Production of human insulin by recombinant DNA technology

Human insulin produced by recombinant DNA technology was first approved for general medical use in 1982, initially in the USA, West Germany, the UK and The Netherlands. As such, it was the first product of recombinant DNA technology to be approved for therapeutic use in humans. From the 1990s on, several engineered insulin products (discussed later) also gained approval (Table 11.3). 

The initial approach to recombinant insulin production taken entailed inserting the nucleotide sequence coding for the insulin A- and B-chains into two different E. coli cells (both strain K12). These cells were then cultured separately in large-scale fermentation vessels, with subsequent chromatographic purification of the insulin chains produced. The A- and B-chains are then incubated together under appropriate oxidizing conditions in order to promote interchain disulfide bond formation, forming human insulin crb  

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  

The starting material loaded onto the column is fairly pure (—92 per cent), and this step yields a final product of approximately 99 per cent purity. Over 95 per cent of the insulin activity loaded onto the column can be recovered. A single column run takes in the order of 1 h. 

The RP-HPLC polishing step not only removes E. coli-derived impurities, but also effectively separates modified insulin derivatives from the native insulin product. The resultant extremely low levels of impurities remaining in these insulin preparations fail to elicit any significant immunological response in diabetic recipients. 

The quantity of purified insulin obtained from the pancreas of one pig satisfies the requirements of one diabetic for 3 days. The supply of pancreatic tissue is dependent upon the demand for meat, which does not necessarily correlate with the increasing worldwide incidence of diabetes. Recombinant DNA technology provides an obvious way to ensure future adequate supply of insulin. 

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Human insulin was the first commercial health care product produced by recombinant DNA technology ZjEliJLilly,.the producer of this synthetic insulin, often relied on HPLC to confirm the structure and to determine the potency of synthetic human insulin. The story behind Lilly s recombinant DNA-produced insulin is described in an article in Science (1) High performance liquid chromatography (HPLC) techniques developed at Lilly can detect proteins that differ by a single amino acid, and HPLC tests show human insulin (recombinant DNA) is identical to pancreatic human insu-... 

The use of restriction enzymes to cleave DNA at specific sequences was mentioned earlier in this chapter in the context of DNA sequence analysis. These enzymes are also important in the field of recombinant DNA technology. We will illustrate this application by describing a method for the production of human insulin. 

Historically, insulin came from either beef or pork sources. Beef insulin differs by three amino acids and pork by one amino acid when compared to human insulin. Manufacturers in the United States have discontinued production of beef and pork source insulins as of December 2003, and now exclusively use recombinant DNA technology to manufacture insulin. Eli Lilly and Aventis currently use... 

The recombinant DNA technology of Cohen and Boyer enabled them to generate the first commercial product in 1978 human insulin expressed in Escherichia coli. These efforts also led to the first biotech company on 15 October 1980 Genen-tech went public on the New York Stock Exchange. Fascination about this modern biopharmaceutical and the huge potential of the new biotechnology caused the stock price to jump from US 35 to 89 in the first 20 minutes by the evening of the same day, the market capitalization was US 66 million ... 

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