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Protein Sciences

Uniting Drug Development Expertise with Antibody & Protein Production Services

Fab, VHH, ScFv Antibody Fragment Production

Supporting Your Antibody Fragments Production Needs

 

At WuXi Biologics, our Protein Sciences (PS) Department produces high-quality antibody fragments to accelerate your research and bring innovative solutions to the market. Our deep experience and optimized production processes ensure high-titer expression of Fab, VHH, scFv, and sdAb (nanobody) using our WuXian™ Transient, WuXian™ Express, and E. coli expression systems.

High-titer expression of premium-quality antibody fragments, including Fab, VHH, scFv, and sdAb (nanobody) using CHO and HEK 293.

WuXian™ Transient (CHO & HEK293) Expression System

 

Over 70% of biologics, and nearly all monoclonal antibodies (mAbs), are produced in Chinese hamster ovary (CHO) cells, as these cells can generate proteins with bioactive post-translational modifications (PTMs) akin to those produced in the human body. This is especially critical when producing therapeutic antibody fragments which need specific PTMs to ensure stability, solubility, and activity in therapeutic applications. Our WuXian™ Transient CHO system is derived from CHO-K1 cells and stands out for its high-titer expression capabilities, ensuring optimal protein yield in a shorter time frame.

Key Features of WuXian™ Transient Expression System

  • High Titer Expression: High-titer expression levels: More than 1 g/L on Day 7
  • Seamless scale-up: WuXian™ Transient and WuXian™ Express (CHO stable cell line system yielding 10-1,000 grams of antibodies) are both derived from the same parental CHO cells ensuring comparable product attributes and titer as you increase your production scale.
  • Short timelines: Gram-level material delivered within 4-5 weeks

From gene synthesis to purified proteins, WuXian™ Transient Expression platform delivers in 3-4 weeks.

WuXian™ Transient Service Details:

Service Item Deliverables Expression Scale Duration QC Request A Quote
Highly customizable package with multi-step purification Any antibody, tagged or untagged protein with need for scale up manufacturing 1-100 L 3-5 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

Case Study: High Titer of WuXian™ Transient Platform Delivers Grams of mAbs in 3 weeks

Figure A: The graph on the left illustrates the production of a commercial antibody using the WuXian Transient 3.0 system with multiple batches as a control. It demonstrates high-titer expression on both Day 4 and Day 7 of culture. The table on the right showcases the production capabilities of the same platform for three monoclonal antibodies, yielding approximately 1.5 g/L of high titer on Day 7 with a 5 mL culture. Following AC purification, the final yield reached approximately 6 g, with SEC Purity exceeding 95%.

Showcase the high titer production capabilities of the WuXian™ Transient platform for 3 monoclonal antibodies.

WuXian™ Transient expression system can be scaled up seamlessly for grams of mAb production in just 3 weeks.

Figure B: Using the WuXian™ Transient expression system, grams of mAb were produced in just 3 weeks. A mIgG2a was expressed in a 10 L culture using the WuXian™ Transient system and purified on Day 7 through AC and CEX. This process yielded 11.9 grams of target antibody. Further analysis with SEC-HPLC confirmed its purity at 98.8%.

WuXian™ Express (CHO Stable Pool) Expression System

 

The WuXian™ Express stable pool protein expression system can rapidly generate large amounts of proteins for early-stage drug discovery in just 4-7 weeks. Both WuXian™ Transient and WuXian™ Express platforms are derived from the CHO-K1 mammalian cell culture system, the same platform used in our WuXia®cell line development platform.

 

Using the same cell lines across all of these platforms provides comparable product quality attribute profiles and cell culture process performance. Therefore, the use of the WuXian™ platforms is ideal for conducting developability studies for the evaluation of multiple lead candidates and the subsequent seamless integration into stable monoclonal cell line development activities or for large-scale protein production required to support CMC product and process development activities.

Key Features of the WuXian™ Express (CHO Stable Pool) Expression System

  • High productivity stable pool: Average >2.8 g/L for various proteins, and up to 6 g/L for hIgG1/4
  • Titer consistency: Pool-to-pool titer variation <10%
  • Seamless integration to CMC: The same cell lines used to achieve high comparability characteristics of the product and cell culture process
  • Flexible scale: 20 mL to 100 L

WuXian™ Express Protein Production Platform: A Workflow and Timeline

The WuXian™ Express stable pool protein expression system can rapidly generate large amounts of proteins starting from gene synthesis in just 7 weeks.

Express (CHO) Service Details:

Service Item Deliverables Expression Scale Duration QC Request A Quote
Gram-level protein generation for animal study and developability study in CHO cells Any antibody, tagged or untagged protein with need for scale-up manufacturing 20 mL-100 L 6-7 weeks A280, Caliper (R/NR) or SDS-PAGE (R/NR),
SEC-HPLC, and endotoxin (<0.5 EU/mg)		 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: High-Titer Expression of WuXian™ Express for Different Protein Types

 

This case study uses the WuXian™ Express platform to evaluate the production (i.e., titer: g/L) of antibodies and proteins at different cell culture scales.

Figure A: Using the WuXian™ Express platform, the expression of various proteins, including hIgG1/4, mIgG2a/2b, bsAb and Fusion proteins, all demonstrated high-titer expression >2 g/L with culture volumes ranging from 20 mL to 100 L.

Consistent high titer expression of various proteins using the WuXian™ Express platform.

  Timeline Average Yield (CHO) Expression Volume Features Request A Quote

Transient

 2-4 weeks 1 g/L 1 mL to 100 L
  • Start from codon optimization or customer provided DNA
  • Deliver product and QC data after 1-2 steps purification
 Request A Quote

Express

 5-7 weeks 3.5 g/L 20 mL to 100 L
  • Start from codon optimization or customer provided DNA
  • Deliver product and QC data after 1-2 steps purification

Frequently Asked Questions

Q: What are the typical yields for Fab, VHH, and scFv production?

A: Our WuXian™ Transient platforms consistently deliver high-titer expression for all antibody fragments: 

  • Regular human Fab: 500 mg/L to 1 g/L 
  • His-ScFv: 100 to 300 mg/L 
  • His-VHH: ~500 mg/L 
  • Fc-VHH: Comparable to regular IgG 

For reference, most regular human IgGs produced in our CHO transient system yield approximately 1 to 1.5 g/L. 

Q: Is it easier to produce human/rabbit/rat Fab?

A: Producing human Fab is relatively straightforward, with consistently higher yields. However, for other species such as rat or rabbit, yields may be lower, and purification can be more challenging due to limited resin options. In these cases, incorporating a His or Flag tag for purification is recommended. 

Q: What are the advantages of developing antibody fragments over the full-length antibodies as therapeutics?

A: Antibody fragments offer several advantages, including improved tissue penetration due to their smaller size, increased engineering flexibility, and reduced immunogenicity. They also serve as versatile building blocks for bispecific antibodies, facilitating the development of innovative therapeutic modalities. However, antibody fragments generally have a shorter half-life, which may be beneficial for certain applications. If extending the half-life is important, incorporating an Fc region or using a bispecific antibody format could be considered. 

Q: Why is deblocking important during the production of Fab’?

A: During Fab’ expression in CHO cells, adducts can form when cysteine or glutathione from the culture medium binds to the Fab’. Deblocking involves using a reducing reagent to remove these adducts, ensuring that the cysteine residues in the Fab’ are free and available for conjugation. 

Q: For antibody fragment expression, which expression system is considered the best?

A: Compared to E. coli, our ultra-high titer CHO expression system offers superior yields and significantly reduced endotoxin levels, making it the preferred choice for producing antibody fragments. Additionally, CHO cells are critical for expressing bispecific antibodies, which are often built from antibody fragments. 

Q: What purity percentage is acceptable for Fc-VHH after affinity purification?

A: A purity greater than 95% after affinity purification is considered excellent. However, the acceptable purity may vary depending on the quantity and properties of the target molecules, with a typical minimum threshold set at 50%. Other factors such as titers, resin recovery, and stability should also be considered. A comprehensive Micro Developability package is recommended to assess the candidate’s suitability for further development into CMC. 

Q: Among Fab, VHH, and scFv, which antibody fragment has the most therapeutic applications?

A: Each fragment has unique characteristics that make it suitable for specific therapeutic applications. Leveraging our expertise, we can assist in refining your therapeutic molecules to enhance their quality and efficacy. Notably, our expertise in scFv molecular design allows us to address aggregation challenges and facilitate the conversion of regular IgGs into scFvs with improved developability. 

Q: What makes the linker design important in the scFv case study, and what's the principle behind it?

A: If a linker is derived from a phage display campaign, it’s typically appropriate for the application. However, when converting a regular IgG into an scFv, the linker must be carefully designed to ensure proper spacing and flexibility between the VH and VL domains. This design is crucial to prevent aggregation and maintain the structural integrity of the scFv. 

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