Stable expression and purification of a functional processed Fab′ fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor

Abstract: Monoclonal antibodies and derivative formats such as Fab′ fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab′ fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab′ fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab′ fragment against the Fas receptor, routinely expressed in 1–2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab′ fragments. [Copyright &y& Elsevier]