Identifying bottlenecks in transient and stable production of recombinant monoclonal‐antibody sequence variants in chinese hamster ovary cells

Abstract

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. In this work, we analyzed a pair of 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, it appears that the amount of available transgenic mRNA becomes the limiting event upon stable integration of the recombinant genes. We also show that amino acid substitution at residue 49 results in production of a non-secreted HC variant and postulate that stable antibody expression is maintained at a level which prevents toxic accumulation of this HC-related protein. This study highlights the need for proper sequence engineering strategies when developing therapeutic antibodies and alludes to the early analysis of transient expression systems to identify the potential for aberrant stable expression behavior.

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

“…It is known that small variations in the amino acid sequence of the variable domain of an antibody can have substantial impact on its affinity to the target antigen. However, it is surprising that variations in the rather small variable domains also dramatically influence expression levels and thus manufacturability (Mason et al, ; Seeliger et al, ). Nonetheless, there is an urgent requirement to prepare current industrial CHO host cell lines for such future manufacturing challenges to provide effective therapeutics and to address unmet medical needs.…”

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

“…2), the stable CHO-DG44 BI-HEX cells expressing MCLA-128 produce both the KK and DEDE molecules in minor amounts (Fig. 5), which may result from suboptimal protein assembly in these cells (19). This figure also shows that these product-related impurities can be efficiently removed in the CEX polishing step that is routinely used in the purification of therapeutic IgG.…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…Amino acid sequences and secondary structures of specific proteins contain features associated with limitations in recombinant protein production in CHO cells 30, 31, 32. Functionally related proteins TIMP‐2 (well‐secreted) and TIMP‐3 (poorly secreted), with significant sequence and structural identity have been used as models to test sequence‐specific determinants of secretion in a transient CHO system 26.…”

“…Reports surrounding production of monoclonal antibodies (mAbs) suggest that unique sequence features influence the effectiveness of recombinant protein production 30, 31, 32. For bacterial expression systems, computational tools have been implemented to predict of surface properties to allow rational design of recombinant targets with increased protein solubility of otherwise insoluble or poorly soluble target proteins 29, 33, 34, 35, 36.…”