Applications of a novel cho glycosylation mutant

APPLICATIONS OF A NOVEL CHO GLYCOSYLATION MUTANT by JOHN GOH SOO YANG (B. Eng. (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Department of Biochemistry NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION “I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously.” …………………. John Goh Soo Yang 22 Jan 2014 ii ACKNOWLEDGMENTS I am very grateful to Prof. Miranda Yap for giving me the opportunity to pursue a PhD under the A*STAR Scientific Staff Development Award. I would also like to express my deepest thanks to Dr. Song Zhiwei for his constant patience and guidance throughout the course of the PhD journey. Without Dr. Song's very capable mentorship, this thesis would not have been possible. Secondly, I would like to thank my collaborators from the Bioprocessing Technology Institute: Mr. Chan Kah Fai, Dr. Zhang Peiqing, Dr. Lee May May, Dr. Muriel Bardor and the Analytics group. I am also thankful to my collaborators from overseas, Prof. Zhang Yuanxing and Mr. Liu Yingwei from the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China and the Shandong E Hua Biopharmaceutical Co., Ltd for graciously allowing the use of their bioreactor facility. I would also like to thank Ms. Tan Xueyu for her technical assistance. Further, I am grateful to all my friends for their friendship, prayers and encouragement in my PhD journey. Finally, I would like to dedicate this thesis to my dear parents, who have always encouraged me in all my endeavors and for their unfailing love and support. iii TABLE OF CONTENTS DECLARATION . ii ACKNOWLEDGMENTS . iii TABLE OF CONTENTS .iv SUMMARY viii LIST OF PUBLICATIONS x LIST OF TABLES xi LIST OF FIGURES xii CHAPTER INTRODUCTION . 1.1 Background . 1.2 Thesis objectives . 1.3 Thesis organization . CHAPTER LITERATURE REVIEW 2.1 Glycosylation 2.2 The impact of glycosylation on recombinant production of therapeutic glycoproteins . 2.3 Glycosylation of recombinant proteins in different industrial host cell lines . 2.3.1 Bacteria hosts . 2.3.2 Yeast cells 2.3.3 Insect cells 11 2.3.4 Mammalian cells 12 2.4 Strategies for improving sialylation in CHO cells 14 2.5 N-acetylglucosminyltransferase I (GnT I) and glycosylation mutants with defective GnT I . 17 2.6 Erythropoietin . 20 CHAPTER MATERIALS AND METHODS 22 3.1 Cell culture 22 3.2 Isolation of RCA-I resistant clones . 22 3.3 Expression constructs 23 iv 3.4 Transient expression of recombinant EPO in CHO cells 23 3.5 RNA extraction and cDNA synthesis . 24 3.6.1 Polymerase chain reaction to amplify wild-type and mutant GnT I coding sequence 24 3.7 Sequencing 25 3.8 SDS-PAGE and western blotting 26 3.9 Endonuclease H, neuraminidase and PnGASE F treatment of cell supernatant samples 27 3.10 Isoelectric focusing and immunoblotting 27 3.11 Expression and purification of EPO-Fc fusion protein . 28 3.12 High pH anion exchange chromatography pulsed amperometric detection (HPAEC-PAD) . 29 3.13 Total sialic acid quantification of EPO-Fc 30 3.14 Construction of zinc-finger nuclease expression plasmid targeting dihydrofolate reductase . 31 3.15 Sorting of zinc-finger nuclease transfected cells 31 3.16 Genomic extraction and PCR amplification . 32 3.17 Western blot of cell lysate to confirm the absence of DHFR . 33 3.17 Transfection, selection and amplification of stably transfected cells with pEGD 33 3.19 Coomassie blue staining of IEF gel 34 3.20 Perfusion culture of CHO-gmt4D-GnT I EPO cells . 34 3.21 Purification of EPO from perfusion culture 35 3.22 Total sialic quantification of purified EPO samples . 36 3.23 Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry . 37 CHAPTER CHARACTERIZATION OF CHO GLYCOSYLATION MUTANTS ISOLATED FROM RCA-I 38 4.1 Overview 38 4.2 Results . 39 4.2.1 Lectin kill curve for CHO-wild type cells . 39 4.2.2 Isolation of RCA-I CHO clones . 40 4.2.3 SDS-PAGE analysis of transiently expressed EPO in JW152 . 41 v 4.2.4 Complementation of EPO expression with co-transfection of glycosylation genes in JW152 . 41 4.2.5 Cloning and sequencing of GnT I open reading frame in glycosylation mutants . 46 4.2.6 CHO-K1 and JW152 EPO-Fc glycan mass spectrometry analyses . 53 CHAPTER HIGHLY SIALYLATED ERYTHROPOIETIN EXPRESSION IN CHO GLYCOSYLATION MUTANTS . 60 5.1 Overview . 60 5.2 Results . 60 5.2.1 Superior sialylation is not due to the overexpression of GnT I . 60 5.2.2 IEF blot of transiently expressed EPO-Fc with and without GnTI function restoration 63 5.2.3 Total sialic acid assay in purified transient EPO . 64 5.2.4 HPAEC profiling of EPO-Fc . 65 5.2.5 Stable expression of EPO in JW152 cells with restored GnT I function also show superior sialylation 66 5.3 Discussion . 68 CHAPTER UTILIZING ZINC-FINGER NUCLEASE TO KNOCK OUT DHFR IN CHO-GMT4 CELLS 71 6.1 Overview . 71 6.2 Results . 71 6.2.1 Knocking out DHFR in JW152 cells (CHO-gmt4) 71 6.2.2 Characterization of DHFR knock out cell line 73 6.3 Discussion . 76 CHAPTER AMPLIFICATION OF ERYTHROPOIETIN EXPRESSION IN CHO GLYCOSYLATION MUTANT CELLS . 78 7.1 Overview . 78 7.2 Results . 78 7.2.1 Stable cell line and amplification . 79 7.2.2 Gene amplification with methotrexate in EPO-producing CHO-gmt4D lines 79 7.2.3 Perfusion bioreactor . 81 7.2.4 IEF comparison of batch supernatant 84 vi 7.2.5 EPO purification 84 7.2.6 IEF comparison of purified EPO . 86 7.2.7 Total sialic acid quantification . 86 7.2.8 MALDI-TOF structural glycan analysis 89 7.3 Discussion . 92 CHAPTER CONCLUSIONS AND RECOMMENDATIONS . 95 8.1 Conclusions . 95 8.2 Recommendations for future research 98 8.2.1 Expression of other recombinant therapeutics which require good sialylation . 98 8.2.2 Expression of monoclonal antibodies with no fucosylation 99 8.2.3 Expression of glucocerebrosidase for therapeutic protein production . 99 8.2.4 Investigation into mechanism behind better sialylation of CHO-gmt4 in presence of restored GnT I function 100 ABBREVIATIONS 102 BIBLIOGRAPHY 104 vii SUMMARY Glycosylation plays an important role in biology but its impact on the quality and biologics manufacturing is just beginning to be appreciated. Studies have shown that the sialylation of glycoproteins result in better circulatory half-life and therefore higher efficacy. In the production of erythropoietin (EPO), almost 80% that is produced is discarded due to insufficient sialylation. The research documented in this thesis centers upon the improvement of sialylation of EPO in a glycosylation mutant isolated through lectin selection. It is proposed that this cell line would result in improving the way EPO and possibly other recombinant therapeutic glycoproteins are produced. In this research, the characterization of glycosylation mutants that survived lectin selection using Ricinus communis agglutinin-I (RCA-I), it was demonstrated that the lectin only selected CHO mutants that were deficient in Nacetylglucosaminyltransferase I (GnT I). The mutations found in these glycosylation mutants shed more light on the structure and function of the glycosylation enzyme. One of these cell lines was named CHO-gmt4. Interestingly, the restoration of functional GnT I in CHO-gmt4 cells seemed to enable the expression of EPO that was better sialylated than the wild-type CHO-K1 cells. This was observed in all the glycosylation mutants isolated from RCA-I selection. The overexpression of GnT I in CHO-K1 cells did not result in the same improvement in sialylation. Further, the sialylation of transiently expressed EPO-Fc fusion protein was also shown to be better when co-expressed with GnT I using CHO-gmt4 cells. viii Transiently expressed EPO-Fc in CHO-gmt4 cells with functional GnT I restored contained 23% more sialic acid than CHO-K1 expressed EPO-Fc as quantified by the thiobarbituric acid assay. HPAEC-PAD analysis also showed that the improvement in sialylation over wild-type expressed recombinant EPO-FC was due to increased proportion of tri- and tetra-antennary sialylated structures. The improvement in sialylation was also observed in the stable co-expression of EPO and GnT I. In order to generate an EPO- producing cell line with a better titer, the CHO-gmt4 cell line was then gene-edited using zinc-finger nuclease to knock out dihydrofolate reductase gene to enable subsequent gene amplification. The successful knock out of DHFR generated a new cell line, CHO-gmt4D. CHO-gmt4D was stably transfected with both EPO and GnT I and after several rounds of methotrexate amplification, a series of clones that produced EPO with superior sialylation was generated. One of these clones, named CHO-gmt4D-EPO-GnT I was cultured in an industrial bioprocess with perfusion-culture based bioreactor and the resulting EPO was purified. The bioreactor studies showed that the superior sialylation of EPO was maintained Using HPAEC-PAD, sialic acid quantification and MALDI-TOF analyses, the purified EPO was shown to contain better sialylated EPO than the existing industrial clone that was used for regular EPO production in that bioprocess. These results demonstrate that the CHO-gmt4 cell line can be applied in the production of recombinant EPO with more superior sialylation, thus paving the way to a more efficient way of producing recombinant therapeutic glycoproteins. ix LIST OF PUBLICATIONS Goh, J. S., Zhang, P., Chan, K.F., Lee, M. M., Lim, S.F., Song, Z. (2010) RCA-Iresistant CHO mutant cells have dysfunctional GnT I and expression of normal GnT I in these mutants enhances sialylation of recombinant erythropoietin, Metab. Eng. 12(4), 360-368 Goh, J.S.*, Liu, Y.*, Liu, H., Chan, K.F., Wan, C., Teo, G., Zhou, X., Xie, F., Zhang, P., Zhang, Y., Song, Z. (2013) Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt-4 (JW152) with restored GnT I function, Biotech. J. 9(1), 100-109 *equal contributions were made by these authors to the publication. x contain two more glycosylation sites. Whilst the individual glycan structures have not been published, it would be possible that the expression of this glycoengineered protein might need a cell line that can sialylate it better. 8.2.2 Expression of monoclonal antibodies with no fucosylation Glycan analysis of EPO-Fc expressed in CHO-gmt4 cells showed a mix of fucosylated and non-fucosylated glycans with four to five mannose residues capping the glycan. However, it has been shown in two different studies that the glycans present in the IgG1 Fc portion of antibodies produced in similar GnT I glycosylation mutants not contain fucose (Sealover et al., 2013; Zhong et al., 2012). The characterization of cells lines that survive RCA-I selection show that only GnT I mutants that produce these oligomannose structures, hence existing antibodyproduction cell lines may be selected for GnT I mutants that would produce the requisite non-fucosylated antibodies. 8.2.3 Expression of glucocerebrosidase for therapeutic protein production Patients who suffer from Gaucher’s disease lack the functional enzyme, glucocerebrosidase, to break down glycolipids completely. As a result, the peripheral macrophage becomes engorged with the glycolipids that cannot be broken down. Hence the current treatment available for these patients is enzyme replacement therapy, which involves administering glucocerebrosidase to the patients so that it can be taken up by the peripheral macrophages through the cell-surface mannose 99 receptors. In order for glucocerebrosidase to be endocytosed by the macrophage cells, the recombinant therapeutic has to undergo carbohydrate remodeling through sequential treatment with glycosidases to trim back the N-glycan to reveal the trimannosyl-core (Riske et al., 2012). This results in improvements mannose receptor binding, macrophage uptake and intracellular half-life and efficacy (Van Patten et al., 2006) . GnT I mutant cell lines such as CHO-gmt4 are perfectly suited to produce recombinant glycoprotein therapeutics such as glucocerebrosidase. Although the resultant glycan structure is a penta-mannose structure, the take-up rate of glucocerebrosidase with this glycan structure has been shown to not be affected (Van Patten et al., 2006). 8.2.4 Investigation into mechanism behind better sialylation of CHO-gmt4 in presence of restored GnT I function The improvement in sialylation of EPO in CHO-gmt4 after restoring GnT I function still remains unknown. Both Lec1 cells and all the GnT I mutants isolated from RCAI are able to sialylate EPO better although they were derived from different lectin selections. It is thus speculated that the mutant might have certain compensatory mechanisms in place due to its genetic deficiency in GnT I. Besides the inability to catalyze the attachment of GlcNAc, the other aspects of the GnT I mutant cell in terms of expression level of other glycosylation enzymes might differ from CHO-K1 wild-type cells. It would be interesting to use microarrays to determine whether there 100 are any changes in mRNA expression on a global level when the GnT I dysfunction occurs. Further, the improvement in branching of N-glycans resulting in better sialylation leads to the speculation that the compensatory mechanism may lie in an upregulation of GnT IV, GnT V or both. This can be tested by performing quantitative real-time PCR to first test the mRNA expression and to quantify the amount of GnT IV or GnT V enzyme present in the cell-lysate via western blot. Activity assays for GnT IV or GnT V may also be set up and optimized to determine whether the glycosylation enzymes contained in the cell lysates are more highly expressed. 101 ABBREVIATIONS ADCC antibody-dependant cell-mediated cytotoxicity Ala alanine Asn asparagine CH3COOH acetic acid CHO Chinese hamster ovary DEAE diethylaminoethanol DHFR Dihydrofolate reductase DMEM Dulbecco's modified eagle's media Dnase deoxyribonuclease EPO erythropoietin Epo-Fc erythropoietin-IgG1 Fc fusion protein ER endoplasmic reticulum FACS Fluorescence-activated cell sorting FBS fetal bovine serum Fc IgG1 constant region Glc glucose GlcNAc N-acetylglucosamine Gluase glucosidase GnT N-acetylglucosaminyltransferase HPAEC-PAD high pH anionic exchange chromatography-pulsed amperometric detection IEF isoelectric focusing/immunoblot IFN-γ Interferon-gamma IgG1 Immunoglobulin IRES Internal ribosome entry site LacNAc N-acetyllactosamine MALDI-TOF Matrix-assisted laser desorption/ionization-Time of Flight Man mannose Manase mannosidase MTX methotrexate mRNA messenger ribonucleic acid NaOH sodium hydroxide 102 Neu Neuraminidase/sialidase PBS phosphate buffered saline PCR polymerases chain reaction PNGase F Peptide -N-Glycosidase F RCA-I Ricinus communis agglutinin I RNAi ribonucleic acid interference SGC SpsA GnT I core domain siRNA small-interfering ribonucleic acid TBA Thiobarbituric acid assay ZFN zinc-finger nuclease 103 BIBLIOGRAPHY Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., Bailey, J.E., 1999. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180. Altmann, F., Schwihla, H., Staudacher, E., Glossl, J., März, L., 1995. Insect cells contain an unusual, membrane-bound beta-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J. Biol. Chem. 270, 17344–17349. Altmann, F.F., Staudacher, E.E., Wilson, I.B.I., März, L.L., 1999. Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J 16, 109–123. Arnold, D.F., Misbah, S.A., 2008. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 358, 2735–2736. Aumiller, J.J., Mabashi-Asazuma, H., Hillar, A., Shi, X., Jarvis, D.L., 2012. A new glycoengineered insect cell line with an inducibly mammalianized protein Nglycosylation pathway. Glycobiology 22, 417–428. Baenziger, J.U., Fiete, D., 1979. Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J. Biol. Chem. 254, 9795–9799. Bardor, M., Faveeuw, C., Fitchette, A.-C., Gilbert, D., Galas, L., Trottein, F., Faye, L., Lerouge, P., 2003. Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 13, 427–434. Batta, S.K., Rabovsky, M.A., Channing, C.P., Bahl, O.P., 1978. Effect of removal of carbohydrate residues upon the half life and in vivo biological activity of human chorionic gonadotropin. Adv Exp Med Biol 112, 749–756. Bertozzi, C.R., Rabuka, D., 2009. Structural Basis of Glycan Diversity. Essentials of 104 Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 2. Bork, K.K., Reutter, W.W., Weidemann, W.W., Horstkorte, R.R., 2007. Enhanced sialylation of EPO by overexpression of UDP-GlcNAc 2-epimerase/ManAc kinase containing a sialuria mutation in CHO cells. FEBS Letters 581, 4195–4198. Burg, M., Müthing, J., 2001. Characterization of cytosolic sialidase from Chinese hamster ovary cells: Part I: Cloning and expression of soluble sialidase in Escherichia coli. Carbohydr. Res. 330, 335-346 Chen, W., Stanley, P., 2002. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology 13, 43–50. Chen, W., Unligil, U.M., Rini, J.M., Stanley, P., 2001. Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI. Biochemistry 40, 8765–8772. Choi, B.K., Bobrowicz, P., Davidson, R.C., 2003. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris, Proc. Natl. Acad. Sci. U S A. 100, 5022-5027. Chung, C.H., Mirakhur, B., Chan, E., Le, Q.T., 2008. Cetuximab-induced anaphylaxis and IgE specific for galactose-α-1, 3-galactose. N. Engl. J. Med. 358, 1109-1117. Ciucanu, I., Kerek, F., 1984. A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 31, 209–217. Costa, A.R., Rodrigues, M.E., Henriques, M., Oliveira, R., Azeredo, J., 2013. Glycosylation: impact, control and improvement during therapeutic protein production. Crit. Rev. Biotechnol. doi:10.3109/07388551.2013.793649 105 Dell, A., Reason, A.J., Khoo, K.H., Panico, M., McDowell, R.A., Morris, H.R., 1993. Mass spectrometry of carbohydrate-containing biopolymers. Methods Enzymol 230, 108–132. Delorme, E., Lorenzini, T., Giffin, J., Martin, F., 1992. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry. 31, 9871-9876 Durocher, Y., Butler, M., 2009. Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 20, 700–707. Egrie, J.C.J., Browne, J.K.J., 2000. Development and characterization of novel erythropoiesis stimulating protein (NESP). Nephrol. Dial. Transplant. 16 Suppl 3, 3– 13. Ferrari, J., Gunson, J., Lofgren, J., Krummen, L., Warner, T.G., 1998. Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant dnase in batch culture with increased sialic acid. Biotechnol. Bioeng. 60, 589–595. Fukuda, M.N., Sasaki, H., Lopez, L., Fukuda, M., 1989. Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. Blood 73, 84–89. Fukuta, K., Abe, R., Yokomatsu, T., Kono, N., Asanagi, M., Omae, F., Minowa, M.T., Takeuchi, M., Makino, T., 2000a. Remodeling of sugar chain structures of human interferon. Glycobiology 10, 421–430. Fukuta, K., Yokomatsu, T., Abe, R., Asanagi, M., Makino, T., 2000b. Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases. Glycoconj. J. 17, 895–904. Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M., 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj. J. 16, 81-97. 106 Graber, S.E.S., Krantz, S.B.S., 1977. Erythropoietin and the control of red cell production. Medicine (Abingdon) 29, 51–66. Grabowski, G.A., Barton, N.W., Pastores, G., 1995. Enzyme therapy in type Gaucher disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann. Intern. Med. 122, 33-39. Green, E.D., Brodbeck, R.M., Baenziger, J.U., 1987. Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin. Anal. Biochem. 67, 62-75. Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H., Choi, B.-K., 2006. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443. Ho, S.C.L., Bardor, M., Feng, H., Mariati, Tong, Y.W., Song, Z., Yap, M.G., Yang, Y., 2012. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J. Biotechnol. 157, 130–139. Hollister, J., Grabenhorst, E., Nimtz, M., Conradt, H., Jarvis, D.L., 2002. Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans. Biochemistry 41, 15093–15104. Hudgin, R.L.R., Pricer, W.E.W., Ashwell, G.G., Stockert, R.J.R., Morell, A.G.A., 1974. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J. Biol. Chem. 249, 5536–5543. Huggett, B., Lähteenmaki, R., 2012. Public biotech 2011—the numbers. Nat. Biotechnol. 30, 751-757. Hull, E., Sarkar, M., Spruijt, M.P.N., Höppener, J.W.M., Dunn, R., Schachter, H., 1991. Organization and localization to chromosome of the human UDP-N107 acetylglucosamine: α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I gene. Biochem. Biophys. Res. Commun. 176, 608–615. Iida, S.S., Misaka, H.H., Inoue, M.M., Shibata, M.M., Nakano, R.R., Yamane-Ohnuki, N.N., Wakitani, M.M., Yano, K.K., Shitara, K.K., Satoh, M.M., 2006. Nonfucosylated therapeutic IgG1 antibody can evade the inhibitory effect of serum immunoglobulin G on antibody-dependent cellular cytotoxicity through its high binding to FcgammaRIIIa. Clin. Cancer Res. 12, 2879–2887. Iskratsch, T., Braun, A., Paschinger, K., Wilson, I., 2009. Specificity analysis of lectins and antibodies using remodeled glycoproteins. Anal. Biochem. 386(2),133-146. Jenkins, N.N., Curling, E.M.E., 1994. Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb. Technol. 16, 354–364. Jeong, Y.T., Choi, O., Son, Y.D., Park, S.Y., Kim, J.H., 2009. Enhanced sialylation of recombinant erythropoietin in genetically engineered Chinese‐hamster ovary cells. Biotechnol. Appl. Biochem. 52, 283–291. Kanda, Y., Imai-Nishiya, H., Kuni-Kamochi, R., Mori, K., Inoue, M., KitajimaMiyama, K., Okazaki, A., Iida, S., Shitara, K., Satoh, M., 2007. Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating completely non-fucosylated recombinant therapeutics. J. Biotechnol. 130, 300–310. Kumar, R.R., Yang, J.J., Larsen, R.D.R., Stanley, P.P., 1990. Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc Natl Acad Sci U S A 87, 9948–9952. Lam, J.S., Huang, H., Levitz, S.M., 2006. Effect of differential N-linked and O-linked mannosylation on recognition of fungal antigens by dendritic cells. PLoS ONE 2, e1009–e1009. Lasne, F., de Ceaurriz, J., 2000. Recombinant erythropoietin in urine. Nature 405, 108 635–635. Lasne, F., Thioulouse, J., Martin, L., de Ceaurriz, J., 2007. Detection of recombinant human erythropoietin in urine for doping analysis: interpretation of isoelectric profiles by discriminant analysis. Electrophoresis 28, 1875–1881. Lim, S.F., Lee, M.M., Zhang, P., Song, Z., 2008. The Golgi CMP-sialic acid transporter: A new CHO mutant provides functional insights. Glycobiology 18, 851– 860. Lim, Y., Wong, N.S.C., Lee, Y.Y., Ku, S.C.Y., Wong, D.C.F., Yap, M.G.S., 2010. Engineering mammalian cells in bioprocessing - current achievements and future perspectives. Biotechnol Appl Biochem 55, 175–189. Lin, F.K., Suggs, S., Lin, C.H., Browne, J.K., Smalling, R., Egrie, J.C., Chen, K.K., Fox, G.M., Martin, F., Stabinsky, Z., 1985. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 82, 7580–7584. Marchal, I., Jarvis, D.L., Cacan, R., Verbert, A., 2001. Glycoproteins from Insect Cells: Sialylated or Not? Biol Chem. 382(2), 151-159. Markely, L.R.A., Ong, B.T., Hoi, K.M., Teo, G., Lu, M.Y., Wang, D.I.C., 2010. A high-throughput method for quantification of glycoprotein sialylation. Anal. Biochem. 407, 128–133. Misaizu, T.T., Matsuki, S.S., Strickland, T.W.T., Takeuchi, M.M., Kobata, A.A., Takasaki, S.S., 1995. Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin. Blood 86, 4097–4104. Morell, A.G.A., Gregoriadis, G.G., Scheinberg, I.H.I., Hickman, J.J., Ashwell, G.G., 1971. The role of sialic acid in determining the survival of glycoproteins in the circulation. J. Biol. Chem. 246, 1461–1467. Morelle, W., Michalski, J.C., 2007. Analysis of protein glycosylation by mass 109 spectrometry. Nat. Protoc. 2, 1585–1602. Nakanishi-Shindo, Y.Y., Nakayama, K.K., Tanaka, A.A., Toda, Y.Y., Jigami, Y.Y., 1993. Structure of the N-linked oligosaccharides that show the complete loss of alpha1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J. Biol. Chem. 268, 26338–26345. Narhi, L.O., Arakawa, T., Aoki, K.H., Elmore, R., Rohde, M.F., Boone, T., Strickland, T.W., 1991. The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem. 5, 23022-23026. Ngantung, F., Miller, P., Brushett, F., 2006. RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol. Bioeng. 5, 106-119. North, S.J., Huang, H.-H., Sundaram, S., Jang-Lee, J., Etienne, A.T., Trollope, A., Chalabi, S., Dell, A., Stanley, P., Haslam, S.M., 2010. Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity. J. Biol. Chem. 285, 5759–5775. Opat, A.S., Puthalakath, H., Burke, J., Gleeson, P.A., 1998. Genetic defect in Nacetylglucosaminyltransferase I gene of a ricin-resistant baby hamster kidney mutant. Biochem J 336 ( Pt 3), 593–598. Park, S.S., Park, J., Ko, J., Chen, L., Meriage, D., Crouse-Zeineddini, J., Wong, W., Kerwin, B.A., 2009. Biochemical assessment of erythropoietin products from Asia versus US Epoetin alfa manufactured by Amgen. J Pharm Sci 98, 1688–1699. Patnaik, S.K., Stanley, P., 2006. Lectin‐resistant CHO glycosylation mutants. Methods Enzymol. 416, 159-182 Reeves, P.J., Callewaert, N., Contreras, R., Khorana, H.G., 2002. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N- 110 acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. U S A. 99, 13419–13424. Riske, F.F., Hamilton, A.A., Zhang, C.C., Hayes, M.M., 2012. Remodeling the oligosaccharides on β-glucocerebrosidase using hydrophobic interaction chromatography and applications of hydroxyl ethyl starch for improving remodeling and enhancing protein stability. Biotechnol. Bioeng. 109, 1217–1227. Saito, M., Robert, K.Y., 1995. Biochemistry and function of sialidases. Biology of the sialic acid. 261-313. Santiago, Y., Chan, E., Liu, P.-Q., Orlando, S., Zhang, L., Urnov, F.D., Holmes, M.C., Guschin, D., Waite, A., Miller, J.C., Rebar, E.J., Gregory, P.D., Klug, A., Collingwood, T.N., 2008. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105, 5809–5814. Sarkar, M., Pagny, S., Unligil, U., Joziasse, D., Mucha, J., Glossl, J., Schachter, H., 1998. Removal of 106 amino acids from the N-terminus of UDP-GlcNAc: alpha-3-Dmannoside beta-1, 2-N-acetylglucosaminyltransferase I does not inactivate the enzyme. Glycoconj J 15, 193–197. Schriebl, K., Trummer, E., Weik, R., Lattenmayer, C., Müller, D., Kunert, R., Katinger, H., Vorauer-Uhl, K., 2007. Applicability of different fluorescent dyes for isoform quantification on linear IPG gels. Electrophoresis 28, 2100–2107. Sealover, N.R., Davis, A., Brooks, J.K., George, H.J., Kayser, K.J., Lin, N., 2013a. Engineering Chinese Hamster Ovary (CHO) Cells for Producing Recombinant Proteins with Simple Glycoforms by Zinc-Finger Nuclease (ZFN)-Mediated Gene Knockout of Mannosyl (alpha-1, 3-)-glycoprotein beta-1, 2-Nacetylglucosaminyltransferase ( Mgat1). J. Biotechnol. 167, 24-32 Shahrokh, Z., Royle, L., Saldova, R., Bones, J., Abrahams, J.L., Artemenko, N.V., Flatman, S., Davies, M., Baycroft, A., Sehgal, S., Heartlein, M.W., Harvey, D.J., Rudd, P.M., 2011. Erythropoietin produced in a human cell line (Dynepo) has 111 significant differences in glycosylation compared with erythropoietins produced in CHO cell lines. Mol. Pharm. 8, 286–296. Shields, R.L.R., Lai, J.J., Keck, R.R., O'Connell, L.Y.L., Hong, K.K., Meng, Y.G.Y., Weikert, S.H.A.S., Presta, L.G.L., 2002. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733–26740. Sinclair, A.M., Elliott, S., 2005. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J. Pharm. Sci. 94, 1626–1635. Sodetz, J.M., Pizzo, S.V., McKee, P.A., 1977. Relationship of sialic acid to function and in vivo survival of human factor VIII/von Willebrand factor protein. J. Biol. Chem. 252, 5538-5546 Son, Y.D., Jeong, Y.T., Park, S.Y., Kim, J.H., 2011. Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes. Glycobiology. 21, 1019-1028. Stanley, P., Siminovitch, L., 1977. Complementation between mutants of CHO cells resistant to a variety of plant lectins. Somat. Cell. Mol. Genet. 3, 391–405. Takeuchi, M., Inoue, N., Strickland, T.W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S., Kobata, A., 1989. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. U S A 86, 7819–7822. Tomiya, N., Howe, D., Aumiller, J.J., Pathak, M., Park, J., Palter, K.B., Jarvis, D.L., Betenbaugh, M.J., Lee, Y.C., 2002. Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-Nacetylglucosaminyltransferase II. Glycobiology 13, 23–34. Urnov, F.D.F., Miller, J.C.J., Lee, Y.-L.Y., Beausejour, C.M.C., Rock, J.M.J., 112 Augustus, S.S., Jamieson, A.C.A., Porteus, M.H.M., Gregory, P.D.P., Holmes, M.C.M., 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651. Ünligil, M., Zhou, S., Yuwaraj, S., Sarkar, M., Harry Schachter, J.M.R., 2000. X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J. 19, 5269–5280. Van Patten, S.M., Hughes, H., Huff, M.R., Piepenhagen, P.A., Waire, J., Qiu, H., Ganesa, C., Reczek, D., Ward, P.V., Kutzko, J.P., Edmunds, T., 2006. Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease. Glycobiology 17, 467–478. Weikert, S., Papac, D., Briggs, J., Cowfer, D., Tom, S., Gawlitzek, M., Lofgren, J., Mehta, S., Chisholm, V., Modi, N., 1999. Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins. Nat. Biotechnol. 17, 1116–1121. Wurm, F., 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22, 1393-1398 Yamane-Ohnuki, N., Kinoshita, S., Inoue-Urakubo, M., Kusunoki, M., Iida, S., Nakano, R., Wakitani, M., Niwa, R., Sakurada, M., Uchida, K., Shitara, K., Satoh, M., 2004. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibodydependent cellular cytotoxicity. Biotechnol. Bioeng. 87, 614–622. Yip, B., Chen, S.H., Mulder, H., Ho ppener, J.W., Schachter, H., 1997. Organization of the human beta-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis. Biochem. J. 21, 465-474. Zhang, M., Koskie, K., Ross, J.S., Kayser, K.J., Caple, M.V., 2010. Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol. Bioeng. 105, 1094–1105. 113 Zhang, P., Lifen Tan, D., Heng, D., Wang, T., Mariati, Yang, Y., Song, Z., 2010. A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines. Metab. Eng. 12, 526–536. Zhong, X., Cooley, C., Seth, N., Juo, Z.S., Presman, E., Resendes, N., Kumar, R., Allen, M., Mosyak, L., Stahl, M., Somers, W., Kriz, R., 2012. Engineering novel Lec1 glycosylation mutants in CHO-DUKX cells: Molecular insights and effector modulation of N-acetylglucosaminyltransferase I. Biotechnol. Bioeng. 109, 1723– 1734. 114 [...]... combinatorial library screening (Choi et al., 2003) Through the same approach of library screening of and introduction of glycosyltransferase fused to mannosidase yeast localization signals, the same group arrived at a strain of Pichia pastoris capable of producing galactosylated complex Nglycans Finally, the biosynthetic pathway for sialic acid and α2,6-sialyltranferase was introduced arriving at a strain... out of CHO- gmt4 cells and details the characterization of that cell line Chapter 7 describes the amplification process and bioreactor run results Chapter 8 gives the conclusions of this research thus far and possible applications and recommendations 4 CHAPTER 2 2.1 LITERATURE REVIEW Glycosylation Glycosylation is a post-translational modification that attaches a sugar moiety to the protein backbone as... becoming increasingly apparent as demonstrated by the immunogenicity of plant expressed recombinant proteins (Bardor et al., 2003) and the administration of Cetuximab leading to patients suffering from anaphylaxis (Arnold and Misbah, 2008) Many sugar epitopes such as galactose-alpha-1,3-galactose (alpha-Gal), Nglycolylneuraminic acid (Neu5Gc) and hyper-mannosylated glycan structures are now known to... the acceptor carbohydrate structure Glycosidases are responsible for trimming the oligosaccharide structure 5 There are ten monosaccharides that are involved in N -glycosylation Of these ten, seven are utilized in mammalian N -glycosylation : N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylneuraminic acid (Neu5Ac) or otherwise known commonly as sialic acid, mannose... the addition of N-acetylglucosamine to different extents of branching up to a maximum of four branches Terminal sugars 6 such as galactose and finally sialic acid are also added in addition to a fucose, which is attached to the N-acetylglucosamine proximal to the asparagine residue 2.2 The impact of glycosylation on recombinant production of therapeutic glycoproteins Since the commercial production of. .. elongation of this sugar chain by various enzymes to give many different glycan forms N -glycosylation is a common post-translational process, which takes place in eukaryotic cells and archaea The initial mechanisms for N -glycosylation is largely conserved through archaea to mammalian systems and the process of this posttranslational modification are dependent on a set of enzymes found in the endoplasmic... Wong et al (2006) also overexpressed the CMP-sialic acid transporter in order to increase the amount of sialic acid available for the sialyltransferases in the Golgi In this study, the IFN-γ producing CHO cell line was also shown to have improved sialylation as analyzed by total sialic acid quantification using the thiobarbituric acid assay Jeong et al (2009) stably expressed a combination of human α2,3... structure lasted till amino acid 105 and that 106 was the start of another beta sheet The sequence homology was also highly similar from amino acid 106, suggesting that the catalytic domain might start from that amino acid Indeed, it was found that by transfecting different lengths of cDNA into mutant cells that the absence of the first 29, 84 and 106 N-terminal amino acids did not affect the activity of GnT... 2.5 N-acetylglucosaminyltransferase I (GnT I) and glycosylation mutants with defective GnT I N-acetylglucosaminyltransferase I, also known as UDP-GlcNAc :a- 3-D-mannoside P1,2-N-acetylglucosaminyltransferase I, is an inverting glycosyltransferase that belongs to the SGC superfamily of transferases (Ünligil et al., 2000) It was first cloned by the complementation of phage lambda library in cells isolated... glycan (Tomiya et al., 2002) Sialyltransferases which attach sialic acid in α2,6 and α2,3 linkages have been cloned into these cell lines together with the use 11 of sialic acid-rich medium to achieve sialylation of the N-glycan (Hollister et al., 2002) Finally, a heterologous and functional sialic acid biosynthetic pathway which consists of UDP-GlcNAc 2-epimerase/ManNAc kinase, N-acetylneuraminate-9phosphate . administration of Cetuximab leading to patients suffering from anaphylaxis (Arnold and Misbah, 2008). Many sugar epitopes such as galactose-alpha-1,3-galactose (alpha-Gal), N- glycolylneuraminic acid. monosaccharides that are involved in N -glycosylation. Of these ten, seven are utilized in mammalian N -glycosylation : N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylneuraminic. bioreactor studies showed that the superior sialylation of EPO was maintained Using HPAEC-PAD, sialic acid quantification and MALDI-TOF analyses, the purified EPO was shown to contain better sialylated