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Bispecific Antibody Production Services

High-Titer, High-Purity Bispecific Antibodies Across Diverse Formats

 

At WuXi Biologics, we specialize in producing high-titer, high-purity bispecific antibodies (bsAbs). Our expert team leverages innovative platforms for bispecific antibody engineering and production, from high-throughput, small-scale production for optimal pairing identification, to overcoming production challenges across various formats such as CrossMab, ScFv-Fab, and Fc-fusion—delivering high-purity, gram-level bsAbs suitable for in vivo and in vitro studies.

 

  • High-titer CHO production with over 98% heterodimer purity and endotoxin levels below 0.1 EU/mg
  • Expertise in diverse bispecific antibody formats like CrossMab, ScFv-Fab, Fc-fusion, and bispecific VHH
  • Custom molecular design and engineering strategies for improved developability
  • Over 3,000 bispecific antibodies delivered, with over 100 into CMC development
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3D model of a bispecific antibody (BsAb), illustrating dual-targeting capabilities for advanced therapeutic applications.

Quick ‘n’ Clean: High-Throughput Bispecific Antibody Production

 

WuXi Biologics’ Quick ‘n’ Clean platform enables high-throughput bispecific antibody (bsAb) production for early-stage drug discovery. Powered by high-titer CHO expression system, it produces 1-7 mg of custom bispecific antibodies with 99% heterodimer purity from 20 mL CHO in just 3-4 weeks. Our fully automated workflow, integrated with high-throughput SPR binding assays, provides a rapid, cost-effective approach for optimal pairing identification. The bsAbs produced are not only suitable for in vitro screening assays, but also applicable for some in vivo studies.

Key Features of Quick ‘n’ Clean Service

  • High-throughput bispecific antibody production for early-stage drug discovery
  • Automated 3-step purification system (including SEC)
  • 1-7 mg bispecific antibodies from 20 mL CHO cell culture in just 3-4 weeks
  • 99% heterodimer purity, >95% monomer, and endotoxin level <0.1 EU/mg
  • Suitable for most in vitro and some in vivo studies

Quick 'n' Clean bispecific antibody production platform yields 1-7 mg high-purity bispecific antibodies from 20 mL CHO cells in just 3-4 weeks.

Quick ‘n’ Clean Service Details:

Service Item Deliverables Turnaround Time Quality Control Request A Quote
Gene synthesis, cloning and plasmid prep all included.
20 mL • 1-7 mg bispecific antibody
• 3-step automatic purification
• 99% heterodimer		 3-4 weeks <0.1 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

Case Study #1: High-Purity Bispecific Antibody Production Across Various 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.

Four bispecific antibody formats, including One Arm, VHH, ScFv, and WuXiBody, with SDS-PAGE analysis for purity assessment.

Case Study #2: High-Throughput Four-Chain Bispecific Antibody Production with the TFC Quick ‘n’ Clean Platform

 

Our True Four Chain Quick ‘n’ Clean platform offers high-throughput production of four-chain bispecific antibodies with excellent yield and purity. Leveraging the WuXiBody™ backbone, this advanced platform eliminates LC mispairing, enabling the production of bispecific antibodies with two LCs. This process ensures >99% heterodimer purity, as verified by Intact Mass analysis.

Figure A: WuXiBody™ formats used in the TFC Quick ‘n’ Clean platform

Bispecific antibody WuXiBody formats (1+1, 2+1) used in the high-throughput production process.

Figure B: The TFC Quick ‘n’ Clean platform was utilized to produce 15 four-chain bispecific antibodies in 20 mL CHO cell cultures. Results showed consistently high monomer purity (mostly above 98%), an average yield of 3.34 mg, and low endotoxin levels averaging 0.06 EU/mg.

Graph illustrating monomer purity 98.23 percent, bispecific antibody yields 3.34 mg, and endotoxin level 0.06 EU per mg.

Figure C: Intact Mass analysis confirms the absence of impurities like homodimers and LC mispairing, with the heterodimer purity of these bsAbs exceeding 99%.

LC-MS confirms larger than 99 percent heterodimer purity of these bispecific antibodies.

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Premium Bispecific Antibody Production Services: LC-MS-Assisted Purification for High Purity

 

WuXi Biologics Premium Bispecific Antibody production services overcome key challenges in bispecific antibody (bsAb) production including chain mispairing, homodimer formation, and incomplete assembly. Leveraging ultra-high-titer CHO expression system, advanced LC-MS-assisted purification, and chain ratio optimization, this platform delivers high-yield, high-purity bispecific antibodies at milligram-to-gram scale within 4-6 weeks. With expertise in diverse bsAb formats, we optimize each stage of production to enhance efficiency, quality and efficacy.

Key Features of Premium Bispecific Antibody Service

  • LC-MS-assisted purification, ensuring >98% heterodimer purity
  • Chain ratio optimization to enhance titer and purity upon request
  • Large-scale production from milligrams to grams, ensuring seamless transition to CMC development
  • Delivered over 3,000 bispecific antibodies across diverse formats, including CrossMab, ScFv-Fab, and Fc fusion
  • Suitable for in vitro and in vivo

Extensive Experience with Many Different Bispecific Antibodies Formats:

 

Visual representation of bispecific antibody formats, including Common LC, Cross mAb, WuXiBody, Different LC, Fc Fusion, ScFv Fab, Charge Pairing, and Complex 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%.

SEC-HPLC and SDS-PAGE demonstrating 98 percent purity of bispecific antibody samples while LC-MS analysis only showing 50 percent purity.

Premium Bispecific Antibody Service Details:

Premium Bispecific Antibody Production (Week) Request A Quote
Week 1 Week 2 Week 3 Week 4 Request A Quote

 
DNA Synthesis and Prep WuXian Transient™ Expression Purification and Analysis
 
  • Additional 2 weeks for ratio study
  • Analysis includes: LC-MS, SDS-PAGE, SEC-HPLC
  • Purification scouting
  • Purification scale up

 

Case Study #1: Optimizing Chain Ratio for Bispecific Antibodies for Higher Purity & Yield

 

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.

Graph comparing yield and purity improvements in bispecific antibody production, demonstrating increased SEC-HPLC purity from 12 percent to 72 percent through chain ratio optimization.

Case Study #2: Removing Homodimers with Mixed Mode & CEX Purification for High-Purity Bispecific Antibodies

 

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 bispecific antibody construct. After affinity purification, the HMW, half antibody and homodimer were observed based on SEC-HPLC, SDS-PAGE gel and Intact MS.

Post Protein A purification quality analysis using SEC-HPLC and SDS-PAGE with half antibody and homodimer observed.

Figure B: Mix mode was used as the first polishing step. After that, most of the whole half antibody was removed.

Mixed mode purification for bispecific antibodies, using SDS-PAGE and intact mass spectrometry to validate fraction purity.

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.

Cation exchange chromatography CEX purification of bispecific antibodies, with SDS-PAGE and intact mass analysis confirming high purity.

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Frequently Asked Questions for Bispecific Antibody Production

Q: Do you use SEC for large-scale bispecific antibody production?

A: No, SEC is not ideal for large-scale bispecific antibody production due to scalability limitations. Instead, we utilize CEX chromatography or mixed-mode purification to efficiently remove aggregates in large-scale production. However, during early-stage antibody discovery and high-throughput antibody production, SEC remains a valuable tool for achieving high-purity bispecific antibodies for research purposes.

Q: Do you use LabChip instead of SDS-PAGE for bispecific antibody analysis?

A: The choice between LabChip and SDS-PAGE depends on throughput requirements. For high-throughput analysis, such as for 200 or more molecules, we use LabChip. For lower-throughput applications, SDS-PAGE is preferred due to its simplicity and ease of handling. 

Q: Do you have experience with light chain mispairing? How do you address this issue (e.g., CrossMab)?

A: Yes, we have extensive experience in mitigating light chain mispairing in bispecific antibody engineering. To address this issue, we first optimize the light chain-to-heavy chain ratio to enhance proper pairing. If mispairing persists, it may indicate poor light chain expression. In such cases, we recommend swapping the VH-VL pair in the Fab arm with the VH-VL pair in the cross arm. Alternatively, switching the VH and VL domains within the same cross arm can further reduce mispairing.

Q: What recommendations do you have to generate heterodimers of four-chain bispecific antibodies?

A: We optimize chain ratios upon request to enhance the purity and titer of four-chain bispecific antibodies. While we have default expression conditions and extensive experience across various bispecific antibody formats, we strongly recommend conducting a chain ratio study, especially for large-scale bispecific antibody production, to achieve optimal yields. However, chain ratio optimization has its limits if one chain is poorly expressed. We suggest early-stage antibody engineering, sequence design, and micro developability testing to improve overall expression and stability.

Q: How do you decide between CEX, HIC, and mixed-mode chromatography for bispecific antibody polishing?

A: The selection of CEX, HIC, or mixed-mode chromatography depends on the pI differences and hydrophobicity of the molecule. CEX chromatography is preferred when there is a sufficient pI difference between product-related variants. If pI differences are minimal, HIC or mixed-mode chromatography is employed to enhance bispecific antibody purification efficiency. When light chain mispairing is a concern, HIC may offer better performance than mixed-mode chromatography.

Q: What purification steps do you use to ensure good viral clearance for WuXiBody™ given its low pI?

A: At the research stage, viral clearance is not a primary concern. However, during the CMC stage, viral clearance is addressed through acid treatment, exploiting WuXiBody’s tolerance to low pH, or by using detergents for molecules sensitive to acidic conditions. 

Q: Do you use KappaSelect or LambdaFabSelect for Fab-scFv purification?

A: We generally avoid KappaSelect for Fab-scFv purification, as it can lead to aggregation issues, especially when handling large numbers of molecules in high-throughput antibody production. While LambdaFabSelect offers an alternative, it often exhibits low elution efficiency, making it less suitable for routine use due to potential bispecific antibody purification challenges. Instead, we employ optimized purification strategies tailored to the specific formats to achieve high purity and yield.

Q: Are Protein A Knockout Mutations Beneficial for Bispecific Antibody Purification?

A: In bispecific antibody (bsAb) production, Protein A knockout (KO) mutations can simplify purification by reducing byproduct binding to Protein A columns, which can be useful for early-stage in vitro studies. However, this approach is not recommended for in vivo studies, as the half-life of bsAbs with one Fc bearing the FcRn-null mutation has not been well understood.

Q: Why is LC-MS analysis so important during the purification of bispecific antibodies?

A: LC-MS analysis is essential for guiding bispecific antibody purification, as it accurately distinguishes heterodimers from homodimers, which often have similar molecular weights. Traditional methods like SDS-PAGE or size-exclusion chromatography (SEC) lack the resolution needed to differentiate these variants effectively. Alternatively, LC-MS analysis ensures high-purity bispecific antibody production, improving both yield and product quality.

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 optimized tag system for bispecific antibody production, as it is designed to enable high-throughput purification, achieving high purity, and in a cost-effective manner. However, if you have preferred custom tags for your bispecific antibodies, we can evaluate their compatibility with our high-throughput antibody production platform and discuss potential integration to meet your specific needs.

Q: For small-scale bispecific antibody 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 bispecific antibodies. 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 observed any discrepancies between the two transfection approaches?

A: Chain ratio studies inherently differ between transient and stable transfection approaches. Transient transfection allows for more extreme chain ratios, such as pairing Fc fusion with Fab at a 6:1 ratio, which can be challenging to achieve in a stable transfection system. However, since chain ratio reflects the molecular nature of the bispecific antibody, transient transfection chain ratio studies generally correlate well with stable pool chain ratio studies. Before transitioning to CMC development, we offer customized options based on project needs. Many clients prefer stable pools for chain ratio studies due to their scalability and consistency. Our bispecific antibody production services are flexible to accommodate specific preferences for your antibody discovery and development projects.

Q: Is the chain ratio optimization achieved by changing the ratio of input DNA constructs?

A: Yes, in our transient transfection system, we clone the heavy chain (HC) and light chain (LC) into separate vectors, allowing flexible adjustment of the input DNA construct ratio to achieve the desired chain ratio efficiently. For stable cell lines, we use molecular biology techniques to integrate multiple copies of the target molecules into one, two, or even three vectors. While this approach increases gene copy number, it offers less flexibility compared to transient transfection and involves a longer development timeline due to the complexities 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: We provide custom bispecific antibody design and engineering services, leveraging our extensive expertise in antibody engineering and drug developability assessment. Beyond simply designing bispecific antibody molecules, we evaluate various bispecific antibody formats to recommend the most suitable candidates for late-stage drug development and manufacturing. Our goal is to help clients identify molecules with the highest potential for therapeutic success, ensuring optimized bispecific antibody production with desired developability attributes.

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