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)
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 different 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. Abstract
Results
Case Study: High-Purity, High-Throughput Production of 15 BsAbs at 20 mL Scale
Capabilities of TFC Quick ‘n’ Clean
• Yield: >1 mg • Scale: 20 mL and up • Timeline: 3-4 weeks
• Heterodimer Purity: >99% • Monomer Purity: >95% • Endotoxin Level: <0.1 EU/mg
Figure 5: The Purification Strategy of TFC Quick ‘n’ Clean
Ni
Anti-DYKDDDDK
SEC
Introduction
Figure 6: Average Purity, Yield, and Endotoxin Level of 15 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 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 efficiently produces four-chain bsAbs in a high-throughput, high-purity manner, serving as an invaluable research tool for optimal paring screening.
Monomer Purity (%)
Yield (mg)
Endotoxin (EU/mg)
110.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
98.23%
100.00
90.00
3.34 mg
80.00
70.00
60.00
0.06 EU/mg
50.00
Figure 1: BsAb Formats Compatible with Quick ‘n’ Clean One Arm VHH ScFv
Common LC
A*
B*
Figure 7: SDS-PAGE (NR/R) Analysis of 15 BsAbs
kDa
**
*
250 75 100 150
* A, B can be any proteins that do not contain a light chain (LC), such as VHH, cytokine, etc.
37 50 20 25 15 10
Figure 2: Byproducts in Four-Chain BsAb Production
Byproducts
HC1 HC2
Non-Reduced
Reduced
Figure 8: Intact Mass Analysis of 15 BsAbs
LC1
LC2
BsAb
½ Antibody
Homodimer
LC mispairing
Figure 3: Advantages of True Four Chain (TFC) Quick ‘n’ Clean
Improbable Byproducts
The WuXiBody Backbone is used to prevent LC mispairing
VH
Fab
VL
TCR C β
TCR C α
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%.
LC mispairing is eliminated
Flag His
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. Method WuXi Biologics has developed TFC Quick ‘n’ Clean, a high-throughput production platform for four-chain bsAbs, designed to identify optimal parings at early-stage drug discovery. • This platform leverages the WuXiBody backbone to eliminate LC mispairing, enabling HTP production of bsAbs with two different LCs. • Four-chain bsAbs are produced with >99% heterodimer purity, as validated by Intact Mass analysis. Conclusions We thank all scientists from the Protein Sciences Department for their great support during the development of the TFC Quick ‘n’ Clean platform. We also thank the legal department, public relations department and the marketing department for their great collaboration. Acknowledgment 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. For more information, visit us at wuxibiologics.com About WuXi Biologics
Byproducts containing only one tag are efficiently removed in the flow-through during affinity purification.
Figure 4: WuXiBody Formats Used in TFC Quick ‘n’ Clean
1+1
2+1
2+1
Poster modified on 12/9/2024
To discuss this poster: PS_BD@wuxibiologics.com
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