Native Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine Residues

Abstract

We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic-mass spectrometric (LC-MS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissociation of conjugated HC and LC during chromatographic separation and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…ESI-MS is also amenable to analyze intact disulfide-linked ADC, as demonstrated by Vallerie-Douglass et al Using mild buffer conditions and by deconvoluting the resulting spectra, they were able to resolve individual forms of MMAF-conjugated antibodies ( Fig. 9) (89). The observed DAR results were consistent with results from an orthogonal HIC analysis.…”

Development of a Native Nanoelectrospray Mass Spectrometry Method for Determination of the Drug-to-Antibody Ratio of Antibody–Drug Conjugates

“…Recently, Valliere-Douglass et al 1 described a native MS method for analysis of reduced disulfide bond cysteine-linked ADCs. Here, we report another approach which, in addition to the native MS, employs limited enzymatic treatment and nanoelectrospray ionization (nano-ESI) to achieve optimized ionization, sensitivity, and determination of DAR for ADCs.…”

Formulation Development for Antibody-Drug Conjugates

“…The cysteine-linked ADC with the conjugation sites at the sulfhydryl groups generated from nonspecific reduction of interchain disulfide bonds of the antibody such as brentuximab vedotin typically generates a mixture of ADCs with zero to eight drugs per antibody (Ducry 2013;Valliere-Douglass et al 2012). The DARs of lysine-linked ADC such as trastuzumab emtansine being conjugated through the lysine residues of the antibody ranges from DAR 0 to DAR 9 (Flygare et al 2013).…”