With years of experience in antibody discovery and development, Creative Biolabs is proud to offer controlled Fab-arm exchange (cFAE) bispecific antibody (BsAb) generation service. Based on the advanced cFAE platform, we are dedicated to provide first-class BsAbs to support your projects in a timely and cost-effective manner.
BsAbs antibody production is actively pursued for therapeutic and research antibody development. On the account of elevated interest in the therapeutic potential of BsAbs, a number of formats have been created to exploit the promise of dual targeting antibodies. Early BsAb formats were formed by co-expression of relevant heavy (H) and light (L) chains or chemical crosslinking with the lack of product homogeneity, and the following purification complexity led to poor product yield. After that, a variety of protein engineering methods allowed the design of formats with improved homogeneity and yield. These strategies are based on enhancing the desired H-H and H-L pairing upon co-expression or integrating both antigen binding sites in a single polypeptide chain (or single HL pair). Although these methods overcame some of the manufacturing problems, it was often at the loss of the physicochemical or pharmacokinetic properties of these agents.
The cFAE technique platform enables to develop stable IgG-like BsAbs. The cFAE strategy refers to separate expression of IgG1 mAbs each comprised a unique matched point mutation at the CH3-CH3 domain interface. When controlled reduction of hinge disulfide bridges in vitro, the matched mutations urge the efficient recombination of binding arms. This process is essentially unidirectional, because the mutations are selected to weaken the non-covalent CH3-CH3 interaction in the parental mAbs, which induces dissociation of HL homodimers, meanwhile, enables the generation of a strongly favored heterodimeric HL interaction. These characteristics greatly enhance bsIgG1 (bispecific IgG1) end product yield and post-exchange stability via reoxidation of the hinge. In addition, the use of a wild-type IgG1 hinge which is resistant to reduction under physiological conditions in vivo further improves the post-exchange stability of the bsIgG1 end product.
There are four major steps for the generation of cFAE BsAbs. Firstly, separate expressing two parental IgG1s comprising single matching point mutations in the cH3 domain. Secondly, mix parental IgG1s under permissive redox conditions in vitro to allow recombination of half-molecules. Then, remove the reductant to let reoxidation of interchain disulfide bonds. Finally, measure the exchange efficiency and final product by chromatography-based or mass spectrometry (Ms)–based methods. The protocol produces BsAbs with common IgG architecture, properties and quality attributes both at bench scale and at a mini-bioreactor scale.
Fig 1. Fab arm exchange among IgG4 antibodies. Part A shows the heavy chains of IgG4 antibodies switch between interchain and intrachain disulphide-bonded configurations. Part B shows the dissociation of the two halves of the IgG4 antibodies enables recombination to form asymmetric, BsAbs. (Cortazar, F. B., 2015)
With the well-established cFAE BsAbs generation platform, the experienced scientists here at Creative Biolabs are dedicated to help you develop unique BsAbs. We also provide other various services regarding BsAbs development. Please feel free to contact us for more information and a detailed quote.
1. Cortazar, F. B.; Stone J. H. “IgG4-related disease and the kidney.” Nature Reviews Nephrology. 2015, 11(10): 599-609.
2. Labrijn, A. F.; et al. “Controlled Fab-arm exchange for the generation of stable bispecific IgG1.” Nature protocols. 2014, 9(10): 2450-2463.
3. Gramer, M. J.; et al. “Production of stable bispecific IgG1 by controlled Fab-arm exchange: scalability from bench to large-scale manufacturing by application of standard approaches.” MAbs, 2013, 5(6): 962-973.