Introduction -Definitions:

hybridoma fusion and cloning supplement (HFCS): is a low serum containing media supplement for the repalcemetn of fetal calf serum (FCS) in cultures of hybridoma cells. HFCS is specifically formulated to optimize cell growth of freshly fused hybridomas during selection and cloning procedures in low serum-contianing cell culture media.

Cell Expression of Bi-specific Antibodies:

Mammalian cells are the predominant workhorses for IgG production in industry. However, the production of bispecific antibodies is more complex and typically requries at least two plasmids for heterodimerized heavy chains and one plasmid for a common light chain or two light chain plasmids if two different light chains are used. Notably, expressing HC and LC on separate plasmids is recommended because the manipulation of the plasmid ratio is an easy and efficient approach to optimize protein assembly for desired products. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)

co-culture method:

To solve the light chain mispairing issue and retain the natural antibody architecture, Speiss propsoed to produce bispecific antibodies by comining two distinct half antibodies expressed form two different cell lines in vitro. Half antiboides are then purified and mixed with 1:1 molar artio in vitro to generate functional bispecific antibodies. While this half-antibody method can be effective, the method is attended with some inherent challenges. using two separate cell ines means that two culture vessels, harvests, and purificaiotn processes must be performed before combining in vitor, potentially increasing costs and the risk of contamination. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)

The co-culture method was first demonstrated in E. coli in which cells were transformed with plasmids containing different half antibodies genes (A and B) containing knobs-into-holes to prevent self dimerization of the H chains prior to association with light chains. After culturing, the cells are lysed to harvest half antibodies. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)