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)
Overcoming the Challenges in Asymmetric Bispecific Antibody Production by Using Quick ‘ n ’ Clean Platform
Gaozhan Zhang, Lupeng Cui, Dawei Zhang, Mengjie Lu, Jiansheng Wu WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China
Bispecific antibody (BsAb) is a large family of molecules designed to recognize two different epitopes or antigens and represents a highly promising class of biotherapeutic modality. Structural diversity has been fast-growing in the bispecific antibody field, ranging from single-chain variable fragment (ScFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike vanilla antibody, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. To support our clients and to overcome the challenges associated with BsAb production, WuXi Biologics has established the HTP BsAb production platform Quick ‘n’ Clean. In this platform, 1 mg of customized BsAb with 99% heterodimer can be produced from 20 mL culture medium through 3-step automated purification in 3-4 weeks. The robustness of WuXi Biologics’ Quick ‘n’ Clean platform can provide high throughput screening of large numbers of customized BsAb at early-stage drug discovery. Abstract
Results
Fig 4. The chromatography profile of 3-step automatic purification and summary of QC data Case study: 102 BsAb production at 20 mL scale In this case study, 102 customized BsAb were purified in the Quick ‘n’ Clean platform through 3-step automatic purification.
Kappa Select
Protein A
SEC
Kappa Elution
Protein A Elution
SEC Elution
Monomer (%)
Yield (mg)
Endotoxin (Eu/mg)
12
110%
0.5
10
100%
99%
0.4
8
90%
Introduction
0.3
6
80%
0.2
4
70%
Production of bispecific antibodies (BsAbs) often requires the separation of impurities formed as a result of HC mispairing, LC mispairing, fragmentation and aggregation, while the purification process is complex and time-consuming. In the Quick ‘n’ Clean platform, customized formats of bispecific antibodies which comprise an ScFv or VHH and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. Using innovative purification strategies, high purity customized BsAbs can be purified in a high throughput, automatic mode.
0.1 Eu/mg
2.3 mg
0.1
2
60%
0
50%
0
0
40
80
120
0
40
80
120
0
40
80
120
Fig 5. Production of four asymmetric BsAbs
Molecule A
Molecule BMolecule C Molecule D
150 100 kDa 250
ScFv
75 50 37 25 15 10 20
Fig 1. Impurities formed in customized asymmetric BsAb production
Desired asymmetric BsAb
Impurities
His6 Capture CH1-Ni-SEC
Fig 6. Production of 12 WuXiBody BsAbs
01
02
03
04
05
06
150 100 kDa 250
½ Abs
homodimer
homodimer ½ Abs
75 50 37 25 20 15 10
VHH/ScFv
β
07
08
09
10
11
12
α
Fig 2. The features of Quick ‘n’ Clean platform
07
08
09
10
11
12
150 100 250
Automated 3-Step Purification
Customized HTP Bispecific Production
WuXiBody KappaSelect-ProteinA-SEC
75 50 37 25 20 15 10
• 1 mg • 20 mL scale
• 99% heterodimer • 3-4 weeks
SDS-PAGE showed that the purity of all samples were >95%.
All experiments were conducted at the department of Protein Sciences, WuXi Biologics. Briefly, the codon of DNA sequence was optimized by WuXi Biologics lab data management system (LDMS) and the plasmid was constructed into WuXi Biologics proprietary vector. WuXian Transient was performed based on SOP and the culture medium was collected after 7 days. After clarification of the culture medium, all supernatant was loaded onto an appropriate tandem Kappa Select, protein A or Nickel and SEC column. Final product was analyzed by CBB-Staining, SEC-HPLC and endotoxin detection. Method • WuXi Biologics has developed a high throughput bsAb production platform Quick ‘n’ Clean, which can support client’s drug discovery projects in early stage. • Quick ‘n’ Clean platform can provide high throughput screening of large numbers of customized BsAb. • The platform is robust, time-saving and reproducible. Conclusions We thank all scientists from WuXi Biologics’ Protein Sciences Department for their great support during the development of the Quick ‘n’ Clean platform. We also thank the legal department, public relationship department and marketing department for their great help in patent filing and poster preparation and revision. 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 see us at wuxibiologics.com About WuXi Biologics
β
α
One Arm
VHH
ScFv
WuXiBody
For various asymmetric BsAbs, the Quick ‘n’ Clean platform can provide 1 mg BsAb with 99% heterodimer which is produced from 20 mL culture medium through 3-step automatic purification in 3~4 weeks.
Fig 3. Purification strategies in Quick ‘n’ Clean platform
VHH/ScFv
Capture CH1/Kappa Select
Ni
SEC
His6
β
α
Kappa Select
Protein A
SEC
WuXiBody
WuXiBody and other customized BsAbs can be purified using different strategies.
Poster modified on 1/29/2024
To discuss this poster: PS_BD@wuxibiologics.com
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