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25 min of adsorption for four of the six adsorbents. The model underestimated the dynamic Ig breakthough on packed beds of rProtein A Sepharose FF, MabSelect, MBI HyperCel, and MabSorbent A1P, applying a feedstock of 20–100% (v/v) clarified rabbit antiserum. However, when employing a maximum adsorption capacity 25% greater than that determined in batch binding studies, excellent agreement was obtained at all antiserum strengths for most adsorbents. Useful insights into scale-up and process design can be obtained by applying the model, without determining tentative parameters specific for each adsorbent and target protein concentration. However, the model parameters are solvent dependent so a prerequisite for its true applicability is that binding is both Langmuirian and essentially independent of the ionic strength of the feedstock applied.
doi:10.1016/j.chroma.2006.04.083 
Copyright © 2006 Elsevier B.V. All rights reserved.
Evaluation and comparison of alternatives to Protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases
Received 8 October 2005;
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Abstract
In this paper Protein A mimetic and hydrophobic charge induction chromatographic (HCIC) stationary phases are characterized in terms of their protein adsorption characteristics and their selectivity is compared with Protein A chromatography using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were employed to compare the selectivities of these resins for both non-IgG model proteins as well as antibodies and the fusion proteins. While none of the non-IgG model proteins were observed to bind to the Protein A resin, most of them did in fact bind to the alternative resins. In addition, while the elution pH was similar for the model proteins and antibodies on the HCIC resin, the mimetic resins did exhibit higher binding for the antibodies under these linear pH gradient conditions. A mixed mode preparative isotherm model previously developed for HCIC was shown to accurately describe the adsorption behavior of the mimetic materials as well. Host cell protein clearance profiles were also investigated under preparative conditions using complex biological feeds and the results indicated that while some selectivity was observed for both the HCIC and the mimetic materials, the purification factors were in general significantly less than those obtained with Protein A. It is important to note, however, that the selectivity of the mimetic and HCIC materials was also observed to be antibody specific indicating that further optimization may well result in increased selectivities for these materials.
Keywords: Antibodies; Protein A alternatives; Hydrophobic charge induction chromatography and mimetic systems; Selectivity
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Materials
- 2.2. Equipment
- 2.3. Determination of pH of elution
- 2.4. Determination of adsorption isotherms
- 2.5. Protein purification experiments starting from Protein A eluate pools
- 2.6. Protein purification experiments starting from cell culture harvest fluids
- 2.7. Analysis
- 3. Theory
- 4. Results and discussion
- 4.1. Comparison of linear retention for model proteins and Fc containing molecules
- 4.2. Adsorption isotherms on mimetic and HCIC resins
- 4.3. Selectivity comparisons for host cell protein removal starting from Protein A eluate
- 4.4. Selectivity comparisons for host cell protein removal starting from cell culture harvest
- 5. Conclusions
- References
