Download (direct link):
• reliability of supply;
• elimination of the risk of accidental transmission of disease, due to the presence of pathogens
in animal pancreatic tissue;
• economically attractive (once initial capital investment has been made).
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.
The initial approach taken (by scientists at Genentech) 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 disulphide bond formation, forming human insulin crb (Box 8.2).
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.
A number of studies have shown conclusively that the recombinant insulins are chemically and functionally identical to native human insulin. Sequencing data confirms the expected amino acid sequence. HPLC analysis also yields identical chromatograms (Figure 8.5). Circular dichromism studies, which evaluate tertiary structure, also yield identical patterns, as does X-ray crystallographic analysis. Radioreceptor- and radioimmuno-assays yield identical results, as does the traditional bioassay for insulin (measure of the hormone’s ability to induce a hypoglycaemic response in rabbits). Prior to its approval for medical use, human clinical trials also found the recombinant insulin to be as effective as previously available products in terms of controlling hyperglycaemia. Recombinant human insulins are now routinely produced in 10000 gallon fermentation vessels and several such products have gained marketing approval (Table 8.3).
While the recombinant product is identical to native insulin, any impurities present will be E. coli-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 which exploit differences in molecular hydrophobicity, e.g. hydrophobic interaction chromatography or reverse-phase chromatography) (Figure 8.6).
A ‘clean-up’ process-scale reverse-phase HPLC (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 801 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 8.7). 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% acetonitrile).
Figure 8.5. HPLC chromatograms of native and recombinant human insulin: (a) the HPLC protocol employed is capable of differentiating between insulins, which differ by as little as a single amino acid (b)
While the starting material loaded onto the column is fairly pure (*92%), this step yields a final product of approximately 99% purity. Over 95% 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.
Formulation of insulin products
Insulin, whatever its source, may be formulated in a number of ways. This directly affects its activity profile upon administration to diabetic patients. Fast (short)-acting insulins are those preparations that yield an elevated blood insulin concentration relatively quickly after their administration, which is usually by s.c. or, less commonly, by i.m. injection. Slow-acting insulins, on the other hand, enter the circulation much more slowly from the depot (injection) site. This is characterized by a slower onset of action, but one of longer duration (Table 8.4).