Biotechnol. Prog.

1092, 8, 469-478 409

REVIEW
Recombinant Human Insulin
Michael R. Ladisch' and Karen L. Kohlmann
Laboratory of Renewable Resources Engineering, A. A. Potter Engineering Center, Purdue University,
West Lafayette, Indiana 47907

Insulin is a well-characterized peptide that can be produced by recombinant DNA

technology for human therapeutic use. A brief overview of insulin production from
both traditional mammalian pancreatic extraction and recombinant bacterial and yeast
systems is presented, and detection techniques, including electrophoresis, are reviewed.
Analytical systems for insulin separation are principally based on reversed-phase
chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin,
proinsulin, and insulin. Process-scale separation is a multistep process and includes
ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or
disadvantages of various separation approaches, as described by the numerous literature
references on insulin purification, are presented.

Contents body. The main functions of insulin are to stimulate

anabolic reactions for carbohydrates, proteins, and fats,
Introduction 469 all of which have the metabolic consequences of producing
Background 470 a lowered blood glucose level (Norman and Litwack, 1987).
Insulin Structure 470 I t was estimated in 1986 that there were 60 million
Traditional Insulin Production and 470 diabetics in the world (Johnson, 1986)and that 12 million
Purification Americans have diabetes (Barfoed, 1987). These numbers
Production of Human Insulin 471 are increasing dramatically. The US. population is
Conversion of Porcine Insulin 471 thought to be growing at a rate of 1%per year, but the
rate of growth of the insulin-using diabetic population is
Insulin Produced by 472 5 4 % per year (Johnson, 1986).
Recombinant Methods Health problems relating to diabetes can be devastating.
Two-Chain Method 472 Barfoed (1987) reports that, although acute symptoms
Proinsulin Method 473 can be treated with insulin, vascular complications in the
(Intracellular) later stages of the disease reduce the life expectancy of
Proinsulin (Secreted) 474 diabetics by one-third. In addition, he reports that
Analytical Separation of Insulin 474 diabetics are 25 times more likely to go blind and have
Reversed-Phase High-Performance 474 twice the risk of heart disease than nondiabetics. In the
Liquid Chromatography US. the direct and indirect costs to the economy are
estimated to be over 5 billion dollars annually.
Affinity Chromatography 474
The administration of insulin as a diabetic treatment
Electrophoresis of Insulins 474 has a long and interesting history. In 1922, Frederick
Large-Scale Purification of Human 475 Banting and Charles Best successfully treated the first
Insulin human patient with an insulin preparation obtained from
Conclusion 476 animal pancreatic extractions (Barfoed, 1987). From 1922
to 1972 the only available insulin was purified from the
pancreases of pigs and cows. This insulin was quite
Introduction valuable in prolonging the lives of diabetics who otherwise
would have slowly died because glucose was unavailable
Insulin is a polypeptide hormone that is essential for to their body cells. Improvements in the purification of
the supply of energy to the cells of the body (Barfoed, insulin during the 1970s were successful in producing a
1987). It has been estimated that the disease diabetes stable drug with a predictable action time (Chance et al.,
mellitus (impaired insulin production and its complica- 1975; Dolan-Heitlinger, 1981; Barfoed, 1987). In 1950,
tions) is the third largest cause of death in industrialized Novo Nordisk A/S (Bagsvaerd, Denmark) modified the
countries after cardiovascular diseases and cancer (Bar- state of the insulin itself to obtain an insulin crystal with
foed, 1987). Produced by the @-cellsof the pancreas in a high zinc content which had a long action time, in
response to hyperglycemia, insulin is a potent hormone comparison to amorphous insulin that was quickly ab-
directly or indirectly affecting every organ or tissue in the sorbed (Dolan-Heitlinger, 1981; Barfoed, 1987). The
additions of protamine and zinc delayed absorption since
* Author to whom correspondence should be addressed. insulin forms a stable hexamer in the presence of zinc,

8756-7938/92/3008-0469$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Engineers
470 Biotechnol. Prog.., 1992, Vol. 8, No. 6

human insulin, and recombinant human insulin is identical

to human insulin. Recombinant human insulin is less
likely to cause immunological reactions during therapeutic
use than animal-derived insulin. Porcine insulin, when
administered over long periods of time, may result in
serious allergic reactions. Sequence variation in insulin
is most common a t positions 8-10 (middle of the A chain
disulfidebond), and these differencescan lead to antigenic
responses (Norman and Litwack, 1987). One impetus for
development of the recombinant human insulin process
was a perceived beef and pork shortage in the 1970swhich,
if it had actually occurred, would have restricted the
availability of the pancreatic tissue from which insulin
was extracted and, therefore, the amount of insulin
available (Hall, 1987). A t that time it was feared that the
insulin demand was increasing faster than the supply.
Michael R. Ladisch is Professor of Bioprocess and Agri- Americans continue to limit meat consumption, thereby
cultural Engineering and group leader of the Research and affecting the availability of pancreatic tissue.
Process Engineering Group in the Laboratory of Renewable
Resources Engineering at Purdue University. He received
Insulin was a particularly good choice to be the first
his B.S. degree in chemical engineeringfrom Drexel University therapeutic protein to be produced with DNA technology,
in 1973 and M.S. and Ph.D. degrees in chemical engineering simply because of the large amount of insulin needed. In
from Purdue University 1974 and 1977, respectively. Dr. comparison to another recombinant product, the human
Ladisch’s research interests include bioseparations, kinetics growth hormone which has a current usage of several
of biochemical reactions, chemical reaction engineering, and kilograms/year, the market for insulin is 2 orders of
biomass conversion. He received the U. S. Presidential Young magnitude higher (Prouty, 1991). Since being developed
Investigator Award in 1984 and the Van Lanen Award of the in 1982, the amount of human insulin used has increased
BIOT Division of the American Chemical Society in 1990. dramatically. It is estimated that in the U.S. 73% of the
His work has resulted in numerous publications and several patients use human insulin (Crossley, 1991).
patents. He was recently chairman of the committee on
bioprocess engineering (NRC), which studied research pri-
orities and policy issues in bioprocess engineering. Background

Insulin Structure. Insulin is a well-defined peptide

with known amino acid sequence and structural charac-
teristics (Watson et al., 1983; Norman and Litwack, 1987).
This hormone consists of two separate peptide chains
which are the A chain (21 amino acids) and the B chain
(30 amino acids) joined by disulfide bridges as indicated
in Figure 1. Proinsulin is the biologicalprecursor of insulin
and is a single peptide chain formed when the A and B
chains are connected by the C peptide (Figure 2). Human
and porcine insulins differ by only one amino acid, while
bovine and human insulins differ by three amino acids, as
indicated in Figure 1.
In a recent text on hormones, Norman and Litwack
(1987) detail the discovery of insulin sequence and
Karen L. Kohilmann is currently a postdoctoral research structure. Because of its small size, insulin has been an
associate in the Laboratory of Renewable Resources Engi- ideal molecule for the study of peptide sequence and
neering (LORRE) at Purdue University. She obtained her structure. In 1955,the primary amino acid sequence was
Ph.D. degree from the Food Science Department at Purdue found by F. Sanger, and in 1967, D. F. Steiner and R.
University in the area of food protein chemistry. Her thesis Chance determined the structure of proinsulin. Work
research was in the study of the plasminogen/plasminogen continued with the elucidation of the three-dimensional
activator enzyme system in bovine milk and the relationship structure through X-ray crystallography by D. Crowfoot-
of proteolytic enzymes to the gelation of milk. Prior to Hodgkin in 1969. Using this technique, insulin was seen
graduate school she worked in the Central Research Group to exist as a monomer, a dimer, or a hexamer. X-ray
of the American Home Foods division of the American Home
Products Corporation. Professionalaffiliationshave included crystallography remains an important technique in con-
the American Dairy Science Association and the Institute of firming the identities of different types of insulin. In the
Food Technologists. Her research interests lie in the devel- structure-function relationship for insulin, three features
opment and improvement of protein separations. have been found to have been conserved: (1)the precise
positions of the three disulfide bonds; (2) the N- and
C-terminal regions of the A chain; and (3)the hydrophobic
with the rate of dissociation of the hexamer being the residues of the B chain (Norman and Litwack, 1987).
rate-controlling step in the absorption of the insulin. Recombinant human insulin is chemically,physically,and
The development of recombinant DNA technologies immunologically equivalent to pancreatic human insulin
resulted in the production and use of the first recombinant and is biologically equivalent to both pancreatic human
product, human insulin, in 1982. Then, as now, recom- insulin and purified pork insulin (Chance et al., 1981a).
binant human insulin has two distinct advantages over Traditional Insulin Production and Purification.
traditional insulin obtained from pancreatic extraction: Historically, insulin has been purified from animal tissue
there is virtually an unlimited supply of recombinant by extraction procedures followed by chromatographic
Bhtechnol. Rog., 1992, Vol. 8, No. 6 471

Structures of Various lnsulins

I 1 I 2 13 14 [ 5 I 6 I 7 I8 19 I10 111 I12 I13 114 I15 [ 16 I17 I18 119 120 I21 122 I 23 I24 I25 I 26 I 27 I 28 I29 I30
A--Chain:

Ala-Gly-Val

Ala -Val

S
I
Gly-lle-Val-GluCln-Cys-Cys-Thr-Ser-lle-Cys-~r-Leu-T~-Gln-Leu-Glu-Asn-Tyr~s-Asn
Rabbil
I I
Human

8.- Chaln:
} I [
Human Phe-Val-Pisn-Gk-Hl~~u~~s-Gly-~~-~ls-L~~Va~Glu-~~-~eu-~~-L~uV~~~s-Gly-Glu-Ar~Cly-Ph~Ph~T~-~hr-~~Ly~-T

Rabbil
Ser I
” }
Sheep
Ala

Figure 1. Comparison of A and B chains of insulin from different sources. Reprinted with permission from Dolan-Heitlinger, J.
Recombinant DNA and Biosynthetic Human Insulin, A Source Book; Eli Lilly and Co.: Indianapolis, IN, 1982. Copyright 1982 Eli
Lilly and Co.

techniques (Dolan-Heitlinger, 1981; Barfoed, 1987). Fro-

zen bovine or porcine pancreases are diced, extracted with
ethanol, and acidified to pH 2 with HC1 or H2S04. These
steps both inactivate and remove the enzyme trypsin, SYNTHETIC
TRINUCLEOTIDE
which could degrade the insulin. Calcium carbonate is
then added to neutralize the solution, the extract is I
concentrated by vacuum extraction a t low temperatures, V
and the insulin is precipitated by salt addition. The
precipitate is redissolved in water and reprecipitated by
adjusting the pH to the isoelectric point of insulin.
Chromatography steps for further purification of the
insulin may includegel filtration followed by ion exchange
systems (Dolan-Heitlinger,1981). Application of insulin
to a gel filtration column gives a major peak composed of
insulin, desamido insulin(s), arginine insulin(s), insulin
ethyl ester(s), glucagon, pancreatic polypeptide, and
somatostatin. A small amount of larger molecular weight
material elutes ahead of the insulin peak and contains
proinsulin, and proinsulin intermediates, plus a mixture FUSION
1 TRANSFORM E COU

PROTEIN
of covalent insulin dimers. Proinsulin-like materials may
be immunogenic and therefore must be removed during
the purification procedure (Chance, 1972; Chance et al.,
1975). Gel filtration followed by ion exchange chroma-
tography separates insulin from these other materials.
Production of Human Insulin. The desire to not be
restricted to animal tissue sources for insulin production
1 CnBr

led to the interest in manufacturing human insulin.

Human insulin can be obtained by extraction from the C-PEPTIDE
human pancreas, chemical synthesisfrom individualamino
acids, conversion of porcine insulin to human insulin
“semisynthesis”,or fermentation of genetically engineered
microorganisms (Barfoed,1987). Extraction from a human
pancreas is only feasible for research purposes, and
,I(.:&:ENZYMATIC

chemical synthesis (although it has been accomplished) is INSULIN

currently not economical. The third and fourth methods
have been developed for commercial production of insulin
for therapeutic use and will be discussed. NH2
Conversion of Porcine Insulin. Semisynthetic hu- Figure2. Production of proinsulin in bacteria (modifiedto show
man insulin was commercially developed in the 1970s by location of the C peptide). Reprinted with permission from
Novo Nordisk A/S and is produced by substituting the Watson, J. D.; et al. Recombinant DNA-A Short Course;W. H.
B-30 alanine residue of porcine insulin with a threqnine Freeman Co.: New York, 1983;pp 231-235. Copyright 1983 W.
residue. Five steps result in human monocomponent H. Freeman Co.
insulin (Barfoed, 1987). Insulin is first extracted from processing entails conventional purification processes,
frozen porcine pancreas glands. The second step of which have been described in the previous section. Next,
472 Biotechnol. Pmg., 1992, Vol. 8, No. 6

the purified porcine insulin is converted to human insulin

in a medium that contains only a small amount of water
and trypsin and a large quantity of organic solvent and
threonine ester. The trypsin hydrolyzes insulin a t LYSBB-
AlaBso, while a t the same time catalyzing the reverse
ATG
SYNTHETIC OLIGONUCLEOTIDESCODING
FOR A AND B CHAINS OF INSULIN
A
_.... .....
63 NUCLEOTIDES
TGA ATG - B
90 NUCLEOTIDES
TGA

reaction in which the threonine ester displaces alanine

from position B-30 in the insulin molecule. This transpep-
tidation of porcine insulin to human insulin was optimized
to 97 % yield using solubletrypsin (Markussenet al., 1983).
Transpeptidation is also catalyzed by immobilizedtrypsin,
although the yield is lower a t 80% (Ueno and Morihara,
1987). This is followed by chromatographic purification
to reduce measurable levels of proinsulin and remove the
other reagents. Finally, the product is formulated and
then filled under sterile conditions, packaged, and dis-
tributed.
Insulin Produced by Recombinant Methods. Hu-
man insulin was the first animal protein to be made in
bacteria in a sequence identical to that of the human
pancreatic peptide (Watson et al., 1983). This was
accomplished by Eli Lilly and Co. (Indianapolis, IN) and
Genentech (San Francisco,CA). These companies worked
together to achieve expression of recombinant human
insulin in 1978 in Escherichia coli (E. coli) K-12 using
genes for the insulin A and B chains synthesized a t the
City of Hope National Medical Center (Chance et al.,
1981a). Scientists at Genentech cloned the genes in frame A-CHAIN PROTEIN B-CHAIN PROTEIN
with the @-galactosidasegene of plasmid pBR322; the
recombinant plasmid was amplified in E. coli (Chance et $ . / -
al., 1981b). The first successfulexpressionwas announced

'L
in 1978, with scale-up and approval by the appropriate
drug regulatoryagenciesachieved by 1982 (Johnson,1983).
Insulin's small size and the absence of methionine (Met)
and tryptophan (Trp) residues in the A and B chains were
critical elements in the decision to undertake the cloning
of this peptide, as well as in the achievement of rapid
ACHAIN
(2, AMINO ACIDS)
777,
..................... ..... ...
INSULIN

/
s +
development of the manufacturing process. The Met and
Trp residues produced as a consequence of engineering NH2
and expression in E. coli are hydrolyzed by the reagents
0 ACIDS)~
used during the insulin recovery process. The presence ~ ~ ~ 0

of these amino acids in insulin would have resulted in the Figure 3. Production of insulin in bacteria by using synthetic
hydrolysis and destruction of the product. insulin genes. The bacteria produce hybrid proteins consisting
of the N-terminal portion of the &gal protein fused to either the
Two-Chain Method. Although recombinant human A or B chain of insulin. Reprintedwith permissionfrom Watson,
insulin is now produced in several ways, the first successful J. D.; et al. Recombinant DNA-A Short Course; W. H. Freeman
method, illustrated schematically in Figure 3 (Watson et and Co.: New York, 1983; pp 231-235. Copyright 1983 W. H.
al., 1983), was accomplished on a laboratory scale by Freeman Co.
Genentech followed by successful scale-up by Eli Lilly similar results with 20% of the total cellular protein
and Company (Chance et al., 1981a,b,c). Each insulin expressed as either the A or B chain fusion protein.
chain was produced as a @-galactosidasefusion protein in Subsequent folding of S-sulfonated chains gave 50-80 %
separate fermentations using E. coli transformed with correct folding.
plasmids containing either the A or B insulin peptide The large size of the @-galfusion protein limited yields
sequence. The products were intracellular and appeared since the fusion protein of @-gal(- 1000 amino acids) and
in prominent cytoplasmic inclusion bodies (Williams et insulin A or B chain (21 or 30 amino acids, respectively)
al., 1982). The method used to extract the peptides from became detached from the cell's ribosome (premature
the inclusion bodies is proprietary information. Recom- chain termination during translation)and thereforeyielded
binant proteins produced in E. coli usually represent 10- incomplete insulin peptides (Burnett, 1983; Hall, 1987).
40% of the total protein (Burgess, 1987). A key improvement to this approach was the use of the
Once removed from the inclusion bodies, chemical tryptophan (Trp) operon in place of the lac operon (@-gal
cleavage by CNBr a t the Met residue between the system)to obtain a smaller fusion protein. The Trp operon
@-galactosidase(abbreviated @-gal)and the A or B chain, consists of a seriesof five bacterial genes which sequentially
followed by purification, gave separate A and B peptides. synthesize the enzymes responsible for the anabolism of
The peptides were then combined and induced to fold a t tryptophan. One of these enzymes, Trp E, has only 190
a ratio of 2:l of A B chains (S-sulfonated forms) in the amino acids compared to @-gal's1000 amino acids. The
presence of limited amounts of mercaptan in order to Trp E gene followed by genes for the A or B chains of
obtain an active hormone (Chance et al., 1981c;Frank and insulin has the added advantage of enhancing fusion
Chance, 1985). After 24 h, the yield was approximately protein production from 5-10% to 20-30% of the total
60% based on the amount of B chain used (Chance et al., protein (Hall, 1987) since the Trp promoter is a strong
1981a; Johnson, 1986). Goeddel et al. (1979) obtained promoter in E. coli. This leads to a t least 10-fold greater
Biotechnol. Prog., 1992, Vol. 8, No. 6 475

A-chain

1004 21

I h
ml 191
A-chain

21

Figure 4. Structure of insulin-codingplasmids: ApR,ampicillii

-
tm

1-
LE

proinsulin

Figure 5. Relative molecular size of various human insulin

chimeric polypeptides. The numbers indicate the number of
amino acids in each fragment. Reprinted with permission from
resistance, TCR,tetracycline resistance; Trp, tryptophan operon Burnett, J. P. In Experimental Manipulation of Gene Expres-
5'regulatory sequences. Reprinted with permissionfrom Burnett, sion; Inouye, M., Ed.; Academic Press, Inc.: New York, 1983;pp
J. P. In Experimental Manipulation of Gene Expression;Inouye, 259-277. Copyright 1983 Academic Press, Inc.
M., Ed.; Academic Press, Inc.: New York, 1983; pp 259-277.
Copyright 1983 Academic Press, Inc.
insulin chains. Further modificationsof the A and B chains
expression of polypeptide when compared to the lac (i.e., include oxidative sulfitolysis, purification, and combina-
&gal) system (Burnett, 1983). The Trp operon is turned tion to produce crude insulin. This crude insulin is
on when the E. coli fermentation runs out of tryptophan subjected to ion exchange, size exclusion, and reversed-
(Hall, 1987;Etienne-Decant, 1988). This characteristic is phase high-performance liquid chromatography to produce
beneficial during fermentation since cell mass can first be the purified recombinant human insulin (Frank and
maximized. Then, when appropriate, the cell's insulin Chance, 1983).
production system can be turned on by allowing the There is a recent patent (Bobbitt and Manetta, 1990)
fermentation media to become depleted in Trp. Since on the chaotropic and sulfitolyticsolubilizationof inclusion
the insulin fusion protein is an intracellular product (i.e., bodies of heterologous proteins. This particular process
inclusion body), productivity is proportional to cell mass. includes sulfitolysis followed by a warming step, allowing
If the inclusion bodies were formed prematurely, cell the protein S-sulfonate to precipitate in high purity: 95 95
growth would stop, and therefore, total cell mass would pure compared to the starting material. This method seeks
be lower than the maximum possible. Consequently, the to avoid improper disulfide bond formation or intermo-
"Trp switch" is a very important practical tool in maxi- lecular cross-linking which can occur in the conventional
mizing production. processing following cell lysis.
The advantage of the Trp LE' system in terms of ProinsulinMethod (Intracellular). Human insulin can
potential productivity is clear from Figure 5. The smaller also be made with recombinant microorganisms that
Trp protein, like the large lac protein, is highly insoluble. produce intact proinsulin instead of the A or B chains
Consequently, the Trp E fusion protein also accumulates separately. The proinsulin route is the current method of
intracellularly in the form of insoluble inclusion bodies, choice for insulin production (Kroeff et al., 1989) and
which help to retard the proteolytic degradation of the entails one sequence of fermentation and purification steps
product during fermentation and initial recovery (Burnett, rather than two seta of sequences (i.e., one for the A chain
1983). The structure of the insulin coding plasmid is shown and one for the B chain). This approach is summarized
in Figure 4, and the relative molecular size of various by Kroeff et al. (1989) with the basis of the recombinant
human insulin chimeric proteins is shown in Figure 5 technology described by Watson (Watson et al., 1983),as
(Burnett, 1983). shown in Figure 2. Initially, mRNA is copied into cDNA,
Details of the two-chain method of insulin production and a methionine codon is chemically synthesized and
are given in the literature. Separate fermentations of attached to the 5' end of the proinsulin cDNA. The cDNA
specific recombinant E. coli strains are performed to is inserted into a bacterial gene in a plasmid vector that
produce each chain with the inclusion bodies processed to is introduced and then grown in E. coli. Proinsulin can
yield partially purified S-sulfonate forms. These are be released from the bacterial enzyme @gal) fragment
subsequently used in the combination reaction (Frank and (or alternatively from the Trp-LE'-Met-proinsulin [Trp
Chance, 1983;Johnson, 1984;Johnson, 1986). Both of the proinsulin]) by destroying the methionine linker. The
fermentations produce a chimeric protein composed of proinsulin chain is subjected to a folding process to allow
the specific A or B chain linked through the amino acid intermolecular disulfides to form, and the C peptide can
methionine (Met) to a Trp-LE' promoter peptide (Frank then be cleaved with enzymes to yield human insulin
and Chance, 1983). (Frank and Chance, 1983). In comparison, the two-chain
After fermentation is completed, the cells are recovered method previously described is more complex (Figure 3).
and disrupted. The cell debris is then separated from the Human insulin, derived from proinsulin generated through
inclusion bodies, and the inclusion bodies are dissolved in the Trp LE promoter, is discussed by Kroeff et al. (1989).
a solvent, although specifics are not known (Wheelwright, After recovery, the Trp proinsulin undergoes CNBr
1991). Inclusion bodies are sometimes dissolved in 6 M cleavage to yield proinsulin. The proinsulin is subjected
guanidine HCl and 0.1 mM dithiothreitol (Burgess, 1987). to oxidative sulfitolysis, folding conditions in the presence
Next, the Trp-LE'-Met-A chain and the Trp-LE'-Met-B of a mercaptan, several purification steps, and then
chain undergo a CNBr cleavage to release the A and B enzymatic treatment to form the crude insulin. Ion
474 Biotechnol. Prog., 1992, Vol. 8, No. 6

exchange, reversed-phase, and size exclusion chromatog- (particularly large proteins) to bond so tightly to the
raphy steps result in the purified recombinant human stationary phase that they are difficult to elute appears
insulin. to be the major limitation of the alkylsilane supports.
Proinsulin (Secreted). Villa-Komaroff etal. (1978)were It is difficult to compare the analytical systems described
first to describe a secretion system for human proinsulin in Tables I and 11. Each particular system was designed
in E. coli. Watson (1983) suggested that the ideal situation and optimized for a specific series of separations. In
for recombinant protein production would be to have large general though, Smith et al. (1985) have summarized the
amounts of the foreign protein efficiently secreted into conditions affecting the RP-HPLC of insulin and related
the medium by the bacteria. In the specificcase of insulin, compounds. Acetonitrile or methanol with various mix-
the recombinant protein could consist of j3-lactamase (an tures of aqueous buffers is usually used for the mobile
enzyme that inactivates penicillin, which is naturally phase in insulin analysis. Capacity factors of insulin
secreted by bacteria into the culture media) and proinsulin. decrease with increasing acetonitrile concentration up to
Yeasts are also attractive for this type of system since 40% (Grego and Hearn, 1981). Since most polypeptides
they secrete only a few of their own proteins. Therefore, and proteins strongly bind to alkylsilicasupports, chloride
fewer extraneous proteins would need to be removed in salt may be substituted for phosphate salt in the mobile
purification. Yeasts also are able to facilitatethe formation phase. Most separations are carried out at ambient
of disulfide bonds. However, for glycosylated proteins, temperatures, with optimal detection of insulin being
yeasts tend to over glycosylate. between 190 and 220 nm. All systems suffer, in varying
Novo Nordisk A/S used baker’s yeast or Saccharomyces degrees, from interferences that are due to preservatives
cerevisiae to screte insulin as a single-chain insulin or other insulin impurities (Smith et al., 1985).
precursor. Both the process and the product were With the advent of recombinant insulins, other ana-
approved by the Danish Parliament in 1986 (Diers et al., lytical systems as well as modifications of the RP-HPLC
1991). Diers et al. (1991) describe the unfolded peptide system have been described for the analytical separation
as a leader or prosegment, next a Lys-Arg sequence of insulin, proinsulin, C peptide, and insulin Aand B chains
(recognizedby the processing enzyme), the B chain (amino from each other and from recombinant fusion proteins.
acids 1-29), a short peptide bridge, followed by the A chain De Guevara (1985) isolated and quantitated the A and B
(amino acids 1-21). In this precursor, amino acid 29 of chains of insulin with a RP-HPLC system (Waters Radial
the B chain of insulin is connected to amino acid 1of the Pak with Bondapak c18 cartridge, Waters, Milford, MA)
A chain by a short connecting peptide containing one basic using ion pairing with trifluoroacetic acid (TFA) as the
amino acid adjacent to the A chain. Human insulin is counterion for the A chain and formic acid for the B chain.
produced through transpeptidation followed by hydrolysis Kakita et al. (1981)used gel chromatography (Biogel P-30,
of the ester bond formed. Several chromatography steps Bio-Rad, Richmond, CA) to separate proinsulin from the
follow for further purification (Diers et al., 1991). C peptide with 1 M acetic acid as the eluting buffer.
Another process for producing human proinsulin in- Welinder (1984) looked a t the homogeneity of crystalline
tracellularly in the yeast S. cereuisiae has recently been insulin using RP-HPLC (Nucleosil 10 CIS) and high-
described (Tottrup and Carlsen, 1990). Using this yeast performance gel permeation chromatography (HP-GPC)
system in an optimized batch-fed fermentation, yields of (1-125 column, Waters). Welinder and Linde (1984)
the fusion protein of superoxide dismutase-human pro- reported the separation of insulin and insulin derivatives
insulin (SOD-PI) were reported to be 1500 mg/L. SOD- using high-performance ion exchange chromatography
PI would be the starting material for the production of (HP-IEC) and gave a comparison to RP-HPLC. Ion
recombinant human insulin; yields of the final product exchange chromatography using a 0-0.29 M NaCl salt
have not been reported. Apparently, the expression of gradient over a DEAE-derivatized polymeric stationary
heterologous polypeptides in yeast has sometimes been phase was recently reported by Ladisch et al. (1990). In
lower than desirable. An Eli Lilly Co. patent (Beckage this ion exchange based separation, insulin was separated
and Ingolia, 1988) describes a process of aerobic culturing from &galactosidase and from the insulin A chain.
of yeast, followed by anaerobic and then back to aerobic Affinity Chromatography, Affinity chromatography
conditions. This method is reported to result in a high may also be used in the purification of fusion proteins. A
expression of product (produced intracellularly in S. one-step affinity purification procedure which gives an
cerevisiae). overallyield of 85-95 ?6 of hybrid protein with @-galactivity
using TPEG-Sepharose under high-salt conditions has
Analytical Separation of Insulin been described (Burgess, 1987). Nilsson et al. (1989)
Reversed-Phase High-Performance Liquid Chro- describe a general gene fusion approach to facilitate
matography. Reversed-phase high-performance liquid purification of recombinant proteins based on the fusion
chromatography (RP-HPLC) over alkylsilane supports of a gene of interest to a gene encoding a protein with a
appears to be the method of choice in the analysis of insulin strong affinity to a ligand (affinity fusion).
(Tables I and 11) (Monch and Dehnen, 1978; Damgaard Smith et al. (1988) report the use of a technique called
and Markussen, 1979; O’Hare and Nice, 1979; Terabe et chelating peptide immobilized metal ion affinity chro-
al., 1979; Dinner and Lorenz, 1979; Kroeff and Chance, matography (CP-IMAC)to purify recombinant proinsulin.
1982; Lloyd and Corran, 1982; Rivier and McClintock, In this method the proinsulin is expressed with a chelating
1983;Parman and Rideout, 1983;McLeod and Wood, 1984, peptide on the NH2 terminus. The proinsulin can then
Knip, 1984;Kalant et al., 1985; Smith and Venable, 1985; be affinity-purified with immobilized metal ions, and the
Vigh, 1987). The high selectivity and high resolving chelating peptide is removed chemically or enzymatically.
capabilities of RP-HPLC using a wide variety of stationary Electrophoresis of Insulins. Electrophoresis is a
and mobile phases allow the separation of insulin species commonly used method for detecting contaminating
which differ by only one amino acid (Koreff et al., 1989). proteins in a previously purified sample. However, unlike
The mechanism for the separation of insulin in RP-HPLC many other proteins, the insulins present a special
systems is based on the hydrophobicities of insulin and challenge. Conventional electrophoretic techniques sep-
related compounds. The tendency of some proteins arate proteins down to molecular weights of about 1 2 OOO
Biotechnol. Rug., 1992, Vol. 8, No. 6 475

Table I. Isocratic &versed-Phase Chromatography Systems for Insulin

separation system comments reference
bovine and porcine insulin LiChrosorb RP-&*elution higher molecular weight Dinner and Lorenz, 1979
from monodesamido with acetonitrile in sulfuric proinsulin impurities
derivatives acid buffer retained until acetonitrile
concentration increased
to 26-27 %
bovine and porcine insulin Nucleosil ODS,a-cUltrasphere the second elution system had Lloyd and Corran, 1982
from monodesamido ODS,'vd Hypersil ODs,".' the advantage of separating
derivatives LiChrosorb RP-18,' Spherisorb bovine and porcine insulin
ODS,'f and Zorbax TMSI from human insulin
elution with (1)tartaric or acetic
acid with acetonitrile and
cetrimide and (2) acetonitrile
in acid phosphate buffer
bovine, porcine, and human Zorbax TMS;g elution with suggests that HPLC techniques Kroeff and Chance, 1982
insulin, monodesamido acetonitrile in an acid can ala0 be used to determine
insulins and insulin dimers phosphate buffer potency of insulin crystals
and formulations
bovine, porcine, pancreatic Vyda$ derivatized three ways: all three columns and both Rivier and McClintock, 1983
human, chicken, ovine, rabbit, (1)C-18, (2) phenyl, (3) C-4; buffers separated the insulins
and rat insulins elution with (1)TFA in
acetonitrile and (2)
triethylammonium
phosphate in acetonitrile
hormones: (21) ACTH Nucleosil 5C-1Rkelution with separated bovine, porcine, and Terabe et al., 1979
analogues, (3) LH-RH tartarate buffer-acetonitrile human insulin with 29.2 %
analogues, and (4) insulins containing sodium + butane acetonitrile in the mobile phase;
sulfonate and sodium sulfate slight changes in pH or buffer
concentration were not critical,
but consistent acetonitrile
concentration was critical
for retention times
bovine, human, porcine, ODS Ultrasphere;a,i isocratic use of insulin analogues allowed McLeod and Wood, 1984
mouse, turkey, rodent insulins; and shallow gradient elution comparison of separations
bovine proinsulins and a series in acid phosphate buffer, with predictions
of bovine insulin analogues included chaotropic salts
bovine, porcine, recombinant LiChrosorb RP-8band p better separation of human Kalant et al., 1985
human, pancreatic human Bondapak C-18;j elution with insulin was obtained with the p
insulins; oxidized bovine three systems: (1)ammonium Bondapak C-18 column
insulin A and B chains sulfate acidified with
sulfuric acid and mixed
with acetonitrile, (2)
tetramethylammonium
hydroxide, orthophosphoric
acid mixed with methanol, (3)
ethanolamine, orthophosphoric
acid mixed with acetonitrile
bovine and porcine Nucleosil C-&kdisplacement a proinsulin contamination target Vigh et al., 1987
insulin chromatography with methanol of 100 ppm was achieved
phosphate buffer and cetrimide
disp1acer
ODS = octadecylsilane. Merck, Darmstadt, FRG. Camlab, Great Britain. Anachem, Great Britain. e Shandon Southern, Great Britain.
fPhase Separations, Great Britain. 8 Hitchin, Great Britain. * Separations Group, Hesparia, CA. Altex. j Waters, Milford, MA. Macherey
and Nagel, FRG.

Da, although modifications of conventional procedures Large-Scale Purification of Human Insulin

are reported to extend the separation range to Mr = 2500
(Fling and Gregerson, 1986). Proteins or peptides smaller Purification of a protein that has been overproduced in
than this migrate and also may stain differently than larger E. coli requires a somewhat different methodology than
proteins. Insulin is a polypeptide (M, = 5800) [A chain
that used in conventional protein purification or tech-
M, = 2300,B chain Mr = 34001 (Sanger, 1949). Using the niques used for analytical purposes (Welinder and Linde,
1984). An exceedingly high degree of purity is required
techniques of Fling and Gregerson (19861,the B chain of as well as the freedom from contaminating solvents and
insulin shows an anomalously high molecular weight, while toxins (from E. coli) and the nutrients, metabolites,
the A chain does not stain due to either anomalous catabolites, and host cell functional molecules which are
migration, lack of banding, or inadequate fixing prior to present as a consequence of cell growth (Johnson, 1986).
staining. There are clearly some difficulties involved in The recombinant protein must also be free of fusion protein
the electrophoretic separation of insulin from ita compo- components. Furthermore, processing conditions must
nent A and B chains. Insulin can, however, be separated be chosen to avoid proteolysis of hybrid proteins, especially
from proinsulin and monodesamido insulin using a poly- the larger proteins which are subject to proteolytic
acrylamide disc-gel electrophoresis system described by degradation (Ullmann, 1984). In particular, insulin must
Chance et al. (1968). In this particular system, however, be separated from other forms of insulin (proinsulin,
insulin and desalanine (des B-30)insulin have the same desamido insulin, etc.) which might be immunogenic. A
net charge and migrate identically. large-scale purification scheme must meet all these re-
476 Blotechnol. Prog., 1992, Vol. 8, No. 6

Table 11. Gradient Reversed-Phase Chromatography Systems for Insulin

separation system comments reference
insulin and calibration proteins ODS Nucleosil 10-C18;avb separates proteins and Monch and Dehnen, 1979
(cytochrome C, bovine serum elution with phosphate peptides
albumin, aldolase, catalase, 2-methoxyethanol and
chymotrypsinogen A, isopropyl alcohol
ferritan, ovalbumin 2-methoxyethanol
bovine and porcine insulin, p Bondapak C-18;celution one of the first reports of Damgaard and Markussen, 1979
porcine insulin from porcine with acetonitrile in an acid the separation of insulin
monodesamido insulin phosphate buffer from monodesamido forms
(32) polypeptides and (9) Hypersil ODS,d Nucleosilti-Cl8,b under optimal conditions all O'Hare and Nice, 1979
proteins Spherisorb ODs,",' LiChrosorb of the polypeptides could be
RP-lg and RP-8, and Zorbax chromatographed; low pH
C8,g elution with acetonitrile (<4.0) and high buffer
in an acid phosphate buffer molarity were important
bovine, porcine, and human Zorbax TMS;h elution with separation at 40 O C gave Smith et al., 1985
insulins; desamido forms acetonitrile in an acid better results than lower
of insulin sulfate buffer temperatures; HPLC
techniques and bioassay
potencies were in agreement
porcine proinsulin from RPC-Md (ODs);"elution a gradient system may better Parman and Rideout, 1983
porcine insulin with acetonitrile or detect spontaneous conversion
methanol-acetonitrile products in insulin and
and phosphate buffer proinsulin preparations than
an isocratic system
pancreatic peptides, insulin Sherisorb S5 ODS2;'se elution system useful for the separation Knip, 1984
and proinsulin with acetonitrile in an acid of peptide hormones
phosphate buffer
0 ODS = octadecylsilane. Macherey and Nagel, FRG. Waters, Milford, MA. Shandon, Runcorn, Great Britain. e Phase Separations,
Queensferry, Great Britain. f Merck, Darmstadt, FRG. 8 Du Pont, Hitchin, Great Britain. h Du Pont Instruments, Wilmington, DE.

quirements, and in doing so, it has been estimated that the RP-HPLC is done fairly rapidly (within a matter of
more than half of the processing costs are in the down- hours) the deamidation can be minimized. This RP-HPLC
stream purification relative to the actual fermentation system was successfullyused in a production-scale system
(Ullmann, 1984). to purify recombinant insulin with a biological potency
Inclusion bodies are formed in more than 80% of the equal to that obtained from the conventional purification
cases where proteins have been overproduced in E. coli system, which employs ion exchange and size exclusion
(Welinder and Linde, 1984). The E. coli fusion protein chromatography (Kroeff et al., 1989).
8-galactosidase-insulin is no exception. Burgess (1987) Welinder and Linde (1984) investigated high-perfor-
gives a procedure to release an insoluble fusion protein mance ion exchange chromatography (HP-IEC)for insulin
involving lysis,centrifugation, denaturation, renaturation, purification and compared this method to RP-HPLC for
recipitation, and ion exchange chromatography steps. insulin separation. HP-IEC allowed rapid fractionation
Many proteins have been purified from insoluble inclusion of crystalline insulin with no salt gradient. They found
bodies by these methods of denaturation and renaturation. HP-IEC gave good recovery and had fewer organic
More conventional purification procedures can be followed contaminants than RP-HPLC, but needed to be performed
for a secreted fusion protein such as 8-lactamase-insulin. under dissociating conditions (7 M urea).
There are few detailed, published procedures for a
complete large-scale purification sequence for recombinant Conclusion
insulin fromE. coli. However, Kroeff et al. (1989) describe Insulin is a therapeutic protein which now has a history
a multimodal chromatography preparative-scale system of production using recombinant DNA methods. Im-
with an integral RP-HPLC step developed to purify and provements in genetic engineeringmethods have facilitated
analyze recombinant human insulin produced from E. coli. both the production and the recovery of this high-value
This RP-HPLC system is placed fairly late in the insulin peptide. The standard method for analysis of insulin and
purification system since the majority of the impurities insulin derivatives is RP-HPLC. Standard methods for
(mainly from E. coli) are removed prior to this step by an the purification of insulin produced by recombinant
ion exchange step. A size exclusion separation follows the methods include ion exchange, gel permeation, and
reversed-phase step. The reversed-phase system is based reversed-phase chromatography. While the current prac-
on a stationary phase of 10 pm Zorbax (Du Pont Instru- tice of these methods yields highly purified insulin, further
ments, Wilmington, DE) process-grade CS for the pro- refinement in separation is an area of continuing research.
duction-scale columns and 5-pm particles for the analytical This reflects the increasing demand for therapeutic
scale columns. Partially purified human insulin zinc proteins with minimal contamination and the need to
crystals prepared at Eli Lilly and Co. were the starting reduce the high cost of purification.
material for this part of the purification sequence. The Notation
insulin was applied in a water-rich mobile phase and then
eluted in a linear gradient of 0.25M acetic acid (eluent A) HCl hydrochloric acid
to 60% aqueous acetonitrile (eluent B). An acidic mobile CNBr cyanogen bromide
phase is recommended sinceit provides excellentresolution RP- reversed-phase high-performance liquid chroma-
of insulin from structurally similar insulin-likecomponents HPLC tography
while promoting insulin solubility. The ideal pH is thought Met methionine
to be in the region of 3.0-4.0, which is well below the TFA trifluoroacetic acid
isoelectric pH of 5.4. Under mildly acidic conditions HP- high-performancegel permeation chromatography
insulin may deamidate to monodesamido insulin, but if GPC
Bbtechnol. Rog.., 1992, Vol. 8, No. 6 477

HP-IEC high-performance ion exchange chromatography Frank, B. H.; Chance, R. E. Two Routes for Producing Human
HzS04 sulfuric acid Insulin Utilizing Recombinant DNA Technology. Munch.
Med. Wschr. 1983,125 (Suppl. l), 514-520.
Mr molecular mass
Frank, B. H.; Chance, R. E. In Quo Vadis? Therapeutic Agents
Produced by Genetic Engineering; Joyeauk, A., Leygue, G.,
Morre, M., Roncucci, R., Schmelck, P. H., Eds.; Sanofi Group:
Acknowledgment Toulouse-Labege, France, 1985;pp 137-146.
The material is this work was supported b y the Purdue Goeddel,D.V.;Kleid,D.G.;Bolivar,F.;Heyneker,H.L.;Yansura,
University Agricultural Experiment Station, NSF Grant D. G.;Crea, R.; Hirose, T.; Kraszewski, A.; Itakura, K.; Riggs,
A. D. Expression in Escherichia coli of Chemically Synthesized
BCS 8912150, and t h e Rohm and Haas Co. We t h a n k Dr. Genes for Human Insulin. Proc. Natl. Acad. Sci. U.S.A. 1979,
Chuck Pidgeon (Purdue PharmacyDept.) andDr. Suzanne 76 (l),106-110.
Nielsen (Purdue Food Science Dept.) for their helpful Grego, B.; Hearn, M. T. J. Role of the Organic Solvent Modifier
comments during review of this manuscript. We also wish in the Reversed Phase High-Peformance Liquid Chromatog-
to t h a n k Dr. Ronald E. Chance,Dr. Eugene P. Kroeff, and raphy of Polypeptides. Chromatographia 1981,14,589-592.
Dr. Walter F. Prouty (Eli Lilly and Co., Indianapolis, IN) Hall, S. S. Znuisible Frontiers--TheRace to Synthesize a Human
for helpful comments and their considerable efforts in Gene; Atlantic Monthly Press: New York, 1987.
thoroughly reviewing this manuscript. Johnson, I. S. Human Insulin from Recombinant DNA Tech-
nology. Science 1983,219,632637,
Literature Cited Johnson, I. S. In Biotechnology and Biological Frontiers; The
Beckage,C. A,;Ingolia, T. D. Method, Vectors,and Transformanta American Association for the Advancement of Science
for High Expression of Heterologous Polypeptides in Yeast. Publishers: Washington, DC, 1984;pp 46-56.
U.S.Patent 4,745,057,May 17, 1988. Johnson, R. D. The Processing of Biomacromolecules: A Chal-
Bobbitt, J. L.; Manetta, J. V. Purification and Refolding of lenge for the Eighties. Fluid Phase Equilib. 1986,29,109-
Recombinant Proteins. US.Patent 4,923,967,May 8, 1990. 123.
Barfoed, H. C. Insulin Production Technology. Chem.Eng. Prog. Kakita, K.; Horino, M.; Tenku, A.; Nishida, S.; Mataumura, S.;
1987,83(lo),49-54. Matauki, M. Gel Chromatographic Separation of Human
Burgess, R. In Protein Engineering; Oxender, D. L., Fox, C. F., C-Peptide and Proinsulin. J. Chromatogr. 1981,222,33-40.
Eds.; Alan R. Liss, Inc.: New York, 1987;pp 71-82. Kalant, D.; Crawhall, J. C.; Posner, B. I. Separation of Biological
Burnett, J. P. In Experimental Manipulation of Gene Expres- Variant Insulin Molecules from Different Speciesby Reversed-
sion; Inouye, M., Ed.; Academic Press: New York, 1983;pp Phase High-Performance Liquid Chromatography (HPLC).
259-277. Biochem. Med. 1985,34,230-240.
Chance, R. E. Amino Acid Sequences of Proinsulins and Knip, M. Analysis of Pancreatic Peptide Hormones by Reversed-
Intermediates. Diabetes 1972,21(Suppl. 2), 461-467. Phase High-Performance Liquid Chromatography. Horm.
Chance, R. E.; Ellis, R. M.; Bromer, W. W. Porcine Proinsulin: Metab. Res. 1984,16,487-491.
Characterization and Amino Acid Sequence. Science 1968, Kroeff, E. P.; Chance, R. E. In Hormone Drugs, Proceedings of
161, 165-167. the FDA-USP Workshop on Drug and Reference Standards
Chance, R. E.; Root, M. A.; Galloway, J. A. In Immunological for Znsulins, Somatropins, and Thyroid-axis Hormones;U.S.
Aspects of Diabetes Mellitus; Anderson, 0. O., Deckert, T., Pharmacopeial Convention,Inc.: Bethesda, MD, 1982;pp 148-
Nerup, J., Eds.; Acta Endocrinologica (Kbh.) (Suppl. 205): 162.
Denmark, 1975;pp 185-196. Kroeff, E. P.; Owens, R. A.; Campbell, E. L.; Johnson, R. D.;
Chance,R. E.; Kroeff, E. P.; Hoffmann, J. A. InZnsulins, Growth Marks, H. I. Production Scale Purification of Biosynthetic
Hormone and Recombinant DNA Technology; Raven Press: Human Insulin by Reversed-Phase High-Performance Liquid
New York, 1981a;pp 71-85. Chromatography. J. Chromatogr. 1989,461,45-61.
Chance, R. E.; Kroeff, E. P.; Hoffmann, J. A.; Frank, B. H. Ladisch, M. R.; Hendrickson, R. L.; Kohlmann, K. L. In Protein
Chemical, Physical, and Biologic Properties of Biosynthetic Purification: From Molecular Mechanisms to Large Scale
Human Insulin. Diabetes Care 1981b,4 (2),147-154. Processes; ACS Symposium Series 427;American Chemical
Chance, R. E.; Hoffmann, J. A.; Kroeff, E. P.; Johnson, M. G.; Society: Washington, DC, 1990;pp 93-103.
Schmirmer, E. W.; Bromer, W. W.; Ross, M. J.; Wetzel, R. In Lloyd, L. F.; Corran, P. H. Analysis of Insulin Preparations by
Peptides: Synthesis-Structure-Function;Rich, D. H., Gross, Reversed-Phase High-Performance Liquid Chromatography.
E., Eds.; Pierce Chemical Co.: Rockford, IL, 1981c;pp 721- J. Chromatogr. 1982,240,445-454.
728. Markussen, J.; Damgaard, U.; Pingel, M.; Snel, L.; Sorensen, A.
Crossley Patient Survey, 1991. R.; Sorensen, E. Human Insulin (Novo): Chemistry and
Damgaard, U.; Markussen, J. Analysis of Insulins and Related Characteristics. Diabetes Care 1983,6 (Suppl. l), 231-235.
Compounds by HPLC. Horm. Metab. Res. 1979,11,580-581. McLeod, A.; Wood, S. P. High-Performance Liquid Chroma-
De Guevara, 0.L. Identification and Isolation of Human Insulin tography of Insulin. J. Chromatogr. 1984,285,319-331.
A and B Chains by High-PerformanceLiquid Chromatography.
J. Chromatogr. 1985,349,91-98. Monch, W.;Dehnen, W. J. High-Performance Liquid Chroma-
Diers, I. V.; Rasmussen, E.; Larsen, P. H.; Kjaersig, 1.L. In Drug tography of Polypeptides and Proteins on a Reversed-Phase
Biotechnology Regulations (ScientificBasis and Practices); Support. J. Chromatogr. 1978,147,415-418.
Chiu, Y. H., Gueriguian, D. L., Eds.; MarcelDekker, Inc.: New Nilsson, B.; Anderson, S.; Uhlen, M. In Advances in Gene
York, 1991;pp 167-177. Technology;Brew, K., Ed.; IRL Press Limited: McLean, VA,
Dinner, A.; Lorenz, L. High-Performance Liquid Chromato- 1988; pp 122-123.
graphic Determination of Bovine Insulin. Anal. Chem. 1979, Norman, A. W.; Litwack, G. In Hormones;Academic Press: New
51 (ll),1872-1873. York, 1987;pp 264-317.
Dolan-Heitlinger, J. In Recombinant DNA and Biosynthetic O’Hare, M. J.; Nice, E. C. Hydrophobic High-PerformanceLiquid
HumanZnsulin, A Source Book; Eli Lilly and Co.: Indianapolis, Chromatography of Hormonal Polypeptides and Proteins on
IN, 1982. Alkylsilane-Bonded Silica. J . Chromatogr. 1979, 171, 209-
Etienne-Decant, J. In Genetic Biochemistry: From Gene to 226.
Protein; Ellis Horwood Limited: Chichester, U.K., 1988;pp Parman, A. U.; Rideout, J. M. Purification of Porcine Proinsulin
125-127. by High-Performance Liquid Chromatography. J. Chro-
Fling, S. P.; Gregerson, D. S. Peptide and Protein Molecular matogr. 1983,256,283-291.
Weight Determination by Electrophoresis Using a High- Prouty, W. F. Production Scale Purification Processes. In Drug
Molarity Tris Buffer System Without Urea. Anal. Biochem. Biotechnology Regulations; Marcel Dekker, Inc.: New York,
1986,155,83-88. 1991;pp 221-262.
478 Biotechnol. Prog.., 1992, Vol. 8, No. 6

Rivier, J.; McClintock, R. Reversed-Phase High-Performance Vigh, G.; Varga-Puchony, Z.; Szepesi, G.; Gazdag, M. Semi-
Liquid Chromatography of Insulins from Different Species. J. Preparative High-PerformanceReversed-Phase Displacement
Chromatogr. 1983,268,112-119. Chromatography of Insulins. J. Chromatogr. 1987,386,353-
Sanger, F. Fractionation of Oxidized Insulin. Biochem. J. 1949, 362.
44, 126-128. Villa-Komaroff, L.; Efstratiadis, A.; Broome, S.; Lomedico, P.;
Smith, D. J.; Venable, R. M.; Collins, J. Separation and Tizard, R.; Naber, S. P.; Chick, W. L.; Gilbert, W. A Bacterial
Quantitation of Insulins and Related Substances in Bulk Clone Synthesizing Proinsulin. Proc. Natl. Acad. Sci. U.S.A.
Insulin Crystals and in Injectables by Reversed-Phase High 1978,75 (81, 3727-3731.
Performance Liquid Chromatography and the Effect of Watson, J. D.; Tooze, J.; Kurtz, D. T. Recombinant DNA-A
Temperature on the Separation. J. Chromatogr. Sci. 1985, Short Course;Scientific American Books, W. H. Freeman Co.:
23,81-88. New York, 1983;pp 231-235.
Smith, M.C.; Furman, T. C.; Ingolia,T. D.; Pidgeon, C. Chelating Welinder, B. S.In CRC Handbook of HPLC for the Separation
of Amino Acids, Peptides and Proteins; Hancock, W. S., Ed.;
peptide-immobilized metal ion affinity chromatography. J.
Biol. Chem. 1988,263 (15),7211-7215. CRC Press: Boca Raton, FL, 1984; pp 413-419.
Welinder, B. S.;Linde, S. In CRC Handbook of HPLC for the
Terabe, S.;Konaka, R.; Inouye, K. Separation of Some Polypep- Separation of Amino Acids, Peptides and Proteins; Hancock,
tide Hormones by High-Performance Liquid Chromatography. W. S., Ed.; CRC Press: Boca Raton, FL, 1984;pp 357-362.
J. Chromatogr. 1979,172,163-177. Wheelwright, S. M. Protein Purification; Oxford University
Tottrup,H. V.; Carlsen,S.A. Process for the Production of Human Press: New York, 1991;p 217.
Proinsulin in Saccharomyces cerevisiae. Biotechnol. Bioeng. Williams, D. C.; Van Frank, R. M.; Muth, W. L.; Burnett, J. P.
1990,35,339-348. Cytoplasmic Inclusion Bodies in Escherichia coli Producing
Ueno, Y.; Morihara, K. Use of Immobilized Trypsin for Semi- Biosynthetic Human Insulin Proteins. Science 1982,215(5),
svnthesis of Human Insulin. Biotechnol. Bioeng. 1989,33, 687-689.
126-128.
Accepted July 3, 1992.
Ullmann, A. One-Step Purification of Hybrid Proteins which
have 8-Galactosidase Activity. Gene 1984,29,27-31. Registry No. Insulin, 9004-10-8.