Purity/impurity testing, structural characterization (e.g., MS- based protein ID and post translational modification analysis), and SPR/ELISA/cell-based activity assays.
Protein Sciences: Early-stage R&D Protein Analytics Platform
Mass Spectrometry
• Intact Mass/PeptideMap/ Disulfide • N-/O- Glycan Analysis • De Novo Antibody Sequence • Proteomics • HCP
Our experience extends beyond standard monoclonal antibodies to more complex biologics like bispecific and multi-specific antibodies. Most common contaminant in bispecific antibody production are homodimers. Other mispairing happens and can be revealed by mass analysis. Detailed characterization of bispecific antibody by intact mass analysis WuXi Biologics’ protein analytics platform provides comprehensive testing during early-stage R&D activities. Our extensive capabilities cover routine testing of the critical quality attributes and additional product characterization aspects of your lead molecule. Capabilities include purity/impurity testing, structural characterization (e.g., mass spectrometry- based protein ID and post translational modification analysis), and SPR/ELISA/ cell-based activity assays. This comprehensive analysis allow our clients to thoroughly assess their molecules much earlier during the discovery stage and triage/optimize the leads more effectively.
Micro Developability Package • Biophysical: DSF, DLS • Polyspecificity/PK Indicating Assay: Baculovirus/DNA/ Insulin Binding
Impurity Test • Residual DNA • Host Cell Protein • Residual Protein A • Endotoxin
Solutions
Binding Activity & MOA Related Bioassay • SPR/BLI Binding Assay • Enzymatic Activity Assay • ELISA/Cell-based Bioassay
Purity Test by LC&CE
• SEC-HPLC • SEC-MALS • CEX-HPLC • NR/R CE-SDS • icIEF
Surface Plasmon Resonance (SPR)
Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions. Benefits of SPR: an ideal tool for evaluating product characteristics such as binding affinity and kinetics and product concentration Versatile: capable of detecting most types of molecules Wide range: analyze from uM to fM binde
Kinetics/A inity
10 15 20 25 30 35 40
HC1 homodimer 150K Da
HC2 homodimer 150K Da
0 5
+
+
LC-HC mispair 150K Da
-5
bsAb
0
-50
5 100150200250300350400450
Epitope Binning/Competition
HC dimer w/o LC 100K Da
300
200
Target peak (HC1+HC2+LC1+LC2)
100
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
159202.1
0
Mispairing (HC1+HC2+LC1+LC1)
0
50 10
150 200 250
HC1+HC2+LC1+Cysteine HC1+HC2
Concentration Analysis
110896.3
4000 6000 8000 10000
Measurement 1 Measurement 2
40 60 80 100 120
100µI/min
LC dimer 51267.0 50000
2 4 6 8 10
136649.4
5µI/min
0.01
0.001
20
2000
100000 Counts vs. Deconvoluted Mass (amu)
150000
-10 0 0
10 20
30 40
0.001 0.01 0.1 1 10 100 1000
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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