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Bispecific Antibody Production
Helping You Overcome Challenges in the Production of Bispecific Antibodies
Bispecific antibodies (bsAbs) are gaining prominence in the field of antibody therapeutics. However, bsAb production comes with certain challenges, such as heterogeneity, production complexity, and stability issues, which can affect the quality of the final product.
At WuXi Biologics, the Protein Sciences (PS) Department excels in producing high-purity bispecific antibodies tailored to your specific needs. With a track record of delivering more than 3,000 bispecific antibodies suitable for both in vitro and in vivo studies, our platforms can handle everything from high-throughput, small-scale production at 1 mg to large-scale operations up to grams.
Quick ‘n’ Clean: A HTP Bispecific Antibody Production Platform
With the growing interest in using bispecific antibodies in therapeutic development and the use of AI in drug discovery, the need for high throughput bsAb production to unlock the optimal pairings of bsAbs has never been higher. Leveraging our drug development expertise and high-tier CHO transient expression platform, WuXi Biologics’ Protein Sciences developed the Quick ‘n’ Clean bispecific antibody production platform to offer a fast and cost-effective solution for the identification of the optimal bsAb pairings. Combining the power of speed and precision, our Quick ‘n’ Clean service can produce 1-7 mg of customized bsAb with high purity (>99% heterodimers) from 20 mL culture in 3-4 weeks.
Key Features of Quick ‘n’ Clean Service
- Optimized to produce many bispecific antibodies at the early stage
- Automated 3-step purification including SEC
- 1-7 mg bsAb from 20 mL cell culture with timeline of 4 weeks
- 99% heterodimer, >95% monomer, <0.5EU/mg endotoxin
- Suitable for most in vitro and some in vivo studies
Quick ‘n’ Clean Service Details:
| Service Item | Deliverables | Duration | QC | Request A Quote |
| Gene synthesis, cloning and plasmid prep all included. | ||||
| 20 mL | • 1-7 mg bsAb • 3-step automatic purification • 99% heterodimer |
3-4 weeks | <0.5 EU/mg endotoxin control, QC incl. Titer, A280, Caliper (R/NR) or SDS-PAGE, SEC-HPLC | Request A Quote |
| Additional QC |
RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing |
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Case Study: Using Quick ‘n’ Clean Platform to Produce High-Purity Bispecific Antibodies in Various Customized Formats
The production of bispecific antibodies typically involves a complex and time-consuming purification process to remove impurities caused by HC mispairing, LC mispairing, fragmentation, and aggregation. With our Quick ‘n’ Clean platform, the customized formats of bispecific antibodies, which comprise a scFv or VHH and a Fab on a heterodimeric Fc, can be purified in a high-throughput and automated manner, thereby eliminating the possibility of light-chain mispairing.
Figure A: A variety of customized format of bispecific antibodies, including One Arm, VHH, scFv and WuXiBody were all produced in high purity (99% heterodimer, 95% monomer) via the Quick ‘n’ Clean platform, which is demonstrated on the non-reduced and reduced gel.
The production of bispecific antibodies presents certain challenges due to the formation of various byproducts resulting from chain mispairing, homodimer formation, and incomplete assembly. Our Premium Bispecific antibody service is capable of generating high-quality bsAbs, ranging from milligrams to grams, in just 4-6 weeks. Having produced over 3,000 different bsAbs, we have accumulated extensive expertise in this field.
During the expression stage, we undertake construct optimization and chain ratio studies to maximize expression levels of the desired target. In the purification stage, a tailored approach is essential, as the byproducts for each specific bsAb differ. To address this challenge, we employ intact mass spectrometry analysis as a powerful tool to identify and monitor these byproducts during the purification process. This information guides us in selecting the most effective purification steps for pooling and polishing. Subsequently, we scale up the process as needed to produce the desired quantity of the final product.
Key Features of Premium Bispecific Antibody Service
- Intact MS analysis to drive the purification process with extensive scouting and optimization
- Chain ratio study upon request (to optimize the expression level)
- Large-scale production of 500 mL and up (transient & Stable pools), 10 mg to grams of bsAb
- 4 to 6 weeks to deliver for transient production
- Suitable for in vitro and in vivo studies
Extensive Experience with Many Different Bispecific Antibodies Formats:
LC-MS Mandatory for the Premium Bispecific Antibody
Figure A: A bispecific antibody purified by AC-SEC. The SEC-HPLC and SDS-PAGE suggest high purity. However, this is a mixture of heterodimer and homodimer indicated by Intact MS, for which the existence of homodimer is ~50%.
Premium Bispecific Antibody Service Details:
| Premium BsAb Production (Week) | Request A Quote | |||
| Week 1 | Week 2 | Week 3 | Week 4 | Request A Quote
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| DNA Synthesis and Prep | WuXian Transient Expression | Purification and Analysis | ||
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Case Study #1: Optimization of Chain Ratio Increases the Percentage of Heterodimers via Premium Bispecific Antibody Service
One challenge for bispecific antibody production is the unbalanced chain expression level. In this case study, a chain ratio study is proposed to improve the expression of bispecific antibodies.
Figure A: The bispecific antibody contains 3 chains: LC, HC1 and HC2, of which HC1 is a Fc fusion protein. The default transfection ratio HC1: HC2: LC = 2:1:1 showed low expression level and low percentage of heterodimer. To overcome this challenge, the HC1 ratio was increased to different ratios. The percentage of heterodimer increased from 12% to 72% after optimization.
Case Study #2: Mixed Mode + CEX Could Remove Half Antibodies & Homodimers
This case study presents a common LC bispecific antibody construct. After affinity purification, the HMW, half antibody and homodimer were observed based on SEC-HPLC, SDS-PAGE gel and Intact MS. Mix mode was used as the first polishing step. After that, most of the whole half antibody was removed. However, the homodimer still existed, indicated by LC-MS. CEX was applied as the second polishing step to eliminate the homodimer and trace amount of half antibody.
Figure A: Here is a common LC BsAb construct. After affinity purification, the HMW, half antibody and homodimer were observed based on SEC-HPLC, SDS-PAGE gel and Intact MS.
Figure B: Mix mode was used as the first polishing step. After that, most of the whole half antibody was removed.
Figure C: However, the homodimer still existed, indicated by LC-MS. CEX was applied as the second polishing step to eliminate the homodimer and trace amount of half antibody.
Frequently Asked Questions for Bispecific Antibody Production
Q: To produce bispecific antibodies, can I use my own tags, or do we have to use WuXi Biologics’ tags?
A: We typically recommend using our tags for bsAb production, because they deliver high throughput and offer greater purity while being cost-effective. However, if you have your own preferred tags for your bispecific antibodies, we can discuss accommodating them if they fit into our HTP platform.
Q: For small-scale bsAb production, can the tags be removed afterwards?
A: For our Quick ‘n’ Clean platform, our primary aim is to generate ~1 mg of highly purified bispecific antibody in a fast and cost-effective manner to help unlock the optimal pairings of bsAbs. The molecules don’t need to be in their final formats, so there’s no need to remove the tag. After the optimal pairings are identified, we can re-clone the chosen molecules into the final CMC-ready format and use our Premium Bispecific Antibody Production Service to generate the bispecific antibodies without a tag.
Q: Do you use antibodies to isolate tag antibodies? If so, are there concerns about anti-tag antibody leakage during purification?
A: Resin usage always poses potential leakage concerns. The key is to understand your molecule’s key quality requirement. For early-stage molecules, leakage isn’t often a significant concern. To our knowledge, we don’t see significant leakage of resin, even with our Quick ‘n’ Clean service. Those seeking extra assurance can opt for our Premium Bispecific Antibody Production Service in which we don’t rely on any tags and use Process Development (PD)-like resin to handle all steps of your purification to achieve very high quality.
Q: Do you use transient or stable transfection for the chain ratio study, and have you seen any discrepancy between the optimal chain ratio determined by the two-transfection approaches?
A: Chain ratio studies have intrinsic differences between transient and stable transfections. Transient transfection allows for more extreme chain ratios, like paring Fc fusion with Fab at a 6:1 ratio, which is challenging in a stable setting. However, chain ratio reflects the nature of your molecule and therefore the transient chain racial study generally correlates well with the stable pool chain ratio study. Before proceeding to CMC, we provide options based on your needs, and many clients prefer stable pools for these chain racial studies. We are very flexible with these preferences.
Q: Is the chain ratio step achieved by changing the ratio of input DNA constructs ratio?
A: In our transient transfection, we clone the heavy chain and light chain in separate vectors. This allows us great flexibility in achieving any chain ratio by simply adjusting the input ratio. For our stable cell lines, however, we typically use molecular biology techniques to clone multiple copies of the molecules into 1-2 or even 3 vectors. This approach increases the copy number but offers less flexibility and the process is lengthier due to the intricacies of molecular biology.
Q: Could the early-stage purification process be transferred to the CMC process?
A: Yes, absolutely. We work very closely with our Process Development (PD) teams to transfer our protocols, knowledge, and experience. For the developability assessment of bispecific antibodies in our PS Department, we use PD-ready resin to screen for different methods. These methods are amenable for scale-up and can be transferred to our PD department for further optimization. However, if your focus is solely on getting a large amount of high-quality bispecific antibodies without a developability study, some methods from our Premium Bispecific Antibody Production Service may not be readily transferred to the PD department.
Q: Do you offer a service to design bispecific antibodies?
A: Absolutely. We offer a dedicated service for bispecific antibody design. Beyond just designing your bsAb molecules, our expertise is rooted in a deep understanding of the drug developability across various formats of bispecific antibodies. Our goal is to recommend the molecules to our clients that have the highest potential on developability.
Q: Why is intact mass so important during the purification of bispecific antibodies?
A: Intact mass analysis is necessary to guide bispecific antibody purification because it can differentiate main products (heterodimers) from byproducts (homodimers) that have similar molecular weights. Standard SDS or SEC methods can’t achieve the level of differentiation that intact mass can achieve.
Related Content
- Webinar: Unlock the Optimal Pairings of Bispecific Antibody Through Small Scale HTP BsAb Production & Efficient Scale-up Strategies
- Poster: Unlock The Optimal Pairings of BsAb Using Quick n’ Clean
- Blog Article: Innovations in Bispecific Antibodies, From Unlocking Optimal Pairings to Scale-up Production