Profiling Antibody Drug Conjugate Positional Isomers: A System-of-Equations Approach

Abstract

Antibody drug conjugates enable the targeted delivery of potent chemotherapeutic agents directly to cancerous cells. They are made by the chemical conjugation of cytotoxins to monoclonal antibodies, which can be achieved by first reducing interchain disulfide bonds followed by conjugation of the resulting free thiols with drugs. This process yields a controlled, but heterogeneous, population of conjugated products that contains species with various numbers of drugs linked to different former interchain disulfide cysteine residues on the antibodies. We have developed a mathematical approach using inputs from capillary electrophoresis and hydrophobic interaction chromatography to determine the positional isomer distribution within a population of antibody drug conjugates. The results are confirmed by analyzing isolated samples of specific drug-to-antibody ratio species. The procedure is amenable to rapid determination of positional isomer distributions and features low material requirements. A survey of several antibody drug conjugates based on the same IgG framework and small molecule drug combination has shown a very similar distribution of isomers among all of the molecules using this technique, suggesting a robust conjugation process.

Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody–Drug Conjugates

“…Our characterization of the IgG1 ADC conforms to previous reports detailing the distribution of the various drug‐loaded species in the intact ADC . To our knowledge, the detailed characteristics of the IgG2 conjugation profile have not been previously reported.…”

“…The expected fragments were detected in both IgG1 and IgG2 ADCs. The IgG1 ADC contains larger relative amounts of fragment species coupled with a decrease of the intact species, similar to profiles reported for other vcMMAE‐conjugated IgG1 antibodies . In contrast, the majority of the IgG2 ADC was detected in its intact form.…”

Developments and Challenges for mAb-Based Therapeutics

“…Drugs can be linked to inter-chain disulfides following reduction [173]. Nonetheless, the use of disulfides for conjugation may still yield a heterogeneous population of conjugated products that contains positional isomers [174]. The heterogeneity derives from the different tendencies of disulfides for reduction during the conjugation process.…”

Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

“…With 80-100 lysine and only 8 interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation this despite the similar overall level of drugs incorporated per antibody (between 0 and 8 with an average of 3-4) [6]. Chromatography and mass spectrometry are at the forefront in ADC analysis and critical characteristics such as drug-to-antibody ratio (DAR), drug distribution and conjugation sites can be extracted out of the generated data [7,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. 1.…”