INSULIN-Ffifififififi Q
`Seite 14 -— 20
`
`
`
`man insulin produced using recom-
`binant DNA technology is of high
`purity, is identical to the human in-
`sulin produced by the human pan-
`creas, and is safe for use in humans.
`The functioning and genetics of
`the bacterium, Escherichia coli, has
`gy is our ability to manipulate this
`been studied for many years, and, as
`a result, most of the recombinant
`bacterial plasmid DNA which is ac-
`DNA research has_been done with
`complished by isolating the plasmid
`
`Page 2
`S14
`
`M'Linch'. med. Wschr. 125 (1983) Suppl. 1
`
`© MMW Medizin Verlag GmbH Miinchen, Miinchen 1983
`
`
`
`
`
`
`
`r
`I
`
`-
`
`1
`
`_
`
`DNA, cleaving ,.,_.with restriction er’,
`zymes
`and inserting the desired.
`DNA. The desired DNA is obtained“.
`either by synthesis,
`isolation fro“
`natural sources,.or by a combination.
`of these procedures. In the human"
`insulin work,
`the A- und B-chain,
`genes were prepared by synthetic nu
`cleotide chemistry, while the huma
`proinsulin gene was semisynthetic
`that is, the gene was constructed us."-
`ing a segment of the natural DN"
`which codes for proinsulin along‘
`with a fragment of synthetic DNA]
`
`The nucleotide synthesis was pe
`formed by Itakura and coworkers a
`the City of Hope, and by Goeddel.
`and coworkers at the Genentech”
`Corporation (5, 9). After the desire‘
`DNA is obtained and inserted, the._
`plasmid DNA is rejoined using a lig--"Q
`ase enzyme and then reintroduced
`_into the host cell thru a process cal-1‘,
`led transformation. The cell then is?
`cloned,
`that
`is many copies are
`made, and, after verifying that the
`desired and correct gene is present,
`the material is stored in ampoules for‘
`future use in production. Thus each
`fermentation is
`started from the : 5;;
`same seed pool which has been ver-
`ified to have the correct gene pre-
`sent.
`In order to maximize the produc-7*‘
`
`.
`tion of the desired protein in the E;_
`coli cells, the gene message that wasj ;_1;*
`inserted into. the’ plasmid also con-—_pf'..
`tains
`a
`so-called promoter. This?" f
`promoter determines the rate at:
`which messenger RNA is formed; Q.
`thus, if one uses" a strong promoter ‘if,
`more messenger RNA is
`formed,-. v_
`and consequently there is greater‘_'_
`production by the cell of the desired
`gene product. For human insulin
`biosynthesis, two promoter systems
`have been used, originally Beta— .
`galactosidase and now tryptophan
`synthetase, or Trp E. The Trp E .
`promoter yields more of the desired; '1
`product as compared to the Beta-
`.
`galactosidase
`promoter
`system.’
`When the E. coli cells are producing '
`
`
`B. H. Frank, Ph. D., R. E. Chance, Ph. D.,
`Lilly Research Laboratories, Eli Lilly and
`Company,
`Indianapolis,
`Indiana
`46285 ‘
`U.S.A.
`
`V
`
`B. H. Frank, R. E. Chance
`
`
`
`
`
`
`1. Human insulin synthesized in bacteria represents an-
`important and safe source of highly purified insulin iorthe
`future treatment of the insulin—dependent diabetic.
`2. _ Biosynthetic human insulin produced by recombinant DNA
`methods and prepared by either chain combination orconver—
`sion of proinsulin is chemically, physically, and biologically
`equivalent to pancreatic human insulin.
`
`
`
`
`'Zwei Wege zur gentechnologischen Ge-
`chemisch, physikalisch und biologisch
`winnung von Humaninsulin:
`identisch mit pankreatischem Humaninsu-
`
`
`1. Die Synthese von Humaninsulin in
`Iin, gleich aufwelchem Wege—entweder
`
`
`Bakterien stellt eine wichtige und sichere
`durch die Kombination getrennt herge-
`Quelle zur Gewinnung von hochgereinig—
`stellter A- und B—Kette oderdurch enzy-
`
`
`tem insulin fiirdie zukiinitige Behandlung
`matische Umwandlung von gentechnolo—
`
`
`insulinabhangiger Diabetiker dar.
`gisch hergestelltem Proinsuiin —es ge-
`
`‘wonnen wird.
`“
`
`2. Biosynthetisches Humaninsuiin ist
`
`
`The preparation of human insulin
`utilizing recombinant DNA technol-
`ogy marks a significant accomplish-
`ment in the field of molecular biolo-
`
`this microorganism. For safety and
`containment reasons‘, Eli Lilly has
`chosen the K-12 strain of E. coli to
`use in our. recombinant DNA re-
`
`
`
`gy and provides a secure source of
`insulin for the future treatment of
`
`the insulin-dependent diabetic. This
`manuscript will review some of the
`molecular biology that Eli Lilly has
`applied in order to accomplish this.
`Further, the procedures used to pre-
`pare and isolate highly purified hu-
`man insulin are described. Finally,
`the results of analytical tests are pre-
`sented to demonstrate that the hu-
`
`search and production, because this
`is a weakened strain of E. coli which
`cannot colonize the intestinal tract of
`
`humans or animals. The functioning
`of the protein synthesis apparatus of
`bacterial cells is obviously central for
`being able to produce human insulin
`in these ‘cells. Although proteins are
`synthesized only on the ribosomes in
`the cytoplasm of the cell, the genetic
`code for production of proteins rc-
`sides in both the chromosomal DNA
`and in the small rings of cytoplasmic
`DNA called plasmids. Both of these
`sources of DNA are transcribed into
`
`messenger RNA, which is subse-
`quently translated into proteins. The
`basis of recombinant DNA technolo-
`
`

`
`iuauuu-Fonscuuue
`wo Routes for Producing Human insulin Utilizing
`R_ecomb_i~.nant DNA Technology
`
`ed product (A chain or B chain or
`proinsulin) — called the chimeric pro-
`tein. The chimeric product can be
`schematically represented as Trp E-
`Met—A Chain (or -B Chain or -Proin-
`sulin). The methionine linkage pro-
`vides a specific chemical cleavage
`site for release of the desired poly-
`peptide from the promoter protein
`Trp—E.
`"
`An interesting fact to note is that
`all of the E. coli cells contain pro-'
`duct, while in contrast, only a small
`fraction of the cells of the pancreas
`contain insulin. Thus, one actually
`has lesser amounts of impurities to
`remove during the isolation of the
`biosynthetic human insulin (BHI) as
`compared to the pancreatic insulins.
`Figure 2 indicates the two schemes
`we have explored for producing
`biosynthetic human insulin. The cur-
`rent method. is to make the A and B
`
`chains in separate E. coli fermenta-
`tions, while the second route is the
`production of proinsulin in a single
`E. coli fermentation and eventually
`to transform it to human insulin. As
`far as we have been able to deter-
`
`mine, both methods yield equivalent
`preparations of biosynthetic human
`insulin (2, 8).
`Figure 3 illustrates in more detail
`
`'_ure 1: Transmission electron micro-
`g aph of inclusion bodies in E. coli cells of
`égculture producing Trp E-met-A-Chain
`lilmerlc protein. (Photograph kindly pro-
`lgied by Dr. D. C. Williams of the Lilly
`Research Laboratories).
` we desired gene product, one can
`p'bserve electron dense bodies (see
`iliigure
`1) which by
`immunocy-
`Tiiichemical
`techniques
`(17) have
`been shown to be the promoter link-
`
`Methods for Producing Biosynthetic Human Insulin
`
`Option 1
`Blosynlhetic A Chaln'+ 8 Chain
`
`Option 2
`Blosynmatlc Human Proinsulin
`
` Cannecling
`Puplldl
`
`the current method used to produce
`biosynthetic human insulin. The
`chimeric protein, Trp E-Met-Chain,
`is produced in the E. coli cells in
`separate fermentations. Methionine
`is used as a cleavage point since it is
`sensitive to the chemical cyanogen
`bromide, or CNBr, and because
`methionine does not occur either in
`the A or B—chains or proinsulin. Af-
`ter the cyanogen bromide cleavage,
`the A and B chains are converted to
`the stable S-sulfonate derivatives,’
`purified and chemically combined to
`yield insulin. The insulin is
`then
`purified by modern gel-filtration and
`ion-exchange chromatographic pro-
`cedures. At this point it should be
`emphasized that all of the biosynthe-
`tic human insulin presently being-
`produced by Eli Lilly is derived from
`this chain combination procedure
`and that all clinical studies have been
`conducted with suchinsulin.
`
`Before reviewing the characteriza-
`tion of the insulin produced by this
`process, a description of the chain
`combination procedure developed at
`the Eli-Lilly Research Laboratories
`is of interest (2). This procedure con-
`sistently gives higher yields of insulin
`than ever reported for this reaction
`(see Figure 4). Optimal yields of hu-
`man insulin are obtained using a 2:1
`weight ratio of A chain to B chain in
`a O._1 M glycine buffer at pH 10.5 and
`at ‘4°C. The S-sulfonate derivatives
`are reduced tosulfhydryl derivatives
`by use of nearly equivalent amounts
`of the thiol reducing agent, dithio—
`threitol, or DTT. The resulting solu-
`tion is stirred for 24 hours at 4°C in
`
`an open vessel to permit the proper
`V disulfide bonds to form as a result of
`air oxidation reactions. As shown,
`the insulin yield is approximately 60
`percent relative to the limiting B
`chain. The biosynthetic human insu-
`lin is ‘purified and isolated by column
`chromatography and crystallization.
`The excess chain materials and by-
`products are then recycled. This
`chain combination procedure is an
`extension of studies from several
`laboratories (7, 12, 13, 15) that were
`conducted during the 1960’s when
`chemically synthesized chains of in-
`sulin were combined to yield synthe-
`
`S15
`
`
`
`»
`
`Biosynthetlc Human Insulin
`
`Sure 2: Two pathways to producing biosynthetic human insulin.
`
`Page 3
`
`ilnch. med. Wschr. 125 (1983) Suppl. 1 © MMW Medizln Verlag GmbH Miinchen,Miinchen1983
`
`

`
`
`
`A Chain
`
`ti) LE'gene
`
`Insulin chain A gene
`
`CE Plasmid
`
`DNA
`E. coli E
`
`B Chain
`353 LE'gene
`~ Plasmid
`DNA
`. coli
`
`Insulin chain 8 gene
`
`ti
`
`production
`
`Separation ‘
`and purification
`
`Separation p
`and purification
`
`3r_gLEf-Met—A Chain
`
`n»_p LE’--Met—-B Chain
`
`CN Br Cleavage
`
`Oxidative Suifitoiysis
`
`B Chain
`
`L
`
`a—(s-so3 - )2
`
`A—(S-SO3‘)4
`
`Combina
`
`tion
`
`Crude insulin
`
`Purified Bios
`Human In
`
`ynthetic
`suiin
`
`(BHI)
`
`Figure 3: General biosynthetic and chemical modification process leading to the
`production of biosynthetic human insulin.
`
`tic insulin preparations." However,
`the yields are significantly better
`than in the earlier studies due in part
`to the availability of modern analyti-
`cal techniques such as high perform-
`ance
`liquid
`chromatography
`(HPLC). The availability of
`this
`technique allowed the examination
`and optimization of the many vari-
`ables of this complex series of reac-
`tions, and thus the achievement of
`excellent yields of human insulin.-
`The preparation of human insulin
`employing
`human
`proinsulin
`is
`shown in Figure 5. The chimeric pro-
`tein produced by the E. coli cells is
`Trp E-Met-Proinsulin. As in the A
`and B chain case, the chimeric pro-
`
`cyanogen
`using
`cleaved
`is
`tein
`bromide. The proinsulin is subse-
`quently converted to its S-sulfonate
`derivative by oxidative sulfitolysis
`and then isolated. Then the proinsu-
`lin—S-sulfonate is treated with a thiol
`reagent,
`beta-mercaptoethanol,
`which allows the proinsulin molecule
`to fold and form the proper disulfide
`bonds. Yields as high as 70 percent
`are achieved in this process, which
`was also developed in The Lilly Re-
`search Laboratories (8). The proin-
`sulin is
`then purified by ion-ex-
`change
`chromatographic methods
`and the by-products of the folding
`reaction are recycled. The proinsulin
`is then converted in greater than 95
`
`percent yield to insulin using a com
`bination of the enzymes t1‘ypsin.,a'n‘
`carboxypeptidase B (see Figure’ ‘
`This process is a modification of
`procedure originally developed
`Kemmler and coworkers (14). Th
`biosynthetic human insulin is subg-
`quently purified by gel-filtrationand
`ion-exchange chromatography, ah"
`by crystallization.
`the bi‘
`As
`indicated earlier,
`synthetic human insulin produc
`via thisvscheme is identical to If
`-insulin made .by the chain combifia..
`tion method. Before turning to a di
`
`cussion of the characterization
`biosynthetic
`human_ insulin,’
`should point out that one of the e"
`citing aspects of
`the proinsul
`scheme is that we are now ableto.
`produce human proinsulin andj
`peptide which may have interesti
`activities of their own and which can‘?
`be investigated clinically for their plot;
`tential usefulness in the treatment of
`diabetes.
`
`The Characterization of
`. Biosynthetic Human Insulin,‘
`A wide variety of evaluative tests‘ I
`have been used to exhaustively ex- ’
`amine biosynthetic human insulin’;
`(see Table 1). The results of all off"
`these tests can be best summarized --
`by saying that biosynthetic human."
`insulin has been shown to be chemi-."_'V
`cally, biologically, immunologically,
`‘
`and physically identical to a na'tive_K
`pancreatic human insulin standard,‘
`(3). Tests in a variety of in vivof
`assays, as well as in many diifer_eI1't__.;
`insulin receptor assays, have demon-
`strated that biosynthetic human insu—--
`lin exhibits equivalent biological ac-
`tivity to pancreatic human insulin.
`The
`same
`conclusion has been
`reached based on the data from the
`insulin radioimmunoassays as well as
`from the results of studies in the
`rabbit hypoglycemia test — it is Ob-
`vious that biosynthetic human insu-
`1in_ is equivalent biologically to both
`pancreatic human insulin and pot-_
`cine insulin.
`The amino acid compositions Of"
`biosynthetic human insulin and pan.‘
`creatic human insulin are identical to, -
`
`S16
`
`.t
`Page 4,
`Munch. med. Wschr. 125 (1983).Suppi.1 © MMW Medizin Verlag GmbH Mi‘1nchen,M'Linchen1983-.
`
`‘
`
`
`
`INSULIN-F@R$@i=WN%
`Two Routes forProducing Human insulin Utilizing Recombinant DNA Teh
`
`
`
`noiof
`
`

`
`
`
`
`
`
`
`
`
`
`. combination of Human Insulin Chains
`sso; sso;
`'
`sso;
`I
`sso;
`sso ‘
`sso;
`.
`;g,_..1_._.____._I_..._.__.___
`’
`2A+1B wlw 5-10 mglml
`rm I
`1
`' _
`1
`.
`SH: sso, l~1.2w1lh orr)
`3°"? 5
`OJMGI
`'
`, H10.5
`-
`oxidation
`24 hr‘ 4°¥:°'”° 9
`
`
`
`
`
`
`‘u"EV‘ £Vflfl'éR1 -‘..5o°/6 yield relatlve to B chaln. isolation and purification by
`
`
` nausea-Fonscnunic
`my
`
`rrwo Routes for Producing. Human lnsulin Utilizing Recombinant DNA Technology
`
`
`
`
`
`
`
`
`
`TrpE-Met-Proinsulin
`1 CNBr
`Prolnsulin (crude)
`
`Oxidative Sulfitolysis
`
`Proinsulin — ssoa
`L Folding + S-—-S Bond Formation
`Proinsulin (crude)
`— 1 Purification
`Proinsulin
`
`Enzymatic Transformation
`
`Insulin (crude)
`Purification
`
`Biosynthetic Human Insulin
`
`
`
`
`
`Figure 5: General biosynthetic and chemi-
`cal modification process via human proin-
`sulin,
`leading to the production of
`bicsynthetic human insulin.
`
`laboratories. These separations are
`truly remarkable when one considers
`that
`the amino acid sequences of
`these four insulins are nearly identi-
`cal, particularly the human and pork
`insulins. The single difference bet-
`ween human and pork insulin is at
`position 30 in the B chain where
`threonine resides in human insulin
`
`Table 2: Comparison of amino acid com-
`position of biosynfhetic human insulin and
`pancreatic human insulin’
`
`
`
`
`
`3.00 (3)
`2.77 (3)
`2.56 (3)
`7.11 (7)
`1.03 (1)
`3.98 (4)
`0.97 (1)
`S'.31(6)
`3.76 (4)
`1.66 (2)
`6.16 (6)
`391(4)
`2.99 (3)
`1.97 (2)
`0.97 (1)
`6.89
`1.00 (1)
`
`2.77 (3)
`2.63 (3)
`7.10 (7)
`0.99 (1)
`3.98 (4)
`' 0.99 (1)
`5.43 (6)
`3.71 (4)
`1.61 (2)
`6.14 (6)
`3.90 (4)
`2.91 (3)
`1.99 (2)
`0.97 (1)
`6.95
`1.00 (1)
`
`Aspartic acid
`Threonine
`Serine
`Glutamic acid
`Proline ‘
`Glycine
`Alanine
`Half-cystine
`Valine
`Isoleucine
`Leucine
`Tyrosine
`Phenylalanine
`Histldine
`Lysine
`Ammonia
`Arginine
`
`“Molar amino acid ratios calcuated using aspar-
`tic acid as unity, which was 160 nmoles/mg for
`biosynthetic human insulin and 156 nmoles/mg
`for pancreatic human insulin. Each value is the
`average of three determinations. Theoretical
`values are listed in parentheses.
`
`S17
`
`times are achieved. Figure 7 is an
`illustration of the physical structure
`of an HPLC column. The column is
`tightly packed with small particles or
`resin (called the stationary phase)
`which is coated with hydrocarbon. In
`order to obtain flow thru the column
`
`and to elute the compounds applied
`to the column, the solvent (mobile
`phase) must be pumped thru the col-
`umn under pressure. The basic‘ prin-
`ciple of the separation is that each
`protein has a different affinity for the
`resin particles and the solvent. Thus,
`as the mixture of proteins flowsthru
`the column, separation occurs be-
`cause each protein spends different
`amounts of time interacting with the
`resin particles or with the solvent.
`Thus, as illustrated here for a mix-
`ture of beef, sheep, human and pork
`insulins, at the bottom of the column
`these four insulins have been resol-
`ved from one another. This example
`has been chosen because reverse-
`
`phase HPLC today is the most sensi-
`tive method available for distinguish-
`ing minor structural differences in
`closely related molecules such as
`these different species of insulin.
`Single
`amino acid differences
`in
`structure can be determined by diffe-
`rent elution times as shown in Figure
`8. This HPLC chromatogram shows
`baseline separation of a mixture of
`beef, sheep, human, and pork insu-
`lins as is routinely ‘achieved in our
`
`27.5 :- 1.7 U/mg"‘ (PS 0.05)
`
`Relative potency 98 i 7% of purified porkeinsulin
`1
`(P $0.05)
`Relative immunopotency 98 i 22°/0 of pancreatic
`human insulin (P S 0.05)
`Comparable to pancreatic human insulin (see Table II)
`PTH-Gly and PTH-Phe equivalent to purified pork
`insulin
`Correct sequences verified
`
`
`
`
`
`
`1 ‘column chromalography and cryslalllzatlon. By-products are
`
`I :fg'cyCiEd-
`
`
`ggure 4: Summary of insulin chain combi-
`lion method.
`
`4 thin experimental error (see Table
`-2). The results of conventional pH
`3,5
`polyacrylamide
`gel
`elec-
`trophoresis
`demonstrate‘
`that
`liiosynthetic human insulin is of high
`purity and electrophoretically equi-
`'_ lentto the pancreatic human and
`pork insulin standard preparations.
`The same conclusion is apparent
`from the results of studies with
`isoelectric
`focusing in polyacryl-
`amide gels.
`One of the most useful tests for
`
`evaluating biosynthetic human insu-
`lin.
`is
`high
`performance
`liquid
`chromatography, or HPLC, whose
`generalyprinciples can be described
`111 the ‘following manner. HPLC1s an
`
`in which higher resolution, higher
`sensitivity, and more rapid elution
`
`
`
`ble 1,:-Evaluative tests on BHI (3)
`
`l, USP rabbit hypoglycemia assay
`. Insulin radioreceptor assay
`
`
`1
`
`
`
`. Insulin radioimmuncassay
`
`. Amino acid composition
`
`V
`5. Quantitative NH; terminal analysis by Edman '
`degradation
`. Amino acid sequence of A— and B-chains used to
`make insulin
`
`.
`(L
`~
`
`7. Absorption and circular dichroic spectra.
`8. Zinc insulin crystallization
`9. HPLC
`
`
`
`
`
`
`
`
`bacterial endotoxin
`
`
`
`2 USP rabbit pyrogen test
`
`UV and CD spectra identical to purified pork insulin
`See Figure 11
`'
`Peak retention identical to pancreatic human insulin
`Electrophoretic migration identical with pancreatic
`human insulin and purified pork insulin
`< 0.6 ng/mg
`
`
`
`Satisfactory
`
`
` nch. med. Wschr. 1,25 (1988) Suppl. 1
`
`© MMW Medizln Verlag GmbH Mflnchén, ML'lnchen1983
`
`
`
`

`
`
`
`
`in‘‘soLIN?-F’eeee‘eu'ne"" ””*_
`Two Routes for Producing Human Insulin Utilizing Recombinant DNA Technolo
`
`Table 3: Limulus amebocyte lysate (LAL) and byrogen data for human insulin lots (11).
`
`
`"-1"
`Fyrogen r"'eni1t.‘—_
`result"
`p
`
`35
`'
`.
`,‘_: (jxrieanivtemp. rise/
`*
`] (ng.e'nd0toxi‘n/ .1 .
`1
`'
`no. ofrabbits)
`'
`.. mg insulin
`"
`
`
`615—70N—174—9
`
`615—84S—30A
`
`989BAO
`46L~295
`
`" 44L~55
`
`4 142CY1
`143CY1
`
`46L—296
`
`44L—79
`46L—297
`
`905CY1
`
`544CJ1
`
`
`
`
`
`. Lot Number
`
`< 0.6
`0.8-1.6
`0.4-0.8
`0.1-0.2
`0.1-0.2
`< 0.05
`' 0.2-0.4
`0.05—0.1
`< 0.05
`0.4-0.8
`0.2—0.4
`0.05—0.1
`
`In order to obtain additional sup-
`porting evidence that biosynthetic
`human insulin is structurally identi-
`cal to pancreatic human insulin, a so-
`called HPLC “fingerprint” analysis
`has been utilized. In this procedure
`the insulin is fragmented by the en-
`zyme Staphylococcal aureus V8 pro-
`tease, an enzyme that hydrolyzes
`peptide bonds on the carboxyl side
`of glutamic acid residues. These
`cleavages yield fragments that con-
`
`Proinsulin
`
`Trypsin
`
`lnsulin—Arg—Arg
`+
`
`lnsu|in——Arg
`
`'
`
`Carboxypeptidase B
`
`Insulin + I-\rg
`
`Figure 6: General steps of enzymatic con-
`version of proinsulinto insulin.
`
`and alanine resides in pork insulin.
`HPLC profiles for biosynthetic hu-
`man insulin, pancreatic human insu-
`lin, and a mixture of the two human
`insulin preparations have identical
`retention times (3). These HPLC re-
`tention studies,
`therefore, strongly
`suggest
`structural equivalence of
`biosynthetic and pancreatic human
`insulins.
`
`
`0.19” C/N = 3 (nonpyrogenic),-'
`O.17° C/N = 3 (nonpyrogeni
`
`0.11" C/N = 3 (nonpyrogeni
`
`0.U1° C/N = 3 (nonpyrogenic)_,;
`
`020° C/N = 3 (nonpyrogeni
`
`0.15“ C/N = 3 (nonpyrogeni
`0.06° C/N = 3 (nonpyrogenic);
`
`0.16“ C/N = 3 (nonpyrogenic)
`
`0.04“ C/N = 3 (nonpyrogenicjf
`
`027° C/N = 3 (nonpyrogenic)
`0.07° C/N = 3 (nonpyrogeni I"
`
`0.07° C/N = 3 (nonpyrogeni
`
`
`
`tain the various disulfide bonds (‘
`shown in Figure 9) which is an im
`.
`portant aspect of this test. The e"
`zyme. digests are then" evaluated" by.
`HPLC as shown in Figure 10. For
`‘
`purpose of illustration, computer-'
`drawn elution profiles are aligned for? '=
`comparison. The bottom profileyis
`from the biosynthetic human insulinj ,
`reference standard made by chain,
`combination. The top profile is from
`a semisynthetic human insulin th”
`was derived by converting pancreat
`pork to human insulin. These
`fragment profiles are identical.
`
`
`
`Figure 11 demonstrates the zi_’I_1_o
`insulin crystals of BHI, which, as f_'<_1I;
`as we know, are the first crystals ever
`formed from a recombinant DN
`product. In addition to their aesthe;._,'_p“
`tically pleasing appearance,
`theses‘
`human insulin crystals indicateiia.‘:_l*’
`
`
`
`;
`
`High~Perlcrmance Liquid Chromatography
`Solvent lmobile phasel
`
`‘Ti
`
`Pork insulin lul
`
`Human Insulin ill
`
`She ep insulin ill
`
`Bovine Insulin ill
`
`
`
`v' Ultraviolet-
`light Source
`
`HPLC column
`
`Packed Resin
`lslationary phasel
`
`High Pressure
`End Filling
`
`Ultraviolet (UV)
`' Detector
`
`__l
`
`S18
`
`L.___
`
`'Figure 7: Schematic for reverse-phase HPLC column.
`Page 6
`
`Munch. med. Wschr. 125 (1983) Suppl. 1 © MMW Medizln Verlag GmbH Milnchen, Mtinchen 19,
`
`Figure 8: HPLC elution profile for a
`ture of pork, human, sheep, and beef in‘
`sulins. See Reference 11 for conditiofl 1’
`
`‘V
`
`

`
` ipmw
`lN$ULlN-FGRSCHUNG
`jwo Routes for Producing Human ‘Insulin Utilizing
`Recombinant DNA Technology
`mm
`n-.—4;;;_,_,;,,9.q,;;-
`
`source that 'are,.used in medicine are
`at risk of being contaminated with
`pyrogenic substances that raise the
`body temperature. Results of two
`tests for pyrogens in several lots of
`biosynthetic human insulin are listed
`in Table 3. The results of
`the
`
`Limulus Amebocyte Lysate test, or
`LAL test,- indicate very low and in-
`significant
`levels of endotoxin. We
`View these as very significant results
`since the LAL is widely viewed ‘as
`the most sensitive in vitro test for
`endotoxin. Also, we would note that
`none of the BHI lots proved to be
`pyrogenic in rabbits.
`
`Finally, let us address the issue of
`potential E. coli polypeptide cont-
`aminants. From the previous data it
`is obvious that very little,
`if any,
`bacterial endotoxin is present in the
`biosynthetic human insulin prepara-
`tions. However, a logical question to i
`ask is how much other contamina-
`
`tion might be present that could be
`derived from the E. coli organisms
`used
`in
`the manufacturing
`of
`biosynthetic human insulin. This is a
`very difficult question to answer
`since we don’t have radioimmunoas-
`says for specific E. coli polypeptides
`as we do for specific polypeptide
`contaminants in pancreatic insulins
`(10). For example, in pancreatic in-
`sulins we can measure the level of
`
`other pancreatic hormones such as
`glucagon and pancreatic polypeptide
`to get an indication of the relative
`purity of these pancreatic insulins.
`We do not yet have the capability of
`determining specific E. coli polypep-
`tides although this is the subject of
`intense
`research
`in
`the
`Lilly
`Laboratories. As an alternative we
`
`have prepared an E. coli polypeptide
`mixture (ECP) that could potentially
`be ‘contaminants in a final BHI pre-
`paration. This ECP preparation is
`being used to develop solid-phase
`radioimrnunoassays in order to aid in
`the estimation of the level of E. coli
`
`(1). During the de-
`polypeptides
`velopment of this assay we found
`that it was difficult to elicit an anti-
`
`body response in rabbits unless com-
`plete Freund’s adjuvant was used.
`This, along with results of other tests
`has indicated that the ECP prepara-
`
`‘vi ‘sulin and semi-synthetic human insulin (see Reference 2 for details).
`
`
`S19
`llnch. med. Wschr. 125 (1983) Suppl. 1 © MMW Medizin Verlag GmbH Milnchen, Milnchen 1983
`
`I
`
`,,,.g, NH2— B-CHAIN
`f?
`
`
`
`wmwfiwymeemwwwamwmwqewzgmwnnmwrwnmusyawggwzegwxwwl..as-rwa:~rA:‘4ae.':.<V‘s!f‘f""'vI_1:_¢t‘zt_?x:MA«"'~'1...«-:~.xr;e~.-,--,-.ii;protease.
`
`
`figure 9: Peptide fragments of human insulin formed by cleavage with S. aureus V8
`
`sulin derived from biosynthetic hu-
`man proinsulin. This X-ray diffrac-
`tion analysis assures us that all of the
`chemical bonds in BHI are identical
`
`to those of pancreatic human insulin
`(4, 6).
`the Limulus
`of
`results
`The
`Amebocyte Lysate assay for py-
`rogenic bacterial endotoxins and the
`U.S.P. Rabbit Pyrogen assay are_
`also given in_ Table 1. As is generally
`known, protein materials from any
`
`ghigh degree of purity since an insulin
`Eiwith
`improperly matched disulfide
`Eébonds would not have crystallized
`jwith zinc. We should also note that
`‘-Drs. Guy Dodson and Tom Blundell
`in England .have recently examined
`‘BHI crystals by X-ray diffraction
`{analysis and have found them to be
`§identical to crystals of natural pan-
`Efireatic human insulin, to crystals of
`“jhuman insulin prepared from pork
`.ii_1sulin, and to crystals of human in-
`
`
`
`HPLG “Fingerprint” Analyses
`
`1351.
`
`B (22-30)
`
`A (5-12)
`.
`an4m
`
`r“
`
`T
`
`“_
`
`/[Ms-17)
`Bung
`
`peas
`snag
`
`A (18-21)
`(14-21)
`
`I8
`
`A (1 '4)
`
`A (13-17)
`
`a_.a...L..L._n
`-J.:_.a_*n_.i._l...t._L...i_.|n
`336. S
`676. E
`1B13. SEC
`
`

`
`EN$UL!NF®R$@Ed§N$G
`
`cubated with the coated tubes so
`to allow the antigen-antibody re
`tion to occur. Finally, 125I—Protein
`is incubated with the mixture
`lowed by exhaustive washing.
`remaining bound radioactivity
`counted and used as a measure
`ECP antibody in the serum sam
`The data obtained using this assay on
`clinical samples indicated that th
`was no statistically significantchang
`in the serum binding in diabetic pa"
`tients after one year of treatme '1
`with BHI (11).
`It should be em
`phasized that these sera were take?
`from patients who have receivedn
`type of insulin other than BHI. T i
`no development of anti-ECP Ig
`
`antibodies has been observed in pa
`tients treated with biosynthetic hu
`man insulin.
`‘
`
`Acknowledgement
`
`The authors Wish to very gratefully acknow-
`_
`ledge the large number of our Lilly associates;
`who have made many important contribution" :f-
`to this work.
`
`
`
`
`
`
`insulin
`11: Zinc
`Figure
`crystals of
`biosynthetic human insulin.
`'
`
`tion is a poor immunogen. In the
`specific ECP assay, polystyrene wells
`are coated with purified rabbit IgG
`
`anti-ECP antibodies (100 111) at 4°C
`for 24 hours and then washed 3
`
`times. The sample or standards (100
`11.1) are incubated in the wells at 4°C
`for 18 hours and washed 3 times.
`Then
`I-125
`anti-ECP antibodies
`
`(IgG fraction) are incubated in the
`wells at room temperature for 6-7
`hours, washed 3 times and counted
`to determine the I-125 bound activi-
`
`‘ty. This assay continues to be under
`active
`investigation and develop-
`ment. One such development is to
`broaden the detection‘ capabilities of
`the assay. Probably the much more
`important point is that along with the
`ECP assay, an assay has been de-
`veloped in order to monitor patient’s
`serum for potential anti—E. coli poly-
`peptide antibodies (16). To date no
`significant ECP antibody formation
`has been observed. The solid-phase
`anti-E. coli polypeptide (ECP) anti-
`body assay is performed in the fol-
`lowing manner. First, polystyrene
`tubes are coated with the ECP mix-
`ture at 4°C for 48-72 hours,
`then
`washed. Next, serum samples are in-
`
`‘ R
`
`12. Katsoyannis, P. G.: Science 154 (1966)I..'
`1509-1514.
`13. Katsoyannis, P. G., Tometsko, A.: Proc. if
`Nat. Acad. Sci.
`(Wash.)
`55
`(1966)
`1554-1561.
`;
`14. Kemmler, W., Peterson, J. D., Steinéré, "
`D. F.:
`J. ' biol. Chem.
`246
`(1971)'
`6786-6791.
`*
`
`15. Meienhofer, J., Schnabel, E., Bremet‘,
`H., Brinkhofi‘, 0., Zabel, R., Sroka, Wu 2
`Klostermeyer, H., Brandenburg, Du.
`Okuda, T., Zahn, H.: Z. Naturforsc1__I-:-
`18b (1963) 1120-1123.
`I;
`._
`16. Ross, J. W., Baker, R. S., Hooker, C. 5'-1
`Johnson, I. S., Schmidtke, J. R., smith;
`W. C.: In: Hormone Drugs. Proceedings H
`of the FDA-USP Workshop on Drugs and '
`Reference Standards for Insulins, S031?’
`tropins and Thyroid-axis Hormones, PP-..
`127-138. U.S. Pharmacopeia Convent’
`tion, Rockville, MD 1982.
`‘
`‘-
`17. Williams, D. C., Van Frank, R. M;-vii.
`Muth, W. L., Burnett, J. P.: Science 215-:
`(1982) 687-89.
`
`
`
`eferences
`
`1. Baker, R. S., Ross, J. M., Schrnidtke, J.
`R., Smith, W. C.: Lancet
`(1981/2)
`- 1139-1142.
`‘
`2. Chance, R. E., Hoffmann, J. A., Kroeff,
`E. P., Johnson,~M. G., Schirmer, E. W.,
`Bromer, W. W., Ross, M. J., Wetzel, R.:
`In: Peptides. Synthesis, Structure and
`Function Proceedings of
`the Seventh
`American Peptide Symposium. Eds.
`Rich, D. H., Gross, E., pp. 721-728.
`Pierce Chemical Company, Rockford, IL.
`1981.
`.
`3. Chance, R. E., Kroeff, E. P., Hoffmann,
`J. A., Frank, B. H.: Diabetes Care 4
`(1981) 147-154.
`4. Chawdhury, S. A., Dodson, E. J., Dod-
`son, G. G., Reynolds, C. D., Tolley, S.,
`Cleasby, A.: In: Hormone Drugs. Pro-
`ceedings of the FDA-USP Workshop on
`Drugs and Reference Standards for Insu-
`lins, Somatropins, and Thyroid-axis Hor-
`mones, pp. 106-115. U.S. Pharmacopeia
`Convention, Rockville, MD 1982.
`
`T. L.:
`
`5. Crea, R., Kraszewski, A., Tadaaki, H.,
`Itakura, K.: Proc. Nat. Acad. Sci.
`(Wash.) 75 (1978) 5765-5769.
`6. Dodson, G. G., Blundell,
`Diabetalogia (in press).
`7. Du, J.-C., Jiang, R.-Q., Tsou, C.-L.: Sci.
`sin. 14 (1965) 229-236.
`8. Frank, B. H., Pettee, J. M., Zirnmer-
`mann, R. E., Burck, P. J.: In:. Peptides.
`Synthesis, Structure and Function. Pro-
`ceedings of the Seventh American Pep-
`tide Symposium. Eds. Rich, D. H.,
`Gross, E., pp. 729-738. Pierce Chemical
`Company, Rockford, IL. 1981.
`A 9. Goeddel, D. v., Kleid, D. G., Bolivar,
`F., Heyneker, H. L., Yansura, D. G.,
`Crea, R., Hirose, T., Kraszewski, A.,.
`Itakura, K., Riggs, A. D.: Proc. Nat.
`Acad. Sci. (Wash.) 76 (1979) 106-110.
`10. Heding, L. G.: In: Insulins, Growth Hor-
`mone and Recombinant DNA,Technolo-
`gy. Ed. Gueriguian, J. E., pp. 87-97.’
`Raven Press, New York 1981.
`11. Johnson, I. S.: Diabetes Care 5, Suppl. 2
`(1.982) 4-12.
`
`S20
`
`Page 8
`
`
` _ .
`Miinch. med. Wschr. 125 (1983) Suppl. 1 © MMW Medizin Verlag GmbH Miinchen. MUnchen1983_.