Antibody fragments (Fab) represent the most important class of therapeutic biopharmaceutical proteins used to treat various immune-oncology diseases. Correct folding and assembly of the HC and LC of Fab are crucial steps to obtain a native and biologically active Fab, which is affected by the ratio of HC to LC gene expression (HC:LC). The balanced expression of each polypeptide chain is one of the critical challenges in large-scale manufacturing of the Fab. These are heterodimeric proteins, and each polypeptide may have its own optimal level of production, which may lead to the unequal expression of these chains. Our investigation is focused on understanding the effect of expression construct (dual promoter vs. co-transformation strategy), codon optimization, and induction strategies on yield and expression stoichiometry of LC and HC genes of Fab. rHu biosimilar Ranibizumab was selected as a model protein for this study. Expression stoichiometry of HC and LC gene at mRNA level was studied using RTqPCR, whereas protein expression level was studied quantitatively using RP-HPLC and SDS-PAGE analysis. In the case of dual promoter expression construct, it was observed that LC gene cloned in the MCS1 of the duet vectors has &gt; 2-fold expression than the HC gene, cloned in the MCS2. Transcript abundance profile of the HC and LC genes determined at different time intervals post-induction shows a difference in the gene expression at the transcriptional level. Comparative analysis of dual promoter and co-transformation strategy shows better stoichiometry in co-transformation (1:1.3), whereas higher protein yield in a dual expression system (&gt;2.4 fold). The use of lactose and galactose as inducers show higher Fab yield of 2.30 ± 0.03 g/L and 2.81 ± 0.06 g/L with expression stoichiometry of 1:1.9 and 1:2 (HC: LC) respectively than IPTG-based induction with a protein yield of 1.40 ± 0.02 g/L.

Understanding expression stoichiometry of heavy and light chain gene in antibody fragments therapeutic

ACS Spring 2022

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