IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains

Area of Science:

• Biotechnology
• Protein Engineering
• Cell Line Development

Background:

• Protein aggregates in therapeutic IgG monoclonal antibodies (mAbs) compromise safety and efficacy.
• Previous studies link mAb aggregation in Chinese hamster ovary (CHO) cells to an overabundance of heavy chain (HC) relative to light chain (LC).

Purpose of the Study:

• To investigate the formation and characteristics of aggregates in CHO cells with excess HC.
• To identify strategies for reducing mAb aggregation in cell culture.

Main Methods:

• Analysis of intracellular and secreted aggregates in CHO cells with varying HC and LC expression levels.
• Genetic modification of CHO cells, including mutation of a free cysteine on HC and re-transfection with BiP (an IgG molecular chaperone) or LC.

Main Results:

• Aggregates, primarily composed of HC, formed intracellularly and were secreted into the supernatant.
• Preventing disulfide bonding via cysteine mutation did not reduce aggregation.
• Overexpressing BiP partially reduced aggregates and fragments by enhancing proper assembly or degradation.
• Successfully increasing LC expression to exceed HC levels completely eliminated aggregates and fragments.

Conclusions:

• mAb aggregation in CHO cells with excess HC results from limited chaperone availability and an imbalanced LC:HC ratio.
• Balancing LC and HC expression is critical for developing CHO cell lines with reduced protein aggregation.
• These findings offer insights for optimizing mAb production and improving therapeutic product quality.