Agitation-Induced Aggregation of Lysine- And Interchain Cysteine-Linked Antibody-Drug Conjugates

• Published in: Journal of pharmaceutical sciences Vol. 113; no. 5; pp. 1265 - 1274
• Main Authors: Johann, Florian, Wöll, Steffen, Winzer, Matthias, Gieseler, Henning
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2024
• Subjects: Antibodies, Monoclonal; Antibody–drug conjugate(s) (ADC); Conjugation; Cysteine; Developability; High-throughput technology(s); Hydrophobic and Hydrophilic Interactions; Immunoconjugates; Lysine; Monoclonal antibody(s); Physical stability; Protein aggregation; Protein aggregation; High-throughput technology(s); Developability; Antibody–drug conjugate(s) (ADC); Conjugation; Monoclonal antibody(s); Physical stability
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/j.xphs.2023.12.003

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody–drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability.