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

A high-throughput production platform for true four-chain bsAbs, designed to identify optimal parings at early-stage drug discovery

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

Gaozhan Zhang, Xiayan Li, 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_BD@wuxibiologics.com)

Abstract 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 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. The production of bispecific antibodies (bsAbs) often 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-effectively identify optimal pairings at early-stage drug discovery. While highly effective for bsAbs with single LC ( Fig. 1 ), the Quick ‘n’ Clean platform is less suitable for bsAbs containing two 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 efficiently produces four-chain bsAbs in a high-throughput, high-purity manner, serving as an invaluable research tool for optimal pairing screening. Introduction

Fig 6. Average Purity, Yield, and Endotoxin Level of BsAbs

Monomer Purity (%)

Yield (mg)

Endotoxin (EU/mg)

Monomer Purity (%)

Yield (mg)

Endotoxin (EU/mg)

0.5

0.5

0 1 2 3 4 5 6 7

100

100

12

98.83%

98.23%

0.4

0.4

90

90

10

8.91 mg

80

80

8

0.3

0.3

3.34 mg

7 0

7 0

6

0.2

0.2

60

60

4

0.1

0.1

0.013 EU/mg

0.06 EU/mg

50

50

0

0

0

Double tag

Non tag

Fig 7. SDS-PAGE (NR/R) Analysis of BsAbs

kDa 250 150 100

kDa 250 150 100

*

**

*

**

75 50 37 25 20 15 10

75 50 37 25 20 15 10

R

R

NR

NR

Double Tag

Non Tag

Fig 8. Intact Mass Analysis of BsAbs

Top/Max Inhibition %

Fig 1. BsAb Formats Compatible with Quick ‘n’ Clean

One Arm

Common LC

VHH

ScFv

A*

B*

* 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

Byproducts

HC1

HC2

Double Tag

LC2

LC1

Homodimer

½ Antibody

LC mispairing

BsAb

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

Unlikely Byproducts

Target

Fab

VH

Fab

VH

The WuXiBody™ backbone is used to prevent LC mispairing.

VL

VL

TCRC

TCRC

DP Cleavage

Occurred in deglycosylation

DP Cleavage

TCRC

TCRC

FcRn null *

*

*

Flag His

Target

Non Tag

Double Tag

Non Tag

Double Tag

Byproducts containing only one tag or Fab arm are efficiently removed in the flow-through during affinity purification.

DP Cleavage

Occurred in deglycosylation

DP Cleavage

Non Tag Intact Mass analysis confirms the absence of impurities, such as homodimers and LC mispairing. The major peaks represent correctly formed heterodimers, confirming the purity of all bsAbs exceeds 99%.

*

**

Double Tag

Non Tag

Method All experiments were conducted at WuXi Biologics’ Protein Sciences Department. Briefly, the codons of DNA sequence were optimized, and the plasmids were constructed into WuXi Biologics proprietary vector. Transfection was performed using transient CHO cells and the culture medium was harvested after 7 days. Following the culture medium clarification, HCCF was loaded onto AKTA Pure with Nickel, Anti-DYKDDDDK, and SEC tandem columns. Final products were analyzed by SDS-PAGE, SEC-HPLC, Intact Mass, and LAL assays.

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

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

Conclusions

2+1

1+1

2+1

Acknowledgment We thank all scientists from the Protein Sciences Department for their great support during the develop- ment of the TFC Quick ‘n’ Clean platform. We also thank the legal department, public relations depart- ment and the marketing department for their great collaboration. 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 LCs. Four-chain bsAbs are produced with >99% heterodimer purity, as validated by Intact Mass analysis. • •

Results

Case Study: High-Purity, High-Throughput Production of 15 BsAbs at 20 mL Scale Capabilities of TFC Quick ‘n’ Clean Yield: >1 mg

Heterodimer Purity: >99% Monomer Purity: >95% Endotoxin Level: <0.1 EU/mg

Scale: 20 mL and up Timeline: 3-4 weeks

Fig 5. The Purification Strategy of TFC Quick ‘n’ Clean

Double Tag

Ni

Anti-DYKDDDDK

SEC

WuXi Biologics is a global leading Contract Research, Development discover, develop and manufacture biologics from concept About WuXi Biologics For more information, visit us at wuxibiologics.com partners to

and Manufacturing Organization (CRDMO) o ffe ring end-to-end solutions to enable

Non Tag

Protein A

SEC

to commercialization.

To discuss this poster: PS_BD@wuxibiologics.com

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