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A summary of the exhaustive analyses of human insulin (recombinant DNA) shows chemical,
structural, and biologic equivalence to pancreatic human insulin. The high degree of purity accounts for
its lack of pyrogenicity and immunogenic contamination. Human insulin from both the proinsulin and
chain combination methods is essentially identical. The role of proinsulin as an alternate route to human
insulin, and as a new treatment for diabetes when used alone or in combination with human insulin or
connecting peptide, warrants further investigation, DIABETES CARE 5 (SUPPL. 2): 4-12, 1982.
1004 21
trpLE'
A-chain
191 21 Purified Blosynthetlc
Human Insulin
trp LE' (BHD
proinsulin
FIG. 2. The general biosynthetic and chemical modification pathway, from
191 86 the chimeric plasmid and the resulting fused'gene product that is cleaved
by cyanogen bromide (CNBr), to the conversion of S'sulfonate derivatives
FIG. J. Comparison of the ratios of promoter amino acids to those of the (S-SOf) that are purified and combined to yield crude insulin and
desired proteins, i.e., A-chain or proinsulin. ultimately purified human insulin (recombinant DNA).
AB
is higher with the tryptophan synthetase promoter than with
the beta galactosidase promoter, as shown in Figure 1. In the
biosynthesis of human proinsulin, the improvement in the
ratio is still greater. Other promoters are being studied in the
hope of finding one that increases the ratio even more.
The plasmids inserted into one E. coli culture contain the
genetic code for tryptophan synthetase promoter, methio-
nine, and A-chain. The other culture contains plasmids that
are identical except for substitution of the genetic code for B-
chain. Once the separate fermentations have been carried out,
cyanogen bromide effectively cleaves at the methionine
i i residue, releasing the chains from the fused-gene product.
The resulting chains are isolated as the S-sulfonates that are
thiolysed and oxidized to form the correct disulfide linkages
that produce the desired insulin molecule (Figure 2).
A large number of thiol-reducing agents were examined,
and dithiothreitol was superior. Different ratios of this agent
to the sulfonates give different yields, but optimum is
between 50 and 60% of the theoretical maximum value.
It is theoretically possible for disulfide linkages to be formed
other than those required to give a product identical to pan-
creatic human insulin. Two such isomers are more probable
FIG. 3. Schematic representation of the configurations possible when than others and are shown below the correct configuration in
the disulfide bonding of the A- and B-chains occurs. The configuration Figure 3. Samples of these two products were made avail-able
at the top is one desired for human insulin; the two below are the most to Eli Lilly by Ciba-Geigy, who obtained them in the course of
probable isomers that could theoretically occur. the total chemical synthesis of insulin. They are easily separable
from the desired molecule by high-perform-ance liquid
chromatography (HPLC) and are not detected in Lilly's human
insulin (recombinant DNA).
-682
1800 2160 2430 2780
FIG. 4. HPLC elution profile of human insulin (recombinant DNA). Abscissa numbers are seconds; ordinate numbers are
percents of solvent B indicated by the gradient line. See text for discussion of peaks. Flow rate was 0.5 mVmin.
RANIT
- 75
XB
50
SEMISYNTHETIC HUMAN INSULIN
- 25
- 0
338.5 078.0 1013. SEC 1351. 1688. 2028. 2383. 2701.
GRADIENT 12. 15. 1. 28. 0 CMPO S.AUREUS DIGEST-HUMAN INSULIN
FIG. 8. HPLC elution profiles of peptide fragments from enzymatic cleavage of semisynthetic human insulin and human
insulin (recombinant DNA), confirming their structural identity and purity.
BIOSYNTHETIC
HUMAN INSULIN
305
200 210 220 230 240 250 260 270 280 290 300 310
WAVELENGTH (mu)
PORCINE INSULIN
and porcine insulin were compared by circular dichroic spec- and active, but also that it is as free as possible from any
trometry.' Figure 9 shows the spectra obtained that indicate adverse effects. Human insulin is a foreign protein to labo-
identical spatial arrangement of the molecules. ratory animals and its main effect is to reduce blood glucose
Finally, polyacrylamide gel electrophoresis and isoelectric levels. This makes assurance of the prolonged survival of
1 animals to which it is administered difficult. Human insulin
focusing techniques have given identical results with human
insulin (recombinant DNA), pancreatic human insulin, and (recombinant DNA) was subjected to acute toxicity studies in
pork insulins (Figure 10). mice, rats, and dogs, to studies over 14-day periods in
In the case of any product such as insulin that is intended monkeys, and to studies over 30-day periods in rats and dogs.
for continuous use over prolonged periods of time, there is a In all these studies, there was evidence only of reduction in
need to ensure not only that the material is pure, authentic, blood glucose levels, and not of any other adverse effects.
TABLE 1
Limulus amebocyte lysate (LAL) and pyrogen data for human insulin lots
NH,— B-CHAIN
be advantageous. The human pancreas secretes proinsulin undesirable contaminants which are achieved by Eli Lilly
along with insulin in the ratio of about 1:100. It is possible and Company in the manufacture of human insulin. Control
that mixtures of insulin and proinsulin, or insulin and C- procedures carried out on every lot ensure that all human
peptide, or even of all three, may ultimately come to be used insulin produced is authentic, pure, and free from
in diabetic therapy. Human proinsulin is, therefore, not only undesirable contaminants, including any capable of causing
of interest as a route to human insulin, but for the immune re-actions.
possibilities it offers of novel therapeutic approaches. The procedures developed for the manufacture of human
insulin by the proinsulin route open up new possibilities for
future investigation in the field of treatment of diabetes.
CONCLUSIONS
Many of these possibilities are now being actively explored
The exhaustive developmental studies described demonstrate by scientists and clinicians throughout the world.
the identity between human insulin (recombinant DNA) made
by either route described, and pancreatic human in-sulin, and From the Lilly Research Laboratories, Eli Lilly and
the extreme degree of purity and freedom from Company, Indianapolis, Indiana.
OB.
10000-
A r100 TABLE 3
8978- -90 Evaluation of human insulin derived from human proinsulin (recombinant
7955- -80 DNA)
6932- -70
5909- -60 Test Results
> 4887-: -50
_ ^ - — •
3864 -i Biosynthetic -40
-~
USP rabbit hypoglycemia assay 28.0 ± 2.2 U/mg
2841-3 Human Insulin -30 (144 rabbits)
1819 -i (Proinsulin Route) i-20
796- i-10
Insulin radioimmunoassay 106 ± 10% of pancreatic human
.997- J Insulin radioreceptorassay
in-sulin standard
96 ± 3% of pancreatic human in-
10000 -g sulin standard
B rlOO Excellent
Amino acid composition
8980-i r90 Gel electrophoresis UV Excellent
7959- r80
and CD spectra HPLC Identical to pork insulin standard
6938- r70
Same retention time as pancreatic
5918-i r60
human insulin
S 4897-i h50
Zinc crystallization Excellent
3878-: Btotynttwtlc ^ -40
Limulus assay for bacterial endo- <0.1 ng/mg
2866 -j HunuHi RisuNn '" E-30
1836-i (A + B Rout*) 1-20
toxin
814 -i J
V -10
USP rabbit pyrogen test Nonpyrogenic
BP proteolytic activity assay Satisfactory
I» 270 540 810 1080 1350 1620 1690 2160 2430 271DO Proinsulin radioimmunoassay 11.3 ppm
C-peptide radioimmunoassay <1 ppm
E. coli peptide radioimmunoassay <4 ppm
FIG. 15. HPLC of human insulin (recombinant DNA) produced by way of
proinsulin (top) and A- and B-chain combination (bottom).
Synthesis—Structure—Function. Proceedings of the Seventh
American Peptide Symposium. Rockford, Pierce Chemical Com-
Address reprint requests to Irving S. Johnson, Lilly Research pany, 1981, pp. 721-28.
Laboratories, 307 East McCarty Street, Indianapolis, Indiana 46285. 3 Ross, J. W., Baker, R. S., Hooker, C. S., Johnson, I. S.,
Schmidtke, J. R., and Smith, W. C : Procedure for detection of
potential E. coli peptides (ECPs) in biosynthetic human insulin
REFERENCES (BHI), antibodies to ECPs in patients treated with BHI and meas-
1 Chance, R. E., Kroeff, E. P., Hoffmann, J. A., and Frank, B. urement of bacterial endotoxins in BHI. Paper presented at "FDA-
H.: Chemical, physical, and biologic properties of biosynthetic hu- USP Symposium" held in Washington, D.C., May 19, 1982.
man insulin. Diabetes Care 4: 147—54, 1981. 4Data on file. Lilly Research Laboratories.
2 5 Baker, R. S., Schmidtke, J. R., Ross, J. W., and Smith, W.
Chance, R. E., Hoffmann, J. A., Kroeff, E. P., Johnson, M. G.,
C.: Preliminary studies on the immunogenicity and amount of Esch-
Schirmer, E. W., Bromer, W. W., Ross, M. J., and Wetzel, R.: The
production of human insulin using recombinant DNA technology erichia coli polypeptides in biosynthetic human insulin produced by
and a new chain combination procedure. In Peptides: recombinant DNA technology. Lancet 2: 1139-42, 1981.