Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry

https://doi.org/10.1016/j.ab.2011.01.004Get rights and content

Abstract

Antibody–drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography–mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB–drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.

Section snippets

Biotinylation of target antigen

A NAP-5 column (GE Healthcare, Piscataway, NJ, USA) was equilibrated with 10 ml of sodium bicarbonate buffer (pH 7.8) (Sigma–Aldrich). A 0.5-ml aliquot of the target antigen (e.g., human MUC16 extracellular domain [ECD], Genentech, South San Francisco, CA, USA) at a concentration of approximately 4 mg/ml was loaded onto the column for buffer exchange and then eluted by 1 ml of sodium bicarbonate. Subsequently, the buffer-exchanged MUC16 ECD (∼1 ml) was mixed with approximately 13 μl of 5 mg/ml

Method development of affinity capture LC–MS

Although site-specific conjugation allows for a tightly controlled stoichiometry of DAR 2 in TDCs, it is possible that the covalently bound drugs may be released with time in vivo, resulting in the formation of conjugates carrying fewer drugs (i.e., DAR 1) or no drugs (i.e., DAR 0). Similarly, other conventional ADCs with higher initial DARs may result in lower DARs in vivo. Fig. 1 shows a schematic of intact ADCs species (using the TDC as a model) in plasma or serum analyzed by affinity

Discussion

Release of conjugated cytotoxic drugs from ADCs can be a concern because this process changes the drug load of the ADC and potentially affects the pharmacokinetics, efficacy, and safety. Using the affinity capture LC–MS method, we were able to directly characterize the drug release process that occurred in a model using engineered site-specific anti-MUC16 TDC in plasma and serum both in vitro and in vivo for the first time. Formation of the DAR 0 and DAR 1 species was effectively detected as

Acknowledgments

The authors thank John Lowe, Valerie Quarmby, An Song, Patricia Siguenza, and Paul Fielder for their assistance and support in this research. We also thank John Stults and Valerie Quarmby for their critical review of the manuscript.

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