Avrupa’da kabul edilen ilaçlara dair bir analiz (İng)


Analysis of the landscape of biologically-derived pharmaceuticals in Europe: Dominant production systems, molecule types on the rise and approval trends

  • Centre for Process Systems Engineering, Department of Chemical Engineering and Chemical Technology, Imperial College London, United Kingdom

Received 11 September 2012

Revised 20 November 2012

Accepted 23 November 2012

Available online 20 December 2012


A thorough sort of the human drugs approved by the European Medicines Agency (EMA) between its establishment in 1995 until June 2012 is presented herein with a focus on biologically-derived pharmaceuticals. Over 200 (33%) of the 640 approved therapeutic drugs are derived from natural sources, produced via recombinant DNA technology, or generated through virus propagation. A breakdown based on production method, type of molecule and therapeutic category is presented. Current EMA approvals demonstrate that mammalian cells are the only choice for glycoprotein drugs, with Chinese hamster ovary cells being the dominant hosts for their production. On the other hand, bacterial cells and specifically Escherichia coli are the dominant hosts for protein-based drugs, followed by the yeast Saccharomyces cerevisiae. The latter is the dominant host for recombinant vaccine production, although egg-based production is still the main platform of vaccine provision. Our findings suggest that the majority of biologically-derived drugs are prescribed for cancer and related conditions, as well as the treatment of diabetes. The approval rate for biologically-derived drugs shows a strong upward trend for monoclonal antibodies and fusion proteins since 2009, while hormones, antibodies and growth factors remain the most populous categories. Despite a clear pathway for the approval of biosimilars set by the EMA and their potential to drive sales growth, we have only found approved biosimilars for three molecules. In 2012 there appears to be a slow-down in approvals, which coincides with a reported decline in the growth rate of biologics sales.

Graphical abstract


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  • European medicines agency;
  • Host cells;
  • Therapeutic molecule class;
  • Therapeutic area;
  • rDNA drugs;
  • Biosimilars

1. Introduction

Biologically-derived drugs represent a growing sector of the pharmaceutical market. A classification of the currently approved drugs can aid in identifying key trends in the European biopharmaceutical industry and market, in terms of production hosts, types of molecules produced and therapeutic areas targeted. In the following article, we analyse the data for the biologically-derived drug approvals by the European Medicines Agency (EMA) until the start of June 2012 in terms of production method (organisms/hosts/methods used), molecule type and therapeutic category. We examine the trends of EMA-approved biologically-derived drugs with respect to approval rate and production method since its establishment in 1995, look at the threats and opportunities and draw conclusions on the landscape of biopharmaceuticals production in Europe.

2. Methodology

A thorough search was performed on the human medicines listed within the EMA database (http://www.ema.europa.eu/ema/) and the scientific discussions provided therein for all 651 drugs approved by the first half of June 2012 (uploaded prior to 3rd August 2012, last drug approved on 15/06/2012). The drugs were initially categorised based on whether their active substance was chemically synthesised or biologically derived. Subsequently, the biologically-derived drugs were further classified based on their production method as being derived from plants, human plasma, microbial or mammalian hosts, and the organism used was recorded. Further categorisation was performed based on the type of molecule used as the active substance, e.g. hormone, monoclonal antibody, etc. Finally, data on main therapeutic categories of the drugs were collated based on the Anatomical Therapeutic Chemical (ATC) categorisation by the World Health Organisation (WHO). ATC is a code-based system to categorise existing drugs into groups based on therapeutic, pharmacological and chemical properties. The methodology for the categorisation presented herein is outlined below.

2.1. Chemical synthesis versus biologically-derived drugs

EMA-approved drugs were classified based on the derivation of their active substance: mainly chemically synthesised drugs and drugs that their active substance is derived from natural sources (microorganism fermentation, excluding protein molecules, or extraction from plants), using recombinant DNA technology methods (proteins and glycoproteins) or virus propagation (for vaccines). This classification is in accordance with previous studies (Newman and Cragg, 2007, Newman and Cragg, 2012 and Newman et al., 2003). The categorisation resulted in the following sections (also summarized in Fig. 1A and B): proteins and glycoproteins, vaccines (listed under the J07 ATC subcategory), natural products (naturally occurring substances produced from fermentation of microorganisms, such as complex organic antimicrobial molecules, or derived from plant cells, such as betaine, not including protein molecules), semi-synthetic drugs containing biologically-derived active substances which have been further modified chemically, and cell therapies.

A further umbrella category is that of recombinant DNA technology drugs, which comprises glycoproteins (excluding those derived from human plasma and the one from the quadroma cell line), protein drugs (excluding two proteins derived from the fermentation of Clostridium bacteria), and recombinant vaccines. Our analysis excludes drugs containing fermentation-derived amino acids (e.g. Carbaglu®, which contains carglumic acid resulting from the reaction of l-glutamic acid and potassium cyanate, and the synthetic peptide containing drugs Signifor®, Byetta® and Budyron®) from the semi-synthetic category. It further excludes medicines under the V04, V08, and V09 ATC categories, which represent imaging and diagnostic tools rather than therapeutics (in total eleven drugs have been authorised by the EMA to date under these categories).

In cases where two or more drug formulations contain the same molecule as the active substance, we have considered them as two separate drugs. Examples of such cases include glycoproteins Xgeva® and Prolia®, both produced in CHO cells by Amgen Europe B.V., and proteins ViraferonPeg® and PegIntron® that contain pegylated interferon and are both marketed by Schering-Plough Europe.

2.2. Production method

This classification is based on the data included in the scientific discussions attached in the EMA website of all the drugs presented herein. The production method and host cell line for each drug are provided in Supplementary material (file 2).

2.3. Drug-molecule type

The classification of drugs with respect to molecule type was based on the format proposed by Aggarwal for protein and glycoprotein drugs (Aggarwal, 2007, Aggarwal, 2008, Aggarwal, 2009, Aggarwal, 2010 and Aggarwal, 2011). Only one category has not been included from that classification, which is that of thrombolytics/anticoagulants. These have been accounted for in three new categories, namely serpins, serine proteases, and non-human occurring peptides and proteins. These categories correspond to the actual molecule types of these drugs and not their therapeutic category or indication of treatment. For vaccines, natural and semi-synthetic drugs a different classification method has been used. Specifically, the chemical classification for natural and semi-synthetic drugs is based on the chemical classification of the substances from Pubchem (http://pubchem.ncbi.nlm.nih.gov/). Classification of vaccines is based on vaccine type, i.e. split virus inactivated, whole virus inactivated, live virus attenuated and antigen containing (recombinant or purified protein), or combinations of these types.

2.4. Therapeutic areas

There are two methods for categorisation into therapeutic areas: one is based on the ATC code and the other is based on the therapeutic category assigned by the EMA. For the purpose of our analysis we have used the former, as in Ferrer-Miralles et al. (2009), as this is the most objective way to classify the drugs. The ATC categories that were identified and the drugs within each category are presented in Supplementary material (file 4). Only one drug, namely Dificlir®, has not yet been assigned an ATC code. Dificlir® contains the antibiotic active substance fidaxomicin isolated from Dactylosporangium aurantiacum. We classified this drug within the J01 ATC category for “antibacterials for systemic use”, because it is prescribed to treat Clostridium infections.

3. Analysis

3.1. Major categories of EMA-approved drugs and their production method

As illustrated in Fig. 1A, chemically-synthesised pharmaceuticals constitute the majority (66.8%) of drugs approved by the EMA by May 2012, while glycoprotein- and protein-based drugs stand at 12.2% and 7.5%, respectively. Vaccines represent 6.1% of approved medicines, followed by semi-synthetic drugs, which contain starting material derived from a living organism, at 5.9%, natural at 1.3%, and, finally, only one approved drug as cell therapy (0.2%). The cumulative number of approvals of biologically-derived EMA-approved drugs for each category is presented in Fig. 1B and is discussed in the Landscape Overview part.

Fig. 1. (A) EMA-approved drugs to date displayed by major category. (B) Cumulative number of approvals of biologically-derived EMA-approved drugs. Recombinant DNA technology (rDNA) drugs have resulted from the summation of 67 glycoprotein drugs (not including the ten human plasma drugs and the one from the quadroma cell line), 46 protein drugs (not including the two drugs produced using Clostridium bacteria) and thirteen recombinant vaccine drugs.

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3.1.1. Glycoproteins

Glycoproteins represent the largest category of biologically-derived medicines, with 78 out of 212 approved biologically-derived drugs. Of these, ten have been filed as orphans and five as biosimilars. Orphans are drugs that do not affect more than five in 10,000 people within the European Union at the time of filing or drugs the production of which would not be financially viable “without incentives”. Seven out of the ten glycoproteins filed as orphans involve therapeutic enzymes targeting rare hereditary diseases, such as Fabry and Gaucher diseases and Mucopolysaccharidosis. The remaining orphan drugs correspond to two monoclonal antibody containing drugs, namely Soliris® and Arzerra®, prescribed to treat ‘infection-associated haemolytic uraemic syndrome’ and ‘chronic lymphocytic leukaemia’, respectively. Finally, the drug Rilonacept Regeneron®, which is a fusion protein, has also received an orphan designation to treat ‘cryopirin-associated periodic syndromes’. All five biosimilar drugs contain erythropoietin, which makes it the only EMA-approved biosimilar of a glycoprotein.

Glycoproteins are produced mainly in mammalian cell culture systems (Fig. 2A), since they are the sole means to deliver proteins with human-like glycosylation patterns and thus ensure reduced immunogenicity and higher in vivo efficacy and stability ( Jefferis, 2009, Wright and Morrison, 1997, Raju, 2008 and Sola and Griebenow, 2009). Specifically, Chinese hamster ovary (CHO) cells are the predominant host cell line for glycoprotein production (61.5%), followed by myeloma cells (14.1%), baby hamster kidney (BHK) cells (3.8%) and fibrosarcoma cell line HT-1080 (3.8%). Additionally, 12.8% of approved therapeutic glycoproteins are derived from human plasma, 2.6% are produced in transgenic animals and one product is derived from a hybrid hybridoma (quadroma) cell line (1.3%, Removab®). Of the aforementioned, only the ten human plasma derived-drugs and the one drug produced from the quadroma cell line are not produced with recombinant DNA technology.

Fig. 2. (A) Breakdown of host cells/organisms and production methods for glycoprotein-based drugs. CHO: Chinese hamster ovary cells; Murine myeloma: mainly correspond to NS0 and Sp2/0 cell lines; BHK: baby hamster kidney cells; HT-1080: a human fibrosarcoma cell line; Quadroma: a cell line that has resulted from a rat/mouse hybrid hybridoma; Human plasma: corresponds to human plasma extracted glycoproteins; Transgenic animals: corresponds to drugs produced from transgenic animals. (B) Host cells/organisms pie for protein-based drugs.

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3.1.2. Proteins

There are 48 approved non-glycosylated therapeutic proteins. Of these, three have been filed as orphans and eight as biosimilars. Orphan drugs involve two drugs in the pituitary and hypothalamic hormones and analogues ATC category, one of which is prescribed for the treatment of Acromegaly (Somavert®) and contains a recombinant human growth hormone analogue, and the other, Increlex®, is prescribed for the treatment of patients with Laron syndrome and contains recombinant human insulin-like growth factor-1. The third orphan drug is Nplate®, which belongs to the antihemorrhagics ATC category and contains a fusion protein. It is prescribed for idiopathic thrombocytopenic purpura. As far as the biosimilar drugs are concerned, two out of the eight are growth hormones, namely: Omnitrope® and Valtropin®. The remaining six are growth factors, specifically granulocyte colony stimulating factors (GCSFs). Namely these drugs are Biograstim®, Ratiograstim®, Tevagrastim®, Filgrastim Hexal®, Zarzio® and Nivestim®.

In contrast with glycoproteins, non-glycosylated protein-based drugs can be produced in microbial hosts, reducing the cost of production. As shown in Fig. 2B, the majority are produced in Escherichia coli (68.8%) or Saccharomyces cerevisiae (27.1%). A further 2.1% is produced in Clostridium botulinum and 2.1% in Clostridium histolyticum. All of the aforementioned protein drugs are produced using recombinant DNA technology except from the two drugs produced in Clostridium, which contain toxins naturally produced from the specific bacterial strain.

3.1.3. Vaccines

Currently, there are 39 EMA-approved vaccines, which correspond to a 6.1% of the total EMA-approved therapeutics. A large number of hosts of mammalian, insect and microbial origin are used to produce the active substances of vaccines as presented in Fig. 3A. These hosts have been segregated according to how many vaccine components are produced with each production platform. Therefore if a vaccine contains two or more components, the host system for each component has been taken into account separately (see also Supplementary material file 2). There are sixteen vaccines that include more than two active substances of biological origin. Twelve of the multiple active substances vaccines contain active ingredients derived from two hosts; two vaccine drugs contain active substances from four hosts, one an active substance derived from five different hosts (Infanrix Penta®) and one from six hosts (Infanrix Hexa®).

Fig. 3. (A) Host cells or methods (in the case of Hen’s eggs) of EMA-approved vaccines. Recombinant vaccines include the following host organisms: Saccharomyces cerevisiae, Escherichia coli (E. coli), Vibrio cholerae and insect cell line Hi-5 derived from Trichoplusia ni. Hen’s eggs and the following cell lines are mainly used for virus propagation: MDCK (Madin Darby canine kidney cells), Vero (kidney epithelial cells from African green monkey), MRC-5 (human foetal lung cell line), WI-38 (human lung fibroblast cell line) and chicken embryo cells. Some vaccines contain components resulting from the fermentation of the following bacterial cell lines: Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Haemophilus influenzae, Neisseria meningitides, Salmonella minesota, Streptococcus pneumonia. Most vaccine drugs contain more than one component from bio-origin. Therefore if a vaccine contains two or more bio-derived components, the host system for each component has been taken into account separately by fractioning the components for this particular vaccine. (B) Fractioned (based on number of active ingredients per drug, as also in (A)) accumulation of vaccines hosts/methods of production per year.

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Recombinant vaccines use rDNA technology to produce viral proteins (e.g. capsid proteins) that are used as active substances in the final vaccine formulation. As shown in Fig. 3A, there are thirteen recombinant vaccines approved to date and these are mainly produced in S. cerevisiae (14.4% of all vaccines). Most vaccine hosts, however, are used for virus propagation, with hen’s eggs being the dominant production platform (29.5% of all vaccines), followed by Vero cells (17.6% of all vaccines).

3.1.4. rDNA drugs

Currently, there are 126 EMA-approved drugs produced via recombinant DNA technology. These drugs result from the summation of 67 glycoprotein drugs (not including the ten human plasma drugs and the one from the quadroma cell line), 46 protein drugs (not including the two drugs produced using Clostridium bacteria) and thirteen recombinant vaccine drugs.

As presented in Fig. 4A 38.1% of total rDNA drugs are produced using CHO cells, 27% in E. coli cells, 18.3% in S. cerevisiae, 8.7% in murine myeloma cells, 2.4% from each of HT-1080 and BHK cell lines and 1.6% of transgenic animals. Our results are very similar with those presented in Ferrer-Miralles et al. (2009).

Fig. 4. (A) rDNA EMA-approved drugs host cells/organisms/production methods pie. CHO: Chinese hamster ovary cells; Murine myeloma: mainly correspond to NS0 and Sp2/0 cell lines; BHK: baby hamster kidney cells; HT-1080: a human fibrosarcoma cell line; Transgenic animals: corresponds to drugs produced from transgenic animals; Insect cell line: corresponds to Hi-5 cell line isolated from Trichoplusia ni. (B) Cumulative numbers of hosts/organisms/production methods used for the production of rDNA EMA-approved drugs.

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3.1.5. Cell therapies

Only one drug has been classified under this category. The brand name of this drug is ChondroSellect® prescribed to treat cartilage diseases. It involves the in vitro cultivation of the patient’s own chondrocytes and their re-injection to the patient.

3.1.6. Natural products (not including proteins)

We have identified eight drugs within this category, resulting to a 1.3% of the total EMA therapeutic drugs pie. Seven of these contain active substances produced naturally from bacteria and one (Cystadane®) containing the active substance betaine anhydrous extracted from sugar beets. Two drugs derived from natural sources are prescribed to treat orphan diseases: Cystadane® for the treatment of homocystinuria, and Tobi Podhaler® containing the aminoglycoside antibacterial tobramycin, prescribed to treat Cystic Fibrosis.

3.1.7. Semi-synthetic drugs

Semi-synthetic drugs correspond to 5.9% of the total number of EMA-approved therapeutics with 38 approvals. Seventeen semi-synthetic drugs are derived from plants, eleven from bacteria, nine from fungi and one is from porcine origin. The latter is a verteporfin-containing drug (Visudyne®) prescribed as an antineovascularisation agent. Four drugs from this category have been filed as orphans. All of these are categorised under the antineoplastics ATC category L01, with three containing rapamycin as an active substance. Thirteen semi-synthetic drugs are generics, i.e. they contain a similar molecule to a reference drug. Five of the aforementioned generics contain docetaxel, which is an active substance extracted from yew needle plants and prescribed as antineoplastics. Three contain the fungus-derived active substance mycophenolate mofetil, while the remaining five involve drugs containing the plant-derived topotecan.

Generic drugs contain active ingredients similar to an existing medicine, the so-called reference medicine. Generic and reference medicines contain the active ingredient in the same doses and are used to treat the same diseases. The active ingredients used for generics have demonstrated similar chemical structure with the reference medicine, as opposed to biosimilars, for which similarity cannot be proven due to the nature of the molecules contained (proteins, glycoproteins) and their complexity.

3.2. Major categories analysed by molecule type

3.2.1. rDNA drugs, human plasma-derived, quadroma-derived and non-human proteins

Fig. 5A and B present the molecule types for glycoprotein- and protein-based drugs, including human plasma-derived glycoproteins and the proteins naturally derived from Clostridium bacteria. In the umbrella category of rDNA drugs (i.e. excluding human plasma-derived, quadroma cell line derived and naturally occurring proteins), we find six blood factors, ten cytokines, six fusion proteins, 23 growth factor, 29 hormones, 25 monoclonal antibodies, two naturally occurring protein and peptides, two serine proteases, two serpins and eight therapeutic enzymes (also illustrated in Fig. 6A and B). These categories are discussed in detail below. Antibodies (Polyclonal and Monoclonal)

This category involves monoclonal antibodies (mAbs) as described in Aggarwal, 2011 and Elvin et al., 2011, i.e. including non-glycosylated antigen binding fragments. The latter are produced in E. coli cells, while all the other antibodies are produced in mammalian cells (CHO, murine myeloma and quadroma cell line). Polyclonal antibodies are antibodies derived directly from human plasma and involve human normal immunoglobulin and human hepatitis B immunoglobulin.

Monoclonal antibodies are proteins defined by a symmetric structure that consists of two heavy and two light chains attached by disulphide bridges and are very important molecules in the modulation of our immune response. Light and heavy chains are divided into constant and variable regions. The constant regions define the different isotypes of the heavy and light chains. Heavy chains, for example, can be of five isotypes, i.e. based on gene and in turn amino acid sequence, namely: A, D, E, G, or M (Jimenez del Val et al., 2010). All therapeutic antibodies are of type G, which is further divided into four subtypes: IgG1, IgG2, IgG3 and IgG4. The most therapeutically relevant isotype is the IgG1 format (Wright and Morrison, 1997, Jimenez del Val et al., 2010 and Walsh, 2010). The complex structure of mAbs is further divided into two regions: the antigen-binding fragment (Fab) and the crystallisable fragment (Fc). The Fab region, which comprises the variable regions of both heavy and light chains, is responsible for binding antigens with high specificity. The Fc region, which consists of the constant parts of the heavy and light chains, determines the mechanism by which the immune response will be mounted to the specific antigen (Jimenez del Val et al., 2010).

On the other hand, polyclonal antibodies contain a variety of antibodies that, in the case of approved therapeutics, are normally found circulating in human blood. Hence, the difference from monoclonal antibody treatment is that mAb formulations contain highly specific antibodies with defined Fab domains and not the variety of antibody molecules contained in human plasma-derived immunoglobulins. Blood and coagulation factors

This category has been expanded from the classification in Aggarwal, 2007, Aggarwal, 2008, Aggarwal, 2009, Aggarwal, 2010 and Aggarwal, 2011, which only includes blood factors VIIa, VIII and IX, to also consider two drugs containing fibrinogen, also known as blood coagulation factor I (Giangrande, 2003). The latter two drugs further include thrombin along with fibrinogen. Thrombin is a serine protease used to promote coagulation (Turgeon and Houenou, 1997). We have chosen to consider drugs containing both fibrinogen and thrombin under this category instead of serine proteases, since they are both considered blood coagulation agents. The two glycoproteins act synergistically in marketed therapeutics and are both contained in human plasma-derived drugs Evicel® and TachoSil®. The remaining EMA-approved drugs in this category involve factors VIIa (one approved drug), VIII (four approved drugs) and IX (two approved drugs). Further details are provided in Supplementary material (file 3). Cytokines

Interferon (IFN) and interleukins (IL) are the main active substances that have been listed in this category based on (Aggarwal, 2007, Aggarwal, 2008, Aggarwal, 2009, Aggarwal, 2010 and Aggarwal, 2011). We have also included two more active substances: tumour necrosis factor (TNF) and anakinra. TNF is a cytokine produced during invasion, injury or infection (Tracey and Cerami, 1994) and TNFα-1a is the active substance of the drug Beromun®. Kineret® is a formulation of anakinra, which is an Interleukin-1 receptor antagonist (IL-1RA). IL-1RA is a member of the IL-1 gene family, responsible for the regulation of IL-1α and IL-1β (Bresnihan et al., 1998). Both aforementioned drugs are produced in E. coli. As far as interferon drugs are concerned, two IFN-β 1α drugs are produced in CHO cells, namely Avonex® and Rebif®, while the remaining six are produced in E. coli. Fusion proteins

Fusion proteins refer to proteins that have been created by the fusion of two main protein molecules. Five of the six approved fusion proteins are produced in CHO cells, while one is produced in E. coli. The fusion proteins produced using CHO cells include the following drugs/molecules: the medicine Enbrel® contains the extracellular ligand-binding portion of human tumour necrosis factor receptor (p75) linked to an analog Fc of human IgG1 mAb. The drugs Orencia® and Nulojix®, both produced by Bristol-Myers Squibb Pharma EEIG, contain human cytotoxic lymphocyte associated antigen 4 (CTLA-4) and an Fc region of human IgG1 mAb. The difference between the aforementioned drugs is that the latter contains a slightly modified amino acid sequence (two different amino acids) for the CTLA-4, which enhances activity (Weclawiak et al., 2010). Rilonacept Regeneron® (previously Arcalyst®) contains extracellular domains of human cytokine receptor (parts of the interleukin-1 receptor) and the Fc portion of human IgG1. Finally, the active substance of Elonva® has resulted from the fusion of follicle-stimulating hormone and chorionic gonadotropin subunits.

The fusion protein-containing drug Nplate® is the only one within this molecule type category produced using bacterial cells and specifically E. coli. The drug consists of two identical subunits of a human IgG1 Fc domain linked to a peptide chain containing two thrombopoietin receptor-binding domains. Growth factors

This class of drugs comprises erythropoietin and Colony Stimulating Factors (CSFs). All ten approved erythropoietin drugs are produced in CHO cells. Growth factors are also produced in bacterial cells. Filgrastim is the active substance of seven approved drugs (six of which are biosimilars), which is a granulocyte colony stimulating factor (G-CSF). All of the aforementioned drugs are produced in E. coli. Other E. coli-derived drugs in this category include the recombinant human keratinocyte growth factor (rHuKGF) containing drug Kepivance® and the recombinant human insulin-like growth factor-1 (rhIGF-1)-containing drug Increlex®. Finally, there is also the platelet derived growth factor (PDGF)-containing drug Regranex® that is produced in S. cerevisiae.

Further to previous studies (Aggarwal, 2007, Aggarwal, 2008, Aggarwal, 2009, Aggarwal, 2010 and Aggarwal, 2011), we have also included in this category bone morphogenetic proteins (BMP), also produced in CHO cells. These drugs are represented by BMP-2 (InductOs®) and BMP-7 (Osigraft® and Opgenra®) proteins. The ten erythropoietins and three BMPs are the only glycoproteins in the growth factor class; hence they are produced in mammalian cell systems. Hormones

In 2010 hormones were the second-highest class of biologics in terms of sales in the USA (Aggarwal, 2011). Out of the 29 hormone-containing approved drugs, 22 are produced in microbial cell systems and seven in CHO cell production platforms. Insulin has received the most approvals out of the microbial cell-derived drugs with 15 brand names. Seven insulin-containing drugs are produced using E. coli and the remaining eight using S. cerevisiae (brand names are provided in Supplementary materialfiles 2 and 3). Growth hormone, also known as somatropin, is the active ingredient of the following three authorised drugs: NutropinAq®, Omnitrope®, and Valtropin®. The former two are produced using E. coli as host, whereas for the production of Valtropin® S. cerevisiae is used as a host. Parathyroid hormone is the active ingredient of two branded drugs, both produced in E. coli (Preotact®, Forsteo®). Modified growth hormone that acts like a growth hormone receptor antagonist (Somavert®, produced in E. coli), and a fragment of the naturally occurring human glucagon-like peptide-1 sequence (active substance known as liraglutide and given the brand name Victoza®, produced in S. cerevisiae) are two other authorised drugs that we have also considered in the hormones category.

The following hormones are produced using mammalian cells (all CHO cells) corresponding to a total of seven drugs: follicle stimulating hormone, also known as follitropin, is branded as Gonal-f®, Puregon® and Fertavid®. Luteinizing hormone (lutropin) is branded as Luveris®. Thyrotropin stimulating hormone (thyrotropin alfa) is branded as Thyrogen®. Chorionic gonadotropin (choriogonadotropin) is branded as Ovitrelle®. Finally, there is also an approved drug (Pergoveris®) that contains two hormones, namely follitropin and lutropin. Non-human peptides and proteins

One drug approved by the EMA is based on the natural anticoagulant found in the saliva of the European leech (Hirudo medicinalis), also called hirudin. Hirudin is a 65 amino acid peptide (Schlaeppi et al., 1990). The marketed drug, namely Revasc®, is produced by rDNA technology in S. cerevisiae.

Natural proteins correspond to a non-human enzyme-containing drug (Fasturtec®) and two toxin-containing drugs. Fasturtec® contains the enzyme urate oxidase (recombinant form of which is named rasburicase), an enzyme that is not naturally present in humans. The drug is produced with rDNA technology using cDNA from Aspergillus flavus in S. cerevisiae. The two toxin-based drugs within this category correspond to the botulinum toxin type B produced by fermentation of C. botulinum, branded as NeuroBloc®, and the collagenase toxin, branded as Xiapex®, produced in C. histolyticum. Serine proteases

We have classified in this category the serine proteases: protein C (isolated from human plasma) in Ceprotin® and the tissue plasminogen activator containing drugs (t-PA) Metalyse® produced in CHO cells, and Rapilysin® produced in E. coli. Protein C is also called blood and coagulation factor XIV (Seegers et al., 1976), so could also have been included in the blood and coagulation factors category. However, we selected to include it in the serine proteases category following the nomenclature in Giangrande (2003). Serine proteases, amongst many other biological roles, play a very important part in the homeostasis of the cardiovascular system, including coagulation, but also fibrinolysis (removal of blood clots, further discussed in part 3.3.1) and tissue regeneration ( Turgeon and Houenou, 1997 and Bouton et al., 2012). Serpins (serine protease inhibitors)

Serpins are a class of protein molecules that represent a whole superfamily of proteins (Hunt and Dayhoff, 1980). These drugs correspond to the following active substances: antithrombin alfa (Atryn®), conestat alfa (Ruconest®), C1 inhibitor (Cinryze®). The first two drugs are produced in transgenic animals (goat and rabbit, respectively), while Cinryze® is extracted from human plasma. It is important to mention here that conestat alfa is the recombinant form of C1 inhibitor (Davis and Bernstein, 2011). Therapeutic enzymes

Therapeutic enzymes are mainly characterised by the use to treat rare diseases (Aggarwal, 2007, Aggarwal, 2008, Aggarwal, 2009, Aggarwal, 2010 and Aggarwal, 2011). All except one have orphan designations. The only therapeutic enzyme containing drug that does not have an orphan designation is Cerezyme®, which is prescribed for patients with Gaucher disease. All drugs within this category are produced using mammalian cells as hosts, with five produced in CHO cells and three in the human cell line HT-1080.

3.2.2. Vaccines

In Fig. 5C we attempt to group vaccines based on their active substance. The four major types are attenuated live virus containing vaccines (six), inactivated split virus vaccines (six), inactivated whole virus and non-recombinant antigen-containing vaccines with five approved products in each category. It should be noted that although thirteen vaccines contain recombinant antigens/proteins, only three of them contain solely the recombinant protein as the active substance. The remaining ten also include some other type of active substance along with the recombinant protein and hence are classified separately in Fig. 5C.

Fig. 5. Breakdown of EMA-approved drugs by molecule class for glycoproteins (A), proteins (B) and vaccines (C). From left to right, the labels and their corresponding number (in brackets) of EMA-approved vaccines are: live virus attenuated (6), whole virus inactivated (5), antigen containing not recombinant (5), solely recombinant vaccines (3), split virus inactivated (6), recombinant antigen, toxoid and whole cell inactivated (1), recombinant antigen and whole virus inactivated (3), antigen, polysaccharides on toxoids (the latter used as a carrier protein), recombinant antigen, toxoids and whole virus inactivated (1), recombinant antigen with a derivative from the lipopolysaccharides of Salmonella minesota as an adjuvant (2), recombinant antigen and whole cell inactivated (1), recombinant antigen and polysaccharides conjugated to toxoids (1), oligosaccharides conjugated to carrier protein (1), antigen, recombinant antigen, toxoid and whole virus inactivated (1), polysaccharides conjugated to carrier protein (2), polysaccharides conjugated on toxoid (1).

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3.2.3. Natural products (not including proteins)

Molecule types of natural drugs involve macrolide (four approved drugs), aminoglycoside (one approved drug), cyclic lipopeptide (one approved drug), macrocycle (one approved drug) antibiotics and trimethyl-glycine (or betaine, one approved drug). These are shown in the tables provided in Supplementary material (file 3).

3.2.4. Semi-synthetic drugs

We identified 16 different molecule types within the EMA-approved semi-synthetic drugs. Taxanes-containing drugs are the largest category with nine approved drugs. The second largest category is alkaloids-containing drugs with six approved medicines, five of which are generics of the reference drug Hycamtin® licensed by the EMA in 1996.

Four of the semi-synthetic drugs are macrolides, while another four morpholinoethyl esters of mycophenolic acid. As discussed earlier three of the latter are generics of the reference medicine CellCept® approved by the EMA in 1996. The fifth largest category of semi-synthetic drugs involves echinocandins, and the remaining categories of semi-synthetic drugs are represented by one drug each, as presented in Supplementary material (file 3).

3.3. Major categories analysed by ATC code

ATC categories of rDNA drugs and vaccines are presented in Fig. 7A and B, respectively. These are further analysed below along with the ATC categories of human plasma- and quadroma-derived, natural protein containing, natural (non-protein-containing) and semi-synthetic drugs.

3.3.1. rDNA drugs

EMA-approved rDNA drugs are classified in 24 different ATC categories (presented in Fig. 7A). Drugs prescribed for diabetes (A10 ATC category) are the largest category with 16 approved products. Diabetes is a metabolic disorder that causes hyperglycemia due to either insufficient production or reduced action of insulin, which is a pancreatic hormone that aids in reducing blood glucose levels (Maritim et al., 2003 and Rorsman, 2005).

Fig. 7. Approved rDNA drugs (A) and vaccines (B) categorised by ATC code. ATC (Anatomical Therapeutic Chemical categorisation) is a code-based system established by the World Health Organisation (WHO) to categorise existing drugs into groups based on their therapeutic, pharmacological and chemical properties.

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The rDNA medicines in the diabetes ATC category include human insulin in various forms. This can be, for example, human insulin with different amino acid sequences, such as rapid-acting insulin glulisine (Apidra®, Sanofi-Aventis), or insulins with another molecule attached to them, such as the long acting insulin drug Levemir® (Novo Nordisk). The latter, contains a fatty acid attached to the insulin molecule. Only one molecule is not insulin-based; Victoza® contains a fragment of the human glucagon-like peptide-1 (GLP-1), which is a glucose-dependent hormone. Among its other biological roles, it lowers the plasma levels of glucagon (peptide hormone that has opposite action of insulin, i.e. raises blood sugar levels), and aids gastric motility (Kieffer and Habener, 1999).

Immunostimulants (L03 ATC category) and immunosuppressants (L04 ATC category) share the first place of approved drugs along with those prescribed for diabetes. Immunostimulants include cytokines (interferons and tumour necrosis factor) and growth factors (granulocyte colony-stimulating factor known as filgrastim). Currently, there are eight approved interferon-containing EMA-approved drugs under the L03 ATC code that are prescribed to treat conditions such as multiple sclerosis, a chronic autoimmune disorder (Dhib-Jalbut and Marks, 2010), conditions associated with cancer (leukemia, follicular melanoma), or hepatitis B and C. Interferons are a wide family of cytokines with anti-proliferative, immunoregulatory and antiviral properties (Kirkwood, 2002). Interferons of alfa type (IFNα) are prescribed for cancer-associated diseases and multiple sclerosis, whereas interferons beta (IFNβ) for multiple sclerosis.

Tumour necrosis factor (TNF) is another cytokine in the L03 ATC category that is prescribed to treat sarcoma. One drug has been approved under this description (Beromun®). Sarcomas involve mesodermal malignancies/cancers (Borden et al., 2003). The functionality of TNF in cell survival, inflammation, immunity and apoptosis is mainly employed locally to reduce the possibility of limb amputation (van Horssen et al., 2006) in such conditions.

The growth factor filgrastim in the immunostimulants ATC category is mainly prescribed to treat neutropenia. Neutropenia is a condition often resulting after cancer treatment and is associated with a low blood count of neutrophil granulocytes (type of white cells of the immune system). The granulocyte colony stimulating factor (the recombinant form of which is termed filgrastim) stimulates the production of such kind of white blood cells (Waller, 2012). The relatively large number of drugs under the immunostimulants category can be attributed to the biosimilars of the reference drug Neupogen® (approved in 1991) containing filgrastim, which entered the market in 2008. There are six filgrastim biosimilars, of which three were marketed in 2008, two in 2009 and one in 2010.

On the other hand, immunosuppressants (L04 ATC category) mainly consist of fusion proteins and monoclonal antibodies. Immunosuppressants are prescribed to treat autoimmune diseases, such as for example arthritis, lupus erythematosus and Crohn’s disease. Specifically, four fusion molecules are prescribed as immunosuppressants, namely: Enbrel®, Orencia®, Rilonacept Regeneron® and Nulojix®. Eleven drugs of the immunosuppressant category are mAbs, one of which consists of only a Fab fragment produced in E. coli (Cimzia®).

In general terms, fusion proteins that contain antibody fragments act similarly to mAbs in order to treat disease. Briefly, monoclonal antibodies act as “magic bullets” against unrecognised invaders (antigens, cells) of one’s organism generating an immune response cascade against them (Jimenez del Val et al., 2010 and Strebhardt and Ullrich, 2008). They can also be engineered to target and stop the action of specific proteins and this is the main philosophy behind their use either as immunosuppressants, antineoplastics, or to treat bone and several other diseases in various ATC codes (described at the end of this section).

The immunosuppressants ATC category also contains the drug Kineret®, which contains a human interleukin-1 receptor antagonist. This molecule blocks interleukin-1 signalling (Fleischmann et al., 2006) and is prescribed to treat rheumatoid arthritis. The interleukin-1 term is used to describe a wide family of cytokines regulating immune response (Sims et al., 1993).

The fourth largest ATC category of approved rDNA drugs are recombinant vaccines. There are thirteen approved recombinant vaccines, of which only three are solely recombinant (two against human papilloma virus and one against hepatitis B), while the rest also contain other viral and bacterial non-recombinant components. Five of the recombinant vaccines are for hepatitis (J07BC ATC category) and the recombinant component is a hepatitis B virus surface antigen. Three are under the J07CA ATC code, i.e. bacterial and viral vaccines combined, with the recombinant component again being a hepatitis B virus surface antigen.

Three more vaccine drugs provide immunisation against the human papilloma virus (J07BM ATC category) and contain the major capsid protein of papillomavirus as the main active ingredient. The remaining two recombinant vaccines are administered for immunisation against cholera (J07AE ATC code), containing the recombinant cholera toxin B subunit, and against pneumococcal infection (J07AL ATC code) containing among other components a recombinant carrier protein produced in E. coli.

Antianemics (B03XA ATC category) follow in the ATC categorisation list with ten approved drugs. All the drugs within the B03XA category contain erythropoietin as the active substance and five of them have been filed as biosimilars, all filed in 2007. Erythropoietin is a growth factor that regulates the production of red blood cells in the bone marrow (Krantz, 1991) and hence directly associated with anaemic treatment.

Antineoplastic drugs (under the L01XC ATC category) and alimentary tract and metabolism drugs (A16AB ATC code) are next with 6.3% each of the rDNA ATC pie. Eight recombinant mAbs are included in the antineoplastics category the active ingredients of which are provided in Supplementary material (files 1 and 4).

Alimentary tract and metabolism drugs include the eight therapeutic enzymes that are prescribed to treat rare diseases such as Fabry, Gaucher diseases and Mucopolysaccharidosis, also discussed in part These diseases are mainly associated with deficiencies of certain metabolic enzymes leading to accumulation of specific metabolites into the lysosomes and hence also called lysosomal storage diseases (Desnick and Schuchman, 2012). The enzyme agalsidase (recombinant form of alpha-galactosidase) is prescribed to treat Fabry disease and is administered by two different forms, namely: alfa (Replagal®) and beta (Fabrazyme®). The lack of the enzyme causes a defect in the degradation of neutral glycosphingolipids, which accumulate progressively in body fluids and tissues (Lidove et al., 2012).

The enzyme glucocerebrosidase (acid-β-glucosidase) in the recombinant forms imiglucerase (Cerezyme®) and velaglucerase alfa (Vpriv®) is prescribed to treat Gaucher disease. This is an inherited lipid storage disease (Panicker et al., 2012). Gaucher disease was the first lysosomal disorder to receive enzyme replacement therapy (Desnick and Schuchman, 2012).

Mucopolysaccharidoses are diseases associated with several enzyme deficiencies that have been classified into seven types involving defects of glycosaminoglycan degradation (Desnick and Schuchman, 2012). Enzyme replacement therapies have been approved for three of the aforementioned types, namely: Mucopolysaccharidosis I, II and VI. The enzyme α-L-iduronidase (its recombinant form is called: laronidase and branded as Aldurazyme®) is prescribed to treat Mucopolysaccharidosis I. The enzyme iduronate-2-sulfatase, called idursulfase in its recombinant form, is prescribed to treat Mucopolysaccharidosis II and is branded as Elaprase®. Finally, Naglazyme® contains galsulfase, which is the recombinant form of the enzyme N-acetylgalactosamine 4-sulfatase and is prescribed to treat Mucopolysaccharidosis VI.

One more therapeutic enzyme is included in the alimentary tract and metabolism ATC code (A16AB ATC code), prescribed to treat Glycogen Storage Disease Type II, also known as Pompe disease. This disease is associated with ineffective glycogen storage mainly in skeletal and smooth muscle (Desnick and Schuchman, 2012). The enzyme is called alpha-glucosidase (its recombinant form is known as alglucosidase alfa) and is branded as Myozyme®.

Antihemorrhagics (B02 ATC code) and sex hormones and genital system modulators (G03GA ATC code) share the same percentage in the ATC classification pie with 5.6%, corresponding to seven drugs each. Antihemorrhagics are represented by the six recombinant blood factor-containing drugs discussed in part, as well as fusion protein Nplate®. Blood factors (VIIa, VIII and IX, as described in part are directly associated with regulating coagulation and are mainly used in the treatment of haemophilia. Haemophilia is a genetic disorder associated with blood factors VIII (haemophilia A) and IX (haemophilia B) deficiencies (Mannucci, 2003). In turn, factor VIIa is prescribed for both types of haemophilia, since it is involved in both factor VIII and IX coagulation cascades (Monroe, 2012).

The fusion protein within the antihemorrhagics category (Nplate®) consists of a thrombopoietin receptor-binding domain attached to the Fc region of an IgG1 mAb, as also discussed in part This enhanced molecule increases platelet production in the bone marrow by mimicking thrombopoietin, a protein produced in the liver and the kidney that stimulates platelet production. Simultaneously, the Fc region portion increases the half-life of the molecule (Kuter, 2008).

The sex hormone and genital system modulators ATC category comprises six hormone-containing drugs and a fusion glycoprotein of two hormones (follicle stimulating hormone and chorionic gonadotropin). All drugs within this ATC category are prescribed to assist female ovulation and are all produced in CHO cells.

Pituitary and hypothalamic hormones and analogues (H01 ATC code) share 4.8% of rDNA drugs. Five drugs contain hormones and one the insulin like growth factor-1-containing drug Increlex®. Three of the hormone-containing drugs contain growth hormone (recombinant form of which is called somatropin), while the remaining two contain thyrotropin stimulating hormone and a growth hormone receptor antagonist (see also part Growth hormone-containing drugs are prescribed to treat diseases such as Dwarfism and Pituitary Turner Syndrome. Both diseases are closely associated with the dysfunction of the pituitary gland, where growth hormone is produced (Isaksson et al., 1985). Growth hormone stimulates cell growth and regeneration (Isaksson et al., 1985).

Thyrotropin stimulating hormone (TSH, recombinant form known as thyrotropin alfa), is another hormone within the H01 ATC category. It is used to treat thyroid neoplasms. TSH acts in the thyroid gland and stimulates the production of other hormones (T3, T4) that regulate the metabolism of nutrients (Danforth and Burger, 1989 and Fisher, 1996). The growth hormone receptor antagonist drug within the H01 ATC category is prescribed to treat patients with Acromegaly. Patients diagnosed with Acromegaly produce excessive amounts of growth hormone and as a result certain parts of their body become unnaturally large (Kopchick et al., 2002). The administration of the growth hormone receptor antagonist blocks the growth hormone receptor in the patients’ cells and reduces irregular growth (Kopchick et al., 2002).

In turn, the insulin-like growth factor-1 (IGF-1)-containing drug Increlex® within the H01 ATC category is prescribed to treat humans with Laron Syndrome. Individuals with this disease present insensitivity to growth hormone (Walker et al., 1991) and for this reason IGF-1 is administered to them. IGF-1 is a hormone produced by the liver as the result of growth hormone action in healthy individuals (Walker et al., 1991).

Drugs to treat bone disease (M05 ATC category) and antithrombotics (B01 ATC category) come next at 4% (five drugs) and 3.2% (four drugs) respectively. The former include three bone morphogenetic proteins and two mAbs. Bone morphogenetic protein drugs involve two bone morphogenetic protein-7 (BMP-7) containing drugs and one bone morphogenetic protein-2 (BMP-2) drug, as discussed in part Bone morphogenetic proteins induce bone formation and regulate tissue reformation (Laflamme and Rouabhia, 2008). Hence, BMPs are directly associated with the treatment of bone diseases.

The mAb formulations within the bone diseases M05 ATC category are both based on denosumab and are marketed under the brand names Xgeva® and Prolia®. These drugs are produced by Amgen Europe B.V. and although contain the same active substance; they are prescribed for different indications. Xgeva® is prescribed for bone-associated cancer metastasis, whereas Prolia for osteoporosis. In this case, the mAb is engineered to target and subsequently stop the action of a protein (receptor activator of nuclear factor kappa-B ligand, RANKL) that is known to regulate the growth and survival of osteoclasts, which are bone cells responsible for bone resorption (Lewiecki and Bilezikian, 2012).

Antithrombotics (B01 ATC code) contain one hirudin (naturally-occurring non-human peptide) drug, two serine proteases, i.e. the tissue plasminogen activator (t-PA) containing drugs Rapilysin® and Metalyse®, and a serpin (ATryn®). Hirudins, also discussed in part, are non-human peptides that act as thrombin inhibitors. Thrombin is an important serine protease in the coagulation cascade (Turgeon and Houenou, 1997). The hirudin containing drug within the B01 ATC category is branded as Revasc® and is prescribed to treat venous thrombosis.

Tissue plasminogen activator (t-PA) drugs within the B01 ATC category are prescribed to treat myocardial infarction. T-PA aids the removal of fibrin from the blood. Fibrin is a protein responsible for the formation of blood clots and hence its removal is critical in order to maintain blood fluidity (Sheehan and Tsirka, 2005). Thrombin, which was discussed earlier, facilitates the production of fibrin and, for this reason, its inactivation (e.g. by hirudins) is crucial for direct antithrombotic action (Sheehan and Tsirka, 2005).

The serpin-based drug within the antithrombotics category (ATryn®) contains antithrombin alfa and is produced using transgenic goats. Antithrombin, as the term also suggests, presents an inhibitory action against thrombin and in this specific case is administered to patients with antithrombin III deficiency (Rodgers, 2009).

Detoxifying agents (V03AF ATC code) for antineoplastic treatment and calcium homeostasis (H05 ATC code) categories contain two rDNA drugs each, corresponding to 1.6% each of the total rDNA drugs ATC pie. The former category includes a human keratinocyte growth factor (Kepivance®) and a non-human naturally-occurring occurring enzyme (Fasturtec®). The human keratinocyte growth factor (KGF) containing drug is prescribed to treat mucositis. Mucositis mainly arises after chemotherapy or radiotherapy treatment in patients with cancer. The disease is associated, among other clinical indications, with the formation of ulcers in the oral, oesophageal and intestinal areas (Blijlevens and Sonis, 2007). KGF enhances the regeneration of epithelial cells, which are the main recipients of the adverse effects associated with the aforementioned condition.

The drug Fasturtec® is another drug prescribed as a detoxifying agent. It contains the non-human enzyme urate oxidase (as also discussed in part, that aids in breaking down uric acid to allantoin, which can be secreted in urine more easily than uric acid (Hochberg and Cairo, 2008). Urate oxidase is prescribed to treat hyperuricemia, which is a disease that often occurs in cancer patients and is associated with the build-up of uric acid in blood (Hochberg and Cairo, 2008).

The drugs within the H05 ATC category are parathyroid hormones. The two parathyroid hormones, one short (Forsteo®) and one that has the full length of the human parathyroid hormone (Preotact®), currently in the EMA market are prescribed to treat osteoporosis. Parathyroid hormones play a crucial role in regulating bone formation by osteoblasts, which are the specialised cells for this kind of purposes in humans (Kraenzlin and Meier, 2011).

Six more ATC categories contain one rDNA drug each. Blood substitutes and perfusion solutions (B05 ATC code) include the recombinant C1 inhibitor (Ruconest®) containing drug produced using transgenic rabbits. C1 inhibitor is further described in part 3.3.2. Dermatologicals (D03 ATC code) contain the rDNA drug Regranex® that contains a platelet derived growth factor (PDGF). The drug is prescribed to treat skin ulcers and to facilitate wound healing. PDGF has direct effects in skin repair, since it enhances the proliferation and migration of connective tissue cells. It also stimulates the synthesis of extracellular matrix by the aforementioned type of cells (Rollman et al., 2003).

The following drugs use the capabilities of mAbs, to treat various diseases. In the immune sera and immunoglobulins (J06 ATC code) category a mAb against the respiratory syncytial virus has been licensed and is branded as Synagis®. Another mAb-containing drug (Xolair®) is prescribed to treat asthma and is categorised in the respiratory system (R ATC code) drugs. Antineovascular agents (S01LA ATC code), also contain the mAb Lucentis® prescribed to treat wet macular degeneration, which is a degenerative disease. Finally, one mAb-containing drug (Zevalin®) is included in the therapeutic radiopharmaceuticals (V10 ATC code) category licensed to treat follicular lymphoma. The drugs described in this part are also presented in detail in the tables of the Supplementary material (files 1 and 4).

3.3.2. Human plasma- and quadroma-derived drugs

Five of the ten human plasma-derived drugs are members of the J06 ATC category, standing for immune sera and immunoglobulins and are all polyclonal antibodies. Four of these contain human normal immunoglobulin and one hepatitis B immunoglobulin. The four drugs containing human normal immunoglobulin are prescribed to treat immunologic deficiency syndromes and mainly contain high purity IgG-type monoclonal antibodies isolated from the plasma of healthy donors (Wood, 2012).

The plasma derived drug that contains hepatitis B immunoglobulin (Zutectra®) is mainly prescribed to provide passive immunisation against Hepatitis B. It again contains primarily IgG-type antibodies specific against hepatitis B virus.

From the remaining human plasma drugs, four are included in the B02 ATC category, which corresponds to antihemorrhagics. Three of these are in the blood and coagulation factors molecule type category and one is a serpin. The plasma-derived blood and coagulation factors in the B02 ATC category include a human coagulation factor IX that is prescribed to treat Hemophilia B (described in more detail in part 3.3.1) and two drugs that contain fibrinogen and thrombin. Fibrinogen and thrombin play an important role in the coagulation cascade and are both prescribed for haemostasis following surgery (Lew and Weaver, 2008).

The plasma-derived serpin in the B02 ATC category involves the C1 inhibitor-containing drug Cinryze®, which is prescribed to treat hereditary angioedemas. Hereditary angioedemas are mainly associated with C1 inhibitor deficiency (Sickels et al., 2010). C1 inhibitor plays an important role in the regulation of the complement system, which is the first line of defence and the mediator between adaptive and innate immune responses as described in Lambris et al. (2008).

The remaining plasma derived drug is a serine protease, listed in the B01 ATC code, i.e. prescribed as an antithrombotic. This drug (Ceprotin®) contains protein C and is prescribed for a disease associated with protein C deficiency termed as Purpura Fulminans. Protein C is another coagulation protein critically associated with thrombin (Goldenberg and Manco-Johnson, 2008) that has antithrombotic actions (thrombin is further discussed in part 3.3.1 above).

As far as the quadroma-derived drug is concerned this is included in the L01XC ATC category, i.e. prescribed as antineoplastic. Specifically, the drug targets ascites cancer and is a trifunctional bispecific antibody (Bokemeyer, 2010). The antibody consists of a rat IgG2b heavy and light chain with the Fab region being able to associate with T lymphocytes (cells of the human immune system) and a mouse IgG2a heavy and light chain with the Fab region this time able to bind to a protein highly expressed in many epithelial tumours (Bokemeyer, 2010). This engineered antibody actually results from the association of two different pairs of heavy and light chains from different organisms. The Fc region of this specific antibody binds to other “accessory” cells of the immune response different than T lymphocytes, e.g. macrophages. Hence, in contrast with ordinary antibodies, this engineered molecule has the ability to employ two different classes of immune response cells to destroy cancerous ones.

3.3.3. Naturally occurring proteins

The two proteins produced from Clostridium bacteria are listed under two ATC codes. The botulinum toxin type B containing drug NeuroBloc® is listed under the M05 ATC category standing for muscle relaxants. This drug is prescribed to treat Torticollis, also known as cervical dystonia, which is a disease associated with involuntary contraction of specific muscles (Brashear et al., 1999). The particular toxin helps alleviate the disease symptoms.

The collagenase-containing drug Xiapex is listed under the “other drugs for the musculoskeletal system” ATC M09 category. The drug is prescribed to treat Dupuytren’s contracture, a connective tissue disease (Wurster-Hill et al., 1988) characterised by problematic growth and proliferation of collagen (main ingredient of connective tissue) in the palm and in the bottom of the foot (area that supports the arch). The active substance consists of two different collagenases that have the ability to cleave collagen and so weaken contracted collagen cords and improve elasticity (Gilpin et al., 2010).

3.3.4. Vaccines

ATC categories of vaccines are presented in Fig. 7B. 41% of marketed EMA-approved vaccines provide immunization against influenza vaccines. Hepatitis vaccines are the second largest ATC category at 12.8%. Three ATC categories share the third place at 7.7% each, which are bacterial and viral combined, papillomavirus and pneumonococcal vaccines. The remaining categories can be seen in Fig. 7B and in the relevant table in Supplementary material (file 4).

3.3.5. Natural products (not including proteins)

From the total of eight natural (no protein based) identified drugs, three drugs are prescribed as immunosuppressants (L04 ATC code) and exactly the same number as antibacterials for systemic use (J01 ATC code). The natural immunosuppressants use macrolides as their active substance and are produced from the fermentation of Streptomyces bacteria. The drugs within the J01 ATC code contain complex antibacterial molecules (cyclic lipopeptide, aminoglycoside and macrocycle). The remaining two drugs are in the alimentary tract and metabolism ATC category A16 (a trimethyl-glycine containing drug) and in the dermatologicals ATC category D (a macrolide-containing drug).

3.3.6. Semi-synthetic drugs

The majority of semi-synthetic drugs are prescribed as antineoplastics (L01 ATC code). Twenty-one of the total 38 semi-synthetic drugs are antineoplastics. Hence, antineoplastics account for 54.1% of the ATC codes assigned to semi-synthetic drugs. Semi-synthetic drugs in the antineoplastics ATC category mainly involve topotecan (synthesized using the plant derived alkaloid camptothecin), taxanes (containing starting material from the plants of the genus Taxus) and molecules (temsirolimus, everolimus) containing rapamycin as starting material.

Four of the semi-synthetic drugs are prescribed as immunosuppressants (L04 ATC code). These drugs contain mycophenolate mofetil, also described in parts 3.1.7 and 3.2.4. Three are prescribed as antibacterials for systemic use (J01 ATC code) and another three as antimycotics for systemic use (J02 ATC code). Two are included in the respiratory system ATC code R. While each of the following ATC categories consist of one semi-synthetic drug: antianemics (B03 ATC code), antineovascularisation agents (S01LA ATC code), antiprotozoals (P01 ATC code), antivirals for systemic use (J05 ATC code) and dermatologicals (D ATC code). The active substances of the aforementioned drugs are included in Supplementary material (files 1 and 4).

4. Landscape overview

Chemically synthesised drugs represent the majority of medicines approved by the EMA. However, rDNA drugs are an increasingly significant class of high-value pharmaceuticals that currently accounts for 19% of the therapeutic drugs in the European market. Recombinant drugs are used for a vast array of diseases predominantly targeted against diabetes, cancer and immunosuppression. The recombinant DNA technology drugs targeting cancer include antineoplastics, i.e. drugs that act directly against cancer, or medicines that target conditions associated with cancer such as anaemia, or treatments working alongside cancer therapeutics, such as immunostimulation. The recombinant drugs used for immunosuppression involve the treatment of autoimmune disorders or are used to facilitate transplantations.

Overall, insulin and insulin analogues together with monoclonal antibodies for cancer and associated conditions dominate the biologically-derived pharmaceuticals landscape. These two drug categories are also displaying high sales growth rates as reported in Aggarwal (2011). In the vaccine arena, a large percentage of commercial products target the prevention of influenza infections, followed by hepatitis and vaccines for children. In recent years, we have also seen the approval of the human papilloma virus vaccine (recombinant), which is offered free-of-charge through the national healthcare systems of nine European countries (since 2009 in the UK), and has grasped a substantial share of the vaccines sector.

Mammalian cells, CHO cells in particular, are still the dominant production system for glycoproteins thanks to their human-like post-translational modification machinery. Non-glycosylated proteins are produced in microbial cell systems, which are a cheaper and easier to manipulate production platform. Our findings are similar to those reported by Ferrer-Miralles et al. (2009), who performed a similar analysis for EMA- and FDA-approved medicines in 2009. This indicates that the landscape has changed little since then, but also that approval rates by the two agencies are comparable.

Despite recent efforts to humanise the protein modification and secretion pathways in the yeast species Pichia pastoris (Hamilton et al., 2006), and the acquisition of GlycoFi, the company that owned this technology, by Merck in 2006, there are still no glycoproteins derived from yeast approved by the EMA. In contrast, the FDA has already approved Kalbitor® produced in P. pastoris modified with the GlycoSwitch system, which makes the production of proteins with human-like glycosylation possible, as well as the drug Elelyso®, which is produced in genetically engineered plant cells. Kalbitor® did not receive authorisation by the EMA because of insufficient clinical studies. Elelyso® was also not authorised by EMA, because the Committee for Medicinal Products for Human Use concluded that the “medicine benefits outweighed the risks”, but also because another product, Vpriv®, marketed by Shire Pharmaceuticals, has exclusivity in the European Union until August 2020. This exclusivity right was granted by the EMA as an incentive for the production of orphan drugs.

Regardless of calls for a more robust production platform that is not susceptible, for example, to avian influenza like hen’s eggs, egg-based virus propagation is still the dominant methodology for vaccine provision (Ng, 2012), and together with Vero cells have shown a high rate of increase in usage. As shown in Fig. 3B, approvals of vaccines produced via propagation in hen’s eggs or Vero cells showed a significant increase in 2005–2006, the time that the avian flu pandemic reached Europe and the Middle East. A further increase is seen in 2009, when a swine flu pandemic was declared by the WHO. Within the recombinant vaccine arena, S. cerevisiae remain the most popular production system.

In terms of approvals trends by major category, glycoproteins, vaccines and semi-synthetics all show steady rates, as shown in Fig. 1B. Glycoproteins are the fastest growing and most populous category, followed by proteins and vaccines. Personalised medicine is hardly represented in the EMA approvals, with only one drug under the cell therapy category having received approval as shown in Fig. 1B. This involves the in vitro cultivation of the patient’s own chondrocytes and the re-injection to the patient. Recently, the EMA approved the first gene therapy drug in Europe, Glybera®, which contains a viral vector carrying the gene for lipoprotein lipase and will be licensed to treat patients with familial lipoprotein lipase deficiency (not yet listed on the EMA website).

Looking at individual molecule types produced via rDNA technology in Fig. 6A, we can see that hormones, growth factors and monoclonal antibodies have the highest number of approved drugs, followed by cytokines and recombinant vaccines, while all other types have fewer than ten drugs approved. The top three categories also show a high rate of approvals, especially during the decade 1999–2009, even though no hormone-based drug has been approved in the last 2 years, and no growth factor has been approved since 2010 (Fig. 6B). Out of the smaller categories, particular mention should be made of fusion proteins, which have seen five drugs approved in the last 6 years.

Fig. 6. (A) Molecule classes and number of EMA-approved rDNA drugs. (B) Accumulation of rDNA drugs categorised by molecule type (non-human peptides and proteins and serine proteases follow similar trends and hence non-human peptides and proteins line is not visible, only the first drug for the latter was approved in 1996, while for the former in 1997).

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Interestingly, in the year 2012 approval has so far been granted for five semi-synthetic drugs and two vaccines (as at the start of June). This slow-down coincides with a low growth rate in sales for biologics as reported for the United States market, which may be fuelled by the loss of exclusivity and the rise in the development and use of generic small molecules (Aggarwal, 2011).

Opposite the threat of small molecules, stands the opportunity of biosimilars, for which there is a clear roadmap both in Europe and, more recently, in the U.S. (Wang and Chow, 2012). Specifically, although the EMA has already set guidelines for six different classes of molecules, namely erythropoietins, insulin, growth hormones, alpha interferon, GCSFs and low molecular weight heparin, with three more to come (beta interferons, follicle stimulating hormone and mAbs) (Wang and Chow, 2012), solely biosimilars of erythropoietin, GCSFs and human growth hormone have received approval (as also discussed in Walsh (2010) 2 years ago). At the same time, patents for EMA-approved interferon alpha drugs have already expired (Ledford, 2007), and patents of many blockbuster drugs are about to expire (e.g. Herceptin®, Remicade® and others reported in Walsh (2010)). It therefore remains to be seen whether high-value biosimilars will drive research and development in the future or if the threat of market fragmentation and price erosion will deter manufacturers.