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

Abstract

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.

Change in the Kinetic Regime of Aggregation of Yeast Alcohol Dehydrogenase in the Presence of 2-Hydroxypropyl-β-cyclodextrin

“…It can be assumed that the mechanism of action of these agents may be similar. Sucrose can also stabilize proteins and suppress aggregation by preferential exclusion of water [56] and shielding of hydrophobic regions [57], and it should be taken into account when interpreting its effects and comparing them with 2-HP-β-CD.…”

“IgG’s: contending with aggregating circumstances”

“…14 Parallel needs exist for tractable and informative approaches for assaying IgG aggregation, together with approaches for preparing aggregated IgG's as comparative controls. For the latter (and as an IgG aggregation model), transient exposure to low pH (as relevant to producer cell culture and IgG purification) is commonly and effectively employed to induce IgG aggregation [15][16][17][18] (with heat, agitation, shear force, and high pressure as additional options). Assays of aggregated IgG's include first those which can be essentially 2-step, and report on relative frequencies of aggregates.…”