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Microbials for the production of monoclonal antibodies and antibody fragments

2013; Elsevier BV; Volume: 32; Issue: 1 Linguagem: Inglês

10.1016/j.tibtech.2013.10.002

0167-9430

Oliver Spadiut, Simona Capone, Florian Krainer, Anton Glieder, Christoph Herwig,

Transgenic Plants and Applications

•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 protein expression holds several drawbacks such as low product yield and growth rate, risk of viral contamination, and requirement for serum. Despite the introduction of serum-free (SF) chemically defined media (CDM) encountering regulatory requirements [56van der Valk J. et al.Optimization of chemically defined cell culture media–replacing fetal bovine serum in mammalian in vitro methods.Toxicol. In Vitro. 2010; 24: 1053-1063Crossref PubMed Scopus (373) Google Scholar], the addition of chemically undefined hydrolysates is still necessary to support cell growth. This, however, highly contradicts QbD guidelines demanding defined growth media [57Kim J.Y. et al.Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates.Appl. Microbiol. Biotechnol. 2011; 89: 1917-1928Crossref PubMed Scopus (18) Google Scholar]. Furthermore, the current standard production process is cumbersome and time-consuming. Cell transfection leads to high clone heterogeneity, necessitating repeated screening procedures at increasing drug concentrations for the isolation of a positive, highly productive clone [8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar]. Clone evaluation and culture condition optimization is then performed in shake flasks and lab-scale bioreactors before production processes can be set up. However, scale-up is also very challenging. The catabolism of the main carbon sources, glucose and glutamine, leads to formation of the inhibiting metabolites lactate and ammonium, respectively; hence batch and fed-batch operation modes, both representing closed cultivation systems, are only possible for a restricted timeframe. Because the metabolism of mammalian cells is highly sensitive and responsive to changing culture conditions, bioprocesses are hard to model – in fact only unstructured models are possible – and to control, which again contradicts QbD guidelines [57Kim J.Y. et al.Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates.Appl. Microbiol. Biotechnol. 2011; 89: 1917-1928Crossref PubMed Scopus (18) Google Scholar]. Consequently, chemostat cultivations, which describe open cultivation systems where substrate is constantly fed and cultivation broth is continuously removed, are generally employed to avoid metabolite inhibition. To avoid a critical wash out of mammalian cells, perfusion systems that provide cell retention by employing membranes are mainly used. However, operating a continuous culture with a perfusion system requires more devices and control systems than a batch or fed-batch system and also bears the elevated risk of contamination. Another drawback associated with scaling-up mammalian cell cultures is their sensitivity to shear stress, creating further challenges to efficient aeration in large vessels. Thus, although mammalian cells can produce mAbs with compatible PTMs, several drawbacks in bioprocessing are yet to be overcome.Nevertheless, a full length antibody with a glycosylated Fc domain is not necessary for antigen recognition. In fact, both the Fv and the Fab region alone (Figure 1) exhibit antigen binding properties. Furthermore, antibody fragments show increased tissue penetration and a lower retention time in non-target tissues compared to mAbs [9Ahmad Z.A. et al.scFv antibody: principles and clinical application.Clin. Dev. Immunol. 2012; https://doi.org/10.1155/2012/980250Crossref PubMed Scopus (435) Google Scholar]. Although the lack of the stabilizing Fc domain causes reduced stability [10Nelson A.L. Antibody fragments: hope and hype.MAbs. 2010; 2: 77-83Crossref PubMed Scopus (326) Google Scholar], the absence of glycosylation on both the Fv and the Fab regions allows their production to be less complex and enables easier engineering and cultivation of microbial host organisms such as bacteria and yeasts.Microbial expression hosts for mAbs and antibody fragmentsThe yeast S. cerevisiaeS. cerevisiae was the first yeast employed in the production of recombinant proteins, and several biopharmaceuticals produced in this yeast have since been successfully marketed [11Walsh G. Biopharmaceutical benchmarks 2010.Nat. Biotechnol. 2010; 28: 917-924Crossref PubMed Scopus (639) Google Scholar]. There are several intrinsic characteristics, like the stability of the expression system and the ease of cultivation, as well as advances in host engineering, that make S. cerevisiae an attractive host for the production of mAbs and antibody fragments. In fact, the production of Llama heavy chain antibody fragments (Hvv) in S. cerevisiae already represents a well-established industrial process, ensuring production titers up to hundreds of mg/l [12Gorlani A. et al.Expression of VHHs in Saccharomyces cerevisiae.Methods Mol. Biol. 2012; 911: 277-286Crossref PubMed Scopus (24) Google Scholar].Expression systemS. cerevisiae is easy to transform either chemically or by electroporation. There are three main types of shuttle vectors in use: (i) yeast episomal plasmids (Yep), which contain the 2 μ origin of replication, allowing gene expression without genomic integration at high copy numbers; (ii) yeast centromeric plasmids (Ycp), which contain an autonomously replicating sequence and replicate with single or very low gene copy number; and (iii) yeast integrative plasmids (Yip), which lack the yeast origin of replication and are integrated into the host genome [13Chee M.K. Haase S.B. New and redesigned pRS plasmid shuttle vectors for genetic manipulation of Saccharomyces cerevisiae.G3 (Bethesda). 2012; 2: 515-526Crossref PubMed Scopus (67) Google Scholar]. Although genomic integration of the target gene leads to a reduced expression level, it is highly desirable in terms of process quality and stability [14Park Y.N. et al.Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.Yeast. 2011; 28: 673-681Crossref PubMed Scopus (18) Google Scholar]. To overcome the disadvantage of low expression, targeted integration of the heterologous gene at the highly transcribed ribosomal DNA locus was developed recently [15Leite F.C. et al.Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae.Plasmid. 2013; 69: 114-117Crossref PubMed Scopus (14) Google Scholar]. In addition, commonly used promoters derived from the native glycolytic pathway, such as the promoters for glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol dehydrogenase1 (ADH1), phosphoglycerate kinase (PGK), and phosphoglycerate kinase (PGK1), allow high transcription levels [16Partow S. et al.Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae.Yeast. 2010; 27: 955-964Crossref PubMed Scopus (234) Google Scholar]. Finally, new cloning strategies introduced recently allow the concomitant expression of two or more genes located on specially designed self-replicating plasmids [17Maury J. et al.Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae.FEBS Lett. 2008; 582: 4032-4038Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar], which also addresses the issue of low expression levels of heterologous genes caused by genomic integration.Strain engineeringDespite continuing advances in genetic manipulation, efficient production of mAbs and antibody fragments in S. cerevisiae can still be impaired by endoplasmic reticulum (ER) misfolding and inefficient trafficking. Although Hvv can be produced successfully in sufficient amounts [12Gorlani A. et al.Expression of VHHs in Saccharomyces cerevisiae.Methods Mol. Biol. 2012; 911: 277-286Crossref PubMed Scopus (24) Google Scholar], the expression of the significantly smaller single chain Fv (scFv) region (Figure 1) leads to intracellular accumulation of misfolded proteins in the ER or in vacuolar-like organelles. A possible explanation for this is the higher hydrophobicity of the variable light and heavy chains of scFv compared to Hvv [18Joosten V. et al.The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi.Microb. Cell Fact. 2003; 2: 1Crossref PubMed Scopus (115) Google Scholar]. However, additional overexpression of chaperones and foldases can correct protein folding and allow subsequent scFv secretion [19Xu P. et al.Analysis of unfolded protein response during single-chain antibody expression in Saccharomyces cerevisiae reveals different roles for BiP and PDI in folding.Metab. Eng. 2005; 7: 269-279Crossref PubMed Scopus (65) Google Scholar].Several strategies have been developed to increase the overall secretory capacity and productivity of S. cerevisiae. These approaches include engineering intracellular protein trafficking by over-expression of soluble N-ethylmaleimide-sensitive factor (NFS) attachment protein receptor proteins (SNAREs) [20Hou J. et al.Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae.Metab. Eng. 2012; 14: 120-127Crossref PubMed Scopus (83) Google Scholar], reduction of proteolytic degradation by multiple protease gene deletions [21Idiris A. et al.Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway.Appl. Microbiol. Biotechnol. 2010; 85: 667-677Crossref PubMed Scopus (55) Google Scholar], and engineering of the heat shock response (HSR) pathway by overexpressing the heat shock transcription factor (Hsf) [22Hou J. et al.Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae.Appl. Microbiol. Biotechnol. 2013; 97: 3559-3568Crossref PubMed Scopus (38) Google Scholar]. Although these engineered strains have not yet been used for the production of mAbs and antibody fragments, they demonstrate the ongoing, intensive strain engineering work that is being done with S. cerevisiae.Production processProduction of antibody fragments in S. cerevisiae is generally done in glucose-limited fed-batch cultivations [12Gorlani A. et al.Expression of VHHs in Saccharomyces cerevisiae.Methods Mol. Biol. 2012; 911: 277-286Crossref PubMed Scopus (24) Google Scholar]. Yeast shows a mixed oxidative/fermentative metabolism, which can result in the undesired production of toxic metabolites. Fermentative mode shift is triggered by oxygen depletion or by elevated carbon source concentration. Limiting glucose is therefore a valid strategy for preventing fermentation during cultivation processes with this yeast. Recently, a fully aerobically engineered strain, in which glucose uptake was reduced, was developed, allowing a full aerobic respiration even at elevated glucose concentrations [23Ferndahl C. et al.Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory.Microb. Cell Fact. 2010; 9: 47Crossref PubMed Scopus (23) Google Scholar].As this discussion indicates, there are ongoing efforts to optimize the yeast S. cerevisiae for the production of mAbs and antibody fragments. Because antibody fragments are not glycosylated, they can be produced successfully in this yeast and are not affected by hypermannosylation, which characterizes S. cerevisiae [24Hamilton S.R. Gerngross T.U. Glycosylation engineering in yeast: the advent of fully humanized yeast.Curr. Opin. Biotechnol. 2007; 18: 387-392Crossref PubMed Scopus (239) Google Scholar]. Furthermore, current studies are investigating the possibility of humanizing the glycosylation machinery in S. cerevisiae [25Chiba Y. et al.Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae.J. Biol. Chem. 1998; 273: 26298-26304Crossref PubMed Scopus (122) Google Scholar], in an attempt to engineer this yeast for the production of full length mAbs.The yeast P. pastorisAs an alternative to S. cerevisiae, the methylotrophic yeast P. pastoris, which is closely related to S. cerevisiae, can be used for the production of mAbs and antibody fragments as it also holds a generally recognized as safe (GRAS) status [26Mattia, A. Diversa Corporation (2006) GRAS notification concerning BD16449 – phospholipase C enzyme preparation from Pichia pastoris, http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000204.pdfGoogle Scholar].Expression systemSimilar to the process in S. cerevisiae, the target gene is integrated into the genome of P. pastoris to guarantee reproducibility and stability of the expression system. However, a major obstacle in P. pastoris is the substantial degree of non-homologous recombination. One solution to this challenge is the use of a recently developed P. pastoris strain with an inactivated non-homologous end joining pathway [27Naatsaari L. et al.Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology.PLoS ONE. 2012; 7: e39720Crossref PubMed Scopus (164) Google Scholar].P. pastoris can use methanol as a sole carbon source, as it is a crucial part of its metabolism (e.g., [28Krainer F.W. et al.Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.Microb. Cell Fact. 2012; 11: 22Crossref PubMed Scopus (137) Google Scholar]). However, instead of the traditional hard-to-control alcohol oxidase promoter system typically used for P. pastoris, alternative adjustable promoters are currently under investigation [29Delic M. et al.Repressible promoters – a novel tool to generate conditional mutants in Pichia pastoris.Microb. Cell Fact. 2013; 12: 6Crossref PubMed Scopus (31) Google Scholar]. Furthermore, the generation of artificial and semi-artificial, tunable promoter variants are the subject of recent synthetic biology approaches [30Ruth C. et al.Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris.Syst. Synth. Biol. 2010; 4: 181-191Crossref PubMed Scopus (37) Google Scholar].Strain engineeringThe genome sequences of the wild type strains NRRL Y-1603 (identical to DSMZ 70382 or CBS704) [7de Marco A. Biotechnological applications of recombinant single-domain antibody fragments.Microb. Cell Fact. 2011; 10: 44Crossref PubMed Scopus (136) Google Scholar], NRRL Y-11430 (identical to ATCC 7673 or CBS7435), and GS115 are available online [31De Schutter K. et al.Genome sequence of the recombinant protein production host Pichia pastoris.Nat. Biotechnol. 2009; 27: 561-566Crossref PubMed Scopus (372) Google Scholar, 32Mattanovich D. et al.Open access to sequence: browsing the Pichia pastoris genome.Microb. Cell Fact. 2009; 8: 53Crossref PubMed Scopus (49) Google Scholar] and a genome-scale metabolic model of P. pastoris was published recently [33Sohn S.B. et al.Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production.Biotechnol. J. 2010; 5: 705-715Crossref PubMed Scopus (90) Google Scholar], allowing straight-forward strain engineering approaches. For example, co-overexpression of helper proteins, such as the protein disulfide isomerase or the transcription factor of the unfolded protein response Hac1 [34Inan M. et al.Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.Biotechnol. Bioeng. 2006; 93: 771-778Crossref PubMed Scopus (158) Google Scholar], as well as inactivation of endogenous proteases (e.g., [35Boehm T. et al.Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin.Yeast. 1999; 15: 563-572Crossref PubMed Scopus (60) Google Scholar]) enhances the production and secretion of recombinant proteins. Engineering the protein trafficking pathway represents another successful approach to improve secretion [36Baumann K. et al.Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris.Microb. Cell Fact. 2011; 10: 93Crossref PubMed Scopus (47) Google Scholar]. In addition, intensive glycoengineering work is ongoing to humanize the glycosylation events in P. pastoris and allow production of full length mAbs in this yeast (Box 2).Box 2Glycoengineering of Pichia pastoris allows mAb productionP. pastoris can be used for the production of both antibody fragments and mAbs (e.g., [58Ning D. et al.Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation.J. Biochem. Mol. Biol. 2005; 38: 294-299Crossref PubMed Google Scholar]). For mAbs, the correct human-type glycosylation is not only essential for proper folding and biological activity, but also for targeting and stability in circulation. P. pastoris lacks the Golgi-resident α-1,3-mannosyltransferase, but harbors four additional β-mannosyltransferases instead [59Wildt S. Gerngross T.U. The humanization of N-glycosylation pathways in yeast.Nat. Rev. Microbiol. 2005; 3: 119-128Crossref PubMed Scopus (271) Google Scholar, 60Mille C. et al.Identification of a new family of genes involved in beta-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans.J. Biol. Chem. 2008; 283: 9724-9736Crossref PubMed Scopus (75) Google Scholar]. The absence of terminal α-1,3-mannoses on P. pastoris-derived glycoproteins is of importance because this glycan structure causes high antigenicity in humans [61Cregg J.M. et al.Recent advances in the expression of foreign genes in Pichia pastoris.Biotechnology. 1993; 11: 905-910Crossref PubMed Scopus (842) Google Scholar]. Thus, the humanization of the N-glycosylation pathway in P. pastoris has been an important goal. The Outer CHain elongation 1 gene (OCH1) coding for an α-1,6-mannosyltransferase was knocked out [62Choi B.K. et al.Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 5022-5027Crossref PubMed Scopus (292) Google Scholar], and an α-1,2-mannosidase, β-N-acetylglucosaminyltransferase I (GnTI) and an UDP-GlcNAc transporter were introduced [63Nett J.H. et al.A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.Yeast. 2011; 28: 237-252Crossref PubMed Scopus (32) Google Scholar]. The Kluyveromyces lactis UDP-GlcNAc transporter, mouse α-1,2-mannosidase IA, Drosophila melanogaster mannosidase II, human GnTI, and rat GnTII were introduced into an och1 knockout strain, resulting in the homogeneous formation of the complex human GlcNAc2Man3GlcNAc2 glycan [64Hamilton S.R. et al.Production of complex human glycoproteins in yeast.Science. 2003; 301: 1244-1246Crossref PubMed Scopus (335) Google Scholar]. In other studies, OCH1 was inactivated via a knock-in strategy [65Bernett M.J. et al.Engineering fully human monoclonal antibodies from murine variable regions.J. Mol. Biol. 2010; 396: 1474-1490Crossref PubMed Scopus (43) Google Scholar], an ER-targeted HEDL (His-Asp-Glu-Leu; C-terminal tetrapeptide involved in the lumen sorting of soluble proteins)-tagged α-1,2-mannosidase from Trichoderma reesei was introduced, and a chimeric human GnTI was fused to the N-terminal part of Saccharomyces cerevisiae Kre2 for Golgi localization [66Callewaert N. et al.Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris.FEBS Lett. 2001; 503: 173-178Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. A further approach included the construction of a strain expressing mouse mannosidase IA, the K. lactis UDP-GlcNAc transporter, human GnTI, and rat GnTII, in which the ALG3 gene, encoding an α-1,3-mannosyltransferase of the ER lumen, was knocked out [67Davidson R.C. et al.Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris.Glycobiology. 2004; 14: 399-407Crossref PubMed Scopus (54) Google Scholar], leading to the formation of GlcNAc2Man3GlcNAc2. Additional coexpression of a fusion protein consisting of the S. cerevisiae Mnn2 Golgi localization domain and the activities of Schizosaccharomyces pombe UDP-Gal 4-epimerase and human β-1,4-galactosyl transferase allowed the production of Gal2GlcNAc2Man3GlcNAc2 glycans. An alternative protocol allowed production of Gal2GlcNAc2Man3GlcNAc2 N-glycans using the GlycoSwitch vector technology [68Jacobs P.P. et al.Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology.Nat. Protoc. 2009; 4: 58-70Crossref PubMed Scopus (174) Google Scholar], where specially designed vectors are used to replace genes of the native glycosylation pathway. Further humanization was achieved by additional biosynthesis of cytidine monophosphate-linked Sia, its transport and the transfer of Sia onto the N-glycans of nascent polypeptides, leading to complex human Sia2Gal2GlcNAc2Man3GlcNAc2 glycans [69Hamilton S.R. et al.Humanization of yeast to produce complex terminally sialylated glycoproteins.Science. 2006; 313: 1441-1443Crossref PubMed Scopus (410) Google Scholar]. Additional glycoengineering studies included the elimination of α-1,2-mannosidase-resistant high Man glycans [70Hopkins D.