Biopharmaceutical discovery and production in yeast

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ScienceDirect Biopharmaceutical discovery and production in yeast Michael A Meehl and Terrance A Stadheim The selection of an expression platform for recombinant biopharmaceuticals is often centered upon suitable product titers and critical quality attributes, including post-translational modifications. Although notable differences between microbial, yeast, plant, and mammalian host systems exist, recent advances have greatly mitigated any inherent liabilities of yeasts. Yeast expression platforms are important to both the supply of marketed biopharmaceuticals and the pipelines of novel therapeutics. In this review, recent advances in yeastbased expression of biopharmaceuticals will be discussed. The advantages of using glycoengineered yeast as a production host and in the discovery space will be illustrated. These advancements, in turn, are transforming yeast platforms from simple production systems to key technological assets in the discovery and selection of biopharmaceutical lead candidates. Addresses GlycoFi, Biologics Research, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA Corresponding author: Stadheim, Terrance A ([email protected]) Current Opinion in Biotechnology 2014, 30:120–127 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Beth Junker and Jamey D Young

http://dx.doi.org/10.1016/j.copbio.2014.06.007 0958-1669/# 2014 Elsevier Ltd. All rights reserved.

Introduction The dawn of recombinant DNA techniques ushered in the modernization of protein-based pharmaceuticals, known as biopharmaceuticals or biologics. In the early 1980s, recombinant human insulin was marketed and provided Type I diabetics with a more homogenous and reliable medicine that no longer required sourcing from animal pancreas. The development of recombinant insulin helped pave the way for the marketing approval of Escherichia coli and Saccharomyces cerevisiae derived products. Three decades later, the current quality of recombinant glycoprotein expression in yeast rivals that of traditional mammalian cell culture systems. Such engineering and process development efforts have occurred in different systems with varying degrees of success. Recent advancements in yeast engineering have provided yeast-based platforms with more than just the ability to Current Opinion in Biotechnology 2014, 30:120–127

manufacture aglycosylated peptides or proteins with low complexity. Considerable effort has focused on strain improvements to develop glycoengineered yeast systems for the production of recombinant glycoproteins with desired physiochemical properties and efficacy. Innovations in yeast genetics and protein secretion will also be discussed that enable yeast to be a state-of-the-art tool for biopharmaceutical research and development.

Yeast production systems and derived products Early biopharmaceuticals originated from animal and human sources. However, the majority of newly marketed products are derived from recombinant expression systems. Yeast, along with bacterial (i.e. E. coli) and mammalian hosts (i.e. Chinese Hamster Ovary) represent the most frequently used expression systems for biopharmaceuticals. In the early 1980s, ZymoGenetics industrialized S. cerevisiae as a host for recombinant human insulin leading to the marketing of Novolin1 with Novo Nordisk in 1987. Today, the S. cerevisiae platform currently delivers half of the world supply of insulin. With the recent interest in biosimilar products, Pichia pastoris has been used to produce recombinant human insulin with titers of 3 g/L [1,2]. In fact, Biocon is the world’s fourth largest supplier of insulin products and utilizes P. pastoris [URL: http://www.biocon.com]. The example of recombinant insulin demonstrates the capabilities of yeast-based biopharmaceutical expression and production. In recent years, many new biopharmaceutical candidates were produced in S. cerevisiae, P. pastoris, and Hansenula polymorpha platforms [3–5,6,7]. The most recent FDA approval of a yeast-expressed biopharmaceutical was in 2012 for ocriplasmin, sold as Jetrea1 by ThromboGenics [8]. Ocriplasmin is an aglycosylated protease expressed in P. pastoris approved for the treatment of symptomatic vitreomacular adhesion [9]. Other notable yeast-based biopharmaceuticals with marketing approval include: a Hepatitis B subunit vaccine using Hepatitis B surface antigen (S. cerevisiae, P. pastoris, and H. polymorpha); recombinant human granulocyte macrophage-colony stimulating factor, Leukine1(sargramostim, S. cerevisiae, 1991); recombinant human platelet derived growth factor, Regranex1 (becaplermin, S. cerevisiae, 1997); human papillomavirus subunit vaccine, Gardasil1 (S. cerevisiae, 2006); and a kallikrein inhibitor, Kalbitor1 (ecallantide, P. pastoris, 2009). Numerous yeast-based biopharmaceuticals are in clinical development. Ablynx reports at least two clinical programs focused on nanobody-based therapeutics www.sciencedirect.com

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expressed in P. pastoris [URL: http://www.ablynx.com]. Alder Biopharmaceuticals reports two clinical antibody leads targeting interleukin-6 (IL-6), named ALD518 (BMS-945429), and calcitonin gene-related peptide (CGRP), named ALD403, in their pipeline using the P. pastoris expression system [URL: http://alderbio.com]. In the case of ALD518 (BMS-945429), the humanized antibody was engineered to lack Fc glycosylation. However, serum half-life is preserved and is comparable to other marketed glycosylated antibodies [10,11]. This latter example of full-length antibody expression in P. pastoris signifies a shift towards the production of more complex biopharmaceuticals. Indeed, there are reports of the production in P. pastoris of biopharmaceuticals with human-like glycosylation patterns, including antibodies and therapeutic proteins [12–14,15,16–18,19,20–22].

suitable for clinical use. Proteins with yeast-type glycosylation, when delivered in vivo, interact with human C-type lectins of the innate immune system resulting in altered pharmacokinetic properties, activation of complement, enhanced anti-drug–antibody formation, or sequestration by circulating anti-glycan antibodies [20,25–27,28]. Furthermore, mannose glycans have been shown to alter the efficacy of recombinant vaccines [29,30]. Many therapeutic glycoprotein candidates require a particular human-like glycoform for optimal efficacy, such as sialic acid for half-life extension [18,31], lack of fucose on antibodies for improved cytotoxic properties [32], and paucimannose for selective targeting to macrophages [33]. While in vitro glycan modifications using processing enzymes are possible, the most robust and efficient methods to produce particular glycosylation profiles are those that can be synthesized in vivo, such as using glycoengineered yeast.

Strain engineering and process improvements N-glycan modifications

Many biopharmaceuticals require proper post-translational modifications, including glycosylation, for optimal pharmacokinetic (PK) and pharmacodynamic (PD) properties [23,24]. Recombinant biopharmaceuticals with yeast-derived high mannose N-glycans are often not

N-glycan humanization centers on the conversion of an extant fungal high mannose oligosaccharide to a complex oligosaccharide that can be further optimized to contain specific terminal sugars (i.e. N-acetylglucosamine (GlcNAc), galactose and sialic acid) (see Figure 1). Over the past several years, this feat of genetic engineering

Figure 1

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Yeast N-glycan engineering. The N-glycosylation pathway of glycoengineered Pichia pastoris was previously reviewed [34]. Glycoproteins harboring predetermined glycoforms [78] are obtained depending on the glycoengineered yeast host used, each of which contains a unique set of gene deletions and glycosylation enzymes, as indicated by arrows. The main glycosylation pathway to obtain mammalian biantennary glycans is shown in the upper left rectangle. As indicated by (*), sialic acid linkages may be exclusively a-2,6 or a-2,3 depending on the chosen sialyltransferase. Other yeast modifications (e.g. beta-linked mannose, mannosylphosphate) are not depicted in the figure. www.sciencedirect.com

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has been achieved most notably in P. pastoris (for reviews, see [17,34]). Through the use of genetic perturbations limiting yeast mannosylation and the introduction of heterologous glycosyltransferases and related pathway machinery, the P. pastoris N-glycosylation pathway was engineered to enable secretion of proteins with complex glycans [16,34–39]. Using glycoengineered P. pastoris, antibodies and therapeutic proteins have been produced with complex glycoforms exhibiting suitable pharmacokinetic and pharmacodynamics properties required for in vivo efficacy [18,19,20,40,41]. N-glycan engineering efforts have also been recently reported in S. cerevisiae [42,43], H. polymorpha [44,45,46], Kluyveromyces lactis [47], and Yarrowia lipolytica [48,49]. These yeast glycoengineering reports are centered on two main stages, namely removal of yeast hypermannosylation and the conversion to complex glycoforms containing terminal GlcNAc, galactose, or sialic acid. In the first stage, varying approaches have been taken to remove or limit hypermannosylation. In one approach, yeast glycosyltransferase genes (e.g. OCH1) were disrupted to eliminate hypermannosylation and mannosidase genes expressed to obtain a human-like Man5GlcNAc2 glycoform [36,42,45,49,50]. In other approaches, the deletion of the ALG3 gene in combination with varying modifications leads to the generation of a Man3GlcNAc2 glycoform [35,39,43,46–48]. In the second stage of humanization, the terminal mannose substrate can be further modified by a specific N-acetylglucosamine transferase, GnT-I, to result in a hybrid-type glycoform containing terminal GlcNAc. In subsequent steps, a second GlcNAc sugar is added to the terminal a-1,6-mannose residue through the activity of GnT-II. The resulting glycan, GlcNAc2Man3GlcNAc2, can be further modified to contain terminal galactose and sialic acid (Figure 1). As evidenced from the profiling of glycoproteins expressed in yeast [51] and the analysis of Leukine1 expressed in S. cerevisiae [52], the occupancy of N-glycan sites is often not complete. Incomplete glycan occupancy may create challenges for certain biotherapeutics by impacting stability and/or efficacy. To eliminate this macroheterogeneity, N-linked glycan site(s) can simply be removed from the molecule if no impact on efficacy or immunogenicity is anticipated. As mentioned earlier, Alder Biopharmaceuticals has used this approach to produce their clinical antibody ALD-518, whereby the Asn residue at position 297 is substituted for an Ala residue [11]. Rather than eliminating N-glycans in yeast-based recombinant proteins, an alternative approach is to maximize N-glycan site occupancy. A genetic solution was provided by heterologous expression of a STT3 enzyme to increase site occupancy of complex glycans to near completion without negatively affecting protein secretion [52]. These recent reports on yeast N-glycan humanization are signaling a shift from the proof of concept phase to implementation by improving characteristics of recombinant proteins suitable for clinical use. Current Opinion in Biotechnology 2014, 30:120–127

O-Glycan modifications Removing wild-type yeast O-glycosylation

In yeast O-glycosylation, the hydroxyl functional groups of serine and threonine residues are modified with a mannose monosaccharide in a non-template driven manner in the endoplasmic reticulum by the PMT (protein O-mannosyltransferase) family of enzymes [53,54]. Mannose residues are then added to the O-glycan in the Golgi apparatus by additional enzymes creating a polydisperse mannose polymer of up to five mannose residues. Proteins made in yeast that are decorated with O-glycans are capable of mediating interactions with human mannose binding lectins, conferring inferior PK properties of biopharmaceuticals [19,55]. Conversely, vaccine antigenicity can be improved by yeast O-glycosylation via targeting to antigen presenting cells [56]. To reduce the presence, or occupancy, of O-glycans, PMT gene disruptions have been employed. Since yeast typically possess multiple protein O-mannosyltransferases, often with overlapping target specificities, a single PMT gene deletion is not sufficient to remove all O-glycosylation [53,57]. Furthermore, chemical PMT inhibitors also demonstrated the ability to significantly reduce O-mannose occupancy in yeast, and interestingly also correlated with improved antibody production [57–59]. Furthermore, expression of mannosidases capable of hydrolyzing a-1,2-mannose linkages was successfully shown to limit O-mannose chain length, and as a result, led to a reduction in lectin binding by the glycoprotein [55]. Overall, data suggests wild-type, or uncontrolled, yeast O-glycosylation may be detrimental for PK properties of biopharmaceuticals, and genetic or process solutions that control O-glycan occupancy and chain length have resulted in proteins with properties undifferentiated from mammalian proteins lacking O-mannosylation [41]. Glycoengineering of yeast O-glycosylation

Rather than limiting the occupancy or extension of O-mannose linkages, an alternative strategy has been described to humanize O-glycans. In humans, two main types of O-glycans are found, the mucin-type characterized by Ser/Thr-O-GalNAc-linkages and the a-dystroglycan-type of Ser/Thr-O-Man-GlcNAc. Efforts to convert yeast O-mannose to O-GalNAc are particularly challenging due to the overlapping functions of the PMT family. Nevertheless, the engineering of mucin-type O-glycosylation in S. cerevisiae has been reported [60]. In an earlier study by the same group, an artificial O-glycosylation pathway was engineered in S. cerevisiae to produce an O-fucosylated epidermal growth factor (EGF) domain, demonstrating the ability to produce a heterologous O-glycoform [61]. The introduction of the enzyme protein-O-linked-mannose b-1,2-N-acetylglucosaminyltransferase 1 (PomGnT1) to P. pastoris led to the production of highly sialylated a-dystroglycan-type www.sciencedirect.com

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O-glycans [15]. In summary, significant remodeling of yeast N-glycosylation and O-glycosylation pathways offers the ability to optimize biopharmaceuticals with desired in vivo properties [19].

Biopharmaceutical R&D using yeast systems The varying efforts of yeast glycoengineering have focused on the development of biopharmaceutical production systems capable of manufacturing proteins with

Figure 2

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Discovery of yeast-based biopharmaceuticals. The advancements in recombinant protein expression in yeast enable end-to-end biopharmaceutical production, from discovery to manufacturing. Novel leads are generated from either randomized approaches, such as cell surface display (Ia) or rational designs of glycan and/or amino acid variants (Ib). Clones expressing variants may be selected using FACS (II) and/or in medium-throughput assays (III). In the case of Fc display [73], yeast expressing cell-surface variants are also capable of co-secretion, shortening the time needed for candidate selection. Following the assessment of preclinical assays (in vitro and in vivo) to support candidate selection (IV), final yeast clones are selected for manufacturing (V). www.sciencedirect.com

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superior pharmacokinetic and pharmacodynamic properties. However, yeast platforms have great potential upstream of manufacturing in the discovery of biopharmaceutical candidate molecules. Yeast offer numerous advantages in the discovery space including ease of genetic manipulation with good transformation efficiency, the ability to increase diversity through mating haploid strains, eukaryotic quality control of the secretory pathway, and the rapid generation of test materials using a clonal population. Whereas some candidate molecules are fairly well defined at the outset of a program (e.g. enzymes, growth factors, cytokines, receptors, etc.), other molecules such as antibodies can require large screening effort to find the optimal lead. Transformation efficiencies for S. cerevisiae and P. pastoris are reported to be in the range of 105–106 CFU/mg DNA [62–64]. These efficiencies are readily suitable for the selection of high expression clones with varying gene copies [65]. Randomized library-based screening efforts may require sequences in the 106–1011 range. For further increases in diversity, yeast has the distinct advantage of mating MATa and MATa haploid clones for generating highly diverse antibody libraries [66,67]. Diploid strains obtained though mating are also capable of robust production of a full-length antibody, as shown in a recent study using wild-type and glycoengineered diploid P. pastoris strains under fermentation conditions [68]. Antibodies and antibody fragments are some of the most efficacious and medically important therapies on the market today, as proven by the robust success of the anti-TNF agents [69]. Popular methods to identify the Fab sequence against an antigen include both in vivo (e.g. through immunization of either naı¨ve or transgenic rodents) and in vitro (e.g. phage, yeast, ribosome, mammalian displays) methods. While murine hybridomas and phage display technologies initially dominated the antibody discovery space, recent advances in yeast display have provided powerful advantages for antibody discovery and engineering when used in combination with fluorescence-activated cell sorting (FACS) (for recent reviews, see [70,71]). A number of approaches have been developed to enable yeast surface display, including a report that described thirteen new GPImodified cell wall proteins in P. pastoris that may be sufficient to display heterologous proteins on the cell surface [72]. In a recent and novel approach, a surfaceanchored ‘bait’ Fc-Sed1p fusion was co-expressed in a glycoengineered P. pastoris strain capable of both surface display and full-length antibody secretion [73]. This approach provides a single system for linking genotype with phenotype while evaluating function/ developability of a Fab in the context of a full-length antibody. Current Opinion in Biotechnology 2014, 30:120–127

In other recent studies, yeast display was utilized to find unique antibody sequences that demonstrated cross-reactive binding to a specific epitope on a target protein using competitive panning [74] and to membrane proteins using detergent-solubilized cell lysates containing antigens with near-native conformations [75,76]. A key determinant to the developability of an antibody is the lack of off-target binding. As such, broad panel tissue distribution experiments are often performed following clone identification, but a recent study detailed a FACSbased high-throughput selection tool using soluble membrane proteins coupled with yeast display to counter select against polyspecific antibodies early in the lead identification process [77]. The importance of yeast surface display in discovery continues to grow, as yeast systems are now capable of covering a diverse human library, display on the cell surface for phenotype–genotype linkages, isolate optimized clones while counter selecting undesirable qualities, and secrete fully human antibodies for lead identification efforts (for an illustration, see Figure 2).

Conclusions Recent innovations in yeast have unlocked new applications for recombinant protein production. Yeast systems are established end-to-end platforms for the discovery, development, and manufacture of biopharmaceuticals. Notable improvements include glycoengineering platforms, display technologies and libraries, and robust scalable manufacturing to support the use of yeast in all aspects of research and development. And as such, the next decade of biopharmaceutical development proves to be fruitful for the yeast platform in the supply of lifealtering medicines around the world.

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reticulum or the Golgi apparatus of Pichia pastoris. Yeast 2011, 28:237-252. 39. Davidson RC, Nett JH, Renfer E, Li H, Stadheim TA, Miller BJ, Miele RG, Hamilton SR, Choi BK, Mitchell TI et al.: Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris. Glycobiology 2004, 14:399-407. 40. Nett JH, Gomathinayagam S, Hamilton SR, Gong B, Davidson RC, Du M, Hopkins D, Mitchell T, Mallem MR, Nylen A et al.: Optimization of erythropoietin production with controlled glycosylationPEGylated erythropoietin produced in glycoengineered Pichia pastoris. J Biotechnol 2012, 157:198-206. 41. Zhang N, Liu L, Dumitru CD, Cummings NR, Cukan M, Jiang Y, Li Y, Li F, Mitchell T, Mallem MR et al.: Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study. MAbs 2011, 3:289-298. 42. Arico C, Bonnet C, Javaud C: N-glycosylation humanization for production of therapeutic recombinant glycoproteins in Saccharomyces cerevisiae. Methods Mol Biol 2013, 988:45-57. 43. Parsaie Nasab F, Aebi M, Bernhard G, Frey AD: A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae. Appl Environ Microbiol 2013, 79:997-1007. 44. Wang H, Song HL, Wang Q, Qiu BS: Expression of glycoproteins  bearing complex human-like glycans with galactose terminal in Hansenula polymorpha. World J Microbiol Biotechnol 2013, 29:447-458. The authors describe for the first time the production of glycoproteins in H. polymorpha with N-glycans containing terminal galactose. Through the use of ALG3 and ALG11 mutations, with heterologous mammalian glycosylation genes, strains were fermented at a 3 L research scale with predominant complex glycans. 45. Cheon SA, Kim H, Oh DB, Kwon O, Kang HA: Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans. J Microbiol 2012, 50:341-348. 46. Oh DB, Park JS, Kim MW, Cheon SA, Kim EJ, Moon HY, Kwon O, Rhee SK, Kang HA: Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly. Biotechnol J 2008, 3:659-668. 47. Liu B, Gong X, Chang S, Yang Y, Song M, Duan D, Wang L, Ma Q, Wu J: Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis. J Biotechnol 2009, 143:95-102. 48. De Pourcq K, Tiels P, Van Hecke A, Geysens S, Vervecken W, Callewaert N: Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core. PLoS ONE 2012, 7:e39976. 49. De Pourcq K, Vervecken W, Dewerte I, Valevska A, Van Hecke A, Callewaert N: Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man(8)GlcNAc(2) and Man(5)GlcNAc(2). Microb Cell Fact 2012, 11:53. 50. Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K, Contreras R: In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol 2004, 70:2639-2646. 51. Jacobs PP, Inan M, Festjens N, Haustraete J, Van Hecke A, Contreras R, Meagher MM, Callewaert N: Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate. Microb Cell Fact 2010, 9:93. 52. Choi BK, Warburton S, Lin H, Patel R, Boldogh I, Meehl M,  d’Anjou M, Pon L, Stadheim TA, Sethuraman N: Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris. Appl Microbiol Biotechnol 2012, 95:671-682. As expression hosts often have varying levels of N-glycan occupancy, the authors describe the use of LmStt3d in glycoengineered Pichia to significantly improve the occupancy of recombinant antibodies to greater than 99%. Current Opinion in Biotechnology 2014, 30:120–127

53. Nett JH, Cook WJ, Chen MT, Davidson RC, Bobrowicz P, Kett W, Brevnova E, Potgieter TI, Mellon MT, Prinz B et al.: Characterization of the Pichia pastoris protein-Omannosyltransferase gene family. PLoS ONE 2013, 8:e68325. 54. Loibl M, Strahl S: Protein O-mannosylation: what we have learned from baker’s yeast. Biochim Biophys Acta 2013, 1833:2438-2446. 55. Cukan MC, Hopkins D, Burnina I, Button M, Giaccone E, Houston Cummings NR, Jiang Y, Li F, Mallem M, Mitchell T et al.: Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris. J Immunol Methods 2012, 386:34-42. This paper evaluates the binding of N-glycoforms and O-glycoforms on recombinant proteins to DC-SIGN, an important human lectin involved in antigen presentation and immune stimulation. The authors demonstrate mannotriose and wild-type yeast N-glycans, as well as O-glycans with a chain length of at least two mannose sugars, are able to significantly interact with DC-SIGN whereas other glycans do not. 56. Ahlen G, Strindelius L, Johansson T, Nilsson A, Chatzissavidou N,  Sjoblom M, Rova U, Holgersson J: Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses. PLoS ONE 2012, 7:e46959. Through the use of a highly O-glycosylated PSGL-1/mIgG2b fusion protein expressed in P. pastoris, the authors demonstrate robust APCtargeting to enhance both humoral and cellular immune responses of vaccines. 57. Argyros R, Nelson S, Kull A, Chen MT, Stadheim TA, Jiang B: A phenylalanine to serine substitution within an O-protein mannosyltransferase led to strong resistance to PMTinhibitors in Pichia pastoris. PLoS ONE 2013, 8:e62229. 58. Orchard MG, Neuss JC, Galley CM, Carr A, Porter DW, Smith P, Scopes DI, Haydon D, Vousden K, Stubberfield CR et al.: Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1). Bioorg Med Chem Lett 2004, 14:3975-3978. 59. Kuroda K, Kobayashi K, Kitagawa Y, Nakagawa T, Tsumura H, Komeda T, Shinmi D, Mori E, Motoki K, Fuju K et al.: Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta. Appl Environ Microbiol 2008, 74: 446-453. 60. Amano K, Chiba Y, Kasahara Y, Kato Y, Kaneko MK, Kuno A, Ito H, Kobayashi K, Hirabayashi J, Jigami Y et al.: Engineering of mucin-type human glycoproteins in yeast cells. Proc Natl Acad Sci U S A 2008, 105:3232-3237. 61. Chigira Y, Oka T, Okajima T, Jigami Y: Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains. Glycobiology 2008, 18: 303-314. 62. Suga M, Hatakeyama T: High-efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr Genet 2003, 43:206-211. 63. Cregg JM: DNA-mediated transformation. Methods Mol Biol 2007, 389:27-42. 64. Wu S, Letchworth GJ: High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 2004, 36:152-154. 65. Aw R, Polizzi KM: Can too many copies spoil the broth? Microb Cell Fact 2013, 12:128. 66. Baek DS, Kim YS: Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating. J Microbiol Biotechnol 2014. (Epub ahead of print). 67. Blaise L, Wehnert A, Steukers MP, van den Beucken T, Hoogenboom HR, Hufton SE: Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene 2004, 342:211-218. 68. Chen MT, Lin S, Shandil I, Andrews D, Stadheim TA, Choi BK: Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production. Microb Cell Fact 2012, 11:91. www.sciencedirect.com

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69. Ioannidis JP, Karassa FB, Druyts E, Thorlund K, Mills EJ: Biologic agents in rheumatology: unmet issues after 200 trials and $200 billion sales. Nat Rev Rheumatol 2013, 9:665-673. 70. Doerner A, Rhiel L, Zielonka S, Kolmar H: Therapeutic antibody  engineering by high efficiency cell screening. FEBS Lett 2014, 588:278-287. In this recent review, the authors focus on antibody engineering using surface display and related high efficiency cell screening methods. 71. Gera N, Hussain M, Rao BM: Protein selection using yeast surface display. Methods 2013, 60:15-26. 72. Zhang L, Liang S, Zhou X, Jin Z, Jiang F, Han S, Zheng S, Lin Y: Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface. Appl Environ Microbiol 2013, 79:5519-5526. 73. Shaheen HH, Prinz B, Chen MT, Pavoor T, Lin S, Houston Cummings NR, Moore R, Stadheim TA, Zha D: A dual-mode surface display system for the maturation and production of monoclonal antibodies in glyco-engineered Pichia pastoris. PLoS ONE 2013, 8:e70190. In an innovative strategy for yeast cell surface display, the authors describe a method using a surface-anchored ‘bait’ Fc that enables simultaneous surface display and secretion of full-length antibodies in the same glycoengineered yeast cell. This method provides a tool for the discovery of optimized mAb sequences while shortening the cycle time needed to produce full-length mAb material for in vitro and in vivo studies. 74. Puri V, Streaker E, Prabakaran P, Zhu Z, Dimitrov DS: Highly efficient selection of epitope specific antibody through

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competitive yeast display library sorting. MAbs 2013, 5: 533-539. 75. Tillotson BJ, Cho YK, Shusta EV: Cells and cell lysates: a direct  approach for engineering antibodies against membrane proteins using yeast surface display. Methods 2013, 60:27-37. The authors describe a direct approach to identify and optimize antibody leads against membrane proteins through the use of yeast cell surface display with solubilized whole-cell lysates of mammalian cells containing membrane-bound antigen. 76. Tillotson BJ, de Larrinoa IF, Skinner CA, Klavas DM, Shusta EV: Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources. Protein Eng Des Sel 2013, 26:101-112. 77. Xu Y, Roach W, Sun T, Jain T, Prinz B, Yu TY, Torrey J, Thomas J,  Bobrowicz P, Vasquez M et al.: Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool. Protein Eng Des Sel 2013, 26:663-670. To address polyspecificity of antibodies derived from yeast-based libraries, the authors report a flow cytometric assay to counter select antibody sequences that interact with non-specific membrane preparations. In this manner, the authors present a protocol that is added onto the existing Adimab workflow to interrogate antibody developability using FACS-based screening. 78. Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM: GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 2008, 7:1650-1659.

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