Purification scheme

Recovery nd Purifica.tion. The production of EH Lilly s human insulin requires 31 principal processing steps of which 27 are associated with product recovery and purification (13). The production process for human insulin, based on a fermentation which yields proinsulin, provides an instmctive case study on the range of unit operations which must be considered in the recovery and purification of a recombinant product from a bacterial fermentation. Whereas the exact sequence has not been pubUshed, the principle steps in the purification scheme are outlined in Figure la. 

The design of bioseparation unit operations is influenced by these governmental regulations. The constraints on process development grow as a recovery and purification scheme undergo licensing for commercial manufacture. 

Interest in synthetic naphthenic acid has grown as the supply of natural product has fluctuated. Oxidation of naphthene-based hydrocarbons has been studied extensively (35—37), but no commercially viable processes are known. Extensive purification schemes must be employed to maximize naphthene content in the feedstock and remove hydroxy acids and nonacidic by-products from the oxidation product. Free-radical addition of carboxylic acids to olefins (38,39) and addition of unsaturated fatty acids to cycloparaffins (40) have also been studied but have not been commercialized. 

The air process has similar purity requirements to the oxygen process. The ethane content of ethylene is no longer a concern, due to the high cycle purge flow rate. Air purification schemes have been used to remove potential catalyst poisons or other unwanted impurities ia the feed. 

FIG. 22-83 Frequency of use of purification methods at different stages in purification schemes published in the literature. Adaptedfi-om Bonnerjea et ah, op. cit.)... 

SEC is very easy to perform up to the production scale and the results are very reproducible. Because no gradient elution has to be applied, no programmable gradient mixing system is necessary and only comparatively simple equipment is needed for the operation. Additionally, the method can be integrated easily in purification schemes and most of the operational steps can be... 

Example of a Protein Purification Scheme Purification of the Enzyme Xanthine Dehydrogenase from a Eungus... 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

Alkynyl ketones 157 were also used for the synthesis of pyrimidines 159 by reacting amidines in acetonitrile at 120 °C in a dedicated microwave synthesizer without the requirement of additional purification (Scheme 56) [104]. 

Extraction of Sodium Channel Blockers. A review of published reports shows that methods for purification of sodium channel blockers from bacterial cultures are similar to techniques for isolation of TTX and STX from pufferfish and dinoflagellates (30, 31, 38, 39). Typically, cell pellets of bacterial cultures are extracted with hot 0.1% acetic acid, the resulting supernatant ultra-filtered, lyo-philized, and reconstituted in a minimal volume of 0.1% acetic acid. Culture media can also be extracted for TTX by a similar procedure (Ji). Both cell and supernatant extracts are analyzed further by gel filtration chromatography and other biological, chemical, and immunological methods. Few reports describe purification schemes that include extraction of control samples of bacteriological media (e.g., broths and agars) which may be derived from marine plant and animal tissues. 

Figure 2. Purification scheme of the polygalacturonases from Sclerotinia sclerotiorum...

After salt break chiral acid 11 was converted to the methyl ketone 13 in essentially quantitative yield via the intermediacy of the Weinreb amide and processed as an oil without further purification (Scheme 9.6). The carbonyl group in 13 was then reduced to the secondary alcohol 16 using L-Selectride as previously described in 97% assay yield. We were pleased to find that an attainable reaction temperature of-50°C was sufficient to obtain high selectivity of >98 2, and that, in the absence of the reactive nitrile group, no issues were observed or during... 

Reaction of hydroxyketone 308 with Lawesson s reagent afforded dithiaphos-pholane 309 as a single P-diastereomer in 52% yield after HPLC purification (Scheme 73) [110]. 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

Figure 11.3 A likely purification scheme for human insulin prb. A final RP-HPLC polishing step yields a highly pure product. Refer to text for details...

Cleavage Purification Scheme 7.1 Strategies for solid-phase oligosaccharide synthesis. 

A purification scheme was devised to isolate and icjlgntify the factor for promoting photochemical reactions, by using C-mexa-carbate as the substrate (Table I). It must be noted that the factor obtained here is only partially purified. Also in some cases it requires the presence of FMN to fully express its stimulatory potency for this substrate. 

Analytical scientists will provide support for many of the activities in a biopharmaceutical company. They are responsible for characterizing the molecules in development, establishing and performing assays that aid in optimization and reproducibility of the purification schemes, and optimizing conditions for fermentation or cell culture to include product yields. Some of the characterization techniques will eventually be used in quality control to establish purity, potency, and identity of the final formulation. The techniques described here should provide the beginning of a palette from which to develop analytical solutions. 

---