“Build your own” ADC mimics: Identification of non-toxic linker/payload mimics for HIC-based DAR determination, high-throughput optimization, and continuous flow conjugation

Abstract

This manuscript reports the identification of hydrophobic interaction chromatography (HIC)-shifting, non-toxic linker-payload surrogates as tool molecules for the optimization of maleimide/cysteine conjugations relevant to antibody-drug conjugates (ADCs). These tool molecules are demonstrated to allow conjugation measurement via HIC with a mAb (mono-clonal antibody) bearing engineered cysteines, and for conjugation to mAb interchain cysteines. The linker/payload (LP) mimics were employed to optimize conjugations via high-throughput experimentation and employed to facilitate the development of continuous flow conjugations in a microfluidic reactor and on larger scale. Putative identification of the novel ADC mimics by HIC was confirmed by mass spectrometry. Overall, our studies provide confidence that commercially available, non-toxic LP mimics can be employed successfully to optimize ADC-type conjugations in batch and flow, while minimizing materials needs and experimental work in specialized facilities required for potent compound handling.

“Build Your Own” ADC Mimics: Identification of Nontoxic Linker/Payload Mimics for HIC-Based DAR Determination, High-Throughput Optimization, and Continuous Flow Conjugation

“…Overall, we consider the work described herein (and in the corresponding preprint) to be a valuable addition to the available toolbox for conjugation reaction development. Beyond reaction optimization and continuous flow development, we anticipate that this approach will find use in the development of other technologies.…”

Electrocatalytic Reduction of Disulfide Bonds in Antibodies

“…Electrochemical reduction followed by bioconjugation with a maleimide N-(Mal-PEG 6 )-N-bis(PEG 3 -Boc) in one pot showed the formation of several new species corresponding to cysteine-maleimide adducts by hydrophobic interaction chromatography (HIC). 43 The ratio of antibody to maleimide, referred to as drug to antibody ratio (DAR), is a key attribute of ADCs typically correlated with potency. 4 Mass spectrometry confirmed the formation of the DAR 8 species as the major species, which confirms that VB 12 can reduce all 4 interchain disulfide bonds in IgG1 mAbs successfully, while not interfering with bioconjugation after electrolysis (Figure 3D).…”

Electrocatalytic Reduction of Disulfide Bonds across Chemical Modalities

“…Upon exposure to a slightly modified reaction protocol (Supporting Information), complete electrocatalytic reduction of Zilovertamab to the mAb light and heavy chains was observed by MS, RPLC, and capillary electrophoresis (97% conversion). Electrochemical reduction followed by bioconjugation with maleimide N -(Mal-PEG 6 )- N -bis­(PEG 3 -Boc) in one pot showed the formation of cysteine–maleimide adducts, as determined by hydrophobic interaction chromatography (HIC) . The antibody:maleimide ratio, termed the drug:antibody ratio (DAR), is a key attribute of ADCs that typically correlates with potency .…”