Leveraging Mixed-Mode Filter During Cell Culture Harvest to Prevent Antibody Reduction.

Abstract

Disulfide bond reduction was observed for a monoclonal antibody (mAb1) produced in a Chinese Hamster Ovary (CHO) process, driven primarily by the thioredoxin (Trx)-thioredoxin reductase (TrxR) system present in harvested cell culture fluid (HCCF). To address this issue, we evaluated the Solventum Polisher ST (PST) depth filter as a secondary harvest-stage filtration step designed to remove redox-active enzymes and improve product stability. PST filtration reduced Trx and glucose-6-phosphate 1-dehydrogenase (G6PD) by approximately 1log at loadings up to 150L/m² and non-detectable TrxR at loadings up to 100L/m², as demonstrated by mass spectrometry. Protein biophysical property calculations indicated strong electrostatic attraction between Trx and mAb1 that may facilitate enzyme-antibody co-elution through conventional quaternary amine filters. Compared with dilution, KMnO₄ oxidation, and reversible inhibition using L-cystine, PST filtration most effectively suppressed disulfide bond reduction over seven days at room temperature. Pre-spiking HCCF with guanidinium-containing compounds prior to PST further extended mAb1 stability in the 100-200L/m² fraction from 19.5h to 72.5-95.3h, confirming the functional role of guanidinium groups in mitigating reduction. Scalability up to 50L pilot batch assessments supported consistent performance at larger volumes, demonstrating PST filtration as a practical and robust approach for controlling enzymatic reduction during bioprocess harvest operations.