Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants

Abstract

Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy-and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.

Culture temperature modulates monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures

“…Contrarily, in fed-batch cultures, mAb concentration was decreased with lowering culture temperature due to the rapid decreased in q mAb . Lowering the temperature can improve specific productivity (Marchant et al 2008); however, this effect varies between target proteins and among both cell lines and expression systems (Mason et al 2014). In this study, q mAb was decreased with lowering culture temperature in both batch and fed-batch cultures ( Table 1).…”

“…In our previous study, carboxypeptidase B and carboxypeptidase H have been confirmed as two major basic carboxypeptidases responsible for C-terminal lysine cleavage in CHO cells (Zhang et al 2015). GAPDH, which was used as an internal control, was reported not to be affected by culture temperature (Mason et al 2014). As shown in Fig.…”

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

“…We anticipated that some culture conditions could potentially alter impurity formation. In particular, culture temperature affects transcript levels in CHO cells (Bedoya‐Lopez et al, ; Gomez et al, ; Mason, Sweeney, Cain, Stephens, & Sharfstein, ; Oguchi, Saito, Tsukahara, & Tsumura, ; Yoon, Kim, & Lee, ) that could potentially mediate B‐Ab folding or correct chain assembly. We found culture temperature modulated two product‐related impurities in cell line X: half B‐Ab and high‐molecular‐weight (HMW) species.…”

The relationship of metabolic burden to productivity levels in CHO cell lines

“…Besides recombinant protein yields, the quality of recombinant proteins is another key consideration factor in the biopharmaceutical industry. Although no work on antibody quality is done in this case, the finding that environmental perturbations make an effect on the quality of antibody products should still be taken into consideration . Recently, Mason et al.…”

“…Recently, Mason et al. showed that differential antibody production responses of closely related antibody mutants were found in response to mild hypothermia. Moreover, when culture temperature was reduced, biophysical properties as indicators of protein structure were also changed in antibody variants, suggesting that environmental perturbations may have a greater influence on product quality than previously thought.…”