Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

Abstract

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

Design and Production of Bispecific Antibodies

“…Indeed, for in vivo assembly, the efficient co-expression of engineered heavy and light chains relies deeply on the selection of stably expressing cell clones. Plus, culture conditions such as temperature also affect the half-antibody expression and aggregate formation [184]. In contrast, following downstream Protein A purification of CH3 mutants, in vitro assembly (separate expression of two different heavy-chain types followed by mixing them at appropriate redox conditions to induce assembly) have demonstrated their capability in generating high-quality molecules.…”

Research Update on Bioreactors Used in Tissue Engineering

“…Although heat shock protein 70 (HSP70) can delay the expression of caspase-3, increase the lifespan of the cells, and protect the cells, it can arrest the G2/M phase cell cycle, which occurs at a temperature no higher than the optimal cell culture temperature. It is also conducive to the production of mAbs [32][33] . When the culture temperature is lower than the optimal temperature, cell viability and lifespan are also affected.…”

Development of Therapeutic Antibody Technology And Recent Perspectives For Leukemia-Lymphoma Treatment

“…A bispecific antibody has two different light chains and two different heavy chains pair via engineered interchain interactions. Furthermore, bispecific antibodies have high efficacy for cytotoxicity, high affinity and small in size (easily penetrable to tissues) [64][65].…”