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 degrees C exhibited =5% aggregates at harvest. Aggregate levels increased 412-fold in 33 degrees C cultures when compared to 37 degrees C, with a corresponding 24-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37 degrees C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 2575% at 33 degrees C versus 37 degrees C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33 degrees C for this cell line. Finally, we identified a 25-fold increase in mAb aggregate formation at 33 degrees C compared to 37 degrees 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. Biotechnol. Bioeng. 2012;109: 125136. (c) 2011 Wiley Periodicals, Inc.