Adv Biochem Eng Biotechnol (2014) 139: 1–9 DOI: 10.1007/10_2013_255 Springer-Verlag Berlin Heidelberg 2013 Published Online: 21 November 2013
Mammalian Cell Cultures for Biologics Manufacturing Anne Kantardjieff and Weichang Zhou Abstract Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.
Keywords Biopharmaceuticals Cell culture operations Mammalian cell culture Production cell lines
Contents 1 Introduction.......................................................................................................................... 2 Licensed Therapeutic Biologics.......................................................................................... 3 Market Size.......................................................................................................................... 4 Industrial Production Cell Lines ......................................................................................... 5 Industrial Cell Culture Operations...................................................................................... 6 Conclusion ........................................................................................................................... References..................................................................................................................................
A. Kantardjieff (&) Upstream Development, Alexion Pharmaceuticals, Cheshire, CT 06410, USA e-mail: [email protected] W. Zhou Biologics process Development, WuXi AppTec Co., Shanghai 200131, China
2 2 3 5 8 9 9
2
A. Kantardjieff and W. Zhou
1 Introduction Biologically derived drugs represent a growing sector of the pharmaceutical market. This class of compounds, known as biologics or biopharmaceuticals, is derived from the genetic manipulation of living organisms. Biologics include recombinant DNA-derived proteins and monoclonal antibodies, along with gene therapies and bioengineered animals and plants. Biopharmaceuticals are used to treat a broad range of diseases, especially in the fields of rheumatology and oncology, as well as cardiology, dermatology, gastroenterology, and neurology. In many medical fields, biologics represent the sole therapeutic option available to patients.
2 Licensed Therapeutic Biologics There are currently 230 approved biologics on the market [5, 7, 8]. Figure 1 shows the distribution of therapies among eight major classes of compounds: recombinant blood factors, including Factor VIII; recombinant thrombolytics and anticoagulants, including tissue plasminogen activator and hirudin; recombinant hormones, including insulin, human growth hormone, and follicle-stimulating hormone; recombinant growth factors, including erythropoietin and granulocyte colony-stimulating factor; recombinant interferons and interleukins, including interferon-a and interferon-b; recombinant vaccines, including hepatitis B; monoclonal antibodies (mAbs) and monoclonal antibody-based products; and other recombinant products, including bone morphogenic proteins, recombinant enzymes, and nucleic acid-based products. As can be seen, recombinant hormones are the most represented class of compounds (51 therapies, 22 % of approved biologics), and mAbs are the second most predominant class with 49 therapies on the market (21 % of approved biologics). Of the 230 approved biologics on the market, 93 have been approved since 2006, which represents more than 40 % of commercially available biopharmaceuticals. Figure 2 shows the number of drugs approved by the US Food and Drug Administration (FDA) since 1998 [7]. This includes both small molecule drugs, filed as New Molecular Entities (NMEs) and biologics, filed under biologics license applications (BLAs). The pace of approval for new biologics has been growing over the past years. A total of 37 drugs was approved by the FDA in 2012. Of these, 14 were biologics, as filed under BLAs. This represents the largest number of approved biologics license applications in the past decade. Among new approvals, 32 % were for orphan disease indications. Although these rare diseases have small patient populations, they have also been shown to have a higher success rate in the clinic and require smaller clinical trials. As a consequence, many pharmaceutical and biopharmaceutical companies have added orphan drug programs to their product pipelines.
Mammalian Cell Cultures
3
Fig. 1 Distribution of the number of approved biologics by compound class
3 Market Size Global sales of biologics reached US $124.9 billion dollars in 2012, a 10.4 % increase over 2011. Among them, sales of monoclonal antibodies and antibody fusion proteins were US $65 billion dollars, over 50 % of the total biologics sales. The growth of the US biologics market has mirrored that of the global biologics market. Growth data for the US market since 2002 is shown in Figure 3. As can be seen, the market has been growing steadily since 2002, albeit at a reduced pace in more recent years [2]. In 2011, total sales of biologics in the US market were US $53.8 billion, which represents a 4.9 % increase over 2010 sales [1]. Monoclonal antibodies are the best-selling class of biologics, with US sales in 2011 of *$20.3 billion. In fact, sales of monoclonal antibodies have almost doubled since 2006, when US sales were $11.4 billion. This dramatic increase can be attributed to two factors: the first is significant sales of new entries into the market. Four monoclonal antibodies approved in 2011 had combined sales of $2 billion. The second is the growth in sales of the three top-selling monoclonal antibodies (Humira, Remicade and Rituxan), which each sold more than $3 billion in the US alone.
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A. Kantardjieff and W. Zhou
Fig. 2 Number of drugs approved by the US food and drug administration (FDA) since 1998
Fig. 3 Growth trends in the US biologics market
Mammalian Cell Cultures
5
A second class of compounds that has been growing significantly in recent years is recombinant hormones, where insulin analogues account for three-quarters of reported sales. Sales of recombinant hormones in 2011 topped US $12.2 billion, up from $5.39 billion in 2006, an increase of more than 126 %. One insulin analogue, Lantus (Sanofi Aventis), was the second–best selling biologic drug in the United States in 2011, with total sales of $3.5 billion, only slightly behind AbbVie’s Humira, with total sales of north of $3.5 billion [1]. There were more than 30 blockbuster antibodies and proteins in 2012, as defined by global sales in excess of US $1 billion dollars [6]. The top-ten highestgrossing biologics are shown in Table 1. Nearly all of the monoclonal antibodies and antibody fusion proteins, along with many blockbuster recombinant proteins such as erythropoietins, granulocyte colony-stimulating factor, coagulation factors, and replacement enzymes are manufactured by mammalian cell cultures.
4 Industrial Production Cell Lines An analysis of industrial production cell lines used for the manufacture of biologics shows that 51 % of currently approved biologics are produced in mammalian cells (Fig. 4). Mammalian cell lines are especially predominant in the production of certain classes of biologics. Notably, 83 % of recombinant blood factors are produced in mammalian cell lines, 95 % of monoclonal antibodies, and 74 % of other recombinant products. Biologics of increased complexity, including products with extensive post-translational modifications, must be produced in mammalian cell lines in order to obtain the desired product quality profile including humanlike glycan profile [5, 7, 8]. Mammalian cell lines used for biologics production include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and mouse myeloma cells, including NS0 and SP2/0, as well as human cell lines (HEK293, HT-1080). Of these, CHO cells are the most widely used, accounting for the production of more than 60 % of mammalian cell culture derived biologics currently on the market (Fig. 5). Chinese hamster ovary cells were first used for an approved biologics by Amgen in 1989, for production of Epogen, a recombinant human erythropoietin used for the treatment of anemia. Since then, CHO cells have become the most widely used mammalian cell line for industrial biologics production. Seven of the eleven monoclonal antibodies approved since 2010 are produced in CHO cells. The productivity of mammalian cells for production of biopharmaceuticals has increased more than 20-fold in the past two decades [3]. Titers in the range of 1–5 g/L are now commonplace, especially for antibody production processes, with productivities as high as 10–15 g/L being reported in a fed-batch culture of 2–3 weeks [4]. This increase has been driven by two factors: the first is a dramatic
Infliximab (Remicade)
3
10
4 5 5 6 7 8 9
Etanercept (Enbrel)
2
TNF alpha6 antagonist TNF antagonist
Target/mechanism of action
TNF alpha antagonist Rituximab (Rituxan/MabThera) CD20 Trastuzumab (Herceptin) HER2 Bevacizumab (Avastin) VEGF Insulin glargine (Lantus) Insulin receptor Pegfilgrastim (Neulasta) G-CSF receptor Ranibizumab (Lucentis) VEGF Epoetin alfa (Epogen/ESPO/ Erythropoietin Procrit/Eprex) receptor Insulin aspart (NovoLog Insulin receptor NovoRapid)
Adalimumab (Humira Pen)
1
Table 1 Blockbuster biologics in 2012 Ranking Product name
mAb mAb mAb Protein Protein mAb Recombinant protein Recombinant protein
Recombinant protein mAb
mAb
Class of compound
Roche (Genentech, Chugai); Biogen-IDEC Roche (Genentech, Chugai) Roche (Genentech, Chugai) Sanofi Amgen Roche (Genentech); Novartis Amgen; Ortho Biotech; Janssen-Cilag; Kyowa Hakko Kirin Pharma Novo Nordisk
7 Centocor; Merck; Mitsubishi Tanabe Pharma
Amgen; Pfizer; Takeda Pharmaceutical
Abbott; Eisai
Company
$7.143 $6.272 $6.139 $6.510 $4.092 $3.975 $3.442
billion billion billion billion billion billion billion US $2.940 billion
US US US US US US US
US $7.468 billion
US $8.406 billion
US $9.534 billion
2012 global sales (vs. 2011)
6 A. Kantardjieff and W. Zhou
Mammalian Cell Cultures
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Fig. 4 Distribution of cell lines used for industrial biologics manufacturing by number of licensed biologics until 2012
increase in the maximum achievable viable cell concentration and longer sustained cell viabilities in a typical fed-batch culture process. As a result, the total cell mass has significantly increased in fed-batch cultures. This is often expressed in terms of the integral of viable cell concentration (IVCC), which is determined by integrating the viable cell concentration over culture duration. The second factor attributable for increased titers is the development of cell lines with higher specific productivities (qp). Specific productivities on the order of 10 pg/cell/day were commonplace a decade ago, and have now been replaced by high-producing cell lines with specific productivities of more than 50 pg/cell/day [3]. These rival or exceed the specific productivities of human plasma cells, the body’s antibody production cells. These two factors combined have contributed to the higher titers now commonplace in biologics production in mammalian cells.
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Fig. 5 Distribution of mammalian cell lines used for industrial biologics manufacturing by number of licensed biologics until 2012
5 Industrial Cell Culture Operations There are three major cell culture operations used in the industry, batch, fed-batch, and perfusion. Both batch and fed-batch cultures are used to produce stable molecules such as monoclonal antibodies due to longer residence time in culture, whereas perfusion cultures are required to produce labile molecules such as recombinant enzymes and coagulation factors. Several antibodies are also produced by perfusion cultures. In a batch culture, all nutrients are added in the beginning. The nutrient concentrations are generally low to limit osmolality. These low nutrient supplies limit the maximum cell concentration, the culture duration, and the product concentration achieved. In a fed-batch culture, additional nutrients are added during the culture to prevent nutrient depletion, thus prolonging the cell growth phase and culture duration. This results in a much higher maximum cell concentration and longer culture lifetime. As a result, a much higher product concentration is achieved. A perfusion culture is a continuous cell culture process with cell retention. Cells are retained in the reactor, while new culture media is continuously added in and culture supernatant is removed at the same rate to keep the reactor volume constant. A number of licensed therapeutic biologics products are produced in perfusion cultures to prevent product degradation and ensure better product quality for labile molecules.
Mammalian Cell Cultures
9
6 Conclusion Biologically derived drugs have continued to drive growth in the pharmaceutical industry. This is due partly to a sustained increase in the number of approved drugs in recent years. Furthermore, improved sales, especially for monoclonal antibodies, have contributed to the observed growth. Mammalian cells are the predominant expression system used for biologics production, partly due to their ability to generate humanlike posttranslational modifications essential for drug safety and efficacy. Improvements in process development over the past decades have resulted in higher titers and specific productivities, making higly robust and productive mammalian cell culture processes common place in the industry.
References 1. Aggarwal S (Rob) (2012) What’s fueling the biotech engine—2011 to 2012. Nat Biotechnol 30(12):1191–1197 2. Aggarwal S (Rob) (2007) What’s fueling the biotech engine? Nat Biotechnol 25(10): 1097–1104 3. De Jesus M, Wurm F (2011) Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors. Eur J Pharm Biopharm 78:184–188 4. Kim JY, Kim Y-G, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microb Biotechnol 93:917–930 5. Kling J (2012) Fresh from the biotech pipeline—2011. Nat Biotechnol 30(2):128–131 6. La Merie Business Intelligence (2013) Blockbuster Biologics 2012. R&D Pipeline News, May 7 2013, Special Edition 1 7. Osborne R (2013) Fresh from the biotech pipeline—2012. Nat Biotechnol 31(2):100–103 8. Walsch G (2010) Biopharmaceutical benchmarks 2010. Nat Biotechnol 28(9):917–924
Mammalian Cell Cultures for Biologics Manufacturing Anne Kantardjieff and Weichang Zhou Abstract Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.
Keywords Biopharmaceuticals Cell culture operations Mammalian cell culture Production cell lines
Contents 1 Introduction.......................................................................................................................... 2 Licensed Therapeutic Biologics.......................................................................................... 3 Market Size.......................................................................................................................... 4 Industrial Production Cell Lines ......................................................................................... 5 Industrial Cell Culture Operations...................................................................................... 6 Conclusion ........................................................................................................................... References..................................................................................................................................
A. Kantardjieff (&) Upstream Development, Alexion Pharmaceuticals, Cheshire, CT 06410, USA e-mail: [email protected] W. Zhou Biologics process Development, WuXi AppTec Co., Shanghai 200131, China
2 2 3 5 8 9 9
2
A. Kantardjieff and W. Zhou
1 Introduction Biologically derived drugs represent a growing sector of the pharmaceutical market. This class of compounds, known as biologics or biopharmaceuticals, is derived from the genetic manipulation of living organisms. Biologics include recombinant DNA-derived proteins and monoclonal antibodies, along with gene therapies and bioengineered animals and plants. Biopharmaceuticals are used to treat a broad range of diseases, especially in the fields of rheumatology and oncology, as well as cardiology, dermatology, gastroenterology, and neurology. In many medical fields, biologics represent the sole therapeutic option available to patients.
2 Licensed Therapeutic Biologics There are currently 230 approved biologics on the market [5, 7, 8]. Figure 1 shows the distribution of therapies among eight major classes of compounds: recombinant blood factors, including Factor VIII; recombinant thrombolytics and anticoagulants, including tissue plasminogen activator and hirudin; recombinant hormones, including insulin, human growth hormone, and follicle-stimulating hormone; recombinant growth factors, including erythropoietin and granulocyte colony-stimulating factor; recombinant interferons and interleukins, including interferon-a and interferon-b; recombinant vaccines, including hepatitis B; monoclonal antibodies (mAbs) and monoclonal antibody-based products; and other recombinant products, including bone morphogenic proteins, recombinant enzymes, and nucleic acid-based products. As can be seen, recombinant hormones are the most represented class of compounds (51 therapies, 22 % of approved biologics), and mAbs are the second most predominant class with 49 therapies on the market (21 % of approved biologics). Of the 230 approved biologics on the market, 93 have been approved since 2006, which represents more than 40 % of commercially available biopharmaceuticals. Figure 2 shows the number of drugs approved by the US Food and Drug Administration (FDA) since 1998 [7]. This includes both small molecule drugs, filed as New Molecular Entities (NMEs) and biologics, filed under biologics license applications (BLAs). The pace of approval for new biologics has been growing over the past years. A total of 37 drugs was approved by the FDA in 2012. Of these, 14 were biologics, as filed under BLAs. This represents the largest number of approved biologics license applications in the past decade. Among new approvals, 32 % were for orphan disease indications. Although these rare diseases have small patient populations, they have also been shown to have a higher success rate in the clinic and require smaller clinical trials. As a consequence, many pharmaceutical and biopharmaceutical companies have added orphan drug programs to their product pipelines.
Mammalian Cell Cultures
3
Fig. 1 Distribution of the number of approved biologics by compound class
3 Market Size Global sales of biologics reached US $124.9 billion dollars in 2012, a 10.4 % increase over 2011. Among them, sales of monoclonal antibodies and antibody fusion proteins were US $65 billion dollars, over 50 % of the total biologics sales. The growth of the US biologics market has mirrored that of the global biologics market. Growth data for the US market since 2002 is shown in Figure 3. As can be seen, the market has been growing steadily since 2002, albeit at a reduced pace in more recent years [2]. In 2011, total sales of biologics in the US market were US $53.8 billion, which represents a 4.9 % increase over 2010 sales [1]. Monoclonal antibodies are the best-selling class of biologics, with US sales in 2011 of *$20.3 billion. In fact, sales of monoclonal antibodies have almost doubled since 2006, when US sales were $11.4 billion. This dramatic increase can be attributed to two factors: the first is significant sales of new entries into the market. Four monoclonal antibodies approved in 2011 had combined sales of $2 billion. The second is the growth in sales of the three top-selling monoclonal antibodies (Humira, Remicade and Rituxan), which each sold more than $3 billion in the US alone.
4
A. Kantardjieff and W. Zhou
Fig. 2 Number of drugs approved by the US food and drug administration (FDA) since 1998
Fig. 3 Growth trends in the US biologics market
Mammalian Cell Cultures
5
A second class of compounds that has been growing significantly in recent years is recombinant hormones, where insulin analogues account for three-quarters of reported sales. Sales of recombinant hormones in 2011 topped US $12.2 billion, up from $5.39 billion in 2006, an increase of more than 126 %. One insulin analogue, Lantus (Sanofi Aventis), was the second–best selling biologic drug in the United States in 2011, with total sales of $3.5 billion, only slightly behind AbbVie’s Humira, with total sales of north of $3.5 billion [1]. There were more than 30 blockbuster antibodies and proteins in 2012, as defined by global sales in excess of US $1 billion dollars [6]. The top-ten highestgrossing biologics are shown in Table 1. Nearly all of the monoclonal antibodies and antibody fusion proteins, along with many blockbuster recombinant proteins such as erythropoietins, granulocyte colony-stimulating factor, coagulation factors, and replacement enzymes are manufactured by mammalian cell cultures.
4 Industrial Production Cell Lines An analysis of industrial production cell lines used for the manufacture of biologics shows that 51 % of currently approved biologics are produced in mammalian cells (Fig. 4). Mammalian cell lines are especially predominant in the production of certain classes of biologics. Notably, 83 % of recombinant blood factors are produced in mammalian cell lines, 95 % of monoclonal antibodies, and 74 % of other recombinant products. Biologics of increased complexity, including products with extensive post-translational modifications, must be produced in mammalian cell lines in order to obtain the desired product quality profile including humanlike glycan profile [5, 7, 8]. Mammalian cell lines used for biologics production include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and mouse myeloma cells, including NS0 and SP2/0, as well as human cell lines (HEK293, HT-1080). Of these, CHO cells are the most widely used, accounting for the production of more than 60 % of mammalian cell culture derived biologics currently on the market (Fig. 5). Chinese hamster ovary cells were first used for an approved biologics by Amgen in 1989, for production of Epogen, a recombinant human erythropoietin used for the treatment of anemia. Since then, CHO cells have become the most widely used mammalian cell line for industrial biologics production. Seven of the eleven monoclonal antibodies approved since 2010 are produced in CHO cells. The productivity of mammalian cells for production of biopharmaceuticals has increased more than 20-fold in the past two decades [3]. Titers in the range of 1–5 g/L are now commonplace, especially for antibody production processes, with productivities as high as 10–15 g/L being reported in a fed-batch culture of 2–3 weeks [4]. This increase has been driven by two factors: the first is a dramatic
Infliximab (Remicade)
3
10
4 5 5 6 7 8 9
Etanercept (Enbrel)
2
TNF alpha6 antagonist TNF antagonist
Target/mechanism of action
TNF alpha antagonist Rituximab (Rituxan/MabThera) CD20 Trastuzumab (Herceptin) HER2 Bevacizumab (Avastin) VEGF Insulin glargine (Lantus) Insulin receptor Pegfilgrastim (Neulasta) G-CSF receptor Ranibizumab (Lucentis) VEGF Epoetin alfa (Epogen/ESPO/ Erythropoietin Procrit/Eprex) receptor Insulin aspart (NovoLog Insulin receptor NovoRapid)
Adalimumab (Humira Pen)
1
Table 1 Blockbuster biologics in 2012 Ranking Product name
mAb mAb mAb Protein Protein mAb Recombinant protein Recombinant protein
Recombinant protein mAb
mAb
Class of compound
Roche (Genentech, Chugai); Biogen-IDEC Roche (Genentech, Chugai) Roche (Genentech, Chugai) Sanofi Amgen Roche (Genentech); Novartis Amgen; Ortho Biotech; Janssen-Cilag; Kyowa Hakko Kirin Pharma Novo Nordisk
7 Centocor; Merck; Mitsubishi Tanabe Pharma
Amgen; Pfizer; Takeda Pharmaceutical
Abbott; Eisai
Company
$7.143 $6.272 $6.139 $6.510 $4.092 $3.975 $3.442
billion billion billion billion billion billion billion US $2.940 billion
US US US US US US US
US $7.468 billion
US $8.406 billion
US $9.534 billion
2012 global sales (vs. 2011)
6 A. Kantardjieff and W. Zhou
Mammalian Cell Cultures
7
Fig. 4 Distribution of cell lines used for industrial biologics manufacturing by number of licensed biologics until 2012
increase in the maximum achievable viable cell concentration and longer sustained cell viabilities in a typical fed-batch culture process. As a result, the total cell mass has significantly increased in fed-batch cultures. This is often expressed in terms of the integral of viable cell concentration (IVCC), which is determined by integrating the viable cell concentration over culture duration. The second factor attributable for increased titers is the development of cell lines with higher specific productivities (qp). Specific productivities on the order of 10 pg/cell/day were commonplace a decade ago, and have now been replaced by high-producing cell lines with specific productivities of more than 50 pg/cell/day [3]. These rival or exceed the specific productivities of human plasma cells, the body’s antibody production cells. These two factors combined have contributed to the higher titers now commonplace in biologics production in mammalian cells.
8
A. Kantardjieff and W. Zhou
Fig. 5 Distribution of mammalian cell lines used for industrial biologics manufacturing by number of licensed biologics until 2012
5 Industrial Cell Culture Operations There are three major cell culture operations used in the industry, batch, fed-batch, and perfusion. Both batch and fed-batch cultures are used to produce stable molecules such as monoclonal antibodies due to longer residence time in culture, whereas perfusion cultures are required to produce labile molecules such as recombinant enzymes and coagulation factors. Several antibodies are also produced by perfusion cultures. In a batch culture, all nutrients are added in the beginning. The nutrient concentrations are generally low to limit osmolality. These low nutrient supplies limit the maximum cell concentration, the culture duration, and the product concentration achieved. In a fed-batch culture, additional nutrients are added during the culture to prevent nutrient depletion, thus prolonging the cell growth phase and culture duration. This results in a much higher maximum cell concentration and longer culture lifetime. As a result, a much higher product concentration is achieved. A perfusion culture is a continuous cell culture process with cell retention. Cells are retained in the reactor, while new culture media is continuously added in and culture supernatant is removed at the same rate to keep the reactor volume constant. A number of licensed therapeutic biologics products are produced in perfusion cultures to prevent product degradation and ensure better product quality for labile molecules.
Mammalian Cell Cultures
9
6 Conclusion Biologically derived drugs have continued to drive growth in the pharmaceutical industry. This is due partly to a sustained increase in the number of approved drugs in recent years. Furthermore, improved sales, especially for monoclonal antibodies, have contributed to the observed growth. Mammalian cells are the predominant expression system used for biologics production, partly due to their ability to generate humanlike posttranslational modifications essential for drug safety and efficacy. Improvements in process development over the past decades have resulted in higher titers and specific productivities, making higly robust and productive mammalian cell culture processes common place in the industry.
References 1. Aggarwal S (Rob) (2012) What’s fueling the biotech engine—2011 to 2012. Nat Biotechnol 30(12):1191–1197 2. Aggarwal S (Rob) (2007) What’s fueling the biotech engine? Nat Biotechnol 25(10): 1097–1104 3. De Jesus M, Wurm F (2011) Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors. Eur J Pharm Biopharm 78:184–188 4. Kim JY, Kim Y-G, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microb Biotechnol 93:917–930 5. Kling J (2012) Fresh from the biotech pipeline—2011. Nat Biotechnol 30(2):128–131 6. La Merie Business Intelligence (2013) Blockbuster Biologics 2012. R&D Pipeline News, May 7 2013, Special Edition 1 7. Osborne R (2013) Fresh from the biotech pipeline—2012. Nat Biotechnol 31(2):100–103 8. Walsch G (2010) Biopharmaceutical benchmarks 2010. Nat Biotechnol 28(9):917–924
