LC-MS driven purification for high-purity bispecific antibodies ranging from 10 mg to grams in 4-6 weeks
Optimizing Bispecific Antibody Production for Higher Titer & Purity Across Diverse Formats Gaozhan Zhang, Li Zhang, Dawei Zhang, Mengjie Lu, Zhumei Feng, Jiansheng Wu WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)
Abstract
Fig 5. Chain Ratio Study in 24-Well Deep Well Plate (DWP) Gene Synthesis with Codon Optimization 3 Formats 10+ Molecules 3 Chain Ratios 24-DWP Expression and Purification
Bispecific antibodies (BsAbs) hold immense therapeutic potential but face production challenges such as low titers and impurities, including homodimers and mispairings. At WuXi Biologics, our CRO Services team leverages advanced molecule design, high-titer CHO expression, chain ratio optimization, and Intact Mass-driven purification to generate grams of BsAbs with >98% heterodimer purity across diverse BsAb formats. These innovations enhance BsAb production efficiency and facilitate more efficient therapeutic development.
Best Constructs and Optimal Chain Ratio
Introduction
The production of bsAbs faces challenges throughout the entire process, starting from expression and extending to purification. Our integrated bsAb production strategies address these challenges at every stage, including transfection, cell culture, purification, and analytical bioassays. These strategies guarantee high yields and improved purity across a wide range of bsAb formats.
Fig 1. Different Formats of BsAb
Optimal chain ratio was determined based on SDS-PAGE, CEX-HPLC, or Caliper results
24-DWP expression & purification Average yield of 24-DWP (3 mL): ~0.5-1 mg/well
3 BsAb formats
Fig 6. Intact Mass Identified the Presence of Homodimer While SEC-HPLC and SDS-PAGE Suggested High Purity Achieving >98% Heterodimer Purity Using Intact Mass-Driven Purification
Common LC
CrossMab
WuXiBody
Different LC
Complex Formats
Charge Pairing
Fc Fusion
ScFv Fab
Fig 2. Premium BsAb Production Strategies
Separation of major impurities
Balanced chain expression
Fig 7. CEX Purification Achieved >98% BsAb Purity Confirmed by Intact Mass and SEC-HPLC
Cell Culture
Purification
LC-missing Byproduct
Half Antibody
Analytics Bioassay
Formulation
H-H Homodimer
K-K Homodimer
Impurity detection method
• Solubility stability • Lyo
We want to express our gratitude to the scientists in the CRO Services Department at WuXi Biologics for their invaluable support in the development of our Premium BsAb platform. Additionally, we extend our appreciation to WuXi Biologics' legal, public relations, and marketing departments for their assistance in patent filings and the preparation and production of written materials. Acknowledgments WuXi Biologics is a global leading Contract Research, Development and Manufacturing Organization (CRDMO) offering end-to-end solutions to enable partners to discover, develop and manufacture biologics from concept to commercialization. About WuXi Biologics All experiments were conducted within the CRO Department at WuXi Biologics. The DNA sequence codon was optimized using WuXi Biologics’ laboratory data management system (LDMS), and the plasmid was then constructed into WuXi Biologics' proprietary vector. A WuXian Transient procedure was then performed according to SOPs and the culture medium was collected after 7 days. After clarifying the culture medium, all supernatant was loaded onto tailored purification platforms, including mixed-mode, ion exchange (IEX), hydrophobic interaction chromatography (HIC), carboxymethyl (CHT), and protein A columns. The final products were analyzed using SDS- PAGE, size-exclusion chromatography (SEC-HPLC), Intact Mass analysis, and endotoxin detection. Method Conclusions WuXi Biologics has optimized bispecific antibody (bsAb) strategies to enable scalable production with higher titer and purity across various bsAb formats. • Optimizing chain ratio during transfection improves both titer and quality. • Employing Intact Mass during purification is crucial, as it identifies impurities and guides purification processes to consistently achieve >98% heterodimer purity.
Fig 3. A Roadmap for Premium BsAb Purification BsAb Asymmetric
Symmetric
Fc Extension
IgG Extension
Protein A-Binding Abolished
Charge Pair
Knob into Hole
Aggregates
Homodimer ½ Ab
½ Ab
Homodimer ½ Ab
Homodimer ½ Ab
ΔpI > 1: Tool B > A ΔpI < 1: Tool A > C
Tool D > A
Tool B > A
Tool E > F / A
< 1g Material: Tool E > 1g Material: Tool B > C
Tool Box A: Mixed Mode B: IEX C: HIC D: Protein A pH Gradient Elution
E: Protein A Dual Gradient Elution F: CHT
Results
Challenges Superior Product Quality Fig 4. Chain Ratio Optimization Results in Higher BsAb Yield and Improved Purity Optimizing Chain Ratio for Improved Titer and BsAb Quality Improving Strategy
• Low transient expression titer • Low SEC-HPLCpurity after one-step purification • Low intact molecular species
Optimize chain ratio
Higher Protein Yield Better Purity
Titer and Product Quality Improvement
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Increase transfection ratio of HC1: HC1 : HC2: LC = 2 :1: 1 HC1 : HC2: LC = 4 :1: 1 HC1 : HC2: LC = 6 :1: 1
14
12
12
10
5
Amount (mg)
Purity by SEC-HPLC (%)
Poster modified on 10/1/2024
HC1:HC2:LC=1:1:2 HC1:HC2:LC=2:1:1 HC1:HC2:LC=4:1:1 HC1:HC2:LC=6:1:1
To discuss this poster: PS_BD@wuxibiologics.com
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