posted on 2023-11-30, 13:34authored byPhillip
Y. Chu, Hui Wang, Emily Ross, Nicole Stephens, Hui-Min Zhang, Nisana Andersen, Wayman Chan, Vittal Shivva, Susan R. Crowell, Christoph Spiess, Patrick G. Holder, Nicholas J. Agard, Chengjie Ji, John Chen, Alavattam Sreedhara, Jianyong Wang, Cong Wu, Yichin Liu, John C. Tran
Biotransformation leading to single residue modifications
(e.g.,
deamidation, oxidation) can contribute to decreased efficacy/potency,
poor pharmacokinetics, and/or toxicity/immunogenicity for protein
therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or
AAPH for oxidation are commonly used for predicting liabilities in
manufacturing and storage and are sometimes considered a predictive
tool for in vivo liabilities. However, reports discussing
their in vivo translatability are limited. Herein,
we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically
relevant compartments for diverse protein therapeutic modalities.
The workflow has low bias of <10% in quantitating degradation in
the relevant pharmacokinetic concentration range for monkey and rabbit
serum/plasma (1–100 μg/mL) and allows for high sequence
coverage (∼85%) for discovery/monitoring of amino acid modifications.
For oxidation and deamidation, the assay was precise, with percent
coefficient of variation of <8% at 1–100 μg/mL and
≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation
on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not
observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false
positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also
tested but resulted in limited success (1 out of 4 molecules) in predicting
oxidation liabilities.