Protein purification process

Londo, T., Lynch, R, Kehoe, T., Meys, M., and Gordon, N., Accelerated recombinant protein purification process development. Automated, robotics-based intergration of chromatographic purification and analysis, /. Chromatogr. A, 798, 73, 1998. 

Compared with isolated enzymes, application of whole cells as biocatalysts is usually more economical since there is no protein purification process involved. Whole cells can be used directly in chemical processes, thereby greatly minimizing formulation costs. Whole cells are cheap to produce and no prior knowledge of genetic details is required. Microorganisms have adapted to the natural environment and produce both simple and complex metabolic products from their nutrient sources through complex, integrated pathways. 

The basic techniques used in the purification of protein drugs have been developed in biochemical laboratories over the past several decades. However, the objectives of protein purification as applied to rDNA products differ from those encountered in the basic research laboratory. Purity of the final product is one of the objectives that is shared by protein purification processes in both situations. An objective critical in the purification of protein drugs is the clinical safety of the final product. In addition, the commercial nature and the scale of the protein pharmaceutical enterprise make cost another objective of great importance (Heinrikson and Tomasselli, 1991). This has led to the adaptation of older techniques to production scales as well as the development of newer methods. 

The development of a qualified down-scale model of a process module is integral to the approach of process validation using bench-scale experiments, as described earlier. We have developed down-scale models of process steps ranging from various types of process chromatography for protein purification to separation by precipitation and filtration. These down-scale models have been utilized to evaluate the effects of relevant process parameters on product-quality attributes. The normal logical sequence of process development, of course, is bench scale to pilot scale to full scale. However, for many plasma protein purification processes, a reverse order needs to be followed. As licensed full-scale processes already exist, the full-scale process steps need to be scaled down to construct small process models in order to evaluate the robustness of process parameters on the product without impacting full-scale production. These models can also be utilized to evaluate process changes, improvements, and optimizations easily and economically. 

Anicetti, V. and Hancock, W. S. (1994) Analytical Considerations in the Development of Protein Purification Processes. In R. Harrison (Ed) Protein Purification Process Engineering. Marcel Dekker, Inc., New York. 

Rational design and optimization of protein purification processes have been developed and optimal operation sequences determined by using expert system (Asenjo et al. 1991 Leser and Asenjo 1992 Mao and Hearn 1996 Lienqueo et al. 1999 Vasquez-Alvarez et al. 2001 Simeonidis et al. 2005). 

Lienqueo ME, Salgado JC, Asenjo JA (1999) An expert system for selection of protein purification processes experimental validation. J Chem Technol Biotechnol 74(3) 293-299 Liu J, Xi W (2006) Purification and characterization of a bifunctional enzyme with chitosanase and cellulase activity from commercial ceUulase. Biochem Eng J 30(l) 82-87 Liu LC, Prokopakis GJ, Asenjo JA (1988) Optimization of enzymatic lysis of yeast. Biotechnol Bioeng 32(9) 1113-1127... 

Vasquez-Alvarez E, Lienqueo ME, Pinto JM (2001) Optimal synthesis of protein purification processes. Biotechnol Prog 17 685-696... 

Harrison, R.G. 1994. Protein Purification Process Engineering. New York Marcel Dekker. 

Fee, C.J. and Chand, A. (2006). Intensification of minor milk protein purification processes. Proceedings of the 2nd Conference on Process Intensification and Innovation, Christchurch, New Zealand, September 24—29. 

I) pH Proteins exist as cations or anions on either side of the isoelectric point At the isoelectric point the protein is least soluble. It has been seen that the pH of minimum solubility (isoelectric point) usually decreases with increasing salt concentration. Suitable buffers should therefore be added for adequate pH control during the protein purification process. 

LA Gathn, SL Nail. Freeze drying a practical overview. In RG Harrison, ed. Protein Purification Process Engineering. New York Marcel Dekker, 1993, pp. 317-367. 

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