Synergistic viral inactivation platform: Rapid enveloped virus inactivation at mildly acidic pH using low non‐ionic detergent in Protein A eluates

Abstract

Low‐pH viral inactivation (LPVI) is a widely used, validated unit operation for enveloped virus clearance in antibody manufacturing. However, acid exposure during Protein A (ProA) elution and subsequent low‐pH holds can destabilize some antibodies and Fc‐containing modalities, increasing aggregation risk and product loss—a challenge that is particularly acute for multi‐specific antibody formats and other pH‐labile Fc‐containing modalities. Mild‐pH ProA capture strategies protect such molecules, but the resulting higher‐pH eluates often require re‐acidification to meet conventional LPVI setpoints, reintroducing low‐pH stress. Here we evaluate a dual‐mechanism strategy in which mild acidity is combined with low concentrations of non‐ionic detergent to accelerate inactivation without prolonged exposure to strongly acidic conditions. Using xenotropic murine leukemia virus (X‐MuLV) spiked into ProA eluates, 0.10% N‐methylglucamide (Mega‐10) at pH 3.91 reduced infectivity to below the assay limit of detection within 2.5 min (log10 reduction factor [LRF] ≥ 5.40), whereas low pH alone or detergent alone produced substantially lower reductions over the same interval. Similarly, 0.15% Mega‐10 at pH 4.05 achieved nondetectable infectivity within 10 min (LRF ≥ 5.81). Comparable time compression was observed with Tween 80 or Tween 20 at pH ~3.97, reducing time‐to‐nondetectable infectivity from 120 min (low pH alone) to 5 min. The operating window was tunable through paired adjustment of detergent concentration and pH, and feasibility was demonstrated at pH 4.56 with low detergent levels, offering a practical solution for pH‐labile products and higher‐pH eluates.