Biotechnology - new directions in medicine

Biotechnology - new directions in medicine

We Innovate Healthcare

Biotechnology – new directions in medicine

Cover picture

The Roche Group, including Genentech in the United States and Chugai in

Japan, is a world leader in biotechnology, with biotech production facilities

around the globe. The cover photo shows a bioreactor at Roche’s Penzberg

facility and conveys at least a rough of idea of the sophisticated technical

know-how and years of experience required to manufacture biopharma-

ceuticals.

Published by

F. Hoffmann-La Roche Ltd

Corporate Communications

CH-4070 Basel, Switzerland

© 2006

Second, revised edition

Any part of this work may be reproduced, but the source should be cited in full.

All trademarks mentioned enjoy legal protection.

This brochure is published in German (original language) and English.

Reported from: Mathias Brüggemeier

English translation: David Playfair

Layout: Atelier Urs & Thomas Dillier, Basel

Printers: Gissler Druck AG, Allschwil

7 000 728-1

Content

ForewordProgress via knowledge 5

Beer for Babylon 7

Drugs from thefermenter 25

Main avenues of research 39

Treatment beginswith diagnosis 51

Progress via knowledge

Over the past few decades biotechnology – sometimes describedas the oldest profession in the world – has evolved into a mod-ern technology without which medical progress would bescarcely imaginable. Modern biotechnology plays a crucial roleboth in the elucidation of the molecular causes of disease and inthe development of new diagnostic methods and better target-ed drugs.These developments have led to the birth of a new economic sec-tor, the biotech industry, associated mostly with small start-upcompanies. For their part, the more established healthcare com-panies have also been employing these modern techniques,known collectively as biotechnology, successfully for manyyears. By studying the molecular foundations of diseases theyhave developed more specific ways of combating diseases thanever before. This new knowledge permits novel approaches totreatment, with new classes of drug – biopharmaceuticals – at-tacking previously unknown targets. Increasing attention is alsobeing paid to differences between individual patients, with theresult that in the case of many diseases the goal of knowing inadvance whether and how a particular treatment will work in agiven patient is now within reach. For some patients this dreamhas already become reality.Diagnosis and treatment are thus becoming increasingly inter-twined. When a disease, rather than being diagnosed on the ba-sis of more or less vague signs and symptoms, can be detectedon the basis of molecular information, the possibility of suc-cessful treatment depends largely on what diagnostic techniquesare available. To the healthcare industry this represents a majordevelopment in that diagnosis and treatment are growing evercloser together, with clear benefits for companies that possesscompetence in both these areas. To patients, progress in medicalbiotechnology means one thing above all: more specific, saferand more successful treatment of their illnesses. To the health-care industry it represents both an opportunity and a challenge.For example, more than 40% of the sales of Roche’s ten best-sell-ing pharmaceutical products are currently accounted for by bio-pharmaceuticals, and this figure is rising.This booklet is intended to show what has already been achievedvia close cooperation between basic biological research, appliedscience and biotechnologically based pharmaceutical and diag-nostic development.

5

Beer for Babylon

For thousands of years human beingshave used microorganisms to makeproducts – and in so doing havepractised biotechnology. Just as inthe past the development of beer,bread and cheese were major breakthroughs, another revolution isnow about to overtake medicine:compounds produced using biotechnological methods are opening up entirely new possibilitiesin medical diagnostics and therapy,and in so doing are bringing about amajor restructuring of markets.

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Babylonian biotechnologists were a highly regarded lot. Theirproducts were in demand among kings and slaves and were ex-ported as far as Egypt. They are even mentioned in the Epic ofGilgamesh, the world’s oldest literary work – the Babylonian brewers, with their 20 different types of beer. Their knowledgewas based on a biological technology that was already thousands

of years old – fermentationby yeast.Though it may sound strange, the brewing of beeris an example of biotechnol-ogy. Likewise, so is the bak-ing of bread. Wine, yogurt,cheese, sauerkraut and vine-gar are all biotechnologicalproducts. Biotechnology ispractised wherever biologi-

cal processes are used to produce something, whether Babylo-nian beers or monoclonal antibodies. The only thing that is relatively new about the biotechnology industry is its name.

The term ‘biotechnology’ was first used in a 1919publication by Karl Ereky, a Hungarian engineerand economist. He foresaw an age of biochemis-

try that would be comparable to the Stone Age and the Iron Agein terms of its historical significance. For him, science was partof an all-embracing economic theory: in combination with po-litical measures such as land reform, the new techniques wouldprovide adequate food for the rapidly growing world population– an approach that is just as relevant today as it was in the pe-riod after the First World War.

Stone Age, Iron Age, Age of Biochemistry

5000–2000 BC

Fermentation processes are used inEgypt, Babylon and China to makebread, wine and beer. Wall paintingfrom an Egyptian tomb built duringthe Fifth Dynasty (c. 2400 BC).

500 BC

The antibiotic effect of tofu mould cultures is discovered and used fortherapeutic purposes in China.

From knowledge to science: the history of biotechnology

Terms

Biopharmaceuticals drugs manufactured using biotech-nological methods.DNA deoxyribonucleic acid; the chemical substance thatmakes up our genetic material.Genes functional segments of our genetic material that servemostly as blueprints for the synthesis of proteins.Genome the totality of the DNA of an organism.Gene technology scientific work with and on the geneticmaterial DNA.Recombinant proteins proteins obtained by recombiningDNA, e.g. by introducing human genes into bacterial cells.

Beer for Babylon 9

Ereky’s vision is all the more astonishing given that at that timethe most important tools of modern biotechnology were yet tobe discovered. Until well into the second half of the 20th centurybiologists worked in essentially the same way as their Babylo-

AD 100

Ground chrysanthemum seeds areused as an insecticide in China.

800–1400

Artificial insemination and fertilisationtechniques for animals and plantsimprove reproduction rates and yieldsin the Middle East, Europe and China.

1595

Hans Janssen, a spectacle maker,builds the first microscope.

© Rijksmuseum van Oudheden, Leiden, The Netherlands

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nian predecessors: They used the natural processes that occur incells and extracts of plants, animals and microorganisms to pro-duce the greatest possible yield of a given product by carefullycontrolling reaction conditions.Thanks to newly developed methods, however, the biotechnol-ogy of the 20th century was able to produce a far greater rangeof such natural products and at far higher levels of purity and quality. This was due to a series of discoveries that permit-ted the increasingly rapid development of new scientific tech-niques:z In the first half of the 19th century scientists discovered the

basic chemical properties of proteins and isolated the firstenzymes. Over the following decades the role of these sub-stances as biological catalysts was elucidated and exploitedfor research and development.

z The development of ever more sophisticated microscopesrendered the form and contents of cells visible and showedthe importance of cells as the smallest units of life on Earth.Louis Pasteur postulated the existence of microorganismsand believed them to be responsible for most of the fermen-tation processes that had been known for thousands of years.This was the birth of microbiology as a science.

z From 1859 Charles Darwin’s theory of evolution revolution-ised biology and set in train a social movement that led ul-timately to a new perception of mankind. For the first timethe common features of and differences between the Earth’sorganisms could be explained in biological terms. As a result,biology changed from a descriptive to a more experimentalscientific discipline.

z The rediscovery of the works of Gregor Mendel at the end of the 19th century ushered in the age of classical genetics.Knowledge of the mechanisms of inheritance permitted targeted interventions. Cultivation and breeding techniquesthat had been used for thousands of years now had a scien-tific foundation and could be further developed.

C. 1830

The chemical nature of proteins is discovered and enzymes are isolated.

C. 1850

The cell is identified as the smallestindependent unit of life.

1665Examining a thin slice of cork underthe microscope, Robert Hooke discov-ers rectangular structures which henames ‘cells’. Two years later Antonivan Leeuwenhoek becomes the firstperson to see bacterial cells.

Beer for Babylon 11

These developments changed the face of biochemistry and bio-technology. In addition to the classical, mostly agricultural,products, more and more new products entered the market-place. Enzymes were isolated in highly purified form and madeavailable for a wide variety of tasks, from producing washing powder to measuring blood glucose. Standardised biochemicaltest methods made their entrance into medical diagnostics andfor the first time provided physicians with molecular measuringinstruments. The structures and actions of many biomoleculeswere elucidated and the biochemical foundations of life therebymade more transparent. Biochemistry progressed from basic re-search to a field of development.

However, it was only with the advent of gene tech-nology that biology and biotechnology reallytook off. From 1953, when James Watson and

Francis Crick presented the double helix model of DNA, workon and with human genetic material took on the attributes of ascientific race. As more was discovered about the structure ofDNA and the mechanisms of its action, replication and repair,more ways of intervening in these processes presented them-selves to researchers. Desired changes in the genetic makeup ofa species that previously would have required decades of system-atic breeding and selection could now be induced within a fewmonths.For example, newly developed techniques made it possible to in-sert foreign genes into an organism. This opened up the revolu-tionary possibility of industrial-scale production of medicallyimportant biomolecules of whatever origin from bacterial cells.The first medicine to be produced in this way was the hormoneinsulin: in the late 1970s Genentech, an American company, de-veloped a technique for producing human insulin in bacterialcells and licensed the technique to the pharmaceutical companyEli Lilly. Hundreds of millions of diabetics worldwide have ben-

Gene technology spurs innovation

1859

Charles Darwin publishes his revolutionary theory of evolution.

1866

The Augustinian monk Gregor Mendeldiscovers the rules governing theinheritance of traits in peas. It will be35 years before his work receives therecognition it deserves and lays thefoundations of modern genetics.

1869

Friedrich Miescher, working in Tübingen, isolates a substance fromwhite blood cells in purulent bandages that he refers to as ‘nuclein’.His description leads later to use of the term ‘nucleic acids’.

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1879

Walther Fleming describes the ‘chromatin’ present in cell nuclei; thiswill later be identified as DNA.

1913

In studies on the fruit fly Drosophilamelanogaster, Thomas Hunt Morgandiscovers more rules of inheritance.

1878While searching for the organism responsible for anthrax, Robert Kochdevelops techniques for the cultivationof bacteria that are still used today.

In 1982 human insulin became the world’s first biotechnolog-ically manufactured medicine. This hormone plays a centralrole in glucose metabolism in the body. In diabetics the bodyeither has lost the ability to produce insulin in sufficient quan-tity (type 1 diabetes) or else no longer responds adequatelyto the hormone (type 2 diabetes). All people with type 1 dia-betes and most people with type 2 diabetes require regulardoses of exogenous insulin.Until 1982 insulin was isolated from the pancreas of slaughtered animals via a complex and expensive process –up to 100 pig pancreases being required per diabetic patientper year. In its day, this classical biotechnological method it-self represented a major medical breakthrough: until 1922,when medical scientists discovered the effect of pancreaticextracts, a diagnosis of type 1 diabetes was tantamount to adeath sentence. The hormone obtained from cattle and pigsdiffers little from the human hormone. However, some patientstreated with it develop dangerous allergic reactions.

In 1978 the biotech company Genentech developed a methodof producing human insulin in bacterial cells. Small rings ofDNA (plasmids), each containing part of the gene for thehuman hormone, were inserted into strains of Escherichia coli.The bacteria then produced one or the other of the two insu-lin chains. These were then separately isolated, combined andfinally converted enzymatically into active insulin. The pharma-ceutical company Eli Lilly acquired an exclusive licence for thismethod from Genentech and introduced the medicine in 1982in the USA and later worldwide – thus firing the starting gunfor medical biotechnology.Some 200 million diabetics worldwide now benefit from theproduction of human insulin. Without gene technology andbiotechnology this would be impossible: in order to meet cur-rent demands using pancreatic extract, around 20 billion pigswould have to be slaughtered annually.

Gene technology: human insulin from bacteria

Beer for Babylon 13

efited from this, the first biotechnologically manufactured med-icine, since its introduction in 1982 (see box, p. 12).

This technology laid the foundation for a new in-dustry. The early start-up biotech companies joined forces with large, established pharmaceu-

tical companies; these in turn used biotechnology to develophigh-molecular-weight medicines.

In the early 1980s very few companies recognisedthe medical potential of the rapidly expandingfield of biotechnology. One such visionary com-

pany was Genentech. This company, which can lay claim tobeing a founder of the modern biotech industry, was formed in1976 by Herbert Boyer, a scientist, and Robert Swanson, an en-trepreneur, at a time when biochemistry was still firmly ground-ed in basic research. However, Genentech did not remain alonefor long. From the late 1970s, and even more after the introduc-tion of recombinant human insulin, more and more companiesthat aimed to exploit the scientific success of gene technologyfor the purposes of medical research and development were formed, especially in the USA. Even today, nine of the ten biggest companies devoted purely to biotechnology are based inthe USA (see box, p. 16).At first these young companies worked in the shadow of thepharmaceutical giants. This was true both in relation to salesand number of companies and also in relation to public profile.The situation changed abruptly, however, when biotech prod-ucts achieved their first commercial successes. In the 1990s pro-gress in gene technological and biotechnological research anddevelopment led to a veritable boom in the biotech sector.Within a few years thousands of new biotech companies sprangup all over the world. Many of these were offshoots of public or

Rapid expansion and stock market boom

1919

Karl Ereky, a Hungarian engineer,coins the term ‘biotechnology’.

1922

Frederick Banting, Charles Best and James Collip observe the beneficial effect of a pancreaticextract on diabetes; the hormoneinsulin is discovered.

1928

Alexander Fleming discovers the antibiotic effect of penicillin.

A new economicsector arises

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private research institutes whose scientists hoped to obtainfinancial benefit from their findings. Fuelled by expectations ofenormous future profits, the burgeoning biotechnology indus-try became, together with information technology, one of thedriving forces behind the stock market boom of the final yearsof the 20th century.Measured on the basis of their stock market value alone, many

young biotech companieswith a couple of dozen em-ployees were worth more atthat time than some estab-lished drug companies withannual sales running intohundred of millions ofdollars. While this ‘investorexuberance’ was no doubtexcessive, it was also essen-tial for most of the start-upsthat benefited from it. Forthe development of a newdrug up to the regulatoryapproval stage is not only

extremely lengthy, but also risky and hugely expensive. Themain reason for this is the high proportion of failures: only onein every 100,000 to 200,000 chemically synthesised moleculesmakes it all the way from the test tube to the pharmacy.Biotechnological production permits the manufacture of com-plex molecules that have a better chance of making it to the mar-ket. On the other hand, biotechnological production of drugs ismore technically demanding and consequently more expensivethan simple chemical synthesis. Without the money generatedby this stock market success, scarcely any young biotech com-pany could have shouldered these financial risks.For this reason many smaller biotech companies – just like Gen-entech in 1982 – are dependent on alliances with major drug

1953

On the basis of Rosalind Franklin’s x-ray crystallographic analyses, JamesWatson and Francis Crick publish amodel of the genetic substance DNA.

From 1961

Various researchers unravel the genetic code.

1944

Oswald Avery, Colin MacLeod andMaclyn McCarthy identify DNA as the chemical bearer of geneticinformation.

This life-size bronze sculpture of Genentech’s foundersis on display at the company’s research centre in SouthSan Francisco.

Beer for Babylon 15

It took courage to found a biotechnology company in 1976.At that time the business world considered the technology tobe insufficiently developed and the scientific world feared thatthe search for financial rewards might endanger basic re-search. Itwas scarcely surprising, therefore, that the respected

biologist Herbert Boyer had intended to grant the young venture capitalist Robert Swanson only ten minutes of histime. Yet their conversation lasted three hours – and by thetime it ended the idea of Genentech had been born. Furtherdevelopments followed rapidly:1976 On 7th April Robert Swanson and Herbert Boyer found-ed Genentech.1978 Genentech researchers produce human insulin in cloned bacteria.1980 Genentech shares are floated at a price of USD 35; anhour later they have risen to USD 88.1982 Human insulin becomes the first recombinant medicineto be approved for use in the USA; the drug is marketed bythe pharmaceutical company Eli Lilly under licence fromGenentech.1985 For the first time, a recombinant medicine produced bya biotech company is approved for use: Protropin, producedby Genentech (active ingredient: somatrem, a growth hor-mone for children).1986 Genentech licenses Roferon-A to Roche.1990 Roche acquires a majority holding in Genentech andby 1999 has acquired all the company’s shares.1987–97 Major new drug approvals: Activase (1987; activeingredient: alteplase, for dissolving blood clots in myocardialinfarction); Actimmune (1990; interferon gamma-1b, for usein chronic immunodeficiency); Pulmozyme (1992; dornasealfa, for use in asthma, cooperative project with Roche);Nutropin (1993; somatropin, a growth hormone); Rituxan(1997; rituximab, for use in non-Hodgkin’s lymphoma, coop-erative project with Idec).1998 The humanised monoclonal antibody Herceptin (tra-stuzumab) is approved for use against a particular type ofbreast cancer.1999 Fortune magazine rates Genentech as one of the ‘hun-dred best companies to work for in America’; Roche refloatsGenentech on the New York Stock Exchange (NYSE).2002 The journal Science rates Genentech as the most popu-lar employer in the field of biotechnology and pharmaceuticals.2003–2004 Approval of Xolair (omalizumab, for use inasthma); Raptiva (efalizumab, for use in psoriasis); Avastin(bevacizumab, for the treatment of cancer).

1973

Stanley Cohen and Herbert Boyer use restriction enzymes and ligases torecombine DNA.

1975

Georges Köhler and César Milsteinpublish their method for the production of monoclonal antibodies.

1976

Herbert Boyer and Robert Swansonfound Genentech, the first modernbiotechnology company.

The first modern biotechnology company: Genentech

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companies or the services of contract manufacturers. As a resultof the changed stock market conditions after 2000 some ofthese alliances evolved into takeovers: the market value of mostbiotech companies collapsed as abruptly as it had risen, and access to additional capital via the stock market was mostly impossible. The modern biotechnology sector is therefore nowin the middle of its first wave of consolidation.

1982

Human insulin becomes the firstmedicine to be produced using genetechnology, ushering in the age ofmodern biotechnology.

1977Walter Gilbert, Allan Maxam and Frederic Sanger present their methodfor sequencing DNA.

1983

Kary Mullis and coworkers developthe polymerase chain reaction (PCR).

1 Amgen (USA) 83602 Genentech (USA) 33003 Serono (Switzerland) 20004 Biogen Idec (USA) 18501

5 Chiron (USA) 17506 Genzyme (USA) 15707 MedImmune (USA) 10508 Invitrogen (USA) 7809 Cephalon (USA) 710

10 Millenium (USA) 430

Source: company reports1 comparative figure after the merger of Biogen and Idec in Nov.

2003

Many of the major healthcare companies are now also involvedin the biotech sector. If these too are taken into account, the following picture emerges:

1 Amgen 78662 Roche Group including

Genentech and Chugai 61913 Johnson & Johnson 61004 Novo Nordisk 35615 Eli Lilly 30436 Aventis 20757 Wyeth 18708 Schering-Plough 17519 Serono 1623

10 Baxter International 112511 Biogen 105712 Schering AG 103513 Genzyme 87914 MedImmune 78015 GlaxoSmithKline 72916 Bayer AG 56317 Pfizer 48118 Abbott Laboratories 39719 Akzo Nobel 37520 Kirin 355

Source: Evaluate Service

World’s largest biotech companies by sales in 2003, in million USD

World’s largest healthcare companies by salesof biotech products in 2003, in million USD

Beer for Babylon 17

This development did not, however, occur inexactly the same way all over the world. Unlike itscounterpart in the USA,the European biotechnol-

ogy industry soon came to be dominated by established compa-nies founded on classical biochemistry, chemistry and phar-macology. The United Kingdom, Germany, France andScandinavia, in particular, have vibrant biotechnology sectors,while Serono, the European market leader, is a Swiss company.However the motors driving development in the world’s secondmost important biotech region are derived almost exclusivelyfrom the classical industrial sectors.Boehringer Mannheim (BM) provides a good example of thistrend.As a supplier of laboratory equipment for use in biochem-ical research and medical diagnostics, this German companyhad possessed an abundance of expertise in developmental andmanufacturing processes for the biotechnology sector since itsvery inception. As early as the 1940s BM had engaged in classi-cal biotechnology, first in Tutzing and later in Penzberg, nearMunich (see box, p. 19). It made the transition to modern bio-technology during the 1980s with the introduction of a numberof recombinant (i.e. genetically engineered) enzymes.In 1990 BM introduced its first genetically engineered medicine,NeoRecormon (active ingredient: erythropoietin, or EPO). In amore recently developed form, this drug still plays an importantrole in the treatment of anemia and in oncology. This makes itone of the world’s top-selling genetically engineered medicines– and an important source of income for the company, whichwas integrated into the Roche Group in 1998.Roche itself has been a pioneer of biotechnology in Europe. LikeBM, Roche had had an active research and development pro-gramme in both therapeutics and diagnostics for decades. It be-gan large-scale production of recombinant enzymes as long agoas the early 1980s. In 1986 it introduced its first genetically en-

From 1984

Genetic fingerprinting revolutionisesforensics.

Europe: Pharma entersthe biotech sector

1994

The first genetically modifiedtomatoes are marketed in the USA.

1990

The Human Genome Project is launched; the German gene technology law is passed.

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gineered medicine, Rofer-on-A, containing interferonalfa-2a. This product for useagainst hairy cell leukemiawas manufactured under li-cence from Genentech. Afterits takeover of BoehringerMannheim, Roche devel-oped the Penzberg site intoone of Europe’s biggest bio-technology centres.Following its acquisition ofa majority stake in Genen-tech in 1990, Roche’s take-over of BM was the Group’ssecond major step into bio-technology. Finally, its ac-quisition of a majority stakein the Japanese pharmaceu-ticalandbiotechnology com-pany Chugai in 2002 put theRoche Group close behindthe world market leaderAmgen in terms of biotechsales.Roche thus provides a goodexample of the developmentof European biotechnology.Its competitors have fol-lowed a similar course,though in some cases lateror with different focuses.

1997For the first time a eukaryotic genome,that of baker’s yeast, is unravelled.

1998The first human embryonic cell linesare established.

2001The first draft of the human genome ispublished.

Beer for Babylon 19

2003The sequencing of the human genomeis completed.

Research could scarcely be more picturesque: one of Eu-rope’s biggest biotech sites is situated 40 kilometers south ofMunich at the foot of the Bavarian Alps. For over 50 yearsresearchers at Boehringer Mannheim, working first in Tutzingand later in Penzberg, developed biochemical reagents forbiological research and medical diagnostics and therapy.Since Roche took over BM in 1998, Penzberg has becomethe Group’s biggest biotechnological research and produc-tion site.1946 Working with a small research group, Dr Fritz Engel-horn, a departmental head at C. F. Boehringer & Söhne, under-takes biochemical work in the former Hotel Simson in Tutzing.1948 The amino acid mixtures ‘Dymal’, ‘Aminovit’ and ‘Lae-vohepan’ become BM’s first biotechnologically producedpharmaceuticals.1955 Under the brand name ‘Biochemica Boehringer’, BM supplies reagents forresearch and enzyme-based diagnostics through-out the world.1968 The isolation ofpolynucleotides launchesresearch into molecularbiology.1972 BM acquires a dis-used mining site in Penz-berg and builds a new pro-duction plant there for itsrapidlyexpandingbiochem-ical and diagnostics prod-uct lines.1977 First work in genetechnology at Tutzing.1980 Establishment of alaboratory for the produc-tion of monoclonal anti-bodies at Tutzing.1981 Large-scale produc-tion of recombinant en-zymes begins at Penzberg.

1985 Roche is awarded German Industry’s Innovation Prizefor Reflotron, an analytical device for determining bloodparameters.1986 Process development work for BM’s first recombinantmedicine, NeoRecormon (active ingredient: erythropoietin)begins.1990 NeoRecormon is approved for use in the treatment ofanemia.1996 Rapilysin (active ingredient: tissue plasminogen activa-tor, for the treatment of myocardial infarction) becomes thefirst recombinant drug to be discovered, developed and pro-duced in Germany.1998 The Roche Group takes over BM; over the followingyears Roche develops the Penzberg site into one of Europe’sbiggest and most modern biotechnology centres.

‘Big biotech’ at the foot of the Alps: Penzberg

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Compared to their counterparts in Europe, thepharmaceutical companies of the various Asiancountries – which are otherwise so enthusiastic

about new technology – were slow to recognise the potential ofthis new industrial sector. This despite the fact that the Japanesepharmaceutical market is the world’s second largest, after that of

Japan: potential inbiotechnology

Roche’s line of biotechnological products dates back to the1940s. The resulting expertise has paid off: The Roche Groupis now the world’s second largest biotechnology companyand has a broader product base than any of its biotech com-petitors. Its three best-selling medicines are biopharmaceuti-cals, and almost half the sales of its top ten pharmaceuticalproducts are accounted for by biopharmaceuticals. Roche’sDiagnostics Division supplies over 1700 biotechnology-basedproducts. PCR technology alone generates annual sales of 1.1billion Swiss francs. Key milestones on the way to this successare listed below:1896 Fritz Hoffmann-La Roche founds the pharmaceuticalfactory F. Hoffmann-La Roche & Co. in Basel.1933 Industrial production of vitamin C begins; within a fewyears Roche becomes the world’s largest producer of vita-mins.1968 With its Diagnostics Division, Roche opens up a for-ward-looking business segment; Roche establishes theRoche Institute of Molecular Biology in Nutley, USA.1971 The Basel Institute for Immunology is set up and fi-nanced by Roche.1976 Georges Köhler (a member of the Institute from 1976to 1985) begins his work on monoclonal antibodies.1980 Cooperation with Genentech begins; over the followingdecades alliances with biotech companies become a centralfeature of the Roche Group’s corporate philosophy.1984 Niels Kaj Jerne and Georges Köhler of the Basel Insti-tute for Immunology are awarded the Nobel Prize for Physiol-ogy or Medicine jointly with César Milstein; their colleagueSusumu Tonegawa (a member of the Institute from 1971 to1981) is awarded the Nobel Prize in 1987.1986 The alliance with Genentech leads to the developmentof Roferon-A (active ingredient: interferon alfa-2a), Roche’sfirst genetically engineered drug; Roche introduces an HIVtest.1991 Roche acquires worldwide marketing rights to thepolymerase chain reaction (PCR) from Cetus Corporation;only two years later this technology forms the basis of the HIVtest Amplicor, the first PCR-based diagnostic test.1992 Hivid, Roche’s first AIDS drug, is introduced.

1994 Roche takes over the US pharmaceutical companySyntex and in 1995 converts it into Roche Biosciences.1998 Roche takes over the Corange Group, to which Boeh-ringer Mannheim belongs. Cooperation with deCODE genet-ics begins.1999 Following its complete takeover of Genentech, Rochereturns 42% of the company’s shares to the stock market; themonoclonal antibody Herceptin is approved for use in breastcancer.2000 The Basel Institute for Immunology is transformed in-to the Roche Center forMedical Genomics.2001 The merger of Nip-pon Roche and Chugairesults in the formation ofJapan’s fifth largest phar-maceutical manufacturerand leading biotech com-pany.2002 Pegasys (activeingredient: peginterferonalfa-2a, for use againsthepatitis C) is approvedfor use in Europe and theUSA; Roche sells its Vita-mins and Fine ChemicalsDivision to DSM.2003 Cooperation withAffymetrix on the pro-duction of DNA chipsbegins; AmpliChip CYP450, the world’s firstpharmacogenomic medi-cal diagnostics product,is introduced.2004 New biotechno-logical production plantsare built in Basel andPenzberg.

No newcomer to biotech: the Roche Group

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the USA; in scarcely any other country are so many drugs pre-scribed, an eighth of worldwide pharmaceutical sales being ac-counted for by Japan alone. Moreover, two Japanese companies,Takeda and Sankyo, rank among the 20 largest pharmaceuticalcompanies in the world.In the 1990s Japan set out on the road to catch up, in particularvia large-scale support programmes and targeted alliances. Theresult is that Japanese pharmaceutical companies are now atleast on a par with their counterparts in most European coun-tries in terms of sales of biopharmaceutical products. However,the country still lags behind in terms of the number of biotechcompanies based there, the period of rapid expansion in the1990s having largely passed Japan by. As yet, Japanese companiesdevoted exclusively to modern biotechnology have an evensmaller slice of the world market than their European competi-tors.Japanese biotechnology is largely in the hands of representativesof classical branches of industry such as the brewery Kirin, thefood manufacturer Takara, the chemical manufacturer KyowaHakko and various pharmaceutical companies.The market lead-er in modern biotechnology in Japan is Chugai Pharmaceutical

Number one in Japanese biotechnology: Chugai Pharma

When the Japanese set themselves a goal, their competitorshave a hard time of it. A few years ago the Japanese phar-maceutical company Chugai set its sights on joining the firstrank of the world’s biotech companies. Since then it has beencatching up at an astonishing rate and is now at the top of theJapanese market, at least. Since its merger with NipponRoche, Chugai has become not only the fifth largest pharma-ceutical company, but also the largest modern biotechnologycompany, in Japan. A brief chronology follows:

1925 Juzo Uyeno founds a small pharmaceuticalcompany in Tokyo that becomes increasingly impor-tant nationally over the coming decades.1986 The present-day company Chugai PharmaEurope takes up headquarters in London.1989 Chugai acquires Gen-Probe, an American bio-tech company and diagnostics manufacturer.1990 Epogin (active ingredient: erythropoietin, a growth factor) becomes the first genetically engi-neered drug produced by Chugai to be approved foruse in Japan.1991 Granocyte (active ingredient: rHuG-CSF, for

promoting the growth of white blood cells) is approved for usein Japan and later also in Europe, Australia and China.1993–96 Chugai enters into a number of alliances for thediscovery, development and marketing of drugs.1995 The Chugai Research Institute for Molecular Medicineis founded.1997 Chugai Diagnostics Science is formed.2002 Chugai and Nippon Roche merge to form Japan’s fifthlargest pharmaceutical company.

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Co., Ltd., a company with an 80-year tradition and one of thefirst companies in Japan to invest in gene technology.Milestones along this company’s development in this area wereits acquisition of the American biotech company Gen-Probe in1989 and, a year later, the granting of regulatory approval for itsfirst genetically engineered drug, Epogin (active ingredient:erythropoietin, for use in anemia). Access to the worldwidemarket for these products is provided by the Roche Group,which acquired a majority stake in Chugai in 2002.The merger between Nippon Roche, Roche’s Japanese subsidi-ary, and Chugai in 2002 led to the formation of Japan’s fifth-largest pharmaceutical company and largest biotech company.Chugai operates as an independent member of the Roche Groupand is listed separately on the stock exchange. It is responsiblefor the sale of all Roche products in Japan and also benefits fromthe Group’s worldwide sales network; for its part, Roche has li-censee rights to all Chugai products marketed outside of Japanor South Korea.

As seen from the example of the Roche Group,small, innovative biotech companies are increas-ingly entering into alliances with big pharma-ceutical companies. At the same time, the big

companies have expanded their portfolios by acquiring majori-ty stakes in biotech companies listed separately on the stockexchange and by entering into alliances in this area. And an im-petus to change is arising from biotech companies themselves:by engaging in takeovers and opening up new business seg-ments, they too are investing beyond their established areas ofoperation.As a result of this development, most biotechnologically manu-factured drugs are marketed by pharmaceutical companies. Andthis trend is likely to become even more pronounced in the fu-ture. Thus, Roche is currently the world’s second biggest sup-plier of biotechnological products and, with more than 50 newdrug projects under way at present, has the world’s strongestearly development pipeline in this area. Aventis and Glaxo-SmithKline, each with 45 drug candidates, share second place inthis ranking. Amgen, currently the world’s largest biotech com-pany, had about 40 drug candidates in the pipeline in 2004.At the same time, worldwide growth in the biotechnology market shows no sign of slackening. Thus, at present 40% of the

Prospects:biotechnology intransition

Beer for Babylon 23

sales of Roche’s ten best-selling pharmaceutical products are ac-counted for by biopharmaceuticals, and this figure is rising. Themany young biotech companies with drug candidates now ap-proaching regulatory approval are also banking on this growth.Both in Europe and in the USA, many such companies formedat the time of the stock market boom in biotechnology will soonbe marketing their first drug or drugs. Sales of these will supporttheir development pipelines – and thereby also intensify com-petition in this field.At present the world’s ten largest biotech companies account forabout 85% of the approximately 37 billion US dollars of sales ofbiotechnological products worldwide. A comparison of the de-velopment pipelines of the big companies with those of the gen-erally smaller companies that are devoted exclusively to bio-technology suggests that this concentration is likely to becomeeven greater in the coming years, though given the spectaculargrowth rate of this sector, the possibility of surprises cannot beruled out. What is clear is that biotechnology has had a decisiveinfluence on the pharmaceutical market – and that the upheavalis not yet at an end.

Works consulted and literature for further reading

Campbell NA, Reece JB: Biologie. Spektrum Akademischer Verlag, Heidelberg, 6th edition2003

Stryer L: Biochemie. Spektrum Akademischer Verlag, Heidelberg, 4th edition 2003Die Arzneimittelindustrie in Deutschland – Statistics 2004. VFA Verband Forschender

Arzneimittelhersteller e.V., editor, Berlin, August 2004Presentations at a media conference: The Roche Group – one of the world’s leaders in

biotech, Basel, November 2004http://www.roche.com/home/media/med_events

Prowald K: 50 Jahre Biochemie und Biotechnologie bei Boehringer Mannheim. 50 Jahr Feier, Evangelische Akademie Tutzing, 1966

Balaji K: Japanese Biotech: A Plan for the Future. Japan Inc., August 2003. See: www.japaninc.net

bio.com – life on the net: www.bio.comGenentech, Inc.: www.gene.comRoche Group: www.roche.comBioJapan: www.biojapan.deChugai Pharmaceutical Co., Ltd.: www.chugai-pharm.co.jpSchmid RD: Pocket Guide to Biotechnology and Genetic Engineering. Wiley-VCH, Weinheim,

2002

Drugs from the fermenter

Biotechnological production of drugsconfronts pharmaceutical researchand development with new challenges. For example, complexbiomolecules such as proteins canonly be produced by living cells incomplex fermentation plants, yetthey have the potential to open upentirely new directions in medicine.

26

Though you might not think so at first glance,modern biotechnology and traditional drug de-velopment have much in common. The aim of

both, for example, is to develop substances able to cure or pre-vent disease. To achieve this they both rely on recent findingsfrom the life sciences. For most patients it is a matter of indiffer-ence whether a drug is obtained by biotechnological or chemi-cal means. The main thing is that it works. However, beneath thesurface there are striking differences between the two kinds ofdrug product.

Almost all traditional drugs are small molecules.They are usually relatively simple organic com-pounds containing a few functional molecular

groups. On the other hand, therapeutic proteins, the largestgroup of biopharmaceuticals, are quite a different kettle of fish.

They are made up of dozens,sometimes hundreds, ofamino acids, each of whichis as big as the acetylsalicylicacid molecule of aspirin.To take an example, the ac-tive ingredient in CellCept,currently Roche’s top sellingtraditional drug, is an organ-ic compound made up of 62atoms with a total molecularweight of 433.5 daltons (onedalton [Da] equals 1.7 · 10-27

kg). Roche’s leading bio-pharmaceutical, the mono-clonal antibody MabThera/Rituxan (rituximab), isnearly 350 times heavier,

weighing in at a hefty 150,000 daltons. No wonder this largemolecule poses entirely different challenges for research, devel-opment and production. And it also acts differently than con-ventional drugs in the body.

Biopharmaceuticalstransform medicine

Terms

Biopharmaceuticals drugs manufactured using biotech-nological methods.Dalton (Da) unit used to express the weight of atoms andmolecules; one dalton is equal to 1.7 · 10-27 kg.Enzymes biocatalysts; proteins able to facilitate and accel-erate chemical reactions.Eukaryotes organisms whose genetic material is enclosed ina cell nucleus; they include all fungi, plants and animals, includingman.Fermentation a chemical reaction in which biological sub-stances are acted upon by enzymes.Fermenter also known as a bioreactor; a cultivation andreaction vessel for living cells.Gene technology scientific techniques for manipulating thegenetic material DNA.Recombinant proteins proteins obtained by recombiningDNA, e.g. by introducing human genes into bacterial cells.Therapeutic proteins proteins used as active agents inpharmaceuticals.

Molecules hundreds of times bigger

Drugs from the fermenter 27

Size comparison: erythropoietin and aspirin

Aspirin

N

N O

O OO

O

H

CH2

CH2

CH3

CH3

C

C

C

C

CCH

N

Phe

N

N

N

O

OH

Tyr

C

AlaH

1 10

3040

20

50 60

7080

90 100

110120

140130

166 160 150

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala

Lys

Glu

Ser

Ser

Ile

Val

Asn

Ala

Thr Asn Glu Asn Leu Ser Cys His Glu Ala Cys Gly Tyr Tyr Ile Asn Glu Ala Glu

AspPro Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val

LeuVal Leu Ala Gin Gly Arg Leu Val Ala Glu Ser Leu Leu Ala Leu Gly Gln Trp Val

SerSer Gln Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly Leu Arg

AspAla Pro Pro Ser Ile Ala Glu Lys Gin Ala Gly Leu Ala Arg Leu Leu Thr Thr Leu

AlaSer Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr

AspArg Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys Leu Lys Gly Arg Leu Phe Asn

Biopharmaceuticals are generally much bigger compounds thantraditional drugs. Each of the amino acid residues in the proteinerythropoietin is comparable to an aspirin molecule in size.

28

The most important consequence of the size dif-ference between traditional and biotechnologicaldrugs relates to their structure. The three-dimen-

sional shape of simple organic molecules, known in chemicalparlance as ‘small molecules’, is essentially determined by fixedbonds between the individual atoms. As a result, traditionaldrugs are usually highly stable compounds that retain theirthree-dimensional shape in a wide range of ambient conditions.Only drastic changes to the milieu – e.g. the presence of strongacids or bases or elevated temperatures – are able to cause per-manent damage to these molecules. Traditional drugs are usual-ly easy to handle and can be administered to patients conve-niently in various forms such as tablets, juices or suppositories.It is true that many traditional drugs were originally derivedfrom natural products. For example, healers used an extract ofthe leaves or bark of certain willow species to treat rheumatism,fever and pain hundreds of years before the Bayer chemist FelixHoffmann reacted the salicylate in the extract with acetic acid in1897 to form acetylsalicylic acid, a compound that is gentler onthe stomach. Today drugs like these are usually produced chem-ically from simple precursors. The methods have been tried and tested for decades, and the drugs can be manufacturedanywhere to the same standard and in any desired amount. Ster-ile conditions, which pose a considerable technical challenge,are rarely necessary. On the other hand, preventing the organicsolvents used in many traditional production processes fromdamaging the environment remains a daunting task.

Biopharmaceuticals require a far more elaborateproduction process. Most drugs manufactured bybiotechnological methods are proteins, and pro-

teins are highly sensitive to changes in their milieu. Their struc-ture depends on diverse, often weak, interactions between theiramino-acid building blocks. These interactions are optimallycoordinated only within a very narrow range of ambient condi-tions that correspond precisely to those in which the organismfrom which the protein is derived best thrives. Because of this,even relatively small changes in the temperature, salt content orpH of the ambient solution can damage the structure. This, inturn, can neutralise the function of the protein, since this de-pends on the precise natural shape of the molecule.This applies analogously to therapeutic proteins used in medi-

Proven methods for small molecules

Unstable structure of proteins


cine. Most of these mole-cules act as vital chemicalmessengers in the body. Thetarget cells that receive andtranslate the signals bearspecial receptors on theirsurface into which the cor-responding chemical mes-senger precisely fits. If thethree-dimensional shape ofthe chemical messenger iseven slightly altered, themolecule will no longer berecognised by its receptorand will be inactive.The situation is similar foranother group of therapeutic proteins, the antibodies. In theirnative state these molecules are components of the immune sys-tem. Their function is to recognise foreign structures, for whichpurpose they have a special recognition region whose shape pre-cisely matches that of the target molecule. Changing just one ofthe several hundred amino acids that make up the recognitionregion can render the antibody inactive. It is possible to produceantibodies to target any desired foreign or endogenous sub-stance. Modern biotechnology makes use of the technique toblock metabolic pathways in the body involved in disease pro-cesses. Like other therapeutic proteins, antibodies must there-fore assume the correct molecular arrangement to be effective.

This structural sensitivity also causes problemsbecause proteins do not always automatically as-sume the required structure during the produc-tion process. Long chains of amino acids in solu-

tion spontaneously form so-called secondary structures,arranging themselves into helical or sheetlike structures, for ex-ample. However, this process rarely results in the correct overallshape (tertiary structure) – especially in the case of large pro-teins where the final structure depends on the interactions ofseveral, often different, amino acid chains.During natural biosynthesis of proteins in the body’s cells, a se-ries of enzymes ensure that such ‘protein folding’ proceeds cor-rectly. The enzymes prevent unsuitable structures from being

Detecting signals: interferon gamma and its receptor

The signal protein interferon gamma (blue) is recognised by aspecific receptor (left and right) located on the surface of itstarget cells. Interferon gamma as a biopharmaceutical is usedto treat certain forms of immunodeficiency. (Source of illustration:

http://arginine.chem.cornell.edu/structures/ifncomplex.html)

Biopharmaceuticals:biological instead of chemical production

30

formed in the early stages, separate signal-processing segmentsfrom the proteins, add non-protein sections, combine severalproteins to form complexes and interlink these as required.Thesestrictly controlled processes make protein production a highlycomplex process that has so far proved impossible to replicate bychemical means. Instead, proteins are produced in and isolatedfrom laboratory animals, microorganisms or special cultures ofanimal or plant cells.

Biological production methods do, however, haveseveral disadvantages. The straightforward ap-

proach, isolating natural proteins from animals, was practisedfor decades to obtain insulin (see article ‘Beer for Babylon’). Butthe limits of this approach soon became apparent in the secondhalf of the 20th century. Not only are there not nearly enoughslaughtered animals to meet global demands for insulin, but theanimal protein thus obtained differs from its human counter-part. As a result, it is less effective and may trigger allergic reac-tions. The situation is similar for virtually every other biophar-maceutical, particularly since these molecules occur in animalsin vanishingly small amounts or, as in the case of therapeutic an-tibodies, do not occur naturally in animals at all.Most biopharmaceuticals are therefore produced in cultures ofmicroorganisms or mammalian cells. Simple proteins can be

Natural sources limited

Diverse and changeable: the structure of proteins

A chain of up to twenty different amino acids (primary struc-ture – the variable regions are indicated by the squares of dif-ferent colours) arranges itself into three-dimensional struc-tures. Among these, helical and planar regions are particularlycommon. The position of these secondary structures in rela-tion to one another determines the shape of the protein, i.e.its tertiary structure. Often, a number of proteins form func-tional complexes with quaternary structures; only whenarranged in this way can they perform their intended func-tions. When purifying proteins, it is extremely difficult to retainsuch protein complexes in their original form.

}}

primary structure

secondary structure

tertiary structure

quaternary structure


obtained from bacteria. For complicated substances consistingof several proteins or for substances that have to be modified bythe addition of non-protein groups such as sugar chains, mam-malian cells are used. To obtain products that are identical totheir human equivalents, the appropriate human genes must beinserted into the cultured cells. These genetically manipulatedcells then contain the enzymes needed to ensure correct foldingand processing of the proteins (especially in the case of mam-malian cells) as well as the genetic instructions for synthesisingthe desired product. The responsible gene is then placed underthe control of a super-active DNA signal element. In this way agenetically modified cell is obtained which produces large quan-tities of the desired product in its active form.

But multiplying these cells poses a technologicalchallenge, particularly when mammalian cells areused to produce a therapeutic protein. Cells are

living organisms, and they react sensitively to even tiny changesin their environment. This concerns not only easily controllablefactors (e.g. temperature and pressure), as in conventional chemical synthesis. From the nutrient solution to the equip-ment, virtually every object and substance the cells touch ontheir way from, say, the refrigerator to the centrifuge can affectthem.

Little helpers: the biological production of drugs

The bacterium Escherichia coli is relatively easy to cultivate.However, it can only be used to manufacture simple pro-teins that require no modifications following biosynthesis.

A cell line that was developed from Chinese hamster ovarycells (CHO cells) is now used in biopharmaceutical pro-duction facilities worldwide.

Biotech production: eachfacility is unique

32

These factors determine not only the yield of useful product butalso the quantity of interfering or undesired byproducts and thestructure of the product itself. As a result, each biopharmaceu-tical production plant is essentially unique: Changing just oneof hundreds of components can affect the result. In extreme cases it may even be necessary to seek new regulatory approval.

Laboratories and manufacturers around theworld work with standard cell lines to producebiopharmaceuticals, enzymes and antibodies.

These cell lines are used because they are well researched and, asfar as is possible with living organisms, are amenable to stan-dardisation. This allows reproducible results to be obtainedworldwide. Important standard organisms used in basic re-search and the biotech industry include bacteria of the species Escherichia coli and eukaryote CHO (Chinese hamster ovary)cells (see figure, p. 31).Biotech researchers insert structural and control genes into thecells of these and similar lines to produce the desired pharma-ceutical. This establishes a new cell line, which is usually treatedas a closely guarded company secret. After all, these cells are theactual factories of the biopharmaceutical concerned. They areallowed to reproduce and are then safely stored at low tempera-tures in what is known as a master cell bank. If the cells need to

Focus on Chinesehamster cells

Large-scale industrial production facilities for biopharma-ceuticals are precisely controlled closed systems. The

smallest impurity can render a batch useless. Biopharma-ceuticals must be produced under strict cleanroom condi-tions.

High-tech cell cultivation: biotechnological production facility in Penzberg


be stored for long periods, they can be kept almost indefinitelyin liquid nitrogen at –196°C.Cells are then drawn from the cell banks and used in biophar-maceutical production. Broadly speaking, the production pro-cess is divided into the following steps:

Cultivation: The cells are transferred from the cryogenic cellbank to a liquid nutrient medium, where they are allowed toreproduce. The length of this step depends on the type of cellused. Under favourable conditions bacterial cells such as Escherichia coli usually divide once every 20 minutes; thus

Industrial cell cultivation: flowchart of biopharmaceutical production

80 l 400 l 2000 l 80 l 400 l 2000 l 80 l 400 l 2000 l

Fermenter12,500 l

Inoculum

Purification

Filling

Harvest tank Separator

Affinity(protein A)

Cationexchange

Anionexchange

Purification train (3 steps)

Dia-/UltrafiltrationAPI Filling& Freezing

Fermentation

The production of biopharmaceuticals starts when a nutri-ent solution is inoculated with cells from a cell bank. Theseare allowed to reproduce in stages up to a scale of several

thousand liters. The cells secrete the desired product,which is then isolated from the solution, purified and trans-ferred to containers.

34

one cell gives rise to 4.7 · 1021 cells within 24 hours. By con-trast, mammalian cells such as CHO cells divide about onceevery 24 hours, and it takes correspondingly longer to obtaina sufficient number of cells. During the growth phase the cellculture is transferred to progressively larger culture vessels.

Fermentation: The actual production of the biopharmaceuticaloccurs during this phase. The culture medium contains sub-stances needed for the synthesis of the desired therapeuticprotein. In total, the medium contains around 80 differentconstituents at this stage, although manufacturers never dis-close the exact composition. The industrial-scale steel vesselsin which fermentation takes place have capacities of 10,000liters or more. There are not only technological but also bio-logical constraints on the size of the reactor vessel: The big-ger a fermenter is, the more difficult it becomes to create uni-form conditions around all the cells within it.

Purification: In technical terms, the production of biopharma-ceuticals in cells is a one-step process and the product can bepurified immediately after fermentation. In the simplest casethe cultured cells will have secreted the product into the am-bient solution. In this case the cells are separated from theculture medium, e.g. by centrifugation or filtration, and thedesired product is then isolated via several purification steps.If, on the other hand, the product remains in the cells follow-ing biosynthesis, the cells are first isolated and digested (i.e.destroyed), and the cellular debris is then separated from thesolution together with the product.The yield from bioproduction processes is usually much lowerthan from chemical synthesis. For example, a 10,000-literfermenter yields only a few kilograms of a therapeutic anti-body such as MabThera/Rituxan (rituximab) or Herceptin(trastuzumab). The production steps, including purifica-tion, take several weeks. Several more weeks are then neededto test the product: Each product batch is tested for purity toavoid quality fluctuations, and a 99.9 percent purity level isrequired for regulatory approval. Only then can the finishedproduct be further processed and shipped.

Formulation: The final steps in the production of biopharma-ceuticals are also demanding. The sensitive proteins are con-verted to a stable pharmaceutical form and must be safelypackaged, stored, transported and finally administered.Throughout all these steps the structural integrity of themolecule has to be safeguarded to maintain efficacy. At pres-


ent this is only possible in special solutions in which the product can be cryogenically frozen and preserved, thoughthe need for low temperatures does not exactly facilitatetransport and delivery. Biopharmaceuticals are thereforeproduced strictly on the basis of demand – even more so thantraditional drugs.Because of the sensitive nature of most biopharmaceuticals,their dosage forms are limited to injectable solutions. Thera-peutic proteins cannot pass the acidic milieu of the stomachundamaged, nor are they absorbed intact through the in-testinal wall. Though work on alternatives such as inhalers isin progress (especially for the relatively stable insulin mol-ecule), injection remains the only option for introducingbiopharmaceuticals into the body.

Nowadays all the steps in the production of biopharmaceuticalsare fully automated. Production staff step in only if problems occur. Because cell cultures react so sensitively to fluctuations inambient conditions, the window for high-yield production isquite narrow: If the physical and chemical properties of the nu-trient medium deviate ever so slightly from the norm, the pro-duction staff must take action to restore optimum conditions.Even trace amounts of impurities can spell considerableeconomic loss, as the entire production batch then has to be dis-carded and the production process has to be restarted fromscratch with the cultivation of new cells.

Despite their elaborate production process, bio-pharmaceuticals offer a number of advantages,two of which are uppermost in patients’ minds:

efficacy and safety. These are determined by the molecular prop-erties of therapeutic proteins.Thanks to their structure, proteins have a strong affinity for aspecific target molecule. Unlike traditional, low-molecular-weight drugs, biopharmaceuticals therefore rarely enter intononspecific reactions. The result is that interference and danger-ous interactions with other drugs as well as side effects are rare.Nor do therapeutic proteins bind nonspecifically to receptorsthat stimulate cell growth and cause cancer. Biopharmaceuticalsare unable to penetrate into the interior of cells, let alone intothe cell nucleus, where many carcinogenic substances exert theirdangerous (side) effects.

Advantages in terms ofefficacy and safety

36

However, this property is also associated with a drawback com-pared to traditional drugs: The number of possible targets islimited. Ultimately, only substances that occur in an unboundstate between cells or on the outer cell surface come into ques-tion.Another ambivalent property is the fact that therapeutic pro-teins strongly resemble endogenous proteins. On the one hand,this means that their breakdown rate can be readily predictedand varies far less between individuals than is the case with tra-ditional drugs. This makes it easier for physicians to determinethe right drug dose for their patients. On the other hand, thera-peutic proteins are more likely than small molecules to triggerimmune reactions. Simply put, proteins present a larger surfacearea for the immune system to attack. Moreover, foreign pro-teins may be interpreted by the immune system as a sign of in-fection. One way in which researchers are trying to prevent these reactions,for example in the case of monoclonalantibodies,is via the use of ‘humanised’ therapeutic antibodies, which areproduced by inserting human antibody genes into culturedcells.

Overall, the virtues of biopharmaceuticals interms of their efficacy and safety also mean an

economic advantage: The likelihood of successfully developinga new biopharmaceutical is significantly greater than in tradi-tional drug development. Not least because interactions, side ef-fects and carcinogenic effects are rare, 25 percent of biophar-maceuticals that enter phase I of the regulatory process are

Bio vs. traditional: advantages and disadvantages of biopharmaceuticals

Traditional drugs Biopharmaceuticals

Unspecific binding Specific bindingInteractions with other drugs Interactions rareCarcinogenic substances possible Not carcinogenicPharmacokinetics difficult Breakdown is predictable for the most partImmune reactions rare Immunogenic effects possibleTheoretically, any target molecule Target molecules limited, only can be reached outside the cell 6% success rate in phases I–III 25% success rate in phases I–IIIDevelopment costs high, Development costs low, production costs low production costs high

Higher success rates

eventually granted approval. The corresponding figure for con-ventional drugs is no more than six percent.However, the lower risk of failure is offset by an investment riskat the end of the development process. The construction of abiopharmaceutical production plant is so technologically, legal-ly and scientifically demanding – and therefore so time-consum-ing – that it must be planned even before the phase III studies.From a medical point of view it seems likely that the current suc-cess of biopharmaceuticals will continue unabated and that these products, especially those used in the treatment of com-mon diseases such as cancer, will gain an increasing share of themarket. However, therapeutic proteins are unlikely ever to fullyreplace their traditional counterparts. In many applicationssmall molecules will remain the drugs of choice. Examples in-clude lipid-lowering drugs and drugs for the treatment of type2 (non-insulin-dependent) diabetes. The future also holds pro-mise for hybrids of conventional and biopharmaceutical drugs.The potential of such ‘small molecule conjugates’ is discussed inthe following article along with other major areas of research.



Brüggemeier M: Top im Abi – Biologie. Schroedel, Braunschweig, 2004Presentations at a media conference: The Roche Group – one of the world’s leaders in bio-

tech, Basel, November 2004http://www.roche.com/home/media/med_events

Schmid RD: Pocket Guide to Biotechnology and Genetic Engineering. Wiley-VCH, Weinheim,2002


Main avenues of research

Modern medical biotechnology is a relatively new discipline. Nevertheless, new discoveries about the molecular causes of diseases and the influence exertedby our genes on the effectiveness of medicines are already leading to the development of specific diagnostic techniques and better targeted treatment for individualpatients.

40

Few sectors of the economy are as research-inten-sive as the healthcare industry. Any findings andmethods discovered by universities and institutesworking in the life sciences usually find their way

immediately into the industry’s development laboratories. Butcompanies do not just borrow findings from academic re-searchers. They also invest a great deal of effort and money intotheir own research. This is true both of biotechnology compa-nies and of major healthcare companies. The exchange betweenindustry and science is vigorous and productive. Just a few ex-amples:z During the 1990s biology was defined by the fields of human

genetics and genomics. By deciphering the human genome re-searchers obtained profound new insights into the hered-itary basis of the human body. From the mass of genetic in-formation now available researchers can filter out potential

target molecules for newbiopharmaceuticals.z Since the late 1990s pro-teomics has attracted in-creasing attention both inbasic research and in drugdevelopment. Because pro-teins can act either as targetmolecules or as drug mole-cules, new findings benefitdrug research doubly. In ad-dition, proteins can serve asmarkers for diagnostic tests.z Modifications of DNAand proteins have recentlymoved into the limelight. Ithas been recognised thatsuch modifications in bio-molecules – some tempo-

rary, some permanent – play a role in many diseases and cantherefore serve as useful diagnostic aids. In addition, modifi-cations of therapeutic proteins strongly influence their effi-cacy and stability.

z In recent years researchers have succeeded in shedding morelight on the key functions of the immune system. These findingshave led to various new diagnostic approaches and more refined methods for developing therapeutic antibodies.

The changing face of biotechnology … and ofmedical science

Terms

Chimeric made up of components from two different species orindividuals. Chimeric antibody a modified antibody molecule whose variablesegments, which bind to an antigen, are derived from a different antibody than its constant region. The technique led to the produc-tion of the first humanised chimeric antibodies, in which variable seg-ments obtained from mouse antibodies are combined with a constantsegment from a human antibody.Copegus (ribavirin) a Roche product used in combination withPegasys for the treatment of hepatitis C.Enzymes biocatalysts; proteins able to facilitate and acceleratechemical reactions.PCR polymerase chain reaction; molecular biological method forcopying sections of hereditary material (DNA) millions of times over.Targets the molecules in our body with which drugs interact; mostare proteins.Therapeutic antibodies antibodies used as agents for the treat-ment of diseases.Therapeutic proteins proteins used as active substances indrugs.


z Techniques such as the polymerase chain reaction (PCR) andthe development of DNA chips have opened up new horizonsin the fields of basic research and drug and diagnostic test de-velopment.

Modern medical biotechnology uses a wide rangeof methods to diagnose and treat diseases – fromthe biotechnological production of simple natu-ral products to gene therapy. The most important

group of biotechnological drugs by far, however, are the thera-peutic proteins. Most therapeutic proteins are chemical mes-sengers, enzymes or, especially in recent times, monoclonal an-tibodies. Some occur naturally in the body. For example, manylong-established biotechnological products such as the hor-mones insulin and erythropoietin (EPO) are natural chemicalmessengers. Now these molecules can be produced in genetical-ly modified cells that carry the hereditary information for pro-ducing the human protein.

Identification ofnew moleculartargets

Assessmentof availableand new targets

Leadidentification

Leadoptimisation

The number of good molecular targets fortherapeutic proteins is limited

Pick the winners; assessment in cellular and animal models

Design of therapeutic proteins, e.g. human antibodies,peptides/proteins with long duration of action

Profiling and selection of the best therapeutic protein inpreclinical models

The four major steps in biotechnological drug development are closely linked to prog-ress in basic biological research.

Research-orientated: development of therapeutic proteins

Most important drug group: therapeuticproteins

42

In addition, new findings from basic research now allow thera-peutic proteins to be coupled with non-protein components toimprove their efficacy and duration of action.

In the human body the hormone erythropoietin(EPO) controls the formation of red blood cells

from precursor cells in the bone marrow. Since the substance isproduced mainly in the kidneys, patients with renal damage areprone to develop anemia. Those affected – often dialysis patients– generally feel weak and tired, because their red blood cells nolonger carry sufficient supplies of oxygen to the body. But themost often unrecognised and severe complication of anemia israpid progressing congestive heat failure (CCF) as the heart hasto pump much more in order to compensate for insufficient redblood cells. CCF is the leading cause of death in patients withanemia of chronic kidney disease.Anemia can also be due to other causes, e.g. chemotherapy,autoimmune diseases, inflammation associated with cancer,bone marrow transplantation or HIV infection.Since the early 1990s recombinant erythropoietin has replacedtime-consuming, costly and risky blood transfusions, previous-ly the standard treatment for anemic patients. Because the hor-mone is a glycoprotein (see illustration), it cannot be producedin bacterial or yeast-cell cultures: the erythropoietin moleculehas several carbohydrate side chains that slow its breakdown inthe body but also modify its intrinsic bioactivity. These side

chains can be attached toproteins only by the synthe-sising apparatus found inmammalian cells. For thisreason, only mammaliancells can be used to producecomplex therapeutic pro-teins. In the case of erythro-poietin, researchers have in-serted the human EPO geneinto Chinese hamster ovarycells, for which reason theproduct is also known asrhEPO (recombinant hu-man erythropoietin).

A glycoprotein: EPO

The hormone erythropoietin (EPO) is used in the treat-ment of chronic anemia. A glycoprotein, it bears severalcarbohydrate side chains. These slow down the break-down of the molecule in the body.

Erythropoietin: the molecule

protein + carbohydrate chain = glycoprotein

carbohydrate chain


Thanks to its numerous uses, rhEPO is one of the top-sellingdrugs worldwide. The Roche Group markets rhEPO under theproprietary names NeoRecormon and Epogin (Chugai).

CERA (continuous erythropoietin receptor ac-tivator) is a chemically modified protein underinvestigation for therapeutic use in patients with

anemia associated with chronic kidney disease and chemo-therapy. Chemically synthesised, it binds to the erythropoietinreceptor differently than EPO and is also broken down more slowly. The result is that EPO receptors on the surface oferythrocyte precursor cells remain permanently active, thusmaintaining the production of new red blood cells. This meansthat patients require fewer intravenous or subcutaneous injec-tions. In renal clinical trials untreated anemic patients can ex-perience a correction of their anemia with one injection twice amonth. Patients who are in maintenance can be managed with asingle monthly injection whether they have reached end stagerenal disease (chronic kidney disease stage 5) or not (typicallychronic kidney disease stages 3 and 4). Less frequent adminis-trations reduce the oscillation in hemoglobin levels outside theoptimal range of hemoglobin as defined by best practice guide-lines, which is often seen with existing short-acting compounds(epoetin, darbepoetin). Such excursions are associated with ad-verse events and considered to contribute to further deterio-ration of cardiac and renal functions. It is believed that less fre-quent administrations represent a significant gain in quality oflife for patients but also allow overworked nephrologists andnurses to concentrate on the other serious medical conditionsaffecting many of these patients such as hypertension, diabetes,chronic heart failure and obesity. CERA is being developed forthe treatment of anemia in patients with chronic kidney diseaseand for the treatment of anemia associated with cancer.

Improved efficacy of proteins can be achievedwith the help of specific modifications. One method for inducing modifications in proteins is

known as pegylation. PEG (polyethylene glycol) is a very largefamily of molecular entities with a common building block.These molecules vary in size and in shape (branching versus linear). It is essential to select the proper moiety that will confer

A new innovative drug:CERA

The principle of pegylation: Pegasys

44

to the active protein the de-sired properties. The choiceof linker is also very impor-tant as its rigidity (or lackthereof) will influence theultimate properties of thenew medicine. Roche hassuccessfully applied thisprinciple to develop a drugfor the treatment of hepati-tis C and B. In this methodthe drug is enveloped in oneor two highly branchedmolecules of polyethyleneglycol. These PEG barriersprotect the molecule fromthe protein breakdown ma-chinery in the body’s cells,thus prolonging the drug’sactivity.Pegasys is a modified inter-feron-alfa-2a molecule. Thisnatural chemical messenger

inhibits hepatitis C virus replication. It has been used for de-cades for treating hepatitis C, a widespread infection which causes inflammation of the liver. To date no treatment exists thatis able to eradicate the hepatitis C virus from the body.The standard treatment requires at least three interferon injec-tions per week. As a result, drug levels in the patients’ blood-stream undergo significant fluctuations in a two-day rhythm,giving rise to side effects and limit efficacy. It is also consideredthat fluctuation is instrumental promoting the appearance ofresistant viruses.Thanks to a carefully selected pegylation with the appropriatebond with the protein, Pegasys is broken down much more slow-ly than simple interferon and therefore remains active in thebody longer. This has several advantages for patients: Firstly,Pegasys only has to be given once weekly. Secondly, the dose does not have to be adjusted gradually – at least not to the samedegree – according to the patient’s age, hepatic status and renalfunction, a time-consuming process. Thirdly, interferon levelsin the bloodstream are subject to less fluctuation, making theside effects more tolerable and improving patient compliance.

A pegylated protein: Pegasys

The molecular structure of interferon with protective PEGshell (red). Pegylation with polyethylene glycol (PEG)slows the breakdown of the drug in the body, thus per-mitting longer intervals between injections and greatlyenhancing the drug’s efficacy.


And fourthly, the efficacy of the drug is increased thanks to themolecular modifications and the relative constancy of drug levels in the blood. A much larger percentage of patients benefitfrom a long-lasting effect.First approved in 2002, Pegasys quickly became the internation-al market leader in the hepatitis C sector. The drug was also thefirst pegylated therapeutic protein in the world to be approvedfor the treatment of chronic hepatitis B.

Therapeutic antibodies form a relatively newdrug class that was only made possible by modernbiotechnology. Antibodies are components of the

immune system. They recognise foreign structures in the body,e.g. molecules on the surface of body cells, bacteria or viruses,and mark them out for elimination by the immune system. Theybelong to a class of proteins known as immunoglobulins (Ig).Several classes of antibodies exist, each of which has a differentfunction. IgG antibodies are the most abundant. These Y-shapedproteins bear on their two short arms two identical regions thatrecognise a specific foreign structure. The long stem of themolecule interacts with other components of the immune sys-tem, which then initiate destruction of the intruders.In 1972 César Milstein and Georges Köhler, who later receivedthe Nobel Prize, found a way to produce copies of identical antibody molecules in unlimited amounts. Within a few yearsthese so-called monoclonal antibodies had revolutionised bio-logical research, allowing any desired molecule to be reliablyidentified and marked. However, it took more than 20 years formonoclonal antibodies to find widespread use in therapy. Notuntil the late 1990s did researchers succeed in exploiting the specificity of monoclonal antibodies for therapeutic purposes.For example, monoclonal antibodies can be designed to bind tospecific molecules and block their disease-causing effects.However, drug developers were unable to use antibodies ob-tained from standard mammalian (usually mouse) cells. Becausethe molecules differ in structure from one species to the next,mouse antibodies proved to be of very limited benefit in humans. In addition, dangerous side effects occurred. Re-searchers therefore turned their attention to what are known aschimeric and humanised antibodies, where only the recognitionregions are based on mouse genes. It is now possible to insert allthe human genes required to produce antibodies into laboratory

A new drug class: therapeutic antibodies

46

animals. As a result, medical science now has at its disposal anarsenal of therapeutic antibodies that are structurally identicalto their natural counterparts in the human body.

A good example of a highly effective chimeric an-tibody is the Roche product MabThera/Rituxan(rituximab). MabThera/Rituxan is the world’sfirst monoclonal antibody for the treatment of

indolent and aggressive forms of non-Hodgkin’s lymphoma(NHL). NHL is a type of malignant lymphoma, i.e. a cancer ofthe lymphoid tissues. The drug rituximab was developed to bind

Example MabThera: hope for patients withlymphoma

A new drug class: therapeutic antibodies

Mouse Chimeric Humanised Human

Each antibody bears on its two short arms identical regionsthat recognise a specific foreign structure, to which theybind. This principle is exploited in therapeutic antibodies inorder to recognise pathogenic and other substances and

render them harmless. Whereas early therapeutic anti-bodies were still partly derived from mouse genes (yellowsegments), therapeutic antibodies of the latest generationare indistinguishable from their human counterparts.

Mouse with human Ig genes

Gene transfer

Human monoclonalantibodies

Immunisation

Fully human therapeutic antibodies are obtained by infect-ing a transgenic mouse that carries human genes for the

production of immunoglobulins (Ig) with the target for theantibodies that one wishes to produce.


specifically to the surface of lymphoma cells. The target proteinof this therapeutic antibody is a receptor located on the surfaceof B lymphocytes (white blood cells), which in lymphomas growuncontrollably. The antibodies bind to the cancer cells, markingthem out for destruction by the body’s immune system. At thesame time rituximab makes the cells more susceptible to certainforms of chemotherapy, thus improving the survival chances ofpatients who previously had no further therapeutic options fol-lowing unsuccessful chemotherapy.

Therapeutic antibodies such as rituximab helpthe patient’s immune system to home in on dis-eased target cells. To achieve this, biotechnology

has exploited a natural function of antibodies: the long segmentof antibodies (the FC region, ‘c’ standing for ‘constant’) interactswith other components of the immune system to initiate a spe-cific immune response against the recognised foreign substance.This immunological effect can be boosted by skilfully modifyingthe molecule, e.g. by adding further sugar molecules to the FCregion of a therapeutic antibody (see box p. 48).

A chimeric monoclonal anti-CD20 antibody

Variable regions

Light chain

Variableregion

Constantregion

Heavy chain

V

C C

C C

CC

V V V

Chimärer monoklonaler Anti-CD20-AntikörperKonstante Region C (Mensch) mit variablen Regionen V (Maus) gekoppelt

A turbocharger for the immune system

The constant region (C) of a human antibody is combined with the variable regions (V) of a mouse antibody.

48

The next step was to link therapeutic antibodieswith small molecules to form what are known assmall molecule conjugates. Antibodies have a disadvantage that they share with other thera-

peutic proteins: they are too bulky to penetrate into the interiorof cells. Potential targets are therefore limited to molecules lo-cated outside of or on the surface of the body’s cells. By contrast,many conventional, chemically synthesised small moleculedrugs can readily pass through the cell membrane to targets within the cell or even the cell nucleus.Small molecule conjugates combine the specificity of therapeu-tic proteins – especially antibodies – with the broad target rangeof small molecules. To produce them, researchers have de-veloped complexes, or conjugates, consisting of therapeuticantibodies coupled to low-molecular-weight drugs. In such con-jugates the antibody’s role is to ferry the actual drug directly toits target in the body.An important area for the use of conjugated antibodies is can-cer therapy. Drugs commonly used to destroy cancer cells alsoattack healthy cells in the body. This results in numerous side ef-fects, some merely unpleasant (e.g. the typical side effect of hairloss) and some life-threatening (e.g. when the drugs attack vitalcells such as the precursors of red and white blood cells).

Engineering of antibody

Wildtype antibody

Engineered negativecontrol antibody

Cel

llys

isin

%

Antibody concentration (ng/ml)

120

100

80

60

40

20

00 10 20 30 40 50

Enhanced immune response: modified therapeutic antibodies

Specifically modified therapeutic antibodies can induce a five to eight times stronger immune response (e.g. lysis of tumorcells) than natural antibodies.

The next drug generation: small molecule conjugates


Among other things, Rocheis working on conjugatedantibodies that will bindspecifically to structures(e.g. a receptor) on the sur-face of cancer cells. Oncethis occurs, the entire conju-gate is internalised in thecell. In cancer cells the anti-body is digested and releasesthe small molecule, whichthen destroys the diseasedcell. In this way cancer cellscan be specifically targetedand adverse effects onhealthy cells can be minim-ised.Small molecule conjugatesrepresent a new generationof biopharmaceuticals. Ifthe findings from tests areborne out, the latest gener-ation of these drugs couldsignal a breakthrough notonly in cancer therapy but inmany other therapeuticareas where medical sciencehas hitherto had to contendwith severe side effects caused by the unspecific actions of conventionaldrugs.



Presentations at a media conference: The Roche Group – one of the world’s leaders in bio-tech, Basel, November 2004http://www.roche.com/home/media/med_events

Schmid RD: Pocket Guide to Biotechnology and Genetic Engineering. Wiley-VCH, Weinheim,2002

Biotechnology Information Secretariat: http://www.i-s-b.orgAssociation of Research-Based Pharmaceutical Manufacturers: http://www.vfa.deMedia information of the Roche Group, 2002-2006: http://www.roche.com/home/media.htm

orzelle

Cancer cell

Linking of antibodies to small drugmolecules (small molecule conjugates)

Complex bindsto cell

Entire complex inside cell

Cancer cell

Complex carriesdrug into cell

Drug killscancer cell

Cancer cell

Conjugated antibodies combine the specificity of thera-peutic proteins with the broad target range of small mole-cules. The antibodies target a specific structure on the surface of cancer cells. Once the antibody has located itstarget and bound to it, the conjugated small molecule drugis released, penetrates the cancer cell and kills it.

Treatment begins with diagnosis

Molecular diagnosis providesmodern medicine with an entirelynew tool. As well as the therapeuticpossibilities it offers, modern biotechnology can lead to novel waysof combating diseases such as diabetes, cancer and rheumaticdiseases. For example, early andspecific diagnosis, and also tests thatcan monitor treatment and thecourse of an illness, can result inmore effective treatment of patients.

52

Medical science can only be as good as its under-standing of disease processes. The more doctorsknow about the causes of diseases, the more ef-

fectively they can deal with them. This realisation may soundsimple, but translating it into practice remains difficult, be-cause the critical part of treatment is often finding the rightdiagnosis. It is precisely in this area that biotechnology has madetremendous strides in recent decades.Thus, for example, alleviating pain should not be the only goalwhen treating patients with chronic pain. It is only when thesource of the pain has been identified that steps can be taken tocounter it in the long term. Yet pain patients in particular oftenhave to undergo veritable medical odysseys as a result of uncer-tain diagnoses, failed treatments and ever increasing pain. De-spite having similar symptoms, painful rheumatic diseases can

be caused by very differentdisorders, each of which re-quires a distinct treatment.Whether a treatment is suc-cessful therefore ultimatelydepends on a rapid, precisediagnosis.The picture is similar withcancer, where the sheer va-riety of causes requires a newdiagnostic approach. A tu-mor can remain completelyharmless or rapidly developinto aggressive malignancy,depending on the tissue oforigin and genetic pattern ofthe cells as well as the immu-nological constitution andlifestyle of the patient.Which therapy is the rightone for an individual casedepends largely on these fac-tors, and whether those fac-

tors are identified in time can spell the difference between lifeand death. In this respect, biotechnology has devised new meansfor identifying the precise molecular causes of such disorders.

The biotechnology marketgrows apace

Terms

Biopharmaceuticals drugs manufactured using biotech-nological methods.Enzymes biocatalysts; proteins able to facilitate and acceler-ate chemical reactions.Epigenomics the science concerned with the variable modi-fications of DNA and their effects.Genome the (largely unalterable) complement of all genes ofan organism.Genomics the science concerned with the form, functionand interaction of the genes of an organism.Genotype the variants of a given gene possessed by an organism; as a rule a human can have no more than two variantsof each gene – one from the father and the other from the mother.Phenotype the physical constitution of an organism withregard to a specific trait as determined by the interaction of itsgenotype and the environment.Proteomics the science that deals with the form, functionand interactions of the proteins of a biological system.Recombinant proteins proteins obtained by recombiningDNA, e.g. by inserting human genes into bacterial or mamma-lian cells.Single nucleotide polymorphisms (SNPs) differencesin individual building blocks (base pairs) randomly distributed inthe genome which are inherited from generation to generation.

Treatment begins with diagnosis 53

In conventional medical diagnostics doctorsmainly observe their patients’ manifest signs andtheir excreta. For thousands of years experienced

doctors have gathered crucial information about their patients’health from visible wounds, bone structure, posture and thecolour of the skin, eyes, blood and excrement. Other methods ofconventional diagnostics include palpation, for example formuscular indurations or masses, and an in-depth exchange ofinformation between the doctor and patient. Modern medicalscience has supplemented this range of methods with imagingtechniques, e.g. x-rays, computed tomography and magneticresonance imaging. These routine methods still form the basisof every successful therapy – even if they often prove inadequatefor the diagnosis of many diseases.

The next level of medical diagnostics concernsthe internal structure of the body and focusesspecifically on the functions and interactions of

organs and tissues. In this area as well, modern diagnostic tech-niques such as sonography, computed tomography, intestinalendoscopy and arthroscopy have added to the arsenal of con-ventional examination methods. Nevertheless, tried-and-testedexamination methods remain important.

Conventional diagnosticsat the body level

From body structure to the genome: the diagnostics playing field

DNA

GenotypeGene alterationsPCR technologyGenomics

PhenotypeProteins ProteomicsDifferences between ‘healthy’ and ‘diseased’

Cellular organisationDiabetes

Traditionalmedicineimaging

Proteinnetworks

Modifiedproteins ProteinsTissueOrgans Body

Diagnostics at the organand tissue level

54

Take liver biopsy tests, for example, which involve the removalof liver cells through a long needle inserted into the abdominalwall. Examining these cells closely under a microscope is still themost reliable way to identify diseases of the liver. However, inmost cases biopsy is the final link in a diagnostic chain that startswith laboratory tests.As early as 1970 Boehringer Mannheim (BM), which was takenover by Roche in 1998, developed the gamma-GT test, which isused to measure a metabolic enzyme whose levels are elevated inpatients with inflammation of the liver. This sensitive, noninva-sive technique is now an important laboratory test for the earlydetection of hepatic infections: Only in cases where the gamma-GT concentration is significantly elevated or slightly elevatedover an extended period do doctors order further tests such assonography or a liver biopsy.Such tests became possible only with the advent of enzymes pro-duced by biotechnological means. Thanks to such screeningtests, which do not require surgical intervention and produce re-liable results quickly and easily, doctors are now able to recog-nise and treat many more functional disorders of organs and tis-sues. An added benefit is that if screening test findings arenegative, patients are spared an unnecessary and relatively riskyintervention.

In the case of diabetes, the advantages of quicktests go even further: such tests are actually an in-tegral part of diabetes therapy. Diabetes is due ei-

ther to deficient insulin production by pancreatic cells or to anacquired insensitivity of certain body cells to insulin. In eithercase, the detection and treatment of the disease require regularmonitoring of blood glucose levels with the help of enzymesproduced by biotechnological methods. On the basis of thesemeasurements, diabetics are able to determine when and howmuch insulin they should inject.Until just a few decades ago diabetics had to visit their doctor forsuch tests, making it all but impossible to adapt insulin doses in-dividually. Instead, diabetics had to adapt their diet and lifestyleto a standard therapy. Today, by contrast, modern diagnostic devices like Roche’s Accu-Chek allow diabetics to check theirblood glucose levels themselves at any time and thus adapt theirtreatment to their individual needs. This advance has not onlyenhanced the quality of life of diabetics but has also led to a

Diabetes: better quality oflife, fewer complications


marked reduction in complications due to inadequate diabetestherapy.The enzymesrequired for measuring such blood or urine param-eters were produced as early as 1954 by Boehringer Mannheimusing conventional biotechnological methods. To this end,microorganisms were cultivated in 50,000-liter batches. Fromthe biomass thus produced enzymes such as glucose oxidase andcholesterol oxidase were obtained for measuring blood glucoseand cholesterol levels, respectively.

Modern biotechnology has recently opened upwhole new prospects in the field of diagnostics:the search for the molecular causes of diseases.

This line of enquiry is based mainly on the sciences of genomics,which deals with our hereditary material, and proteomics,which deals with its manifestations in individuals at the proteinlevel. This has led in recent decades to many fresh insights, withthe result that we now know far more about the development,progression and treatment of most diseases than was the case ageneration ago.In fact, these profound insights into molecular relationshipswithin our bodies allowed the term ‘disease’ to be comprehen-sively defined for the first time as a state caused by an alteredflow of information in a biological system.

This comprehensive definition of disease formsthe basis for molecular diagnoses, which can be

divided into two groups:❚ Genotype: DNA, which makes up our hereditary material, acts

as the main store of information in biological systems. Gene-tic factors not only causehereditary diseases butare also implicated in thedevelopment and pro-gression of noninheriteddiseases. The genotypecan make a person sus-ceptible or resistant tocertain disorders, endowhim/her with a strong orweak immune system

Molecular diagnosis:What is a ‘disease’?

Genotype and phenotype

What is a ‘disease’?

The ‘disease’ state is the consequence of an al-tered flow of information in a biological system.Information carriers include proteins.Only if we know what proteins are present in abiological system and at what concentrationscan we describe the balance between healthand disease. Proteomics is a powerful tool fordescribing protein variety.

56

and determine how he/she responds to drugs. Researchersworldwide are searching for the genes and gene segments re-sponsible for these phenomena with a view to developingtests that will enable doctors to detect such predispositions intheir patients. Such tests would make it possible to delay oreven prevent the onset of disease and to select the best treat-ment for a particular patient.

❚ Phenotype: It is not always possible to draw direct conclu-sions from the genome about how a genotype is expressed,i.e. the phenotype. Various and variable signals help deter-mine whether, how and how frequently individual genes areactually read. Only at the level of gene products, namely pro-teins, can a patient’s state of health be accurately determined.

Large and small differences in the genome makeeach of us a unique individual – not only in ap-pearance and behaviour but also in terms of our

health risks and response to treatments. Because the reasons forthese differences were poorly understood, medical science was

unable to respond to themexcept to a very limited ex-tent. Finding the right treat-ment for a given patient wastherefore often a matter oftrial and error. However, ifthe genetic basis of individu-ality in terms of disease andtreatment is known, doctorswill be better able to tailortherapies to patients’ needs.But our genome is not as im-mutable as was believed formany decades: By modifyingtheir DNA, cells are able todisable or activate specificgenes or even alter the waythey are read (see box).A person’s genotype istherefore not the immutablelink in the informationchain of biological systemsthat it was long thought to

Changeable hereditary material: methylation

Methylation is the most important long-term, but not ir-reversible, modification of the hereditary material in ourbody’s cells. In this process, small organic moleculesknown as methyl groups are attached to the backbone ofthe DNA molecule. DNA sections marked in this way areno longer read and therefore are not expressed in theform of proteins. Although DNA methylation patterns arehereditary, they can be altered by environmental condi-tions –unlike the DNA itself. This enables cells to respondswiftly and on a long-term basis to changes in their envi-ronment.

N

C

N

C

CC

O N

H

H

H

Genomics: diagnosticgoal information store


be. This fact makes the hereditary material all the more impor-tant for the molecular diagnosis of diseases. If a person’s life cir-cumstances are reflected at the DNA level, this could offer keyinsights to help find the right treatment. Moreover, gene modi-fications of this nature may present suitable sites for drugs to actupon.

Molecular diagnostics is especially advanced inthe field of single nucleotide polymorphisms, orSNPs for short (pronounced ‘snips’). These are

randomly distributed variations of individual building blocks ofthe genome, which can differ between individuals. Most SNPshave no effect on the phenotype, i.e. the physical constitution ofan individual. However, if one building block is replaced withanother within a gene, the consequences can be far-reaching.Often in such cases the corresponding gene product – usually aprotein or a protein complex – is altered with the result that itacts faster or slower or reacts differently to external influences.In extreme cases, the exchange of a single building block canrender the gene product useless, usually resulting in a severehereditary disease.That’s why researchers, doctors, and the pharmaceutical indus-try have been paying greater attention to SNPs in recent years.Many polymorphisms are widespread in the population withoutcausing any actual damage. The effects are not noticeable unlessthe affected gene products perform important functions, i.e. ifthey favour the development of certain diseases or are involvedin the breakdown of toxins or drugs in the body. In such casesthe choice of the right drug – and above all its dosage – can de-pend on the SNP variants a patient carries.

The first diagnostic product that allows patientsto benefit from these findings has been commer-cially available in Europe since 2004. Roche’s Am-

pliChip CYP450 test rapidly identifies the most important vari-ants of two genes involved in the breakdown of many drugs. Ifthe drug breakdown process proceeds too quickly, it leads to aloss of drug efficacy; if it proceeds too slowly, it leads to an in-creased risk of side effects. Doctors can use the AmpliChip testto predict how their patients will react to a drug and adjust theirtherapy optimally.

Markers of individuality:SNPs

New to medical science:DNA chips

58

The AmpliChip CYP450 testis an example of a new groupof tools in modern diagnos-tics, the DNA chip. Most are thumbnail-size siliconwafers on which short frag-ments of DNA are deposit-ed. If a solution containinglonger DNA strings, for ex-ample one obtained by PCRamplification of DNA ex-tracted from a patient’sblood cells, is applied to aDNA chip, the fragmentsbind to complementary, i.e.mirror-image, sections ofthe longer DNA strings. Inthis way specific genes, generegions and even SNPs canbe detected.

Another technique used for detecting DNA seg-ments is the polymerase chain reaction, or PCR.The PCR is used to make any desired number of

copies of specific DNA segments. This is also an important pre-requisite for DNA analysis on the AmpliChip CYP 450. Since itsinvention in 1982, the PCR has been a major factor in the rise ofbiotechnology. No genetics laboratory could do without it, andgenome sequencing projects, of which there are many, would beinconceivable without it. It has even revolutionised forensicscience with the introduction of genetic fingerprinting, which isbased on the PCR. In medical science the technique forms thebasis of nearly all genetic investigations:❚ Scientists engaged in research into the molecular basis of dis-

eases depend on the PCR. DNA, as a ubiquitous quantity inthe information system of all life forms, can only be analysedwith its help. Many pioneering findings are based, for ex-ample, on the Human Genome Project, in the course ofwhich the human genome was sequenced. A number of

A tool for genetic diagnostics: the PCR

The first pharmacogenomic product: AmpliChip CYP450

The AmpliChip CYP450 test, which was launched in Eu-rope in 2004, was the first commercially available phar-macogenomic product. (Pharmacogenomics describeshow the effects of drugs depend on a patient’s genome.)The AmpliChip can distinguish between the most impor-tant variants of two genes involved in the breakdown ofmany drugs, enabling doctors to determine the right dos-age of a drug for a given patient even before the treat-ment is started.


follow-on projects are now looking for genetic variation relevant to the development and treatment of diseases.

❚ The PCR is also important in drug development. Every bio-tech drug has to undergo several development phases inwhich the PCR is required. To manufacture a therapeuticprotein, for example, it is first necessary to identify the cor-responding gene in the human genome – an extremely diffi-cult task without the PCR. The gene is then transferred to aspecial cell line, and this step too requires DNA ‘amplifica-tion’ with the PCR to determine if the gene was successfullyinserted.

❚ In most genetic testing the PCR is needed to make copies of apatient’s DNA so that enough of it is available to be analysedby other methods. In this way, patients can be tested for sus-ceptibility to a certain hereditary disease, for example. Pre-natal and preimplantation diagnostic tests also make use ofthe same process. And finally, the PCR can be used to quick-ly and accurately detect SNPs and other medically relevantgenetic variations. Molecular diagnostic tests at the DNA lev-el will continue to rely on the PCR as an essential tool for theforeseeable future.

As the most important group of biological sub-stances, proteins (gene products) are key targetsof molecular diagnostics.

❚ Various metabolic proteins serve as the targets of diagnostictests, because their activity may indicate the presence of cer-tain diseases. One example is the previously mentioned gam-ma-GT test to detect liver damage.

❚ Restriction enzymes (used for accurately cutting DNA stringsinto shorter lengths) and proteases (which cut proteins atspecific sites) are basic tools used by molecular biology insti-tutes everywhere. Molecular diagnostics uses these tools,among others, to identify genes and proteins associated withdiseases.

❚ Antibodies are another powerful tool used in modern biolo-gy. They form the basis of the ELISA, the most important method for identifying biomarkers in solutions and bodyfluids (see box, p. 60).

❚ As active substances, proteins are attracting increasing atten-tion. In comparison to most conventional drugs, therapeuticproteins can be used to target the molecular causes of many

Proteins – informationcarriers par excellence

60

diseases with great specificity, making a precise diagnosis ofthe underlying disorder all the more important. Particularlyin the field of biotechnology, treatment and diagnosis go to-gether hand in glove.

A protein that is suitable for detecting altered in-formation flow in a biological system is called a

biomarker. The aim of diagnostic research is to find such dis-ease-specific proteins (DSPs) and develop tests to detect them inpatients’ body fluids, e.g. blood, urine or saliva (see box abovefor a description of the ELISA principle).The main areas of research are the major prevalent diseases forwhich only unsatisfactory diagnostic tests and therefore treat-ment options are available – mainly malignant diseases such asintestinal, lung or breast cancer, and systemic diseases such asrheumatic diseases and diseases affecting the central nervoussystem, e.g. Alzheimer’s disease. What all these disorders have incommon is that they lack a clearly defined cause. Rather, they arecaused by an unfortunate chain of multiple genetic and envi-ronmental factors. It is therefore all the more important to rec-ognise the early phases of these diseases and break the chain

Proteins as biomarkers

Specific antibodybound tosolid support

Analytes (= antigens presentin serum)Specific binding of antigento antibody

Unbound analyteswashed away

Second antibody withlabel attached facilitatingdetection

One principal method of measuring biomakers in peripheral fluid: ELISA Enzyme-Linked Immunosorbent Assay (simplified)

1 2 3 4

ELISA (enzyme-linked immunosorbent assay) is one of theforemost tools in the search for biomarkers. In this methodantibodies against the specific protein being sought areattached to a carrier (1). Any biomarker molecules presentin the test solution bind to the antibody (2). The surplus so-

lution is washed away (3) and a solution with labelled an-tibodies, which also bind to the biomarker, is added (4). Byobserving the labels it can be determined whether and howmuch biomarker the sample contains.


through targeted treatment. If the disease does develop, earlyand specific treatment is often life-saving, and this, in turn, de-pends on finding the right diagnosis. Biomarkers can thereforebring about progress at four levels:❚ Screening markers can help even in the asymptomatic phase

to detect the start of the disruption of information flow thatis responsible for disease. For this purpose entire populationgroups are examined, e.g. everyone above a certain age witha familial predisposition or carriers of other broadly definedrisk factors. To ensure that as many people as possible bene-fit from such preventive examinations, the proceduresshould be as painless, simple and safe as possible. This cate-gory includes various cancer screening tests.

❚ Prognostic markers indicate how fast a disease is progressingin an individual. Forms of the same disease that differ in theirvirulence often require entirely different therapies. For ex-ample, early rheumatic symptoms are usually treated by con-servative methods such as physiotherapy or the use of anti-inflammatory ointments and drugs. In especially rapidlyprogressing cases, aggressive therapeutic intervention maybe indicated, even in early stages, despite an increased like-lihood of side effects.

Genomics

DNA

What information is stored in abiological system?

Predisposition

Small changes in a person’s DNA (SNPs = SingleNucleotide Polymorphisms) can predict the onset ofdisease

Patient stratification

How effectively will a particular drug be metabolised?

Proteomics

Proteins

What pieces of stored information are beingexpressed at a given point in time?

What proteins are present in ‘health’, and whatones are present in ‘disease’?

SNPs versus DSPssingle nucleotide polymorphisms disease-specific proteins

Target

Molecular diagnosis: new prospects opened up by genomics and proteomics

62

❚ Stratification markers enable doctors to predict whether andhow well a patient responds to a certain type of drug. This de-pends largely on genetic variations of drug metabolising en-zymes, which in most cases can be detected at the gene levelusing modern techniques, Roche’s AmpliChip CYP450 testbeing a prime example.

❚ Efficacy markers, finally, describe how well a drug is workingin an individual patient. Here again it is often not enough torely on the improvement of symptoms. Only at the molecu-lar level can the effects of many drugs really be assessed. Forexample, the success of HIV therapy must be continuouslymonitored in order to be able to transfer a patient to otherdrugs if the virus starts replicating again rapidly because ithas developed resistance to the drugs being used.

The fight against cancer is one of the greatest chal-lenges facing modern medicine. According to anestimate by the International Agency for Researchon Cancer, part of the World Health Organization,

over 1.7 million people died from cancer in Europe in 2004,mostly lung cancer, followed by intestinal and breast cancer.Intestinal cancer is one of the most underestimated forms. Al-though screening programs are in place in most industrialisedcountries, people do not avail themselves of them to the neces-sary extent. Yet up to 90 percent of all fatal cases of intestinalcancer, says the German Felix Burda Foundation, could be pre-vented in the space of ten years by instituting a program of reg-ular endoscopic checks. The major misgiving is that althoughintestinal endoscopy is effective, it is also unpleasant and, beinginvasive,not without its risks.To date there is no screening meth-od that is able to identify high-risk patients simply and safely.The early detection of intestinal cancer still relies for the mostpart on the results of an occult blood test, which detects hidden(‘occult’) blood in the stool. Depending on the study con-cerned, however, this test fails to identify up to half of positivecases. In addition, one in five patients proves to be healthy aftersubsequent endoscopy. Given the large number of patients withintestinal cancer, medical researchers are therefore working in-tensively on alternatives to the occult blood test. Strong hopesare pinned on biotechnology to find an answer. Suitable screen-ing tests based on protein biomarkers could become availablewithin just a few years.

Example of cancer prevention: early intesti-nal cancer detection


Different causes, similar symptoms: Arriving atan accurate diagnosis is especially difficult in thecase of complex diseases, which include rheumat-ic diseases. It is now known that over 100 different

disorders – some degenerative, some inflammatory – are sub-sumed under the umbrella term ‘rheumatism’. That alone shows to what extent doctors have to depend on modern diag-nostic testing, especially since the right treatment often dependson the actual cause of the pain symptoms.The most common inflammatory form of rheumatic disease isrheumatoid arthritis (RA). RA is thought to be due to an au-toimmune reaction, where the immune system attacks tissues ofthe body itself, often causing destruction of the joints. Womenare affected more often than men. Patients usually have to con-tend with severe pain and considerable impairment of move-ment. The causes of the disease are still unknown, but it appearscertain that genetic predisposition, previous diseases and prob-ably also lifestyle are all factors.

Example of a complex disease: rheumatoid arthritis

Marker selection = Search for additional information

1

3

42

Markers 2, 3, 4 provide additional information not provided by marker 1

By themselves, markers 2, 3, and 4 would not be specific enough to be used as stand-alone markers

1% of the information needed to completely describe a disease

Often one biomarker is not enough to detect a disorderwith certainty, particularly in the case of complex diseasessuch as rheumatoid arthritis (circle 1). For this reason re-searchers look for an optimum combination of markerswhich together describe as many disease factors as possible (circles 2 to 4). This approach is based on amathematical model known as Regularised DiscriminantAnalysis (RDA).

RDA is less concerned with testing the suitability of individ-ual markers than with determining how much additionalinformation each provides. The best marker combinationstherefore do not necessarily contain the best individualmarkers.

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The fact that diverse factors contribute to the development andprogression of rheumatoid arthritis is also reflected in the search for suitable biomarkers. Not a single protein is knownwhich can be used to diagnose a disease with absolute reliability– a fact that has become increasingly clear in recent years. All themolecular candidates so far tested either do not occur in all pa-tients or occur also in other inflammatory diseases. Biologistshave therefore teamed up with mathematicians to develop a model to help in the search for an optimum combination ofmultiple markers (see box, p. 63).

Biotechnology has made key contributions notonly to therapy but also to diagnostics. Armedwith molecular diagnostic tests at the gene andprotein levels, doctors can already search much

more effectively for the causes of a patient’s illness and adapt thetreatment accordingly, and not just in the early phases. Bio-marker analyses can also be used to monitor the success of atreatment. Diagnostics, treatment and treatment monitoringare evolving together, and research in this area is being inten-sively pursued.An example that illustrates this development is the detectionand treatment of HIV-positive individuals. The behavior of theHI virus is highly variable: Without treatment some people in-fected with the virus develop AIDS symptoms within just a fewmonths, while others remain healthy for decades. The reasonsfor this are varied, ranging from differences in the immune re-sponse between individuals to significant variations in the ge-nome of the virus.Biotechnology therefore figures prominently in the diagnosisand treatment of AIDS. Thus, the HI virus is routinely detectedindirectly with the help of specific antibodies which are usuallypresent in sufficient quantities for testing some six to twelveweeks after infection.The antibodies therefore serve as biomark-ers for the infection.However, as early as 1996 Roche introduced a far more sensitivediagnostic test. At the time, the Amplicor HIV-1 Monitor testwas the first diagnostic PCR test, and it is still used today for de-tecting viral RNA directly. PCR has two advantages: First, HIVRNA can be measured with reliable and consistent quantitationdown to 50 copies/mL. This sensitivity enables physicians to ac-curately quantitate and track HIV viral load levels – even at ex-

Prospects: diagnosticsand treatment evolve together


tremely low levels. This en-ables physicians to confi-dently initiate proper thera-peutic regimens that willdeliver more effective viralload suppression, and tran-sition to alternate therapiesif and when viral outbreakshould occur. Secondly, PCRtests e.g., the Amplicor HIV-1 Monitor test Version 1.5,provide quantitation of avariety of HIV-1 subtypes.Since the HIV-1 virus iscomprised of multiple sub-types, accurate quantitationof HIV-1, regardless of sub-type or genetic diversity, iscritical to ensuring effectivepatient management.Because HI viruses changevery rapidly and become re-sistant to the drugs used,HIV therapy must be continuously adapted to the individual pa-tient’s needs. HIV-positive patients usually take a cocktail ofthree or four different drugs to keep the viruses in their body un-der control. Modern molecular diagnostic methods are there-fore needed not only at the start of the therapeutic process butthroughout treatment. The treatment regimen must be contin-uously adapted to the patient’s current viral status, and in thecase of HIV that means for the rest of the patient’s life.

As in the case of HIV, the diagnosis and treatmentof other diseases are also merging together. Themore specifically a drug is directed against the

cause of a disease, the more important it is for doctors to identify the cause accurately. For pharmaceutical companiesthat are active in both areas, this development has opened up aunique opportunity: Now diagnosis and therapy can be con-sidered together to help patients individually.Progress in the treatment of complex diseases in particular shows that molecular diagnostics holds new promises for med-

Treatment begins withdiagnosis

Diagnosis and treatment evolve together:example of HIV

The HI virus specifically attacks those immune cells thatare meant to prevent such attacks, and victims usually dieas a result of an acquired immunodeficiency syndrome,hence the acronym AIDS. Thanks to molecular diagnos-tic methods and new biopharmaceuticals, the pathogenscan now be held in check for decades.To ensure that HIV-positive people in poor countries,where HIV is rampant, can also benefit from this medicaladvance, Roche decided in 2003 to sell its drugs in suchcountries at a ‘nonprofit price’.

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ical science. In this area, biotech drugs and diagnostic agents arenot competing with conventional therapies but in many casespermit specific therapy for the first time where before the aim oftreatment was merely to relieve unspecific symptoms – a realblessing for patients.



Presentations at a media conference: The Roche Group – one of the world’s leaders in bio-tech. Basel, November 2004http://www.roche.com/home/media/med_events

Genes and Health. Roche Series, Basel, 2003Media Information of the Roche Group, 2002-2005:http://www.roche.com/home/media.htmBoyle P, Ferlay J: Cancer incidence and mortality in Europe, 2004. Annals of Oncology, 2005Darmkrebs-Information (Felix Burda Stiftung): http://www.darmkrebs.deMedicine-Worldwide: http://www.m-ww.de

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