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 Characterization Services
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. The most common contaminant in bispecific antibody production are homodimers. Other mispairings can happen that can be revealed by mass intact mass analysis. Detailed characterization of bispecific antibodies by intact mass analysis At WuXi Biologics, our Protein Sciences (PS) Department provides comprehensive protein characterization 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 enables 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
Kinetics/A inity
10 15 20 25 30 35 40
HC1 homodimer 150 kDa
HC2 homodimer 150 kDa
0 5
+
+
LC-HC mispair 150 kDa
-5
bsAb
0
-50
5 100150200250300350400450
Epitope Binning/Competition
HC dimer w/o LC 150 kDa
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
affinity and kinetics and product concentration
0
Mispairing (HC1+HC2+LC1+LC1)
0
50 10
150 200 250
HC1+HC2+LC1+Cysteine HC1+HC2
Concentration Analysis
Versatile: capable of detecting most types of molecules
110896.3
4000 6000 8000 10000
Measurement 1 Measurement 2
40 60 80 100 120
100 µL/min
2 4 6 8 10
LC dimer 51267.0 50000
136649.4
5 µL/min
Wide range: capable of analyzing from μM to fM binde
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 cysteine
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
Labeled Glycan Pool + Dye
Glycans with Free Reducing End
Clean
Identification Software Search Manual Interpretation
Liquid Chromatography MS/MS
Component Identification
Glycan Distribution
Quantification 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
Database Search
LC-MS/MS
Peak feature detection based on MS1 only
MS2/MS3
LC-MS/MS (tMS2) CID/HCD/ETD
Un-identified Small Peaks
Manual Assignments
m/z
PTM 9%
Variants 2%
Adducts, non-specific digest 89%
1000-2000 components
Learn more: Protein Sciences: Early-Stage R&D Protein Characterization Services
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
5-9-2024
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