Surface Energetics of Protein Adsorption on to Chromatographic Supports

Muhammad Aasim(1), Poondi Rajesh Gavara(2), Rami Reddy Vennapusa(3), Marcelo Fernandez Lahore(4*)

(1) Jacobs University Bremen, Germany
(2) Jacobs University Bremen, Germany
(3) Jacobs University Bremen, Germany
(4) Jacobs University Bremen, Germany
(*) Corresponding author

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Protein separation behavior during adsorption chromatography is governed by system thermodynamics and kinetic factors. Hydrophobic interaction chromatography (HIC) is widely utilized since many important biological products present a quite hydrophobic character. In this work, the interaction between a set of model proteins (n = 9) and a commercial adsorbent (Phenyl Sepharose FF, high substitution, GE Healthcare) was studied via extended DLVO (XDLVO) calculations. Psychochemical properties of both separand and adsorbent were gathered by contact angle determination and zeta potential measurements. Proteins were subjected to the mentioned measurements in the hydrated and the dehydrated state, so as to simulate protein properties in a low vs. high salt concentration milieu, respectively. In HIC, protein adsorption usually take place at high concentrations of ammonium sulphate (up to 1.7M) and protein desorption occurs by decreasing salt concentration in the mobile phase. The mentioned XDLVO approach allowed the calculation of the free energy of interaction vs. distance profiles between the interacting surfaces, in the aqueous environment provided by the operating mobile phase. XDLVO calculations were correlated with the actual chromatography behavior of the studied model proteins. This correlation revealed that these proteins can be segregated in two main groups, according to surface energy calculations and elution position during chromatography: i) strong binding showing a deeper secondary minimum energy >|0.20| kT ii) and weak binding having a small secondary minimum energy <|0.12| kT, thus calculations were able to predict early or late elution from a gradient chromatography experiment; the more the calculated interaction energy, the stronger will be protein binding and the later will be the elution time. The knowledge generated from these studies will generate a better understanding of real downstream bioprocess behavior which could, in turn, facilitate process design and optimization.
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Contact Angle of Proteins; Protein Adsorption; Surface Energies of Proteins

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R. R. Vennapusa, C. Tari, R. Cabrera, M. Fernandez-Lahore, Surface energetics to assess biomass attachment onto hydrophobic interaction adsorbents in expanded beds, Biochem. Eng. J. 43 (2009) 16-26.

R. R. Vennapusa, S. M. Hunegnaw, R. B. Cabrera, M. Fernandez-Lahore, Assessing adsorbent-biomass interactions during expanded bed adsorption onto ion exchangers utilizing surface energetics, J. Chromatogr. A 1181 (2008) 9-20.

A. Jungbauer, C. Machold, R. Hahn, Hydrophobic interaction chromatography of proteins: III. Unfolding of proteins upon adsorption, J. Chromatogr. A 1079 (2005) 221-228.

M. E. Lienqueo, A. Mahn, J. C. Salgado, J. A. Asenjo, Current insights on protein behavior in hydrophobic interaction chromatography, J. Chromatogr. B 849 (2007) 53-68.

J. A. Queiroz, C. T. Tomaz, J. M. S. Cabral, Hydrophobic interaction chromatography of proteins, J. Biotechnol. 87 (2001) 143-159.

B. Lee, F. M. Richards, The interpretation of protein structures: estimation of static accessibility, J. Mol. Biol. 55 (1971) 379-400.

C. J. van Oss, R. J. Good, M. K. Chaudhury, Solubility of proteins, J. Protein Chem. 5 (1986) 385-405.

H. Stellan, Some general aspects of hydropbobic interaction chromatography, J. chromatogr. B. (1973) 325-331.

C. J. Van Oss, L. L. Moore, R. J. Good, M. K. Chaudhury, Surface thermodynamics properties and chromatographic and salting out behavior of IgA and other serum proteins, J. Protein Chem. 4 (1985) 245-263.

C. J. van Oss, R. F. Giese, Role of the properties and structure of liquid water in colloidal and interfacial systems, J. dispersion Sci. Tech 25 (2004) 631-655.

P. K. Sharma, R. K. Hanumantha, Analysis of different approaches for evaluation of surface energy of microbial cells by contact angle goniometry, Adv. Colloid Interface Sci. 98 (2002) 341-463.

A. H. Weerkamp, H. M. Uyen, H. J. Busscher, Effect of zeta potential and surface energy on bacterial adhesion to uncoated and saliva-coated human enamel and dentin., J. Dent. Res. 67 (1988) 1483-1487.

C. J. van Oss, R. J. Good, Orientation of the water molecules of hydration of human serum albumin, J. Protein Chem. 7 (1988) 179-183.

C. J. van Oss, R. J. Good, Surface tension and the solubility of polymers and biopolymers: The role of polar and apolar interfacial free energies, J. Macromolec. Sci. 26 (1989) 1183-1203.

C. J. van Oss, Interfacial forces in aqueous media (Taylor and Francis, 2006).

A. E. Wiacek, E. Chibowski, Zeta potential and droplet size of n-tetradecane/ethanol (protein) emulsions, Colloids Surf. B. Biointerfaces 25 (2002) 55-67.

H. Bak, O. R. T. Thomas, Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum, J. Chromatogr. B 848 (2007) 116-130.

C. J. van Oss, Interfacial forces in aqueous media (Marcel Dekker, 1994).

H. C. van der Mei, R. Bos, H. J. Busscher, Physico-chemistry of initial microbial adhesive interactions: its mechanisms and methods for study, FEMS Microbiol. Rev. 23 (1999) 179-230.

C. D. Volpe, S. Siboni, Some reflections on acid-base solid surface free energy theories, J. Colloid Interface Sci. 195 (1997) 121-136.

C. J. van Oss, Hydrophobicity and hydrophilicity of biosurfaces, Curr. Opin. Colloid. Interfac. Sci 2 (1997) 503-512.

C. J. van Oss, R. J. Good, M. K. Chaudhury, Determination of the hydrophobic interaction energy-application to separation processes, Separation Science and Technology 22 (1987) 1-24.

K. Michael, Protein liquid chromatography (Elsevier science BV, 2005).

A. J. Rowe, Probing hydration and the stability of protein solutions - a colloid science approach, Biophys. Chem. 93 (2001) 93-101.

C. M. Roth, A. M. Lenhoff, Electrostatic and van der Waals contributions to protein adsorption: computation of equilibrium constants, Langmuir 9 (1993) 962-972.

C. J. van Oss, R. F. Giese, P. M. Bronson, A. Docoslis, P. Edwards, W. T. Ruyechan, Macroscopic-scale surface properties of streptavidin and their influence on aspecific interactions between streptavidin and dissolved biopolymers, Colloids Surf. B. Biointerfaces 30 (2003) 25-36.


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