Protein Characterization: Testing to Triage Lead Candidates

Disulfide mapping analysis to determine unknown disulfide linkage

Deliverables: • Confirm known disulfide linkage information • Report free cysteine amount and any mis-linked disulfide bond with >5% abundance • Determine unknown disulfide linkage for every cystein

Purified Protein Samples

Ethanol Precipitation

Alkylation

Digest by Specific Proteases (pH<6.7)

Non-reduced Fraction

LC-MS

Deglycosylation if Necessary

DTT

Profile Comparison to Find Different Peaks

Reduced Fraction

LC-MS

N-glycan analysis by LC-Fluorescence-MS/MS for complex glycosylation

Deliverables: • Report any glycan relative abundance >1% based on fluorescence • Report glycan ID based on mass spec results

N-glycanase Release Glycans HLB Plates

Glycan Pool

Glycoprotein

high mannose hybrid complex

M6P

Convert

Fluorescent 2-AB/Procainamide Labeling

Glycans with Free Reducing End

Labeled Glycan Pool + Dye

Clean

Identification Software Search Manual Interpretation

Component Identification

Glycan Distribution

Liquid Chromatography MS/MS

Quatification by Byomap

Detailed peptide mapping for sequence coverage, PTM, sequence variant analysis

Deliverables: • Confirm protein sequence: sequence coverage map • Software-based identification for PTM, SV and fragment et al analysis • Check sequence homogeneity: manual check for all components with user defined intensity threshold (>1%) for detailed PTM, SV and fragment et al analysis

Identified Major Peaks

Confirmed Peak Identity Sequence Coverage

LC-MS/MS

Database Search

Peak feature detection based on MS1 only

Un-identified Small Peaks

LC-MS/MS (tMS2) CID/HCD/ETD

Manual Assignments

MS2/MS3

m/z

PTM 9%

Variants 2%

Adducts, non-specific digest 89%

1000-2000 components

Learn more: Protein Sciences: Early-stage R&D Protein Analytics Platform

About WuXi Biologics

WuXi Biologics is a leading contract research, development and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.

The world’s leading global single-source platform from concept to commercialization.

wuxibiologics.com | info@wuxibiologics.com

3-10-2023

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