Microbial host systems remain the most efficient and cost-effective chassis for biotherapeutics production. Escherichia coli is often the preferred host due to ease of cloning, scale-up, high product yields, and most importantly, cost-effective cultivation. E. coli often experience difficulties in producing biologically active therapeutics such as Fab fragments, which require protein folding and subsequent three-dimensional structure development. This paper outlines the recent improvements in upstream and downstream unit operations for producing Fab fragments in E. coli. Monoclonal antibody fragments (Fab) are a rising class of biotherapeutics and their production has been optimised using coexpression of molecular chaperones such as DsbC or DnaK–DnaJ–GrpE, as well as strain engineering for post-translational modifications such as disulphide bridging. Different media systems such as EnBase and combining nitrogen source supplementation with low-temperature cultivation have resulted in improvement in cell integrity, protein expression, and protein refolding. The recovery of native proteins from insoluble inclusion bodies can be improved by adjusting refolding conditions, as well as by incorporating multimodal and affinity chromatography for achieving high product yields in purification. Recent developments summarised in this review may tune the E. coli expression system to produce more complex and glycosylated proteins for therapeutic use in the near future.

Separation of charge variants during purification of monoclonal antibodies is important for obtaining the desired distribution of charge variants |