Human Insulin from
`
`Recombinant DNA Technology
`
`Irving S. Johnson
`
`During 1982, human insulin of recom—
`binant DNA origin was approved by the
`appropriate drug regulatory agencies in
`the United Kingdom,
`the Netherlands,
`West Germany, and the United States.
`This new source guarantees a reliable,
`expandable, and constant supply of hu—
`man insulin for diabetics around the
`world.
`
`University of Toronto to develop a stan-
`dardized and clinically acceptable insulin
`product. Banting had just begun to ex—
`tract relatively crude insulin from ani—
`mals and inject it into his diabetic pa—
`tients.
`In the early 1970’s we began to be
`concerned about a possible shortage of
`insulin. Until now,
`the world’s insulin
`needs have been derived almost exclu—
`The research, development, and pro-
`sively from pork and beef pancreas
`duction of human insulin by recombinant
`glands, which were collected as by-prod-
`DNA technology ushers in a new era in
`ucts from the meat industry. This supply
`pharmaceuticals, agricultural products,
`
`Summary. Human insulin produced by recombinant DNA technologyyis the first
`commercial health care product derived from this technology. Work on this product
`was initiated before there were federal guidelines for large—scale recombinant DNA
`work or commercial development of recombinant DNA products. The steps taken to
`facilitate acceptance of large—scale work and proof of the identity and safety of such a
`product are described. While basic studies in recombinant DNA technology will
`continue to have a profound impact on research in the life sciences, commercial
`applications may well be controlled by economic conditions and the availability of
`investment Capital.
`
`and industrial chemicals by establishing
`the feasibility of commercial production
`of a gene product initiated at a laboratory
`level of expression.
`1 shall review how
`human insulin became the first human
`health product of this technology. I will
`also discuss some of the special prob-
`lems, in terms of regulatory environment
`and public opinion, that had to be over~
`come in order to bring it to the current
`stage of development.
`
`Sources of Insulin
`
`Eli Lilly and Company has been in—
`volved in the development and manufac-
`ture of insulin and other products for
`diabetics since 1922.
`In that year our
`scientists began working with Frederick
`G. Banting and his associates at
`the
`
`Irving S. Johnson is vice president of research.
`Lilly Research Laboratories. a division of Eli Lilly
`and Company, Indianapolis, Indiana 46285.
`632
`
`changes with the demand for meat and is
`not
`responsive to the needs of
`the
`world‘s diabetics. Indeed, from 1970 to
`1975, the supply of pancreas glands in
`the United States declined sharply (I)
`and remained on a plateau at that lower
`level
`in succeeding years. There is no
`accurate way to predict availability of
`future supplies of glands, although we
`predicted that
`the demand for insulin
`would continue to increase. Our ‘concern
`was whether or not there Would be a time
`when the supply of bovine and porcine
`pancreas glands might not be sufficient
`to meet the needs of insulin-dependent
`diabetics. Although it is difficult to ob-
`tain substantiated figures for a nonreport—
`able disease, we estimate that there are
`60 million diabetics in the world—more
`
`than half of them in less developed coun-
`tries. In the developed countries, some 4
`million diabetics, 2 million in the United
`States, are treated with insulin.
`Today,
`the diabetic population is
`
`growing more rapidly than the total pop-
`ulation. While the US. population is
`increaSing at a rate of about 1 percent per
`year and the world population at slightly
`more than 2 percent, the annual rate of
`increase 0f insulin-using diabetics in this
`country has been 5 to 6 percent in recent
`years, and a similar pattern may hold
`true worldwide (2).
`Several factors contribute to this ac-
`celerated growth of the insulin-using dia-
`betic population. One factor, of course,
`is the availability of insulin, which en—
`ables diabetics, who often did not sur—
`vive beyond their teens,
`to live long,
`productive—and
`reproductive—lives.
`Because of the genetic etiologic compo-
`nent of diabetes, the offspring of diabet-
`ics are likely to suffer from the disease as
`well. Other factors that contribute to
`growth of the diabetic insulin-using pop—
`ulation include improved methods of de-
`tection, greater public awareness of the
`disease and its symptoms, less reliance
`on oral forms of therapy, and changes in
`dietary habits.
`Because of the uncertainty of the insu-
`lin supply and the forecasts of rising
`insulin requirements, it seemed not only
`prudent but a responsibility as well for
`the scientific community and insulin
`manufacturers to develop alternatives to
`animal sources for supplying insulin to
`the world’s diabetics. Lilly established
`several internal committees of scientists
`to examine various solutions to the prob-
`lem. They considered augmentation of
`insulin production from pancreas glands,
`transplantation of islet of Langerhans
`cells, chemical synthesis, beta cell cul-
`ture, directed-cell synthesis, and cell—
`free biosynthesis, as well as insulin re—
`placements. These discussions touched
`on the technology called genetic engi-
`neering.
`is to
`The function} of DNA in a cell
`serve as a stable repository of coded
`information that can be replicated at the
`time of cell division to transmit the ge-
`netic information to the progeny cells
`and to encode the information necessary
`to synthesize proteins and other cell
`components. There are several ways of
`performing genetic engineering, some of
`which have been practiced for many
`years by geneticists. The first is muta-
`tiOn. Mutations in DNA can be either
`spontaneous, due to environmental fac—
`tors and errors in DNA replication, or
`they can be induced in the laboratory by
`physical and chemical agents. Mutations
`can lead to a change in the structure of
`the product coded for by the gene in
`question;
`sometimes
`this
`change
`in
`structure is so great that the product is
`SCIENCE. VOL. 219
`
`Page1
`
`KASHIV EXHIBIT 1016
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`|PR2019-00791
`
`Page 1
`
`KASHIV EXHIBIT 1016
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`

`

`-
`
`vastly different, Other mutations may
`affect the regulatory elements that con-
`trol the expression of the structural gene,
`leading, in some instances, to increased
`or decreased production of gene prod—
`ucts. A key point is that mutagcnesis is
`an essentially random technique.
`A second type of genetic engineering,
`recombination, has also been used for a
`long time. Recombination refers to ex-
`change of a section of DNA between two
`DNA molecules. Recombination
`of
`DNA fragments from diiferent organisms
`can occur by the mating of two orga—
`nisms-——a process called conjugation——
`where DNA is physically transferred
`from one organism to another. This is a
`process occurring in nature, which can
`be duplicated in the laboratory. Two
`other natural processes whereby cells
`exchange DNA in nature are transforma-
`tion and transduction. Recombination
`
`may also occur following the use of a
`technique known as protoplast fusion,
`where one literally strips olf the outer
`cell wall of cells of fungi and bacteria.
`This phenomenon only occurs in the
`laboratory and allows the remaining pro-
`toplasts, which now have just a cell
`membrane enclosing the components of
`the cell,
`to fuse together. The fused
`protoplasts contain the DNA molecules
`of both parents, and exchange of sec-
`tions of DNA can now occur as these
`cells regenerate and divide. All
`these
`recombination processes involve ran-
`dom exchange of DNA sequences, and
`this exchange is generally, but not al—
`ways,
`limited to members of a single
`species of organism.
`In 1972, Jackson, Symons, and Berg
`(3) described the biochemical methods
`for cutting DNA molecules from two
`different organisms, using restriction en—
`zymes, and recombining the fragments
`to produce biologically functional hybrid
`DNA molecules. In 1973. Cohen, Chang,
`and Boyer (4) reported that they could
`make a hybrid molecule that would ex-
`press the foreign DNA within it as
`though it were a part of the original
`molecule’s natural heritage (5). That pro—
`foundly significant accomplishment also
`generated major concern over potential
`biohazards.
`
`Regulating DNA Research
`
`The Berg Committee was formed, and
`it responded to concerns about conjec—
`tural risk associated with recombinant
`DNA research in 1974 by calling for a
`moratorium and deferral of certain types
`of recombinant DNA research until the
`11 FEBRUARY 1983
`
`scientific community could evaluate the
`risks and benefits associated with it (6).
`Work was halted—including work in my
`own organization—until after the Asilo—
`mar Conference in 1975 (7).
`The majority of scientists invited to
`the Asilomar Conference were molecular
`biologists from government and acade-
`mia;
`those with expertise in infectious
`disease or those from industry with ex-
`tensive knowledge of large-scale fermen-
`tation processes and other techniques
`that require careful methods of contain-
`ing potentially harmful materials were
`underrepresented. As a result, many of
`us believe that the guidelines defined at
`Asilomar were unnecessarily restrictive.
`An example was the establishment of the
`10—liter limit. To those of us in industry,
`this restriction was never considered
`reasonable. We were accustomed to han-
`dling containment problems at much
`larger volumes. But few of the scientists
`at Asilomar conceived of performing
`large-scale fermentation with recombi-
`nant organisms; as noted in the British
`journal Nature, “[In 1975] even opti—
`mists would have predicted that it would
`be a decade before genetic engineering
`would be cognnercially exploited” (8).
`This volume limit was established be—
`cause it was regarded as probably the
`largest volume that could be convenient-
`ly handled in an experimental laboratory
`by conventional laboratory centrifuges.
`It was clear that the 10-liter limit exclud-
`ed industrial—level activity. It was not
`suggested that
`increased volume of a
`culture of a safe organism would result in
`any increased risk. The conjectural na—
`ture of the early concerns soon became
`
`clear, and broader participation in re-
`search decisions by those expert in infec—
`tious diseases, containment, and risk-
`assessment, as well as more practical
`experience,
`led inevitably to revisions
`and continued relaxation of restrictions
`around the world.
`In June 1976, the National Institutes of
`Health (NIH) announced guidelines for
`recombinant DNA work, marking the
`end of the 2-year moratorium on this
`type of research. Only research financed
`by the federal government was subject to
`the guidelines, for which Lilly, with oth-
`er companies, NIH, the Pharmaceutical
`Manufacturers Association (PMA),
`the
`Food and Drug Administration (FDA),
`and the Department of Health, Educa-
`tion and Welfare (HEW), was actively
`involved in developing compliance pro-
`cedures.
`
`At Lilly, work on DNA recombina-
`tion, which had been under way before
`Jackson, Symons, and Berg (3) pub-
`lished their work, resumed vigorously.
`We contracted with a new California
`company, Genentech, Inc., for specific
`work on human insulin. Genentech sub-
`contracted the synthesis of the human
`insulin gene to the City of Hope Medical
`Center, which succeeded in its mission.
`Scientists at Genentech inserted the
`genes for both chains of insulin into a K-
`12 strain of Escherichia coli and after
`isolation and purification, the A and B
`chains were joined by disulfide bonds to
`produce human insulin. In the Lilly Re-
`search Laboratories, we have also used
`recombinant DNA technology to pro-
`duce human proinsulin, the insulin pre-
`cursor.
`
`Pork
`
`Rabbit
`Hum n
`
`L”WM...“
`
`1. HPLC chromato-
`Fig.
`grams of insulins which dif»
`fer by one or more amino
`acids.
`
`Beef
`
` W
`
`1.00 338.5 676
`
`1013
`
`1351 1688 2026 2363 2701
`Seconds
`
`633
`
`Page 2
`
`Page 2
`
`

`

`Human Insulin
`
`The successful expression of human
`insulin (recombinant DNA) in E. coli
`was announced on 6 September 1978.
`This was a first step. Although we had
`been successful in obtaining expression
`of the hormone under laboratory condi-
`tions and scale, we still faced the equally
`diflicult challenge of achieving satisfac—
`tory production of the purified product
`on a commercial scale. The process we
`used in accomplishing large—scale pro-
`duction has been described (9-11), but it
`may be useful to touch on some of the
`methods that we employed to prove that
`the product produced was indeed human
`insulin.
`High-performance liquid chromatogra-
`phy (HPLC)
`techniques developed at
`Lilly can detect proteins that diifer by a
`single amino acid (10), and HPLC tests
`showed that human insulin (recombinant
`DNA) is identical to pancreatic human
`insulin and that it is close to, but not the
`same as, pork insulin, which differs from
`the human by one amino acid; beef.
`which differs by three amino acids; and
`sheep, which diifers at four residue posi-
`tions (Fig. l). A chrornatogram of human
`insulin (recombinant DNA), pancreatic
`human insulin, and a mixture of the two,
`showed that they were superimposable
`and identical (Fig. 2). HPLC has become
`an important analytical tool to determine
`structure and purity and is now consid-
`ered to be a more precise measurement
`of potency than the rabbit assay,
`al—
`though most government
`regulatory
`agencies around the world still empha-
`size the rabbit potency assay.
`tertiary
`A measure of the correct
`structure and appropriate folding is the
`circular dichroic spectrum. The spec-
`trum for porcine insulin and for human
`insulin (recombinant DNA) were found
`to be identical. X—ray crystallographic
`studies further revealed the structural
`
`integrity of the recombinant molecule
`(12). We also found the amino acid com-
`position of human insulin (recombinant
`DNA) and pancreatic human insulin to
`be identical (Table 1). In addition, we
`compared polyacrylamide gel
`electro—
`phoresis for human insulin (recombinant
`DNA), pancreatic human insulin, and
`pork insulin, as well as isoelectric focus-
`ing gels for these three insulins.
`Another technique that we found use-
`ful for ensuring that we had the appropri-
`ate disulfide bonds and lacked other
`types of protein or peptide contaminants
`was HPLC of a specifically degraded
`sample. There is a staphylococcal prote-
`ase that cleaves insulin in a specific way
`at
`five sites—always next
`to glutamic
`acid, except for one site between serine
`and leucine. After treating the insulin
`with the protease, we looked for and
`identified the various peptide fragments
`by HPLC (Fig, 3) and found none that
`were not derived from insulin.
`In the end, we employed 12 different
`tests to establish that what we had pro-
`duced was human insulin. We believe
`the correlation among three of the tests
`was particularly importantethe radio-
`receptor assay,
`the radioimmunoassay,
`and HPLC. Moreover, the pharmacolog-
`ic activity ofhuman insulin (recombinant
`DNA), as demonstrated by a rabbit hy-
`poglycemia test, showed a response es-
`sentially identical
`to pancreatic human
`insulin.
`Another serious question remained to
`be answered—namely that of the poten-
`tial contamination of the product with
`trace amounts of antigenic E. coli pep—
`tides. Relevant to this question is the
`difference in starting materials between
`human insulin of recombinant DNA ori-
`gin and pancreatic animal insulins. The
`glandular tissue is collected in slaughter-
`houses, with no control over bacterial
`contamination. The desired gene product
`is isolated from a few cells of the islets of
`
`A Human Insulin (recombinant
`
`DNA)
`
`B Pancreallc human insulin
`
`AV"
`C Pancreatic + human insulin
`(recombinant DNA) mix
`
`\
`
`
`
`MMw—g
`l
`i
`i
`L.
`L
`188.5
`1.00
`376
`563.5
`751
`Seconds
`
`I
`938.5
`
`l
`1126
`
`l
`1313
`
`1501
`
`634
`
`Fig. 2. HPLC chromato-
`grams of human insulin
`(recombinant DNA), pan-
`creatic human insulin, and
`mixtures of the two show—
`ing identity.
`
`Page 3
`
`Langerhans, which make up less than 1
`percent of the glands; thus more than 99
`percent of the tissue represents tissue
`contaminants and undesirable materials.
`The common protein contaminants of
`the animal insulins are other pancreatic
`hormones or proteins, many of which are
`highly immunogenic.
`In contrast, with recombinant DNA
`production of human insulin, almost 100
`percent of the cells (E. coli) produce the
`desired gene product. Because of the
`method of manufacture, none of the pan-
`creatic contaminants of the animal insu—
`lins are found in the human insulin of
`recombinant origin. The issue of protein-
`aceous contamination derived from the
`bacterial host cell was addressed through
`some experiments that were made possi—
`ble by running large-scale fermentations
`of the production strain ofE. coli, which
`contains the production plasmid with the
`code for the insulin chain sequence de-
`leted. The small quantities of peptides
`isolated after applying the chain purifica-
`tion and disulfide linking process to the
`“blank” preparation were shown not to
`be antigenic except in complete Freund’s
`adjuvant (13); in addition, no changes in
`amount of antibody to E. coli peptides
`were detected in serum from patients
`who had been treated with human insulin
`for more than a year (14).
`
`Commercial Production
`
`As we were scaling up this new tech-
`nology for commercial production, we
`recognized that there would be external
`problems and forces with which to con-
`tend. Because this would be the first
`human health care product
`resulting
`from recombinant DNA techniques, we
`expected that many people would per-
`ceive that
`there were risks associated
`with this new scientific tool. We also
`
`recognized that the existing regulatory
`systems had not been designed to cope
`with the new technology. The public’s
`concern reached such levels that some
`communities, most notably Cambridge,
`Massachusetts, passed ordinances regu-
`lating recombinant DNA research (15).
`In Congress, several bills were intro-
`duced to regulate the research. Some of
`these would have subjected all recombi—
`nant DNA research, public or private, to
`federal regulation (16). It was probably
`fortunate that none of the bills was en-
`acted into law, as former Representative
`Paul Rogers (D—Fla.) noted: “I think
`Congress was right
`[in not regulating
`rDNA research]. Congress did a good _
`service in airing the issue, but
`there
`wasn’t a necessity to pass a law” (17).
`SCIENCE. VOL. 219
`
`Page 3
`
`

`

`too, adapted
`The regulatory system,
`well to this unexpected challenge to its
`flexibility. On 22 December 1978,
`the
`FDA had published in the Federal Regis—
`ter a “Notice of Intent to Propose Regu—
`lations” governing recombinant DNA
`work. But, by the time that the FDA’s
`Division of Metabolism and Endocrine
`Drug Products convened a conference
`on the development of
`insulin and
`growth hormone by recombinant DNA
`techniques (11) in mid—1980, attitudes
`had changed, and the regulations were
`never promulgated.
`the containment of
`Concerns about
`potentially harmful organisms fell under
`the purview of the Recombinant DNA
`Advisory Committee (RAC) and the Na-
`tional Institute of Occupational Safety
`and Health (NIOSH). RAC, established
`in 1974 by the secretary of HEW, had 11
`members, all of whom were scientists. In
`December 1978, 14 more members were
`added to RAC; all of the new members
`were nonscientists. It was apparent that
`the nonscientists would have to rely
`heavily on the scientists to develop their
`understanding of the new teChnology.
`Lilly scientists participated actively in all
`aspects of public discussion, through tes-
`timony in both houses of Congress, par-
`ticipation in the open forum of the Na-
`tional Academy of Sciences (18), and in
`meetings of RAC, and by submitting-
`comments and amendments to NIH for
`its guidelines.
`In June 1979, Lilly made the first ap-
`plication to RAC for an exception to the
`rule limiting recombinant DNA work to
`lO-liter volumes. At its meeting in Sep-
`tember 1979, RAC recommended that
`our request to scale up production of
`bacteria-derived insulin be approved,
`and a month later the director of the
`NIH granted us permission to use 150-
`liter containers. In 1980, permission to
`expand to 2000-liter containers was
`granted. This was a major step toward a
`production type of operation;
`the sub-
`mission to RAC contained detailed engi-
`neering specifications for equipment and
`monitoring systems as well as descrip-
`tions of the proposed operating proce-
`dures. Because of the unprecedented
`volume increase in the handling of cul-
`tures of
`recombinant organisms,
`the
`scale—up request was preceded by a visit
`to our plant by a group consisting of
`RAC representatives and NIH officials;
`they came to see for themselves how we
`could handle containment problems.
`With the experience gained at these in-
`termediate levels, we are now routinely
`using 10,000-gallon fermentors.
`Throughout 1980, there were several
`other positive developments. NIH pub-
`11 FEBRUARY 1983
`
`.
`A(5—12)
`l
`
`ANS-21)
`3(14—21)
`\
`
`
`
`5(22 30)
`
`— 100
`_
`—
`_
`‘
`~ 75
`_
`
`A(5-17)
`I
`-
`5(1-13)
`A(5-21)‘
`— so 3;. v
`[
`_
`
`
`/B(1 20'
`m
`LN”;‘1
`‘i 25
`
`
`
`
`4.700 '
`g
`. /
`.
`r
`
`3.525
`
`M14)
`
`£
`-
`z
`-
`.:
`
`g 2.350 —m -
`
`E
`_
`-
`
`1.175 ‘—
`
`
`Aha-17)
`.
`
`1
`
`Semisynthetic
`
`human insulin
`
`_
`
`0.000 —1_|
`1.00
`
`"‘ mman insulin
`w (recomblnant DNA)
`1
`1
`L
`1
`h . a 1
`.
`.
`.
`338.5
`676
`1013
`1351
`Seconds
`
`'
`
`1
`
`1
`1688
`
`1
`
`1
`I
`2026
`
`L
`
`.
`1
`2363
`
`Z
`L ,
`.
`H 0
`2701
`
`Fig. 3. HPLC chromatograms of peptide fragments from the A and B chains of semisynthetic
`human insulin and human insulin (recombinant DNA) after treatment with a specific staphylo—
`coccal protease. These chromatograms indicate the correctness of the disulfide bridges and the
`lack of any other major peptide components.
`
`lished in the Federal Register draft
`guidelines on physical containment rec-
`ommendations for large-scale uses of or-
`ganisms containing recombinant DNA
`molecules. This draft was not formally a
`part of the guidelines, but it did serve as
`a model for persons preparing submis-
`sions to RAC for large-scale fermenta-
`tions with
`recombinant
`organisms.
`About the same time, the National Insti—
`tute of Allergy and Infectious Diseases
`sponsored a workshop on risk assess-
`ment. Among the issues discussed were
`risks associated with pharmacological
`action of hormones from recombinant
`
`organisms populating the human intesti-
`nal tract, medical surveillance of work-
`ers involved in large-scale fermentation
`
`'
`
`Table 1. Amino acid compositions of human
`insulins. Molar amino acid ratios with aspartic
`acid as unity [actual aspartic acid yields were
`160 nanomoles per milligram for human insu—
`lin (recombinant DNA) and 156 nanomoles
`per milligram for pancreatic human insulin
`(10)].
`
`Recom-
`Pan—
`Amino acid
`binant
`.
`
`DNA
`creatlc
`
`Aspartic acid
`Threonine
`Serine
`Glutamic acid
`Proline
`Glycine
`Alanine
`Half-cystine
`Valine
`Isoleucine
`Leucine
`Tyrosine
`Phenylalanine
`Histidine
`Lysine
`Ammonia
`Arginine
`
`3.00
`2.77
`2.56
`7.11
`1.03
`3.98
`0.97
`5.31
`3.76
`1.66
`6.16
`3.91
`2.99
`1.97
`0.97
`6.89
`
`3.00
`2.77
`2.63
`7.10
`0.99
`3.98
`0.99
`5.43
`3.71
`1.61
`6.14
`3.90
`2.91
`1.99
`0.97
`6.95
`1.00
`
`of recombinant organisms, pathogenesis
`of approved recombinant hosts, and con
`tainment practices in commercial—scale
`fermentation facilities. Most participants
`indicated that there was little or no risk
`involved in these practices. A few
`months later,
`the industrial practices
`subcommittee of the Federal Interagen-
`cy Advisory Committee (FIAC), a work-
`ing group of representatives from all the
`cabinet-level departments as well as all
`federal agencies that are in any way
`affected by recombinant DNA issues in-
`vited Lilly to make a formal statement.
`Bernard Davis of the Harvard Medical
`School and I submitted a document on
`the safety of E. coli K-12, the reliability
`of commercial-scale equipment, opera-
`tor training, and other topics; this was
`favorably received. NIOSH also pub-
`lished a favorable report of its on-site
`inspection of Lilly Research Labora—
`tories’ recombinant DNA research facili-
`ties and procedures for large-scale fer-
`mentations of recombinant organisms.
`In July 1980. we began clinical trials of
`our human insulin in the United King-
`dom. Within weeks, similar tests were
`under way in West Germany and Greece
`and, finally, in the United States. Plants,
`specifically designed for the large-scale
`commercial production of human insulin
`(recombinant DNA), were built at India-
`napolis (Fig. 4) and at Liverpool in the
`United Kingdom. On 14 May 1982, we
`filed our new drug application for human
`insulin with the FDA.
`Clinical studies with human insulin
`(recombinant DNA) indicate its efficacy
`in hyperglycemic control. It appears to
`have a slightly quicker onset of action
`than animal
`insulins.
`In double-blind
`
`transfer studies with animal insulins, pa-
`tients previously treated with mixed
`635
`
`Page 4
`
`Page 4
`
`

`

`
`
`
`at. m vac-m
`‘ “pa-M W
`
`Fig. 4. The new production plant in Indianapolis for human insulin produced by recombinant
`DNA technology.
`
`beef-pork insulin had a 70 percent de-
`crease of bound insulin in comparison
`with a base line. Species-specific binding
`of human, pork, and beef insulin at 6
`months decreased by 61. 58. and 57
`percent, respectively. In patients previ-
`ously treated with pork insulin.
`the
`bound insulin decreased by 30 percent in
`control subjects treated with pork insu-
`lin. and by 51 percent in patients trans-
`ferred to human insulin. Species-specific
`binding of beef and human insulins de-
`creased equally whether patients were
`maintained on purified pork insulin or
`switched to human insulin. Species-spe-
`cific binding for pork insulin. however.
`remained constant in both groups ([9).
`The clinical importance of these findings
`remains to be clarified in long-term stud-
`ies. Occasional‘patients hypersensitive
`to animal insulins and semisynthetic hu-
`man insulin derived from pork insulin
`tolerated human insulin (recombinant
`DNA) well. Recombinant
`technology
`now permits us to study human proinsu-
`lin and mixtures of human proinsulin and
`insulin much as they are secreted by the
`beta cell. These studies may provide an
`improved modality of therapy in diabe-
`tes.
`
`The power of recombinant DNA tech-
`nology resides in its high degree of speci-
`ficity. as well as the ability it provides to
`splice together genes from diverse orga-
`nisms—organisms that will not normally
`exchange DNA in nature. With this tech-
`nology, it is now possible to cause cells
`to produce molecules they would not
`normally synthesize. as well as to more
`efficiently produce molecules that they
`do normally synthesize. The logistic ad-
`vantages of synthesizing human insulin.
`growth hormone. or interferon in rapidly
`636
`
`dividing bacteria. as opposed to extract-
`ing these from the tissues in which they
`are normally produced. are obvious.
`We have shown the practicality of
`using recombinant
`technology ' to pro-
`duce proteins of pharmacological inter-
`est as fermentation products. This was
`accomplished without adverse environ-
`mental impact or increased risk to work-
`ers. At this point it seems reasonable to
`speculate about the future of this new
`technology.
`
`Impact of the Technology on Industry
`
`A whole growth industry largely de-
`pendent on investor interest has devel-
`oped. Through newsletters. conferences
`to develop research strategies. market
`estimates. and so forth. these investors
`supposedly predict which projects will
`be brought to fruition through this new
`biotechnology. It is difficult to estimate
`the extent to which these prognostica-
`tions will reflect economic and scientific
`reality. but there are some items of fact
`that appear to be supported by fairly
`simple logic.
`In the biomedical area there will cer-
`tainly be other proteins and peptides of
`pharmacological
`interest
`produced.
`Some of these are likely to result from
`new discoveries as additional genes are
`cloned. As an example. perhaps the most
`interesting aspect of the cloning of the
`interferon genes is that they represent a
`family of genes that code for a large
`number of interferons.
`leading to the
`possibility of producing hybrid mole-
`cules that have not been seen in nature.
`It seems unlikely that interferons should
`be unique in this respect among cyto-
`
`Page 5
`
`kines or other biologically interesting
`messengers.
`The technology will probably permit
`the mapping of the entire human genome
`during the next decade. Medical geneti-
`cists have laboriously mapped human
`genes by studying electrophoretic vari-
`ants or phenotypic expression of disease
`tracked through family trees. It is now
`possible to isolate individual human
`chromosomes on a preparative scale.
`followed by establishment of gene banks
`or libraries for each chromosome. The
`work should advance rapidly with an
`enormous potential impact on new medi-
`cal research and the understanding of
`human biology.
`In addition,
`it seems
`likely that eventually we will understand
`the mechanism of gene control and regu-
`lation which. combined with information
`now being unraveled concerning potent
`tumor-specific
`oncogenic DNA se-
`quences. clearly suggests major applica-
`tions in our understanding of oncology
`and dilferentiation. Consider. for exam-
`plc.
`the recent
`finding that
`the point
`mutation in a normal human gene that
`leads to the acquisition of transforming
`properties is due to a single nucleotide
`change from guanylate to thymidylate.
`This codon change results in a single
`amino acid substitution of valine for gly-
`cine in the 12th amino acid residue of the
`T24 oncogene encoded p25 protein;
`it
`appears to be suflicient to confer trans—
`forming properties on the T24 human
`bladder oncogene (20).
`Assumptions can be made about appli-
`cations to agriculture as well. It seems
`incontrovertible that in some areas. for
`example. the amount of productive land
`is decreasing because of the fall of water
`tables and sometimes increasing salinity
`of ground water. Moreover. the number
`of people producing crops is decreasing
`while the population dependent upon
`them continues to increase. Recombi-
`nant
`technology.
`in combination with
`conventional plant breeding. plant cell
`culture. and regeneration. may well re-
`sult
`in the production of new plants.
`Such plants could increase the produc-
`tivity of existing farmland as well as
`permit farming on land currently consid-
`ered to be nonproductive. Equally im-
`portant applications are technically fea-
`sible in the animal husbandry area. and
`many other types of applications—in the
`fermentation industry. industrial chemi-
`cals.
`environmental
`clean-up—have
`been suggested.
`We can certainly debate how rapidly
`these further developments will occur
`and whether or not they will be economi-
`cally feasible. However. we must all be
`SCIENCE. VOL. 219
`
`Page 5
`
`

`

`impressed with the speed with which the
`technology has progressed since 1974
`and can be confident that if we invest
`
`this rate Will be maintained or
`wisely,
`even increased.
`
`References and Notes
`1. U.S. Department of Agriculture estimates. Live—
`stock and Slaughter Reports (Bulletin of Statis-
`tics 522, Economic. Statistic and Cooperative
`Services, Washington. D.C., 1980).
`2. National Institutes of Health Pub]. 78-1588
`(April 1978), p. 9.
`D. A. Jackson, R. H. Symons, P. Berg, Proc.
`Natl. Acad. Sci. U.S.A. 69, 2904 (1972).
`S. N. Cohen, A. C. Y. Chang. H. W. Boyer, R.
`B. Helling, ibid. 70, 3240 (1973).
`US. patent number 4,237,224.
`P. Berg et al., Science 185, 303 (1974).
`P. Berg, D. Baltimore, S. Brenner, R. O. Roblin
`III, M. F. Singer. ibid. 188, 991 (1975).
`
`\onyuAw
`
`8. B. Hartley, Nature (London) 283. 122 (1980).
`9. R. E. Chance et al.,
`in Peptides: Synthesis~
`StruetiirevFunetion, D. H. Rich and E. Gross,
`Eds.
`(Proceedings of the Seventh American
`Peptide Symposium. Pierce Chemical Compa»
`ny, Rockford. [||., 1981). pp. 721—728.
`10. R. E. Chance, E. P. Kroetf, J. A. Hotfmann. B.
`H. Frank, Diabetes Care 4, 147 (1981).
`11.
`I. S. Johnson,
`in Insulins, Growth Hormone,
`and Recombinant DNA Technology,
`J. L.
`Gueriguian, Ed. (Raven, New York, 1981), p.
`183.
`12. S. A. Chawdhury. E. J. Dodson, G. G. Dodson.
`C. D. Reynolds, S. Tolley, A. Cleasby, in Hor-
`mone Drugs: Proceedings ofthe FDA-USP
`Workshop on Drugs and Reference Standards
`for lnsulins, Somatotropins. and Thyroid-axis
`Hormones (U.S. Pharmacopeia, lne., Roekville.
`Md, in press).
`13. R. S. Baker, J. M. Ross, J. R. Schmidtke, W. C.
`Smith, Lancet 1981-11. 1139 (1981).
`14. J. W. Ross, R. S. Baker, C. S. Hooker, I. S.
`Johnson, J. R. Schmidtke, W. C. Smith,
`'
`in
`Hormone Drugs: Proeeedings of the FDA-USP
`
`Workshop on Drugs and Reference Standards
`for Insulins, Somatotropins, and Thyroid-axis
`Hormones (U.S. Pharmacopeia, lnc., Rockville,
`Md., in press).
`‘
`15. Bull. At. Sci. 33, 22 (1977).
`16. 95th Congress. 2d sess. amended to 5.1217;
`Calendar No, 334, H. Rep. No. 95359 (1977).
`17. A. J. Large. Wall Street Journal, 25 January
`1982, p. 18.
`'
`18.
`I. S. Johnson.
`in Research with Recombinant
`DNA, an Academy Forum (National Academy
`of Sciences, Washington. DC, 1977), p. 156.
`l. S. Johnson, Diabetes Care 5 (Suppl. 2), 4
`19.
`(Novem