Part I Modern Biopharmaceuticals: Research Is the Best Medicine – Sanitas Summum Bonus

Part I Modern Biopharmaceuticals: Research is the Best Medicine – Sanitas Summum Bonus

Modern Biopharmaceuticals: Recent Success Stories, First Edition. Edited by Jörg Knäblein. # 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

“In all things of nature there is something of the marvelous.” Aristotle (384–322 BC)

1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology” J€org Kn€ablein

1.1 Biotechnology – The Science Creating Life

“Biotechnology” is a combination of the Greek words bios, techne, and logos, meaning life, technology, and knowledge. Hence, biotechnology is the technical application of existing knowledge on bacteria for the weal of humankind. Or, according to a less- impassioned definition from the University of Hohenheim: “ . . . technical (fermentation) processes applying organisms, cells or parts thereof with the aim to manufacture products for the pharmaceutical, food or cosmetics industry as well as sustainable waste reduction in the environmental biotechnology field.” Even more complex is the 1989 European Federation of Biotechnology (EFB) definition: “ . . . the integrated application of natural and engineering sciences in order to technically use organisms, cells, parts thereof and their molecular analo- gous. Thus, biotechnology deals with the application of biological processes for technical and industrial production and hence is a very application-oriented science of microbiology and biochemistry in tight conjunction with technical chemistry and process engineering.” As we can see, although definitions of the term “biotechnology” can be somewhat different, one thing they all have in common: biotechnology improves our daily life (e.g., biopharmaceuticals or genetically engineered food) – in some cases biotechnology is the only enabler of life!

1.2 The Inauguration of Biotechnology

Since thousands of years people manipulated nature in order to make the maximum use of it – ﬁrst biological, then technological,andﬁnally biotechnological (Figure 1.1). Already eighteen thousand years before Christ, people in the Middle East had successfully domesticated sheep and deer – later (about 5000 BC) pigs by the Chinese. At the same time, the Sumerians in Mesopotamia (the area between

Modern Biopharmaceuticals: Recent Success Stories, First Edition. Edited by Jörg Knäblein. # 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA. Jesus 4 18.000 BC 1.000 BC 10.000 BC 5.000 BC Mesopotamia: Egypt: Babylon: Hsia-Dynasty: Hammurabi‘s Christ 1500 AD j Kasch Irep Henket Rice wine Laws: Dates wnyTosn er fBiotech of Years Thousand Twenty 1 Roquefort Strong beer Whiskey

1500 AD 1850 1900 1950 1960 1970 1980 1990 2000 2011

ca. 1500: Sauerkraut 1953: Watson & Crick “3 D structure of DNA double helix” 1516: Purity requirement for beer 1956: Arthur Kornberg “Discovery of DNA polymerase” 1673: Anton Leeuwenhoek “Microskope” 1958: John C. Kendrew “3 D structure of myygoglobine” 1826: J & E Sturge “Citrate-production” 1960: Jacob & Monod “Transkription and mRNA” 1838: Justus von Liebig “Citrate-structure” 1963: Max F. Perutz “3 D structure of hemoglobin” Watson, Knäblein 2004 1963: Jacob & Monod “Operator-model and operon” 1859: Charles Darwin “Survival of the fittest” –

1860: Louis Pasteur “Vinegar with Acetobacter” 1964: Marshall W. Nirenberg “Genetic code” From 1866: Johann Gregor Mendel “Pea-genetics” 1968: Werner Arber “Restriction enzymes”

1869: Johann Friedrich Miescher “Nucleus” 1970 : Ti&BliTemin &Baltimore “RiReverse transcriptase“” “ Hwang, Knäblein 2005* Traditional 1893: C. Wehmer “Citrate from fungi” 1973: Maxam, Gilbert & Sanger “DNA sequencing” 1909: Wilhelm Johannsen “Genotype” 1973: Cohen & Boyer “Recombinant insulin” 1913: Michaelis & Menten “Enzyme kinetics” 1975: Köhler & Milstein “Monoclonal antibodies” 1921: Banting & Best “Insulin mode of action” 1980: 1st biotech-company “Genentech goes public” ” to 1922: Theodor Escherich “E. coli strain K12” 1981: Robert C. Gallo “Discovery of AIDS / HIV” “ 1923: Pfizer “Citrate large scale fermentation” 1983: Kary Mullis “Polymerase chain reaction, PCR” Biotechnology Modern 1926: Whipple, Minot, Murphy “Anemia treatment” 1984: Huber et al. “Photo-reaction-center” 1928: Alexander Fleming “Penicillin discovery” 1996: Fast insulin “Lispro Humalog ®” 1928: Fred Griffith “Genetic transformation” 1996: Ian Wilmut “SCNT: clone-sheep Dolly” 1933: Tadeusz Reichstein “Vitamin C synthesis” 1998: Personalized medicine “Herceptin ®” 1934: Roche‘s Vitamin-C-drug “Redoxon ®” 2001: UNESCO-Report “Human Cloning” 1944: Oswald Avery “DNA = genetic information” 2004: Global Pharma Specialists (GPS)®

1945: Florey & Chain “Penicillin production” 2005: Knäblein Modern Biopharmaceuticals® (I-IV) ” *2005 press conference, shortly before Huang was 2011: Knäblein Modern Biopharmaceuticals® (V) pledged guilty of fraud in “human cloning” Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ®,” Knäblein 2005 www.get-gps.net

Figure 1.1 Twenty thousand years of biotech: “Traditional Biotechnology” exists since 20 000 years –“Modern Biotechnology” since the advent of genetic engineering. 1.3 From “Traditional” to “Modern Biotechnology” j5

Euphrates and Tigris, in which according to the Holy Bible “milk and honey was streaming”–today Iraq) were capable of brewing beer as depicted in detail on the Monument Bleu – a Sumerian stone drawing kept in the Paris Louvre – showing the process of wheat preparation for beer production. In some years, almost half of the entire wheat harvest was used for brewing the typical beer (Kasch or Bufa). It should be mentioned though that these early biotechnologies were very empirical and the underlying processes far from being reproducible. Another thousand years later, the Egyptians developed the art of winemaking (Irep) almost to perfection, and about 3000 BC, the Babylonians were able to brew 20 different types of beer. In Egypt, in 2600 BC, beer (Henket) was considered a staple food and the breweries were a royal monopoly – the situation was similar in 2200 BC with rice beer during the Chinese Hsia Dynasty. Another biotechnology was mentioned around 1700 BC in the “Laws of Sumerian King Hammurabi” (1728–1686 BC; one of the most influential leaders in the orient): a hand-fertilization technique for date palm trees. Specific fermentation processes were established in areas in which all required ingredients were available: wheat beer in Middle Europe (malt, hops, Saccharomyces cerevisiae), rice wine (rice, Aspergillus oryceae), rice liquor sake in East Asia (rice, koji, S. cerevisiae), kvass in Russia (wheat malt, rye flour, Lactobacillus spec.), pombe in South America and Central Africa (millet mash and yeast, Schizosaccharomyces pombe), and pulque by the Aztecs in Middle America (fruits, Zymomonas mobilis). People having cows produced yogurt (Streptococcus lactis), kefir(Lactobacillus kefir), and cheese (e.g., Streptococcus salivarius) – although these products were created more or less randomly by spontaneous fermentation due to the approximately 500 000 microbes that naturally occur in just 1 ml of milk. In contrast, the Sumerians developed specific fermentation processes to selectively produce certain different kinds of cheese. Later (around 250 AD), the “Roquefort” (Penicillium roquefortii) was shipped to Rome as a “Gallic specialty.” In contrast, brewing of beer was only developed in the sixth century into a stable and reproducible fermentation process when monks became more and more interested in this new type of “liquid bread.” Although during the Lenten season, the monks were prohibited to eat, according to the sentence “liquida non fragunt ieiunum” (“liquids do not infringe the abstinence order”), they were allowed to drink – and thanks to that, we nowadays have very delicious and aromatic strong beers. Today in Germany alone, 100 million hectoliters of beer is brewed annually in compliance with the Bavarian purity law from 1516 using exclusively barley malt, hops, water, and special yeast strains. Worldwide, beer is still the top selling biotech product with a yearly consumption of approximately 1.5 billion hectoliters worth more than D50 billion.

1.3 From “Traditional” to “Modern Biotechnology”

Already at the beginning of the last millennium, “traditional” biotechnologies were developed in order to produce “high value traits”: in 1276, the ﬁrst whiskey distillery was established in Ireland, and two hundred years later certain fermentation 6j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

processes were optimized using special microorganisms in order to produce sauerkraut (Leuconostoc mesenteroides) and different yogurt types (e.g., Lactobacillus bulgaricus). With a size of only a couple of micrometers, the microbes are not visible to the naked eyes; in 1676, Antonie van Leeuwenhoek (1632–1723) was the first person to watch a microorganism at a 200 magnification using his self-made microscope. In 1684, he published his first drawings of the observed microbes [1] and as a result became scientific member of the very prestigious London Royal Society – albeit he had never visited a university. Fantastic discoveries paved the way to modern biotechnology in a fast pace. The foundation of classical genetics was laid by the English scientist Charles Darwin (1809–1882) in his revolutionary theories on the principle of natural selection. On July 1, 1859, Darwin presented his seminal paper on the “Survival of the fittest” [2] on the development of animals to the Royal Linnean Society, which still prides itself as the repository of natural history expertise in Britain. There was very little reaction in the room, but the real furor did not begin until Darwin published “On the Origin of Species” the following year: “There is a grandeur in this view of life ... that whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved” are the concluding lines of the revolutionary book. Darwin, knowing the reaction he was likely to receive, held back for years from airing and then publishing his theories on the principle of natural selection – and his fears were correct: he was pilloried by the scientific and religious establishment of the time. One famous caricature depicted him with the body of a monkey, so angered were people by the suggestion that they might have descended from the monkeys rather than having been created in the image of God. His theories are still rejected by some, notably creationists in the United States, and are less than welcome in the Middle East. Even in Britain a poll in 2006 showed that only 48% of the people believed in Darwin’s evolutionary theories. Today, we honor the man for the 150th anniversary of his presentation to the Royal Linnean Society, and we celebrate Darwin’s 200th birthday and the 150th anniversary of the publication of “On the Origin of Species.” So, obviously, Darwin is still causing waves after 150 years, and most likely he would have been happy to get a fraction of this enthusiasm during his life time. . . . In 1860, it was the French scientist Louis Pasteur (1822–1895) (founder of microbiology and biotechnology) who was using for the first time “pure isolates” (phenotypically identical strains) of Acetobacter to convert alcohol into vinegar. Some years later, in 1866, it was Johann Gregor Mendel (1822–1884) who showed with his experiments on hybrid peers that some of the phenotypic characteristics can be transferred from one generation to the next [3].

1.3.1 Molecular Genetics and Enzymatic Kinetics

The Swiss pathologist Johann Friedrich Miescher (1811–1887) was the ﬁrst who stained the “nucleus” of a cell and in 1869 at University Tubingen€ isolated its “nucleic 1.3 From “Traditional” to “Modern Biotechnology” j7 acid.” Although this was essentially the beginning of molecular genetics only, 40 years later, it was the Danish geneticist Wilhelm Johannsen (1857–1927) who coined in 1909 the term “genotype” with its inheritable characteristics – the “genes.” Another important milestone toward modern biotechnology was the kinetic descrip- tion of fermentation processes, that is, the time it took to convert a certain substrate S with a speciﬁcenzymeE into the product P. Enzymes can catalyze chemical reactions by factors from 100 million up to 1 trillion (108–1012). Assuming a 1012 enzyme-catalyzed reaction takes 1 second to be completed, without enzyme it would theoretically take 300 000 years! Obviously, most of our metabolic reactions would not be feasible without enzymes – hence these biological catalysts enable our life. The enzymes’ catalytic activity stems from their ability to bring substrates into a favored steric orientation – the so-called enzyme–substrate (ES) complex, which reduces the activation energy required to convert the substrate into the respective product. Since one enzyme can only catalyze the reaction of a restricted number of substrate molecules (because at one point all active sites are occupied – the so-called saturation effect), there is a maximum reaction velocity depending on substrate type and enzyme. Chemical reactions without enzymes obviously do not have such kinetics as it was shown already in 1913 by the Berlin biochemists Leonor Michaelis (1875–1949) and Maud L. Menten (1879–1960). From their kinetic experiments, Michaelis and Menten concluded the existence of an ES complex by measuring the maximum velocity for enzyme-catalyzed reactions [4]. This was the earliest evidence (indirectly though) of this phenomenon and therefore today it is called the “Michaelis–Menten kinetic.” Applying this knowledge to the fermentation process – by feeding the substrate continuously (rather than batch) to avoid the saturation effect – it was possible to control the reaction and maximize the time–space yield in a bioreactor. In the following sections, we will see some examples showing that this was really imperative – in combination with genetic optimization – to produce the required amounts of vital and lifesaving substances on a large scale.

1.3.2 Penicillin and Other Lifesaving Antibiotics

The immense importance for humankind of such biotechnological developments is illustrated nicely in the ﬁrst example: Alexander Fleming (1881–1955) was working as a bacteriologist at St. Mary’s Hospital in London when in 1928 he discovered a clear zone around a fungus, which was (accidentally) growing on an agar dish with Staphylococcus. Obviously, this fungus produced some agent preventing the bacteria to grow – and since he named it as Penicillium notatum, Fleming called the compound Penicillin. When Fleming published his work in the British Journal of Experimental Pathology [5], he did not realize the huge medical potential of this compound against a variety of hazardous bacteria: streptococci, staphylococci, anthrax, diphtheria, glandular fever, and tetanus. Ten years later, in 1938, the Ukrainian biochemist Ernst Boris Chain (1906–1979) at Oxford University under- stood the general antibacterial potential of Penicillin. 8j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

With the beginning of World War II in 1939 all of a sudden there was an enormous demand for compounds fighting bacterial infections to treat the great number of casualties. Chain and the Australian pathologist Howard Florey (1898–1998) saw the great need for antibiotics, the potential of Fleming’s work, and continued where he stopped. Two years later, Penicillin was used to treat a 43-year-old patient suffering from a severe staphylococci infection. After a short recovery, the patient died one month later due to the lack of sufficient amount of Penicillin, although Chain and Florey tried to recover the substance from his urine – so, obviously there was an urgent need for improved fermentation processes. One issue though with Fleming’s strain was that it grew exclusively on surfaces and yielded only 2 mg Penicillin per milliliter of culture broth. Joining forces with the National Academy of Science and the fabulous Northern Regional Research Labora- tory (NRRL), scientists were now looking for a strain that could also be grown as a submerse culture. In 1943, Penicillium chrysogenum was isolated with a yield of at least 40 mg/ml Penicillin. Rather than using a surface fermentation the so-called deep-tank process was applied and with a developed mutant of P. chrysogenum this technique yielded 150 mg/ml. The production rate could even be enhanced using residual water from maize processing as a substrate. Nevertheless, to further optimize the strains by applying random UV mutagenesis (evolution in the Petri dish), the War Production Board in the United States assigned universities and other research laboratories. Eventually, also Stanford University, Cold Spring Harbor Laboratory, and twenty US companies got involved. Optimized fermentation processes, mutated strains, and feeding strategies (see Michaelis– Menten) finally led to a production rate of 1.5 mg/ml Penicillin – from 2 mg/ml in the beginning that was a factor of 750! On March 1, 1944, the first large-scale fermentation was run and further technological improvements led to 90% recovery in a 30 000 l reactor (rather than 1% in a 1 l flask), increasing the yearly produced doses from 210 million to 6.8 trillion! Only by the joint research and development (R&D) activities of a large number of very enthusiastic scientists, it was possible to save the life of up to 1500 people during that war period. Subsequently, on D-day (June 6, 1944) innumerable victims were wounded but could fortunately be treated with Penicillin. For this success, and for their achievements in developing Penicillin, Sir Howard Walter Florey, Boris Chain, and Sir Alexander Fleming were awarded the Nobel Prize for Physiology or Medicine in 1945. To treat a variety of other infectious diseases, a number of additional antibiotics were developed since then, that is, streptomycin, tetracycline (both from Streptomy- ces spec.), cephalosporin (Cephalosporium acremonium), and rifamycin (Nocardia mediterranei). They all have in common their selectivity: these antibiotics specifically interfere with essential microbial metabolism, but not with any human pathways. As we will see later, antibiotics are not only lifesaving, they are also essential tools for molecular biology, genetic engineering, and cloning. Perorating, it should be mentioned that nowadays optimized fermentation processes, using genetically 1.3 From “Traditional” to “Modern Biotechnology” j9 optimized high-producing strains, are capable of yielding 20 000 times more Penicillin in 1 l of culture broth compared to what Fleming’s initial P. notatum did!

1.3.3 The Triumphal Procession of Vitamin C

Another very striking example of biotechnological development is the production of vitamin C (L-ascorbic acid) at large scale. Since humans and other primates do not have the essential enzyme L-gulonolactone-oxidase, which is required for biosynthesis of vitamin C, they rely on exogenic supply by food (100 mg/day) to avoid any deficiency signs. The most popular vitamin C deficiency is scurvy, leading to general bleeding, distorted development of connective tissue, gingivitis, and loss of teeth. This phenomenon was known since quite some time for sailors who were not provided with sufficient vegetables and fruits during their long journeys. This was why around 1900 the German emperor gave the order for all sailors to eat a couple of spoons of citrate every day, which unfortunately did not have the desired results: rather than prevention of scurvy the sailors were suffering from diarrhea. Why? Although it is correct that citrus fruits prevent scurvy, we know today it is their vitamin C content and not the citrate as was believed at those times. And so, twofold Nobel Prize laureate Linus Pauling (1901–1994) was eating a couple of grams of vitamin C every day making use of the protective properties of the antioxidants to catch free radicals (Pauling is one of only four individuals to have won multiple Nobel Prizes: Nobel Prize in Chemistry in 1954 and Nobel Peace Prize in 1962. With that he is one of only two people awarded two Nobel Prizes in different fields – the other being Marie Curie for Chemistry and Physics – and the only person awarded two unshared prizes). As a consequence of the high vitamin C diet, Pauling never caught a flew and lived up to 93 years, even though he died of cancer of the urinary bladder as a consequence of over-acidification of his urine. In 1933, Tadeus Reichstein (1897–1996) at ETH Zurich was able to chemically synthesize vitamin C for the first time: in more than ten steps he converted glucose into L-xylose followed by addition of hydrocyanic acid to form L-ascorbic acid [6]. Nevertheless, this complex chemical synthesis was obviously not suitable for large-scale production of vitamin C (although Reichstein received the Nobel Prize for Medicine in 1950, it was not for the vitamin C synthesis, but rather for his merits on cortisones). Reichstein and colleagues [7] improved the synthesis by using sorbose as intermediate. This is gained with 100% yield by the reduction of glucose with hydrogen at a pressure of 150 atm applying a nickel catalyst [8]. The next step, which is the chemical oxidation of sorbit into the precursor of vitamin C, sorbose, is very difficult though, because both asymmetric carbon atoms of L-ascorbicacidstemfromglucosealready– hence the key step in vitamin C synthesisistheregio-specific oxidation. And again, the problem can be solved using bacteria: as early as 1896, the French chemist Gabriel Bertrand (1867–1962) had described the microbial conversion of sorbit into sorbose with Acetobacter 10j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

suboxydans (according to the present nomenclature Gluconobacter oxydans). Reich- stein used its enzyme sorbitol-dehydrogenase to stereo-selectively convert D-sorbit into L-sorbose followed by chemical oxidation into 2-keto-L-gulonic acid (2-KLG) and subsequent water cleavage through acid treatment into L-ascorbic acid, vitamin C. Named after these “biotech pioneers,” the method is called “Reichstein-and- Grussner€ ” process, which was bought by Roche and marketed the vitamin Ccompound 1 Redoxon in 1934. Also before, Roche was interested in another method to produce vitamin C. This procedure to isolate vitamin C from paprika was established by the Hungarian scientist Albert von Szent-Gyorgyi€ Nagyrapolt (1893–1986) who in return received the Nobel Prize in Physiology or Medicine in 1937. For years vitamin C was produced according to the process of Reichstein and Grussner,€ until lately by means of genetic engineering a pure biotech process was developed. Bacteria of the species Erwinia use three enzymes to subsequently convert D-glucose into 2,5-diketo-D-gluconic acid (2,5-DKG), which in turn can serve as a substrate for Corynebacterium spec. to produce 2-KLG using its 2,5-DKG-reductase (again, using acid treatment this can easily be transformed into vitamin C). Although, on ﬁrst sight, it seems obvious to carry out a co-fermentation with these two species, this is not possible for multiple reasons: both strains have very different demands on optimum pH, temperature, and composition of the media. Hence, Erwinia (Gram negative) and Corynebacterium spec. (Gram positive) have very different growth rates and hence cannot be grown in the same bioreactor at the same conditions; these are just a few of the manifold problems that render a co- fermentation impossible. Again, it is biotechnology that can solve the manifold problems by “metabolic engineering”: the genetic information to express the enzyme 2,5-DKG-reductase is transferred from Corynebacterium into the genome of Erwinia (Figure 1.2). Applying such genetic engineering procedures, it was possible to generate a recombinant artiﬁcial “Erwinia hybrid-strain” that produces in only 120 hours impressive 120 g 2-KLG per liter fermentation broth with a yield of more than 60%(!), which is then directly converted into vitamin C. In this case of metabolic engineering, the manufacturing costs could be reduced by a factor of 50 and in the meantime more than 80,000 ton vitamin C are produced every year worth more than US$600 million! From this exciting example, it can be demonstrated how a very complex and sequential low-yield chemical synthesis can be replaced by a pure “one-pot” high- yield biotech fermentation process for cost-effective large-scale production. Using similar approaches, it was possible at BASF to simplify the eight-step chemical vitamin B2 synthesis into a one-step fermentation process and at DSM to substitute a chemical ten-step production of the antibiotic Cephalexin with the one-pot fermentation. As a result, the production costs could be cut in half and the energy consumption reduced by even 65%. The same is true for environmental pollution, because rather than using organic solvents for chemical production, only water is used for fermentation. Hence, these processes are good examples for “white biotechnology.” 1.4 A Small Molecule from Bacteria – A Huge Importance for Mankind j11

Transformation of glucose into vitamin C is catalyzed by four (subsequent) reactions: 3 enzymes from Erwinia 1 enzyme from Corynebacterium

1 4

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.2 Direct conversion of glucose into vitamin C by artificial bacteria: recombinant hybrid of Erwinia and Corynebacterium.

1.4 A Small Molecule from Bacteria – A Huge Importance for Mankind

1.4.1 Plasmids: Transformation by Gene Transfer

Now we have seen some striking examples of genetic engineering – but how does that work? In 1928, the English physician and bacteriologist Frederick Griffith (1877–1941) performed the experimentum crucis, the key experiment for genetic engineering, when he worked with two different strains of Streptococcus pneumo- niae, the causing agent of pleuropneumonia.Onestrain,containedinarough capsule (R), is harmless to mice, whereas the other, with a smooth capsule (S), kills them. Injection of heat-inactivated lethal S-strains into mice has no consequence, but in combination with the living harmless R-strains the mixture kills the animals. The “killing agent” was obviously transferred from the dead R-strain to the (former harmless) S-strain and transformed it into a lethal strain. With this experiment Griffith was able to genetically transform one strain into the other: by transfer of genes from S-strains he created transformed R-strains with S-genotype and S-phenotype. This was the first case of purposely perform- ing genetic engineering by transferring genetic material from one organism to another [9]. 12j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

Based on this exciting experiment, the Canadian physician Oswald Theodore Avery (1877–1955) at Rockefeller Institute in New York went one step further: he took cell extracts from heat-inactivated S-strains, which he purified step by step. All fractions (cell wall, protein fractions, nucleic acids) were analyzed for their ability to perform the transformation of R-strains into S-trains. By adding ethanol, a precipitate was formed, and the transformation did not work anymore. Carbohydrates (e.g., the capsules) do not precipitate, but nucleic acids do, and protease treatment showed no effects. Transformation is obviously only possible with intact nucleic acid fractions and when adding the RNA-degrading enzyme ribonuclease the principle still works. Finally, Avery performed a chemical test for the DNA sugar deoxyribose, which resulted in the expected blue color. He then concluded that the “transforming agent” was not a protein, it was not RNA, and it was not carbohydrate – it was DNA. In 1944, Avery could identify “DNA” as the carrier for all genetic information when he transformed bacteria by injecting chemically synthesized DNA to gain new properties and hereditary characteristics [10]. Combining these two smart genetic experiments from Griffith and Avery to identify DNA as “transforming principle and carrier of genetic information” were the first molecular genetic experiments in history.

1.4.2 DNA: The Molecule of Life

Another quantum leap in molecular biology was in 1953 when the biochemist James D. Watson (1928–today) and the physicist Francis H. Crick (1916–2004) elucidated the structure of “themoleculeoflife” [11]. As spriritus rectors of the 3D structure of DNA, they herald the start of modern biotechnology: this molecule was not just compelling in its sheer beauty, but more importantly, understanding the double-helical structure led to the fundamental mechanisms of transcription and translation (Figure 1.3). This process of copying DNA, namely replication, was the “first three-dimensional Xerox machine” (Kenneth E. Boulding, 1910–1993). Three years later, Arthur Kornberg (1918–2007; Nobel Prize in Physiology or Medicine in 1959) isolated the enzyme that synthesizes the molecule of life: DNA- polymerase [12]. During that time, scientists were also working on the more complex structures of proteins: John C. Kendrew (1917–1997) and coworkers described the structure of myoglobin in 1958 [13] and two years later Max F. Perutz (1914–2002) and colleagues described the structure of hemoglobin [14]. Both shared the Nobel Prize in Chemistry in 1962 for their “studies on the structures of globular proteins.” Also in the field of DNA, one important discovery was followed by the next: the first plasmid was isolated in 1959 and one year later Franzois Jacob (1920–today; Nobel Prize in Physiology or Medicine in 1965) and Jacques Monod (1919–1976; Nobel Prize in Physiology or Medicine in 1965) defined the general architecture of an operon and mRNA as the carrier/blueprint of entire proteins [15]. Another year later, Marshall W. Nirenberg (1927–2010; Nobel Prize in Physiology or Medicine in 1.4 A Small Molecule from Bacteria – A Huge Importance for Mankind j13

By splicing-out the introns, mature mRNA is created to yield in the mature protein of choice

mature protein nucleus DNA nucleotide growing polypeptide chain release of ribosome Transcription mRNA by RNA-polymerase tRNA

intron Translation of mRNA into protein tRNA loaded with exon amino acids mature mRNA

subunits of the ribosome ribosome starts reading of mRNA ribosome with growing cytltoplasm polypeptide chain

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.3 Transcription and translation: in the nucleus DNA is transcribed into mRNA, which is released into the cytoplasm – mRNA is then translated into protein.

1968) started decoding the genetic alphabet by identifying that the codon UUU on mRNA level was encoding the amino acid phenylalanine [16]. Now the mystery of transcription and even of translation was solved, and Watson and Crick, along with their colleague Maurice Wilkins (1916–2004), were awarded the Nobel Prize in Physiology or Medicine in 1962. Then in 1968 “gene scissors,” discovered by Werner Arber (1929–today; Nobel Prize in Physiology or Medicine in 1978), revolutionized molecular biology, since these restriction enzymes were capable of specifically cutting bacterial DNA with specific overhanging nucleotides [17]. These “sticky ends” enabled scientists for the first time to prepare recombinant DNA. Two years later the “central dogma” of biochemistry was proven wrong, namely that the genetic flow is unidirectional from DNA via mRNA to protein: Howard M. Temin (1934–1994; Nobel Prize in Physiology or Medicine in 1975) and David Baltimore (1938–today; Nobel Prize in Physiology or Medicine in 1975) discovered the viral enzyme reverse transcriptase [18] synthesizing cDNA from an mRNA template [19]. This breakthrough discovery eventually allowed the expression of eucaryotic genes, because the non-translated segments (introns) in the genome are spliced-out by this process yielding mature, completely coding templates for protein expression: from reading to writing the genetic code for mammalian gene expression (Figure 1.4). In 1971, the Protein Data Bank (PDB) was established at Brookhaven National Laboratory, New York, and became a repository for protein coordinates, which are 14j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

The enzyme reverse transcriptase uses mRNA as template to create cDNA. Using restriction enzymes and ligase, this insert can subsequently be cloned into a DNA expression vector

mammalian cleavage of insert ligase recombinant expression cell with restriction enzymes vector with mammalian gene

mRNA with gene coding combination of insert fthtifhifor the protein of choice and plasmid by ligase

isolation of mRNA cleavage of plasmid with restriction enzymes generation of cDNA by reverse transcriptase

introduction of expression vector into bacterial cell

plasmid bacterial cell plasmid production expression of mammalian gene in bacterial cells

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.4 Expression of eucaryotic genes: mammalian genes can be introduced into E. coli and functionally expressed by the bacterial protein synthesis machinery.

shared by scientists worldwide. PDB became a very important tool and the basis for rational, structure-based drug-design – a prerequisite for the development of modern (bio)pharmaceuticals [20] (see also Chapters 13, 15–19, 22, 23, 25), [21–49]. In 1973, a new era in biotechnology started with the advent of gene technology, when Allan Maxam (1942–today) and Walter Gilbert (1932–today) at Harvard [50] and Frederick Sanger (1918–today; Nobel Prize in Chemistry in 1958 for protein sequencing and the structural elucidation of insulin [51]) at Cambridge [52] independently developed a “DNA sequencing method.” For these efforts, Frederick Sanger and Walter Gilbert (but not his student Allan Maxam) were awarded the Nobel Prize in Chemistry in 1980. Combining all these fascinating ﬁndings, in 1973, Stanley N. Cohen (1935–today) and Herbert W. Boyer (1936–today) for the ﬁrst time recombined in vitro DNA pieces to a new gene [53].

1.4.3 Immortalized Cells: The Source of Monoclonal Antibodies

Atthesametime,GeorgesJ.F.Kohler€ (1946–1995) and Cesar Milstein (1927– 2002) worked together at the Medical Research Council Laboratory of Molecular Biology in Cambridge, where in 1975 they discovered a technique to produce monoclonal antibodies. Previously, to prepare substantial quantities of antibodies, 1.4 A Small Molecule from Bacteria – A Huge Importance for Mankind j15 scientists had to inject an antigen into an animal, wait for antibodies to form, draw blood from the animal, and isolate polyclonal (a mixture of different types of) antibodies. The only way to obtain monoclonal antibodies was to clone lymphocytes, secreting one single form of antibody molecules. Lymphocytes, however, are short-lived and cannot be cultivated easily. By fusing lymphocytes with myeloma cells (an accumulation of malfunctioning or “cancerous” cells that grow and multiply uncontrollably), Kohler€ and Milstein obtained hybrid cells synthesizing a single species of antibody while perpetuating themselves indeﬁnitely [54]. A great number of modern biopharmaceuticals [20,24] (i.e., therapeutic and diagnostic proteins) today are antibody-based molecules [22,23,33,34], and this is why the development of monoclonal antibodies revolutionized medicine and paved the way for new and target-speciﬁc approaches, where pure, uniform, and highly sensitive protein molecules are used as biopharmaceuticals for diagnosis and therapy. For their achievements, Kohler€ and Milstein were awarded the Nobel Prize in Physiology or Medicine in 1984.

1.4.4 Insulin: The First Biotech Blockbuster

The recombinant DNA technology of Cohen and Boyer [55,56] enabled them in 1978 1 to generate the first commercial product: human insulin (Humulin ) expressed in Escherichia coli. These efforts also led to the first biotech company: on October 15, 1980, “Genentech” went public on New York Wall Street. Fascination about this modern biopharmaceutical and the huge potential of the new biotechnology made the stock price jump from US$35–89 in the first twenty minutes: by the evening of the same day, the market capitalization was US$66 million! [57]. Insulin is a naturally occurring peptide hormone produced by the b cells in the islets of Langerhans of the pancreas in response to hyperglycemia. Insulin facilitates entry of glucose into target tissues – such as muscle, adipose tissue, and liver – via binding to membrane and “facilitated diffusion,” or activation of specific receptors on these cells (Figure 1.5). Malfunction leads to diabetes mellitus, a group of metabolic diseases character- ized by high blood sugar (glucose) levels, which result from defects in insulin secretion, or action, or both. In type 1 diabetes,thismaybeduetob-cell destruction and in type 2 diabetes to a combination of b-cell failure and resistance of target tissues to insulin action (insulin resistance). The latter disease can in its early stages be controlled by low-calorie nonsugar diet and/or treatment with oral antidiabetic drugs, while in the later stages and type 1 diabetes require insulin treatment. The World Health Organization (WHO) estimated that some 170 million people suffer from diabetes, a figure that is likely to double by 2030. Although only a minority of these sufferers actually require daily insulin injec- tions, the current world market for insulin is valued at in excess of US$10 billion. In this context, for example, for Novo Nordisk (one of the leading insulin producers), the insulin business is continuously growing and the company expects an overall growth of about 20% annually. 16j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

facilitated diffusion

porines Äußereinner membrane Membran

O Periplasmaperiplasm O22 respiratoryAtmungskette chain fdiffifreeFreie diff Diffusionusion Innereinner membrane Membran

The easiest kind of passive transport is free diffusion

– Water, O2, or CO2 can freely pass through the membrane Facilitated diffusion is enabled by two different membrane proteins – Transport proteins, for example permeases, facilitate the transport of molecules through the membrane by conformational changes (e.g., uptake of glucose from blood into blood cells) – Channel proteins, for example porines, are pores filled with water , which mediate molecules up to 600 Da to pass through the membrane

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.5 Membranes of a cell: free and facilitated diffusion. Besides free diffusion of O2, bigger molecules pass through the membrane by facilitated diffusion via permeases or porines.

In the first 60 years after the discovery of insulin by Frederick G. Banting (1891–1941; Nobel Prize in Physiology or Medicine in 1923) and Charles H. Best (1899–1978) in 1921 and the successful treatment of diabetics, only insulin extracted from bovine or porcine pancreas was available to treat type 1 diabetics [58]. Unfortunately, with the rapid increase in the incidence of diabetes, it was no longer possible to satisfy the pharmaceutical requirement (estimated to be more than 25 metric tons per year) from animal sources. Furthermore, the animal-extracted insulin is slightly different from human insulin, which might cause formation of anti-insulin antibodies and allergic reactions. Porcine insulin, which only deviates by a single amino acid in position B30 (last amino acid of the B chain; Figure 1.7) from human, can be converted to “authentically” human insulin in a trans-peptidation reaction, in which the alanine is replaced with a threonine. The developments in molecular biology and biotechnology opened up for new possibilities, among these, the biosynthesis of human insulin in E. coli. Insulin is composed of two disulfide-linked peptide chains referred to as the A-chain and B- chain, and the first recombinant approach used E. coli as host for the expression as fusion proteins. In a later approach in E. coli, pro-insulin (B-chain-connecting-C- peptide-A-chain) was expressed, also as a fusion protein. In both of these systems, the fusion proteins were isolated as inclusion bodies and several chemical steps were needed for dissolution, cleavage, folding, and formation of disulfide bridges (Figure 1.6). 1.4 A Small Molecule from Bacteria – A Huge Importance for Mankind j17

Usually transformation is achieved by introducing foreign plasmid DNA. In nature, these are vehicles, which are used to easily transfer resistance genes from one bacterium to another F usion B C A DNA coding for pro-insulin B-Kette I) expression

II) disrup tion C-Peptid cell with gene for pro-insulin III) purification native cell plasmid-DNA A-K ette

A-chain I) CNBr I) trypsin A-chain

II) sulfitolysis II) carboxy B-chain peptidase B B-chain III) O ; pH10,6 2 ACTIVE INSULIN pro-iliinsulin (authentical human)

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.6 Production of recombinant insulin: native cells are transformed with plasmid-DNA coding for pro-insulin, which is then expressed, isolated, and chemically altered.

Later, a single-chain insulin-precursor with a mini-C-peptide could successfully be produced (also containing the correct disulﬁde bridges) and secreted in the yeast S. cerevisiae. Eventually, other mini-C-peptide insulin precursors of human insulin, with minimal post-fermentation chemistry and puriﬁcation, could be achieved with the S. cerevisiae expression system [31]. The demand for a more optimal treatment of the patient has called for the design and development of new fast- and slow-acting insulin analogs and has required alterations of the molecule. For example, by switching the order of K and P at positions 28 and 29 in the B-chain into P and 1 K results in a fast-acting Insulin lispro (the same is true for mutating P at position 1 28 into D resulting in Insulin aspart , whereas fusion of two additional arginines 1 yields the slow-acting Insulin glargine (Figure 1.7)). 1 Another such example is Levemir from Novo Nordisk, an unusual long-acting insulin product that has just recently gained marketing approval. The major structural alteration characteristic of this insulin analog is the attachment of a

C14 fatty acid via the side chain of lysine residue number 29 of the insulin B-chain. This promotes binding of the insulin analog to albumin, both at the site of injection and in the plasma, which in turn leads to a constant and prolonged release of free insulin into the blood, resulting in a duration of action of up to 24 hours. After this short excursion on today’s biotech blockbusters, coming back to 1984, there was another hallmark discovery: the ﬁrst 3D structure of a transmembrane protein, the photosynthetic reaction center from Rhodopseudomonas viridis, was solved. A-chain 18 10 20 j

H N Biotech of Years Thousand Twenty 1 2 G I V E Q C C T S I C S L Y Q L E N Y C N COOH human insulin

10 20 30

H2N F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K T COOH B-chain “native”

10 20 H N 2 G I V E Q C C T S I CLYS L Y Q E N C N COOH Insulin lispro

10 20 30

H2N F V N Q HLC G S HLV E A LYLV C G E R G F F Y T K P T COOH

“fast” – From 10 20 H N 2 G I V E Q C C T S I CLYS L Y Q E N C N COOH IliInsulin aspart “ Traditional 10 20 30

H2N F V N Q HLC G S HLV E A LYLV C G E R G F F Y T D K T COOH ”

“fast” to “ 10 20 Biotechnology Modern H N 2 G I V E Q C C T S I CLYS L Y Q E N C G COOH Insulin glargine

10 20 30 COOH H2N F V N Q HLC G S HLV E A LYLV C G E R G F F Y T P K T R R “slow” ”

Global Pharma Specialists (GPS) From: “Modern Biopharmaceuticals ® ,” Knäblein 2005 www.get-gps.net

Figure 1.7 Engineered insulin variants with improved properties. The onset of insulin’s pharmacologically effect can be modulated by genetically engineered insulin variants: slow and fast acting. 1.5 Biopharmaceuticals – The Mainstay of Modern Biotechnology j19

The challenge in solving the structure of this huge (150 kDa) protein was that it consisted of 11 membrane-spanning, hydrophobic a-helices. Solving the 3D structure was a major breakthrough, since many of the most interesting drug targets are membrane-bound proteins [59]. Together with his colleagues Johann Deisenhofer (1943–today) and Hartmut Michel (1948–today), Robert Huber (1937–today), my PhD supervisor at the Max-Planck-Institute, was awarded the Nobel Prize for Chemistry in 1988.

1.4.5 Polymerase Chain Reaction: How to Infinitely Amplify DNA

Then there is the advent of a surprisingly simple tool that readily revolutionized molecular biology and heavily influenced modern biotechnology. In 1983, Kary B. Mullis (1944–today) invented a process he called polymerase chain reaction (PCR), which solved a core problem in molecular genetics, namely gene amplification. In other words, “How to make copies of a strand of DNA of interest?” PCR turns the job over to the very biomolecules that nature uses for copying DNA as well: two “primers” that flag the beginning and end of the DNA stretch to be copied and an enzyme, called polymerase, that walks along the segment of DNA, reading its code and assembling a copy. To complete the PCR cocktail, a pile of DNA building blocks are added, which the polymerase needs to make that DNA copy in vitro [60]. This process revolutionizes biotechnology and nowadays is essential for ample applications in modern biotechnology, for example, in diagnostic [46,61–65] and drug development [20,34,48,66–70]. Kary B. Mullis was awarded the Nobel Prize in Chemistry in 1993 for this discovery. Now we have recaptured some twenty thousand years of biotechnology – from the very early beginning when people manipulated nature in order to make the maximum use of it until today. Although the aim is still the same, the methods have changed dramatically. The main focus of today’s modern biotechnology is primarily to produce biopharmaceuticals “toward human good,” as stated by Nobel laureate James D. Watson in one of the author’s books: “The making of pharmaceutical and diagnostic agents in cells has moved from edge to the center of their respective commercial development. ‘Modern Biopharmaceuticals’ comprehensively describes the many novel ways cells so are being deployed toward human good” [71]. Some examples of modern biopharmaceuticals will be discussed next.

1.5 Biopharmaceuticals – The Mainstay of Modern Biotechnology

Biopharmaceuticals are currently the mainstay products of the biotechnology market and represent the fastest growing and, in many ways, the most exciting sector within the pharmaceutical industry. The term “biopharmaceutical” was coined in the 1980s, when a general consensus evolved that it represented a class of therapeutics produced by means of modern biotechnologies. As we have seen, 20j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

already a quarter of a century ago, Humulin (recombinant human insulin, produced in E. coli, and developed by Genentech in collaboration with Eli Lilly) was approved and received marketing authorization in the United States in 1982 [20] followed by products from Novo Nordisk, sanofi-aventis, and Biocon. As the production of insulin in bacteria had been already established in 40 000 l scale, the era of large- scale mammalian manufacturing still was to come [24]. In fact, mammalian cell culture got popular when highly glycosylated products 1 1 like tPA (tissue Plaminogen Activator), EPO (erythropoietin), and factor VIII [42] (see also Chapters 19 and 20) became attractive as therapeutics, because these molecules could not be produced in E. coli. In the 1990s, monoclonal antibodies as biopharmaceuticals were developed, for example, to treat several targets for rheumatoid arthritis and cancer. Following the aforementioned invention of Kohler€ and Milstein (which made monoclonal antibodies available as a pharmaceutical resource and allowed the production of the first monoclonal 1 antibody Orthoclone OKT3 from Johnson & Johnson in 1986), several other monoclonal antibody-based biopharmaceutical products have been developed and marketed. Although the OKT3 antibody had a murine structure, eight years later the first 1 chimeric antibody fragment ReoPro (Eli Lilly) was approved in 1994 followed by other 1 1 1 chimeric molecules, like Rituxan /Mabthera (Roche/Biogen Idec), Simulect 1 1 (Novartis), and Remicade (Johnson & Johnson). In 1997 Roche created Zenapax , the first humanized antibody, and successfully launched it in the market. With this 1 1 1 technology trend Synagis (Astra Zeneca), Herceptin (Roche) [34], Mylotarg 1 (Wyeth), Campath (Bayer), and many others were developed. Finally, the technology 1 for fully human antibodies was firstly introduced with Humira (Abbott) in 2002 and 1 1 1 meanwhile Vectibix (Amgen), Simponi (Johnson & Johnson), and Prolia (Amgen) also were on the market. These biopharmaceutical inventions target certain disease indications, for example, OKT3, Zenapax, and Simulect were developed for organ rejection prophy- laxis, whereas Rituxan was developed for cancer-related treatments and rheu- 1 matoid arthritis. The top six products Enbrel , Rituxan, Remicade, Humira, 1 Avastin , and Herceptin alone gained sales of more than US$30 billion in 2009. Whereas the first four mentioned are biopharmaceuticals to treat rheumatoid 1 arthritis, the latter products are dedicated for cancer indications. Rituxan, Zevalin , 1 and Bexxar target non-Hodgkin’s lymphoma, Campath chronic lymphatic leuke- 1 mia, Herceptin breast cancer, and Avastin, Erbitux , and Vectibix colorectal cancer. Currently 31 monoclonal antibodies are approved and marketed for therapeutic use [22,23,33,34]. To further enhance the bioavailability (especially for tumor penetration), also smaller human protein mimetics and artificial, non-antibody-binding proteins based on scaffolds have been invented [23,37,72]. These molecules can be produced in either bacteria or yeasts as alternative to mammalian cell culture. Today the market for biopharmaceuticals already represents 10–15% of the total global pharmaceutical market by value, with a total global sales value of US$86 billion in 2008, and is growing two to three times as fast annually. From 1982 to 1.5 Biopharmaceuticals – The Mainstay of Modern Biotechnology j21

2010, a total of 131 biopharmaceutical proteins, generated by recombinant DNA technology, were registered in the US and European markets (not counting the vaccines, blood products, and other biopharmaceuticals, which are of about the same number [73]). Looking at antibodies, 31 monoclonals have been developed and, although the molecular targets still seem to be limited, the biopharmaceutical market has grown with a compounded annual growth rate (CAGR) of 14% compared to only 4% for the pharmaceutical market. From 1998 to 2009, the market size has multiplied by almost a factor of eight from US$14 billion to 105 billion, and it is estimated that the market value will reach US$136 billion in 2015. Altogether, modern biopharmaceuticals such as Genentech’s human growth factor 1 1 Somatropin or Amgen’s EPO have shown that biopharmaceuticals can benefita huge number of patients, and also generate big profits for these companies at the 1 same time. The single most lucrative product is EPO , and combined sales of the 1 1 recombinant erythropoietin products Procrit (Ortho biotech) and Epogen (Amgen) have almost surpassed the US$10 billion mark. Biopharmaceuticals now represent an estimated 30% of the global pharmaceutical industry development pipeline. This growth is driven by considerable unmet medical need, as these complex products enable binding to specific targets in ways that are simply not possible with conventional medicines. The great potential of biopharmaceuticals is represented in the development pipeline: in 2009, there were 162 monoclonal antibodies, 102 other recombinant proteins, 122 biotechnologically produced vaccines, and 33 other molecules evaluated in clinical phases I–III.

1.5.1 Modern Biopharmaceuticals in Europe

With a focus on Europe, modern biotechnology and its applications generate almost 2% of the EU’s gross value added, indicating that its importance is comparable to Europe’s largest industry sector, and the European-dedicated biotechnology industry directly employs almost 100 000 people, mostly in small- and medium-sized enter- prises (SMEs) – however, given biotechnology’s “enabling effect,” employment in industries using biotechnology products is many times higher. Interestingly, revenues for biotech vaccines jumped from D65 million in 1996 to D259 million in 2009, and for example, in industrial biotech, the EU produces about 75% of the world’s enzymes, and 20% of the agro industry is related to biotech as well. Today, the majority of innovative medicine is made available by applying modern biotechnology to their development and/or manufacturing processes. Beginning with the technologies used in discovering the cause of a disease, to those used in diagnostic or therapy development – each and every step of today’s procedures is based on biotechnology. Biotechnology is no longer limited to the “omics”– genomics, proteomics, and metabolomics. It also provides and detects the receptors, antibodies, ligands, antagonists, hormones, and cytokines that keep our bodies in balance and protect us from harm. Hundreds of millions of patients have beneﬁted from approved biopharmaceuticals manufactured through biotechnology and gene 22j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

technology to treat or prevent heart attacks, stroke, multiple sclerosis, breast cancer, cystic ﬁbrosis, leukemia, hepatitis, diabetes, and other (also rare) diseases. “Modern biotechnology” continues to grow twice as fast annually as the traditional pharmaceutical industry. Today it is already seven times larger than it was 10 years ago. Currently, the leading classes of biopharmaceuticals are growth factors for blood cells. These are used in treating anemia, resulting from a variety of conditions including chronic kidney disease, chemotherapy, radiation treatments, as well as from other critical illnesses. For the ﬁrst time in the history of human healthcare, biotechnology is enabling the development and manufacture of biopharmaceuticals for a number of rare and very rare genetic diseases. In total, these illnesses affect some 20–30 million Europeans and their families. Biotechnology has a major impact on the provision of safe and effective vaccines against infectious diseases (see also Chapters 15 and 16) [35,74]. It also provides safer recombinant alternatives to proteins derived from human blood or tissue. Section 1.6 is largely based on the transcript of the opening speech at Biotechnica on October 4, 2010, in Hannover, Germany: “The Economic Impact of the Bio- technology Industry and its Potential to Transform the Pharma Industry” kindly provided by Peter Heinrich.

1.6 Transformation of the Pharma Industry Through Biotechnology

Biotechnology is setting the course of modern medicine. Biotech innovations help to reveal disease mechanisms, targets, and drug candidates. In addition, they improve advanced therapies, clinical trials, diagnosis, personalized medicine, and therapy monitoring by providing markers, models, platforms, tools, and IT. It is no secret that big pharma’s R&D engine needs a complete overhaul. Despite a number of bold efforts to bring big pharma’s R&D back to higher productivity levels, the pace of innovation remains anemic: the long-term average lags at one new molecular entity (NME) a year per company. Despite R&D spending at a high of 18% of revenues, big pharma’s R&D productivity declined by 20% between 2001 and 2007. The cost of bringing a new drug to market currently runs at more than US$2 billion. Personalized medicine is shifting the focus of medicine toward R&D and biotechnology companies in the interests of patients: before personalized medicine came in fashion, pharma’s focus along the value chain was in clinical development, regulatory affairs, reimbursement, and marketing – this is still the case. However, with the trend for more patient-tailored medicine, the value chain moved back toward R&D and technology transfer, areas that are the strengths of the biotech industry. This shift within the value chain will bring the biotech and pharma industries closer together. And the real question is, whether drug makers can cherry- pick enough biotech candidates to fill their new-product baskets or whether big pharma is just too big to deliver real innovation? In the 1980s, when big pharma produced blockbusters with much greater frequency, internal champions often led innovations. These leaders could rally 1.6 Transformation of the Pharma Industry Through Biotechnology j23 troops across functions and shift the focus of R&D efforts nimbly. Then, the industry’s quest for repeatability and efficiency, an approach focused on “throughput” and “risk mitigation,” began. Repeatable processes delivered a host of benefits for big pharma, for example, the industry found a steady source of revenue in marginally differentiating products and making them “evergreen” through extended releases or co-formulations. Unfortunately, innovation suffered in the process. Pharma companies learned that they were in need of a higher degree of medical differentiation to successfully introduce new products into the market and to meet the patients’ demand for better and affordable medication. This is not a new idea: in the 1990s, the pipeline for cancer treatments became crowded with pharma companies developing chemotherapies, mostly with little therapeutic difference. The difference came from the biotech industry: instead of becoming “me-too,” the biotech pioneer company Genentech concentrated on changing the way cancer was treated. They developed treatments based on humanized monoclonal antibodies – a technology that most pharma companies considered to be too complicated. Gen- entech’s researchers focused on understanding tumor biology and set goals to take patient outcomes to a new level. With its innovative approach, Genentech gained market leadership. In the following sections are some facts and indicators, which drive the transformation process of the pharma industry.

1.6.1 The Market as Motivation for Transformation

The top five pharma companies have lost 20% of their market value in the past five years, while biotechnology companies have gained 18%, as shown for the market capitalization of the following examples: Genentech (US$100 million) vs. Pfizer (US$98 million) Amgen (US$52 million) vs. BristolMeyersSquibb (US$42 million) Gilead (US$40 million) vs. Eli Lilly (US$37 million). Comparing the market value of the top five US-pharma companies with the entire market value of the biotech industry shows that they are almost the same: about US$290 billion. If the value of those biotech companies is added (which were acquired by pharma companies), the market value of the biotech industry would be about US$450 billion and the market capitalization of the top five pharma companies would be down to US$122 billion. As mentioned before, the biopharma market is growing at 14% CAGR compared to only 4% of the pharma industry. The global turnover of biopharmaceuticals has grown between 1998 and 2010 from US$20 billion to US$160 billion (Wood Mackenzie Product review) [21]. Faced with patent expirations, rising expenses, competition from generics (see also Chapter 18), and pressure on branded drug prices, big pharma’s revenue gap could balloon up to almost US$100 billion by 2014. For the 20 biopharma companies in the world, this represents an annual earnings decline of 8% (Bain & Company). 24j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

1.6.2 Innovations and Where They do Come From

Of the 25 compounds approved by the Food and Drug Administration (FDA) in 2009 – new biological entity (NBE) and new chemical entity (NCE) approvals without vaccines – only 8 new drug applications (NDAs) were discovered, developed, and ﬁled by big pharmaceutical companies (32%). The other 17 compounds (68%) were subject to alliances, partnerships, and licensing with biotechs, or to acquisition transactions. Innovations also require huge investments over a long period of time. According to Roche, to develop one drug, 1 million hours of work needs to be invested, with about 420 researchers involved, who have to conduct about 6500 experiments. This costs about US$1 billion of investments and takes at least 12 years. In other words, about 5000 human-years are required to get a drug developed. Looking at the global biotech industry ﬁnancing, biotech funding increased by 84% to US$62 billion in 2009 from just US$33 billion in 2008. Interestingly, the biotech funding derived from pharma partnerships unpropor- tionally increased over the last years and accounted for 60% of the biotech funding in 2009. This funding is expected to further increase and will have a positive impact on the biotech industry, which will further stimulate the pharma transformation.

1.6.3 Mergers and Acquisitions in the Biopharmaceutical Industry and the Impact on Innovation

With the innovation burden hanging heavily over the pharma industry, many pharma companies have started to experiment with new R&D models: Glaxo- SmithKline restructured its R&D centers to emulate biotech R&D principles and hopes to replicate an entrepreneurial culture in a large pharma organization. Eli Lilly acquired ImClone to in-source innovation from outside the company and then left it as a standalone unit operating independently, basically as Roche did with Genen- tech. Pfizer also has set-up a biotech incubator to foster their growth, and meanwhile, AstraZeneca has established a biotech focus, merging purchases Cambridge Antibody Technology and MedImmune into one “biologics powerhouse.” The history of the pharmaceutical industry is closely linked to such business practices of the combination and purchase of corporations. From the creation of the combined entity of Warner–Lambert in 1955, to the purchase of Warner–Lambert by Pfizer in 2000, to the recent acquisition of Wyeth, pharmaceutical and biotech companies have expanded their pipelines, portfolios, and sales forces through mergers and acquisitions (M&A). M&As have defined the landscape of the pharmaceutical and biotechnology industries, both historically and today. The strategic targeting behind corporate acquisitions, as well as mergers, is focused on intellec- tual property (IP), sales force efficiency, streamlining R&D, and reorganizing other key business areas. This M&A trend is obviously continuing (including hostile takeovers), because it seems that big pharmaceutical companies cannot effectively develop their 1.6 Transformation of the Pharma Industry Through Biotechnology j25 desperately needed new drugs on their own: they rather need to extend their pipelines with new and highly innovative biopharmaceuticals. Looking for the ongoing convergence of biotech and big pharma, such mega-acquisitions of mature biotechnology companies (as also nearly seen with Biogen Idec in 2007) is likely to continue. And in fact, in November 2010, sanofi-aventis and Genzyme were still at war: sanofi-aventis had told Genzyme to stand aside and let the shareholders decide on whether an acquisition should take place – finally, in February 2011, Genzyme was purchased for more than $20 billion. Another current change: Roche’s pipeline review could also set the stage for some new biotech companies to take shape. According to Bloomberg, the Swiss pharma giant intends to slim down its pipeline and venture capital (VC) companies are keenly interested in seeing if some of the most promising programs and scientific talent could be packaged and launched as independent companies. In fact, Roche has been assessing VC groups’ interest in financing startups. These new corporate spinouts are gaining considerable momentum on the global pharma industry. Earlier GlaxoSmithKline launched Convergence Pharmaceuticals with some impressive venture-backing and programs that no longer qualified as a core asset. And more startups like these are expected to take shape in coming months. It also would not be a first for Roche, which has successfully spun out assets in collaborative efforts to create Basilea and Actelion. While Bloomberg concentrates on the potential to expand the Swiss biotech scene with new players, there is no reason why Roche would not be interested in playing on a global field. The analysts point to Roche’s cancer and metabolism programs as two areas that would be ripe for a new company launch. Some companies like the UK-based Vernalis or big pharma–backed initiatives like Chorus have established virtual development as a viable and often more effective and efficient development model. Obviously, there is a clear trend from fully integrated pharma company (FIPCO) to a virtually integrated pharma company (VIPCO), outsourcing the different steps of the value chain to third parties, such as contract manufacturing organizations (CMOs), contract research organizations (CROs), contract service organizations (CSOs), and biotechs. Such companies do not just manage costs better by limiting full-time employees, reducing fixed assets, or clamping down on overheads. Rather, their flexibility and lean structures help them to quickly move on to the next promising idea – thus driving innovation. But also the patent situation is accelerating transformation of big pharma: expiring patents and competition from imitation products are increasing the pressure on the pharma industry (“bitter medicine for big pharma”), and it is especially struggling as patents for some best-selling drugs expire and price pressures rise in the United States and Europe due to clashing of the healthcare systems. A total of 40% of the patents from the 50 most important pharma products will expire within two years from 2011 – back in 2007 this was only 15% and in 2002 11%. For example, the Swiss drugmaker Novartis AG will have to deal with a number of 1 key drugs such as multi-billion dollar seller Diovan losing patent protection over the next few years. In November 2010, the Sunday newspaper Sonntag said Novartis 26j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

was planning a cost-cutting program similar to cross-town rival Roche Holding AG. Roche, the world’s biggest maker of cancer drugs, said it would slash 4800 jobs worldwide, hacking CHF2.4 billion (US$2.5 billion) from annual costs. One option to compensate for the patent expirations and cope with declining revenues could be biosimilars and biobetters (see also Chapter 18). Those could also develop into an option for biotechs; and biosimilars are only at the beginning [21] (see also Chapter 17). Interesting to note in this context is a very recent decision of the U.S. District Court for the Middle District of Louisiana, which is the latest jurisdiction to rule that brand manufacturers cannot be held responsible for injuries suffered by patients taking generic versions of their drugs. The court granted summary judgment to Wyeth and Schwarz Pharma in a case involving their gastroesophageal reflux 1 disease treatment Reglan (metoclopramide HCl). The plaintiff in the case took a generic version of Reglan from January 1998 to July 2009 and subsequently developed the neurological disorder tardive dyskinesia, which causes uncontrollable body movements, particularly around the face. This decision is obviously also true (and timely) for biosimilars, as within the next five years the first biopharmaceutical 1 1 blockbusters, including Aranesp , Enbrel, and Neulasta will go off-patent opening the market for biopharmaceutical follow-ons.

1.6.4 A Focus on the Opportunities of European Biotech Industry

The current situation for Europeans is like that of their US-based counterparts. European biotech companies demonstrated considerable resilience in the economic downturn. The number of public companies decreased by only 4%, from 179 companies in 2008 to 171 in 2009 — a much smaller drop than most industry watchers had expected. Revenues of publicly traded European companies grew from D11.0 billion in 2008 to D11.9 billion in 2009 — an 8% increase that was well below the 17% growth seen in 2008. While several of Europe’s leading companies — including Actelion, Crucell, Elan, QIAGEN, and Meda — continued to post double-digit revenue growth rates, UK- based Shire saw a signiﬁcant slowdown on its top line. This was largely the result of 1 the introduction of generic competitors to its blockbuster drug Adderal . Excluding Shire, Europe’s other large companies — those with revenues greater than D200 million — saw their combined top line expand by a robust 14%. However, smaller public companies below the D200 million threshold saw revenues decline by 1%, dragging down the overall sector’s performance. As in the United States, R&D expenditures failed to keep pace with revenue growth in Europe. European public companies’ R&D expenditures were essentially ﬂat, posting a modest 2% decrease in 2009. This was driven not by a few large companies, but rather by R&D cutbacks across much of the industry. Similar to the situation in the United States, close to 60% of public companies reduced their R&D expenditures in 2009. The cost-cutting helped to boost the sector’s net income by a remarkable 68%, as combined net loss fell from 1.7 Biopharmaceutical Production – Uncorking Bottlenecks or Wasting Surplus Capacity? j27

D913 million in 2008 to only D88 million in 2009. Innovations come from dynamic entrepreneurs. In the EU, 23 million SMEs stand for 99% of the European under- takings, 100 million jobs, and 60% of the gross domestic product (GDP). Of this, D625 million came from improvement on the bottom line and D147 million came from the decrease in public company count, since most of the companies that ceased operations or were acquired during the year were in a net loss position. Despite slowing revenue growth, Shire was able to deliver strong growth on the bottom line, and a number of other companies — including Genmab, Meda, Photomed, Q-Med, and QIAGEN — posted strong increases in net income. But again according to Bain & Company, for most pharma companies there is no real rescue in sight. Most companies will ﬁnd that even shopping for innovation externally cannot help to close the gap. A recent analysis of 6000 biotech projects available for late-stage licensing showed that only about 200 are likely candidates for a large pharma company. Of these, fewer than 100 show potential to become top- sellers. Taken together, they account for only about US$30 billion in potential revenue.

1.7 Biopharmaceutical Production – Uncorking Bottlenecks or Wasting Surplus Capacity?

The ripe and blooming market of biopharmaceuticals, on the one hand, is exciting, but, on the other, it became obvious that production capacities for biopharmaceuticals would become a bottleneck and that worldwide fermentation capacities are limited to produce all biopharmaceuticals needed. In the first years of this millennium, pharmaceutical companies were indeed competing for production slots, as illustrated in a seminal Nature Biotechnology article in 2002 [75]. Keeping in mind that the annual demand for a first-generation biopharmaceutical like, for example, Betaferon is 2 kg vs. 300 kg for, for example, a second-generation antibody like 1 Genentech’s Rituximab (factor of 100!) makes these capacity crunches even more obvious. As a result, a lot of efforts were spent on alternative expression systems, like transgenic animals [30] and plants [32,36,76–84]. For example, human anti-thrombin (AT) functions to keep blood from clotting in the veins and arteries of healthy individuals, but this is required in large amounts as a substitute for patients with hereditary AT deficiency to prevent life-threatening clots. Human anti-thrombin, 1 ATryn , is produced in transgenic goats and harvested from their milk. ATryn was approved in 2006 by the European Medicines Agency (EMA) for use in preventing clotting conditions during surgical procedures in patients with hereditary AT deficiency. In 2009, ATryn received also market authorization by the FDA and signifies the United States’ first approval for a biopharmaceutical produced in genetically engineered (GE) animals. But apart from this success, no other biopharmaceutical from either transgenic animals or plants ever made it to the market. Hence, the biopharmaceutical success story created the need for investments in much more (traditional) production capacity. The biggest volume drivers for 28j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

mammalian cell culture, in terms of sales and of production amounts, are monoclonal antibodies and derivatives thereof. With the evolution of the product portfolio, the need for commercial production capacities arose. One example is Amgen, which bought Enbrel innovator Immunex for US$16 billion. Amgen got the approval for their Rhode Island facility that was going to produce Enbrel in 2002. The facility housed eight 8000 l bioreactors, which was not sufficient to provide the market on the long run. Thus, they used additional capacity from a CMO that helped to serve the market demand. Having seen such an example of market dynamics, other big product company organizations (PCOs) decided to timely invest into manufacturing capacities on their own, or to acquire companies who possessed commercial manufacturing capacity (n.b., the life cycle of the products runs in parallel to the life cycle of manufacturing). Many companies have transformed from the stage of having no good manufacturing practice (GMP) capacity over having own clinical capacity, to even owners of commercial capacities (see also Chapter 23). PCOs who heavily invested in mammalian cell culture-manufacturing capacity besides Amgen were Genzyme Bayer, AstraZeneca with MedImmune, Merck KgaA with Serono, BMS, Genmab, Pfizer with Wyeth, Eli Lilly with Imclone, Merck Inc. with Schering- Plough, and Roche with Genentech. Roche and Pfizer already faced the situation of surplus capacities following their respective acquisitions, and as a consequence Roche closed the brand new 200 000 l facility in Vacaville, CA, USA. Pfizer recently announced the closure of their new Shanbally plant in Ireland. Following the life cycle of biopharmaceuticals, there will be a time point for each product when it will be off-patent in the major markets and the sales and production volume will decrease (see also Chapter 18). If the innovator company is not able to compensate the emerging gap, it will have spare capacity up to the dimension by which the facility gets commercially unprofitable. Facilities from PCOs lacking a timely pipeline with an adequate facility-fit will be offered for opportunistic contract manufacturing. Already classical PCOs, like Abbott, for example, have begun to offer GMP capacity. Most of the existing large-scale facilities are designed as mono-product plants and that often is an insurmountable hurdle in transferring approved products from one facility to another. Nevertheless, some PCO collaborations may occur. Not every company will find the right partner and in consequence there will be some facilities offered for sale. Genmab, for example, announced the intention to sell their Brooklyn Park facility [85]. Awaiting a wave of biosimilars between 2013 and 2018, the PCOs have the chance to enter this business segment and fill their capacities according to their capabilities and success, or to outsource the remaining production. CMOs can act as suitable partners for PCOs as their facilities are designed for multi-product use and they always try to maximize the capacity utilization. By nature of their business they possess the right technologies and capabilities. The currently existing capacity will compete with future trends of higher overall yields and thus building smaller scale bioreactors or using disposable technologies according to the fragmentation of certain indications and the titer improvements that the market has frequently seen with product titers of more than 5 g/l [24,86]. 1.8 Conclusion and Outlook j29

In the future, further improvements in titer and downstream yield will be seen that lessen the need for building more capacity [87]. Up to 2009, the PCOs held about 1.85 million liters of commercial mammalian cell culture-manufacturing capacity and therewith 81% of the total capacity of bioreactors that were equal or bigger than 8000 l. CMOs such as Boehringer Ingelheim, Lonza, Celltrion, Diosynth, and Sandoz had a total volume of 422 000 l. In 2003, Boehringer Ingelheim invested more than US$330 million (D255 million) in their large-scale plant and thereby doubled the Biberach capacity in Germany, now being the market leader with 180 000 l (since Lonza sold their 80 000 l Singapore facility to Genentech) (see also Chapter 23). Lonza already has invested in another 80 000 l facility, close to the first one. However, the total CMO capacity will decline to 380 000 l in 2014 in this forecast as Celltrion decided to use more than 80% of their capacity for their biosimilars, and Diosynth has been acquired by the PCO Schering-Plugh (Merck Inc.) keeping their large-scale capacity in Oss (Netherlands) internally. Also the remaining available capacity from Sandoz is not clear, as Sandoz seems to predominantly operate in the biosimilars area (see also Chapter 18). The uptake of Celltrion, Diosynth, and Sandoz into the PCO league lets the total PCO capacity swell to 2.44 billion liters and leaves the CMO playing field to Lonza and Boehringer Ingelheim. Altogether, the FDA approved 44 biopharmaceuticals since 2002, with 32% of them currently manufactured by CMOs. But also the capacity of the CMOs is not unlimited, and building stainless steel bioreactors takes 5 years, and investment volumes of several hundred million US dollars for a green-field plant from engineering to process qualification [88]. Currently, it looks like that the global production capacity for biopharmaceuticals is sufficient, but this can change immediately if new and quantity-demanding treatment paradigms arise on the horizon.

1.8 Conclusion and Outlook

“Men love to wonder, and that is the seed of science” (Ralph Waldo Emerson, 1803– 1882) – this is basically the driving force and enabler for the continuous advance- ment of biotechnologies over the last twenty thousand years. People manipulate nature in order to make the maximum use of it – this started already eighteen thousand years before Christ, but still holds true for today. The difference though today is that we need to safeguard to use nature, but not to abuse our planet. Very early people in the Middle East had successfully domesticated sheep and deer, and later also pigs by the Chinese. At the same time, the Sumerians in Mesopotamia were capable of brewing beer, and in some years almost half of the entire wheat harvest was used for brewing the typical beer (Kasch or Bufa). Egyptians produced wine (Irep), and the Babylonians were able to brew 20 different types of beer. Speciﬁc fermentation processes were established in areas in which all required ingredients were available: wheat beer in Middle Europe, rice wine and rice 30j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

liquor sake in East Asia, kvass in Russia, pombe in South America and Central Africa, and pulque by the Aztecs in Middle America. People having cows produced yogurt, kefir, and cheese, and the Sumerians even developed specific fermentation processes to selectively produce certain different kinds of cheese – Roquefort was shipped to Rome as a “Gallic specialty.” In the sixth century, monks developed beer brewing into a stable and reproducible fermentation process, which leads to a kind of “blockbuster of traditional biotechnology,” keeping in mind that worldwide beer is still the top selling biotech product with a yearly consumption of approximately 1.5 billion hectoliters worth more than D50 billion. Later, at the beginning of the last millennium, “traditional” biotechnologies were developed in order to produce “high value traits” such as whiskey, sauerkraut, and different types of yogurt; Antonie van Leeuwenhoek makes the respective microbes visible using his self-made microscope with 200 magnification. Charles Darwin published his revolutionary theories on the principle of natural selection, Louis Pasteur converted alcohol into vinegar, Johann Gregor Mendel showed that phenotypic characteristics can be transferred from one generation to the next, Johann Friedrich Miescher stained the “nucleus” of cells, and Wilhelm Johannsen coined the terms “genotype” and “genes.” Michaelis and Menten postulated the existence of an ES complex and defined the saturation effect, which subsequently revolutionized fermentation processes to produce the required amounts of vital and lifesaving substances on a large scale. For example, the production of Penicillin (initially discovered by Alexander Fleming with yields of only 2 mg/ml Penicillin of culture broth) can be improved with the help of Ernst Boris Chain and Howard Florey, based on optimized fermentation processes with feeding strategies (Michaelis–Menten kinetic), and mutated strains (evolution in the Petri dish), and finally lead to a production rate of 1.5 mg/ml Penicillin – from the initial 2 mg/ml this is a factor of 750. On March 1, 1944, the first large-scale Penicillin fermentation was run and further technological improvements led to 90% recovery in a 30 000 l reactor (rather than 1% in a 1 l flask) increasing the yearly produced doses from 210 million to 6.8 trillion, which enabled to save the life of up to 1500 people during the war period, and subsequently, on D-day innumerable victims who were wounded could fortunately now be treated with Penicillin. Nowadays optimized fermentation processes, using genetically optimized high-producing strains, are capable of yielding 20 000 times more Penicillin in 1 l of culture broth compared to what Fleming’s initial P. notatum did! Then, another very striking example of biotechnological development is the large- scale production of vitamin C (L-ascorbic acid). Reichstein was able to chemically synthesize vitamin C for the first time, when in more than ten steps he converted glucose into L-xylose followed by addition of hydrocyanic acid to form L-ascorbic acid. Later, Reichstein and colleagues improved the synthesis by using sorbose as intermediate. This was gained with 100% yield by the reduction of glucose with hydrogen at a pressure of 150 atm applying a nickel catalyst. Using bacteria, Reichstein used the enzyme sorbitol-dehydrogenase to stereo-selectively convert D-sorbit into L-sorbose followed by chemical oxidation into 2-KLG, and subsequent water cleavage through acid treatment into L-ascorbic acid, vitamin C. 1.8 Conclusion and Outlook j31

Other intriguing experiments follow: combining two smart genetic experiments, Griffith and Avery identified DNA as “transforming principle and carrier of genetic information,” Watson and Crick elucidated the structure of “the molecule of life,” DNA, and Arber isolated restriction enzymes capable of specifically cutting bacterial DNA with specific overhanging nucleotides. Mullis invented the PCR, which solved a core problem in molecular genetics, namely gene amplification. With this biotechnology, it is now possible to indefinitely create copies of a certain DNA strand, using two “primers” that flag the beginning and end of the DNA stretch of interest. Temin and Baltimore discovered the viral enzyme reverse transcriptase, synthesizing cDNA from an mRNA template. This breakthrough discovery eventually allowed the expression of eucaryotic genes, because the non-translated segments (introns) in the genome are spliced-out by this process yielding mature, completely coding templates for protein expression. Finally, Cohen and Boyer for the first time recombined in vitro DNA pieces to a new gene. Using these new biotechnologies, all of a sudden, it became possible to use bacteria of the species Erwinia to subsequently convert D-glucose into 2,5-DKG, which in turn can serve as a substrate for Corynebacterium spec. to produce 2-KLG using its 2,5-DKG-reductase (acid treatment is used to transform it into vitamin C). Metabolic engineering yields a recombinant artificial Erwinia hybrid-strain, which produces in only 120 hours an impressive 120 g 2-KLG per liter fermentation broth with a yield of more than 60%. The manufacturing costs could be reduced by a factor of 50 and in the meantime more than 80,000 t vitamin C are produced every year worth more than US$600 million! The recombinant DNA technology of Cohen and Boyer enables them also to generate the first commercial product: human insulin (Humulin) expressed in E. coli. Although, in the first 60 years after the discovery of insulin by Banting and Best in 1921, successful treatment of diabetics could only be achieved with insulin extracted from bovine or porcine pancreases, porcine insulin could now be converted to “authentically” human insulin in a trans-peptidation reaction. But recombinant biotechnologies now opened up for new possibilities, among these, the biosynthesis of human insulin in E. coli: single-chain insulin-precursor with a mini-C-peptide could successfully be produced (also containing the correct disulfide-bridges) and secreted in the yeast S. cerevisiae. With minimal post-fermentation chemistry (and purification), fast-acting Insulin lispro, Insulin aspart, slow-acting Insulin glargine, and long-acting Levemir could be created. Kohler€ and Milstein discovered a technique to produce monoclonal antibodies, and in fact, a great number of modern biopharmaceuticals (i.e., therapeutic and diagnostic proteins) today are antibody-based (and hence target-specific) molecules, with the first monoclonal antibody OKT3 being marketed in 1986. Although this antibody had a murine structure, eight years later the first chimeric antibody fragment ReoPro was approved in 1994 followed by other chimeric molecules, like Rituxan/ Mabthera, Simulect, and Remicade. In 1997, Zenapax was the first humanized antibody, followed by Synagis, Herceptin, Mylotarg, and Campath. Finally, the technology for fully human antibodies was firstly introduced with Humira in 2002 and meanwhile Vectibix, Simponi, and Prolia were on the market as well. 32j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

As mentioned before, currently 31 monoclonal antibodies are approved and marketed for therapeutic use, and the top six products Enbrel, Rituxan, Remicade, Humira, Avastin, and Herceptin alone gained sales of more than US$30 billion in 2009. To further enhance the bioavailability (especially for tumor penetration), also smaller human protein mimetics and artificial, non-antibody-binding proteins based on scaffolds, have been invented. The great potential is represented with an impressive 162 monoclonal antibodies in the development pipeline (in clinical phases I–III). Altogether, today the market for biopharmaceuticals already represents up to 15% of the total global pharmaceutical market by value (and an estimated 30% of its development pipeline) with a total global sales value of US$86 billion in 2008. From 1982 to 2010, a total of 131 biopharmaceutical proteins (including monoclonal antibodies) were registered in the US and European markets with a CAGR of 14% compared to only 4% for the pharmaceutical market. From 1998 to 2009, the market size has multiplied by almost a factor of eight from US$14 billion to 105 billion, and it is estimated that the market value will reach US$136 billion in 1 2015. Modern biopharmaceuticals such as Somatropin or EPO have shown that biopharmaceuticals can benefit a huge number of patients, and also generate big profits for these companies at the same time. The single most lucrative product is 1 EPO , and combined sales of the recombinant erythropoietin products Procrit 1 and Epogen have almost surpassed the US$10 billion mark. At the same time though, it has also become obvious that big pharma’s R&D needs to be transformed to higher productivity levels again, because despite 18% spending, R&D productivity declined by 20% due to a lack of in-house innovations. Strikingly, the entire pharma market is only growing at 4% CAGR, whereas biopharma grows at 14% – hence, now the focus is on in-licensing. But there is no real rescue and most companies will find that even shopping for innovation externally cannot help close the gap, since a recent analysis of 6000 biotech projects available for late-stage licensing showed that only about 200 are likely candidates for big pharma. Of these, fewer than 100 showed potential to become top-sellers, and that they account for only about US$30 billion in potential revenue. A total of 25 compounds were approved in 2009, of those, only 8 were discovered, developed, and filed by big pharmaceutical companies alone. The rest (17 compounds) came from alliances, partnerships, licensing with biotechs, or acquisition transactions. Many pharma companies now have new R&D models to emulate biotech principles, for example, acquiring complete biotechs and leaving them as a stand-alone unit operating independently. Another obvious trend to participate from external know-how and to share risks is the transformation from FIPCOs to VIPCOs. Since outsourcing continues to swell, the different steps of the value chain are contracted to third parties, such as CMOs, CROs, CSOs, and biotechs. Also the patent situation is accelerating the desperately needed transformation of the pharma industry: expiring patents and competition from imitation products are increasing. Currently, 40% of the patents from the 50 most important pharma products are expiring. Back in 2007 this was only 15%, and in 2002 it was only 11%. References j33

Within the next five years the first biopharmaceutical blockbusters, including Aranesp, Enbrel, and Neulasta, will go off-patent. One option to compensate for the patent expirations and the declining revenues could in fact be biosimilars and biobetters, which again brings big pharma and biotechs closer together. This would guarantee that, in the future, we will have innovative modern biopharmaceuticals that in many novel ways are being deployed toward human good. Having said that, we must support innovative technologies, further expand its applications, and use the impetus that modern biotechnology gives us, leading to better lives and a sustainable economy. In the words of Max Planck (1858–1947) “How far advanced Man’s scientific knowledge may be, when confronted with Nature’s immeasurable richness and capacity for constant renewal, he will be like a marveling child and must always be prepared for new surprises,” we will definitely discover more fascinating biotechnologies. In fact, this should be our focus, because, then, at the dawn of the new millennium, for the first time we could yield large-enough amounts of biopharmaceuticals to treat everybody on our planet!

References

1 van Leeuwenhoek, A. (1684) Some 10 Avery, O.T. and Dubos, R. (1930) The specific microscopical observations about animals action of a bacterial enzyme on pneumococci in the scurf of the teeth. Phil. Trans. Roy. of Type III. Science, 72, 151–152. Soc. London, 14, 568–574. 11 Watson, J.D. and Crick, F.H. (1953) 2 Darwin, C. (1859) On the Origin of Species, Molecular structure of nucleic acids; a 1st ed, John Murray, London. structure for deoxyribose nucleic acid. 3 Mendel, G. (1866) Versuche uber€ Pflanzen- Nature, 171, 737–738. Hybriden. Verhandlungen des 12 Kornberg, A., Kornberg, S.R., Simms, E.S., naturforschenden Vereines. Abhandlungen, et al. (1956) Metaphosphate synthesis by an Brunn€ ,4,3–47. enzyme from Escherichia coli. Biochim. 4 Michaelis, L. and Menten, M.L. (1913) Biophys. Acta, 20, 215–227. Kinetik der Invertinwirkung. Biochem. Z., 13 Kendrew, J.C., Bodo, G., Dintzis, H.M., 49, 333–369. Parrish, R.G., Wyckoff, H., and Phillips, 5 Fleming, A. (1929) On the antibacterial D.C. (1958) A three-dimensional model of action of cultures of a penicillium with the myoglobin molecule obtained by X-ray special reference to their use in the analysis. Nature, 181, 662–666. isolation of B. influenzae. Brit. J. Exp. 14 Perutz, M.F., Rossmann, M.G., Cullis, Pathol., 10, 226–236. A.F., Muirhead, H., Will, G., and North, 6 Reichstein, T. (1933) Synthesis of d- and A.C. (1960) A three-dimensional Fourier l-ascorbic acids (vitamin C). Helv. Chim. synthesis of reduced human haemoglobin Acta, 16, 1019. at 5.5 A resolution. Nature, 185, 416–422. 7 Reichstein, T., Grussner,€ A., and 15 Jacob, F., Perrin, D., Sanchez, C., and Oppenauer, R. (1933) Die synthese der D- Monod, J. (1960) Operon: a group of genes ascorbins€aure (D-Form des C-vitamins). with the expression coordinated by an Helv. Chim. Acta, 16, 561–565. operator. C.R. Hebd. Seances Acad. Sci., 250, 8 Reichstein, T. and Grussner,€ A. (1934) Eine 1727–1729. ergiebige synthese der L-ascorbins€aure 16 Nirenberg, M.W., Matthaei, J.H., and (C-Vitamin). Helv. Chim. Acta, 17, 311–328. Jones, O.W. (1962) An intermediate in the 9 Griffith, F. (1928) The significance of biosynthesis of polyphenylalanine directed pneumococcal types. J. Hygiene, 27, by synthetic template RNA. Proc. Natl. 113–159. Acad. Sci. USA, 48, 104–109. 34j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

17 Linn, S. and Arber, W. (1968) Host Kn€ablein), Wiley-VCH Verlag GmbH, speciﬁcity of DNA produced by Escherichia Weinheim, pp. 1021–1031. coli. In vitro restriction of phage fd 26 See also Weber, W. and Fussenegger, M. replicative form. Proc. Natl. Acad. Sci. USA, (2005) Baculovirus-based production of 59, 1300–1306. biopharmaceuticals using insect cell 18 Baltimore, D. (1970) RNA-dependent DNA culture processes, in Modern polymerase in virions of RNA tumour Biopharmaceuticals – Design, Development viruses. Nature, 226, 1209–1211. and Optimization, vol. 3, part IV (ed. 19 Temin, H.M. and Mizutani, S. (1970) RNA- J. Kn€ablein), Wiley-VCH Verlag GmbH, dependent DNA polymerase in virions Weinheim, pp. 1045–1058. of Rous sarcoma virus. Nature, 226, 27 See also Rose, T. (2005) Alternative 1211–1213. strategies and new cell lines for high-level 20 See also Walsh, G. (2005) Current status of production of biopharmaceuticals, in modern biopharmaceuticals: approved Modern Biopharmaceuticals – Design, products and trends in approvals, in Development and Optimization, vol. 3, part Modern Biopharmaceuticals – Design, IV (ed. J. Kn€ablein), Wiley-VCH Verlag Development and Optimization, vol. 1 GmbH, Weinheim, pp. 761–776. (ed. J. Kn€ablein), Wiley-VCH Verlag 28 See also Yallop, C., Crowley, J., Cote, J., GmbH, Weinheim, pp. 1–34. Hegmans-Brouwer, K., Lagerwerf, F., 21 See also Moscho, A., Sch€afer, M.A., and Gagne,R.,Martin,J.C.,Oosterhuis,N., Yarema, K. (2005) Healthcare trends and andOpstelten,D.-J.,andBout,A. 1 their impact on the biopharmaceutical (2005) PER.C6 Cells for the industry: Biopharmaceuticals come of manufacture of biopharmaceutical age, in Modern Biopharmaceuticals – proteins, in Modern Biopharmaceuticals – Design, Development and Optimization, Design, Development and Optimization, vol. 4, part VIII (ed. J. Kn€ablein), Wiley- vol. 3, part IV (ed. J. Kn€ablein), Wiley-VCH VCH Verlag GmbH, Weinheim, Verlag GmbH, Weinheim, pp. 1711–1738. pp. 780–803. 22 See also Gottschalk, U. and Mundt, K. 29 See also Birch, J.R., Mainwaring, D.O., and (2005) Thirty years of monoclonal Racher, A.J. (2005) Use of the glutamine antibodies: a long way to pharmaceutical synthetase (GS) expression system for the and commercial success, in Modern rapid development of highly productive Biopharmaceuticals – Design, Development mammalian cell processes, in Modern and Optimization, vol. 3, part V (ed. Biopharmaceuticals – Design, Development J. Kn€ablein), Wiley-VCH Verlag GmbH, and Optimization, vol. 3, part IV (ed. Weinheim, pp. 1105–1145. J. Kn€ablein), Wiley-VCH Verlag GmbH, 23 See also Moroney, S. and Pluckthun,€ A. Weinheim, pp. 809–830. (2005) Modern antibody technology: the 30 See also Echelard, Y. (2005) The ﬁrst impact on drug development, in Modern biopharmaceutical from transgenic 1 Biopharmaceuticals – Design, Development animals: ATryn ,inModern and Optimization, vol. 3, part V (ed. J. Biopharmaceuticals – Design, Development Kn€ablein), Wiley-VCH Verlag GmbH, and Optimization, vol. 3, part IV (ed. Weinheim, pp. 1147–1186. J. Kn€ablein), Wiley-VCH Verlag GmbH, 24 See also Wurm, F. (2005) Manufacture of Weinheim, pp. 995–1016. recombinant Biopharmaceuticals, in 31 See also Andersen, A.S. and Diers, I. Modern Biopharmaceuticals – Design, (2005) Advanced expression of Development and Optimization, vol. 3, part biopharmaceuticals at industrial scale, in IV (ed. J. Kn€ablein), Wiley-VCH Verlag Modern Biopharmaceuticals – Design, GmbH, Weinheim, pp. 723–759. Development and Optimization, vol. 3, part 25 See also Apeler, H. (2005) Producing IV (ed. J. Kn€ablein), Wiley-VCH Verlag modern biopharmaceuticals, in Modern GmbH, Weinheim, pp. 1033–1042. Biopharmaceuticals – Design, Development 32 See also Klimyuk, V., Marillonnet, S., and Optimization, vol. 3, part IV (ed. J. Kn€ablein, J., McCaman, M., and Gleba, References j35

Y. (2005) Production of recombinant vol. 4, part VI (ed. J. Kn€ablein), Wiley-VCH proteins in plants, in Modern Verlag GmbH, Weinheim, pp. 1445–1460. Biopharmaceuticals – Design, Development 40 See also Brorson, K., et al. (2005) and Optimization,vol.3,partIV(ed. Consideration for developing J. Kn€ablein), Wiley-VCH Verlag GmbH, biopharmaceuticals: FDA perspective, in Weinheim, pp. 893–909. Modern Biopharmaceuticals – Design, 33 See also Hempel, J.C. and Hess, P.H. Development and Optimization, vol. 4, part (2005) Contract manufacturing of VII (ed. J. Kn€ablein), Wiley-VCH Verlag biopharmaceuticals including antibodies GmbH, Weinheim, pp. 1637–1667. or antibody fragments, in Modern 41 See also Harris, J. (2005) G-CSF and Biopharmaceuticals – Design, Development bioequivalence: the emergence of health and Optimization, vol. 3, part IV (ed. economics, in Modern Biopharmaceuticals – J. Kn€ablein), Wiley-VCH Verlag GmbH, Design, Development and Optimization, Weinheim, pp. 1083–1142. vol. 4, part VIII (ed. J. Kn€ablein), 34 See also Gutjahr, T. (2005) The Wiley-VCH Verlag GmbH, Weinheim, 1 development of Herceptin : paving the pp. 1755–1769. way for individualized cancer therapy, in 42 See also Riedel, N. and Dorner, F. (2005) A Modern Biopharmaceuticals – Design, new technology standard for safety and Development and Optimization, vol. 1, part I efﬁcacy in factor VIII replacement therapy: (ed. J. Kn€ablein), Wiley-VCH Verlag designing an advanced category rFVIII GmbH, Weinheim, pp. 127–149. concentrate, in Modern Biopharmaceuticals 35 See also Vazquez, F.L. and Oya, R. (2005) – Design, Development and Optimization, AIDS gene therapy: a vector selectively able vol. 2, part II (ed. J. Kn€ablein), Wiley-VCH to destroy latently HIV-1-infected cells, in Verlag GmbH, Weinheim, pp. 419–447. Modern Biopharmaceuticals – Design, 43 See also Moscho, A., Sch€afer, M.A., and Development and Optimization, vol. 2, part Yarema, K. (2005) Healthcare trends and II (ed. J. Kn€ablein), Wiley-VCH Verlag their impact on the biopharmaceutical GmbH, Weinheim, pp. 549–559. industry: biopharmaceuticals come of age, 36 See also Gorr, G. and Wagner, S. (2005) in Modern Biopharmaceuticals – Design, Humanized glycosylation: production of Development and Optimization, vol. 4, part biopharmaceuticals in a moss bioreactor, VIII (ed. J. Kn€ablein), Wiley-VCH Verlag in Modern Biopharmaceuticals – Design, GmbH, Weinheim, pp. 1711–1739. Development and Optimization, vol. 3, part 44 See also Rarbach, M., et al. (2005) IV (ed. J. Kn€ablein), Wiley-VCH Verlag Design of modern biopharmaceuticals by GmbH, Weinheim, pp. 919–927. ultra-high-throughput screening and 37 See also Silacci, M. and Neri, D. (2005) directed evolution, in Modern Ligand-based targeting of disease: from Biopharmaceuticals – Design, Development antibodies to small organic (synthetic) and Optimization,vol.2,partIII(ed. ligands, in Modern Biopharmaceuticals – J. Kn€ablein), Wiley-VCH Verlag GmbH, Design, Development and Optimization, Weinheim, pp. 583–603. vol. 3, part V (ed. J. Kn€ablein), Wiley-VCH 45 See also Reilly, R.M. (2005) Verlag GmbH, Weinheim, pp. 1271–1289. Biopharmaceuticals as targeting 38 See also Wenzel, A.F. and Sonnega, C.E.A. vehicles for in situ radiotherapy of (2005) The regulatory environment for malignancies, in Modern biopharmaceuticals in the EU, in Modern Biopharmaceuticals – Design, Biopharmaceuticals – Design, Development Development and Optimization,vol.2, and Optimization, vol. 4, part VII (ed. part II (ed. J. Kn€ablein), Wiley-VCH J. Kn€ablein), Wiley-VCH Verlag GmbH, Verlag GmbH, Weinheim, pp. 497–526. Weinheim, pp. 1669–1701. 46 See also Bagowski, C.P. (2005) Target 39 See also Modi, P. (2005) The evolving role validation: an important early step in the of oral spray insulin in the treatment of development of novel biopharmaceuticals diabetes, in Modern Biopharmaceuticals – in the post-genomic era, in Modern Design, Development and Optimization, Biopharmaceuticals – Design, Development 36j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

and Optimization, vol. 2, part III (ed. 56 Bolivar, F., Rodriguez, R.L., Greene, P.J., J. Kn€ablein), Wiley-VCH Verlag GmbH, Betlach, M.C., Heyneker, H.L., Boyer, Weinheim, pp. 621–645. H.W., Crosa, J.H., and Falkow, S. 47 See also McCaman, M., et al. (2005) (1977) Construction and characterization Adenovirus-based gene therapy: of new cloning vehicles. II. A therapeutic angiogenesis with adenovirus 5 multipurpose cloning system. Gene,2, fibroblast growth factor-4 (Ad5FGF-4) in 95–113. patients with chronic myocardial ischemia, 57 Dickson, D. (1980) Genentech makes in Modern Biopharmaceuticals – Design, splash on Wall Street. Nature, 287, Development and Optimization, vol. 1, part I 669–670. (ed. J. Kn€ablein), Wiley-VCH Verlag 58 Banting, F.G., Best, C.H., Collip, J.B., GmbH, Weinheim, pp. 151–178. Campbell, W.R., and Fletcher, A.A. (1922) 48 See also Schmidt, M., Volz, B., and Pancreatic extracts in the treatment of Wittig, B. (2005) MIDGE vectors and diabetes mellitus. Can. Med. Assoc. J., 12, dSLIM immunomodulators: DNA-based 141–146. molecules for gene therapeutic strategies, 59 Deisenhofer, J., Epp, O., Miki, K., in Modern Biopharmaceuticals – Design, Huber, R., and Michel, H. (1984) X-ray Development and Optimization, vol. 1, part I structure analysis of a membrane protein (ed. J. Kn€ablein), Wiley-VCH Verlag complex. Electron density map at 3 A GmbH, Weinheim, pp. 183–198. resolution and a model of the 49 See also Szymanski, M., Barciszewski, J., chromophores of the photosynthetic and Erdmann, V.A. (2005) Nonprotein- reaction center from Rhodopseudomonas coding RNAs and their potential as viridis. J. Mol. Biol., 180, 385–398. biopharmaceuticals, in Modern 60 Saiki, R.K., Scharf, S., Faloona, F., Mullis, Biopharmaceuticals – Design, Development K.B., Horn, G.T., Erlich, H.A., and and Optimization, vol. 1, part I (ed. Arnheim, N. (1985) Enzymatic J. Kn€ablein), Wiley-VCH Verlag GmbH, amplification of beta-globin genomic Weinheim, pp. 213–223. sequences and restriction site analysis for 50 Gilbert, W. and Maxam, A. (1973) The diagnosis of sickle cell anemia. Science, nucleotide sequence of the lac operator. 230, 1350–1354. Proc. Natl. Acad. Sci. USA, 70, 3581–3584. 61 See also Cech, T.R. (2005) Beginning to 51 Sanger, F. (1945) The free amino groups of understand the end of the chromosome, in insulin. Biochem. J., 39, 507–515. Modern Biopharmaceuticals – Design, 52 Sanger, F., Donelson, J.E., Coulson, A.R., Development and Optimization, vol. 1 Kossel,€ H., and Fischer, D. (1973) Use of (ed. J. Kn€ablein), Wiley-VCH Verlag DNA polymerase I primed by a synthetic GmbH, Weinheim, pp. 37–45. oligonucleotide to determine a nucleotide 62 See also Geistlinger, J. and Ahnert, P. sequence in phage fl DNA. Proc. Natl. (2005) Large-scale detection of genetic Acad. Sci. USA, 70, 1209–1213. variations – The key to personalized 53 Cohen, S.N., Chang, A.C., Boyer, H.W., medicine, in Modern Biopharmaceuticals – and Helling, R.B. (1973) Construction of Design, Development and Optimization, biologically functional bacterial plasmids vol. 1, part I (ed. J. Kn€ablein), Wiley-VCH in vitro. Proc. Natl. Acad. Sci. USA, 70, Verlag GmbH, Weinheim, 3240–3244. pp. 71–93. 54 Kohler,€ G. and Milstein, C. (1975) 63 See also Gould Rothberg, B.E., Pena,~ C.E. Continuous cultures of fused cells A., and Rothberg, J.M. (2005) A systems secreting antibody of predefined specificity. biology approach to target identification Nature, 256, 495–497. and validation for human chronic 55 Bolivar, F., Rodriguez, R.L., Betlach, M.C., disease, in Modern Biopharmaceuticals – and Boyer, H.W. (1977) Construction and Design, Development and Optimization, characterization of new cloning vehicles. I. vol. 1, part I (ed. J. Kn€ablein), Ampicillin-resistant derivatives of the Wiley-VCH Verlag GmbH, Weinheim, plasmid pMB9. Gene,2,75–93. pp. 99–123. References j37

64 See also Hildebrand, M. (2005) Analytics in (ed. J. Kn€ablein), Wiley-VCH Verlag quality control and in vivo,inModern GmbH, Weinheim, ISBN 3-527-31184-X. Biopharmaceuticals – Design, Development 72 Hey, T., Fiedler, E., Rudolph, R., and and Optimization, vol. 4, part VII Fiedler, M. (2005) Artificial, non-antibody (ed. J. Kn€ablein), Wiley-VCH Verlag binding proteins for pharmaceutical and GmbH, Weinheim, pp. 1557–1577. industrial applications. Trends Biotechnol., 65 See also Murray, M. (2005) MettoxTM:a 23, 514–522. suite of predictive in silico and in vitro 73 Genetic Engineering and Biotechnology assays for metabolic and genotoxicological News (Mar 15, 2008). profiling of preclinical drug candidates, in 74 See also Lingnau, K., Klade, C., Buschle, Modern Biopharmaceuticals – Design, M., and von Gabain, A. (2005) Novel Development and Optimization, vol. 4, part vaccine adjuvants based on cationic peptide VII (ed. J. Kn€ablein), Wiley-VCH Verlag delivery systems, in Modern GmbH, Weinheim, pp. 1603–1636. Biopharmaceuticals – Design, Development 66 See also Boese, Q. and Khvorova, A. (2005) and Optimization, vol. 4, part VII, Rational siRNA design for RNA pp. 1419–1440. interference: Optimization for therapeutic 75 Dove, A. (2002) Unkorking the use, in Modern Biopharmaceuticals – Design, biomanufacturing bottleneck. Nature Development and Optimization, vol. 1, part I Biotechnol., 20, 777–779. (ed. J. Kn€ablein), Wiley-VCH Verlag 76 Schillberg, S. (2004) Molecular Farming: GmbH, Weinheim, pp. 243–264. Plant-made Pharmaceuticals and Technical 67 See also Morris, K.V. and Rossi, J.J. (2005) Proteins, Wiley-VCH Verlag GmbH, Combinatorial RNA-based therapies for Weinheim, ISBN: 3-527-30786-9. HIV-1, in Modern Biopharmaceuticals – 77 Kn€ablein, J. (2003) Biotech: A new era in Design, Development and Optimization, vol. the new millennium – fermentation and 2, part II (ed. J. Kn€ablein), Wiley-VCH expression of biopharmaceuticals in plants. Verlag GmbH, Weinheim, pp. 569–577. Screening - Trends Drug Discovery,4,14–16. 68 See also Rarbach, M., et al. (2005) Design 78 Kn€ablein, J. and McCaman, M. (2003) of modern biopharmaceuticals by ultra- Modern biopharmaceuticals – high-throughput screening and directed recombinant protein expression in evolution, in Modern Biopharmaceuticals – transgenic plants. Screening - Trends Drug Design, Development and Optimization, Discovery,6,33–35. vol. 2, part III (ed. J. Kn€ablein), Wiley-VCH 79 Kn€ablein, J. (2004) Biopharmaceuticals Verlag GmbH, Weinheim, pp. 583–603. expressed in plants – a new era in the new 69 See also Sobek, H., et al. (2005) Millennium, in Applications in Accelerating diagnostic product Pharmaceutical Biotechnology (eds R. Muller€ development process with molecular and O. Kayser), Wiley-VCH Verlag GmbH, rational design and directed evolution, in Weinheim, pp. 35–56, ISBN 3-527-30554-8. Modern Biopharmaceuticals – Design, 80 Kn€ablein, J. (2005) Plant-based expression Development and Optimization, vol. 2, part of biopharmaceuticals, in Encyclopedia of III (ed. J. Kn€ablein), Wiley-VCH Verlag Molecular Cell Biology and Molecular GmbH, Weinheim, pp. 703–719. Medicine, 2nd edn, vol. 10 (ed. R.A. 70 See also Chesnut, J.D. (2005) Learning Meyers), Wiley-VCH Verlag GmbH, from viruses: high-throughput cloning Weinheim, pp. 489–410, ISBN 3-527- 1 using Gateway System to transfer genes 30552-1. without restriction enzymes, in Modern 81 Kn€ablein, J. (2006) Pflanzliche Biopharmaceuticals – Design, Development Expressionssysteme – eine “reife” and Optimization, vol. 2, part III Technologieplattform, in Biotechnologie fur€ (ed. J. Kn€ablein), Wiley-VCH Verlag Einsteiger (ed. R. Renneberg), Elsevier GmbH, Weinheim, pp. 605–620. GmbH, Munchen,€ pp. 196–197, ISBN 71 Watson, J.D. (2005) Modern 3-8274-1538-1. Biopharmaceuticals – Design, Development, 82 See also Baez, J. (2005) and Optimization, vol. I–IV Biopharmaceuticals derived from 38j 1 Twenty Thousand Years of Biotech – From “Traditional” to “Modern Biotechnology”

transgenic plants and animals, in and Optimization, vol. 3, part IV Modern Biopharmaceuticals – Design, (ed. J. Kn€ablein), Wiley-VCH Verlag Development and Optimization,vol.3, GmbH, Weinheim, pp. 967–989. part IV (ed. J. Kn€ablein), Wiley-VCH 85 Genetic Engineering and Biotechnology Verlag GmbH, Weinheim, pp. 833–873. News (Nov 5, 2009). 83 See also Huang, N. and Yang, D. (2005) 86 Jagschies, G. (2008) Where is ExpressTec: high-level expression of biopharmaceutical manufacturing biopharmaceuticals in cereal grains, in heading? BioPharm. Int., 21 (10), Modern Biopharmaceuticals – Design, p. 72. Development and Optimization, vol. 3, part 87 Aldridge, S. (2009) Bringing greater IV (ed. J. Kn€ablein), Wiley-VCH Verlag efﬁciency to antibody manufacturing. GmbH, Weinheim, pp. 931–946. Genet. Eng. Biotechnol. News, 29 (14), 84 See also Gepstein, S., Grover, A., and p. 14. Blumwald, E. (2005) Producing 88 Werner, R. (2004) Economic aspects of biopharmaceuticals in the desert: building commercial manufacture of an abiotic stress tolerance in plants for salt, biopharmaceuticals. J. Biotechnol., 113, heat, and drought, in Modern 171–182. Biopharmaceuticals – Design, Development