Microbials for the production of monoclonal antibodies and antibody fragments

Abstract

•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;