TFC Quick ‘n’ Clean: HTP Production of Four-Chain BsAbs

High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.

TFC Quick ‘n’ Clean: Next-Gen Platform for High-Throughput Production of True Four-Chain BsAbs

Gaozhan Zhang, Xiayan Li, Chun Fan, Dawei Zhang, Mengjie Lu, Zhumei Feng, Jiansheng Wu Protein Sciences Department, WuXi Biologics CRO Services, No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_Marketing@wuxibiologics.com )

Abstract

Fig.9 Intact Mass Analysis of BsAbs

Bispecific antibodies (BsAbs) hold significant therapeutic potential; however, their production at the research stage presents notable challenges in throughput, yield, and purity. These challenges are especially pronounced for four-chain BsAbs containing two di ff erent light chains (LCs), which increase the risk of chain mispairing. To overcome these challenges, the True Four Chain (TFC) Quick ‘n’ Clean platform was developed for high-throughput BsAb production at the early drug discovery stage. Utilizing the WuXiBody™ format, this platform ensures correct LC-HC pairing, producing BsAbs that are essentially free of LC mispairing. TFC Quick ‘n’ Clean is a next-generation research tool for high-throughput BsAb production, enabling the rapid identification of optimal pairings of four-chain BsAbs.

Target

Target:-K

Target:Glycation

Introduction

Fig.10 WuXiBody™ with Non-Tag Format in TFC Quick ‘n’ Clean

The production of bispecific antibodies (BsAbs) o ft en requires the removal of impurities such as heavy chain (HC) and light chain (LC) mispairing, fragmentation, and aggregation, all of which complicate the purification process. To address these challenges, we introduced the Quick ‘n’ Clean platform, which enables high-throughput BsAb production to rapidly and cost-e ff ectively identify optimal pairings at early-stage drug discovery. While highly e ff ective for BsAbs with single LC (Fig. 1), the Quick ‘n’ Clean platform is less suitable for BsAbs containing two distinct LCs, where the risk of HC-LC mispairing is significantly higher. To overcome this limitation, we further developed the TFC Quick ‘n’ Clean platform, which incorporates the WuXiBody™ backbone to eliminate LC mispairing. Using ultra-high titer CHO expression systems and advanced purification strategies, the platform e ff iciently produces four-chain bsAbs in a high-throughput, high-purity manner, serving as an invaluable research tool for optimal paring screening.

Non Tag

Fab

TCR C

Proprietary technology to force overexpression of the Fab arm Byproducts containing only Fab arm are e ff iciently removed in the flow-through during a ff inity purification.

Fig.11 The Purification Strategy of TFC Quick ‘n’ Clean

SEC

Anti TCR α

Non Tag

Fig.1 BsAb Formats Compatible with Quick ‘n’ Clean One Arm VHH ScFv

Case Study 2: High-Throughput Production of 26 Non-Tag BsAbs for ADC Conjugation

Common LC

HTP Conjugation

FACS Binding, Internalization

Cytotoxicity

Quick ‘n’ Clean Production

Fig.14 SDS-PAGE (NR/R) Analysis of BsAbs

Fig.12 5×5 Anti-EGFR and Anti-c-MET BsAb Design

E1 E2 E3 E4 E5

C1 C2 C3 C4 C5

* A, B can be any proteins that do not contain a light chain (LC), such as VHH, cytokine, etc.

Fig.2 Byproducts in Four-Chain BsAb Production

5× Anti-EGFR

5× Anti-c-MET

Byproducts

HC1

HC2

HCCF

Proprietary technology to force overexpression of the Fab arm Fig.13 Average Purity, Yield, and Endotoxin Level of BsAbs

LC1

LC2

110

0.3

FinalNR

9.6mg

BsAb

LC mispairing

½ Antibody

Homodimer

105

0.2

99.6%

100

Fig.3 Advantages of True Four Chain (TFC) Quick ‘n’ Clean

0.07EU/mg

0.1

Unlikely Byproducts

FinalR

Fab

The WuXiBody™ backbone is used to prevent LC mispairing.

TCR C

Fig.15 Screen BsAb ADC with In Vitro Assays

TCR C

FACS Binding to HCC827

Cytotoxicity on HCC827

Internalization on HCC827

10000

Dxd

20 40 60 80 100

E1/E5/E3

E1/E5

8000

E2 E3

Fig.4 WuXiBody™ Formats Used in TFC Quick ‘n’ Clean

6000

4000

E4 Isotype

2000

0.001 0.01 0.1 1 10 100 1000 -20 0

0.001 0.01 0.1 1 10 100 1000 0

0.01 0.1

10 100 1000

ADCs (nM)

1+1

2+1

2+1+1

BsAb Production: BsAbs with C4 tend to form aggregates. Conjugation: BsAbs with C1 and C3 have more aggregation a ft er conjugation. Potency: High: E1, E5; Medium: E2, E3; Low: E4 The a ff inity of EGFR antibody is a key driver for e ff icacy. Binding, internalization, and cytotoxicity show good correlation.

These formats work for both non tag and double tag versions.

Results

Method

Capabilities of TFC Quick ‘n’ Clean

· Monomer Purity: >95% · Endotoxin Level: <0.1 EU/mg

· Timeline: 3-4 weeks · Heterodimer Purity: >99%

· Yield: >1 mg · Scale: 20 mL and up

All experiments were conducted at WuXi Biologics CRO Services. Codon optimization, plasmid construction, and WuXian Transient™ expression followed SOPs. Culture supernatant was purified via tailored chromatography platforms and analyzed by SDS-PAGE, SEC-HPLC, Intact Mass, and endotoxin assays.

Fig.5 WuXiBody™ with Double Tag Format in TFC Quick ‘n’ Clean

Double Tag

Conclusions

Fab

TCR C

About WuXi Biologics CRO Services WuXi Biologics is a leading global CRDMO, with the R represented by our premium-quality CRO Services. With expertise in diverse drug modalities, we deliver comprehensive research services, including antibody discovery, protein production and engineering, and in vitro/in vivo testing, to seamlessly advance our clients’ molecules from concept to CMC development. WuXi Biologics has developed TFC Quick ‘n’ Clean, a high-throughput production platform for four-chain BsAbs, designed to identify optimal pairings at early-stage drug discovery. · This platform leverages the WuXiBody™ backbone to eliminate LC mispairing, enabling HTP production of BsAbs with two di ff erent LCs. · Four-chain BsAbs are produced with >99% heterodimer purity, as validated by Intact Mass analysis. Acknowledgment Thanks to WuXi Biologics' CRO, legal, PR, and marketing teams for their support in BsAb platform development and patent work.

TCR C

Flag His

Byproducts containing only one tag are e ff iciently removed in the flow-through during a ff inity purification.

Fig.6 The Purification Strategy of TFC Quick ‘n’ Clean

SEC

Anti-DYKDDDDK

Double Tag

Case Study 1: High-Throughput Production of 15 Double-Tag BsAbs at 20 mL Scale

Fig.7 Average Purity, Yield, and Endotoxin Level of BsAbs

Fig.8 SDS-PAGE (NR/R) Analysis of BsAbs

250 75 kDa 100 50 150

110

0 1 2 3 4 5 6

0 0.05 0.1 0.15 0.2 0.25 0.3

105

3.34 mg

98.23%

37 20 25 15 10

100

0.06EU/mg

Drug Development Expertise Empowering Research Services for Biologics For more information see us at https://www.wuxibiologics.com/cro-services

TFC_Poster_202511_V2

To discuss this poster: PS_Marketing@wuxibiologics.com

Page 1