Resolution pilot production

The physicochemical and other properties of any newly identified drug must be extensively characterized prior to its entry into clinical trials. As the vast bulk of biopharmaceuticals are proteins, a summary overview of the approach taken to initial characterization of these biomolecules is presented. A prerequisite to such characterization is initial purification of the protein. Purification to homogeneity usually requires a combination of three or more high-resolution chromatographic steps (Chapter 6). The purification protocol is designed carefully, as it usually forms the basis of subsequent pilot- and process-scale purification systems. The purified product is then subjected to a battery of tests that aim to characterize it fully. Moreover, once these characteristics have been defined, they form the basis of many of the QC identity tests routinely performed on the product during its subsequent commercial manufacture. As these identity tests are discussed in detail in Chapter 7, only an abbreviated overview is presented here, in the form of Figure 4.5. 

The enzymatic resolution of (R,S)-2-ethoxycarbonyl-3,6-dihydropyran has been carried out repeatedly in a pilot plant on a 100-125 kg scale. The conditions of pH, agitation, concentration, and enzyme/substrate ratio have been further optimized, but for the most part conditions developed during the laboratory optimization studies have been found to work well. This technology is currently being used to produce ton quantities of (S)-4. It has to be pointed out that no major difficulties were encountered during the scale-up. The work-up of the product in this process, unlike in many other enzyme-catalyzed processes, is quite simple and volume efficiencies are better than some chemical reactions. The recovery and recycling of the enzyme is not needed since it is commercially available and relatively inexpensive. 

Although there are many reports in the literature of enzyme Catalyzed resolutions at laboratory scale (72,73), there are still few examples of processes operating at pilot and production scale (40,57,74—76). The cloning and overexpression of the enzyme, under the control of a high ievel inducible promoter, was essential to the development of a scalable process. Cytidine deaminase has been cloned from both Bacillus (77,78) and E. coli (79), but only the B. subtilis enzyme had previously been overexpressed (from the lac promotor) in a high copy number plasmid (78). 

Currently, both FT-Raman and dispersive Raman spectrometers are being used within the pharmaceutical industry. Dispersive Raman spectroscopy in the form of Raman microprobes are heavily employed in the research area to map active-excipient distribution using the diffraction limited spatial resolution attainable with the microprobe. In this subsection, it is inappropriate to describe the varied applications of Raman microscopy to the study of pharmaceuticals thus, the reader is referred to the literature [108,109] and Chapter 14. Dispersive Raman analyzers are also being used for reaction analysis, pilot-plant batch analysis, and process monitoring. FT-Raman spectrometers have been adopted for formulated product analysis and for incoming goods testing. 

The oxidation of 130 by oxygen under pressure was developed af lab scale. Under optimal conditions, the substrate/bisulfite mixture was added to a solution of 130 (3.9 g/1), MAON401 and catalase over 20h. Substrate 130 (65g/l) was converted to sulfonate 133 with a small amount of 131 (<10%). The enzyme reaction stream was telescoped for cyanation to afford only trans-nitrile 134 in 90% yields from 130. Subsequenfly the nitrile was transformed to the methyl ester and the product was converted to the free base 132. In the final step the free base was crysfallized to afford 135 in 56% yield and >99% ee. The conditions of the procedure were successfully applied to pilot plant scale. Compared with the resolution method in the enzymatic process the product yield was increased by 150%, raw material use was reduced by 59.8%, consumption of water was reduced by 60.7%, and the overall process waste was reduced by 63.1% [167]. 

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