Production-scale electrophoresis

The final purification steps are responsible for the removal of the last traces of impurities. The volume reduction in the earlier stages of the separation train are necessarv to ensure that these high-resolution operations are not overloaded. Generally, chromatograjmy is used in these final stages. Electrophoresis can also be used, but since it is rarely found in process-scale operations, it is not addressed here. The final product preparation may require removal of solvent and drying, or lyophihzation, of the product. 

At present moment, no generally feasible method exists for the large-scale production of optically pure products. Although for the separation of virtually every racemic mixture an analytical method is available (gas chromatography, liquid chromatography or capillary electrophoresis), this is not the case for the separation of racemic mixtures on an industrial scale. The most widely applied method for the separation of racemic mixtures is diastereomeric salt crystallization [1]. However, this usually requires many steps, making the process complicated and inducing considerable losses of valuable product. In order to avoid the problems associated with diastereomeric salt crystallization, membrane-based processes may be considered as a viable alternative. 

Over the past 40 years, capillary electrophoresis (CE) has advanced significantly as a technique for biomolecular characterization. It has not only passed the transition from a laboratory curiosity to a mature instrumental-based method for micro-scale separation, but also emerged as an indispensable tool in the biotech and pharmaceutical industries. CE has become a method of choice in research and development (R D) for molecular characterization, and in quality control (QC) for the release of the therapeutic biomolecules.In the biopharmaceutical industry, more and more CE methods have been validated to meet International Conference on Harmonization (ICH) requirements. In this chapter, we present real industrial examples to demonstrate the role of CE in R D of pharmaceutical products. The focus in this chapter is on method development analytical control for manufacturing and release of therapeutic proteins and antibodies. 

The recovery of products from biotechnological processes has traditionally been focused on bench-scale separation approaches, such as electrophoresis or column liquid chromatography. These methods are dilScult to scale up to production levels and often become prohibitively expensive for medium-and low-value products. 

Capillary electrophoresis is not used as much as liquid chromatography. Advantages of electrophoresis relative to chromatography include (1) higher resolution, (2) low waste production, and (3) generally simpler equipment. Drawbacks of electrophoresis include (1) higher limits of detection, (2) run-to-run irreproducibility of migration times, (3) insolubility of some analytes in common electrolyte solutions, and (4) inability to scale up to a preparative separation. 

Figure 10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of products of limited proteolysis of the debranching enzyme with trypsin. The molecular weights shown of the various bands were determined by the methodology described previously (26). The ratio of debrancher to trypsin was 100 to 1. The incubation was conducted for 60 minutes at 25°. The gel stain was Coomassie Brilliant Blue and the absorbance was measured at 600 nm using a Gilford gel scanner with a 0-1 O.D. chart scale (36).

From a strict biochemical point of view a clear-cut definition of the role of the liver in the biosynthesis of any particular plasma protein can be made only when the particular protein has been clearly and cleanly isolated, as in the case of fibrinogen. The practical difficulties of effecting such isolations on a small scale from isotopic labeling studies of the plasma proteins, such as we have described, seriously militate against such a detailed demonstration at present. The use of fractionation techniques with greater resolving power such as acrylamide gel electrophoresis already show some promise in our laboratory toward affording a more definitive picture of the biosynthetic role of the liver and the nonhepatic tissues in plasma protein production. 

The analysis of PCR products is usually carried out by electrophoresis, and the desired product is identified by length. This is not necessarily the best approach for large scale routine analysis. Alternative sequence-specific procedures are available in which the product is hybridised to labelled oligonucleotides that can be detected by fluorimetric antibody-coupled colorimetric procedures. 

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