Authenticity(recombinant

and Purity of Human Insulin

DNA)
IRVING S. JOHNSON

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.

H The promoter chosen to maximize production of the de-

uman insulin (recombinant DNA) is currently made by sired protein in the E. coli cells is an important facet of
joining A- and B-chains of human insulin produced recombinant production of gene products. A promoter is
by separate strains of Esche-richia coli K12.
Concentrations of the proteins inserted into the plasmid that determines the rate at which
that ultimately give rise to insulin are very much higher than messenger RNA (mRNA) is formed. The stronger the pro-
the concentration of insulin and its precursor in the pancreas. moter, the more mRNA formed and the more protein syn-
Only 1 % of the gland is islets of Langerhans and only a few thesized.
cells in each islet are beta-cells producing insulin, but every Promoters also have the effect of increasing the ratio of
E. coli cell is capable of making the gene product we want the desired protein to associated carrier protein. This ratio
through recombinant DNA technology. Therefore, the po-
tential degree of contamination of the A- and B-chains with Trp E—Met—A Chain | |_ TrpE—Met—B Chain
foreign protein and other extraneous matter is very much less I CN Br Cleavage
than the potential degree of contamination of insulins of
animal origin. A Chain B Chain

Human Insulin Chimeric Proteins

A-(S^O3")4
(3 -gal
A-chain

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).

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

AB
i i
FIG. 3. Schematic representation of the configurations possible when
the disulfide bonding of the A- and B-chains occurs. The configuration
at the top is one desired for human insulin; the two below are the most
probable isomers that could theoretically occur.
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
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
than others and are shown below the correct configuration in
Figure 3. Samples of these two products were made avail-able
to Eli Lilly by Ciba-Geigy, who obtained them in the course of
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).

HIGH-LOW HPLC BIOSVNTHETIC HUMAN INSULIN

-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.

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

HPLC is one of the most sensitive methods now available

for detecting minor structural differences between
substances in a mixture. Figure 4 shows such an HPLC
analysis of human insulin. The main peak that is off the
A Biosynthetic Human Insulin
chart represents the human insulin (recombinant DNA) and
is identical to that produced by pancreatic human insulin.
Further proof of the identity of human insulin (recombinant
DNA) is shown in Figure 5, when HPLC of a mixture of
B Pancreatic Human Insulin human insulin (re-combinant DNA) and pancreatic human
1
insulin gave only one peak.
The minor peaks observed in Figure 4 represent insulin-
related substances, typically monodesamido insulin, some
C Pancreatic + Biosynthetic Human
Insulin Mix.
carbamoyl glycine or phenylalanine derivatives. Each has
been found to have 90—100% of the biologic activity of
insulin itself. The small amount of polymerized human in-
sulin (recombinant DNA) has activity approximately 15%
1.00 188.5 376.0 563.5 751.0 938.5 1126. 1313. 1501. that of insulin.
Sec
In contrast to the procedure for the production of animal
FIG. 5. Comparison of HPLC elution profiles for human insulin insulins, the recombinant DNA method of producing human
{re' combinant DNA), pancreatic human insulin, and a mixture of insulin rules out contamination with other hormones, such
the two human insulin preparations. Sample loads were 20 \ig. as glucagon, somatostatin, or even proinsulin.
Numbers on the abscissa are seconds. Flow rate was 1.0 mllmin. Having shown chemical equivalence, it was essential to
show that biologic activity of human insulin (recombinant
DNA) was also identical to that of pancreatic human insulin.
The rabbit hypoglycemia test is regarded by many regulatory
groups as a standard procedure for measuring insulin potency.
The comparative hypoglycemic effects of human insulin (re-
combinant DNA), pancreatic human insulin, and porcine

RANIT

l.M 338. 3 isis. sec lasi. im*.

CMPO INSULIN HIXTURE

FIG. 6. HPLC elution profiles for various mixtures

of pancreatic insulins that illustrate the sensitivity of
the assay to separate proteins differing by only one or
two amino acids. Numbers on the abscissa are
CMPD INSULIN MIXTURE seconds.

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

FIG. 7. Three-dimensional diagram of the pep-

tide fragments that occur with enzymatic cleavage
of the human insulin molecule.

1 isynthetic human insulin by Staphyhcoccus aureus protease

insulin were identical. Results of HPLC and rabbit assay are
generally in agreement, but HPLC is more sensitive and
capable of separating insulins from different species. Figure
6 illustrates the separation of several insulins that differ by
l
only one or two amino acids.
Hydrolysis of human insulin (recombinant DNA) and sem-
yields peptide fragments that are shown diagramatically in
2
Figure 7. HPLC "fingerprint" analysis of these digests showed
identical peptide fragment profiles (Figure 8), confirming
identical structures.
The stereochemistry of human insulin (recombinant DNA)

HPLC "Fingerprint" Analyses

-, 100

- 75
XB

50
SEMISYNTHETIC HUMAN INSULIN

- 25

BIOSYNTHETIC HUMAN INSULIN

- 0
338.5 078.0 1013. SEC 1351. 1688. 2028. 2383. 2701.
GRADIENT 12. 15. 1. 28. 0 CMPO S.AUREUS DIGEST-HUMAN INSULIN

COLUMN ZORBAX C-8 (40 DEC)

CONDITIONS F. R.-1.0 20 UL IN. 210NM CONC 2.0 MC/ML. IN BUFFER
1.0AUFS

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.

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

BIOSYNTHETIC
HUMAN INSULIN

305

200 210 220 230 240 250 260 270 280 290 300 310
WAVELENGTH (mu)

PORCINE INSULIN

245 255 265 275 285

WAVELENGTH (mu)

FIG. 9. Circular dichroic spectra of human in-

200 210 220 230 240 250 260 270 280 290 300 310 sulin (recombinant DNA) (above) and purified
WAVELENGTH (mu) porcine insulin (below).

and porcine insulin were compared by circular dichroic spec-
trometry.' Figure 9 shows the spectra obtained that indicate
identical spatial arrangement of the molecules.
Finally, polyacrylamide gel electrophoresis and isoelectric
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
pork insulins (Figure 10).
In the case of any product such as insulin that is intended
for continuous use over prolonged periods of time, there is a
need to ensure not only that the material is pure, authentic,
and active, but also that it is as free as possible from any
adverse effects. Human insulin is a foreign protein to labo-
ratory animals and its main effect is to reduce blood glucose
levels. This makes assurance of the prolonged survival of
(recombinant DNA) was subjected to acute toxicity studies in
mice, rats, and dogs, to studies over 14-day periods in
monkeys, and to studies over 30-day periods in rats and dogs.
In all these studies, there was evidence only of reduction in
blood glucose levels, and not of any other adverse effects.

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

test for endotoxin. Table 1 shows the results of both tests on

a number of human insulin (recombinant DNA) lots. None of
3
the lots proved to be pyrogenic.
Another test relating to pyrogenicity is used to detect what
is sometimes known as endogenous human pyrogen, though
the precise nature of this pyrogen is unknown. The method
of detection involves incubation of human peripheral leu-
kocytes with the test preparation. After incubation and cen-
trifugation, a rabbit pyrogen assay is done on the
supernatant. Muramyl dipeptide is an inducer of endogenous
human pyr-ogen and is used to show that the assay is capable
of re-sponding. Pork insulin alone is nonpyrogenic but
becomes pyrogenic if combined with the inducer. Proinsulin
and hu-man insulin (recombinant DNA), whether made by
the proinsulin route or by the combination of the A- and En-
chains, are noninducers.
Theoretically, a possible source of protein contamination
of human insulin could be derived from the E. coli organ-
isms used in its manufacture. To test this, plasmids identical
to those used in the production of human insulin but lacking
the genetic code for either the A- or B-chains were inserted
into the production strain of E. coli; the organisms were
cultured on a large scale and the S-sulfonates prepared. A
mixture of the polypeptide S-sulfonates was fractionated by
gel filtration and fractions combined having peptides smaller
than 15,000 daltons. These were designated E. coli polypep-
tides (ECPs).
By using complete Freund's adjuvant, it was possible, though
difficult, to obtain antibodies and to sensitize animals to ECPs.
A significant immune response to ECPs was obtained only
when guinea pigs were sensitized with ECPs incorporated into
complete Freund's adjuvant. No significant immuno-genicity
and antigenicity were observed when the ECPs were given in
incomplete Freund's adjuvant. No significant Ar-thus-type
reactivity was noted when human insulin or pork insulin was
administered to ECP-sensitized rats and guinea
4,5
pigs

TABLE 1
Limulus amebocyte lysate (LAL) and pyrogen data for human insulin lots

LAL result Pyrogen result

(ng endotoxin/ (mean temp, rise/
Lot number mg insulin) no. of rabbits)
615-70N-174-9 <0.6 0.19°C/N = 3 (nonpyrogenic)
FIG. 10. Polyacrylamide gel electrophoresis and isoelectric focusing of
615>84S-30A 0.8-1.6 0.17°C/N = 3 (nonpyrogenic)
human insulin (recombinant DNA), pancreatic human insulin, and pur-
989BA0 0.4 -0.8 0.11°C/N = 3 (nonpyrogenic)
ified pork insulin.
46L-295 0.1-0.2 0.01°C/N = 3 (nonpyrogenic)
44L-55 0.1-0.2 0.20°C/N = 3 (nonpyrogenic)
Many protein materials used in medicine are at risk of 142CY1 <0.05 0.15°C/N = 3 (nonpyrogenic)
contamination with pyrogens, i.e. substances which raise the 143CY1 0.2 -0.4 0.06°C/N = 3 (nonpyrogenic)
body temperature. Two tests for pyrogens have been used in 46L-296 0.05 -0.1 0.16°C/N = 3 (nonpyrogenic)
44L-79 <0.05 0.04°C/N = 3 (nonpyrogenic)
the study of human insulin (recombinant DNA). One is the
46L-297 0.4-0.8 O.27°C/N = 3 (nonpyrogenic)
conventional method of measuring elevation of temperature 905CY1 0.2 -0.4 0.07°C/N = 3 (nonpyrogenic)
in rabbits. The other is the limulus amebocyte lysate (LAL) 544CJ1 0.05 -0.1 0.07°C/N = 3 (nonpyrogenic)
test, which is widely viewed as the most sensitive in vitro

DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

Solid-Phase E. coli Polypeptide TABLE 2

Analysis for change in ECP antibodies (IgG) in sera from controls and
(ECP) Assay patients treated with human insulin (recombinant DNA)

Wells are coated with purified Mean

IgG anti-ECP antibodies (100ul)
at 4°C for 24 hours. Washed 3
times.
Samples and standards (100/* I)
are incubated in the wells at 4°C
for 18 hours. Washed 3 times.
1251-anti-ECP antibodies (IgG
fraction) are incubated in the wells
at room temperature for 6-7 hours.
Washed 3 times and counted
125
to determine I activity.
FIG. 11. Procedure for detection of immunoreactive E. coli
polypep-tides.
An assay method for detection of immunoreactive ECPs in
human insulin was developed (Figure 11). A 96-well plate is
coated with purified immune gamma globulin (IgG) against the
ECPs. The ECP standards and human insulin samples are added
and incubated, as shown. After washing, a purified IgG for
ECPs containing a radioactive ligand is added to react with any
ECP bound in the wells. By measuring the residual radioactivity
after washing, ECP levels in the sample can be determined by
comparison to known standards.
This assay is done on all lots of human insulin and is
sensitive to 1-3 ppm of immunoreactive ECPs. The human
insulin preparations used in clinical trials contained less than
4 An alternate route to human insulin is via proinsulin,
4 ppm by this assay.
As previously mentioned, ECPs were nonimmunogenic in
laboratory animals unless incorporated in an adjuvant. Sim-ilar
assays were developed to assess the immunologic response to
low levels of contaminants, if present, in the serum of patients
using human insulin (recombinant DNA). In pre-liminary tests,
very little evidence of serum binding to ECPs
Solid-Phase Anti-E. coli Polypeptide (ECP)
Antibody Assay
(D Tubes are coated with ECP (200 ul)
at 4°C for 48-72 hours. Washed.
(2) Serum samples (10 fi\ in 200 pi)
are incubated at 4°C for 24 hours.
Washed.
(3) i25|.protein A (-30,000 CPM) in
200 fi\ is incubated at 25 °C for 1
hour. Washed and counted to
determine ^-activity (CPM)
FIG. 12. Procedure for detection of and-ECP antibodies.
Interval of Patient change 95% C.I.t
treatment group No. (CPM)* mean
0-2 mo Control 31 -2.9 -10.9, +5.1
0-6 mo Treated 97 -4.5 -18.1, +9.0
0-12 mo Treated 64 -30.6 -43.4, -17.8
'Counts per minute.
tConfidence interval.
was seen, but the numbers of patients were small, and the
4
results did not allow conclusions to be drawn.
A new method with greater sensitivity was used to deter-
mine if IgG antibodies to ECPs developed in patients re-
ceiving human insulin (recombinant DNA). This method is
outlined in Figure 12. The results of analyses on sera from
patients who have received no other type of insulin than
human insulin (recombinant DNA) are in Table 2. These
results show that after 6-12 mo of treatment, IgG antibodies
to ECPs are not induced. In other clinical studies on patients
transferred from therapy with beef or pork insulins to human
insulin (recombinant DNA), the results after 6 mo of therapy
in over 350 patients are the same. An effort was made to
detect IgE antibodies to ECPs in sera from a limited number
of patients after 12 mo of treatment. No IgE-class antibodies
were found among these 20 patients; however, the assay has
not been properly validated due to the lack of a positive IgE
anti-ECP serum.
obtained by inserting the proinsulin plasmids into E. coli
K12. The end product is treated with cyanogen bromide to
give crude proinsulin that is subjected to oxidative
sulfitolysis to produce proinsulin S-sulfonate. After
appropriate folding of the molecule and formation of the
disulfide bonds, the re-sulting proinsulin is purified by gel
filtration. The connecting peptide of purified proinsulin can
be enzymatically cleaved to yield insulin (Figure 13).
Trypsin and carboxypeptidase B are used for the
enzymatic conversion of proinsulin to insulin that occurs
rapidly, as shown by HPLC of the reaction mixture in Figure
14- At the beginning of the reaction, only proinsulin is
present. Five minutes later, the reaction is clearly beginning,
and after 15 min, substantial conversion has occurred. After
30 min, the reaction is complete, and insulin is obtained.
Figure 15 shows the HPLC results of human insulin made
via proinsulin and chain combination methods. They are
essentially identical.
Studies and results with human insulin obtained from
proinsulin are summarized in Table 3. Note the additional
tests used to determine the levels of C-peptide, proinsulin,
and cleavage enzymes in the final insulin product.
The presence of human proinsulin in human insulin may

10 DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982

 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

Primary Structure of Human Proinsulin

53 52 51 50

NH,— B-CHAIN

FIG. 13. Schematic of the human proinsuUn

molecule.

be advantageous. The human pancreas secretes proinsulin
along with insulin in the ratio of about 1:100. It is possible
that mixtures of insulin and proinsulin, or insulin and C-
peptide, or even of all three, may ultimately come to be used
in diabetic therapy. Human proinsulin is, therefore, not only
of interest as a route to human insulin, but for the
possibilities it offers of novel therapeutic approaches.
CONCLUSIONS
The exhaustive developmental studies described demonstrate
the identity between human insulin (recombinant DNA) made
by either route described, and pancreatic human in-sulin, and
the extreme degree of purity and freedom from
undesirable contaminants which are achieved by Eli Lilly
and Company in the manufacture of human insulin. Control
procedures carried out on every lot ensure that all human
insulin produced is authentic, pure, and free from
undesirable contaminants, including any capable of causing
immune re-actions.
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.
Many of these possibilities are now being actively explored
by scientists and clinicians throughout the world.
From the Lilly Research Laboratories, Eli Lilly and
Company, Indianapolis, Indiana.

OB.

FIG. 14- HPLC of the reaction mixture of

proinsulin being enzymaticaUy converted to
insulin.

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 AUTHENTICITY AND PURITY OF HUMAN INSULIN (RECOMBINANT DNA)/IRVING S. JOHNSON

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.

12 DIABETES CARE, VOL. 5, SUPPL. 2, NOVEMBER-DECEMBER 1982