Protein-solute interactions affect the outcome of ultrafiltration/diafiltration operations

Protein production operations often involve a final diafiltration of the protein into formulation buffer. For several Amgen product proteins, post-diafiltration assays revealed a significant difference in molar excipient concentrations on the retentate and the permeate side of the membrane. For example, post-diafiltration assays of formulated 200 mg/mL human interleukin-1 receptor antagonist showed molar chloride concentrations up to 30% lower than those of the diafiltration buffer. Deviations from expected results were also observed in cases where a fusion conjugate protein (AMG-719) was formulated by dialysis in 10 mM acetate and where PEGylated soluble tumor necrosis factor receptor (PEG-sTNF-RI) was formulated in 270 mM glycine and 10 mM histidine. Classical thermodynamic theory describing intermolecular interactions predicts that the partitioning of small solutes during dialysis will be dependent on the protein concentration, charge, and surface area. This study illustrates methods to approximate these effects using readily available protein data (theoretical titration curves based on protein sequence, density information, etc.). Additionally, guidelines are provided to determine when intermolecular interactions are likely to significantly impact the outcome of dialysis/diafiltration operations. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.