A HTP bispecific antibody (BsAb) production platform designed to unlock the optimal pairing of BsAb, with the ability to produce 1mg customized BsAb with 99% heterodimer from 20 mL culture in a fast and cost-effective way (~3-4 weeks)
Quick ‘n’ Clean: High-Throughput BsAb Production for Optimal Pairing Screening at Discovery Stage
Gaozhan Zhang, Dawei Zhang, Mengjie Lu, Jiansheng Wu Protein Sciences Department, WuXi Biologics CRO Services No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)
Abstract
Results
Discovering optimal bispecific antibody (bsAb) pairings requires producing numerous parental antibody combinations, followed by screening through cell-based assays that demand low milligram-level production with high purity. BsAb production at the early discovery stage has thus been challenging due to the need for throughput and purity, coupled with the molecular complexity of bsAbs. To overcome these challenges, WuXi Biologics has developed Quick ‘n’ Clean, a high-throughput (HTP) bsAb production platform. This platform enables rapid, cost-e ff ective production of large numbers of bsAbs at small-scale with high purity, significantly streamlining the screening process to identify optimal pairings in early-stage drug discovery.
Fig. 5: SDS-PAGE Analysis of Purified BsAbs with Overexpressed ScFv or Fab Arm Using Protein A vs. Quick ‘n’ Clean Case Study: Quick ‘n’ Clean Enables High-Purity Heterodimer Production, Independent of Arm Overexpression
Protein A
Excessive ScFv
Excessive Fab
150 100 kDa 250
75 50 37 25 15 10 20
Introduction
Excessive ScFv
Excessive Fab
150 100 kDa 250
Producing asymmetric bsAbs involves removing byproducts such as half antibodies, homodimers, light chain (LC) mispairing, fragmentation, and aggregation, making the purification process complex and time-consuming. The Quick ‘n’ Clean platform addresses these challenges by enabling HTP production of diverse bsAb formats (Fig. 2). This platform yields >1 mg of customized bsAbs with 99% heterodimer purity from 20 mL of culture medium within 3–4 weeks. Using a 3-step automated purification strategy, His and Flag tags at the Fc C-terminus facilitate e ff icient purification, providing high-purity bsAbs suitable for optimal pairing screening. While Quick ‘n’ Clean is an invaluable tool for early-stage drug discovery research, it is not intended for producing bsAbs for animal studies or CMC development.
75 50 37 25 15 10 20
Fig. 6: Average Purity, Yield & Endotoxin Level of 25 BsAbs at 20 mL Scale Case Study: HTP, High-Yield Production of 25 ScFv-Fab BsAbs Adjusting DNA ratios alone cannot e ff ectively control overexpression levels of each bsAb arm. In this case study, the default DNA ratio was set to HC:LC:ScFv-Fc= 1:1:3. The Quick ‘n’ Clean platform consistently produced high-purity heterodimers, regardless of which arm was overexpressed.
Monomer Purity (%)
Fig. 1: The Production Workflow of Quick ‘n’ Clean Customized BsAb Designs Fab VHH/ScFv
Endotoxin (EU/mg)
Yield (mg)
12 15
90% 92% 94% 96% 98% 100%
0.2
98.1%
ScFv
0 3 6 9
7.8mg
0.1
Ni
0.032 EU/mg
0.0
Flag His Ni-Anti DYKDDDDK-SEC
Anti-DYKDDDDK
Remove Aggregates
SEC
Flag His
Fig. 7: SDS-PAGE Analysis of 25 Common LC BsAbs at 20 mL Scale Case Study: HTP Production of 25 Common LC BsAbs with >99% Heterodimer Purity
Fig. 2: The features of Quick ‘n’ Clean
NH NH
Ni-Anti-DYKDDDDK-SEC Flag His
Intact Mass analysis confirmed >99% heterodimer purity.
Methods All experiments were conducted at WuXi Biologics’ Department of Protein Sciences. Briefly, codon optimization was applied to the DNA sequence, and the plasmid was constructed using WuXi Biologics’ proprietary vector. Transient transfection of CHO cells was performed, and the culture was harvested a ft er 7 days. A ft er clarifying the culture medium, HCCF was automatically loaded onto Ni, Anti-DYKDDDDK, and SEC columns. The final product was analyzed using SDS-PAGE, SEC-HPLC, and LAL assays, with Intact Mass analysis conducted as needed. Conclusions WuXi Biologics has developed Quick ‘n’ Clean, a high-throughput (HTP), high-quality bsAb production platform designed to support clients’ early-stage drug discovery projects. • The platform facilitates HTP production of large quantities of bsAbs. • It provides a robust, e ff icient, and reproducible solution for optimal pairing screening. • Purified bsAbs show superior purity and low endotoxin levels, making them ideal for cell-based assays. Acknowledgments We thank all scientists in WuXi Biologics' Protein Sciences Department for their invaluable support in developing the Quick 'n' Clean platform. We also thank the legal, public relations, and marketing departments for their assistance with patent filing, poster preparation, and revisions. About WuXi Biologics WuXi Biologics is a global leading Contract Research, Development and Manufacturing Organization (CRDMO) o ff ering end-to-end solutions to enable partners to discover, develop and manufacture biologics from concept to commercialization. ݠ
N-
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XFFLT FFLT
Customized HTP BsAb Production
Fig. 3: BsAb Formats Compatible with Quick ‘n’ Clean
VHH
One Arm
ScFv
Common LC
A*
B*
* A, B can be any proteins that do not contain a LC, such as VHHs, cytokines, etc.
Fig. 4: The Purification Strategy of Quick ‘n’ Clean
SEC
Anti-DYKDDDDK
Ni
For more information, visit us at wuxibiologics.com
Postermodified on 1/29/2024
To discuss this poster: PS_BD@wuxibiologics.com
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