Abstract In the downstream processing of antibody‐based therapeutics, ultrafiltration/diafiltration (UF/DF) is commonly applied for concentration and buffer exchange in the final formulation. For a given molecule, various factors such as membrane type, feed flux, and transmembrane pressure (TMP) can significantly influence the performance of UF/DF, impacting yield, buffer exchange efficiency, and product quality. Conventional membrane pore size selection is based on product molecular weight to ensure high retention. While working on an Fc‐fusion protein, we found that the pH of load material had a critical effect on the retention of the molecule due to conformational changes at different pH values, as evidenced by the size‐exclusion chromatography (SEC). Meanwhile, optimization of the UF/DF process underscored the importance of concentration polarization to protein retention. Approaches to reduce concentration polarization, such as increasing feed flux and lowering TMP, resulted in less protein loss in the permeate stream. High retention of this Fc‐fusion protein during the UF/DF step can be achieved not only by utilizing a 5 kDa membrane but also by employing a 10 kDa membrane with optimized process parameters such as load conditions, feed flux, and TMP. These observations provide important insights on the factors impacting protein retention beyond the molecular weight cutoff (MWCO) of UF/DF membrane.