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A systematic selection of different transmembrane pressures (TMP) and levels of diafiltration (DF) was studied to optimize these critical process parameters during the manufacturing of micellar casein concentrate (MCC) using spiral-wound polymeric membrane filtration. Three TMPs (34.5, 62.1, and 103.4 kPa) and four DF levels (0, 70, 100, and 150%) were applied in the study. The effect of the TMP and DF level on flux rates, serum protein (SP) removal, the casein-to-total-protein ratio, the casein-to-true-protein ratio, and the rejection of casein and SP were evaluated. At all transmembrane pressures, the overall flux increased with increases in the DF level. The impact of DF on the overall flux was more pronounced at lower pressures than at higher pressures. With controlled DF, the instantaneous flux was maintained within 80% of the initial flux for the entire process run. The combination of 34.5 kPa and a DF level of 150% resulted in 81.45% SP removal, and a casein-to-true-protein ratio of 0.96. SP removal data from the lab-scale experiments were fitted into a mathematical model using DF levels and the square of TMPs as factors. The model developed in this study could predict SP removal within 90–95% of actual SP removal achieved from the pilot plant experiments.