Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography.
ABSTRACT A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.
- SourceAvailable from: Rahul Bhambure[show abstract] [hide abstract]
ABSTRACT: This paper describes the development of a HTPD platform for process development of chromatography steps. An assessment of the platform as a guidance tool for establishing the "characterization space" for an ion exchange chromatography step has been performed by using design of experiments (DOE). Case studies involving use of a biotech therapeutic, Granulocyte Colony Stimulating Factor (GCSF), have been used to demonstrate the performance of the platform. We discuss the various challenges that arise when working at such small volumes along with the solutions that we propose to alleviate these challenges in order to make the HTPD data suitable for empirical modeling. Further, we have also validated the scalability of this platform by comparing the results from the HTPD platform (2 µl and 6 µl resin volumes) against those obtained at the traditional laboratory scale (0.5 mL resin volume). We find that after integration of the proposed correction factors, the HTPD platform is capable of performing the process optimization studies at 170 fold higher productivity. The platform is capable of providing semi-quantitative assessment of the effects of the various input parameters under consideration. We think that platform such as the one presented is an excellent tool for examining the "characterization space" and reduce the extensive experimentation at the traditional lab scale that is otherwise required for establishing the "design space". Thus, this platform will specifically aid in successful implementation of Quality by Design (QbD) in biotech process development. This is especially significant in view of the constraints with respect to time and resources that the biopharma industry faces today. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.Biotechnology Progress 02/2013; · 1.85 Impact Factor