Antibody Drug Conjugate Characterization


An antibody is a challenging molecule to characterize due to its size and complexity. Coupling a drug payload is an attractive approach to fighting diseases such as cancer but increases yet further the complexity and risk for heterogeneity. Antibody drug conjugates (ADCs) require careful and detailed characterization in the same way as any other IgG based biopharmaceutical, and appropriate analytical workflows are required. FabRICATOR digestion of ADCs into their subunits allows detailed characterization of this complex class of drugs. The endoglycosidases IgGZERO and GlycINATOR also makes it possible to study the intact ADC but reducing the complexity and increasing the resolution.



Subunit Analysis of ADCs

The characterization of ADCs can be challenging due to the complexity of the conjugated antibody, while analysis at the intact level limits the resolution of LC-MS analysis. The FabRICATOR enzyme is a site-specific protease that digests IgG below hinge for middle-level analysis of native or conjugated antibodies. The ADCs are simply incubated with FabRICATOR enzyme and reduced to generate smaller (25 kDa) Fc/2, Fc' and LC antibody fragments with reduced complexity for high-resolution middle-level LC-MS (Fig. 1). The homogenous antibody fragments generated using FabRICATOR allows for analysis of critical quality attributes such as the drug-antibody ratio (DAR), site-occupancy and ADC biotransformation.

Blog Highlights on ADC Subunit Analysis

Figure 1. Middle-level LC-MS analysis of trastuzumab site-specifically conjugated with MMAE (monomethyl auristatin E) using  GlyCLICK. The antibodies were digested with FabRICATOR and reduced before the analysis showing a)  native, b)  deglycosylated, c) conjugated trastuzumab.

Intact ADC Characterization

Characterization can also be improved using endoglycosidases for trimming of the Fc N-glycans to further reduce the sample complexity in ADC characterizations. By applying IgG-specific endoglycosidases such as IgGZERO or GlycINATOR, the ADC can rapidly be stripped from the major glycan complexity and the intact ADC can be studied using native-MS, IM-MS or top-down MS to determine the ratio of drugs per antibody. The reduced complexity in this approach leads to increased resolution when studying the intact ADC and have been applied by several research groups. The endoglycosidases can also be combined with FabRICATOR digestion for reduced sample complexity in middle-level strategies.





Figure 2. Schematic representation of intact LC-MS analysis of ADCs after deglycosylation using GlycINATOR.

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