Protein engineering of antibody fragments for pharmaceutical production

Abstract

Antibody fragments without the Fc region are attracting attention in the pharmaceutical industry due to their high ability to penetrate solid tissues, cost-effective expression using microbial expression systems, and distinctive modes of action compared to those of full-size antibodies. Based on these characteristics, several antibody fragment agents have been approved. However, developing platform engineering methodologies to accelerate their development is important. In this review, we summarize and discuss protein engineering strategies for preparing therapeutic antibody fragments composed of antibody variable domains. Three (introduction of high-solubility tag systems, complementarity-determining region grafting, and domain arrangements) and two (introduction of purification tag systems and mutagenesis studies for protein L- or protein A-binding) protein engineering strategies have been reported for the cultivation and purification processes, respectively. Fusion tags might negatively impact molecular folding, function, immunogenicity, and final yield. If the production behavior of antibody fragments is not improved through complementarity-determining region grafting, domain arrangements, or human sequence-based mutagenesis, using additional fusion tag systems should be considered, with careful attention to the points described above. This summarized knowledge regarding protein engineering strategies for effectively producing antibody fragments will further accelerate therapeutic antibody fragment development.

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

“…Over the past few decades, many studies have explored the use of peptide- or protein-based tags to minimize aggregation and enhance solubility of heterologous proteins expressed in E . coli with minimal or no impact on enzyme activity [ 32 ]. Tag systems such as MBP (maltose binding protein) [ 33 ] and NEXT (originating from N-terminus of Hydrogenovibrio marinus CA) [ 34 ] are widely used.…”

“…Tag systems such as MBP (maltose binding protein) [ 33 ] and NEXT (originating from N-terminus of Hydrogenovibrio marinus CA) [ 34 ] are widely used. NEXT tag was shown to increase the soluble expression level in E. coli BL21 (DE3) by up to 5.6–8.3 fold [ 32 ]. It is possible that CA4-LOGACA and CA6-SazCA would show improved activity and/or protein titer with properly optimized fusion tag strategies.…”