Frontiers in Bioengineering and Biotechnology (Aug 2023)

Decreasing hydrophobicity or shielding hydrophobic areas of CH2 attenuates low pH-induced IgG4 aggregation

  • Qiang Wu,
  • Qiang Wu,
  • Chunlai Cao,
  • Chunlai Cao,
  • Suzhen Wei,
  • Hua He,
  • Kangyue Chen,
  • Lijuan Su,
  • Qiulian Liu,
  • Shuang Li,
  • Yongjie Lai,
  • Jing Li

DOI
https://doi.org/10.3389/fbioe.2023.1257665
Journal volume & issue
Vol. 11

Abstract

Read online

Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.

Keywords