Assuring Production and Purity

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa- 

Liquid-liquid partitioning is a convenient and often economical method for bioseparations. L. Gu (personal communication, 1999) has shown that an acetonitrile-water system can be used for separation of proteins. This system partitions into two phases under subzero temperatures with the top phase containing more acetonitrile and water. The low temperature and the presence of water in both phases help reduce protein denaturation. An added advantage is that sample solution can be directly applied to reversed-phase high-performance liquid chromatography (HPLC) for further purification. Aqueous liquid-liquid partitioning is likely to remain an attractive choice for the separation of proteins, and exploration of new systems will continue. 

Shiloach (personal communication, 1999) believes that ion-exchange adsorbents are a powerful tool in the purification of proteins. They can be used in the initial steps of the protein recovery process by capturing the proteins, enrichment, or polishing. It is likely that new and improved adsorbents will continue to be introduced. 

Low-abundance proteins are of considerable interest in proteomics. This calls for improvement in detection capabilities. The separations of large proteins, on the other hand, would require development of new support matrices. Efforts are likely to center on the development of new and improved polymers for gel formation. 

Displacement chromatography offers an attractive alternative to the elution mode of operation for preparative purification (S. Cramer, personal communication, 1999). Further investigations must be carried out to identify more cost-effective, nontoxic, and readily detectable displacers that are commercially available to the biotechnology industries. Displacers with high affinities in a range of commercially available stationary phases must be identified to facilitate the development of displacement steps on these materials. This will require significant advances in our understanding of the nature of affinity of these systems. 

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