Skip to content

Advertisement

Open Access

Recombinant pharmaceuticals from microbial cells: a 2015 update

Microbial Cell Factories201615:33

https://doi.org/10.1186/s12934-016-0437-3

Received: 3 December 2015

Accepted: 1 February 2016

Published: 9 February 2016

Abstract

Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment.

Keywords

Recombinant proteinsProtein drugsRecombinant DNAFusion proteinsBiopharmaceuticals

Background

Human cells produce thousands of proteins that integrated into an extremely complex physiologic network perform precise actions as catalysers, signalling agents or structural components. Then, dysfunction of proteins with abnormal amino acid sequences or the absence of a given protein often results in the development of severe pathologies such as diabetes [1], dwarfism [2], cystic fibrosis [3], thalassaemia [4] or impaired blood clotting [5], among many others [6, 7]. In the absence of standardized gene therapy treatments that would genetically reconstitute functional protein production within the patient, protein deficiencies must be treated by the punctual or repeated clinical administration of the missing protein, so as to reach ordinary functional concentrations. These therapeutic proteins are produced ex vivo mostly in biological systems [8], which must guarantee not only full protein functionalities but also a cost-effective industrial fabrication and the absence of hazardous contaminants. Protein drugs have to necessarily conform to quality constrains stricter than those expected in the production of enzymes for chemical industries, which consequently defines the choice of recombinant hosts, protocols and production strategies. Nowadays, there are over 400 marketed recombinant products (peptides and proteins) and other 1300 are undergoing clinical trials (figures updated on May 2015 [9]).

In this context of expanding protein drug markets, there is a generic consensus about the need to enable drugs for cell- or tissue-targeted delivery to reduce doses, production costs and side effects. While increasing protein stability in vivo can be reached by discrete modifications in the amino acid sequence, generating fusions between therapeutic proteins and specific peptide ligands or antibodies that interact with particular cell receptors might allow acquiring specificity in the delivery process. In this regard and also pushed by the convenience to combine diagnosis and therapy in theranostic agents [10, 11], contemporary research on protein pharmaceuticals tends towards engineered versions functionally more sophisticated than plain natural polypeptides.

Review

Cell factories

Since early recombinant DNA times, ever-increasing understanding of cell physiology and stress, and of factors involved in heterologous gene expression and protein production empowered the use of different living factories, namely prokaryotic and eukaryotic cells, plants or animals [12, 13]. By using these systems, recombinant production solves source availability problems, is considered a bio-safe and green process and confers the ability to modify amino acid sequences and therefore protein function, to better adjust the product to a desired function [14]. There is a wide and growing spectrum of expression systems that are becoming available for the production of recombinant proteins [15, 16]. Escherichia coli was the prevalent platform when the biopharmaceutical sector emerged in the 1980s, and it was followed by the implementation of the yeast Saccharomyces cerevisiae. Both systems and the associated genetic methodologies exhibit an unusually high versatility, making them adaptable to different production demands [17]. Despite the exploration of insect cells as initially successful system especially for vaccine-oriented proteins, mammalian cell lines (most notably CHO cells) are nowadays the prevailing animal-derived cell system due to their suitability to produce conveniently glycosylated proteins [18, 19] (Fig. 1). The ability to carry out post translational modifications contrasts with complex nutritional requirements, slow growth and fragility, and relatively high production timing and costs. Thus, among many conventional and emerging cell-based systems for protein production, bacteria, yeast and mammalian cell lines are the most common in biopharma, and both prokaryotic and eukaryotic systems are constantly evolving and competing to improve their properties and intensify as platforms of choice for protein drug production [14]. While bacteria has lost its early leading role in the field [19], about 30 % of marketed biopharmaceuticals are still produced in this system [20], as supported by the unusual physiological and genetic manipulability of prokaryotic cells [21].
Figure 1
Fig. 1

Number of recombinant protein products approved for use as drugs in humans, depending on the type of production platform

In fact, the main purpose in the development of new protein production platforms is to enhance drug functionality through reaching successful protein folding and post-translational modifications, while keeping the low complexity and high flexibility associated to prokaryotic cell culture. In this context, Gram-positive bacteria such as Bacillus megaterium [22] and Lactococcus lactis [23] allow efficient protein secretion in absence of endotoxic cell wall components, while filamentous fungi (such as Trichoderma reesei, [24]), moss (Physcomitrella patens, [25, 26]) and protozoa (Leishmania tarentolae, [2729]) promote glycosylation patterns similar to those in mammalian proteins but being still cultured through methods simpler than those required by mammalian cells. Extensive descriptions of emerging (bacterial and non-bacterial) platforms specifically addressed to the production of high quality protein drugs can be found elsewhere [15, 16, 21]. The recent development of an endotoxin-free strain of E. coli [30] and its application to the fabrication of proteins and protein materials [3032] paves the road for a cost-efficient and versatile production of proteins intended for biomedical uses by skipping endotoxin removal steps, thus gaining in biosafety and reducing production costs [33]. Hopefully, all these new systems would soon offer improved products in still simple and fully controlled biofabrication approaches.

Trends in protein biopharmaceuticals

Nearly 400 recombinant protein-based products have been successfully produced and are approved as biopharmaceuticals [9], a term that refers to therapeutic products generated by technologies that involve living organisms [34]. Other 1300 protein candidates are under development, of which around 50 % are in pre-clinical studies and other 33 % in clinical trials [9] (Fig. 2). In this context, an increase in the number of approvals in next years is predictable. Developed by Eli Lilly & Co in the 70’s, Humulin, a recombinant human insulin fabricated in the bacterium E. coli [35], was the first approved biopharmaceutical (by the FDA) in 1982 [36, 37]. Other natural proteins such as hormones, cytokines and antibodies (Orthoclone OKT3) were among the single nine products approved in 1980s (Table 1). Nowadays, the therapeutic areas that have benefited more from recombinant biopharmaceuticals are metabolic disorders (e.g. diabetes type 1, type 2, obesity or hypoglycaemia), haematological disorders (e.g. renal anaemia, haemophilia A, bleeding or clotting disorders) and oncology (e.g. melanoma, breast or colorectal cancer), with 24, 18 and 15 % of the approvals respectively (Fig. 3). In this regard, oncology is a clearly expanding market. In the period 2010–2014, 9 out of 54 approved biopharmaceuticals were antitumoral drugs, cancer representing the most common indication within this period. Digging into the molecular bases of biopharmaceuticals, there is a clear trend towards antibody-based products. Over the same period (2010–2014), 17 of the 54 protein drugs approved were monoclonal antibodies (31.5 %), compared with 11 % over 1980–1989 [22]. Furthermore, among the top ten selling protein biopharmaceuticals globally in 2014 (Table 2), six are antibodies or antibody-derived proteins (Humira, Remicade, Rituxan, Enbrel, Avastin, Herceptin; http://qz.com/349929/best-selling-drugs-in-the-world/).
Figure 2
Fig. 2

Workflow involved in the development of a new drugs and approximate percentage (bars and numbers) of recombinant proteins currently in each step [9]

Table 1

Recombinant biopharmaceuticals approved in the 1980s

Product

Cell factory

Therapeutic indication

Year

Humulin

E. coli

Diabetes

1982

Protropin

E. coli

hGH deficiency

1985

Roferon A

E. coli

Hairy cell leukaemia

1986

IntronA

E. coli

Cancer, genital warts and hepatitis

1986

Recombivax

S. cerevisiae

Hepatitis B

1986

Orthoclone OKT3

Hybridoma cell line

Reversal of acute kidney and transplant rejection

1986

Humatrope

E. coli

hGH deficiency

1987

Activase

CHO

Acute myocardial infarction

1987

Epogen

CHO

Anaemia

1989

Figure 3
Fig. 3

Amount of marketed recombinant proteins (expressed in percentages) applied to each therapeutic area. Coloured in pink, other therapeutic areas (<5 % each) include diseases related to cardiology, central nervous system, ophthalmology and dermatology among others

Table 2

Top ten selling protein biopharmaceuticals in 2014

Druga

Active ingredient

Molecule

Sales in billions

Origin

Humira

Adalimumab

Recombinant human monoclonal antibody

12.54

CHO

Sovaldi

Sofosbuvir

Nucleotide analogue polymerase (NS5B) inhibitor

10.28

Chemical

Remicade

Infliximab

Recombinant chimeric, humanized tumor necrosis factor alpha (TNF) monoclonal antibody

9.24

Hybridoma cell line

Rituxan

Rituximab

Recombinant humanized monoclonal antibody

8.68

CHO

Enbrel

Etanercept

Recombinant soluble dimeric fusion protein

8.54

CHO

Lantus

Insulin glargine

Insulin receptor agonist

7.28

E. coli

Avastin

Bevacizumab

Recombinant humanized antibody

6.96

CHO

Herceptin

Trastuzumab

Recombinant humanized monoclonal antibody

6.79

CHO

Advair

Fluticasone propionate and salmeterol xinafoate

Glucocorticoid receptor agonist and β-2 adrenergic receptor agonist

6.43

Chemical

Crestor

Rosuvastatin calcium

Antihyperlipedemic agent

5.87

Synthetic

aData according to www.medtrack.com, November 2015

Formerly, biopharmaceuticals were recombinant versions of natural proteins, with the same amino acid sequence as the respective native versions (with only minor modifications, often resulting from the cloning strategy). Since 1990s, a meaningful proportion of the approvals are based on highly modified forms of recombinant proteins. This novel alternative, based on protein or domain fusion and on truncated versions, offers a wide spectrum of possible combinations to obtain novel biopharmaceuticals with different joined activities that are not found together in nature.

Protein drugs for cancer treatment

Oncology is one of the therapeutic indications that dominate the biopharmaceutical market, as cancer is a major cause of morbidity and mortality worldwide. Surgery and radiotherapy are effective in curing cancer at early disease stages; however, they cannot eradicate metastatic disease. The presence of micrometastases or clinically evident metastases at diagnosis requires their use in combination with genotoxic chemotherapy to increase cure rates [38]. Nevertheless, the success of chemotherapy has been hampered because of its lack of selectivity and specificity, so that the toxicity to normal tissues limits the dose that could be administered to patients. The development of biopharmaceuticals capable of inhibiting specific molecular targets driving cancer (for instance, monoclonal antibodies anti-Her2—Trastuzumab- or anti-VEGF—Bevacizumab-) goes in this direction [39].

Among marketed protein biopharmaceuticals, almost 24 % (94 products) are used in antitumoral therapies. Most of these products are used for supportive purposes intended to minimize the side effects of chemotherapy, usually neutropenia or anaemia (some representative examples are shown in Table 3). Nineteen out of those 94 products are true antitumoral drugs, 69 % of which are produced in E. coli (Fig. 4) and are based on engineered amino acidic sequences, protein fusions and single protein domains (Table 4).
Table 3

Representative examples of supportive protein drugs in cancer

Drug name

Cell factory

Biological role

Mechanism of action

Indications

Filgrastim (Scimax)

E. coli

Cytokine

Stimulates hematopoiesis

Bone marrow transplantation and cancer chemotherapy induced neutropenia

Pegfilgrastim (Neupeg)

E. coli

Cytokine

Stimulates differentiation, proliferation and activation of the neutrophilic granulocytes

Cancer chemotherapy induced neutropenia

Darbepoetin alfa (Aranesp)

CHO cells

Hormone

Stimulates processes of erythropoiesis or red blood cell production

Anemia associated with chronic renal failure, cancer chemotherapy or heart failure. Myelodysplastic syndrome

Lenograstim (CERBIOS)

CHO cells

Cytokine

Stimulates differentiation, proliferation and activation of neutrophilic granulocytes

Neutropenia associated with cytotoxic therapy or bone marrow transplantation

Epoetin alfa (Binocrit)

CHO cells

Hormone

Stimulates production of oxygen carrying red blood cells from the bone marrow

Anemia associated with chronic renal failure and cancer chemotherapy induced anemia

Figure 4
Fig. 4

Cell factories used for the production of recombinant biopharmaceuticals against cancer (expressed in percentages)

Table 4

Anticancer recombinant biopharmaceuticals approved until March 2015

Drug name

Cell factory

Source

Biological role

Indications

Denileukin diftitox

E. coli

Fusion protein

Diphtheria toxin fused to cytokine

Cutaneous T-cell lymphoma

Endostatin

E. coli

Modified

Collagen derivative

Non-small cell lung cancer, metastatic colorectal cancer

Aldesleukin

E. coli

Modified

Cytokine

Metastatic renal cell carcinoma, metastatic melanoma, kidney cancer, angiosarcoma

Interleukin-2

E. coli

Modified

Cytokine

Metastatic melanoma, metastatic renal cell carcinoma

Filgrastim

E. coli

Modified

Cytokine

Acute lymphocytic leukaemia, solid tumour

Interferon alpha-2a

E. coli

Modified

Cytokine

AIDS-related Kaposi’s sarcoma, follicular lymphoma, cutaneous T-cell lymphoma, melanoma, chronic myelocytic leukaemia, hairy cell leukaemia, renal cell carcinoma, kidney cancer

Interferon alpha-2b

E. coli

Modified

Cytokine

AIDS-related Kaposi’s sarcoma, pancreatic endocrine tumour, melanoma, non-Hodgkin lymphoma, leukaemia, hairy cell leukaemia, renal cell carcinoma, multiple myeloma, CML, follicular lymphoma, melanoma

Interferon alpha-1b

E. coli

Modified

Cytokine

Renal cell carcinoma, hairy cell leukaemia

Interferon gamma-1a

E. coli

Modified

Cytokine

Kidney cancer, sezary syndrome, mycosis fungoides

Tasonermin

E. coli

Natural

Cytokine

Soft tissue sarcoma

Molgramostim

E. coli

Modified

Growth factor

Myelodysplastic syndrome

Nartograstim

E. coli

Modified

Growth factor

Solid tumour

Palifermin

E. coli

Fraction

Growth factor

Metastatic renal cell carcinoma, metastatic melanoma

Sargramostim

S. cerevisiae

Modified

Growth factor

Acute myelocytic leukaemia

Ziv-aflibercept

CHO cells

Fusion protein

Growth factor receptor fused to IgG1

Metastatic colorectal cancer

Thyrotropin alpha

CHO cells

Modified

Hormone

Thyroid cancer

Trastuzumab biosimilar

CHO cells

Modified

Monoclonal antibody

Breast cancer, gastric cancer, metastatic breast cancer

Rituximab biosimilar

CHO cells

Modified

Monoclonal antibody

Non-Hodgkin lymphoma, chronic lymphocytic leukaemia

Interferon alpha

Human lymphoblastoid cells

Modified

Cytokine

AIDS-related Kaposi’s sarcoma, multiple myeloma, non-Hodgkin lymphoma, CML, hairy cell leukaemia, renal cell carcinoma

Clearly, modified protein versions are the most abundant in cancer therapies over natural polypeptides. As relevant examples, Ziv aflibercept is a recombinant fusion protein produced in CHO cells used against colorectal cancer. It consists of portions of each Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) fused to the constant fraction (Fc) of a human IgG1 immunoglobulin (Fig. 5). This construct acts as a decoy by binding to VEGF-A, VEGF-B and placental growth factor (PlGF), which activate VEGFR. This trap hinders the interaction between the growth factors and the receptors, inhibiting the VEGF pathway which is involved in the angiogenic process [40]. Denileukin diftitox is a recombinant protein composed of two diphtheria toxin fragments (A and B) and a human interleukin-2 (Fig. 5). Diphteria toxin is a potent exotoxin secreted by Corynebacterium diphteriae. Due to its peculiar structure, the whole complex, produced in E. coli, is capable of delivering a cytotoxic agent directly to a specific target. There are two main active blocks whose function is firstly to selectively deliver the biopharmaceutical (IL-2) and secondly cause cytotoxicity (toxin A and B) [41]. The fusion protein binds to the IL-2 receptor, which is expressed in cancerous cells (cutaneous T cell lymphoma). Once the toxin moiety is internalized, the catalytic domain promotes cell death through protein synthesis inhibition [42].
Figure 5
Fig. 5

Schematic molecular structure of two marketed recombinant biopharmaceuticals

As targeted drug delivery for cancer is a most recent and expanding area of research, other non-recombinant, protein-based biopharmaceuticals are also heavily represented. Those mainly include antibody-drug conjugates (ADCs) such as Brentuximab vedotin, Trastuzumab emtansine, or nanoparticle-drug conjugates such as nab-paclitaxel [39, 43]. In these cases, the protein counterpart acts as a targeted vehicle for conventional chemical drugs. Again, this approach pursues the selective drug delivery to specific target cells, aimed to increase antitumoral activity while reducing toxicity on normal cells and the associated side effects.

Products against cancer that provided the highest revenues in 2013 are represented in Fig. 6. Sixty percent of those products are recombinant proteins, supporting the idea that recombinant protein production is still a rising and promising platform, offering room for important advances in the biopharmaceutical sector.
Figure 6
Fig. 6

Income provided by recombinant (top) and chemical drugs (bottom) against cancer in 2013. Figures according to Global Data [9]

Conclusions

In summary, the market and potential for recombinant drugs is expanding by taking advantage of a steady growing spectrum of protein production platforms. Despite the strength of mammalian cell lines as factories, microbial cells and specially E. coli are still potent protein factories essentially supported by their versatility and cost-effective cultivation. Recombinant drugs are moving from plain recombinant versions of natural products to more sophisticated protein constructs resulting from a rational design process. Combining protein domains to gain new functionalities is being exploited in drug discovery by exploiting the structural and functional versatility that merge in proteins as extremely versatile macromolecules.

Abbreviations

AIDS: 

acquired immune deficiency syndrome

ADCs: 

antibody-drug conjugates

CHO: 

chinese hamster ovary

CML: 

chronic myelogenous leukemia

Fc: 

constant fraction

FDA: 

food and drug administration

hGH: 

human growth hormone

IL: 

interleukin

PlGF: 

placental growth factor

VEGF: 

vascular endothelial growth factor

VEGFR: 

vascular endothelial growth factor receptor

Declarations

Authors’ contributions

LSG performed most of the bibliographic search and prepared part of the text, tables and figures, under the supervision of NFM and EV. LM and RM contributed with additional information and revised the manuscript. AV coordinated the whole revision, prepared part of the text and figures and the final manuscript version. All authors read and approved the final manuscript.

Acknowledgements

The authors appreciate the funding for protein drug development received from MINECO (BIO2013-41019-P), AGAUR (2014SGR-132, 2014PROD-00055) CIBER de Bioingeniería, Biomateriales y Nanomedicina (NANOPROTHER), Marató de TV3 foundation (TV32013-132031, TV32013-3930) and ISCIII FIS (PI12/00327, PI15/00272, PI15/00378). LSG received a Lanzadera fellowship from CIBER-BBN, and AV received an ICREA ACADEMIA award.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain
(2)
Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain
(3)
CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, Cerdanyola del Vallès, Spain
(4)
Technology Transfer Office, Edifici Eureka, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain
(5)
Institut d’Investigacions Biomèdiques Sant Pau, Josep Carreras Research Institute and CIBER-BBN, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

References

  1. Vajo Z, Fawcett J, Duckworth WC. Recombinant DNA technology in the treatment of diabetes: insulin analogs. Endocr Rev. 2001;22:706–17.View ArticleGoogle Scholar
  2. Takeda A, Cooper K, Bird A, Baxter L, Frampton GK, Gospodarevskaya E, et al. Recombinant human growth hormone for the treatment of growth disorders in children: a systematic review and economic evaluation. Health Technol Assess. 2010;14:1–4.View ArticleGoogle Scholar
  3. Cutting GR. Modifier genetics: cystic fibrosis. Annu Rev Genomics Hum Genet. 2005;6:237–60.View ArticleGoogle Scholar
  4. Weatherall DJ. Phenotype–genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet. 2001;2:245–55.View ArticleGoogle Scholar
  5. Powell JS. Lasting power of new clotting proteins. Hematology Am Soc Hematol Educ Program. 2014;2014:355–63.View ArticleGoogle Scholar
  6. Hirschhorn JN, Lohmueller K, Byrne E, Hirschhorn K. A comprehensive review of genetic association studies. Genet Med. 2002;4:45–61.View ArticleGoogle Scholar
  7. Savic S, McDermott MF. Clinical genetics in 2014: new monogenic diseases span the immunological disease continuum. Nat Rev Rheumatol. 2015;11:67–8.View ArticleGoogle Scholar
  8. Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol. 2013;23:393–402.View ArticleGoogle Scholar
  9. Global Data 2015. http://www.globaldata.com. 2015.
  10. Lammers T, Aime S, Hennink WE, Storm G, Kiessling F. Theranostic nanomedicine. Acc Chem Res. 2011;44:1029–38.View ArticleGoogle Scholar
  11. Pene F, Courtine E, Cariou A, Mira JP. Toward theragnostics. Crit Care Med. 2009;37:S50–8.View ArticleGoogle Scholar
  12. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv. 2009;27:297–306.View ArticleGoogle Scholar
  13. Adrio JL, Demain AL. Recombinant organisms for production of industrial products. Bioeng Bugs. 2010;1:116–31.View ArticleGoogle Scholar
  14. Walsh G. Biopharmaceutical benchmarks 2014. Nat Biotechnol. 2014;32:992–1000.View ArticleGoogle Scholar
  15. Ferrer-Miralles N, Villaverde A. Bacterial cell factories for recombinant protein production; expanding the catalogue. Microb Cell Fact. 2013;12:113.View ArticleGoogle Scholar
  16. Corchero JL, Gasser B, Resina D, Smith W, Parrilli E, Vazquez F, et al. Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. Biotechnol Adv. 2013;31:140–53.View ArticleGoogle Scholar
  17. Ferrer-Miralles N, Domingo-Espin J, Corchero JL, Vazquez E, Villaverde A. Microbial factories for recombinant pharmaceuticals. Microb Cell Fact. 2009;8:17.View ArticleGoogle Scholar
  18. Zhu J. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv. 2012;30:1158–70.View ArticleGoogle Scholar
  19. Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed M, Ramadan HI, et al. Cell factories for insulin production. Microb Cell Fact. 2014;13:141.View ArticleGoogle Scholar
  20. Overton TW. Recombinant protein production in bacterial hosts. Drug Discov Today. 2014;19:590–601.View ArticleGoogle Scholar
  21. Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv. 2012;30:1102–7.View ArticleGoogle Scholar
  22. van Dijl JM, Hecker M. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb Cell Fact. 2013;12:3.View ArticleGoogle Scholar
  23. Cano-Garrido O, Rueda FL, Sanchez-Garcia L, Ruiz-Avila L, Bosser R, Villaverde A, et al. Expanding the recombinant protein quality in Lactococcus lactis. Microb Cell Fact. 2014;13:167.View ArticleGoogle Scholar
  24. Su X, Schmitz G, Zhang M, Mackie RI, Cann IK. Heterologous gene expression in filamentous fungi. Adv Appl Microbiol. 2012;81:1–61.View ArticleGoogle Scholar
  25. Decker EL, Reski R. Current achievements in the production of complex biopharmaceuticals with moss bioreactors. Bioprocess Biosyst Eng. 2008;31:3–9.View ArticleGoogle Scholar
  26. Decker EL, Reski R. Moss bioreactors producing improved biopharmaceuticals. Curr Opin Biotechnol. 2007;18:393–8.View ArticleGoogle Scholar
  27. Basile G, Peticca M. Recombinant protein expression in Leishmania tarentolae. Mol Biotechnol. 2009;43:273–8.View ArticleGoogle Scholar
  28. Kushnir S, Gase K, Breitling R, Alexandrov K. Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif. 2005;42:37–46.View ArticleGoogle Scholar
  29. Breitling R, Klingner S, Callewaert N, Pietrucha R, Geyer A, Ehrlich G, et al. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif. 2002;25:209–18.View ArticleGoogle Scholar
  30. Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, Kohl TA, et al. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microb Cell Fact. 2015;14:57.View ArticleGoogle Scholar
  31. Ueda T, Akuta T, Kikuchi-Ueda T, Imaizumi K, Ono Y. Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide. Protein Expr Purif. 2016;120:99–105.View ArticleGoogle Scholar
  32. Rueda F, Cano-Garrido O, Mamat U, Wilke K, Seras-Franzoso J, Garcia-Fruitos E, et al. Production of functional inclusion bodies in endotoxin-free Escherichia coli. Appl Microbiol Biotechnol. 2014;98:9229–38.View ArticleGoogle Scholar
  33. Taguchi S, Ooi T, Mizuno K, Matsusaki H. Advances and needs for endotoxin-free production strains. Appl Microbiol Biotechnol. 2015;99:9349–60.View ArticleGoogle Scholar
  34. Rader RA. (Re)defining biopharmaceutical. Nat Biotechnol. 2008;26:743–51.View ArticleGoogle Scholar
  35. Walsh G. New biopharmaceuticals. Biopharm Int. 2012;25:34–8.Google Scholar
  36. Anonymous. Human insulin receives FDA approval. FDA Drug Bull. 1982;12:18–9.Google Scholar
  37. Johnson IS. Human insulin from recombinant DNA technology. Science. 1983;219:632–7.View ArticleGoogle Scholar
  38. Chabner BA, Roberts TG Jr. Timeline: chemotherapy and the war on cancer. Nat Rev Cancer. 2005;5:65–72.View ArticleGoogle Scholar
  39. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12:278–87.View ArticleGoogle Scholar
  40. Patel A, Sun W. Ziv-aflibercept in metastatic colorectal cancer. Biologics. 2014;8:13–25.Google Scholar
  41. Manoukian G, Hagemeister F. Denileukin diftitox: a novel immunotoxin. Expert Opin Biol Ther. 2009;9:1445–51.View ArticleGoogle Scholar
  42. Ho VT, Zahrieh D, Hochberg E, Micale E, Levin J, Reynolds C, et al. Safety and efficacy of denileukin diftitox in patients with steroid–refractory acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Blood. 2004;104:1224–6.View ArticleGoogle Scholar
  43. Lohcharoenkal W, Wang L, Chen YC, Rojanasakul Y. Protein nanoparticles as drug delivery carriers for cancer therapy. Biomed Res Int. 2014;2014:180549.View ArticleGoogle Scholar

Copyright

© Sanchez-Garcia et al. 2016

Advertisement