Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography.
Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption-desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.
Muca
Renata
R
Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Str., 2, 35-959 Rzeszow, Poland.
Marek
Wojciech
W
Piatkowski
Wojciech
W
Antos
Dorota
D
D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000295:instrumentation* / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D017510:Protein Folding; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry*; D013816:Thermodynamics
D012997:Solvents; D012710:Serum Albumin, Bovine; D009113:Muramidase