Development and Optimization of a Hollow Fiber‐Based Countercurrent Dialysis Process for Continuous Manufacturing of Monoclonal Antibodies

ABSTRACT Countercurrent dialysis using hollow fiber modules offers several advantages over both inline diafiltration (ILDF) and hemodialyzer based countercurrent dialysis in terms of buffer requirements and compatibility with GMP manufacturing of biologics. In this work, we evaluate GMP‐grade hollow fiber modules of varying lengths (20, 41.5, and 65 cm) and benchmark their performance against an ILDF unit as a reference process for continuous exchange of buffer in manufacturing of monoclonal antibodies. Hollow fiber modules outperformed the ILDF configuration in terms of buffer consumption and pressure build‐up, particularly for higher‐titer processes. The GMP hollow fiber modules, which are characterized using protein‐based molecular weight cut‐off (MWCO) specifications, achieved buffer exchange comparable to the reference processes when using endogenous excipients (histidine, methionine) as tracers, while providing a bioprocess‐oriented format and documentation suitable for GMP implementation. A D‐optimal experimental design was used to define the design space for the hollow fiber countercurrent dialysis step by varying the ratio of dialysate buffer to feed flow (“alpha factor”, 2–5), normalized feed flow rate (0.05–1.5 L·m −2 ·h −1 ), protein concentration (30–70 mg/mL), and feed conductivity (2–15 mS/cm). The results showed that higher feed concentrations narrowed the acceptable range of normalized feed flow rates and required higher alpha factors to achieve the target buffer exchange. A 5‐day continuous run operated at the selected process set‐point achieved the desired buffer exchange targets with > 95% product yield, no significant impact on aggregation, and transmembrane pressures below 12 psi (≈80 kPa). Scale‐up analysis indicated that the process is suitable for GMP manufacturing at scales up to 3000 L perfusion bioreactors with perfusate titers ranging from 0.6 to 5 mg/mL, while operating within a robust design space.