Scar-free tag removal by CASPON® enzyme with broad physicochemical stability in biomanufacturing – A case study of five proteins

Abstract

Manufacturing of recombinant proteins in microbial systems, in particular in E. coli, generally requires thorough process development due to the absence of a platform process. The caspase-based fusion process (CASPON®) offers a platform manufacturing process using special protease cleavable fusion-tags. These tags allow the implementation of His-tag based affinity chromatography for facile target protein capture and offer expression and solubility enhancing capabilities. The tags are intended as N-terminal fusion motifs that can be fully cleaved using a modified caspase-2 protease, the CASPON® enzyme. Here, we systematically explore the influence of various physicochemical parameters on its enzymatic activity. This characterization was performed in parallel in two independent research laboratories using different assays, i.e. a Förster resonance energy transfer-based assay using small peptide substrates and a reversed phase high performance liquid chromatography method using model proteins. Both assays demonstrated great agreement and reveal that CASPON® enzyme is highly active at a wide range of temperatures, pH and is resistant to a variety of chemical substances that are commonly employed in bioprocessing, e.g. NaCl, kosmotropes, chaotropes and others. The presence of imidazole at concentrations commonly found in the elution fraction of immobilized metal affinity chromatography does not seem to affect the activity of CASPON® enzyme, enabling its use directly after the capture step in downstream processing (DSP). A case study of five biopharmaceuticals is presented. The platform process exhibits consistently high performance in up- and downstream processing, achieving high soluble titers, high yield and purity. The presence of host cell proteins during the DSP in particular was investigated in-depth using process proteomics, revealing that a core of host cell proteins is process dependent and can commonly be expected to be found in the platform DSP. The information presented here can serve as a guide on how to implement the CASPON® platform process for the production of various recombinant proteins.