Glycosylation of recombinant proteins: Problems and prospects

1::3

Glycosylation of recombinant proteins: Problems and prospects

Nigel Jenkins and Elisabeth M. A. Curling

Research School of Biosciences, University of Kent, Canterbury, Kent, UK

Keywords: Glycosylation, biosynthetic pathways; control of; enzymes involved in; biological significance of; commercial implications of; role in pharmacokinetics; role in antigenicity; role in solubility. Oligosaccharides, methods used in analysis of. Glycosyltransferases, cell distribution.

Introduction

Glycosylation, the addition of sugar residues to a peptide backbone, is the most extensive posttranslational modifica- tion made to proteins by eukaryotic cells. The majority of recombinant proteins manufactured for human therapy are glycoproteins derived from animal cells, and it is essential to fully characterize and, if possible, control the glycosylation profile of these products. Indeed, the Food and Drug Ad- ministration (FDA) in the United States and the Commit- tee for Proprietary Medical Productions (CPMP) of the European Community are demanding increasingly sophis- ticated carbohydrate analysis on all new glycoproteins des- tined for human therapy, t

A complete survey of protein glycosylation is beyond the scope of this article, and the reader is referred to the excellent reviews recently published on glycoprotein biosynthesis, 2-7 regulation of biosynthetic pathways, 1,8-13 biological functions of glycan structures, 1.14-19 and glycoprotein analysis. 2°-29 This review will focus on the aspects of glycoprotein research that are of direct relevance to the biotechnology industry, i.e., the reasons and methods for defining the glycosylation patterns of recombinant glycoproteins.

Why is protein g!ycosylation significant?

Commercial importance

At present, most recombinant proteins intended for human therapy are accepted with some degree of glycosylation heterogeneity. However, as pointed out by Ken Seamon of the FDA, it is important to "define what critical recombinant protein heterogeneity is important to the efficacy of a product, and to design an appropriate analytical test to ensure that the heterogeneity is produced consistently between lots."1 This implies that the glycosylation status of products

Address reprint requests to Dr. Jenkins at the Research School of Bio- sciences, University of Kent, Canterbury, Kent CT2 7N J, UK Received 1 October 1993; revised 8 November 1993

must be clearly established, although there is, as yet, no specific FDA or CPMP document that deals specifically with glycosylation.

With these restrictions in mind, engineering the production of specific glycoforms (such as in erythropoietin, EPO) has enabled some companies to issue patents based on increased effectiveness under the Orphan Drug Act. This legislation was passed in the United States 10 years ago to promote the development of drugs that are needed to treat rare diseases or conditions (in < 200,000 people). For a given glycosylation variant of a therapeutic protein to be accepted under the Orphan Drug Act, it must be shown to be sufficiently different in clinical efficacy and safety from an alternative form of the protein with the same primary structure. As an example, Amgen has marketed EPO ex- clusively under the Orphan Drug Act, but is in patent liti- gation with Chugai-Upjohn who intend to market EPO that is biologically different due to changes in its glycosylation profile?Thus, the need to define and monitor the glycosylation pattern of a recombinant product is of importance both to protect a patent and to maintain biological efficacy and safety.

Methods' used to study the effects of glycosylation

Attempts have been made to assess the myriad biological functions of glycan structures present on glycoproteins in several ways. Firstly, the activity of nonglycosylated recombinant forms of glycoproteins expressed in prokaryotes (such as Escherichia coli) can be compared to their glycosylated counterparts expressed in animal cells. 3°-33 Secondly, oligosaccharide structures can be chemically or enzymati- cally removed, and the deglycosylated protein compared to its native form. 34-36 Both of these methods run the risk of producing misfolded proteins that have reduced activity due to conformational changes in the polypeptides rather than a direct effect arising from the absence of glycosylation. Thirdly, the cellular process of glycosylation can be inhibited by several drugs such as tunicamycin. 34,35,37.3s However, the intracellular presence of nonglycosylated or

354 Enzyme Microb. Technol., 1994, vo116, May ¢~ 1994 Butterworth-Heinemann

Glycosylation of recombinant proteins: N. Jenkins and E. M, A. Curling

misfolded proteins induced by this antibiotic can stimulate the production of chaperone-type proteins such as GRP78 that prevent secretion, 39 so reducing the yield of nonglycosylated products. Fourthly, site-directed mutagenesis (SDM) can be used to eliminate or create glycosylation sites in glycoproteins where the full cDNA sequence is known. 4~)-43 However, caution must be exercised where SDM is used to prevent glycosylation, since the resulting changes in biological activity may be due to the importance of the asparagine (Asn) residue for ligand binding, and not due to changes in the~lycosylation status. This was shown elegantly by Liu et a / .~who studied the effect of substitut- ing amino acid residues within the Asn-X-Thr N-glycosylation site on the lutropin-choriogonadotropin receptor. Substitution of Asfi 173 with Gln 173 caused a vastly reduced hormone binding to the receptor, but a Thr175 to Ala175 substitution maintained high-affinity binding, even though the glycosylation consensus sequence had been destroyed in both cases. It was therefore apparent that the Asn173 residue itself and not the presence of N-linked carbohydrates was crucial to binding.

Effects on protein solubility and stability

The presence of oligosaccharide structures (glycans) on glycoproteins often ensures good solubility and prevents aggregation. 45 For example, the physicochemical properties of human granulocyte colony-stimulating factor (hG- CSF) depend on the presence of O-linked glycan structures: if these are removed chemically, there is an increased degree of self-aggregation leading to complete biological inactivation. 46 Similarly, when fibrinogen, an N-linked glycoprotein, is deglycosylated, insoluble complexes are formed. 47 Moreover, the presence of altered or even in- complete glycan structures can lead to significant self- association of proteins. Taking IgG as an example, the absence of sialic acid terminal residues 48 or of galactose residues 49 on the IgG heavy chain often results in immune complex formation and is a striking feature of many disease pathologies including rheumatoid arthritis, Crohn's disease, and tuberculosis. 5°

The stability of a given glycoprotein is often a function of its resistance to protease attack, for example, the presence of terminal sialic acid protects EPO, 51 tissue plasminogen activator (tPA) 52 and interferons (IFN) 3° from proteolytic degradation. The role ofglycan structures at individual sites within a glycoprotein can be assessed by SDM (see Meth- ods used to discover the effects of glycosylation, above). Depending on which site is targeted, protein folding, the position of disulfide linkages, macromolecular assembly, and intracellular transport can be adversely affected, as shown by studies on simian viral hemagglutinin neuraminidase. 53

Effects on biological activity

For many glycoproteins, full glycosylation equates with full biological activity, 36,54-56 but there are several instances where glycosylation status has no major impact on the biological efficacy of a protein in vitro. For example, although desialylated human EPO shows a thousand-fold reduction

in specific activity in vivo compared to its native form, there is little effect in vitro. 42'57 Similarly, E. coli-derived human IFN-~/has full antiviral and anti-proliferative activity in vitro. 58

Glycosylation is often used to regulate biological processes, 16 particularly in the immune response. For example, the synthesis of IgE is regulated by the glycosylation status of an IgE-binding protein. 9 In the case of IgG, the interaction of membrane-bound immunoglobulin with cell- surface receptors is dependent on correct glycosylation, whereas fluid phase interactions such as antibody-antigen interactions remain largely unaffected. Glycan structures are also partially responsible for stabilizing intercellular interactions during the immune response, e.g., the glycoprotein CD2 binds CD-58(LFA-3) on many cell types only if Ash65 is glycosylated with a high mannose structure. 59 The selectin cell adhesion molecules provide another example of carbohydrates acting as recognition molecules. 6° The presence of an c~2,3-1inked terminal sialic acid on the membrane glycoproteins of some leukocytes enables the selectin ELAM-1 (expressed on postcapillary venules) to recognize and contribute to the trapping of these cells at sites of inflammation. The shedding of this antigen by leukocytes (allowing them to escape recognition) may be con- trolled by the level of c~- 1,3 -fucosyltransferase activity. 61 In view of these findings, several biotechnology companies are looking to specific carbohydrate structures as therapeutic agents to manipulate the immune response. 1,9,16,62

Glycosylation at position Ash 184 of tPA (generating type I tPA) inhibits the proteolytic conversion of single-chain to two-chain tPA, by plasmin. 52 Since single-chain tPA has a lower catalytic activity than two-chain tPA, this glycoform difference has a profound effect on the catalytic efficiency of tPA. 4°

Effects on pharmacokinetics

The residence time in vivo of a given glycoprotein is often dependent on its glycosylation status; it has even been sug- gested that selective glycosylation may have evolved as a means of controlling the persistence of proteins in the circulation. For example, sulfated oligosaccharides are thought to regulate clearance of the hormones chorionic gonadotropin (CG) and luteinizing hormone (LH) from the blood. 63 Over 75% of LH carries sulfated terminal N-acetylgalactosamine (GalNAc) residues and is removed from the circulation rapidly via a hepatic receptor found on endothelial and Kupffer cells. 64-66 In contrast, CG contains mostly terminal sialic acid residues in place of sulfated residues, and has a five- to sevenfold longer residence time in blood. 67 Interestingly, when LH is expressed in Chinese hamster ovary (CHO) cells, only terminally sialylated structures are added, and thus it does not display authentic human glycosylation. 65 Glycosylation variants carrying terminal galactose, GalNAc, or N-acetylglucosamine (GlcNAc) instead of sialic acid are removed from serum via a different

18 68 hepatic asialoglycoprotein receptor on parenchymal cells. , A third type of receptor recognizing high-mannose structures is present on hepatic endothelial and Kupffer cells. 69,70

Extensive studies on tPA mutants have also highlighted the importance of peptide conformation, rather than carbohydrate structures, for the clearance of recombinant

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glycoproteins. 69,71,72 In summary, few generalizations can be made about the clearance of a recombinant glycoprotein from a knowledge of its glycan structure alone, and each case must be tested individually.

Effects on immunogenicity

Carbohydrates can affect the immunogenicity of a protein either by being a part of the epitope itself 19 or by masking existing antigenic sites on the peptide backbone. Thus, a mutant form of the H3 influenza virus can escape monoclonal antibody recognition by forming a new glycosylation site. 73 Of particular importance for human therapeutics are the species differences in the expression of al-3- Galactosyltransferase (EC 2.4.1.151), an enzyme that controls the synthesis of Gal(al-3)-Gal, 131-4GlcNAc residues on surface and secreted glycoproteins of most mammals. The exceptions are humans, apes, and curiously, CHO cells, and the gene coding for this enzyme has become inactivated in hu ma ns . 74-76 Over 1% of human serum IgG is directed against the Gal(o~l-3)-Gal, [~I-4GlcNAc epitope, 77 and the antibody is thought to be induced by the presence of this antigen on enteric bacteria. Caution is therefore needed when selecting a host cell type that may confer this epitope, although when direct comparisons of mouse- and CHO- cell-derived glycoproteins have been made in humans, the pharmacokinetic differences induced by interaction with these natural antibodies are minor . 78,79

Oligosaccharide structures

Oligosaccharides attach to lipids or proteins in three ways:

1. Via an N-glycosidic bond to the R-group of an Asn residue within the consensus sequence Asn-X-Ser/Thr (N- glycosylation; Figures 1A and 1B);

2. Via an O-glycosidic bond to the R-group of Set or Thr (O-glycosylation, Fig. 1C). Small glycan structures can also be O-linked to the side chain of hydroxylysine or hydroxyproline;

3. As a component of the glycophosphatidylinositol (GPI) membrane anchor.

The presence of these peptide sequences within a protein by no means guarantees their glyeosylation, i.e., the sites can always be substituted with glycan structures, always ignored, or variably occupied. This ambiguity leads to macroheterogeneity (see Influences on glycosylation: Protein

A B C

N-acetylneurarninic acid (~ Mannose /~ Galactose ~ N-acetylgalactoumine [ ] N-acetylglucosamima ~ . Fucose

Figure 1 Types of glycan structures commonly found on mammalian glycoproteins. (A) N-linked oligomannose glycan; (B) N-linked complex glycan (triantennary structure); (C) O-linked glycan

356 Enzyme Microb. Technol., 1994, vol. 16, May

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Figure 2 Changes in the glycosylation profile of recombinant IFN-~, during batch culture of CHO cells. The percentage of asparagine (Asn) N-glycosylation sites filled by 91ycan structures was determined by polyacrylamide gel electrophoresis. Glycan distribution between each glycosylation site was assigned using FAB-MS. (C]) Percentage Asn28 occupation; (Q) percentage ASnlOO occupation; (~) unglycosylated IFN-~/. See Glycan analysis: Mass spectrometry, and Influences on 91ycosylation: Culture environment for further details

structure, below), whereas the more subtle changes in individual carbohydrate residues within a glycan structure are termed the microheterogeneity. To understand how these differences arise, it is necessary to briefly describe the glycosylation process.

N-Glycosylation

The initial core structure of all N-linked glycans (Glc3 Man9GlcNAc2) is built from the sequential addition of GIcNAc, rnannose (Man), and glucose (Glc), residues onto the lipid dolichol using phosphorylated intermediates, first in the cytoplasm then in the lumen of the endoplasmic reticulum (e.r.). 2,s°-82 The whole oligosaccharide group is then transferred en bloc from the dolichol carrier to the nascent peptide using the enzyme oligosaccharyl transferase (OST) in the e.r. lumen, s3,84 often before the protein detaches from the ribosome. This core structure is trimmed to varying degrees by e.r. glucosidases, and then by man- nosidases in the e.r. and cis-Golgi, s5-87 In some cases the high-mannose core is left relatively intact, with no further additions of other sugar residues (Figure 1A). The preser- vation of a high mannose structure may result from its location within the folded protein that is inaccessible to man- nosidases, since chemical inhibition of these enzymes also results in high mannose structures.SS

Alternatively, complex outer structures can be built on a trimmed glycan core by a series of GlcNAc, galactose, sialic

Glycosylation of recombinant proteins: N. Jenkins and E. M. A. Curling

acid, or fucose additions using Golgi-resident glycosyltransferases and nucleotide-sugar intermediates (Figure 1B). 3,5,835,89 Fucose may be added to the core structure via an al-6 linkage, 9° and tends to be glycosylation site- specific.9l, 92

Microheterogeneity results from the many products of these different modifications to the basic core structure. N-linked glycans are usually classified as:

1. Complex: containing the disaccharide Gall3(1,4) GlcNAc; 2. Oligomannose: i.e., mannose only present in the outer

arms; 3. Hybrid: mannose residues in one arm and complex

structures [containing Gall3(1,4)GlcNAc] on the other arm(s). Further classification of complex-type glycans is based

upon the number of arms (antennae) emanating from the core structure and the types of sugar residue present. 5°

O-Glycosylation O-Glycosylation of Ser or Thr residues does not require dolichol-linked intermediates, and is thought to occur relatively late in the biosynthetic pathway (i.e., mainly in the Golgi, although the exact location of the enzymes responsible is a matter of controversy). 4,8 The O-linked glycan structures attached to proteins can be as simple as one sugar (e.g., fucose, glucose, GlcNAc, or GalNAc) 93,94 or may contain several sugars (Gal131-3GalNAc is a common core motif, Figure IC). Other additions to this core may follow, catalyzed by glycosyltransferases that are, in gen- eral, distinct from those involved in N-glycosylation. 435

To date, only some of the glycosyltransferase enzymes have been cloned, and the structural details of these genes have been reviewed recently. 3,95,96

care must be taken to analyze the purified protein (or alternatively use a very specific antibody for immunoprecipita- tion of the protein) to avoid detection of oligosaccharides in other cell or media components. This is particularly a problem when analyzing products secreted into serum-based media, or alternatively intracellular components recovered from cell lysates. The gel approach is also capable of detect- ing large macroheterogeneity (e.g., variable N-glycosylation) changes in relatively small proteins. 34,99 However, this approach has limited value for assessing microheterogeneity, since the negative charge on many glycan structures may result in inaccurate mass assignments deduced from migration on SDS-acrylamide gels. The same draw- backs apply to isoelectric focusing gels, although these have been used extensively for assessing gross heterogeneity in different batches of product. 100,101

An emerging technique for profiling glycoprotein heterogeneity is capillary electrophoresis, known as CE. 102-106 This technique uses narrow-bore capillaries to perform sep- arations, facilitated by high voltages that generate elec- troosmotic and electrophoretic flow of buffer solutions and ionic species, respectively. The technique can be adapted to run in various modes (e.g., zone electrophoresis, isoelectrie focusing, isotachophoresis, or micellar electrokinetic chromatography), and combined with the use of alternative buffers, offers a wide range of analytical capabilities for resolving charged molecules such as glycoproteins and glycopeptides. Minimal quantities of sample are required for CE, and glycoforms can be quantified by integrating the peaks detected by the UV analyzer. We have found that analysis of recombinant IFN-~ macroheterogeneity using capillary zone electrophoresis 91 is a more sensitive and re- producible technique than conventional gel analysis, 34,99 and the whole process (from sampling to quantitation) can be completed in 1-2 h.

Glycan analysis In recent years there has been a rapid development of analytical methods for complex carbohydrate analysis, stimu- lated by the heightened interest in producing recombinant glycoproteins for human therapy. It is beyond the scope of this review to provide details of each type of carbohydrate analysis, and the reader is referred to the recent excellent reviews for specialist descriptions. 5,9,1°,15,16,2°-29,97 Future developments may well focus on more rapid analytical methods, such that glycan structures can be defined in 1 or 2 days, and will therefore be used to monitor carbohydrate changes during fermentation. Even definitive structural analysis by mass spectrometry is now becoming a reality within a few days of sampling, and this may significantly affect the requirements of regulatory authorities for glyeo- protein batch consistency.

Electrophoresis Basic information about the presence or absence of glycans and their class can be gained by a combination of polyacrylamide gels, endo- and exoglycosidase treatment, and West- ern or dot blots employing specific lectins as detection agents. 9s All the reagents required for this type of prelimi- nary analysis are now available commercially. However,

Chromatography More detailed structural analysis usually requires the cleavage of glycoproteins into smaller components. Either the protein is proteolytically digested and resolved using chromatography to generate glycopeptides, and/or the oligosaccharide moieties are cleaved enzymically or chemically from the peptide backbone. Generating glycopeptides has the advantage that site-specific glycan information is produced, and this is becoming increasingly important as more products are shown to exhibit clear glycan differences between individual glycosylation sites on the same pro- rein. 1°7A°8 However, it must be noted that the glycosylation profile may well influence the efficiency of proteolytic di- gestion, and this may lead to distortions in quantifying the relative amounts of glycoforms within each glycoprotein. 26 Glycopeptides are commonly resolved from other peptides using reverse-phase high-performance liquid chromatography (HPLC).

The alternative approach is to cleave the carbohydrate from the protein. This can be achieved using the enzyme peptide N-glycosidase F (PNGaseF), which cleaves most common mammalian N-linked oligosaccharides (except oligosaccharides containing an ul-3 fucose substitution) at the N-glycosidic bond. Again, caution needs to be applied

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to ensure quantitative release of different glycan structures. Alternatively, hydrazinolysis has become a common method of releasing both N-linked and O-linked glycans, 109 and this technique can now be automated. 26,28

Many glycan structures have been elucidated by high pH anion exchange chromatography (HPAE) 90,110-114 or gel permeation chromatography. Neutral monosaccharides, amino sugars, and charged sugars can be analyzed using HPAE (employing an HPLC apparatus usually linked to a highly sensitive pulsed amperometric detector) with a sensitivity in the picomolar range. For gel permeation chromatography, Biogel P4 is often used to fractionatc radiola- beled glycan structures cleaved from glycoproteins using hydrazinolysis or PNGaseF. Structures are assigned by comparing the elution times with defined standards. These techniques may be used in combination with exoglycosidase digests and other fractionation methods such as HPLC. 115.116

Nuclear magnetic resonance (NMR)

A one-dimensional 1H-NMR spectrum can be used as a fingerprint to identify a specific glycan structure by comparing it to a database of NMR spectra. 92,117 This requires about 50 nmol of material and is used widely. More sophis- ticated forms of NMR, using extra dimensions and dipolar corrections, can be used to determine specific linkages between saccharide units, but these require considerably more sample. 2°,28 This NMR technique is one of the few methods capable of assigning an unambiguous structure to a completely unknown oligosaccharide.

Mass spectrometry

Recent years have seen a marked increase in the popularity of mass spectrometry methods for glycan analysis. Fast- atom bombardment mass spectrometry (FAB-MS) has the highest mass accuracy of the spectrometric methods, and has been used to deduce glycan structures of a number of glycoproteins.90,118 122 However, this expensive equipment is found in few laboratories, and requires a relatively large amount of pure sample. The exceptional mass accuracy of FAB-MS can also be used to semiquantify the degree of glycan occupation at specific glycosylation sites within a protein, since a glycan-substituted Ash residue is converted to Asp when cleaved by PNGaseF, resulting in a mass increase of 1Da compared to an unconjugated site. 28 This type of analysis has also enabled us to monitor the glycan occupation status of individual N-glycosylation sites within recombinant IFN--/(Figure 2).

Alternatives to FAB-MS include instruments based on electrospray (ES-MS) or laser desorption mass spectrometry (LD-MS) techniques. Although not quite achieving the mass accuracy of FAB-MS, these machines are still able to generate mass accuracies in the range of 0.05-0.1%, and require less sample. 25 ES-MS has the added advantage that it can be coupled directly to CE or HPLC separation of glycoforms. 29,123,124 LD-MS instruments have become within the budget of many more laboratories than FAB-MS, and no derivatization of the sample is required. 25,97,125-127 The technique is also reasonably tolerant of buffer salts. Used in combination with the fast sequence exoglycosidase array method of determining exoglycosidase suscepti-

bility,128,129 we have found the LD-MS technique to be an excellent method of determiningglycan structures on small amounts (50-100 Ixg) of protein. 91

Influences on glycosylation

Protein structure

As mentioned in Oligosaccharide structures, above, the presence of the consensus sequence Asn-X-SerFFhr does not, by itself, guarantee N-glycosylation. Thr at position 3 leads to an increased chance of glycosylation compared to Ser at this position, and a proline residue within or near this sequence reduces the likelihood of glycosylation. 130 In addition, potential glycosylation sites nearer the N-terminus are more likely to be occupied, which may reflect temporal competition between protein folding and initiation of N-glycosylation.13°

In contrast to N-glycosylation, there does not appear to be a defined consensus sequence for O-glycosylation, although some patterns have been deduced from a study of human von Willebrand Factor flanking sequences around the O-glycosylated Thr residue. TM Substitution of any amino acid at positions + 3, - 3 , and - 2 was found to markedly decrease the level of O-glycosylation, as did the presence of a charged residue at position - 1.

The mutagenesis by SDM of individual glycosylation sites has enabled the study of glycan interactions within the same protein. For example, SDM of each of the four glycosylation sites on human protein C has revealed that the core glycosylation status of Asn329 is influenced by the presence of a glycan structure on Asn97.132 However, not enough individual glycan structures on recombinant proteins have been manipulated to determine if this type of domain interaction is common.

Host cell type

With the wide availability of different expression systems for producing recombinant proteins, the choice of host cell type should include considerations of the cell's likely glycosylation capabilities. In contrast to human cells, the most widely used animal host, the CHO cell, is known to lack the functional enzyme a2,6-sialyltransferase, leading to exclu- sively a2,3-1inked terminal sialic acid residues. 133 The CHO cell is also unable to sulfate GalNAc residues, which are a common motif on certain glycoprotein hormones and may modulate their clearance in vivo. 65"67'134 Furthermore, the absence of a functional al,3-fucosyltransferase in CHO cells 135 prevents the addition of peripheral fucose residues (fuc-d,3-GlcNAc), as found in authentic human thyroid- stimulating hormone (TSH).1°8 Because the CHO cell line can readily express a wide variety of recombinant proteins, its glycosylation machinery can be modified to resemble more closely the human profile by transfection of the ap-

133 135 an mu propriate glycosyltransferases. - , In addition, m y - tants of CHO cells have been isolated that display altered glycosylation properties that may prove useful hosts for expressing glycoproteins with minimal heterogeneity. 136-139

Other host cell lines have not been studied in sufficient depth to completely define their glycosylation capabilities. Mouse cell lines are able to synthesize sulfated sugars and


Glycosylation of recombinant proteins: IV. Jenkins and E. 114. A. Curling

possess both functional c~2,3- and e~2,6-sialyltransferases, as in humans. 14°,141 However, unlike humans and CHO cells, they express the al,3-galactosyltransferase gene, 142,143 which may result in glycoproteins that are potentially antigenic to humans (see Oligosaccharide structures). Even the use of human cell lines as hosts is not without difficulties, since the transformation event required in most cases to produce stable cell lines may itself result in altered glycosylation profiles. 144-147

The effects of changing the physical parameters of cell culture have also been investigated. Mild or even severe hypoxia has minimal effects on the glycosylation of tPA produced by recombinant CHO ceUs. 158 Similarly, pH changes within the range 6.9-8.2 in the cell culture medium do not have a dramatic effect on the glycosylation profile of a recombinant glycoprotein hormone expressed in CHO cells; however, there is some evidence for under- glycosylation at pH levels outside this range. 159

Culture environment

Perhaps the most difficult problem for the production of authentic recombinant glycoproteins is the growing realiza- tion that the culture environment itself can change the glycosylation profile of a protein. 831,13 We have found that the macroheterogeneity of IFN--,/glycoforms changes dramat- ically during batch culture of recombinant CHO cells (Fig- ure 2), resulting in substantial amounts of nonglycosylated IFN--/(30%) by the end of the culture period. 34 Cells held at low growth rates in glucose-limited chemostat cultures showed glycosylation impairment: there was an increase in the levels of nonglycosylated IFN--/secreted compared to faster growing cells. 148 This change may have resulted from changes in glucose metabolism (due to limiting the culture on glucose) or a lack of key nucleotide sugars (e.g., UDP- GlcNAc) essential to the assembly of oligosaccharides on glycoproteins. Pulsed additions of glucose caused a rapid improvement in the proportion of fully glycosylated IFN--,/ secreted and cell growth increased, but the glycosylation deteriorated once the injected glucose had been depleted. 99 The expression levels of glycosyltransferase may also be influenced by culture conditions; for example, the activity of GlcNAc-transferase V has been correlated with the growth rate of HepG2 cells. 149

Growth factors are often added to increase the produc- tivity of cells in serum-free medium,15° but these may influence glycosyltransferase activity. For example, it was shown that interleukin-6 added to the medium of a myeloma cell line reduced N-acetylglucosaminyltransferase III (GnT- III) activity, but increased GnT-IV and GnT-V activity, leading to altered glycan structures.151 Activation of an endogenous fucosyltransferase can occur during the process of adaptation to suspension growth in serum-free medium, which can result in the expression of the oncofetal antigen sialylated Lewis-X. 151

Improvements in product yields have been reported using butyrate induction of several recombinant and endogenous proteins. 152-154 However, this compound also affects glycosylation by inducing a core-2-GlcNAc-transferase activity of the O-glycosylation pathway, 155 and reducing the level of ¢~2,6-sialyltransferase by a posttranscriptional mechanism.156 The fact that butyrate treatment does not always lead to improved product yields may be related to its induction of chaperone-type proteins such as GRP78 and GRP94 that bind to under-glycosylated or misfolded proteins. 157 Together, the evidence suggests that culture conditions that cause an increase in cell growth rate can lead to the induction of certain glycosyltransferases, thereby altering the sequence of the carbohydrate structures expressed.

Method of cell culture

Most comparisons of the glycosylation profiles resulting from different methods of cell culture have been made using immunoglobulins produced by hybridomas. 160,161 Mon- oclonal IgG1 produced in ascites tumors was found to be deficient in sialic acid, whereas hybridomas grown in serum-based media produced under-galactosylated glycan structures. 162 In contrast, cells grown in serum-free medium had higher levels of terminal sialic acid and galactose residues. In a separate study, IgM antibody was compared in ascites, serum-free suspension, or serum-containing hollow-fiber perfusion culture. 163 The ascitic material again showed less sialylation, but interestingly showed an 80-fold increase in residence time after injection into rats. However, a closely related IgM monoclonal antibody was found to have a similarly long residence time when grown in airlift suspension, suggesting that multiple factors affect antibody pharmacokinetics. The common practice of initi- ating clinical trials on ascites-derived antibody may therefore lead to misleading conclusions, should the eventual culture method be large-scale production in serum-free medium.

Summary and future prospects

The science of glycobiology has become highly significant to the biotechnology industry in recent years, due to the large number of recombinant glycoproteins now in clinical use or under development. Intense efforts are underway to im- prove both the analytical techniques used to measure glycoprotein heterogeneity and to control the biosynthetic pathways of glycosylation. Improvements in analytical pro- cedures now offer the prospect of producing detailed glycan analysis during or soon after fermentation of recombinant cells. This, together with a more detailed knowledge of the glycoprotein biosynthetic pathways, may lead to methods of controlling glycan heterogeneity using special media for- mulations or supplements during fermentation.

The cloning of glycosyltransferases is likely to continue at a fast pace, and will highlight the differences between the glycosylation capabilities of different cell types. Supple- menting the cell's endogenous machinery with cloned glycosyltransferase genes has already been achieved for some

3 133 135 164 ,, enzymes,- . . . . and this type of glycosylation engineering" will complement the use of glycosylation mutants

136 O for the production of specific glycoforms. As the biol g- ical roles of specific glycan structures become apparent, it may become desirable to manipulate genetically or physio- logically the host cells for the biased production of certain glycoforms. Alternatively, downstream processing techniques may be developed to select for different glycoforms in the cell culture medium.


Reviews

In the m o r e d i s tan t fu ture , advances in ca rbohydra te chemistry, 165 a ided pe rhaps by r e c o m b i n a n t glycosyltransferases, may lead to complex ol igosacchar ides be ing bui l t synthet ical ly a n d graf ted o n t o r e c o m b i n a n t p ro te ins m a d e in prokaryot ic systems. Un t i l this proves efficient, an ima l cells will r e m a i n the first choice for the p roduc t i on of most h u m a n r e c o m b i n a n t glycoproteins .

Acknowledgement

T h e a u t h o r s wish to a c k n o w l e d g e the s u p p o r t o f the S E R C B i o t e c h n o l o g y D i r e c t o r a t e for f u n d i n g the i r re- s ea rch p r o g r a m .

Abbreviations

C E C G C H O C P M P

e . r .

E P O E S - M S F A B - M S

F D A

G a l G a l N A c Glc G l c N A c G P I h G - C S F

H P A E - P A D

I F N L D - M S L H M a n N M R O S T P N G a s e F S D M t P A T S H

capil lary e lec t rophores is chor ion ic g o n a d o t r o p i n Ch inese h a ms t e r ovary C o m m i t t e e for P ropr i e t a ry Medica l P roduc t ions of the E u r o p e a n C o m m u n i t y endop la smic r e t i cu lum ery th ropo ie t in e lec t rospray mass spec t romet ry fas t -a tom b o m b a r d m e n t mass spec t romet ry F o o d a n d D r u g A d m i n i s t r a t i o n ( F D A ) in the U n i t e d States galactose N-ace ty lga lac tosamine glucose N-ace ty lg lucosamine glycophosphat idyl inosi to l h u m a n granulocyte colony- s t imula t ing factor high p H a n i o n exchange c h r o m a t o g r a p h y in t e r f e ron laser deso rp t ion mass spec t romet ry lu te in iz ing h o r m o n e m a n n o s e n u c l ea r magne t i c r e sonance ol igosaccharyl t ransferase pep t ide N-glycosidase F s i te -d i rec ted mu tagenes i s t issue p l a s m i n o g e n act ivator thyro id-s t imula t ing h o r m o n e

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Glycosylation of recombinant proteins: Problems and prospects

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