45seconds per sample—makes this method valuable for use in high throughput screening of antibody glycosylationpatterns 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 containprimarily neutral N-linked glycans consisting ofcomplex, hybrids, and high-mannose typeoligosaccharides.Features• Fast separation time – less than 45 secondsper sample, about 1.5 hours for 96 samples.• Software determines the relative amounts ofeach glycan present.• All included reagent set, providespredictable, reproducible results.• Reagents provided in 96-well plate formatfor convenience and ease of automation.• Automatic sampling from a 96-well plate.Performance SpecificationsGlycosylation profile of a therapeutic MAb. Relative abundanceof the major glycan types can be reliably quantified.Amount of sample required:8μl with concentration range of 1.25mg/ml to 7.5mg/ml(10μg to 60μg of MAb total).Reproducibility of %area: CV < 10% for a peak >= 2.5% of total glycan.Limit of detection:1ng of G0f standard. (Smallest amount of labeled G0fstandard that can be detected.)Deglycosylation: >95% of all N-Linked glycans will be released from MAb.Usable size range:Appropriate for neutral glycans found on MAbs. Somecharged glycans may run outside of our usable range.Sizing reproducibility: CV < 2.5%Carryover:< 0.5% of previous sample can be detected in followingsample.Sample prep, chip prep, and < 8 hours for a 96-well plate.analysis time:Number of samples per chip prep:Up to 192 samples before chip needs to be reloaded withfresh gel.Number of samples per chip Up to 384 samples before chip needs to be replaced.lifetime:Caliper Life Sciences Page 2 of 21 PN: 45070968 Elm StreetHopkinton, MA 01748-16681-877-LABCHIP (522-2447)
Kit ContentsThe ProfilerPro Glycan Profiling system consists of:ProfilerPro Glycan Profiling Kit, Ver 2, User GuideGlycan Release and Labeling Kit (part number 760523)• Denaturing Plate (96-samples) for denaturing glycoprotein prior to digestion by PNGase F.• PNGase F Plate (96-samples) for releasing the oligosaccharides.• Dye Plate (96-samples) for labeling the oligosaccharides.• Store at -20CHT High Resolution LabChip (part number 760524)• HT High Resolution Chip. Good for 400 samples.• Chip cleaning consumables kit.• Store at 4C.HT Glycan LabChip Reagent Kit (part number 760525)• HT Glycan Gel Matrix Solution, 2 tubes.• HT Glycan Marker (dry), 4 tubes.• HT Glycan Marker Diluent, 1 tube.• HT Glycan Ladder (dry), 4 tubes.• HT Glycan Ladder Diluent, 1 tube.• Enough for 4 chip preps.• Store at 4C.Additional Items Required• LabChip GXII instrument with LabChip GX software version 2.2 or greater. May be downloaded fromhttp://www.caliperls.com/support/software-downloads.htm• GX Plate Adapter for Deep-Well PCR Plates (part number 126209).• Heating device: any PCR thermocycler is best. A heat block that accepts a 96-well PCR plate will alsowork. An incubator may work, but is less desirable.• Adhesive Plate seals. (PCR type with a minimum thermal range of -20C to 80C. For example, BeckmanCoulter 538619, Axygen PCR-AS-200).• Centrifuge with rotor that accepts a 96-well PCR plate.• Optional: Plate shaker (for example: VarioMag Magnetic Shaker, Cat. 51110, 2,000 rpm, Amplitude =2mm).Safety Warning & Precautions! WARNING ! For Research Use Only. Not recommended or intended for diagnosis of disease inhumans 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 beentrained in laboratory techniques and that it is used in accordance with the principles of good laboratorypractice. As all chemicals should be considered as potentially hazardous, it is advisable when handlingchemical reagents to wear suitable protective clothing, such as laboratory overalls, safety glasses, andgloves. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, washimmediately with water.! WARNING ! Denaturing Buffer, Ladder, and Gel Matrix contain SDS. Avoid inhalation and contactwith skin and eyes.Caliper Life Sciences Page 3 of 21 PN: 45070968 Elm StreetHopkinton, MA 01748-16681-877-LABCHIP (522-2447)
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ProfilerPro Glycan Profiling User Guide - PerkinElmer

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