Fab Antibody Fragment Expression and Purification

What is Fab Antibody

Fab (fragment antigen-binding) fragments are the antibody binding regions of an antibody. It contains one complete L chain in its entirety and the V and CH1 portion of one H chain. The Fab can be further divided into a variable fragment (Fv) composed of the VH and VL domains, and a constant fragment (Fb) composed of the CL and CH1 domains. The Fab fragment has 440-450 amino acids and its molecular weight is approximately 55 kDa.

Antibody fragments (Fabs), lacking the glycosylated Fc constant regions, have the advantage over whole antibodies in applications requiring rapid tissue penetration and rapid clearance from the blood or kidney. In contrast to some of the heavily engineered antibody fragments, the Fab has native sequence; subsequently, it is less likely for the Fab to be immunogenic when used as therapy.

Due to these features, the production of antibody fragments is industrially highly demanded and the antibody fragment pipeline is expanding, with three therapeutic Fabs approved by FDA, and many in the active clinical pipeline and preclinical research.

Structure of full-length antibody and antibody fragments
Fig 1. Structure of full-length antibody, scFv and Fab fragments

scFv vs. Fab Antibody

What's the difference between scFv and Fab fragment? scFv fragment consists of the variable domain of the heavy and light chains of a monoclonal antibody, usually expressed as one polypeptide chain with a linker in between the chains. Fab fragment is the variable domain of each chain, plus the first constant region. In the case of the Fab format, a linker is not essential because the CH1 and CL regions will be fused by disulfide bonds.

  Fab scFv
Composition VH, CH1, VL, CL VH, VL
Molecular weight (kDa) 55 28
Tumor penetration Good Great
Stability High stability in long-term storage Unstable over longer periods

Production of Anti-Fab Antibodies

Fab antibody fragments can be produced by phage display antibody library. Fab antibody library has many advantages. Firstly, it can rapidly and efficiently select new antibodies that are difficult to gain through hybridoma technology. Besides, like scFv antibodies, it could be easier to generate Fab antibodies with higher affinities.

Sino Biological has developed transient transfection technology in HEK293 cells for Fab fragment antibody expression and production, and has generated excellent quality of Fab fragment for antigen-Fab co-crystallization. The recombinant antibody Fab fragment protein produced by our transient transfection in mammalian cells is of superior quality than antibody fragment protein generated from enzyme cleavage of full antibodies. If customers prefer, we can also use our E. coli expression system for Fab antibody production.

Because many antibody Fab fragment proteins do not bind well to protein A, protein G, or protein L affinity resin, we typically add a poly-histidine tag at the C-terminus of the antibody heave chain for purification. A protease cleavage site can be designed between the His-tag and the Fab to allow tag-removal after purification if needed.

For customers that have set up a contract service account with us, we typically do not require an upfront payment for antibody production orders. For antibody production request, we usually quote our price based on successful completion of the production project, and you only need to pay upon receiving the purified antibodies and invoice. However, for some risky antibody production projects, for example, production of antibodies with modified constant regions, we would need to charge a small amount of cost for feasibility pilot study and process development (if needed).

Construction and production details about our Fab fragment antibody service

Host cells • HEK293 or CHO cell (please specify, cost for production in CHO cell is higher)
• We provide Fab antibody production in E. coli expression system as well, if you prefer E. coli system, please specify in your request.
Production • Recombinant technology
transient transfection
• Serum-free cell culture
Purification method • His-tag or FLAG-tag affinity chromatography column purification
• Protein A or G affinity purification maybe feasible for some Fabs without a His-tag/FLAG tag
Timeline 3-6 weeks total from receiving gene sequence to product delivery
Deliverable Purified Fab antibody (quantities to be specified by customer)
Quality specification as specified by customer (cost may be adjusted based on quality specification)
Antibody types Human, mouse, rat, rabbit, canine, rhesus IgG antibodies
Applications Drug discovery: antibody antigen co-crystalization, antibody binding epitope

References

1. Hust, M., Jostock, T., Menzel, C., Voedisch, B., Mohr, A., Brenneis, M., ... & Dübel, S. (2007). Single chain Fab (scFab) fragment. BMC biotechnology, 7(1), 14.
2. Crivianu-Gaita, V., & Thompson, M. (2016). Aptamers, antibody scFv, and antibody Fab'fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements. Biosensors and Bioelectronics, 85, 32-45.
3. Nelson, A. L. (2010, January). Antibody fragments: hope and hype. In MAbs (Vol. 2, No. 1, pp. 77-83). Taylor & Francis.

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