Culture temperature modulates aggregation of recombinant antibody in cho cells

Abstract

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

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

“…It has a direct influence on cell growth, nutrient consumption and productivity (Fan et al 2010;Marchant et al 2008). Moreover, temperature is to be associated with product quality, including glycosylation patterns (Ahn et al 2008), aggregation (Gomez et al 2012) and cysteine variants (Gomez et al 2010). However, the effect of culture temperature on mAb charge variation and the mechanism of how hypothermic condition affects charge variation are rarely known.…”

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

“…In the case of LC and HC mRNA abundance, the assembly and folding of antibody can be limited by the low expression of the ER proteins. It has been shown that low expression of ER proteins in a small ER volume can result in antibody aggregation [34].…”

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

“…Our experiments supported these results by demonstrating that low H chain polypeptide synthesis limits Ab production. Several other publications have studied some effects of H and L chain imbalance on Ab folding and protein production (Gomez et al 2012;Li et al 2005; Schlatter et al 2005; Sung et al 2004). Li et al (2005) have demonstrated using bi-cistronic constructs of the encephalo myocarditis virus internal ribosome entry site, a significant increase in Ab production in HEK293T cells when the expression of H chains exceeded that of L ones.…”