Introduction Current Status of Biopharmaceuticals ... · Upstream and Downstream Processing...

Abstract

Biopharmaceuticals represent the fastestgrowing and, in many ways, the most ex-citing sector within the pharmaceutical in-dustry. Within this Introduction we firstconsider what category of product fallswithin the description of a biopharmaceu-tical. An overall global snapshot of the cur-rent status of the biopharmaceutical sectoris then presented, followed by an overviewof upstream and downstream processingoperations typical of protein-based biophar-maceuticals. General trends in product ap-provals are next overviewed and this is fol-lowed by a summary of the main actualbiopharmaceutical products that havegained approval to date (within the EUand/or US). These are considered by prod-uct type, the most significant of which areblood-related products, hormones, cyto-kines, vaccines and monoclonal antibodies.Biopharmaceuticals that have gained ap-proval for veterinary application are thenconsidered, and the Introduction con-cludes by considering some of the innova-

tions and trends likely to influence theshape of the biopharmaceutical sector inthe future.

Abbreviations

AIDS aquired immunodeficiencysyndrome

BHK baby hamster kidneyBHV bovine herpes virusBMP bone morphogenic proteinCHO chinese hamster ovaryCSF colony-stimulating factordsRNA double-stranded RNAEL eurifelEPO erythropoietinEU Europian UnionFSH follicle-stimulating hormoneG-CSF granulocyte colony-stimulating

factorGH growth hormoneGM-CSF granulocyte macrophage colony-

stimulating factorHAMA human anti-mouse antibodiesHBsAg hepatitis B surface antigen

1

Modern Biopharmaceuticals. Edited by J. KnäbleinCopyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31184-X

Current Status of Biopharmaceuticals: Approved Productsand Trends in ApprovalsGary Walsh

Introduction

HER2 herceptinHIV human immunodeficiency virusIB inclusion bodyIFN interferonIL interleukinLH luteinizing hormonemAb monoclonal antibodyMS multiple sclerosisNPV nuclear polyhedrosis virusPhRMA Pharmaceutical Research and

Manufacturers of AmericaPDGF platelet-derived growth factorPEG polyethylene glycolr recombinantrh recombinant humanRNAi RNA interferencesiRNA small interfering RNATNF tumor necrosis factortPA tissue plasminogen activator

1What are Biopharmaceuticals?

What exactly is a biopharmaceutical? Theterm has now become an accepted one inthe pharmaceutical vocabulary, but it canmean different things to different people.A clear, concise definition is absent frompharmaceutical dictionaries, books on thesubject, or even in the home pages of reg-ulatory agencies or relevant industry orga-nizations. The term “biopharmaceutical”appears to have originated in the 1980s,when a general consensus evolved that itrepresented a class of therapeutic productproduced by modern biotechnological tech-niques. These incorporated protein-basedproducts produced by genetic engineeringor, in the case of monoclonal antibodies(mAbs), produced by hybridoma technol-ogy (see also Part IV, Chapter 16 and PartV, Chapters 1 and 2). During the1990s theconcept of nucleic acid-based drugs foruse in gene therapy and antisense technol-

ogy came to the fore (see also Part I,Chapters 6–8 and Part VI, Chapter 6).Such products are also considered to bebiopharmaceuticals. On that basis biophar-maceuticals may be defined – or at leastdescribed – as proteins or nucleic acid-based pharmaceuticals, used for therapeu-tic or in vivo diagnostic purposes (see alsoPart III, Chapter 7 and Part V, Chapters4–7), and produced by means other thandirect extraction from a non-engineeredbiological source. By defining the methodof manufacture in negative terminology,proteins obtained by direct extraction fromnative sources are excluded. The descrip-tion also encompasses nucleic acid-basedproducts, be they produced by biotechnolo-gical means or by direct chemical synthesis– as is the case for most antisense-basedproducts (see also Part II, Chapters 7 and8 and Part III, Chapter 3). Also, small inter-fering RNAs (siRNAs) and decoy oligonu-cleotides are of course considered biophar-maceuticals, regardless of how they wereproduced (see also Part I, Chapters 9 and10). However, even that definition is becom-ing somewhat restrictive as, for example,cell-based products become more promi-nent (see also Part I, Chapters 11–15).

2A Global Snapshot

It is now 22 years since approval of “hu-mulin” (recombinant human insulin), pro-duced in Escherichia coli and developed byGenentech in collaboration with Eli Lilly[1]. Lilly received marketing authorizationin the US for the product in 1982. Thismarked the true beginning of the biophar-maceutical industry. Currently some 142biopharmaceuticals have gained approvalfor general human use in the EU and/orUS (see also Part II, Chapter 4, Part VII,

Current Status of Biopharmaceuticals: Approved Products and Trends in Approvals2

Chapter 4 and Part VIII, Chapter 1). Themajor companies marketing one or moreapproved biopharmaceutical products inthese regions are listed in Tables 1–9, aspresented later. Additional relevant com-pany and product information is generallyavailable via the company web pages, thedetails of which are also provided in Tables1–9. Approximately one in four of all gen-uinely new drugs currently coming on tothe market is a biopharmaceutical and thebiopharmaceutical sector is estimated tobe worth in excess of $ 30 billion, approxi-mately double its global value in 1999 [2].

Some 250 million people worldwidehave been treated to date with biopharma-ceuticals. The vast majority are proteinbased – either recombinant proteins ormonoclonal/engineered antibodies [3]. Asmall number of cell-based products con-tinue to gain marketing approval and oneantisense-based product (Vitravene, ISISPharmaceuticals) has also been approvedfor general medical use (see also Part III,Chapter 3). Thus far only a single gene-therapy product has gained approval any-where. The product, trade name Gendi-cine, is a human adenovirus engineered tocontain the human p53 tumor suppressorgene. It was approved in October 2003 inChina, and is indicated for the treatmentof head and neck squamous cell carcino-ma [4].

The major categories of product indica-tions are as one might expect; mirroringmajor killers in the “first” world, includingvarious forms of cancer and heart attacks.The single most lucrative product is thatof erythropoietin (EPO). Combined salesof the recombinant EPO products “Procrit”(Ortho biotech) and “Epogen” (Amgen)have reportedly surpassed the $ 6.5 billionmark. The biopharmaceutical sector hasmatured rapidly over the last decade andis set to continue to grow into the foresee-

able future – and follow-on biopharmaceu-ticals are also coming onto the scene (seealso Part VIII, Chapter 3).

3Upstream and Downstream Processing

Protein-based biopharmaceuticals are in-variably produced by an initial cell culture/microbial fermentation step (upstreamprocessing), followed by product recovery,purification and formulation into finalproduct format (downstream processing)[5, 6]. In the region of 40% of all proteinbiopharmaceuticals approved to date areproduced by recombinant means in E. coli.E. coli displays several advantages as a pro-duction system. Its molecular genetics arewell characterized. It is easy to grow, andgrows rapidly and on relatively inexpensivemedia. Furthermore, high product expres-sion levels are generally achieved. Many ofthe earlier approved E. coli-based productsaccumulate intracellularly in the form ofinclusion bodies. This complicated subse-quent downstream processing as it neces-sitated inclusion body recovery, solubiliza-tion and renaturation of the product. How-ever, some E. coli product expression sys-tems now used promote export of thedesired protein into the periplasmic spacein fully folded format, from where it canbe conveniently recovered without the ne-cessity for cellular disruption (see also PartIV, Chapters 7 and 12).

Several products are produced using en-gineered Saccharomyces cerevisiae. These in-clude various insulin-based products man-ufactured by Novo [7] (see also Part IV,Chapter 13), recombinant hepatitis B sur-face antigen (rHBsAg) produced bySmithKline Beecham as well as a recombi-nant form of the anticoagulant hirudin [8,9]. The majority of approved biopharma-

3 Upstream and Downstream Processing 3

ceuticals are, however, expressed in animalcell lines, mainly Chinese hamster ovary(CHO) (see also Part IV, Chapters 1 and4), but also baby hamster kidney (BHK)cells [10] (see also Part IV, Chapter 12).

Although expression in animal cell linesis more technically complex and expensivewhen compared to E. coli-based systems,eukaryotic cell lines, unlike prokaryoticones, are capable of carrying out post-translational modifications such as glycosy-lation (see also Part IV, Chapters 2 and 7).Many key biopharmaceuticals are naturallyglycosylated. Examples include EPO, many(although not all) interferons (IFNs), bloodfactor VIII (see also Part II, Chapter 3),and gonadotrophins such as follicle-stimu-lating hormone (FSH) and luteinizing hor-mone (LH). In some instances unglycosy-lated versions of a naturally glycosylatedprotein retain the therapeutic properties ofthe native protein and several such prod-ucts produced in E. coli have gained regu-latory approval. A prominent example isthat of “Filgrastim” – a recombinant gran-ulocyte colony-stimulating factor (G-CSF)produced in E. coli and which displays abiological activity similar to the native gly-cosylated protein [11] (see also Part VIII,Chapter 3). Additional examples include“Betaferon” (Schering, Berlin) and “Neu-mega”, nonglycosylated versions of IFN-�and interleukin (IL)-11, respectively, bothof which are produced in E. coli [12, 13].The glycocomponent of many glycopro-teins, however, may be necessary for/im-pact upon the biological activity of a pro-tein, or may influence protein stability orits circulating half-life [14]. In such in-stances, expression in a eukaryotic systembecomes desirable, if not necessary. Whileexpression in lower eukaryotes such as S.cerevisiae is possible, glycosylation patternsmore similar to a native human proteinare obtained if the protein is expressed in

an animal cell line. Although glycosylationrepresents the most common post-transla-tional modification characteristic of suchmodified biopharmaceuticals, some otherforms of post-translational modificationcan also occur and be relevant to the thera-peutic/biological activity of the protein. Aprominent example is that of the anticoag-ulant activated protein C (trade name Xi-gris), which harbors several �-carboxylatedglutamic acid residues and one �-hydroxy-lated aspartic acid residue [15]. Both formsof post-translational modification are nec-essary to underpin full functional anticoag-ulant activity.

Although the research literature con-tains numerous examples of high-level re-combinant protein production using insectcell lines, this approach has not been usedthus far to produce any commercial bio-pharmaceutical for human use. Insect-based systems are, however, employed inthe manufacture of several protein-basedveterinary biopharmaceuticals, as de-scribed later. Insect cell line culture isusually straightforward and inexpensive,and cell growth is rapid (see also Part IV,Chapter 14). Many insect cell lines aresensitive to infection by baculovirus. Uponinfection, up to 50% of all cell protein pro-duced is that of the viral protein polyhe-drin. A common recombinant productionstrategy used therefore entails introducingthe gene coding for the protein of interestinto an engineered baculovirus, under theinfluence of the polyhedron promoter [16].

Downstream processing for virtually allprotein biopharmaceuticals follows a fairlypredictable sequence of events (outlined inFig. 1) [17]. Following initial product recov-ery and concentration, multiple chromato-graphic steps are undertaken (usually be-tween three and six individual fraction-ation steps). While gel filtration and ionexchange are particularly common, down-


3 Upstream and Downstream Processing 5

Fig. 1 Generalized overview of the down-stream processing procedures applied to theproduction of therapeutic proteins. Note thatadditional/alternative chromatographic stepsare undertaken for different proteins and thatviral inactivation steps are often also included.Final product sterilization is usually under-taken by filtration. (Reproduced from [17],by kind permission of the publisher.)

stream processing of several products alsoentail the use of the more bioselectivetechnique of affinity chromatography. Ex-amples include the incorporation of an im-munoaffinity step in the purification of re-combinant factor VIII (see also Part II,Chapter 3) and the use of Protein A affin-ity columns in the purification of someantibody-based products. Several otherdownstream processing procedures employat least one pseudoaffinity step, e.g., thepurification of the veterinary product Vi-bragen � (discussed later), which involvesthe use of both dye affinity and immobi-lized metal affinity chromatography. Prep-arative high-performance liquid chromato-graphy systems have also been included inthe downstream processing of some prod-ucts, including some recombinant insulinsand “Leukine”, a recombinant granulocytemacrophage colony-stimulating factor(GM-CSF) sold by Schering.

Inclusion of a viral inactivation step isgenerally also characteristic of downstreamprocessing procedures, especially if theproduct is derived from an animal cell line[18]. Chromatographic steps are them-selves usually quite effective in removingviruses from product streams (e.g., gel fil-tration will separate most viruses quite ef-fectively from much smaller therapeuticproteins), and, of course, the ability of thedownstream processing procedure to re-move typical animal cell viruses from theproduct will have been tested and validatedduring the purification design stage (seealso Part I, Chapter 6 and Part VII, Chap-ter 1). Amongst the various “safety net”viral removal approaches often included indownstream processing procedures aremultiple repeat filtration through a 0.1 �mfilter, heat/UV treatment or treatment withchemical inactivation agents such as �-pro-piolactone (used for some veterinary prod-ucts at least).

Final product may be formulated in liq-uid or freeze-dried form, and virtually allbiopharmaceutical products are sterilizedby filtration followed by aseptic processing.The most commonly employed excipientsinclude human serum albumin, polysor-bate 20 or 80, mannitol, sucrose or mal-tose, amino acids (usually glycine, arginineor histidine) and a buffer (often citrate,acetate or phosphate based).

4Trends in Approvals

4.1Protein Engineered Products

The bulk of first-generation (early ap-proved) biopharmaceuticals were unalteredmAbs or simple replacement proteinssuch insulin, blood factor VIII and IFNs.An increasing number of modern biophar-maceuticals, however, have been engi-neered in order to tailor their therapeuticproperties. The most common form ofprotein engineering involves the alterationof amino acid sequence in order to achieveone or more of the following goals:� Alteration of biological half-life of the

protein.� Reduction or elimination of issues of

product immunogenicity.� Generation of either fast- or slow-acting

product (e.g., variants of insulin).� Generation of novel, hybrid protein ther-

apeutics.

Second-generation tissue plasminogen ac-tivator (tPA) products represent the mostprominent example of a biopharmaceuticalengineered in order to alter biological half-life [19]. Unmodified tPA, although an ef-fective thrombolytic agent, displays a half-life of some 3 min after i.v. administration.


From a practical standpoint this necessi-tated product administration by continu-ous infusion over a 90-min period. Do-main-deleted engineered variants (tradenames Ecokinase and Retavase), however,display half-lives in the region of 15–20 min, facilitating product administrationby a single i.v. injection.

First-generation mAbs approved formedical use were invariably unmodifiedmurine monoclonals produced by classicalhybridoma technology [20]. These sufferedfrom a number of clinical disadvantages,not least the fact that they were highly im-munogenic when administered to man.Administration elicited the production ofhuman anti-mouse antibodies (the HAMAresponse) that limited efficacy, particularlyupon repeat administration. Murine mono-clonals also displayed relatively short half-lives (typically 30–40 h) when administeredto man and they proved to be poor triggersof human immune effector functions,such as the activation of complement. Themajority of antibody-based biopharmaceu-ticals gaining marketing approval in recentyears are engineered in order to reduce oreffectively eliminate such problems [21,22]. Chimeric antibodies are murine–hu-man hybrid antibodies produced by spli-cing the gene sequences coding for themouse-derived antibody variable regions(which contain the antigen binding site) tonucleotide sequences coding for the con-stant regions of a human antibody (seealso Part IV, Chapter 16 and Part V, Chap-ters 1 and 2). Such chimeric products,when compared to first generation murinemAbs, display significantly extended half-lives (of up to 250 h), are capable of acti-vating human immune effector functionsand are significantly less immunogenic.Humanized antibodies are more exten-sively engineered, effectively produced bygrafting (at the DNA level) the actual mur-

ine antibody antigen binding regions (thecomplementarity-determining regions) intoa human antibody sequence. Such prod-ucts display half-lives essentially identicalto fully native antibodies and are signifi-cantly less immunogenic in man, evenwhen compared to chimeric antibodies.

Engineered insulin analogs representthe most prominent group of second-gen-eration biopharmaceuticals modified in or-der to generate either short- or long-actingforms of the native therapeutic protein(see also Part IV, Chapter 13 and Part VI,Chapter 4). Both long- and short-acting in-sulin, and the engineering principles un-derpinning their generation are consideredsubsequently in this Introduction. A num-ber of novel hybrid proteins have also beengenerated by protein engineering and havegained medical approval for various condi-tions. Examples include “Enbrel” [a tumornecrosis factor (TNF) receptor fragmentlinked to an antibody fragment, indicatedin the treatment of rheumatoid arthritis]and “Ontak” (an IL-2–diphtheria toxin fu-sion protein used to treat cutaneous T celllymphoma).

4.2Engineering via Post-translationalModification

Although the majority of engineered bio-pharmaceuticals have been altered specifi-cally in terms of their amino acid se-quence, several products have now comeon stream that are engineered post-synthe-sis. The changes introduced normally en-tail the covalent attachment of a chemicalgroup to the protein’s polypeptide back-bone or the alteration of a specific pre-ex-isting post-translational modification, i.e.,the glycocomponent of glycoproteins (seealso Part IV, Chapters 2 and 7).

4 Trends in Approvals 7

Thus far, engineering by attachment ofa chemical group has centered aroundPEGylation and, to a lesser extent, attach-ment of fatty acid groups. PEGylationentails the covalent attachment of one ormore molecules of polyethylene glycol(PEG) to the polypeptide backbone [23].PEGylation is technically straightforwardto achieve and chemically activated PEGmolecules for conjugation can be gener-ated in-house or purchased commercially.PEGylation generally increases the plasmahalf-life of a protein, by decreasing the rateof systemic clearance. This decreases thefrequency of administration required, withconsequent economic savings and im-proved patient experience, often along withreduced treatment side-effects.

Native IFNs have relatively short plasmahalf-lives, typically of the order of 4 h.PEGylation can increase this value up to24 h (see also Part VI, Chapter 2). IntronA, for example, is a recombinant humanIFN-�2b produced by Schering Plough. Itis approved for the treatment of variouscancers including leukemia, as well as forsome viral infections such as hepatitis Band C. Generally, administration schedulesentail product injection 3 times a week.PEGylated Intron A, however, need onlybe administered once weekly to achievethe same effect [24].

Levemir is a recently approved insulinanalog (Table 3) whose principal engineer-ing feature related to the covalent attach-ment of a fatty acid side-chain, as de-scribed later.

Thus far, at least two approved therapeu-tic proteins are engineered by modificationof their glycocomponent. Nespo (Aranespin the US) is a recombinant human EPOmolecule expressed in a CHO cell line.Native EPO harbors three N-linked carbo-hydrate side-chains, whereas the engi-neered recombinant product displays five

such carbohydrate side-chains. The in-creased carbohydrate content extends theproduct’s serum half-life significantly,again facilitating once weekly administra-tion [25].

Cerezyme is the trade name given torecombinant human glucocerebrosidase, alysosomal enzyme central to the metabo-lism of glucocerebrosides (glycolipids foundnaturally in the body). Lack of glucocerebro-sidase activity triggers Gaucher’s disease, agenetic condition characterized by accumu-lation of glucocerebrosides, particularly intissue-based macrophages. An obvious ther-apeutic strategy in treating Gaucher’s dis-ease would be the direct administration ofthe missing enzyme. However, injected glu-cocerebrosidase is quickly removed fromthe bloodstream by the liver. Cerezyme isproduced in an engineered CHO cell line.However, downstream processing includesan enzyme-based processing step using anexoglucosidase enzyme. The exoglucosidaseremoves the sialic acid sugar caps of the oli-gosaccharide side-chains. This exposes side-chain mannose residues, which in turn pro-motes macrophage-specific enzyme uptake,mediated by mannose-specific receptorspresent on the macrophage cell surface. Inthis way the sugar engineering promotestargeted delivery of the biopharmaceuticalto the cell type most affected [26] (see alsoPart VI, Chapters 5 and 3, and Part VI,Chapter 1).

5Declining Number of Approvals

A marked decrease in the number of newbiopharmaceuticals gaining marketing ap-proval has become evident over the last 2–3 years, both in Europe and the US (seealso Part II, Chapter 4, Part VII, Chapter 4and Part VIII, Chapter 1). Fig. 2 presents


numbers of biopharmaceuticals approvedwithin the EU per year since the introduc-tion of the centralized European applica-tions procedure in 1995 (see also Part VII,Chapter 5). The first product approved un-der that new centralized evaluation systemwas Gonal F, Serono’s follicle-stimulatinghormone product. It was one of three bio-tech products approved that year. Approvalnumbers peaked in 2000, when 18 newbiopharmaceutical products gained mar-keting approval. In 2001 the European fig-ure was 12. Although 14 distinct productswere approved in 2002, eight of these werevarious formulations of the same active in-gredient (recombinant insulin produced byNovo in an engineered strain of S. cerevi-siae) (see also Part IV, Chapter 13). There-fore, in reality only seven genuinely differ-ent biopharmaceuticals were approved thatyear. The downward trend continued in2003, with the approval of just four bio-pharmaceuticals, although eight productswere approved within the EU in 2004. Thereason this “decline” in number ofapprovals over the last few years is notimmediately apparent, but the large num-bers currently under clinical evaluationlikely renders this decline a short-termphenomenon. New approaches for the ac-

celerated development of biopharmaceuti-cals (see also Part III, Chapter 2 and PartIII, Chapter 1) will most likely alsosupport a healthy increasing trend inapproval.

6Products Approved for Human Use

Here, an overview of biopharmaceuticalproducts thus far approved (within the EUand US at least) is presented. The prod-ucts have been grouped into nine catego-ries: recombinant blood factors, recombi-nant thrombolytics, recombinant insulins,additional recombinant hormones, recom-binant hematopoietic growth factors, re-combinant IFNs and ILs, recombinant vac-cines, monoclonal and engineered anti-bodies, and additional biopharmaceuticals(e.g., cell therapy, gene therapy, siRNA).

6.1Recombinant Blood Factors

A total of seven recombinant blood factorshave gained marketing approval, mainlythroughout the 1990s (Table 1). All aim totreat either hemophilia A or B and all are

6 Products Approved for Human Use 9

Fig. 2 Biopharmaceutical numbers approved for human use per year sincethe introduction of the centralized approvals system in 1995.

produced in engineered animal cell linesin order to facilitate product glycosylation.

Blood factor VIII-based products are in-dicated for the treatment and prophylaxisof patients with hemophilia A (see alsoPart II, Chapters 1–3). This is a geneticdisease characterized by the total lack orpresence only at low levels of blood clot-ting factor VIII. Lack of adequate levels ofthis clotting factor results in prolongedbleeding episodes, occurring sponta-neously or after trauma/surgery.

Recombinant blood factor products haveproven to be as effective as the plasma-de-rived product, without suffering the disad-vantage of the potential risk of transmis-

sion of human blood-borne pathogens (seealso Part II, Chapter 3).

Blood factor IX-based products are indi-cated for the control and prevention ofbleeding episodes in patients with hemo-philia B. Hemophilia B again is a heredi-tary disorder caused by a deficiency in cir-culating levels of coagulation factor IX, re-sulting in impaired blood clotting ability(see also Part III, Chapter 6).

NovoSeven is an unusual product in thatit is (a recombinant form of) human coag-ulation factor VII (FVII). The product isconverted in an autocatalytic fashion intothe active two-chain form (FVIIa) duringits chromatographic purification. NovoSe-


Table 1 Recombinant blood factors approved to date

Product Company Therapeuticindication

Approved

Bioclate (rhFactor VIII producedin CHO cells)

Centeon hemophilia A 1993 (US)

Benefix (rhFactor IX producedin CHO cells)

Genetics Institute hemophilia B 1997 (US, EU)

Kogenate (rhFactor VIII producedin BHK cells; also sold as Helixateby Centeon via a license agreement)

Bayer hemophilia A 1993 (US),2000 (EU)

Helixate NexGen (octocog �; rhFactorVIII produced in BHK cells

Bayer hemophilia A 2000 (EU)

NovoSeven (rhFactor VIIa producedin BHK cells)

Novo-Nordisk some formsof hemophilia

1995 (EU);1999 (US)

Recombinate (rhFactor VIII producedin an animal cell line)

Baxter Healthcare/Genetics Institute

hemophilia A 1992 (US)

Advate (octocog-�, rhFactor VIIIproduced in CHO cell line; the productis similar to Recombinate except it isexpressed in culture media free fromanimal-derived proteins and formulatedwithout plasma-derived humanalbumin)

Baxter hemophilia A 2004 (EU)

ReFacto (Moroctocog-�, i.e., B-domain-deleted rhFactor VIII produced inCHO cells)

Genetics Institute hemophilia A 1999 (EU),2000 (US)

ven is employed to stimulate the coagula-tion process in hemophilic patients withinhibitors to factor VIII and IX. It achievesits therapeutic effect by inducing the acti-vation of factor X to factor Xa, which con-verts prothrombin into thrombin (see alsoPart II, Chapters 1 and Part III, Chapters6). This, in turn, triggers the final clottingstep, where fibrinogen is converted into fi-brin to form the hemostatic plug. Thewhole clot-triggering process is thereforeachieved by bypassing the action of factorVIII and IX. This activation occurs only inthe presence of tissue factor (a membraneprotein not present in plasma), calciumand phospholipids, so that coagulation isstimulated only when an injury has oc-curred to a vessel, with resulting local he-mostasis. This complex process is nicelyshown in a video animation on the supple-mentary CD-ROM.

6.2Recombinant Thrombolytics

Six tPA-based thrombolytic products havegained approval thus far (Table 2). NativetPA is a 527-amino-acid glycosylated serineprotease synthesized predominantly invascular endothelial cells, from where itenters the bloodstream. It is the majoractivator of the natural thrombolytic pro-cess and therefore has obvious applicationin the accelerated removal of blood clotsthat form under inappropriate conditions.A recombinant form of native human tPAwas first marketed by Genentech in 1987.Most subsequent tPA-based products areengineered in some way, in order to ex-tend their plasma half-lives, as previouslymentioned.


Table 2 Recombinant tPA-based products thus far approved


Approved

Activase (Alteplase, rhtPA producedin CHO cells)

Genentech acute myocardialinfarction

1987 (US)

Ecokinase (Reteplase, rtPA; differs fromhuman tPA in that three of its fivedomains have been deleted; producedin E. coli)

Galenus Mannheim acute myocardialinfarction

1996 (EU)

Retavase (Reteplase, rtPA; see Ecokinase) BoehringerMannheim/Centocor

acute myocardialinfarction

1996 (US)

Rapilysin (Reteplase, rtPA;see Ecokinase)

BoehringerMannheim

acute myocardialinfarction

1996 (EU)

Tenecteplase (also marketed as Metalyse;TNK-tPA, modified rtPA producedin CHO cells)

Boehringer Ingel-heim

myocardialinfarction

2001 (EU)

TNKase (Tenecteplase; modified rtPAproduced in CHO cells; see Tenecteplaseentry above)

Genentech myocardialinfarction

2000 (US)

6.3Recombinant Insulins

In many ways insulin remains the proto-typic biopharmaceutical. Used to treat dia-betes mellitus, early commercial prepara-tions were extracted directly from the pan-creatic tissue of slaughterhouse animals.The WHO estimates that some 170 mil-lion people suffer from diabetes, a figurethat is likely to double by 2030. Althoughonly a minority of these sufferers actuallyrequire daily insulin injection, the currentworld market for insulin is valued at in ex-cess of $ 4.5 billion, a figure that is likelyto reach $ 8 billion before the end of thedecade (see also Part IV, Chapter 13 andPart VI, Chapter 4). Commercially feasiblemethods of enzymatically converting por-cine insulin into a product identical to hu-man insulin were developed in the 1970s,but recombinant DNA technology has hadthe greatest impact upon this sector. Thebiosynthesis of insulin in the human bodyand the procedure to enzymatically convertporcine insulin is nicely shown on thesupplementary CD-ROM.

Initially produced in 1978, recombinanthuman insulin (trade name Humulin) wasthe first biopharmaceutical to gain ap-proval in any world region (approved in1982). Since then a number of additionalrecombinant insulin products have comeon the market (Table 3). The modern insu-lin industry is dominated by Lilly, Novoand, to a lesser extent, Aventis, and thesecompanies manufacture and market arange of both first-generation and engi-neered (second-generation) insulin prod-ucts (Table 3). Engineered second-genera-tion insulin analogs display an amino acidsequence altered in order to generate either“fast-acing” or “slow-acting” product. Un-modified human insulin molecules, whenstored at typical commercial therapeutic

dose concentrations (around 10–3 M), existprimarily in oligomeric form, as zinc-con-taining hexamers. Each hexamer consistsof three identical dimers, exhibiting stronginter-subunit interactions. Three dimersare coordinated to central zinc ions. Upons.c. administration, hexamers must first dis-associate into monomeric form before entryinto the bloodstream. As a result, injectedinsulin has a slower onset (and a longerduration) of action when compared to en-dogenous insulin secretion. A practical con-sequence is that such insulins must beadministered to the diabetic 30 min or sobefore meal times and the planned mealtime should not subsequently be altered.In addition to such traditional “short-acting”insulins, insulin may be formulated in orderto actually retard the rate of insulin entryinto the bloodstream from the injection site.Such “long-acting” insulins are usually ad-ministered (in combination with short-act-ing insulins) in order to mimic low baselineendogenous insulin levels.

Insulin lispro (sold under the tradenames Humalog and Liprolog) exemplifiesengineered short-acting insulin products.This product displays an amino acid se-quence identical to native human insulin,with the exception that the natural pro-line–lysine sequence characteristics of po-sitions 28 and 29 of the insulin B chainhave been reversed. The sequence inver-sion leads to local conformational changes,eliminating hydrophobic interactions criti-cal to dimer stabilization. As a result, de-oligomerization occurs rapidly upon injec-tion and the product can be administeredat meal times rather than 30 min before.The different forms and formulations ofinsulin are shown on the supplementaryCD-ROM.

Levemir is the trade name given to anunusual long-acting insulin product thathas just recently gained marketing ap-



Table 3 Recombinant insulins/insulin analogs thus far approved


Approved

Humulin (rhInsulin produced in E. coli) Eli Lilly diabetes mellitus 1982 (US)

Novolin (rhInsulin produced inS. cerevisiae)

Novo Nordisk diabetes mellitus 1991 (US)

Humalog (Insulin lispro, an insulinanalog produced in E. coli)

Eli Lilly diabetes mellitus 1996(US and EU)

Insuman (rhInsulin produced in E. coli) Hoechst diabetes mellitus 1997 (EU)

Liprolog (Bio Lysprol, short-actinginsulin analog produced in E. coli)

Eli Lilly diabetes mellitus 1997 (EU)

NovoRapid (Insulin Aspart, short-actingrhInsulin analog produced inS. cerevisiae)

Novo Nordisk diabetes mellitus 1999 (EU)

Novomix 30 [contains insulin Aspart,short-acting rhInsulin analog producedin S. cerevisiae (see NovoRapid) as oneingredient]


Novolog (Insulin Aspart, short-actingrhInsulin analog produced inS. cerevisiae; see also NovoRapid)


Novolog mix 70/30 (contains insulinAspart, short-acting rhInsulin analogproduced in S. cerevisiae as oneingredient; see also Novomix 30)


Actrapid/Velosulin/Monotard/Insula-tard/Protaphane/Mixtard/Actraphane/Ultratard (all contain rhInsulin pro-duced in S. cerevisiae formulated asshort/intermediate/long-acting product)


Lantus (Insulin glargine, long-actingrhInsulin analog produced in E. coli)

Aventis diabetes mellitus 2000(US and EU)

Optisulin (Insulin glargine, long-actingrhInsulin analog produced in E. coli,see Lantus)

Aventis diabetes mellitus 2000 (EU)

Levemir (Insulin detemir, long-actingrhInsulin analog produced inS. cerevisiae)


Apidra (Insulin Glulisine, rapid-actinginsulin analog produced in E. coli)

Aventis diabetes mellitus 2004 (US)

proval (Table 3). The major structural al-teration characteristic of this insulin ana-log is the attachment of a C14 fatty acidvia the side-chain of lysine residue num-ber 29 of the insulin B chain. This pro-motes binding of the insulin analog to al-bumin, both at the site of injection and inthe plasma. In turn, this promotes a con-stant and prolonged release of free insulininto the blood, giving it a duration of ac-tion of up to 24 h.

6.4Additional Recombinant Hormones

Nineteen additional recombinant hor-mones have been approved thus far. Theseinclude a number of recombinant versionsof human growth hormone (hGH), variousgonadotropins, glucagon, parathyroid hor-mone and calcitonin (Table 4). Amongstthese “Somavert” is notable in that it is arecombinant PEGylated analog of hGH. It


Table 4 Additional recombinant hormones approved for general medical use


Approved

Protropin (rhGH, differs from humanhormone only by containing an addi-tional N-terminal methionine residue;produced in E. coli )

Genentech hGH deficiencyin children

1985 (US)

Glucagen (rhGlucagon produced in S.cerevisiae)

Novo Nordisk hypoglycemia 1998 (US)

Thyrogen (Thyrotrophin-�, rhTSH pro-duced in CHO cells)

Genzyme detection/treatmentof thyroid cancer

1998 (US),2000 (EU)

Humatrope (rhGH produced in E. coli) Eli Lilly hGH deficiencyin children

1987 (US)

Nutropin (rhGH produced in E. coli) Genentech hGH deficiencyin children

1994 (US)

Nutropin AQ (rhGH produced in E. coli) Schwartz Pharma growth failure,Turner’s syndrome

2001 (EU)

BioTropin (rhGH produced in E. coli) BiotechnologyGeneral

hGH deficiencyin children

1995 (US)

Genotropin (rhGH produced in E. coli) Pharmacia &Upjohn

hGH deficiencyin children

1995 (US)

Saizen (rhGH produced in an engi-neered mammalian cell line)

Serono hGH deficiencyin children

1996 (US)

Serostim (rhGH produced in anengineered mammalian cell line)

Serono Laboratories treatment of AIDS-associated catabo-lism/wasting

1996 (US)

Norditropin (rhGH produced in E. coli) Novo Nordisk treatment of growthfailure in childrendue to inadequateGH secretion

1995 (US)

has been engineered so as to introducenine mutations into the hGH amino acidsequence. It binds the hGH cell surface re-ceptor, but fails to trigger an intracellularresponse. As such it functions in an antag-onistic fashion, reducing the effects of en-dogenous hGH, underlining its use forthe treatment of acromegaly. The moleculeis PEGylated so as to increase its serumhalf-life (see also Part VI, Chapter 2).

6.5Recombinant Hematopoietic Growth Factors

Recombinant hematopoietic growth factorsconsist of several EPOs and colony-stimu-lating factors (Table 5). EPO represents thesingle most lucrative biopharmaceutical of

all and is indicated for the treatment ofanemia associated with various medicalconditions (see also Part VIII, Chapter 3).As previously described, “Nespo” (Aranespin the US) is an engineered EPO analogdisplaying an extended serum half-life.Leukine (owned by Schering) is a recombi-nant human GM-CSF, a 127-amino-acidglycosylated hematopoietic growth factorproduced in an engineered strain of S. cer-evisiae. It was initially approved for use fol-lowing induction of chemotherapy in adultpatients with acute myelogenous leukemia(or acute nonlymphocytic leukemia) in or-der to shorten time to neutrophil recoveryand reduce the incidence of severe infec-tion. Neupogen (filgrastim) is a recombi-nant human G-CSF (see also Part VIII,


Table 4 (continued)


Approved

Gonal F (rhFSH produced in CHOcells)

Serono anovulation andsuperovulation

1995 (EU),1997 (US)

Puregon (rhFSH produced in CHOcells)

Organon anovulation andsuperovulation

1996 (EU)

Follistim (Follitropin-�, rhFSH producedin CHO cells)

Organon some forms ofinfertility

1997 (US)

Luveris (lutropin-�; rhLH producedin CHO cells)

Ares-Serono some forms ofinfertility

2000 (EU)

Ovitrelle (also termed Ovidrelle;rhCG produced in CHO cells)

Serono used in selected as-sisted reproductivetechniques

2001 (EU),2000 (US)

Forcaltonin (rSalmon calcitoninproduced in E. coli)

Unigene Paget’s disease 1999 (EU)

Forteo (Forsteo in EU; teriparatide;recombinant shortened form of humanparathyroid hormone, produced inE. coli).

Eli Lilly treatment of osteo-porosis in selectedpostmenopausalwomen

2002 (US),2003 (EU)

Somavert [pegvisomant; recombinantengineered hGH analog (antagonist),produced in E. coli]

PharmaciaEnterprises

treatment ofselected patientssuffering fromacromegaly

2002 (EU)

Chapter 3) produced by recombinantmeans in E. coli. It regulates the produc-tion of neutrophils and is indicated for thetreatment of neutropenia associated withvarious medical conditions. Neulasta is aPEGylated form filgrastim, also used totreat neutropenia. It exhibits an extendedduration of action, due to the PEG-mediated reduction in product renal clear-ance rate.

6.6Recombinant IFNs and ILs

Quite a number of IFN-based productshave gained marketing approval over thelast decade and a half (Table 6). The �

IFNs have found application mainly in thetreatment of certain cancer types and viraldiseases. The trend in this case is towardthe development of PEGylated forms ofthese products. As described earlier, theextended plasma half-life associated withsuch PEGylated forms renders possibletheir administration as single as apposedto thrice weekly injection. IFN-� prepara-tions (Betaferon, Schering) have foundapplication in the treatment of multiplesclerosis (MS), a chronic disabling diseaseof the central nervous system. The major-ity of MS patients develop significant dis-abilities, either gradually or due to relaps-ing/remitting symptoms, which involves aworsening of the disease followed by atemporary recovery (see also Part V, Chap-


Table 5 Recombinant hemopoietic growth factors approved for general medical use


Approved

Epogen (rhEPO producedin a mammalian cell line)

Amgen treatmentof anemias

1989 (US)

Procrit (rhEPO producedin a mammalian cell line)

Ortho Biotech treatmentof anemias

1990 (US)

Neorecormon (rhEPO producedin CHO cells)

BoehringerMannheim

treatmentof anemias

1997 (EU)

Aranesp (Darbepoetin-; long-actingrEPO analog produced in CHO cells)

Amgen treatmentof anemia

2001(EU and US)

Nespo (Darbepoetin-; see also Aranesp;long-acting rEPO analog producedin CHO cells)

Dompe Biotec treatmentof anemia

2001 (EU)

Leukine (rGM-CSF, differs from thenative human protein by 1 amino acid,Leu23; produced in S. cerevisiae)

Immunex autologous bonemarrowtransplantation

1991 (US)

Neupogen (Filgrastim, rG-CSF, differsfrom human protein by containing anadditional N-terminal methionine;produced in E. coli)

Amgen chemotherapy-in-duced neutropenia

1991 (US)

Neulasta (Pegfilgrastim, recombinantPEGylated filgrastim – see Neupogen;also marketed in the EU as Neupopeg)

Amgen neutropenia 2002(US and EU)


Table 6 Recombinant IFNs and ILs approved for general medical use


Approved

Intron A (rIFN-�2b produced in E. coli) Schering Plough cancer, genitalwarts, hepatitis

1986 (US),2000 (EU)

PegIntron A (PEGylated rIFN-�2bproduced in E. coli)

Schering Plough chronic hepatitis C 2000 (EU),2001 (US)

Viraferon (rIFN-�2b produced in E. coli) Schering Plough chronic hepatitis Band C

2000 (EU)

ViraferonPeg (PEGylated rIFN-�2bproduced in E. coli)

Schering Plough chronic hepatitis C 2000 (EU)

Roferon A (rhIFN-�2a, producedin E. coli)

Hoffmann-La Roche hairy cell leukemia 1986 (US)

Actimmune (rhIFN-�1b producedin E. coli)

Genentech chronic granulo-matous disease

1990 (US)

Betaferon (rIFN-�1b, differs fromhuman protein in that Cys17 is replacedby Ser; produced in E. coli)

Schering MS 1995 (EU)

Betaseron (rIFN-�1b, differs fromhuman protein in that Cys17 is replacedby Ser; produced in E. coli)

Berlex Laboratoriesand Chiron

relapsing/remittingMS

1993 (US)

Avonex (rhIFN-�1a, producedin CHO cells)

Biogen relapsing MS 1997 (EU),1996 (US)

Infergen (rIFN-�, synthetic type I IFNproduced in E. coli)

Amgen (US) andYamanouchi Europe(EU)

chronic hepatitis C 1997 (US),1999 (EU)

Rebif (rh IFN-�1a producedin CHO cells)

Ares-Serono relapsing/remittingMS

1998 (EU),2002 (US)

Rebetron (combination of ribavirinand rhIFN-�2b produced in E. coli)

Schering Plough chronic hepatitis C 1999 (US)

Alfatronol (rhIFN-�2b producedin E. coli)

Schering Plough hepatitis B and C,and various cancers

2000 (EU)

Virtron (rhIFN-�2b produced in E. coli) Schering Plough hepatitis B and C 2000 (EU)

Pegasys (Peginterferon-�2a producedin E. coli)

Hoffmann-La Roche hepatitis C 2002(EU and US)

Proleukin (rIL-2, differs from humanmolecule in that it is devoid of an N-terminal alanine and Cys-125 has beenreplaced by a Ser; produced in E. coli)

Chiron renal cell carcinoma 1992 (US)

Neumega (rIL-11, lacks N-terminalproline of native human molecule;produced in E. coli)

Genetics Institute prevention of che-motherapy-inducedthrombocytopenia

1997 (US)

Kineret (anakinra; rIL-1 receptorantagonist produced in E. coli)

Amgen rheumatoid arthritis 2001 (US)

ter 3). The underlining mechanism is notcompletely understood, but it is knownthat it is mediated by specific cell receptorsand involves the modulation of the im-mune response.

In addition to IL-2 and -11, an IL-1 re-ceptor antagonist (trade name Kineret) hasalso gained general marketing approval inthe US for the treatment of rheumatoid ar-thritis (Table 6). Kineret binds IL-1� andIL-1� cell surface receptors, but without in-ducing a biological response. The producttherefore blocks IL-1 biological activity,

which is a critical mediator of the inflam-mation and joint damage characteristic ofthis condition.

6.7Vaccines

A number of recombinant vaccines havealso gained marketing approval for generalmedical use. By far the most prominent ex-ample is that of rHBsAg, which is used tovaccinate against hepatitis B. While the re-combinant antigen can be used on its


Table 7 Recombinant vaccines approved for general medical use

Product Company Therapeutic indication Approved

Recombivax (rHBsAg producedin S. cerevisiae)

Merck hepatitis B prevention 1986 (US)

Comvax (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

Merck vaccination of infantsagainst hemophilusinfluenzae type B andhepatitis B

1996 (US)

Engerix B (rHBsAg producedin S. cerevisiae)

SmithKlineBeecham

vaccination againsthepatitis B

1998 (US)

Tritanrix-HB (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

SmithKlineBeecham

vaccination againsthepatitis B, diphtheria,tetanus and pertussis

1996 (EU)

Lymerix (rOspA, a lipoprotein foundon the surface of Borrelia burgdorferi,the major causative agent of Lyme’sdisease; produced in E. coli)

SmithKlineBeecham

Lyme disease vaccine 1998 (US)

Infanrix-Hep B (combination vaccine,containing rHBsAg produced inS. cerevisiae as one component)

SmithKlineBeecham

immunization againstdiphtheria, tetanus,pertussis and hepatitis B

1997 (EU)

Infanrix–Hexa (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

SmithKlineBeecham

immunization againstdiphtheria, tetanus,pertussis, polio,hemophilus influenzae Band hepatitis B

2000 (EU)

Infanrix-Penta (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

SmithKlineBeecham

immunization againstdiphtheria, tetanus,pertussis, polio andhepatitis B

2000 (EU)

own, it more generally represents one com-ponent of multicomponent vaccine prepara-tions (Table 7). The emphasis upon hepati-tis B no doubt reflects the global signifi-cance of this condition. Two billion peopleare infected worldwide, with 350 million in-

dividuals suffering from lifelong chronic in-fections. In excess of 1 million sufferers dieeach year from liver cancer and/or cirrhosistriggered by the condition.


Table 7 (continued)


Ambirix (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

Glaxo SmithKline immunization againsthepatitis A and B

2002 (EU)

Twinrix (adult and pediatric formsinEU; combination vaccine containingrHBsAg produced in S. cerevisiae asone component)

SmithKlineBeecham (EU) andGlaxo SmithKline(US)

immunization againsthepatitis A and B

1996(EU, adult),1997 (EU,pediatric),2001 (US)

Primavax (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

Pasteur MerieuxMSD

immunization againstdiphtheria, tetanus andhepatitis B

1998 (EU)

Pediarix (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

Glaxo SmithKline immunization of children against variousconditions, includinghepatitis B

2002 (US)

Procomvax (combination vaccine,containing rHBsAg as onecomponent)

Pasteur MerieuxMSD

immunization againsthemophilus influenzaetype B and hepatitis B

1999 (EU)

Hexavac (combination vaccine,containing rHBsAg producedin S. cerevisiae as one component)

Aventis Pasteur immunization againstdiphtheria, tetanus, per-tussis, hepatitis B, polioand hemophilusinfluenzae type B

2000 (EU)

Triacelluvax [combination vaccinecontaining recombinant (modified)pertussis toxin]

Chiron immunization againstdiphtheria, tetanus andpertussis

1999 (EU)

Hepacare [rS, pre-S and pre-S2HBsAgs, produced in a mammalian(murine) cell line]

Medeva immunization againsthepatitis B

2000 (EU)

HBVAXPRO (rHBsAg producedin S. cerevisiae)

Aventis immunization of childrenand adolescents againsthepatitis B

2001 (EU)

Dukoral (Vibrio cholerae andrecombinant cholera toxin B subunit)

SBL Vaccine active immunizationagainst disease caused byV. cholerae subgroup O1

2004 (EU)

6.8Monoclonal and Engineered Antibodies

Antibody-based products represent the sin-gle largest category of biopharmaceuticalapproved for general medical use (Table 8)(see also Part IV, Chapter 16 and Part V,Chapters 1 and 2). As previously outlined,first-generation products were invariablymurine monoclonals produced by classicalhybridoma technology. The developmentof engineered products, i.e., chimeric andhumanized antibodies, overcame many ofthe therapeutic difficulties associated withsuch early preparations and the majorityof recently approved products are engi-neered in this way. The majority of anti-body-based products are used to either de-tect or treat various forms of cancer. Theantibodies are raised against specific tu-mor-associated antigens found on the sur-face of specific cancer types and thereforewill bind specifically to those cell typesupon administration. Conjugation of radio-isotopes, toxins or other chemotherapeuticagents should allow selective delivery ofthese agents directly to the tumor surface,courtesy of antibody-binding specificity(see also Part II, Chapter 5 and Part V,Chapter 6). In practice, some difficultiescan arise with this approach, e.g., due toantibody cross-reactivity with nontrans-formed cells or the presence of the tumor-associated antigen, even in low numbers,on the surface of unrelated, healthy cells.

Herceptin is the trade name given toone such antibody-based cancer therapyproduct (Table 8). It is a humanized mAbwith binding specificity for the human epi-dermal growth factor receptor 2 (HER2),which is overexpressed on the surface of25–30% of metastatic breast cancers.HER2 overexpression induces abnormalproliferation of cells (see also Part I, Chap-ter 5). The mAb binds specifically to the

tumor cells overexpressing HER2, thus in-hibiting their proliferation and inducingantibody-directed cell-mediated cytotoxicity.This results in reducing metastasis whilenot affecting normal cells, therefore limit-ing side-effects.

Detection (as opposed to treatment) oftumors is facilitated by the conjugation ofa �-emitting radioactive tag to an appropri-ate antibody. The radioactivity congregatedat the tumor site can penetrate outwardfrom the body, facilitating its detection byequipment such as a planar �-camera (seealso Part V, Chapters 4, 5 and 7). Examplesof such products approved include Leukos-can and Prostascint (Table 8).

Several antibody-based products are indi-cated for non-cancer applications. Zena-pax, for example, is used for the preven-tion of acute kidney transplant rejection.The product is a humanized mAb thatspecifically binds the �-chain (also knownas CD25 or Tac) of the IL-2 receptor. Thisreceptor is expressed on the surface of ac-tivated lymphocytes. It acts as an antago-nist of the receptor, thus blocking thebinding of IL-2 that in turn prevents thestimulation of lymphocytes mediating or-gan rejection.

6.9Additional Biopharmaceuticals

A number of additional products that donot fall into any of the categories dis-cussed thus far have also gained market-ing approval. Amongst these are severalenzymes, including glucocerebrosidase(used for the treatment of Gaucher’s dis-ease, discussed previously), DNase (usedto treat cystic fibrosis), �-galactosidase(used to treat Fabry disease) and enzymesfor amino acid depletion (see also Part II,Chapter 6). Recombinant �-galactosidase isproduced in mammalian cell lines. The



Table 8 Monoclonal/engineered antibody-based products approved for general medical use


CEA-scan [Arcitumomab; murinemAb fragment (Fab), directed againsthuman carcinoembryonic antigen]

Immunomedics detection of recurrent/metastatic colorectalcancer

1996(US and EU)

MyoScint (Imiciromab-Pentetate;murine mAb fragment directedagainst human cardiac myosin)

Centocor myocardial infarctionimaging agent

1996 (US)

OncoScint CR/OV (SatumomabPendetide; murine mAb directedagainst TAG-72, a high-molecular-weight tumor-associated glycoprotein)

Cytogen detection/staging/follow-up of colorectaland ovarian cancers

1992 (US)

Orthoclone OKT3 (Muromomab CD3;murine mAb directed against the Tlymphocyte surface antigen CD3)

Ortho Biotech reversal of acute kidneytransplant rejection

1986 (US)

ProstaScint (Capromab Pentetate;murine mAb directed against thetumor surface antigen PSMA)

Cytogen detection/staging/follow-up of prostateadenocarcinoma

1996 (US)

ReoPro (Abciximab; Fab fragmentsderived from a chimeric mAb, direc-ted against the platelet surface recep-tor GPIIb/IIIa)

Centocor prevention of blood clots 1994 (US)

Rituxan (Rituximab; chimeric mAbdirected against CD20 antigen foundon the surface of B lymphocytes)

Genentech/IDECPharmaceuticals

non-Hodgkin’slymphoma

1997 (US)

Verluma [Nofetumomab; murinemAb fragments (Fab) directed againstcarcinoma associated antigen]

Boehringer-Ingel-heim/NeoRx

detection of small celllung cancer

1996 (US)

Zenapax (Daclizumab; humanizedmAb directed against the � chain ofthe IL-2 receptor)

Hoffmann-La Roche prevention of acute kid-ney transplant rejection

1997 (US),1999 (EU)

Simulect (Basiliximab; chimeric mAbdirected against the � chain of theIL-2 receptor)

Novartis prophylaxis of acuteorgan rejection inallogeneic renaltransplantation

1998(EU and US)

Remicade (Infliximab, chimeric mAbdirected against TNF-�)

Centocor treatment of Crohn’sdisease

1998 (US),1999 (EU)

Synagis (Palivizumab; humanizedmAb directed against an epitope onthe surface of respiratory syncytialvirus)

MedImmune (US)and Abbott (EU)

prophylaxis of lowerrespiratory tract diseasecaused by respiratorysyncytial virus inpediatric patients

1998 (US),1999 (EU)


Table 8 (continued)


Herceptin (Trastuzumab; humanizedantibody directed against HER2)

Genentech (US) andRoche Registration(EU)

treatment of metastaticbreast cancer if tumoroverexpresses HER2protein

1998 (US),2000 (EU)

Indimacis 125 (Igovomab; murinemAb fragment (Fab2) directed againstthe tumor-associated antigen CA 125)

CIS Bio diagnosis of ovarianadenocarcinoma

1996 (EU)

Tecnemab KI [murine mAbfragments (Fab/Fab2 mix) directedagainst high-molecular-weightmelanoma-associated antigen]

Sorin diagnosis of cutaneousmelanoma lesions

1996 (EU)

LeukoScan [Sulesomab; murine mAbfragment (Fab) directed against NCA90, a surface granulocyte nonspecificcross-reacting antigen]

Immunomedics diagnostic imaging forinfection/inflammationin bone of patients withosteomyelitis

1997 (EU)

Humaspect (Votumumab; humanmAb directed against cytokeratintumor-associated antigen)

Organon Teknika detection of carcinomaof the colon or rectum

1998 (EU)

Mabthera (Rituximab; chimeric mAbdirected against CD20 surface antigenof B lymphocytes)

Hoffmann-La Roche(see also Rituxan)


1998 (EU)

Mabcampath (EU) or Campath (US)(Alemtuzumab; humanized mAbdirected against CD52 surface antigenof B lymphocytes)

Millennium andILEX (EU), and Ber-lex, ILEX Oncologyand MillenniumPharmaceuticals(US)

chronic lymphocyticleukemia

2001(EU and US)

Mylotarg (Gemtuzumab zogamicin;humanized antibody-toxic antibioticconjugate targeted against CD33antigen found on leukemic blastcells)

Wyeth Ayerst acute myeloid leukemia 2000 (US)

Zevalin (Ibritumomab Tiuxetan;murine mAb, produced in a CHOcell line, targeted against the CD20antigen)

IDEC pharmaceuti-cals (US) and Scher-ing (EU)


2002 (US),2004 (EU)

Humira [EU and US; also sold asTrudexa in EU; Adalimumab;recombinant (anti-TNF) human mAbcreated using phage displaytechnology]

Cambridge Anti-body Technologiesand Abbott (US),and Abbott (EU)

rheumatoid arthritis 2002 (US),2003 (EU)

429-amino-acid glycoprotein spontaneouslydimerizes, yielding the 100-kDa biologi-cally active enzyme. Fabry disease is a raregenetic condition characterized by a defi-ciency of the lysosomal enzyme �-galacto-sidase A. As a result, sufferers exhibit aninability to break down certain glycolipids,particularly the glycosphingolipid ceramidetrihexoside or globotriaosylceramide (GL-3). Glycolipid accumulates in the walls ofvascular cells, particularly in the kidney,heart and nervous system.

Regranex is an interesting product inthat it is administered not by direct in-

jection as is the case for most biopharma-ceuticals. The active product ingredientis a recombinant human platelet-derivedgrowth factor (PDGF). Active PDGF is ahomodimer. Each 109-amino-acid, glycosy-lated polypeptide is aligned in an antipar-allel fashion relative to the other, yieldingthe 24.5-kDa mature molecule. Regranexis produced by recombinant DNA technol-ogy in S. cerevisiae and the product is pre-sented in a gel formulation containing0.1% active ingredient for external topicaluse. Regranex is indicated for the treat-ment of chronic diabetic ulcers that do not


Table 9 Additional biopharmaceuticals approved for general medical use


Approved

Beromun (rhTNF-� produced in E. coli) Boehringer-Ingelheim

adjunct to surgeryfor subsequent tu-mor removal, to pre-vent or delayamputation

1999 (EU)

Revasc (anticoagulant; recombinanthirudin produced in S. cerevisiae)

Ciba NovartisEuropharm

prevention of venousthrombosis

1997 (EU)

Refludan (anticoagulant; recombinanthirudin produced in S. cerevisiae)

Hoechst MarionRoussel (US) andBehringwerke (EU)

anticoagulation ther-apy for heparin-asso-ciated thrombocyto-penia

1998 (US);1997 (EU)

Cerezyme (r�-glucocerebrosidaseproduced in CHO cells; differs fromnative human enzyme by 1 amino acid,Arg495 is substituted with a His, also hasmodified oligosaccharide component)

Genzyme treatment ofGaucher’s disease

1994 (US);1997 (EU)

Pulmozyme (Dornase-�, rDNaseproduced in CHO cells)

Genentech cystic fibrosis 1993 (US)

Fabrazyme (rh�-Galactosidase producedin CHO cells)

Genzyme Fabry disease(�-galactosidase Adeficiency)

2001 (EU)

Replagal (rh�-Galactosidase producedin a continuous human cell line)

TKT Europe Fabry disease(�-galactosidase Adeficiency)

2001 (EU)

heal with normal wound care practice. Itis usually administered daily for up to amaximum of 20 weeks.

Bone morphogenic proteins (BMPs) rep-resent another interesting class of biophar-maceutical (Table 9). As their name sug-gests, these proteins can promote the de-position and growth of new bone, and are

often administered by implantation as partof a medical device. InductOs, for exam-ple, consists of a recombinant humanBMP-2 that promotes the differentiation ofmesenchymal cells into bone cells (seealso Part I, Chapter 13). The biologicallyactive form is a glycosylated heterodimer,consisting of 114- and 131-amino-acid


Table 9 (continued)


Approved

Fasturtec (Elitex in US; rasburicase;recombinant urate oxidase producesin S. cerevisiae)

Sanofi-Synthelabo hyperuricaemia 2001 (EU),2002 (US)

Aldurazyme (Laronidase; rh-�-l-iduronidase produced in an engineeredCHO cell line)

Genzyme long-term enzymereplacement therapyin patients sufferingfrom mucopolysac-charidosis

2003 (EU)

Regranex (rhPDGF producedin S. cerevisiae)

Ortho-McNeilPharmaceuticals(US) and Janssen-Cilag (EU)

lower extremitydiabetic neuropathiculcers

1997 (US),1999 (EU)

Vitravene (Fomivirsen; an antisenseoligonucleotide)

ISIS Pharmaceuti-cals

treatment of cytome-galovirus retinitis inAIDS patients

1998 (US)

Ontak (rIL-2-diphtheria toxin fusionprotein which targets cells displayinga surface IL-2 receptor)

Seragen/LigandPharmaceuticals

cutaneous T celllymphoma

1999 (US)

Enbrel (rTNF receptor–IgG fragmentfusion protein produced in CHO cells)

Immunex (US) andWyeth Europa (EU)

rheumatoid arthritis 1998 (US),2000 (EU)

Osteogenic protein 1 (rhOsteogenicprotein-1 : BMP-7 produced in CHO cells)

Howmedica (EU)and Stryker (US)

treatment of non-union of tibia

2001(EU andUS)

Infuse (rhBMP2 produced in CHO cells) Medtronic SofamorDanek

promotes fusion ofvertebrae in lowerspine

2002 (US)

Inductos (dibotermin-�; rBone morpho-genic protein-2 produced in CHO cells)

Genetics Institute treatment of acutetibia fractures

2002 (EU)

Xigris [drotrecogin-�; rh activated proteinC produced in a mammalian (human)cell line]

Eli Lilly severe sepsis 2001 (US),2002 (EU)

polypeptide subunits. It is produced in aCHO cell line. InductOs is used in skele-tally mature patients for the treatment ofacute tibia fractures in adjunct to standardcare using fracture reduction and intrame-dullary nail fixation. It is applied duringsurgical procedure at the site of fracture.The use of a bovine collagen sponge en-sures retention of the active substance atthe site of the fracture for the time re-quired for healing, with the matrix com-pletely dissolving over time.

7Products Approved for Veterinary Use

While the majority of pharmaceuticals pro-duced by modern biotechnological meansare destined for human use, several veter-inary biopharmaceuticals have also gainedapproval (Table 10). One of the earliest

such examples is bovine somatotropin (re-combinant bovine GH), used to boost themilk yields of dairy cattle. The majority ofveterinary biopharmaceuticals, however,are engineered vaccines. The importanceof effective vaccination to prevent rapidspread of disease through high-density ani-mal populations characteristic of modernagricultural practice is obvious and mostvaccines are destined for use in agricultu-rally important species. Porcillis Porcoli,for example, is a multisubunit vaccine con-taining a combination of recombinant E.coli-derived adhesin proteins. These pro-teins are essential for colonization of thegut by pathogenic E. coli. Immunization ofsows effectively provides passive immunityto progeny via colostrum for the first fewdays of life, when piglets are particularlysusceptible to E. coli infections. Twoadditional veterinary biopharmaceuticals,which are particularly interesting, are

7 Products Approved for Veterinary Use 25

Table 10 Recombinant veterinary medicinal products approved in EU via the centralized application process


Porcilis Porcoli (combination vaccinecontaining recombinant E. coliadhesins)

Intervet active immunization ofsows

1996

Fevaxyn Pentofel (combinationvaccine containing recombinantfeline leukemia viral antigen as onecomponent)

Fort DodgeLaboratories

immunization of catsagainst various felinepathogens

1997

Neocolipor (vaccine containing fourinactivated E. coli strains; two wild-type strains expressing E. coliadhesins F6 and F41, and tworecombinant strains, engineered toexpress F4 and F5 adhesins)

Merial reduction of neonatalenterotoxicosis of youngpiglets caused by E. colistrains expressing F4,F5, F6 or F41 adhesins

1998

Porcilis AR-T DF (combinationvaccine containing a modified toxinfrom Pasteurella multocida expressedin E. coli)

Intervet reduction in clinicalsigns of progressiveatrophic rhinitis inpiglets: oraladministration

2000

Ibraxion and Vibragen �, as discussedbelow.

The advent of genetic engineering hasfacilitated the development of engineeredvaccines capable of allowing subsequentimmunological differentiation between in-fected and vaccinated animals. Using this

form of vaccination allows veterinary in-spectors to tell if a seropositive animal hassimply been vaccinated (and is noninfec-tious) or if it has been infected with thewild-type pathogen (and is likely to be in-fectious, thereby requiring treatment/isola-tion).


Table 10 (continued)


Porcilis pesti (vaccine containingrecombinant classical swine fever virusE2 subunit antigen produced in aninsect cell baculovirus expressionsystem)

Intervet immunization of pigsagainst classical swine fe-ver

2000

Ibraxion (vaccine consisting of aninactivated, BHV type 1 engineeredby removal of the viral glycoproteingE gene)

Merial active immunization ofcattle against infectiousbovine rhinotracheitis

2000

Bayovac CSF E2 (vaccine consistingof recombinant classical swine fevervirus E2 subunit antigen producedusing a baculovirus vector system)

Bayer immunization of pigsagainst classical swinefever virus

2001

Eurifel FELV (vaccine consisting ofan engineered canarypox virus intowhich the gag, env and a partial polgene of feline leukemia virus havebeen inserted)

Merial Immunization of catsagainst feline leukemiavirus

2000

Vibragen � (rFeline IFN-�) Virbac reduce mortality/clinicalsigns of canineparvovirusis

2001

Eurifel RCPFEVL (multicomponentvaccine containing as one componentan engineered canarypox virus intowhich the gag, env and a partial polgene of feline leukemia virus havebeen inserted (see Eurifel FELVabove)

Merial active immunization ofcats against viralpathogens, includingfeline leukemia virus

2002

Gallivac HVT IBD (live multic-omponent vaccine containing as onecomponent an engineered herpes virusof turkeys housing a gene coding forthe protective VP2 antigen of theinfectious bursal disease virus)

Merial active immunization ofchickens against,amongst others, the viralcausative agent ofinfectious bursal disease

2002

Ibraxion is an example of such an engi-neered vaccine. It is an engineered bovineherpes virus (BHV) from which one struc-tural gene (the gE gene) has been deleted.BHV induces infectious bovine rhinotra-cheitis, a condition characterized by lossesin animal production and abortions. Ibrax-ion induces immunological protection incattle, but the serum of Ibraxion-vacci-nated animals is devoid of anti-gE antibod-ies, whereas infected animals will havehigh titers of such antibodies (Fig. 3).

Vibragen � is IFN-�, a novel type 1IFN. Like other type 1 IFNs, it displaysantiviral activity which is the basis of itsuse in treating parvoviral infections, espe-cially in young dogs – for whom such aninfection can be fatal. Vibragen � is alsosomewhat unusual in that it is manufac-tured using insect-based biosynthesis oc-curring in whole silkworms (Fig. 4). Theprocess entails the use of the silkworm nu-clear polyhedrosis virus (NPV), engineeredto carry cDNA for feline IFN-�. Initial vi-ral amplification is first undertaken to pro-duce sufficient quantities of virus to seedthe process. Amplification is undertakenby viral incubation with an insect cell line(originated from Bombix mori) grown inconventional culture flasks. The product is

then manufactured by rearing severalthousand silkworms on heat-treated, syn-thetic chow in sterile cabinets. After 24–48 h each silkworm is inoculated withNPV using an automatic microdispenser.Five days later the silkworms are mechani-cally incised and the acid-stable IFN is ex-tracted from the body parts. Downstreamprocessing is more conventional, and em-ploys both dye and metal affinity chroma-tography to achieve product purification.

8Likely Future Directions

The main aim of this Introduction is toprovide the reader with a snapshot of theprofile of biopharmaceutical products ap-proved to date. How the sector will devel-op over the next decade or two will likelyecho, at least in part, many of the in-novations discussed within the remainingchapters of this book. A summary over-view of some such likely innovations anddirections is presented below.

8 Likely Future Directions 27

Fig. 3 Diagrammatic representation of the altera-tion made to the engineered BHV in order to pro-duce the product Ibraxion. By means of geneticengineering, the structural gene gE is deleted fromthe genome. BHV induces infectious bovine rhino-tracheitis, a condition characterized by losses in

animal production and abortions. Ibraxion inducesimmunological protection in cattle, but the serumof Ibraxion-vaccinated animals is devoid of anti-gEantibodies, whereas infected animals will havehigh titers of such antibodies.

8.1What is in the Pipeline?

Globally, in excess of 500 candidate bio-pharmaceuticals are undergoing clinicalevaluation. The Pharmaceutical Researchand Manufacturers of America (PhRMA),which represents the US drug industry, es-timates that some 371 biotech medicinesare undergoing trials in the US [27]. Of

these, around half (178) aim to treat can-cer (see also Part II, Chapter 4), and othernotable target indications include infec-tious diseases (47 products) (see also PartVI, Chapter 3), autoimmune disorders (26products) (see also Part V, Chapter 3), neu-rological disorders (22 products) (see alsoPart I, Chapter 14) and AIDS/HIV relatedconditions (21 products) (see also Part II,Chapters 7 and 8). The single largest cate-


Fig. 4 Overview of the manufacture of the veterin-ary medicinal product Vibragen �. The processentails inoculation of whole silkworms (grown onsynthetic food in pre-sterile cabinets) with an en-

gineered silkworm NPV housing the feline IFN-�gene. Product extraction, purification and formula-tion ensues.

gory is vaccines, of which there are 98 indevelopment (see also Part I, Chapter 7).Fifty-three of these vaccines aim to treat orprevent cancers, whereas an additional 29aim to treat various infectious diseases, in-cluding hepatitis and HIV. The secondlargest product category is that of mono-clonal/engineered antibodies (see also PartIV, Chapter 16 and Part V, Chapters 1 and2). Of the 75 such products in develop-ment, 39 (52%) target cancers and 10 aimto treat various autoimmune conditions,most notably rheumatoid arthritis. Thenumber of IL- and antibody-based prod-ucts in trials has increased modestly overthe past 2–3 years. The past few years hasalso witnessed a significant decrease in thenumber of growth factors and gene-thera-py-based products undergoing clinical eval-uation by PhRMA-associated companies, atleast (see also Part VI, Chapter 6). The lat-ter reflects the continued difficulties asso-ciated with making nucleic acid-basedproducts a therapeutic reality [28, 29] (seealso Part I, Chapters 6–9).

8.2Alternative Production Systemsfor Biopharmaceuticals

Essentially all recombinant therapeuticproteins approved thus far are expressedeither in E. coli, S. cerevisiae (see also PartIV, Chapters 12 and 13), or in an engi-neered animal cell line (see also Part IV,Chapters 1 and 4), hybridoma (mouse/hu-man) cells (see also Part IV, Chapter 2) oreven human cells (see also Part IV, Chap-ter 3).

Research continues into the develop-ment of alternative production systemsand of particular note is the use of trans-genic animals or plants (see also Part IV,Chapter 5). A number of recombinanttherapeutic proteins (including �1-antitryp-

sin, �-glucosidase and antithrombin III)have been successfully produced in trans-genic animals, mainly in the milk of mice(proof-of-concept stage) or goats (putativeproduction-scale systems) (see also Part IV,Chapter 11). While this approach has prov-en to be technically possible, a range ofproblems has thus far delayed/preventedapproval of any product produced in thismanner, although ATryn® (antithrombinIII) will most likely be approved at thetime when this book will be published.Difficulties have included modest/variableproduction levels, regulatory issues andcost. The major companies – besides GTCBiotherapeutics (US) – sponsoring thistechnology are PPL therapeutics (Scotland)and Pharming (The Netherlands). Workalso continues on the development oftransgenic plant-based systems for the pro-duction of, for example, oral vaccines orother therapeutic proteins. Again, regula-tory and cost issues are complicating fac-tors, as are issues such as the significantdifference between glycosylation patternscharacteristic of plant versus animal cell-based production systems. A comprehen-sive overview is given by Knäblein in twoexcellent reviews [30, 31]. Due to the im-portance of such emerging systems, thisbook has dedicated a complete section toalternative expressions systems for bio-pharmaceuticals – especially plant-basedexpression systems (see also Part IV, Chap-ter 6, Part IV, Chapters 7 and 8, Part IV,Chapter 9 and Part IV, Chapter 10). Thesection concludes with the engineering ofplant expression systems for abiotic stresstolerance. By making plants tolerant forhigh salt concentrations, heat and drought,this might in the near future lead to grow-ing plants in areas which today cannot beused for agriculture at all.


8.3Alternative Delivery Methodsfor Biopharmaceuticals

Thus far, therapeutic proteins are invari-ably administered parenterally. Drug ad-ministration by nonparenteral means isgenerally less invasive, requires less tech-nical training and is normally associatedwith improved patient compliance (seealso Part VI, Chapter 1, and Part VI, Chap-ters 5 and 3). Some progress has also beenrecorded relating to the development ofnonparenteral delivery routes for biophar-maceuticals. Most prominent in this re-gard is pulmonary delivery [32, 33]. Macro-molecules are absorbed from the lung sur-prisingly well, likely due to the lung’slarge surface area, thin diffusional layerand the presence of proteolytic inhibitors.Nebulizer technology allows product deliv-ery into the deep lung and drugs adsorbedin this way avoid first-bypass metabolism(see also Part VI, Chapter 4). Exubera isthe name given to an insulin product ad-ministered by pulmonary means (see alsoPart IV, Chapter 13). Developed by Pfizer,Aventis and Nektar (see also Part VI,Chapter 2), this product has completedphase III clinical trials, although addi-tional safety studies are currently beingundertaken.

8.4The Advent of Generic Biopharmaceuticals?

Patent protection for many early biophar-maceuticals, such as recombinant insulin,EPO, hGH and IFN-�, is now nearing orat an end (see also Part VIII, Chapter 3).Most of these are blockbuster products –each commands annual sales well in ex-cess of $ 1 billion and hence these repre-sent attractive targets for the fledgling bio-pharmaceutical generics industry [34] (see

also Part II, Chapter 4 and Part VIII,Chapter 1). Companies producing genericbiopharmaceuticals include Sicor (Irvine,CA), Ivax (Miami, FL), Dragon (Vancouver,Canada), Genemedix (Suffolk, UK) andBioGenerix (Mannheim, Germany). Sicoralready markets hGH and �-IFN-� in East-ern Europe, whereas Genemedix marketsa recombinant colony-stimulating factor inChina and is soon to manufacture EPO(see also Part VIII, Chapter 3). Major gen-erics companies such as Teva, Sandoz andMerck will also likely develop/consider de-veloping biopharmaceutical portfolios.However, the regulatory framework re-quired to underpin generic biopharmaceu-tical approvals within Europe and NorthAmerica is not yet finalized, although itappears to be at a more advanced stage inEurope as compared to the US (see alsoPart VII, Chapter 4). The concept of “simi-lar biological medicinal products” is onenow enshrined in the EU regulatory fra-mework. Within the US the regulatoryterminology includes phrases such as “fol-low-on biologics” and “well-characterizedprotein”. Although generics will probablybe reviewed by regulators on a case-by-casebasis, substantial in vitro work as well assome clinical data will almost certainly berequired to show comparability/productequivalence.

8.5Genomics and Proteomics

Most pharmaceutical companies have re-search programs in genomics/proteomics.The “omics” revolution was initially hailedas a revolution in drug discovery. Whilethese modern technologies may well helpidentify a host of putative new biopharma-ceuticals (see also Part I, Chapters 4 and5), they almost certainly will have a farmore significant impact upon identifying


new drug targets as well as disease diag-nostic markers (see also Part I, Chapters 2and 3 and Part V, Chapter 8).

8.6Gene Therapy

There have been well in excess of 400gene-therapy-based trials undertaken todate. The vast majority have reported a dis-appointing lack of efficacy. Safety concernshave also been raised and these concernswere heightened in 1999 when one partici-pant in a gene-therapy trial died. Largelyprompted by this event, the US NationalInstitute of Health requested detailed in-formation from a large number of trialsand uncovered several hundred reports ofserious trial-associated adverse events inthe process. In addition, allegations en-sued that other gene-therapy trial deathswent unreported/misreported, particularlyin trials using retroviral-based delivery vec-tors. As a result, more stringent reportingrequirements were introduced.

Gene therapy did receive a much-neededboost in 2002 when French scientists re-ported that they had apparently correctedsevere combined immunodeficiency in anumber of children using a retroviral-mediated protocol [35]. Severe combinedimmunodeficiency is a genetic conditioncaused by a deficiency of the enzymeadenosine deaminase, which triggers se-vere B and T lymphocyte dysfunction.However, celebrations were halted when anumber of trial participants developed un-controlled lymphoproliferation, a conditionsimilar to leukemia. This halted – at leasttemporarily – several gene-therapy trials invarious regions of the world [36].

Up until then retroviruses had beenused as the delivery vectors in over 75% ofall gene-therapy clinical trials, mainly be-cause their molecular biology was well un-

derstood, the efficiency of gene transfer tosensitive cells was extremely high, subse-quent gene expression is usually high andhigh level stocks of replication-deficientretroviral particles can be produced. De-spite such undoubted advantages, retro-viruses also display certain disadvantagesin the context of gene delivery, includingtheir ability to infect only actively replicat-ing cells and the fact that their proviralDNA integrates randomly into the hostchromosome [37].

The emphasis is now shifting somewhataway from retroviruses and towards alter-native viral as well as nonviral vectors.Adenoviruses are receiving attention dueto their stability, easy manufacture, abilityto infect nondividing cells and their abilityto promote high-level gene expression (seealso Part I, Chapters 6 and 7). However,this category of vector is not without itsown difficulties, as adenoviruses tend tobe highly immunogenic in humans, dis-play broad cell specificity and the durationof resultant gene expression can be transi-ent. The most prominent nonviral vectortype remains liposome based [38] (see alsoPart VI, Chapters 7 and 8).

While genetic diseases constitute an ob-vious target for gene therapy, cancer re-mains the major indication (see also PartI, Chapter 1). A wide range of strategiescontinue to be pursued in this regard, in-cluding selected delivery of toxins/tumor-suppressor genes/suicide genes into tumorcells, modifying tumor cells to increasetheir immunogenicity or modifying lym-phocytes in order to enhance their antitu-mor activity [39].

8.7Antisense and RNA Interference (RNAi)

Antisense technology is based upon themanufacture of short single-stranded


stretches of nucleic acids (DNA or RNAbased) or chemically modified versionsthereof (see also Part I, Chapter 8). Thenucleotide sequence specificity of theseantisense molecules allows them to bindto specific gene or (more commonly)mRNA sequences, thereby preventinggene expression by blocking either tran-scription or translation [40]. The therapeu-tic rationale underlining this approachstems from the fact that many diseases aretriggered or are exacerbated by inappropri-ate expression/overexpression of specificgenes. Antisense, in principle, provides amechanism by which this can be blocked.While the underlining concept is straight-forward, like gene therapy, it is provingmore difficult to apply in practice. Majordifficulties have arisen in relation to prod-uct nuclease sensitivity, product targeting,delivery and cellular uptake.

Vitravene (fomivirsen sodium, ISISPharmaceuticals; see Table 9) remains theonly antisense-based biopharmaceutical ap-proved for general medical use (see alsoPart III, Chapter 3). The product is a 21-base phosphorothioate nucleotide that dis-plays a base sequence complementary tocertain human cytomegaloviral mRNAtranscripts. Its administration inhibits viralreplication through an antisense mecha-nism. Approved in the US in 1998 and inthe EU in 1999, the product is indicatedfor the treatment of cytomegalovirus reti-nitis by intraocular injection in AIDS pa-tients. It was withdrawn from the EU mar-ket in May 2002 for commercial reasons.

RNAi represents an alternative andmore recently pursued mechanism ofdownregulating gene expression ([41] andreferences therein) (see also Part II, Chap-ter 8 and Part I, Chapter 1 and 10). TheRNAi pathway was first discovered inplants, but it is now known to function inmost if not all eukaryotes. RNAi repre-

sents the sequence-specific post-transla-tional inhibition of gene expression, in-duced ultimately by double-stranded RNA(dsRNA). Be it produced naturally orsynthesized in vitro and introduced into acell by researchers, the (sequence-specific)dsRNA is then cleaved into short (20- to25-nt) fragments. The RNA strands thereinare separated – one is degraded and theother binds to a cellular protein complex.This strand will bind to target (comple-mentary) mRNA, which is then cleaved byan endonuclease within the complex. Be-cause of its ability to downregulate geneexpression, RNAi technology has obvioustherapeutic potential, and initial therapeu-tic targets of RNAi include viral infection,neurological diseases and cancer therapy.The synthesis of dsRNA displaying the de-sired nucleotide sequence is straightfor-ward. However, as in the case of additionalnucleic acid-based therapeutic approaches,major technical hurdles remain to be over-come before RNAi becomes a therapeuticreality.

8.8Stem Cell-based Therapies

The therapeutic application of stem cellshas long been a dream of medicalsciences, but recent discoveries and techni-cal advances have brought this dreammuch closer to being a reality. Stem cellsare usually defined as undifferentiatedcells capable of self-renewal, which can dif-ferentiate into more than one specializedcell type. Pluripotent stem cells are cap-able of essentially differentiating into anycell type, whereas multipotent stem cells,often found (be it in low numbers) withinspecific organs, give rise to lineage-re-stricted, tissue-specific cell types (see alsoPart I, Chapter 13). Human embryonicstem cells, harvested from the inner mass


of the blastocyst, are the most convenientsource of pluripotential cells. A quantumleap was taken in 2004 with the generationof an unlimited source of human embryo-nic stem cells by Woo Suk Hwang fromSeoul University. Hwang et al. were ableto obtain pluripotent embryonic stem cellsfrom somatic cell nuclear transfer of re-programmed human adult cells (see alsoPart I, Chapter 11). When cultured in thepresence of various specific growth factorsthese pluripotential cells have be inducedto differentiate into various mature celltypes, including liver, hematopoietic cells,neurons, pancreatic, skeletal and endothe-lial cells. Therefore stem cells harbor thepotential to form replacements for dam-aged/diseased body cells, tissue or evenentire organs (see also Part I, Chapters 12and 15). This technology could eventuallygive rise to cell-based therapies for variousneurological diseases, kidney, heart, lungor other organ failure, diabetes, cardiacdamage, etc. The scope and limitationscurrently underpinning stem cell technol-ogy are well beyond the scope of Introduc-tion, but will be discussed in the respec-tive chapters in this book. The interestedreader is also referred elsewhere [42–45]for sources of additional information.

9Concluding Remarks

Overall, the biopharmaceutical sector isone that is now maturing rapidly. The factthat biopharmaceuticals now generate inexcess of $ 30 billion revenue annually –from a zero starting point just over 20years ago – illustrates the medical and, in-deed, commercial importance of thesedrugs. Even more excitingly, continued ad-vances in both pure and applied medicalresearch will fuel continued growth within

the biopharmaceutical sector for manyyears to come.

Many of the concepts described in hereare explored in more detail in subsequentchapters. It was a real pleasure for me towrite this Introduction and I am im-pressed, because this book brings togetherworld-class contributions from world-classscientists, drawn from both industry andacademia. This compilation is one of themost comprehensive books published todate in this area and it is a “must-read” foreverybody working in this field.

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