1. Academic Validation
  2. Agitation-Induced Aggregation of Lysine- and Interchain Cysteine-Linked Antibody-Drug Conjugates

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

  • J Pharm Sci. 2023 Dec 7:S0022-3549(23)00511-7. doi: 10.1016/j.xphs.2023.12.003.
Florian Johann 1 Steffen Wöll 2 Matthias Winzer 2 Henning Gieseler 3
Affiliations

Affiliations

  • 1 Friedrich-Alexander University (FAU) ErlangenNürnberg, Department of Pharmaceutics, Freeze Drying Focus Group (FDFG), Cauerstraße 4, 91058 Erlangen, Germany; Merck KGaA, Global CMC Development, Frankfurter Straße 250, 64293 Darmstadt, Germany.
  • 2 Merck KGaA, Global CMC Development, Frankfurter Straße 250, 64293 Darmstadt, Germany.
  • 3 Friedrich-Alexander University (FAU) ErlangenNürnberg, Department of Pharmaceutics, Freeze Drying Focus Group (FDFG), Cauerstraße 4, 91058 Erlangen, Germany; GILYOS GmbH, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany. Electronic address: info@gilyos.com.
Abstract

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.

Keywords

antibody–drug conjugate(s) (ADC); conjugation; developability; high-throughput technology(s); monoclonal antibody(s); physical stability; protein aggregation.

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