Effects of Monoclonal Antibody Sequence on Production of a Recombinant Antibody In CHO Cells

The increasing demand for antibody-based therapeutics has emphasized the need for technologies to

improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody

therapeutics address an increasing spectrum of indications, interest has increased in antibody

engineering to improve affinity and biological activity. However, the cellular mechanisms that

dictate expression and the relationships between antibody sequence and expression level remain

poorly understood. Fundamental understanding of how mammalian cells handle high levels of

transgene expression and of the relationship between sequence and expression are vital to the

development of new antibodies and for increasing recombinant antibody titers. To address these issues, we

created a panel of antibody mutants that vary by a single amino acid at Kabat position 49 (heavy

chain framework), resulting in differential transient and stable titers with no apparent loss of antigen

affinity. Through analysis of mRNA, gene copy number, intracellular antibody content, and

secreted antibody, we found that while translational/post-translational mechanisms are limiting in

transient systems, transcription becomes the limiting event upon stable integration of the transgene

following application of selective pressure. We also show that the presence of certain amino acids

at residue 49 results in problematic protein folding and assembly. We postulate that stable antibody

expression is transcriptionally limited to prevent toxic accumulation of misfolded and aggregated

protein within the cell. This study highlights the need for proper sequence engineering strategies

when developing therapeutic antibodies and alludes to the use of transient expression as a predictor

of stable expression behavior.