Competitive diffusion between protein and virus in continuous biopharmaceutical virus filtration

초록

Viral contamination represents a critical risk in the downstream processing of biopharmaceuticals. To ensure the viral clearance, virus filtration is widely used due to its excellent capacity of virus removal via a size exclusion mechanism. However, under low-flux conditions, where Brownian diffusion becomes the predominant process, a decrease in viral clearance has been observed. The objective of this study is to investigate the virus breakthrough behavior in such low-flux conditions and to develop a mechanistic model based on experimental data for ensuring robust viral safety. The protein-modulated virus breakthrough was systematically quantified as a function of protein and virus concentrations, as well as filtrate flux, using a double-layer virus filter and MS2 bacteriophage. The results indicated that under diffusion-dominated low-flux conditions (i.e., ∼10 LMH), the co-filtration of high protein concentrations (i.e., 10–20 g/L) effectively reduced virus breakthrough, maintaining below 1 log10 throughout the filtration. In the absence of protein, a significant virus breakthrough occurred. Notably, this observation differs from conventional high-flux operations (∼500 LMH), where the co-filtration of protein resulted in immediate virus breakthrough (> 2 log10). Furthermore, an internal polarization model incorporating protein-dependent effective virus sieving yielded a diffusion-limited breakthrough threshold of ∼8.6 log10 PFU/m2. This phenomenon is attributed to a competitive diffusion mechanism, wherein protein molecules reversibly occupy and dominate the effective pathways due to their higher diffusivity. These results provide important insights into the mechanisms underlying virus breakthrough behavior in low-flux conditions for continuous biomanufacturing processes. © 2026 Elsevier B.V.

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