Abstract
Antibody fragments (Fabs) represent a highly interesting class of biopharmaceuticals with a broad range of medical applications. They bind antigens comparable to full length monoclonal antibodies, but are smaller in size allowing increased tissue penetration, and lack the glycosylated Fc domain, which is why they can be produced in the prokaryotic expression host E. coli. Due to the presence of disulfide bonds Fabs are usually produced in the oxidative environment of the E. coli periplasm. Even though recombinant production in E. coli is cheaper and easier to realize than in mammalian cells, this intracellular production of the Fab in E. coli entails difficulties in subsequent product analytics. Whereas Fabs are produced extracellularly by mammalian cells and thus can be analyzed in the environment of a defined medium, recombinantly produced Fabs in E. coli have to be analyzed in crude cell lysate containing a lot of host cell proteins, host cell DNA and lipids. Thus, robust and sensitive HPLC analytics for Fab quantification is still scarce today and recombinant Fabs from E. coli are conventionally quantified by expensive and cumbersome immunoassays. In this study we developed a sensitive and robust affinity-based HPLC method for the quantification of recombinant Fabs in E. coli crude cell lysates with a limit of quantification down to 46 μg/mL and high reproducibility. This method will definitely be of key importance for strain generation as well as for early steps in upstream process development for recombinant E. coli producing Fabs.
Published Version
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