Therapeutic hormones - Pharmaceutical Biotechnology: Concepts and Aapplications - page 299

11.2.5 Production of human insulin by recombinant DNA technology

Human insulin produced by recombinant DNA technology was fi rst approved for general medical
use in 1982, initially in the USA, West Germany, the UK and The Netherlands. As such, it was the
fi rst 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 purifi cation of the insulin chains produced. The A- and B-chains are then in-
cubated together under appropriate oxidizing conditions in order to promote interchain disulfi de
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 pu-
rifi cation 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 purifi cation 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 purifi cation of the recombinant product must thus be undertaken. This
entails several chromatographic steps (often gel fi ltration 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 insu-
lin 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 purifi cation run (Figure 11.4). Separation is
achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value suffi ciently
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 typi-
cally 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 fi nal 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 modifi ed insulin derivatives from the native insulin product. The resultant extremely
low levels of impurities remaining in these insulin preparations fail to elicit any signifi cant im-
munological response in diabetic recipients.

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11.2.6 Formulation of insulin products

Insulin, whatever its source, may be formulated in a number of ways, generally in order to alter its
pharmacokinetic profi le. 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