View source for Biotechnology in pharmaceutical manufacturing

[[Image:Escherichia coli (MCC).jpg|thumb|Escherichia coli bacteria, which are often utilized in production of pharmaceutical products.]] 

[[Biotechnology]] is the use of living organisms to develop useful products. Biotechnology is often used in [[pharmaceutical manufacturing]]. Notable examples include the use of bacteria to produce things such as insulin or human growth hormone. Other examples include the use of [[Transgene|transgenic]] pigs for the creation of hemoglobin in use of humans. <!-- needs to be referenced before uncommenting While the genetic modification of microorganisms to produce pharmaceutical products is generally uncontroversial, the insertion of human DNA into farm animals to produce human hormones and enzymes has attracted significant ethical controversy. -->

==Human Insulin==
[[Image:Insulincrystals.jpg|thumb|Insulin crystals]] Amongst the earliest uses of biotechnology in pharmaceutical manufacturing is the use of [[recombinant DNA]] technology to modify ''[[Escherichia coli]]'' bacteria to produce human [[insulin]], which was performed at [[Genentech]] in 1978.<ref name=SNAu/> Prior to the development of this technique, insulin was extracted from the [[pancreas]] glands of cattle, pigs, and other farm animals. While generally efficacious in the treatment of [[diabetes]], animal-derived insulin is not indistinguishable from human insulin, and may therefore produce allergic reactions.<ref>Brar, Deepinder: "The History of Insulin" http://www.med.uni-giessen.de/itr/history/inshist.html {{Webarchive|url=https://web.archive.org/web/20181101104524/http://www.med.uni-giessen.de/itr/history/inshist.html |date=2018-11-01 }}, accessed June 14, 2006</ref> Genentech researchers produced artificial [[gene]]s for each of the two [[protein]] chains that comprise the insulin molecule. The artificial genes were "then inserted... into plasmids... among a group of genes that"<ref name=SNAu>{{cite journal |title=Human Insulin: Seizing the Golden Plasmid |journal=Science News |volume=114 |issue=12 |pages=195–196 |date=1978-09-16 |doi=10.2307/3963132|jstor=3963132 }}</ref> are activated by [[lactose]]. Thus, the insulin-producing genes were also activated by lactose. The recombinant [[plasmids]] were inserted into ''Escherichia coli'' bacteria, which were "induced to produce 100,000 molecules of either chain A or chain B human insulin."<ref name=SNAu/> The two protein chains were then combined to produce insulin molecules.

==Human growth hormone==
[[Image:Somatotropine.GIF|thumb|Growth hormone]]Prior to the use of recombinant DNA technology to modify bacteria to produce [[growth hormone|human growth hormone]], the hormone was manufactured by extraction from the [[pituitary gland]]s of cadavers, as animal growth hormones have no therapeutic value in humans. Production of a single year's supply of human growth hormone required up to fifty pituitary glands,<ref>{{cite journal |title=Labs tie for human growth hormone |journal=Science News |volume=116 |issue=2 |pages=22 |date=1979-07-14 |doi=10.2307/3964172|jstor=3964172 }}</ref> creating significant shortages of the hormone.<ref>{{cite journal |author=Walgate R |title=Pituitary slump |journal=Nature |volume=290 |issue=5801 |pages=6–7 nbgcyt5 |date=March 1981 |pmid=7207586 |doi=10.1038/290006b0 |doi-access=free }}</ref> In 1979, scientists at Genentech produced human growth hormone by inserting DNA coding for human growth hormone into a plasmid that was implanted in [[Escherichia coli]] bacteria. The gene that was inserted into the plasmid was created by [[reverse transcription]] of the mRNA found in pituitary glands to complementary DNA. HaeIII, a type of restriction enzyme which acts at restriction sites "in the 3' noncoding region"<ref name=Goeddel79>{{cite journal |vauthors=Goeddel DV, Heyneker HL, Hozumi T |title=Direct expression in ''Escherichia coli'' of a DNA sequence coding for human growth hormone |journal=Nature |volume=281 |issue=5732 |pages=544–8 |date=October 1979 |pmid=386136 |doi=10.1038/281544a0 |bibcode=1979Natur.281..544G |s2cid=4237998 |display-authors=etal}}</ref> and at the 23rd [[codon]] in [[complementary DNA]] for human growth hormone, was used to produce "a DNA fragment of 551 base pairs which includes coding sequences for amino acids 24–191 of HGH."<ref name=Goeddel79/> Then "a chemically synthesized DNA 'adaptor' fragment containing an ATG initiation codon..."<ref name=Goeddel79/> was produced with the codons for the first through 23rd [[amino acid]]s in human growth hormone. The "two DNA fragments... [were] combined to form a synthetic-natural 'hybrid' gene."<ref name=Goeddel79/> The use of entirely synthetic methods of DNA production to produce a gene that would be translated to human growth hormone in Escherichia coli would have been exceedingly laborious due to the significant length of the amino acid sequence in human growth hormone. However, if the cDNA reverse transcribed from the mRNA for human growth hormone were inserted directly into the plasmid inserted into the Escherichia coli, the bacteria would translate regions of the gene that are not translated in humans, thereby producing a "pre-hormone containing an extra 26 amino acids"<ref name=Goeddel79/> which might be difficult to remove.

==Human blood clotting factors==
Prior to the development and FDA approval of a means to produce human blood [[Coagulation|clotting]] factors using recombinant DNA technologies, human blood clotting factors were produced from donated blood that was inadequately screened for [[HIV]]. Thus, HIV infection posed a significant danger to patients with [[hemophilia]] who received human blood clotting factors: <blockquote>Most reports indicate that 60 to 80 percent of patients with hemophilia who were exposed to factor VIII concentrates between 1979 and 1984 are seropositive for HIV by [the] Western blot assay. As of May 1988, more than 659 patients with hemophilia had AIDS...<ref>{{cite journal |vauthors=White GC, McMillan CW, Kingdon HS, Shoemaker CB |title=Use of recombinant antihemophilic factor in the treatment of two patients with classic hemophilia |journal=N. Engl. J. Med. |volume=320 |issue=3 |pages=166–70 |date=January 1989 |pmid=2492083 |doi=10.1056/NEJM198901193200307}}</ref></blockquote>

The first human blood clotting factor to be produced in significant quantities using recombinant DNA technology was [[Factor IX]], which was produced using [[transgenic]] Chinese hamster ovary cells in 1986.<ref name=Kaufman86>{{cite journal |vauthors=Kaufman RJ, Wasley LC, Furie BC, Furie B, Shoemaker CB |title=Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells |journal=J. Biol. Chem. |volume=261 |issue=21 |pages=9622–8 |date=July 1986 |doi=10.1016/S0021-9258(18)67559-3 |pmid=3733688 |url=http://www.jbc.org/cgi/pmidlookup?view=long&pmid=3733688|doi-access=free }}</ref> Lacking a map of the human genome, researchers obtained a known sequence of the RNA for Factor IX by examining the [[amino acid]]s in Factor IX:<blockquote>Microsequencing of highly purified... [Factor IX] yielded sufficient amino acid sequence to construct oligonucleotide probes.<ref name=Toole84>{{cite journal |vauthors=Toole JJ, Knopf JL, Wozney JM |title=Molecular cloning of a cDNA encoding human antihaemophilic factor |journal=Nature |volume=312 |issue=5992 |pages=342–7 |year=1984 |pmid=6438528 |quote=page 343 |doi=10.1038/312342a0|bibcode=1984Natur.312..342T |s2cid=4313575 |display-authors=etal}}</ref></blockquote>

The known sequence of Factor IX RNA was then used to search for the gene coding for Factor IX in a library of the DNA found in the human liver, since it was known that blood clotting factors are produced by the human liver:<ref name=Toole84/><blockquote>A unique oligonucleotide... homologous to Factor IX mRNA... was synthesized and labeled... The resultant probe was used to screen a human liver double-stranded cDNA library... Complete two-stranded DNA sequences of the... [relevant] cDNA... contained all of the coding sequence COOH-terminal of the eleventh codon (11) and the entire 3'-untranslated sequence.<ref name=Kaufman86/></blockquote>

This sequence of cDNA was used to find the remaining DNA sequences comprising the Factor IX gene by searching the DNA in the X chromosome:<blockquote>A genomic library from a human XXXX chromosome was prepared... and screen[ed] with a Factor IX cDNA probe. Hybridizing recombinant phage were isolated, plaque-purified, and the DNA isolated. Restriction mapping, Southern analysis, and DNA sequencing permitted identification of five recombinant phage-containing inserts which, when overlapped at common sequences, coded the entire 35kb Factor IX gene.<ref>Kaufman, pages 9622–3</ref></blockquote>

Plasmids containing the Factor IX gene, along with plasmids with a gene that codes for resistance to methotrexate, were inserted into Chinese hamster ovary cells via transfection. Transfection involves the insertion of DNA into a eukaryotic cell. Unlike the analogous process of transformation in bacteria, transfected DNA is not ordinarily integrated into the cell's genome, and is therefore not usually passed on to subsequent generations via cell division. Thus, in order to obtain a "stable" transfection, a gene which confers a significant survival advantage must also be transfected, causing the few cells that did integrate the transfected DNA into their genomes to increase their population as cells that did not integrate the DNA are eliminated. In the case of this study, "grow[th] in increasing concentrations of methotrexate"<ref>Kaufman, page 9623</ref> promoted the survival of stably transfected cells, and diminished the survival of other cells.

The Chinese hamster ovary cells that were stably transfected produced significant quantities of Factor IX, which was shown to have substantial coagulant properties, though of a lesser degree than Factor IX produced from human blood:<blockquote>The specific activity of the recombinant Factor IX was measured on the basis of direct measurement of the coagulant activity... The specific activity of recombinant Factor IX was 75 units/mg... compared to 150 units/mg measured for plasma-derived Factor IX...<ref>Kaufman, page 9626</ref></blockquote>

In 1992, the FDA approved Factor VIII produced using transgenic Chinese hamster ovary cells, the first such blood clotting factor produced using recombinant DNA technology to be approved.<ref>United States Food and Drug Administration: "The licensing of the first recombinant DNA-derived clotting factor", https://www.fda.gov/bbs/topics/NEWS/NEW00312.html, accessed June 17, 2006</ref>

==Transgenic farm animals==
[[Image:Penn state university pigs.jpg|thumb|A pig, transgenic generations of which might be used to produce blood substitutes for use in humans.]]Recombinant DNA techniques have also been employed to create [[transgenic]] farm animals that can produce pharmaceutical products for use in humans. For instance, pigs that produce human hemoglobin have been created. While blood from such pigs could not be employed directly for transfusion to humans, the hemoglobin could be refined and employed to manufacture a blood substitute.<ref>{{cite journal |vauthors=O'Donnell JK, Martin MJ, Logan JS, Kumar R |title=Production of human hemoglobin in transgenic swine: an approach to a blood substitute |journal=Cancer Detect. Prev. |volume=17 |issue=2 |pages=307–12 |year=1993 |pmid=8402717 }}</ref> <!-- needs to be referenced before uncommenting: Such transgenic farm animals have been a source of significant ethical objections, which are primarily centered around the perceived creation of human-animal hybrids and an alleged danger to the integrity of the human species. To some extent these concerns may arise from a misunderstanding of the nature of the transgenic animals produced for medical purposes. Such animals might contain, at most, a few human genes, insufficient to imbue them with substantially human characteristics. Some bioethicists object to even the limited transfer of one or several human genes to animals, asserting that this practice is in and of itself a threat to the human species, or that it poses a danger of a "slippery slope" which will eventually lead to the production of human-animal hybrids. Other bioethicists contend that humanity is defined by the entire human genome, and that the transfer of an extraordinarily small fraction of this genome to animals does not create a result that has any meaningful degree of humanity. These bioethicists also hold that the putative "slippery slope" towards human-animal hybrids is not a sufficient ethical objection to transgenic animals since nearly every medical technology presents at least some theoretical danger of a "slippery slope" to an immoral practice. For instance, the use of morphine as an analgesic might lead -- and, on rare occasions, has led -- to the use of morphine to euthanize patients. Yet concerns over preventing euthanasia would not justify the elimination of morphine for analgesic purposes. -->

==Paclitaxel (Taxol)==
[[Bristol-Myers Squibb]] manufactures paclitaxel using ''Penicillium raistrickii'' and plant cell fermentation (PCF).{{Citation needed|date=January 2016}}

==Artemisinin==
Transgenic yeast are used to produce [[artemisinin]], as well as a number of [[insulin analog]]s.<ref>{{cite web|author=Mark Peplow |url=http://www.rsc.org/chemistryworld/2013/04/sanofi-launches-malaria-drug-production |title=Sanofi launches malaria drug production &#124; Chemistry World |publisher=Rsc.org |date= |accessdate=2013-12-17}}</ref>

== See also == 
* [[Molecular Biotechnology (journal)|''Molecular Biotechnology'' (journal)]]
*[[Bacillus isolates|''Bacillus'' isolates]]
*[[Fungal isolates]]
*[[Medicinal molds]]
*[[Sponge isolates]]
*[[Streptomyces isolates|''Streptomyces'' isolates]]

==References==
{{Reflist}}

[[Category:Drug manufacturing]]
[[Category:Biotechnology]]