Antibody purification processe

Tables 2 and 3 show an antibody purification process scale-up from laboratory scale (1 mL) to intermediate scale (500 mL) to large scale of 10-85 L column volumes, maintaining the column bed height constant. Product quality and biocontaminant levels were maintained throughout the scale-up, though operational flow rates were significantly changed, demonstrating the consistency of the overall purification process. Thorough analysis of each coliunn performance is essential in order to sustain the process robustness at different scales of operation.

Table 2 Antibody Purification Process Scale-Up and Performance for Different Bioreactor Scales...

Antibody purification processes are rational combinations of two or more chromatographic separations techniques with the aim of removing all relevant impurities from the antibody preparation according to the demands of the application. 

Iyer, H., Henderson, F., Cunningham, E., Webb, J., Hanson, J., Bork, C., Conley, L., Considerations during development of a protein A-based antibody purification process. Biopharm 2002, 20, 14—20. 

Protein A sorbents used in antibody purification processes are typically the most expensive but have also seen the most improvement with time. To decrease costs, the lifetime of media has to be increased while keeping the adsorption characteristics. 

Aldington, S. and Bonnerjea, J. (2007) Scale-up of monoclonal antibody purification processes. J. Chromatosr- B, 848, 64-78. 

Follman, D. K. and Fahrner, R. L. Factorial screening of antibody purification processes using three chromatography steps without protein A. /. Chromatogr. 1024 79-85, 2004. 

Recombinant proteins with unique properties can potentially generate new markets and penetrate into existing markets if they can be supplied on a large scale. An ideal system would produce the safest biologically active material at the lowest cost, and would be used in combination with an inexpensive and simple purification process. So far, there have been several examples of the high-yield production of recombinant proteins in transgenic crop plants, mainly in the area of molecular medicines such as antibodies, enzymes and vaccines [45, 48-50]. Modern agricultural practices offer... 

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... 

Columns, dialysis membranes, capillaries or beads may be used in immunoaffinity application which is a non-covalent, irreversible purification process based on highly specific interactions between analyte and antibody [11]. 

Highly specific antibodies directed against minor proteins, present in small amounts in biological fluids, or against nonsoluble cytoplasmic or membranous proteins, are often difficult to obtain. The main reasons for this are the small amounts of protein available after the various classical purification processes and the low purity of the proteins. 

The preparation of chiral alcohols can be carried out very simply because the regeneration of NADPH is possible by the addition of isopropanol. Unpurified crude extract samples of the ADH from L. kefir were found to be a useful catalyst for the synthesis of (P)-alcohols [160] some examples for the preparation of some chiral alcohols using this enzyme are given in Table 8. Though this ADH becomes unstable to such a degree during the purification process, enough material of the pure enzyme could be prepared to produce polyclonal antibodies and to screen for related (R)-specific enzymes. 

Affinity chromatography techniques have shown less utility in analytical testing than in preparative separations for a variety of reasons, including cost and the difficulty of validating consistent operation as the column changes over time. Protein A affinity has been commonly used to quantitate the total antibody content of either ascites or cell culture fluids. To provide guidance in the development of a purification process, specific immunoaffinity resins are either available or can be readily prepared to quantitate the levels of unrelated protein contaminants. To rapidly determine what the active species in a mixture is, a monoclonal antibody that... 

In a study of the production of a humanized mAb produced by NSO cells, Rodriguez et al. (2005) compared two different operation strategies (Figure 9.13). The optimized strategy provided a significant decrease in the ratio of the flow rates of product harvest and medium feed, with a twofold increase in the total amount of antibody harvested and a sixfold decrease in the harvested volume. Thus, antibody concentration in the product stream was increased by over 10-fold, with a positive impact on the purification process of this mAb. 

Purity, safety, potency, and cost-effectiveness are some of the main factors that should be considered when designing an expression method and, more importantly, when defining the purification processes. The purification of antibodies was most likely initiated with the separation of proteins, mainly paraproteins, several decades ago. A plethora of protocols have now been described involving precipitation with a variety of chemical agents, electrophoretic separation, membrane methodologies, and liquid chromatography. The latter probably represents the most popular technique because of the ease of implementation, the capability to play on the selectivity, and the... 

The preparation of pure antibody for human therapeutic use is a long process that commences by in vitro use and last for several years of intensive work at various clinical stages. Risks related to the noneffectiveness of the antibody for the target application are relatively high at the early stage of the project. In this context, it would be irrational to search at the beginning for an optimized sophisticated separation and purification process to make the antibody at a large scale. 

Three main situations can distinguished. The first is the preparation of antibodies for diagnostic use in vitro, the second is related to the therapeutic use for relatively modest amounts of antibodies, and the third addresses very large amounts of antibodies for both therapeutic and prophylactic applications. In the second and third situations, although antibodies would meet similar requirements of safety, purity, and potency, the issue of market must be addressed. The production cost resulting from both the method of expression and the purification process must be considered. 

Finally, gel filtration is most often considered an appropriate polishing method when the target antibody is already pure and the only impurities are foreign protein traces or fragments or aggregates that must be eliminated. In this context, separation processes for antibody purification are logical orthogonal combinations of methods. 

Godfrey, M. A., Kwasowski, P., Clift, R., and Marks, V. (1993). Assessment of the suitability of commercially available SpA affinity solid phases for the purification of murine monoclonal antibodies at process scale. J. Immunol. Methods 160, 97-105. 

Hubbard, B. 2005. Platform approaches to monoclonal antibody purification. IBC Conference Antibody Production Downstream Processing, Mar 8-11, San Diego, CA. 

---