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•Glycosylated full length antibodies are currently produced in mammalian cells.•Antibody fragments can be produced in microbial organisms.•Strain engineering allows production of full length antibodies in microbials.•Microbials provide several advantages over mammalian cells. Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. IntroductionOver the past three decades, the biopharmaceutical market has become a significant component of the global pharmaceutical market accounting for around 40% of its sales. The use of organisms as biopharmaceutical production factories offers several advantages over chemical synthesis. Microorganisms can produce high molecular weight compounds such as proteins 1Lee J.Y. Bang D. Challenges in the chemical synthesis of average sized proteins: sequential vs. convergent ligation of multiple peptide fragments.Biopolymers. 2010; 94: 441-447Crossref PubMed Scopus (21) Google Scholar and carry out highly enantio- and regio-selective reactions by their native enzymatic machinery – these reactions are hard to achieve by chemical synthesis. The use of microorganisms also enables repeated implementation of immobilized enzymes or cells resulting in the reduction of the overall production costs 2Bolivar J.M. et al.Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts.Trends Biotechnol. 2013; 31: 194-203Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar. Finally, processes employing microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene 3Chelliapan S. Sallis P.J. Removal of organic compound from pharmaceutical wastewater using advanced oxidation processes.J. Sci. Ind. Res. 2013; 72: 248-254Google Scholar.The biopharmaceutical market originated in the late 1970s with the establishment of recombinant DNA techniques. The industrial interest materialized almost immediately and in 1982 the US Food and Drug Administration (FDA) approved the commercialization of humulin, the human insulin analog, recombinantly produced in the bacterium E. coli 4Walsh G. New biopharmaceuticals: a review of new biologic drug approvals over the years, featuring highlights from 2010 and 2011.Process Development Forum. BioPharm International, 2012http://www.processdevelopmentforum.com/articles/new-biopharmaceuticals-a-review-of-new-biologic-drug-approvals-over-the-years-featuring-highlights-from-2010-and-2011/Google Scholar. For a while the FDA only allowed the transformation of bacteria and the expression of small, non-glycosylated proteins, like insulin, due to concern about introducing new toxicities such as contaminating bacterial substances, which raise immunogenic reactions in patients. However, with the development of selectable resistance markers, like antibiotic resistance markers, and the possibility of production in eukaryotic organisms, the FDA began showing increasing flexibility towards biotechnological innovation, leading to a continually increasing number of approved new biological entities (NBEs). In 2012, the biopharmaceutical market turnover was estimated at around 100–120 billion US dollars per year 5Butler M. Meneses-Acosta A. Recent advances in technology supporting biopharmaceutical production from mammalian cells.Appl. Microbiol. Biotechnol. 2012; 96: 885-894Crossref PubMed Scopus (124) Google Scholar, with more than 200 biopharmaceutical proteins already on the market 6Berlec A. Strukelj B. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.J. Ind. Microbiol. Biotechnol. 2013; 40: 257-274Crossref PubMed Scopus (139) Google Scholar, and is expected to reach 170 billion US dollars in 2014. This exceptionally high market turnover is largely derived from the marketing of mAbs and antibody fragments that currently represent the fastest growing class of approved biopharmaceutical products. In fact, production of full length mAbs (Figure 1) is the most important biopharmaceutical venture to date, with several therapeutic products reaching blockbuster status (e.g., Avastin, Herceptin, Remicade, Rituxan, Humira, and Erbitux).More recently, interest has grown in the production of antibody fragments that can be used not only in therapeutic applications but also in immunodetection, purification, and bioseparation applications 7de Marco A. Biotechnological applications of recombinant single-domain antibody fragments.Microb. Cell Fact. 2011; 10: 44Crossref PubMed Scopus (136) Google Scholar. Antibody fragments still exhibit antigen binding properties and can be produced in microbials, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression system, strain engineering, and production process for the three main microbials for antibody fragment production, namely S. cerevisiae, P. pastoris, and E. coli, and highlight ongoing research that may allow full length antibody production in these organisms in the future.mAbs and antibody fragments: an overviewA full length mAb consists of the constant Fc (crystallizable fragment) domain and an antigen binding domain, comprising the Fv (variable fragment) and the Fab region (antibody binding fragment; Figure 1). Native full length mAbs are glycosylated during their synthesis. Although the glycosylated Fc domain does not directly interact with antigens, it stabilizes the antibody and is important for antibody-dependent, cell-mediated cytotoxicity. Moreover, glycosylation strongly impacts the clearance rate of the recombinant mAb from the body, and incompatible glycoforms can cause severe immunogenic effects in patients. Thus, much current work is focused on optimizing and controlling glycosylation events in mammalian cells 8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar, which at this time are the most often used cell type for the production of mAbs (Box 1).Box 1Production of mAbs in mammalian cells: advantages and drawbacksMammalian cells are used most often for production of mAbs due to their ability to perform post-translational modifications (PTM), especially human-like N-glycosylation. Their use simplifies subsequent medical applications by eliminating the risk of an immunogenic response in patients due to incompatible N-glycans on the protein. Chinese Hamster Ovary (CHO) cell lines are used most frequently to generate full length mAbs with human-like Fc N-glycosylation and production titers of around 10 g/l 8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar. However, the use of mammalian cells for heterologous expression several drawbacks such as and risk of and for the of et of cell culture in mammalian in In 2010; PubMed Scopus Google Scholar], the of is still to cell highly J.Y. et of of recombinant Chinese cells in with Microbiol. Biotechnol. 2011; PubMed Scopus Google Scholar]. the current production process is and Cell to high repeated at increasing drug for the of a highly 8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar. and culture is in and production processes can be However, is also The of the main and to of the and and systems, are only for a Because the of mammalian cells is highly and to culture are hard to – in only are – and to which J.Y. et of of recombinant Chinese cells in with Microbiol. 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For this pharmaceutical are and in E. coli to their production and length mAbs as as antibody fragments represent the most important and class of today. to the for mAbs are still produced in mammalian cells, which several drawbacks to bioprocessing and By contrast, antibody fragments, which are not glycosylated but antigen binding properties, can also be produced in microbial Recent advances in the production of full length mAbs and antibody fragments with mammalian cells and microbials are in advances in the production of full length mAbs and antibody fragments with and expression engineering and of M. et of for therapeutic cell Biotechnol. 2010; PubMed Scopus Google et using 2010; Google et in mammalian cell development for recombinant 2013; Scopus Google E. et of a cell engineering to effects in cells.J. 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Biotechnol. 2012; PubMed Scopus Google to the et of antibody fragment the Escherichia coli by with the in the 2013; PubMed Scopus Google et of cell culture in mammalian in In 2010; PubMed Scopus Google S. of three cell culture 2011; et cell in Saccharomyces recombinant the use of a strain as a microbial cell Cell Fact. 2010; PubMed Scopus Google et to a for recombinant Pichia Cell Fact. 2011; 10: PubMed Scopus Google the native the et engineering for in 2012; PubMed Scopus Google et of an Escherichia coli strain in the for culture at high and cell an to PubMed Scopus Google in a new in current are towards optimizing the production of mAbs and antibody fragments in microbial organisms, mammalian cells in several such as the of and high cell processes on and Although mAbs are still most frequently produced in mammalian cells, ongoing with yeasts (Box and E. coli et of and in Escherichia Microbiol. 2011; PubMed Scopus Google et glycosylation of antibody fragments in Escherichia 2011; PubMed Scopus Google Scholar] are the for the production of glycosylated full length mAbs in microbial IntroductionOver the past three decades, the biopharmaceutical market has become a significant component of the global pharmaceutical market accounting for around 40% of its sales. The use of organisms as biopharmaceutical production factories offers several advantages over chemical synthesis. Microorganisms can produce high molecular weight compounds such as proteins 1Lee J.Y. Bang D. Challenges in the chemical synthesis of average sized proteins: sequential vs. convergent ligation of multiple peptide fragments.Biopolymers. 2010; 94: 441-447Crossref PubMed Scopus (21) Google Scholar and carry out highly enantio- and regio-selective reactions by their native enzymatic machinery – these reactions are hard to achieve by chemical synthesis. The use of microorganisms also enables repeated implementation of immobilized enzymes or cells resulting in the reduction of the overall production costs 2Bolivar J.M. et al.Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts.Trends Biotechnol. 2013; 31: 194-203Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar. Finally, processes employing microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene 3Chelliapan S. Sallis P.J. Removal of organic compound from pharmaceutical wastewater using advanced oxidation processes.J. Sci. Ind. Res. 2013; 72: 248-254Google Scholar.The biopharmaceutical market originated in the late 1970s with the establishment of recombinant DNA techniques. The industrial interest materialized almost immediately and in 1982 the US Food and Drug Administration (FDA) approved the commercialization of humulin, the human insulin analog, recombinantly produced in the bacterium E. coli 4Walsh G. New biopharmaceuticals: a review of new biologic drug approvals over the years, featuring highlights from 2010 and 2011.Process Development Forum. BioPharm International, 2012http://www.processdevelopmentforum.com/articles/new-biopharmaceuticals-a-review-of-new-biologic-drug-approvals-over-the-years-featuring-highlights-from-2010-and-2011/Google Scholar. For a while the FDA only allowed the transformation of bacteria and the expression of small, non-glycosylated proteins, like insulin, due to concern about introducing new toxicities such as contaminating bacterial substances, which raise immunogenic reactions in patients. However, with the development of selectable resistance markers, like antibiotic resistance markers, and the possibility of production in eukaryotic organisms, the FDA began showing increasing flexibility towards biotechnological innovation, leading to a continually increasing number of approved new biological entities (NBEs). In 2012, the biopharmaceutical market turnover was estimated at around 100–120 billion US dollars per year 5Butler M. Meneses-Acosta A. Recent advances in technology supporting biopharmaceutical production from mammalian cells.Appl. Microbiol. Biotechnol. 2012; 96: 885-894Crossref PubMed Scopus (124) Google Scholar, with more than 200 biopharmaceutical proteins already on the market 6Berlec A. Strukelj B. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.J. Ind. Microbiol. Biotechnol. 2013; 40: 257-274Crossref PubMed Scopus (139) Google Scholar, and is expected to reach 170 billion US dollars in 2014. This exceptionally high market turnover is largely derived from the marketing of mAbs and antibody fragments that currently represent the fastest growing class of approved biopharmaceutical products. In fact, production of full length mAbs (Figure 1) is the most important biopharmaceutical venture to date, with several therapeutic products reaching blockbuster status (e.g., Avastin, Herceptin, Remicade, Rituxan, Humira, and Erbitux).More recently, interest has grown in the production of antibody fragments that can be used not only in therapeutic applications but also in immunodetection, purification, and bioseparation applications 7de Marco A. Biotechnological applications of recombinant single-domain antibody fragments.Microb. Cell Fact. 2011; 10: 44Crossref PubMed Scopus (136) Google Scholar. Antibody fragments still exhibit antigen binding properties and can be produced in microbials, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression system, strain engineering, and production process for the three main microbials for antibody fragment production, namely S. cerevisiae, P. pastoris, and E. coli, and highlight ongoing research that may allow full length antibody production in these organisms in the
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