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The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins
The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins Dana C Andersen and Charles F Goochee Stanford U n i v e r s i t y , Stanford, USA Glycoprotein oligosaccharide structure influences numerous important protein properties. In recent years, a number of studies have demonstrated that cell-culture methodology can significantly affect the oligosaccharide structures of recombinant proteins and antibodies, and, in the past year in particular, several of the specific environmental variables responsible for these effects have been identified. Current Opinion in Biotechnology 1994, 5:546-549
Introduction The majority of proteins secreted by mammahan cells, including many proteins of pharmacological importance, are glycoproteins. These proteins possess oligosaccharides covalently attached either to the side-chain nitrogen in asparagine (N-linked) or to serine/threonine hydroxyls (O-linked) (reviewed in [1,2]). The oligosaccharide structures of glycoproteins can have significant effects on important protein properties, including specific activity, plasma clearance rate, antigenicity, immunogenicity, solubility and resistance to thermal inactivation or protease attack [3-5]. A key feature of the glycosylation process is that many of the reactions are often incomplete, leading to the secretion of a mixture of differently glycosylated forms or 'glycoforms' of a given protein. Common sources of heterogeneity in glycosylation include variation in the initial site of attachment of the oligosaccharide to the protein, variation in the branching reactions ofN-hnked glycosylation, and variation in the addition of terminal siahc acid residues. Each of these sources ofoligosaccharide heterogeneity can have significant effects on protein properties (reviewed in [3]); thus, the distribution of glycoforms can be of great importance in the therapeutic efficacy of a glycoprotein. The array of glycosylation enzymes present in a given host cell line defines the types of oligosaccharide that can be produced on glycoproteins within ceils of that line. Many of the glycosylation differences among different eukaryotic cell lines, including mammalian cell hnes of biotechnological interest, have now been well documented [3,4,6]. Furthermore, a variety of factors in the cell-culture environment can also affect the distribution of glycoforms produced. The effects of various factors on glycosylation, including glucose starvation, hormonal
effects, acidotropic amines, culture methods and a variety of agents not normally found in cell culture, have been reviewed previously [7]. The majority of these studies employed mammalian cell systems that are not directly applicable to common biotechnological applications. This review focuses on more recent studies demonstrating that culture conditions can significantly affect the oligosaccharide structures of recombinant proteins and antibodies. These studies fall into two categories. Those in the first category demonstrate that glycoprotein oligosaccharide structure can be influenced by the general cell-culture methodology. Those in the second category identify specific culture variables that can affect oligosaccharide structure. In the following review, we consider each of these categories in turn.
The effect of culture methodology on oligosaccharide structure A number of different techniques are commonly used for the growth of recombinant mammalian cells and antibody-~producing hybridoma cells~ including suspension growth in batch or perfused stirred tanks, attachmentdependent growth in roller bottles or on microcarriers in stirred tanks, hollow fiber reactors, and ascites culture for monoclonal antibody production. Numerous recent studies have demonstrated that the specific choice of method used to culture cells can influence the oligosaccharide structure of the protein produced. Furthermore, for a particular choice of culture methodology, glycoprotein oligosaccharide structure can change as a function of time.
Abbreviations Ig--immunoglobulin; C H ~ h i n e s e hamster ovary; TSH--human thyrotropin; HuTK---human tissue kallikrein; IFN-y~interferon-y; hFSH--human follicle-stimulating hormone; GIcNAc-T V--N-acetylglucosaminyl transferase V.
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© Current Biology Ltd ISSN 0958-1669
The effect of cell-culture conditions on glycosylation Andersen and Goochee 547 For example, cell-culture methodology has been shown to significantly affect immunoglobulin oligosaccharides in several different antibody-producing hybridoma systems. A monoclonal human IgM produced in ascites culture has been shown to possess lower fractions of sialylated complex-type oligosaccharides and different proportions of other structures than the same IgM produced by the same cell line in serum-free airlift suspension culture [8",9"]. Correspondingly, a lower level of sialylation was found for a murine IgG produced in ascites compared with the same antibody produced in serum-free culture [10]. Differences in glycosylation were also noted for this same IgG when it was produced in either serumsupplemented medium or serum-free medium [10]. Similar effects have been observed in the production of recombinant antibodies. For instance, chimeric mouse-human IgGs expressed in a mouse cell line, J558L, have been found to have higher galactosylation when produced in serum-supplemented 'still' culture than when produced in ascites or hollow fiber cultures [11]. Production of a recombinant human monoclonal IgG in the routine myeloma cell line NS0 grown in a serum-free fed-batch culture has revealed significant reductions in glycosylation over the course of the culture [12"]. Specifically, the proportion oflgG containing truncated complex-type and high mannose oligosaccharides secreted by the cells increases relative to the proportion of more fully 15rocessed complex-type structures as the culture progresses [12"]. Effects of the cell-culture methodology on the oligosaccharide structure of Chinese hamster ovary (CHO)produced glycoproteins have also been identified. For instance, recombinant human thyrotropin (TSH) produced in a hollow fiber bioreactor possesses significantly less sialic acid (0.4 sialic acid residues per oligosaccharide compared with 1.9 sialic acid residues per oligosaccharide) and galactose (1.4 sialic acid residues per oligosaccharide compared with 2.1 sialic acid residues per oligosaccharide) than TSH produced on microcarrier beads in a large-scale bioreactor [13"]. The sialic acid level of the TSH produced in the hollow fiber bioreactor also varies significantly over the different bioreactor operational phases [13"]. Additionally, recombinant human tissue kallikrein (HuTK) produced by attachment-dependent C H O cells on microcarrier beads possesses less sialic acid than H u T K produced in attachment-independent C H O cells in free suspension [14°]. In another set of serum-free suspension cultures of recombinant C H O cells, Curling et al. [15] demonstrated that the occupancy of N-linked glycosylation sites decreases as a function of time [15].
The effects of specific culture factors on oligosaccharide structure The studies noted above have unambiguously demonstrated the effects of the cell-culture methodology on
glycoprotein oligosaccharide structure under conditions relevant to biotechnological processes. A variety of recent studies have attempted to identify the specific factors in the cell-culture environment responsible for such variations. Ammonium ions are a natural product of glutamine metabolism or breakdown in cell culture and can rise to levels of 5 - 1 0 m M in mammalian cell culture. Thorens and Vassalli [16] demonstrated that NH4 + at a concentration of 10 mM can significantly reduce the sialylation of an immunoglobulin produced by mouse plasma cells. More recently, NH4 + at levels of as low as 2 mM have been found to reduce the final sialylation reaction in the O-linked glycosylation pathway for CHO-produced recombinant human granulocyte colony-stimulating factor [17°]. Although a variety of reports have demonstrated culture effects on N-linked glycosylation, these results confirm that the O-linked glycosylation of recombinant glycoproteins is also influenced by cell-culture factors. Additional results suggest that NH4 + may be reducing sialylation via a weak base related mechanism involving a pH change in the trans-Golgi compartments, where the sialyltransferases involved in glycosylation are located (DC Andersen, CF Goochee, unpublished data). In another system, the N-hnked glycosylation of recombinant mouse placental lactogen-I produced in C H O cells has been found to be significantly reduced by NH4 + at a level of 9 raM. Inhibition was pH-dependent and related to the concentration of neutral ammonia in the medium [18"]. Although, this result is apparently consistent with a weak base related mechanism, detailed interpretation of this result is complicated by the unknown nature of the changes in glycosylation observed. Glycosidase degradation of oligosaccharides after secretion represents another potential source of glycoform variability in cell culture. In particular, sialidase activity has been identified in C H O batch and perfusion cultures at levels that could hypothetically cause significant degradation of the sialic acid content of glycoproteins [19",20]. Recently, removal of sialic acid from a C H O produced glycoprotein in batch culture has been demonstrated at levels that correlate with the removal calculated from the sialidase activity in the culture (MJ Gramer, CF Goochee, unpublished data). Fucosidase, [~-galactosidase and [3-hexosaminidase activities have also been characterized in C H O cell supernatants as well as in supernatants or lysates from 293, NS0 and hybridoma cell lines [21,22]. The activities, stabilities and pH-dependence of these enzymes are very cell-line specific, but may be responsible for significant oligosaccharide degradation under certain conditions, particularly cell culture operational configurations involving high cell densities and low cell viabilities [21]. Recent work has demonstrated that the glycosylation of transferrin secreted by attachment-dependent HepG2 cells differs between rapidly dividing pre-confluent cells and slowly dividing confluent cells. This change was cor-
548
Expressionsystems related with growth-state dependent changes in the activity of the intracellular branching enzyme N-acetylglucosaminyl transferase V (GlcNAc-T V) [23]. Although these results are intriguing, their relevance to cell lines ofbiotechnological significance has not been assessed. Although CHO-produced glycoproteins contain primarily the N-acetylneuraminic acid form of sialic acid, approximately 3% of the sialic acid on several C H O glycoproteins has been identified to be of the N-glycolylneuraminic acid form [24]. Increases in the partial pressure of dissolved carbon dioxide, ranging from 1 0 m m H g to 160mmHg, were shown to increase the proportion of N-glycolylneuraminic acid by approximately threefold for each of three separate recombinant CHO-produced glycoproteins (GE Grampp, TK Blumen, K Kelly, P Derby, LA Sleeman, D Hettwer, abstract, Cell Culture Engineering IV, San Diego, California, March 1994). Many factors have been identified that may affect the oligosaccharide site occupancy of CHO-produced interferon (IFN)- 7. For example, IFN-7 site occupancy increased during transient periods of glucose excess in glucose-limited continuous cultures, suggesting that glucose depletion may be involved in the observed reduction in glycosylation [25]. Even so, the addition of glucose to batch cultures failed to prevent decreased site occupancy, indicating that the glycosylation change observed in batch cultures was more complex than simple glucose starvation [15]. Other factors in batch cultures, including the initial glutamine concentration, the concentration and purity of bovine serum albumin, the medium lipid composition and the common medium surfactant Pluronic F68, also appear to affect IFN-~t site occupancy in an inter-related manner [26]. Taken together, these results illustrate the potential complexity of the effects of several cell-culture variables on IFN-y glycosylation. Overall, these findings suggest that several different mechanisms may be responsible for the effects of culture factors on glycoprotein oligosaccharide structure. The accumulation of NH4 + in cell cultures appears to affect sialylation by directly altering the trans-Golgi environment. Conditions that cause cell lysis can lead to the degradation of glycoprotein oligosaccharides by glycosidases released from the cells. Culture factors can also affect metabolic pathways, leading to changes in glycosyltransferase activities or, potentially, to the availability of the oligosaccharide precursor required to initiate the N-linked glycosylation pathway, as has been shown for glucose starvation. In other cases, a variety of components in the medium have been shown to affect glycosylation by mechanisms that are not entirely clear.
Conclusions In recent years, a number of studies have demonstrated that the cell-culture environment can exert significant el-
fects on the glycosylation of recombinant proteins and antibodies. Glycoprotein oligosaccharide structure can vary as a result of different cell-culture methodologies and as a function of time over the course of a culture. Such results are not surprising because, unlike protein translation, oligosaccharide processing is not template driven. Rather, glycosylation occurs as a result of the sequential actions of several enzymes in different intracellular compartments, and these reactions can be affected by extracellular factors that influence enzyme activity or substrate availability. Furthermore, the oligosaccharides of secreted glycoproteins can be modified extracellularly by glycosidases released from cultured cells. In the development of processes for the manufacture of therapeutic glycoproteins, the potential effect of the cellculture environment on glycosylation must be viewed on two levels. On the first level, it is necessary to develop a process that results in a reproducible distribution of glycoforms. This goal can be readily achieved for any given bioreactor and purification strategy as long as the process can be reproducibly operated. In addition, the implementation of minor process alterations is even possible without having any effect on glycoform distribution [27]. At a higher level, the goal should be to develop a process that produces the most desirable distribution of glycoforms. For example, it is generally desirable to have a high level of sialic acid to ensure maximum glycoprotein circulatory half-life and solubility in the body. Two key objectives are integral to the optimization of glycoform distribution for a given glycoprotein produced by a given cell type: first, an understanding of the specific environmental factors affecting oligosaccharide structures, and second, the control of these factors at the cellular level. Recent studies described in this review have certainly contributed to our understanding of these environmental factors and suggest that the use of well controlled homogeneous cell-culture environments will increasingly enable the production of secreted glycoproteins with desirable reproducible glycosylation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: ° of special interest °* of outstanding interest t.
Kornfeld R, Kornfeld S: Assembly of Asparagine-Linked Oligosaccharides. Annu Rev Biochem 1985, 54:631-664.
2.
Kobata A: The Carbohydrates of Glycoproteins. In Biology of Carbohydrates, vol 2. Edited by Ginsburg V, Robbins PW. New York: John Wiley & Sons; 1984:87-161.
3.
Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR:
The Oligosaccharides of Glycoproteins: Factors Affecting their Synthesis and their Influence on Glycoprotein Properties. In Frontiers in Bioprocessing II. Edited by Todd P, Sikdar SK, Bier M. Washington: American Chemical Society; 1992:199-240.
The effect of cell-culture conditions on glycosylation Andersen and G o o c h e e 4.
Goochee CF, Gramer MJ, Andersen DC, Bahr IB, Rasmussen JR: The Oligosaccharides of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure and their Effect on Glycoprotein Properties. Biotechnology 1991, 9:1347-1355.
5.
Varki A: Biological Roles of Oligosaccharides: All of the Theories are Correct. Glycobiology 1993, 3:97-130.
6.
Stanley P: Glycosylation Engineering. Glycobiology 1992, 2:99-107.
7.
Goochee CF, Monica T: Environmental Effects on Protein Glycosylation. Biotechnology 1990, 8:421-427.
8. •
Monica TJ, Goochee CF, Maiorella, BL: Comparative Biochemical Characterization of a Human IgM Produced in Both Ascites and in Vitro Cell Culture. Biotechnology 1993, 11:512-515. This study and [9",10] demonstrate significant differences in the level of sialylation, the types of oligosaccharide, and the distribution of oligosaccharide structures between monoclonal antibodies produced in ascites and those produced in cell culture. These glycosylation differences have significant implications for the characteristics of the antibodies produced.
16.
Thorens B, Vassalli P: Chloroquine and Ammonium Chloride Prevent Terminal Glycosylation of Immunoglobulins in Plasma Cells Without Affecting Secretion. Nature 1986, 321:618-620.
17. •
Andersen DC, Goochee CF, Cooper G, Weitzhandler M: Monosaccharide and Oligosaccharide Analysis of Isoelectrlc Focusing-Separated and Blotted Granulocyte Colony-Stimulating Factor Glycoforms using High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection. Glycobiology 1994, in press. This study demonstrates that the cell culture can affect the O-linked glycosylation pathway. Specifically, NH4+ are shown to inhibit the completion of the final O-linked sialylation reaction. 18. •
Borys MC, Linzer DIH, Papoutsakis ET: Ammonia Affects the Glycosylation Patterns of Recombinant Mouse Placental Lactogen-I by Chinese Hamster Ovary Cells in a pH-Dependent Manner. Biotechnol Bioeng 1994, 43:505-514. Ammonia is shown to affect N-linked glycosylation by CHO cells in a pH-dependent manner.
19.
Gramer MJ, Goochee CF: Glycosidase Activities in Chinese
Maiorella BL, Winkelhake J, Young J, Moyer B, Bauer R, Hora M, Andya J, Thomson J, Patel 1", Parekh R: Effect of Culture Conditions on IgM Antibody Structure, Pharmacokinetics and Activity. Biotechnology 1993, 11:387-392. See [8•].
•
10.
Patel TP, Parekh RB, Moellering BJ, Prior CP: Different Culture Methods Lead to Differences in Glycosylation of a Murine IgG Monoclonal Antibody. Biochem J 1992, 285:839-845.
20.
Warner TG, Chang J, Ferrari J, Harris R, McNerney T, Bennett G, Burnier J, Sliwkowski MB: Isolation and Properties of a Soluble Sialidase from the Culture Fluid of Chinese Hamster Ovary Cells. Glycobiology 1993, 3:455-463.
11.
Lund J, Takahashi N, Nakagawa H, Goodall M, Bentley 1-, Hindley SA, Tyler R, Jefferis R: Control of IgG/Fc Glycosylation: A Comparison of Oligosaccharides from Chimeric Human/Mouse and Mouse Subclass Immunoglobulin Gs. Mol Immunol 1993, 30:741-748.
21.
Gramer MJ, Goochee CF: Glycosidase Activities of the 293 and NS0 Cell Lines, and of an Antibody-Producing Hybridoma Cell Line. Biotechnol Bioeng 1994, 43:423-428.
22.
Gramer MJ, Schaffer DV, Sliwkowski MB, Goochee CF: Purification and Characterization of c(-t-Fucosidase from Chinese Hamster Ovary Cell Culture Supernatant. Glycobiology 1994, in press.
23.
Hahn TJ, Goochee CF: Growth-Associated Glycosylation of Transferrin Secreted by HepG2 Cells. J Biol Chem 1992, 267:23982-23987.
24.
14okke CH, Bergwerff AA, Van Dedem GWK, Van Oostrum J, Kamerling JP, Vliegenthart JFG: Sialylated Carbohydrate Chains of Recombinant Human Glycoproteins Expressed in Chinese Hamster Ovary Cells Contain Traces of N-Glycolylneuraminic Acid. FEBS Lett 1990, 275:9-14.
25.
Hayter Strange ture of Human
26.
Castro PML: Optimization of CHO Cell Growth and Recombinant Interferon-7 Production [PhD Thesis]. London: University of London; 1993.
27.
Maiorella BL, Ferris R, Thomson J, White C, Brannon M, Hora M, Henriksson T, Triglia R, Kunitani M, Kresin L, et aL: Evaluation of Product Equivalence during Process Optimization for Manufacture of a Human IgM Monoclonal Antibody. giologicals 1993, 21:197-205.
9. •
Robinson DK, Chan CP, Yu Ip C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M: Characterization of a Recombinant Antibody Produced in the Course of a High Yield Fed-Batch Process. Biotechnol Bioeng 1994, in press. This study reveals that cell-culture factors can influence the glycosylation of the increasingly widely used NS0 cell line.
Hamster Ovary Cell Lysate and Cell Culture Supernatant. Biotechnol Prog 1993, 9:366-373. This study identifies significant levels of sialidase in CHO cultures, indicating another mechanism by which the cell culture can influence glycosylation.
12. •
13. •
Szkudlinski MW, Thotakura NR, Bucci I, loshi LR, Tsai A, EastPalmer J, Shiloach J, Weintraub BD: Purification and Characterization of Recombinant Human Thyrolropin (TSH) Isoforms Produced by Chinese Hamster Ovary Cells: The Role of Sialylation and Sulfation in TSH Bioaclivity. Endocrinology 1993, 133:1490-1503. TSH produced in hollow fiber bioreactors possesses lower levels of sialic acid and galactosethan TSH produced using CHO cells on microcarrier beads in a large-scale bioreactoc (See also [14"].) 14. •
Watson E, Shah B, Leiderman L, Hsu YR, Karkare S, Lu HS, Lin FK: Comparison of N-Linked Oligosaccharides of Recombinant Human Tissue Kallikrein Produced by Chinese Hamster Ovary Cells on Microcarrier Beads and in Serum-Free Suspension Culture. giotechnol Prog 1994, 10:39-44. HuTK produced using attachment-dependent CHO cells on microcartier beads possesses less sialic acid than HuTK produced in attachmentindependent suspension cultures. These results, together with the those of [13"], demonstrate the importance of the cell-culture methodology for glycosylation in recombinant CHO cultures. 15.
Curling EMA, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG, Jenkins N: Recombinant Human Interferon-y: Differences in Glycosylation and Proteolytic Processing Lead to Heterogeneity in Batch Culture. Biochem J 1990, 272:333-337.
PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, PG, Tong JM, Bull AT: Glucose-Limited Chemostat CulChinese Hamster Ovary Cells Producing Recombinant Interferon-y. Biotechnol Bioeng 1992, 39:327-335.
D C Andersen and CF Goochee, Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
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Introduction The majority of proteins secreted by mammahan cells, including many proteins of pharmacological importance, are glycoproteins. These proteins possess oligosaccharides covalently attached either to the side-chain nitrogen in asparagine (N-linked) or to serine/threonine hydroxyls (O-linked) (reviewed in [1,2]). The oligosaccharide structures of glycoproteins can have significant effects on important protein properties, including specific activity, plasma clearance rate, antigenicity, immunogenicity, solubility and resistance to thermal inactivation or protease attack [3-5]. A key feature of the glycosylation process is that many of the reactions are often incomplete, leading to the secretion of a mixture of differently glycosylated forms or 'glycoforms' of a given protein. Common sources of heterogeneity in glycosylation include variation in the initial site of attachment of the oligosaccharide to the protein, variation in the branching reactions ofN-hnked glycosylation, and variation in the addition of terminal siahc acid residues. Each of these sources ofoligosaccharide heterogeneity can have significant effects on protein properties (reviewed in [3]); thus, the distribution of glycoforms can be of great importance in the therapeutic efficacy of a glycoprotein. The array of glycosylation enzymes present in a given host cell line defines the types of oligosaccharide that can be produced on glycoproteins within ceils of that line. Many of the glycosylation differences among different eukaryotic cell lines, including mammalian cell hnes of biotechnological interest, have now been well documented [3,4,6]. Furthermore, a variety of factors in the cell-culture environment can also affect the distribution of glycoforms produced. The effects of various factors on glycosylation, including glucose starvation, hormonal
effects, acidotropic amines, culture methods and a variety of agents not normally found in cell culture, have been reviewed previously [7]. The majority of these studies employed mammalian cell systems that are not directly applicable to common biotechnological applications. This review focuses on more recent studies demonstrating that culture conditions can significantly affect the oligosaccharide structures of recombinant proteins and antibodies. These studies fall into two categories. Those in the first category demonstrate that glycoprotein oligosaccharide structure can be influenced by the general cell-culture methodology. Those in the second category identify specific culture variables that can affect oligosaccharide structure. In the following review, we consider each of these categories in turn.
The effect of culture methodology on oligosaccharide structure A number of different techniques are commonly used for the growth of recombinant mammalian cells and antibody-~producing hybridoma cells~ including suspension growth in batch or perfused stirred tanks, attachmentdependent growth in roller bottles or on microcarriers in stirred tanks, hollow fiber reactors, and ascites culture for monoclonal antibody production. Numerous recent studies have demonstrated that the specific choice of method used to culture cells can influence the oligosaccharide structure of the protein produced. Furthermore, for a particular choice of culture methodology, glycoprotein oligosaccharide structure can change as a function of time.
Abbreviations Ig--immunoglobulin; C H ~ h i n e s e hamster ovary; TSH--human thyrotropin; HuTK---human tissue kallikrein; IFN-y~interferon-y; hFSH--human follicle-stimulating hormone; GIcNAc-T V--N-acetylglucosaminyl transferase V.
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© Current Biology Ltd ISSN 0958-1669
The effect of cell-culture conditions on glycosylation Andersen and Goochee 547 For example, cell-culture methodology has been shown to significantly affect immunoglobulin oligosaccharides in several different antibody-producing hybridoma systems. A monoclonal human IgM produced in ascites culture has been shown to possess lower fractions of sialylated complex-type oligosaccharides and different proportions of other structures than the same IgM produced by the same cell line in serum-free airlift suspension culture [8",9"]. Correspondingly, a lower level of sialylation was found for a murine IgG produced in ascites compared with the same antibody produced in serum-free culture [10]. Differences in glycosylation were also noted for this same IgG when it was produced in either serumsupplemented medium or serum-free medium [10]. Similar effects have been observed in the production of recombinant antibodies. For instance, chimeric mouse-human IgGs expressed in a mouse cell line, J558L, have been found to have higher galactosylation when produced in serum-supplemented 'still' culture than when produced in ascites or hollow fiber cultures [11]. Production of a recombinant human monoclonal IgG in the routine myeloma cell line NS0 grown in a serum-free fed-batch culture has revealed significant reductions in glycosylation over the course of the culture [12"]. Specifically, the proportion oflgG containing truncated complex-type and high mannose oligosaccharides secreted by the cells increases relative to the proportion of more fully 15rocessed complex-type structures as the culture progresses [12"]. Effects of the cell-culture methodology on the oligosaccharide structure of Chinese hamster ovary (CHO)produced glycoproteins have also been identified. For instance, recombinant human thyrotropin (TSH) produced in a hollow fiber bioreactor possesses significantly less sialic acid (0.4 sialic acid residues per oligosaccharide compared with 1.9 sialic acid residues per oligosaccharide) and galactose (1.4 sialic acid residues per oligosaccharide compared with 2.1 sialic acid residues per oligosaccharide) than TSH produced on microcarrier beads in a large-scale bioreactor [13"]. The sialic acid level of the TSH produced in the hollow fiber bioreactor also varies significantly over the different bioreactor operational phases [13"]. Additionally, recombinant human tissue kallikrein (HuTK) produced by attachment-dependent C H O cells on microcarrier beads possesses less sialic acid than H u T K produced in attachment-independent C H O cells in free suspension [14°]. In another set of serum-free suspension cultures of recombinant C H O cells, Curling et al. [15] demonstrated that the occupancy of N-linked glycosylation sites decreases as a function of time [15].
The effects of specific culture factors on oligosaccharide structure The studies noted above have unambiguously demonstrated the effects of the cell-culture methodology on
glycoprotein oligosaccharide structure under conditions relevant to biotechnological processes. A variety of recent studies have attempted to identify the specific factors in the cell-culture environment responsible for such variations. Ammonium ions are a natural product of glutamine metabolism or breakdown in cell culture and can rise to levels of 5 - 1 0 m M in mammalian cell culture. Thorens and Vassalli [16] demonstrated that NH4 + at a concentration of 10 mM can significantly reduce the sialylation of an immunoglobulin produced by mouse plasma cells. More recently, NH4 + at levels of as low as 2 mM have been found to reduce the final sialylation reaction in the O-linked glycosylation pathway for CHO-produced recombinant human granulocyte colony-stimulating factor [17°]. Although a variety of reports have demonstrated culture effects on N-linked glycosylation, these results confirm that the O-linked glycosylation of recombinant glycoproteins is also influenced by cell-culture factors. Additional results suggest that NH4 + may be reducing sialylation via a weak base related mechanism involving a pH change in the trans-Golgi compartments, where the sialyltransferases involved in glycosylation are located (DC Andersen, CF Goochee, unpublished data). In another system, the N-hnked glycosylation of recombinant mouse placental lactogen-I produced in C H O cells has been found to be significantly reduced by NH4 + at a level of 9 raM. Inhibition was pH-dependent and related to the concentration of neutral ammonia in the medium [18"]. Although, this result is apparently consistent with a weak base related mechanism, detailed interpretation of this result is complicated by the unknown nature of the changes in glycosylation observed. Glycosidase degradation of oligosaccharides after secretion represents another potential source of glycoform variability in cell culture. In particular, sialidase activity has been identified in C H O batch and perfusion cultures at levels that could hypothetically cause significant degradation of the sialic acid content of glycoproteins [19",20]. Recently, removal of sialic acid from a C H O produced glycoprotein in batch culture has been demonstrated at levels that correlate with the removal calculated from the sialidase activity in the culture (MJ Gramer, CF Goochee, unpublished data). Fucosidase, [~-galactosidase and [3-hexosaminidase activities have also been characterized in C H O cell supernatants as well as in supernatants or lysates from 293, NS0 and hybridoma cell lines [21,22]. The activities, stabilities and pH-dependence of these enzymes are very cell-line specific, but may be responsible for significant oligosaccharide degradation under certain conditions, particularly cell culture operational configurations involving high cell densities and low cell viabilities [21]. Recent work has demonstrated that the glycosylation of transferrin secreted by attachment-dependent HepG2 cells differs between rapidly dividing pre-confluent cells and slowly dividing confluent cells. This change was cor-
548
Expressionsystems related with growth-state dependent changes in the activity of the intracellular branching enzyme N-acetylglucosaminyl transferase V (GlcNAc-T V) [23]. Although these results are intriguing, their relevance to cell lines ofbiotechnological significance has not been assessed. Although CHO-produced glycoproteins contain primarily the N-acetylneuraminic acid form of sialic acid, approximately 3% of the sialic acid on several C H O glycoproteins has been identified to be of the N-glycolylneuraminic acid form [24]. Increases in the partial pressure of dissolved carbon dioxide, ranging from 1 0 m m H g to 160mmHg, were shown to increase the proportion of N-glycolylneuraminic acid by approximately threefold for each of three separate recombinant CHO-produced glycoproteins (GE Grampp, TK Blumen, K Kelly, P Derby, LA Sleeman, D Hettwer, abstract, Cell Culture Engineering IV, San Diego, California, March 1994). Many factors have been identified that may affect the oligosaccharide site occupancy of CHO-produced interferon (IFN)- 7. For example, IFN-7 site occupancy increased during transient periods of glucose excess in glucose-limited continuous cultures, suggesting that glucose depletion may be involved in the observed reduction in glycosylation [25]. Even so, the addition of glucose to batch cultures failed to prevent decreased site occupancy, indicating that the glycosylation change observed in batch cultures was more complex than simple glucose starvation [15]. Other factors in batch cultures, including the initial glutamine concentration, the concentration and purity of bovine serum albumin, the medium lipid composition and the common medium surfactant Pluronic F68, also appear to affect IFN-~t site occupancy in an inter-related manner [26]. Taken together, these results illustrate the potential complexity of the effects of several cell-culture variables on IFN-y glycosylation. Overall, these findings suggest that several different mechanisms may be responsible for the effects of culture factors on glycoprotein oligosaccharide structure. The accumulation of NH4 + in cell cultures appears to affect sialylation by directly altering the trans-Golgi environment. Conditions that cause cell lysis can lead to the degradation of glycoprotein oligosaccharides by glycosidases released from the cells. Culture factors can also affect metabolic pathways, leading to changes in glycosyltransferase activities or, potentially, to the availability of the oligosaccharide precursor required to initiate the N-linked glycosylation pathway, as has been shown for glucose starvation. In other cases, a variety of components in the medium have been shown to affect glycosylation by mechanisms that are not entirely clear.
Conclusions In recent years, a number of studies have demonstrated that the cell-culture environment can exert significant el-
fects on the glycosylation of recombinant proteins and antibodies. Glycoprotein oligosaccharide structure can vary as a result of different cell-culture methodologies and as a function of time over the course of a culture. Such results are not surprising because, unlike protein translation, oligosaccharide processing is not template driven. Rather, glycosylation occurs as a result of the sequential actions of several enzymes in different intracellular compartments, and these reactions can be affected by extracellular factors that influence enzyme activity or substrate availability. Furthermore, the oligosaccharides of secreted glycoproteins can be modified extracellularly by glycosidases released from cultured cells. In the development of processes for the manufacture of therapeutic glycoproteins, the potential effect of the cellculture environment on glycosylation must be viewed on two levels. On the first level, it is necessary to develop a process that results in a reproducible distribution of glycoforms. This goal can be readily achieved for any given bioreactor and purification strategy as long as the process can be reproducibly operated. In addition, the implementation of minor process alterations is even possible without having any effect on glycoform distribution [27]. At a higher level, the goal should be to develop a process that produces the most desirable distribution of glycoforms. For example, it is generally desirable to have a high level of sialic acid to ensure maximum glycoprotein circulatory half-life and solubility in the body. Two key objectives are integral to the optimization of glycoform distribution for a given glycoprotein produced by a given cell type: first, an understanding of the specific environmental factors affecting oligosaccharide structures, and second, the control of these factors at the cellular level. Recent studies described in this review have certainly contributed to our understanding of these environmental factors and suggest that the use of well controlled homogeneous cell-culture environments will increasingly enable the production of secreted glycoproteins with desirable reproducible glycosylation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: ° of special interest °* of outstanding interest t.
Kornfeld R, Kornfeld S: Assembly of Asparagine-Linked Oligosaccharides. Annu Rev Biochem 1985, 54:631-664.
2.
Kobata A: The Carbohydrates of Glycoproteins. In Biology of Carbohydrates, vol 2. Edited by Ginsburg V, Robbins PW. New York: John Wiley & Sons; 1984:87-161.
3.
Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR:
The Oligosaccharides of Glycoproteins: Factors Affecting their Synthesis and their Influence on Glycoprotein Properties. In Frontiers in Bioprocessing II. Edited by Todd P, Sikdar SK, Bier M. Washington: American Chemical Society; 1992:199-240.
The effect of cell-culture conditions on glycosylation Andersen and G o o c h e e 4.
Goochee CF, Gramer MJ, Andersen DC, Bahr IB, Rasmussen JR: The Oligosaccharides of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure and their Effect on Glycoprotein Properties. Biotechnology 1991, 9:1347-1355.
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Varki A: Biological Roles of Oligosaccharides: All of the Theories are Correct. Glycobiology 1993, 3:97-130.
6.
Stanley P: Glycosylation Engineering. Glycobiology 1992, 2:99-107.
7.
Goochee CF, Monica T: Environmental Effects on Protein Glycosylation. Biotechnology 1990, 8:421-427.
8. •
Monica TJ, Goochee CF, Maiorella, BL: Comparative Biochemical Characterization of a Human IgM Produced in Both Ascites and in Vitro Cell Culture. Biotechnology 1993, 11:512-515. This study and [9",10] demonstrate significant differences in the level of sialylation, the types of oligosaccharide, and the distribution of oligosaccharide structures between monoclonal antibodies produced in ascites and those produced in cell culture. These glycosylation differences have significant implications for the characteristics of the antibodies produced.
16.
Thorens B, Vassalli P: Chloroquine and Ammonium Chloride Prevent Terminal Glycosylation of Immunoglobulins in Plasma Cells Without Affecting Secretion. Nature 1986, 321:618-620.
17. •
Andersen DC, Goochee CF, Cooper G, Weitzhandler M: Monosaccharide and Oligosaccharide Analysis of Isoelectrlc Focusing-Separated and Blotted Granulocyte Colony-Stimulating Factor Glycoforms using High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection. Glycobiology 1994, in press. This study demonstrates that the cell culture can affect the O-linked glycosylation pathway. Specifically, NH4+ are shown to inhibit the completion of the final O-linked sialylation reaction. 18. •
Borys MC, Linzer DIH, Papoutsakis ET: Ammonia Affects the Glycosylation Patterns of Recombinant Mouse Placental Lactogen-I by Chinese Hamster Ovary Cells in a pH-Dependent Manner. Biotechnol Bioeng 1994, 43:505-514. Ammonia is shown to affect N-linked glycosylation by CHO cells in a pH-dependent manner.
19.
Gramer MJ, Goochee CF: Glycosidase Activities in Chinese
Maiorella BL, Winkelhake J, Young J, Moyer B, Bauer R, Hora M, Andya J, Thomson J, Patel 1", Parekh R: Effect of Culture Conditions on IgM Antibody Structure, Pharmacokinetics and Activity. Biotechnology 1993, 11:387-392. See [8•].
•
10.
Patel TP, Parekh RB, Moellering BJ, Prior CP: Different Culture Methods Lead to Differences in Glycosylation of a Murine IgG Monoclonal Antibody. Biochem J 1992, 285:839-845.
20.
Warner TG, Chang J, Ferrari J, Harris R, McNerney T, Bennett G, Burnier J, Sliwkowski MB: Isolation and Properties of a Soluble Sialidase from the Culture Fluid of Chinese Hamster Ovary Cells. Glycobiology 1993, 3:455-463.
11.
Lund J, Takahashi N, Nakagawa H, Goodall M, Bentley 1-, Hindley SA, Tyler R, Jefferis R: Control of IgG/Fc Glycosylation: A Comparison of Oligosaccharides from Chimeric Human/Mouse and Mouse Subclass Immunoglobulin Gs. Mol Immunol 1993, 30:741-748.
21.
Gramer MJ, Goochee CF: Glycosidase Activities of the 293 and NS0 Cell Lines, and of an Antibody-Producing Hybridoma Cell Line. Biotechnol Bioeng 1994, 43:423-428.
22.
Gramer MJ, Schaffer DV, Sliwkowski MB, Goochee CF: Purification and Characterization of c(-t-Fucosidase from Chinese Hamster Ovary Cell Culture Supernatant. Glycobiology 1994, in press.
23.
Hahn TJ, Goochee CF: Growth-Associated Glycosylation of Transferrin Secreted by HepG2 Cells. J Biol Chem 1992, 267:23982-23987.
24.
14okke CH, Bergwerff AA, Van Dedem GWK, Van Oostrum J, Kamerling JP, Vliegenthart JFG: Sialylated Carbohydrate Chains of Recombinant Human Glycoproteins Expressed in Chinese Hamster Ovary Cells Contain Traces of N-Glycolylneuraminic Acid. FEBS Lett 1990, 275:9-14.
25.
Hayter Strange ture of Human
26.
Castro PML: Optimization of CHO Cell Growth and Recombinant Interferon-7 Production [PhD Thesis]. London: University of London; 1993.
27.
Maiorella BL, Ferris R, Thomson J, White C, Brannon M, Hora M, Henriksson T, Triglia R, Kunitani M, Kresin L, et aL: Evaluation of Product Equivalence during Process Optimization for Manufacture of a Human IgM Monoclonal Antibody. giologicals 1993, 21:197-205.
9. •
Robinson DK, Chan CP, Yu Ip C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M: Characterization of a Recombinant Antibody Produced in the Course of a High Yield Fed-Batch Process. Biotechnol Bioeng 1994, in press. This study reveals that cell-culture factors can influence the glycosylation of the increasingly widely used NS0 cell line.
Hamster Ovary Cell Lysate and Cell Culture Supernatant. Biotechnol Prog 1993, 9:366-373. This study identifies significant levels of sialidase in CHO cultures, indicating another mechanism by which the cell culture can influence glycosylation.
12. •
13. •
Szkudlinski MW, Thotakura NR, Bucci I, loshi LR, Tsai A, EastPalmer J, Shiloach J, Weintraub BD: Purification and Characterization of Recombinant Human Thyrolropin (TSH) Isoforms Produced by Chinese Hamster Ovary Cells: The Role of Sialylation and Sulfation in TSH Bioaclivity. Endocrinology 1993, 133:1490-1503. TSH produced in hollow fiber bioreactors possesses lower levels of sialic acid and galactosethan TSH produced using CHO cells on microcarrier beads in a large-scale bioreactoc (See also [14"].) 14. •
Watson E, Shah B, Leiderman L, Hsu YR, Karkare S, Lu HS, Lin FK: Comparison of N-Linked Oligosaccharides of Recombinant Human Tissue Kallikrein Produced by Chinese Hamster Ovary Cells on Microcarrier Beads and in Serum-Free Suspension Culture. giotechnol Prog 1994, 10:39-44. HuTK produced using attachment-dependent CHO cells on microcartier beads possesses less sialic acid than HuTK produced in attachmentindependent suspension cultures. These results, together with the those of [13"], demonstrate the importance of the cell-culture methodology for glycosylation in recombinant CHO cultures. 15.
Curling EMA, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG, Jenkins N: Recombinant Human Interferon-y: Differences in Glycosylation and Proteolytic Processing Lead to Heterogeneity in Batch Culture. Biochem J 1990, 272:333-337.
PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, PG, Tong JM, Bull AT: Glucose-Limited Chemostat CulChinese Hamster Ovary Cells Producing Recombinant Interferon-y. Biotechnol Bioeng 1992, 39:327-335.
D C Andersen and CF Goochee, Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
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