Investigation into temperature-induced aggregation of an antibody drug conjugate.
Conjugation of an antibody to a drug can produce heterogeneous species that may have different physical stabilities and safety profiles. We explored the effect of thermal stress on the physical stability, specifically aggregation, of an antibody drug conjugate (ADC), ADC 1, wherein the antibody was linked to the val-cit-Monomethyl Auristatin E (vc-MMAE) linker drug through the reduction of interchain disulfides. We also explored the effects of conjugation on the secondary and tertiary structures of ADC 1. Circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry showed that for species with high drug loading, conjugation does not measurably alter the secondary structure, but it does render the CH2 domain less stable to thermal stress such that ADC 1 rapidly forms high molecular weight species (HMWS) at 40 °C. Characterization of the HMWS using chromatographic and electrophoretic methods showed that it is an irreversible, noncovalent, and structurally altered form of ADC 1 primarily composed of molecules with six or eight drugs. Furthermore, the variable domain of the antibody may contribute to the extent of aggregation, since eight ADCs with over 90% sequence homology exhibited monthly rates of HMWS formation that differ by up to a factor of 2.
Early Stage Pharmaceutical Development, ‡Purification Development, and §Late Stage Pharmaceutical and Process Development, Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
Nia S
NS
Beckley
Kathlyn P
KP
Lazzareschi
Hung-Wei
HW
Chih
Vikas K
VK
Sharma
Heather L
HL
Flores
D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
D061067:Antibodies, Monoclonal, Humanized; D004220:Disulfides; D018796:Immunoconjugates; D007074:Immunoglobulin G; D009842:Oligopeptides; C495575:monomethyl auristatin E