Abstract
Although improvements in antibody expression by mammalian cells are nearing maturation, efforts to improve antibody efficacy through glycoengineering are rapidly expanding. For example, the production of full length monoclonal antibodies with uniform human N-linked glycans by glycoengineered yeast has been used to optimize antibody effector function. The glycoengineered yeast expression platform not only enables elucidation of structure function relationships but also offers a robust and economically viable alternative to mammalian cell expression. This chapter provides an overview of glycobiology, engineering of P. pastoris to secrete recombinant proteins with uniform human N-linked glycans as well as bioprocess considerations in the production of full length monoclonal antibodies by a yeast based expression system.
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References
Anspach, F. B. C., R. Hartmann, et al. (1999). “Expanded-bed chromatography in primary protein purification.” J Chromatogr 865:129–144.
Azevedo, A. M., P. A. J. Rosa, et al. (2009). “Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing.” Trends Biotechnol 27:240.
Bailey, J., D. Ollis (1986). Biochemical Engineering Fundamentals, New York, NY, McGraw-Hill.
Barnard, G. C., A. R. Kull, et al. (2010). “High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.” J Ind Microbiol Biotechnol 37:961–971.
Barton, N. W., R. O. Brady, et al. (1991). “Replacement therapy for inherited enzyme defficiency – macrophage targetted glucocerebrosidase for Gaucher’s disease.” N Eng J Med 324(May 23, 1991):1464.
Beck, A., O. Cochet, et al. (2010). “GlycoFi’s technology to control the glycosylation of recombinant therapeutic proteins.” Expert Opin Drug Discov 5(1):95–111.
Beck, A., T. Wurch, et al. (2008). “Therapeutic antibodies and derivatives: from the bench to the clinic.” Curr Pharm Biotechnol 9(6):421–422.
Bobrowicz, P., R. C. Davidson, et al. (2004). “Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose.” Glycobiology 14(9):757–766.
Boettner, M., C. Lang (2004). “High-throughput expression in microplate format in Pichia pastoris.” Methods Mol Biol 267:277–286.
Boettner, M., B. Prinz, et al. (2002). “Highthroughput screening for expression of heterologous proteins in the yeast Pichia pastoris.” J Biotechnol 99:51–62.
Brankamp, R. G., K. Sreekrishna, et al. (1995). “Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris.” Protein Expr Purif 6(6):813–820.
Brief, B. (2010). “Deal watch: BMS acquires rights for IL-6 inhibitor.” Nat Rev Drug Discov 9:10.
Browne, S. M., M. Al-Rubeai (2007). “Selection methods for high producing cell lines.” Trends Biotechnol 25:425–432.
Ceaglio, N., et al. (2008). “Novel long-lasting interferon alpha derivatives designed by glycoengineering.” Biochimie 90:437–449.
Choi, B. K., P. Bobrowicz, et al. (2003). “Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris.” Proc Natl Acad Sci USA 100(9):5022–5027.
Cooney, C. L., D. I. C. Wang, et al. (1968). “Measurement of heat evolution and correlation with oxygen consumption during microbial growth.” Biotechnol Bioeng 11:269–281.
Cox, K. M., J. D. Sterling, et al. (2006). “Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.“ Nat Biotechnol 24(12):1591–1597.
Cunha, A. E., J. J. Clemente, R. Gomes, F. Pinto, M. Thomaz, S. Miranda, R. Pinto, D. Moosmayer, P. Donner, M. J. Carrondo (2004). “Methanol induction optimization for scFv antibody fragment production in Pichia pastoris.” Biotechnol Bioeng 86(4):458–467.
Damasceno, L. M., K. A. Anderson, et al. (2007). “Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris.” Appl Microbiol Biotechnol 74(2):381–389.
Durocher, Y., M. Butler (2009). “Expression systems for therapeutic glycoprotein production.” Curr Opin Biotechnol 20:700–707.
Endo, T. (2004). “Structure, function and pathology of O-mannosyl glycans.” Glycoconj J 21:3–7.
Fahrner, R. L., H. Iyer, et al. (1999). “The optimal flow rate and column length for maximum production rate of protein A affinity chromatography.“ Bioproc Eng 21(4):287–292.
Fahrner, R. L., H. L. Knudsen, et al. (2001). “Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes.” Biotechnol Gen Eng Rev 18:301–327.
Feldman, M. F., M. Wacker, et al. (2005). “Engineered N-linked protein glycosylation with diverse O antigen liposaccharide structures in Eschercichia coli.” Proc Natl Acad Sci USA 102(8):3016–3021.
Gagnon, P. (1995). Purification Tools for Monoclonal Antibodies. Validated Biosystems, Vol. 33. Tucson, AZ, Omstead, Biopharm. Int.
Gasser, B., M. Maurer, et al. (2006). “Engineering of Pichia pastoris for improved production of antibody fragments.” Biotechnol Bioeng 92(2):353–361.
Goto, M. (2007). “Protein O-glycosylation in fungi: diverse structures and multiple functions.” Biosci Biotechnol Biochem 71:1415–1427.
Hamilton, S. R., P. Bobrowicz, et al. (2003). “Production of complex human glycoproteins in yeast.” Science 301(5637):1244–1246.
Hamilton, S. R., R. C. Davidson, et al. (2006). “Humanization of yeast to produce complex terminally sialylated glycoproteins.” Science 313(5792):1441–1443.
Harty, C., S. Strahl, et al. (2001). “O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation.” Mol Biol Cell 12:1093–1101.
Hensing, M. C. M., R. J. Rouwenhorst, et al. (1995). “Physiological and technological aspects of large scale heterologous protein production with yeast.” Anton Leeuw Int J G 67(3):261–279.
Hjorth, R. (1997). “Expanded-bed adsorption in industrial bioprocessing: recent developments.” Trends Biotechnol 15:230–235.
Holz, C., O. Hesse, et al. (2002). “A microscale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae.” Protein Expr Purif 25:372–378.
Hossler, P., S. F. Khattak, et al. (2009). “Optimal and consistent protein glycosylation in mammalian cell culture.” Glycobiology 19(9):936–949.
Huether, C. M., O. Lienhart, et al. (2005). “Glyco-engineering of moss lacking plant-specific sugar residues.” Plant Biology 7(3):292–299.
Ihssen, J., M. Kowarik, et al. (2010). “Production of glycoprotein vaccines in Escherichia coli.” Microb Cell Fact 9:61.
Jahic, M., J. Knoblechner, et al. (2006). “Interfacing Pichia pastoris cultivation with expanded bed adsorption.” Biotechnol Bioeng 93:1040–1049.
Jeffries, R. (2005). “Glycosylation of recombinant antibody therapeutics.” Biotechnol Prog 21:11–16.
Jeffries, R. (2007). “Antibody therapeutics: isotype and glycoform selection.” Expert Opin Biol Ther 7:1401–1413.
Jenzch, M., M. Lange, et al. (2004). “Bioreactor retrofitting to avoid aeration with oxygen in Pichia pastoris cultivation processes for recombinant protein production.” Chem Eng Res Des 82(A9):1144–1152.
Jones, D., N. Kroos, et al. (2003). “High level expression of recombinant IgG in human cell line PER.C6.” Biotechnol Prog 19:163–168.
Jungo, C., J. Schenk, et al. (2007). “A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris.” J Biotechnol 131(1):57–66.
Kelley, B. (2007). “Very large-scale monoclonal antibody purification – the case for conventional unit operations.” Biotechnol Prog 23:995–1008.
Kelley, B. (2009). “Industrialization of mAb production technology – the bioprocessing industry at a crossroads.” MAbs 1:1–10.
Kobayashi, K., S. Kuwae, T. Ohya, T. Ohda, M. Ohyama, K. Tomomitsu (2000). “High level secretion of recombinant human serum albumin by fedbatch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategy.” J Biosci Bioeng 90(3):280–288.
Kozlowski, S., P. Swann (2006). “Current and future issues in the manufacturing and development of monoclonal antibodies.” Adv Drug Deliv Rev 58:707–722.
Kula, M. R. (1990). “Trends and future prospects of aqueous two-phase extraction.” Bioseparation 1:181–189.
Kuroda, K., K. Kobayashi, et al. (2008). “Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta.” Appl Environ Microbiol 74(2):446–453.
Li, H., N. Sethuraman, et al. (2006). “Optimization of humanized IgGs in glycoengineered Pichia pastoris.” Nat Biotechnol 24(2):210.
Li, H., M. d’Anjou (2009). “Pharmacological significance of glycosylation in therapeutic proteins.” Curr Opin Biotechnol 20:678–684.
Linden, T., D. Roush, et al. (2010). Optimization and Scale-up of Downstream Processing of Biopharmaceutical Proteins Produced bv Glycoengineered Pichia Pastoris. Recovery of Biological Products XIV, Lake Tahoe, California, USA.
Liu, C., W. Downey (2009). “Contract manufacturing demands remain strong.” GEN 29:53–59.
Low, D., R. O’Learly, et al. (2007). “Future of antibody purification.” J Chromatogr B 848(1):48–63.
Malhotra, R., M. R. Wormald, et al. (1995). “Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose binding protein.” Nat Med 1:237–243.
Nakatsukasa, K., S. Okada, et al. (2004). “Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast.” J Biol Chem 279:49762–49772.
Natsume, A., R. Niwa, et al. (2009). “Improving effector functions of antibodies for cancer treatment: enhancing ADCC and CDC.” Drug Des Devel Ther 3:7–16.
Nimmerjahn, F., J. V. Ravetch (2008). “Fc gamma receptors as regulators of immune responses.” Nat Rev Immunol 8:34–47.
Petricciani, J., R. Sheets (2008). “An overview of animal cell substrates for biological products.” Biologicals 36:359–362.
Plantz, B. A., J. Andersen, et al. (2003). “Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths.” J Ind Microbiol Biotechnol 30(11):643–650.
Potgieter, T. I., M. Cukan, et al. (2009). “Production of monoclonal antibodies by glycoengineered Pichia pastoris.” J Biotechnol 139(4):318–325.
Potgieter, T. I., S. D. Kersey, et al. (2010). “Antibody Expression Kinetics in Glycoengineered Pichia Pastoris.” Biotechnol Bioeng 106(6):918.
Raju, T. S. (2008). “Terminal Sugar of Fc glycans influence antibody effector functions of IgGs.” Curr Opin Immunol 20:471–478.
Reitinger, S., T. Boroviak, et al. (2008). “High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris.” Protein Expr Purif 57(2):226–233.
Rito-Palomares, M. (2004). “Practical application of aqueous two-phase partition to process development for the recovery of biological products.” J Chromatogr B 807(1):3–11.
Rosa, P. A. J., A. M. Azevedo, et al. (2007). “Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies.” J Chromatogr A 1141(2):50–60.
Schenerman, M. A., J. N. Hope, et al. (1999). “Comparability testing of a humanized monoclonal antibody (synagis) to support cell line stability process validation and scale-up for manufacturing.” Biologicals 27:203–215.
Schenk, J., K. Balazs, C. Jungo, J. Urfer, C. Wegmann, A. Zocchi, I. W. Marison, U. von Stockar (2008). “Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris mut+ strain.” Biotechnol Bioeng 99(2):368–377.
Schilling, B. M., J. C. Goodrick, et al. (2001). “Scale-up of a high cell density continuous culture with Pichia pastoris X33 for the constitutive expression of rh-Chitinase.” Biotechnol Prog 17:629–633.
Shepard, S. R. B., A. Gregory, et al. (2001). “Routine manufacture of recombinant proteins using expanded bed adsorption chromatography.” Bioseparation 10:51–56.
Shepard, S. R., B. Robert, et al. (2000). “Large scale purification of recombinant human angiostatin.” Protein Expression Purif 20:216–227.
Sheridan, C. (2010). “Fresh from the biologic pipeline – 2009.” Nat Biotechnol 28(4):307–310.
Shukla, A. (2007). “Downstream processing of monoclonal antibodies -application of platform approaches.” J Chromatogr B 848:28–39.
Shukla, A., J. Kandula (2008). “Harvest and recovery of monoclonal antibodies from large-scale mammalian cell culture.” Biopharm Int 7:34–45.
Spiro, R. G. (2002). “Protein glycosylation: nature, distribution, enzymatic formation and disease implications of glycopeptide bonds.” Glycobiology 12(4):43R–56R.
Srinivas, N. D., A. V. Narayan, et al. (2002). “Mass transfer in a spray column during two-phase extraction of horseradish peroxidase.” Process Biochem 38(3):387–391.
Thoemmes, J., M. Halfar, et al. (2001). “Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2.” Biotechnol Prog 17:503–512.
Trinh, L. N., B. Santosh, et al. (2000). “Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption.” Bioseparation 9:223–230.
Trousdale, R. K., et al. (2009). “Efficacy of native and hyperglycosylated follicle-stimulating hormone analogs for promoting fertility in female mice.” Fertil Steril 91:265–270.
Umana, P., J. Jean-Mairet, et al. (1999). “Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody dependent cellular cytotoxicity activity.” Nat Biotechnol 17(2):176–180.
Veide, A., L. Torgny, et al. (1984). “Continuous extraction of â-D-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer.” Enzyme Microb Technol 6:325–330.
Walsh, G. (2010). “Biopharmaceutical benchmarks 2010.” Nat Biotechnol 28(9):917–924.
Wang, A., R. Lewus, et al. (2006). “Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth.” Biotechnol Bioeng 94:91–104.
Weis, R., R. Luiten, et al. (2004). “Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.” FEMS Yeast Res 5:179–189.
Wildt, S., T. U. Gerngross (2005). “The humanization of N-glycosylation pathways in yeast.” Nat Rev Microbiol 3(2):119–128.
Willer, T., M. C. Valero, et al. (2003). “O-mannosyl glycans: from yeast to novel associations with human disease.” Curr Opin Struct Biol 13:621–630.
Wright, A., S. L. Morrison (1997). “Effect of glycosylation on antibody function: implications for genetic engineering.” Trends Biotechnol 15:26–32.
Yigzaw, Y. (2006). “Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification.” Biotechnol Prog 22:288–296.
Zhang, W. H., M. A. Bevins, B. A. Plantz, L. A. Smith, M. M. Meagher (2000b). “Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A.” Biotechnol Bioeng 70(1):1–8.
Zhu, Y., et al. (2005). “Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the cation-independent mannose 6 phosphate receptor demonstrates improved delivery of muscles of Pompe mice.” Biochem J 389:619–628.
Zhu, Y., et al. (2008). “Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.” Mol Ther 17:954–963.
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Zha, D., Linden, T., Potgieter, T. (2011). Production of Monoclonal Antibodies in Glycoengineered Pichia pastoris . In: Al-Rubeai, M. (eds) Antibody Expression and Production. Cell Engineering, vol 7. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-1257-7_4
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