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'.

Although recombinant product produced by either method is identical in sequence to native insulin, any impurities present will be host microbial-cell-derived and, hence, potentially highly immunogenic. Stringent purification of the recombinant product must thus be undertaken. This entails several chromatographic steps (often gel filtration and ion exchange, along with additional steps that exploit differences in molecular hydrophobicity, e.g. hydrophobic interaction chroma-tography or reverse-phase chromatography) (Figure 11.3).

A 'clean-up' process-scale RP-HPLC step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 80 l or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 11.4). Separation is achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30 per cent acetonitrile).

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.

Supplements For Diabetics

Supplements For Diabetics

All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.

Get My Free Ebook


Post a comment