Feasibility Assessment of a Single-pH Connected Polishing Train for Monoclonal Antibody Purification Using Mildly Acidic Protein A Elution and an Acidic-Window Anion Exchange Membrane.

Monoclonal antibody downstream processing often requires multiple pH adjustments between Protein A capture and polishing steps. These adjustments increase process complexity and make direct unit-operation connection more difficult. We evaluated a connected post-capture polishing block operated at approximately pH 5.0 using mild-pH Protein A elution, an acidic-window anion exchange (AEX) membrane, and cation exchange (CEX) chromatography. Praesto Jetted A50 HipH generated IgG1 eluates near pH 5.0 with conductivity suitable for direct AEX loading. Under standard condition (pH 5.0, 5.0 mS/cm), the AEX membrane reduced xenotropic murine leukemia virus to the assay limit (≥ 3 log10) and minute virus of mice by approximately 2-3 log10. In a connected proof-of-concept run without intermediate pH adjustment, the AEX-CEX polishing block reduced host cell proteins from > 25 to < 10 ng/mg-IgG and high-molecular-weight species from 1.3% to < 1% in the final product pool, maintained low-molecular-weight species below 1%, and achieved 97.6% product recovery. For two additional IgGs, archived strong-AEX flow-through records at pH 7.5 and sequence-derived charge descriptors supported the plausibility of product flow-through at pH 5.0. In a same-IgG1 retrospective comparison, the connected configuration used fewer vessels and conditioning operations and had shorter recipe-derived programmed chromatography time. These findings support process-representative evaluation of single-pH connected polishing.