Optimizing BsAb Production for Improved Purity & Yield

LC-MS driven purification for high-purity bsAbs ranging from 10 mg to grams in 4-6 weeks

Optimizing Bispecific Antibody Production: Strategies for Improved Purity and Yield 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 Deep Well Plate (DWP) Gene Synthesis with Codon Optimization 3 Formats 10+ Molecules 3 Chain Ratios

Bispecific antibodies (BsAbs) are a rapidly evolving and promising category of biotherapeutics. They come in various structural forms, including single-chain variable fragment (ScFv)-based antibodies (no Fc fragment), and full-length IgG-like asymmetric antibodies. Unlike monoclonal antibodies, the production of bsAbs presents significant challenges in terms of quantity, quality, and stability, which could potentially limit their wider adoption in clinical applications. At WuXi Biologics, our Protein Sciences (PS) Department has developed integrated strategies to overcome these challenges in bsAb production. Our industry-leading approaches result in improved purity and yield, ranging from milligrams to grams. This, in turn, facilitates more effective early-stage drug discovery efforts.

24DWP Expression and Purification

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 cell culture, purification, formulation, and analytical bioassays. These strategies guarantee high yields and improved purity across a wide range of bsAb formats.

Fig 1. Different Formats of BsAb

24DWP expression & purification Average yield of 24DWP (3 ml): ~0.5-1 mg/well

Optimal chain ratio was determined based on SDS-PAGE, CEX-HPLC, or Caliper results

3 BsAb formats

Fig 6. Intact Mass Confirmed the Existence of Mispaired Molecules While SEC-HPLC and SDS-PAGE Suggest High Purity Using Intact Mass to Identify Byproducts in Each Step of Purification SEC-HPLC SDS-PAGE Intact MS

Common LC

Cross mAb

WuXiBody

Different LC

M R NR

KDa

250 100 150

37 50 20 25 15

Complex Formats

Charge Pairing

Fc Fusion

ScFv Fab

Fig 2. Premium BsAb Production Strategies

M: Marker R: Reduced NR: Non reduced

Separation of major impurities

Balanced chain expression

Fig 7. Intact Mass Confirmed the Existence of Homodimers While SEC-HPLC and SDS-PAGE Suggest High Purity

SEC-HPLC

SDS-PAGE

Intact MS

Cell Culture

Purification

LC-missing Byproduct

Half Antibody

Analytics Bioassay

Formulation

H-H Homodimer

K-K Homodimer

Impurity detection method

• Solubility stability • Lyo

Method

Fig 3. A Roadmap for Premium BsAb Purification BsAb Asymmetric

All experiments were conducted within the Protein Sciences (PS) 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 suitable purification platforms, including mixed-mode, ion exchange (IEX), hydrophobic interaction chromatography (HIC), carboxymethyl (CHT), and protein A columns. The final product underwent analysis using SDS-PAGE, size-exclusion chromatography (SEC-HPLC), intact Mass analysis, and endotoxin detection.

Symmetric

Fc Extension

IgG Extension

Protein A-Binding Abolished

Charge Pair

Knob into Hole

Conclusions

Aggregates

Homodimer ½ Ab

½ Ab

Homodimer ½ Ab

We want to express our gratitude to the scientists in the Protein Sciences (PS) 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 • WuXi Biologics has developed bsAb production strategies that ensure higher yield and improved purity. • Optimizing chain ratio during transfection results in improved titer and quality. • Utilizing intact mass is essential for bsAb purification.

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 Protein Yield and Improved Purity Optimization of Chain Ratio of Transfection Improves 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

0 10 20 30 40 50 60 70 80

For more information see us at wuxibiologics.com

Increase transfection ratio of HC1: HC1 : HC2: LC = 2 :1: 1 HC1 : HC2: LC = 4 :1: 1 HC1 : HC2: LC = 6 :1: 1

Amount (mg)

Purity by SEC-HPLC (%)

Poster modified on 10/25/2023

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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