Fast Glycan Labeling and Analysis Kit Application Guide

1 Fast Glycan Labeling and Analysis KitApplication GuideJuly 2017 RUO-IDV-05-4092-BThis document is provided to customers who have purchased SCIEX equipment to use in the operation of such SCIEX equipment. This document is copyright protected and any reproduction of this document or any part of this document isstrictly prohibited, except as SCIEX may authorize in that may be described in this document is furnished under a license agreement. It is against the law to copy, modify,or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the licenseagreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. Warrantiesare as stated of this document may make reference to other manufacturers and/or their products, which may contain parts whosenames are registered as trademarks and/or function as trademarks of their respective owners.

2 Any such use is intended onlyto designate those manufacturers' products as supplied by SCIEX for incorporation into its equipment and does not implyany right and/or license to use or permit others to use such manufacturers' and/or their product names as warranties are limited to those express warranties provided at the time of sale or license of its products and are SCIEX ssole and exclusive representations, warranties, and obligations. SCIEX makes no other warranty of any kind whatsoever,expressed or implied, including without limitation, warranties of merchantability or fitness for a particular purpose, whetherarising from a statute or otherwise in law or from a course of dealing or usage of trade, all of which are expressly disclaimed,and assumes no responsibility or contingent liability, including indirect or consequential damages, for any use by the purchaseror for any adverse circumstances arising research use only.

3 Not for use in diagnostic Sciex is doing business as trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective SCIEX is being used under license. 2017 AB SciexAB Sciex LLC500 Old Connecticut PathFramingham, Massachusetts 01701 USAA pplication GuideFast Glycan Labeling and Analysis KitRUO-IDV-05-4092-B2 / 40 Fast Glycan Labeling and Analysis and Materials Equipment and the Reagents and Stock the Bracketing Standard (BST)..8 Reconstitute the Internal Standard (IST)..8 Reconstitute the Fluorophore Label (L6)..8 Denature the the Denaturation the and Label the for Best the Digestion the the Labeling the Released the Excess Dye from the (Optional) Prepare and Label the Glucose Ladder the the Interface the Capillary the Buffer the Sample the Sequence and Start the the the Cartridge After and Identify the Value A Hazardous Substance B Ordering Glycan Labeling and Analysis KitApplication Guide3 / 40 RUO-IDV-05-4092-BContentsFast Glycan Labeling and Analysis Kit (Part Number B94499 PTO).

4 39 Components Available GuideFast Glycan Labeling and Analysis KitRUO-IDV-05-4092-B4 / 40 ContentsNote: Refer to the PA 800 Plus Pharmaceutical Analysis System Overview Guide for instructionsabout the safe use of the : For accurate results, we strongly recommend using the Fast Glycan Labeling and Analysis Kit with a PA800 Plus system that has been qualified with an Operational Qualification 3 .SafetyRefer to the Safety Data Sheets (SDS), available at , regarding the proper handling ofmaterials and reagents. Always follow standard laboratory safety SCIEX Fast Glycan Labeling and Analysis Kit provides reagents and supplies required to label, separate, andidentify reducing carbohydrates for 100 samples using the PA 800 Plus Pharmaceutical Analysis System.

5 This is awidely applicable sample preparation and Analysis method for N- Glycan profiling of glycoproteins. Carbohydrateprofiling of therapeutic glycoproteins provides valuable data toward understanding the activity and efficacy ofthese molecules. Glycans strongly influence circulation half-life, immunogenicity, and receptor-binding activity aswell as physicochemical and thermal stability of proteins of therapeutic interest. Identifying disease-relatedalterations to N- Glycan structures may lead to the discovery of new biomarkers for early protocol consists of the following release of the N-glycans from bead-mediated capture of the released the released glycans with a charged bead-mediated capture of the fluorophore-labeled glycans and dye removal, followed by release ofthe labeled of the labeled glycans by capillary electrophoresis using LIF of the capillary electrophoresis separation results to identify the type of glycans in the sample.

6 Usingthe GU Value softwareFast Glycan Labeling and Analysis KitApplication Guide5 / 40 RUO-IDV-05-4092-BFast Glycan Labeling and AnalysisKitWorkflowIntended UseThe Fast Glycan Labeling and Analysis Kit is for laboratory use and Materials RequiredNote: Unless a manufacturer name is specified, part numbers are for items available from SoftwareIn addition to the Fast Glycan Labeling and Analysis Kit and the equipment described in Customer-SuppliedEquipment and Supplies on page 6, the GU Value software must be installed on the PA 800 Plus PharmaceuticalAnalysis System a computer with internet access, go Search for"Fast Glycan Software" and download the installation file that contains the release notes and example data alongwith the GU Value software.

7 Follow the installation instructions in the release Equipment and Supplies Powder-free gloves (neoprene or nitrile recommended) Safety glasses Laboratory coat mL flat-cap PCR tubes (VWR USA PN 20170-012 or VWR EUR PN 732-0548) Application GuideFast Glycan Labeling and Analysis KitRUO-IDV-05-4092-B6 / 40 Fast Glycan Labeling and Analysis Kit Universal vials (PN A62251) and blue caps (PN A62250) Micro vials (PN 144709) Table-top mini centrifuge Microcentrifuge (or equivalent) Heat block capable of maintaining 60 C Vortex mixer Thermometer for measuring the heat block temperature Pipettors (2 L, 10 L, 20 L, 100 or 200 L, and 1 mL) and appropriate tips Miscellaneous lab glassware for buffers and reagentsCustomer-Supplied Reagents Peptide-N-glycosidase F enzyme (PNGase F) (Prozyme, 200 mU (PN GKE5006B)) HPLC-grade acetonitrile 1 M sodium cyanoborohydride in THF (Sigma-Aldrich PN 296813) Double-deionized (DDI) water (MS grade water filtered through a m filter and with resistance above18 M) To calibrate the LIF detector.

8 LIF Performance Test Mix (Beckman Coulter PN 726022) Capillary Performance Run Buffer A (PN 338426)Prepare the Reagents and Stock SolutionsFast Glycan Labeling and Analysis KitApplication Guide7 / 40 RUO-IDV-05-4092-BFast Glycan Labeling and Analysis KitReconstitute the Bracketing Standard (BST) 100 L of DDI water to the BST vial and then vortex to makes a 50 nM the solution into 20 L not in use, store at 35 C to 15 C for up to six the Internal Standard (IST) 500 L of DDI water to the IST vial and vortex to makes a 440 M limit repeated freezing and thawing, aliquot portions of the solution into mL microfuge vials. Choose avolume appropriate for the experimental 20 L aliquot is sufficient for a sequence of up to 96 not in use, store at 35 C to 15 C for up to six the Fluorophore Label (L6) Add 240 L of L5 to the L6 vial and then vortex to not in use, store at 35 C to 15 C for up to three the ProteinsApplication GuideFast Glycan Labeling and Analysis KitRUO-IDV-05-4092-B8 / 40 Fast Glycan Labeling and Analysis KitNote: The following instructions give quantities for 20 samples.

9 For reference, quantities for 1 sample are alsogiven. For experiments with a different number of samples, modify the quantities as the Denaturation the D2 50 L of DDI water to the D2 the vial to reconstituted D2 can be stored at 4 C for up to 24 a new mL flat-cap PCR tube and then label it "Denaturation Solution". the reagents specified in the following table to the Denaturation Solution tube and then vortex the tubeto mix. This procedure produces enough for 20 up to 20 SamplesFor 1 SampleReagent20 L1 LD120 L1 LReconstituted D220 L1 LD3100 L5 LD4 Discard the unused portion after the the heat block to 60 100 g of glycoprotein sample in 10 L of DDI : For highly-glycosylated samples, a smaller quantity of protein may be the magnetic the M1 vial at maximum speed until all beads are in a pipette, add 200 L of M1 to a new flat-cap mL PCR tube (referred to as the "sample tube" inthe rest of this document).

10 Fast Glycan Labeling and Analysis KitApplication Guide9 / 40 RUO-IDV-05-4092-BFast Glycan Labeling and Analysis the sample tube on a magnetic stand. After the beads are pulled to the side of the vial, use a pipetteto carefully remove the supernatant from the bottom of the sample tube without touching the beads withthe pipette ! Make sure that only the supernatant is removed. To prevent the removal of beads, consider removingsmaller volumes until the all of the supernatant is the tube from the magnetic 10 L of the glycoprotein sample solution (prepared in step 2) to the sample tube. Do not touch the beadswith the pipette the sample tube for 10 sec at maximum speed. Make sure that the magnetic beads are mixed with 5 L of the previously-prepared denaturation solution to the sample tube, vortex briefly, and then incubatefor 8 min at 60 C in the heat block to denature the sample.