METHOD FOR THE PRODUCTION OF DOMAIN ANTIBODIES

摘要

The present disclosure relates to a method for producing a domain antibody in a host other than E. coli, preferably yeast, comprising a) applying conditions that promote the formation of disulfide bridges in domain antibodies, or b) removing domain antibodies lacking at least one disulfide bridge, or c) a combination of (a) and (b). More specifically, the present disclosure relates to a method, wherein said conditions that promote the formation of disulfide brides are selected from one or more of the following: a) addition of oxidizing agents, preferably oxidizing metal ions, preferably one or more selected from Cu2+, Fe2+, Fe3+ and Zn2+; b) co-expression of the domain antibody with a thiol lsomerase,- c) adapting the culturing conditions by one or more selected from the following: lowering culturing temperature and/or optimizing the culturing medium, including but not limited to reduction of methanol feed for hosts requiring a methanol feed, lowering conductivity of the culture medium, addition of yeast extract and/or peptone, or any combination thereof; d) refolding the domain antibody in the presence of denaturant and redox-buffer e) treating the domain antibody by oxygenation, increasing temperature, increasing pH, high pressure or any combination thereof, and f ) combinations of any of a) through e), or wherein conditions that remove domain antibodies lacking at least one disulfide bridge are selected from a) binding domain antibodies comprising free thiol groups to suitable reactive groups, including but not limited to immobilized thiol groups, optionally under denaturing conditions; b) reverse phase high performance chromatography.