4 Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands; and 5 Pharming Technologies BV, Archimedesweg 4, P.O. Box 451, NL-2300 AL Leiden, The Netherlands
Received on April 1, 2004; revised on July 5, 2004; accepted on July 6, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C1 inhibitor / glycosylation / lactation / transgenic rabbit milk
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant glycoprotein production in the mammary gland of transgenic animals is very promising for large-scale economic production, especially for recombinant proteins that require mammalian glycosylation as provided by the host's mammary gland. Recent examples that have reached clinical trials include antithrombin III and tissue plasminogen activator from transgenic goat milk, -glucosidase from transgenic rabbit milk, and
-antitrypsin from sheep milk (Niemann and Kues, 2003
).
The ultimate requirements for any therapeutic glycoprotein are safety, efficacy, and consistency in therapeutic potential. For glycosylation this implies consistency across recombinant glycoprotein batches, with nonimmunogenic glycans that facilitate the required efficacy in vivo. As opposed to cell culture approaches (Werner, 1998), little is known about the glycosylation machinery of the mammary gland in the various animal species used for recombinant glycoprotein production. In view of the hormonal regulation of protein glycosylation throughout lactation (Vijay, 1998
), it is important to investigate whether glycosylation of a transgenic glycoprotein remains consistent throughout lactation, even at the level of the individual animal. Subtle variations in glycan profiles might arise due to differences between individual animals that are used to breed the transgenic colony, reflecting their unique genetic make-up (e.g., transgene copy number, genomic integration site).
Human serum C1 inhibitor (hC1INH) has six N- and seven O-glycosylation sites (Perkins, 1993). Recently, we have analyzed in detail the N- and O-glycosylation pattern of recombinant human C1 inhibitor (rhC1INH) expressed in the milk of transgenic rabbits using a pooled milk sample. The carbohydrate content of rhC1INH was found to be 14%, and Fuc, Man, Gal, GalNAc, GlcNAc, and Neu5Ac were present in the molar ratio of 0.2:3.0:2.4:1.7:2.6:1.3. Neu5Gc did not occur. An ensemble of at least 20 different N-glycans, comprising oligomannose-, hybrid-, and [(
2-6)-sialylated, fucosylated] diantennary complex-type structures, was found. The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicated that besides the usual N-glycan processing route the alternative endo-mannosidase pathway is also followed. For the O-glycans, four core 1 type structures were established (Koles et al., 2004
).
The aim of our present study was to systematically explore the N-glycosylation pattern of rhC1INH throughout lactation in low- and high-expressor lines and to compare these with the glycan profiles observed on seven large-scale batches of rhC1INH produced for (pre)clinical evaluation of the biopharmaceutical. In addition, to demonstrate glycosylation consistency, the monosaccharide composition of 20 additional batches was investigated. For comparison, attention was also paid to differences in N-glycan profiles of hC1INH, rhC1INH, and hC1INH expressed in the mammary glands of mice (rm-hC1INH).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
N-Glycosylation profiles of hC1INH, rm-hC1INH, and rhC1INH
In Figure 1 the high-performance liquid chromatography (HPLC) profiles of the peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F (PNGase F) released and 2-aminobenzamide (2-AB)-labeled N-glycans of hC1INH, rm-hC1INH, and rhC1INH (rabbit J, samples of early and late stages of lactation) are depicted. Fractionation on weak anion-exchange phase GlycoSepC (Figure 1A) and on normal phase GlycoSepN (Figure 1B) columns showed that the degree of sialylation is higher for the N-glycans of hC1INH and rm-hC1INH than for those of rhC1INH. The occurrence of trisialylated components on hC1INH (Figure 1A) is in agreement with earlier findings (Perkins et al., 1990), but these structures were absent on rm-hC1INH and rhC1INH. In Figure 1B, the four major N-glycans have been indicated: mono- and di-(
2-6)-sialylated partially core-fucosylated complex-type structures (codes: 14). They were verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and sequential exoglycosidase digestions (data not shown; see also Koles et al., 2004
). The linkage details for the most complete structure is illustrated below.
|
|
Sialylation throughout lactation
To further explore the details of sialylation throughout lactation, the GlycoSepC N-glycan profiles of rhC1INH from two different lactations of the high-expressor rabbits AF and the low-expressor rabbits GL were compared. Typical N-glycan profiles of rhC1INH from a low-expressor line (rabbit L, 1st lactation) and a high-expressor line (rabbit F, 3rd lactation) are shown in Figures 2A and 2B, respectively. Comparison of these profiles shows that the N-glycans from high-expressor rabbits contain a relatively higher portion of neutral glycans (eluting near the void volume, Vo). For both groups, with the progress of lactation, a steady decrease in the ratio of di- to monosialylated N-glycans was observed (Figures 2C and 2D; circles). Total sialic acid analysis of the various rhC1INH samples revealed a similar pattern (Figures 2C and 2D, triangles), supporting that the overall sialic acid content decreased with the progress of lactation in both groups. It should be noted that the total sialic acid analysis comprises sialic acids on both the N- and O-linked glycans of rhC1INH.
|
|
|
High-expressor lines produce rhC1INH with higher amounts of oligomannose-type N-glycans
Evaluation of all GlycoSepN N-glycan profiles of the 12 individual rabbits indicated a trend for higher amounts of Man5GlcNAc2 in the rhC1INH samples of the high-expressor lines when compared with the samples of low-expressor lines. In low-expressor lines, the relative amount of Man5GlcNAc2 is very low or strongly reduced throughout lactation. These data suggest that expression levels influence the amount of oligomannose-type N- glycans on rhC1INH. To examine this relationship further, an additional series of seven animals were selected with rhC1INH expression levels ranging from 1 g/L to 16 g/L, and the proportion of Man5GlcNAc2 in the respective N-glycan pools was determined at the early (days 26) and the late (days 2327) stages of lactation. The results, shown in Figure 5, suggest a relationship between rhC1INH expression levels and oligomannose-type N-glycan content. As opposed to the earlier stages of lactation, where no significant correlation could be found (r2 = 0.33, p = 0.23), later stages of lactation showed a significant correlation between expression level and Man5GlcNAc2 content (r2 = 0.84, p = 0.01). The lack of correlation at early stages could be explained by the higher individual variations during this period (see Figures 3 and 4).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The various results show that sialylation levels and total rhC1INH-bound sialic acid content decreased with the progress of lactation independent of expression levels. These findings are in accordance with earlier data reported for a few native human and bovine milk glycoproteins. A decrease in protein-bound sialic acid after the first 2 weeks of lactation has been reported for pooled human milk glycoproteins (Carlson, 1985). A change in the glycosylation status of the human milk bile saltstimulated lipase during lactation has been described, whereby sialic acid levels were at highest at the early stages of lactation (Landberg et al., 2000
). The two bovine milk glycoproteins MGP 57 and MGP 53 were more negatively charged immediately after parturition than at later stages, probably due to higher sialylation at the early stages (Aoki et al., 1994
). The observations suggest that a decrease in sialic acid content during lactation in mammals might be a general phenomenon. Because sialylation level plays an important role in the pharmacokinetic properties of many glycoproteins, understanding the biochemical details of this down-regulation might help maximize the degree of sialylation of recombinant proteins.
Two interesting observations were made in relation to the oligomannose-type N-glycans found on rhC1INH. First, the amount of oligomannose-type glycosylation strongly decreased during lactation, even though the expression levels remained constant. Because oligomannose (and neutral hybrid) type N-glycans are readily recognized by the mannose receptors present on epithelial cells and macrophages, resulting in enhanced clearance from the circulation, for therapeutic applications, where a long plasma residency is of importance, a low amount of such structures is desirable. In addition to a decrease in the oligomannose and sialic acid content, a significant decrease in core fucosylation was also seen during lactation. This could be an advantage when considering expression of recombinant monoclonal antibodies in transgenic animals, because recently it has been shown that the absence of core fucose on human IgG strongly increases its antibody-dependent cellular cytotoxicity (Shinkawa et al., 2003). Second, a correlation between the oligomannose-type N-glycan content and expression levels of rhC1INH, particularly at late stages of lactation, was noticed. This suggests that at a high level of expression, the glycosylation machinery of the mammary epithelial cells may become limiting.
It is worth pointing out that on pooled rhC1INH the total amount of oligomannose-type glycan is on average 15% of total, and the majority of the N-glycans (55%) are of the complex type (Koles et al., 2004). These findings are remarkable for a heavily glycosylated glycoprotein expressed at such high levels (12 g/L), illustrating the huge glycosylation capacity of the rabbit mammary gland.
Finally, the differences that were observed at the individual level were virtually absent when analyzing large pools of purified rhC1INH. Indeed, both the N-glycan profiles and monosaccharide composition of different batches were remarkably consistent.
Further detailed studies in other transgenic species might help unravel the contribution of species-specific factors to the glycosylation profile of individual recombinant glycoproteins, like rhC1INH, and this in turn might aid the choice for the most suitable host animal. In this context the observed differences in sialylation between recombinant hC1INH produced in the mammary glands of mice and rabbits are quite striking indeed.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The hC1INH and rm-hC1INH (from pooled milk of a transgenic mouse line expressing the glycoprotein on a g/L scale) samples were gifts of Pharming Technologies BV.
Sialic acid quantification
rhC1INH (20 µg) in 20 µl 20 mM sodium phosphate buffer, pH 7.2, was incubated with 40 mU Arthrobacter ureafaciens sialidase (Roche Molecular Biochemicals, Mannheim, Germany) in 20 µl of the same buffer for 1 h at 37°C. Then 70 µl water and 50 µl 40 µM 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid (internal standard; Toronto Research Chemicals, Toronto, Canada) were added, and 20 µl of the mixture was analyzed by HPAEC-PAD on a Dionex DX-500 system, equipped with a CarboPac PA-1 column (4.0 x 250 mm) and a CarboPac PA-100 guard column (4.0 x 50 mm). Sialic acids were eluted isocratically with 125 mM sodium acetate in 150 mM NaOH for 15 min at a flow rate of 1.0 ml/min. Neu5Ac was quantified using a linear calibration curve of standard Neu5Ac (Sigma, St. Louis, MO).
Monosaccharide analysis
To 50 µg rhC1INH in 267 µl 20 mM sodium phosphate buffer, pH 7.2, was added 133 µl 3638% HCl (final concentration, 4 M HCl). The mixture was incubated for 3 h at 80°C and then dried under vacuum in a SpeedVac; the residue was dissolved in 50 µl water. Prior to chromatography, 50 µl 2-deoxy-D-glucose (15.0 µg/ml) was added as internal standard. Each sample (20 µl) was analyzed by HPAEC-PAD (CarboPac PA-1), using a sodium acetate gradient from 075 mM in 150 mM NaOH (10 min; flow rate, 1.0 ml/min). For the quantification of the monosaccharides, calibration curves were used. All peak areas were corrected for the peak area of the internal standard.
Enzymatic release of N-glycans
Samples of 100 µg rhC1INH (50 µl) were denatured in 10% sodium dodecyl sulfate (w/v) for 5 min at 100°C and digested with 5 U/mg PNGase F (Roche Molecular Biochemicals, Indianapolis, IN) in 20 mM phosphate buffered saline, pH 7.2, containing 10 mM ethylenediamine tetra-acetic acid, 10 mM ß-mercaptoethanol, and 6.4 mg/ml 3-[(3-cholamidodopropyl)dimethylammonio]-1-propane sulfonate (Fluka, Buchs, Switzerland) for 24 h at 37°C. Digests were filtered through 30-kDa cutoff centrifugal filters (Nalgene), and filtrates were treated with Calbiosorb beads (Calbiochem, San Diego, CA) to remove detergents. Released N-glycans were purified on graphitized carbon columns (Alltech, Breda, The Netherlands) and then fluorescently labeled using 0.35 M 2-AB (Sigma) and 1 M sodium cyanoborohydride in a mixture of dimethylsulfoxide:acetic acid (7:3) for 2 h at 65°C (Koles et al., 2004). The 2-AB-labeled glycans were purified via paper chromatography on acid-pretreated quartz fiber filter paper strips (Millipore, Dublin, Ireland), eluted with water, and analyzed by HPLC.
HPLC
N-Glycan profiles at 30°C were obtained on GlycoSepC (4.6 x 100 mm) and GlycoSepN (4.6 x 250 mm) columns (Glyko, Novato, CA), using a Waters 2690 HPLC system equipped with a fluorescence detector (exc.max = 373 nm,
em.max = 420 nm). For weak anion-exchange chromatography on GlycoSepC, two solvent systems were used: solvent A, 50% 500 mM ammonium formate, pH 4.5, 30% HPLC-quality water, and 20% acetonitrile (Biosolve, Valkenswaard, The Netherlands); and solvent B, 80% HPLC-quality water and 20% acetonitrile. After running 100% B for 5 min, charged N-glycans were eluted with a linear gradient of 0100% A over 35 min at a flow rate of 0.4 ml/min. For normal phase chromatography on GlycoSepN, solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was 20% 50 mM ammonium formate, pH 4.4, in acetonitrile. The gradient consisted of a linear increase of A from 6.5% to 43.8% over 100 min at a flow rate of 0.8 ml/min, followed by 100% B for 10 min at a flow rate of 1 ml/min, and a reequilibration step to starting conditions for 25 min at a flow rate of 0.8 ml/min.
MALDI-TOF MS
For experimental details, see Koles et al. (2004).
Exoglycosidase digestions
The following enzymes and digestion conditions were used: ß-galactosidase (Streptococcus pneumoniae; Calbiochem), 24 mU per 25 µl digest in 100 mM sodium acetate buffer, pH 6.0; ß-N-acetylhexosaminidase (jack bean; Glyko), 0.250.5 U per 25 µl digest in 100 mM sodium phosphate/citrate buffer, pH 5.0; -fucosidase (bovine kidney, Glyko), 5 mU per 25 µl digest in 100 mM sodium citrate buffer, pH 6.0; sialidase (A. ureafaciens, Glyko), 25 mU per 25 µl digest in 100 mM sodium acetate buffer, pH 5.0. Combined digestions (i.e., more than one exoglycosidase in the digestion mixture) were performed in 100 mM sodium acetate buffer, pH 5.5. Approximately 3080 pmol of 2-AB-labeled glycans were used for the digestions, which were performed for 18 h at 37°C. Prior to HPLC analysis the samples were deproteinated through a 5-kDa cut-off centrifugal filter (Millipore, Bedford, MA), and the digested glycan mixtures, still containing the buffer salts, were analyzed by HPLC on GlycoSepN. A 2-AB-labeled dextran hydrolysate served as external calibration standard.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
1 Present address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128
2 Present address: GenMab BV, Jenalaan 18d, NL-3584 CK Utrecht, The Netherlands
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aoki, N., Ujita, M., Kuroda, H., Urabe, M., Noda, A., Adachil, T., Nakamura, R., and Matsuda, T. (1994) Immunologically cross-reactive 57 kDa and 53 kDa glycoprotein antigens of bovine milk fat globule membrane: isoforms with different N-linked sugar chains and differential glycosylation at early stages of lactation. Biochim. Biophys. Acta, 1200, 227234.[ISI][Medline]
Archer, D.B. (1994) Enzyme production by recombinant Aspergillus. Bioproc. Technol., 19, 373393.
Bakker, H., Bardor, M., Molthoff, J.W., Gomord, V., Elbers, I., Stevens, L.H., Jordi, W., Lommen, A., Faye, L., Lerouge, P., and Bosch, D. (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc. Natl Acad. Sc. USA, 98, 28992904.
Carlson, S.E. (1985) N-acetylneuraminic acid concentrations in human milk oligosaccharides and glycoproteins during lactation. Am. J. Clin. Nutr., 41, 720726.[Abstract]
Cregg, J.M., Lin Cereghino, J., Shi, J., and Higgins, D.R. (2000) Recombinant protein expression in Pichia pastoris. Mol. Biotechnol., 16, 2352.[ISI][Medline]
Fischer, R. and Emans, N. (2000) Molecular farming of pharmaceutical proteins. Transgenic Res., 9, 279299.[CrossRef][ISI][Medline]
Frenken, L.G.J., Hessing, J.G.M., van den Hondel, C.A.M.J.J., and Verrips, C.T. (1998) Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. Res. Immunol., 149, 589599.[CrossRef][ISI][Medline]
Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., and Dwek, R.A. (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem., 240, 210226.[CrossRef][ISI][Medline]
Houdebine, L.M. (2000) Transgenic animal bioreactors. Transgenic Res., 9, 305320.[CrossRef][ISI][Medline]
Jenkins, N. and Curling, E.M.A. (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microbial Technol., 16, 354364.[CrossRef][ISI][Medline]
Jenkins, N., Parekh, R.B., and James, D.C. (1996) Getting the glycosylation right: implications for the biotechnology industry. Nature Biotechnol., 14, 975981.[ISI][Medline]
Koles, K., van Berkel, P.H.C., Pieper, F.R., Nuijens, J.H., Mannesse, M.L.M., Vliegenthart, J.F.G., and Kamerling, J.P. (2004) N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits. Glycobiology, 14, 5164.
Landberg, E., Huang, Y., Strömqvist, M., Mechref, Y., Hansson, L., Lundblad, A., Novotny, M.V., and Påhlsson, P. (2000) Changes in glycosylation of human bile-salt-stimulated lipase during lactation. Arch. Biochem. Biophys., 377, 246254.[CrossRef][ISI][Medline]
Larrick, J.W. and Thomas, D.W. (2001) Producing proteins in transgenic plants and animals. Curr. Opin. Biotechnol., 12, 411418.[CrossRef][ISI][Medline]
Lin Cereghino, G.P., Lin Cereghino, J., Illgen, C., and Cregg, J.M. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol., 13, 329332.[CrossRef][ISI][Medline]
Ma, J.K.-C., Drake, P.M.W., and Christou, P. (2003) The production of recombinant pharmaceutical proteins in plants. Nature Rev. Genet., 4, 794805.[CrossRef][ISI][Medline]
Molowa, D.T. and Mazanet, R. (2003) The state of biopharmaceutical manufacturing. Biotechnol. Annu. Rev., 9, 285302.[Medline]
Niemann, H. and Kues, W.A. (2003) Application of transgenesis in livestock for agriculture and biomedicine. Animal Reprod. Sci., 79, 291317.[CrossRef][ISI]
Panda, A.K. (2003) Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli. Adv. Biochem. Eng. Biotechnol., 85, 4393.[Medline]
Perkins, S.J. (1993) Three-dimensional structure and molecular modelling of C1 inhibitor. Behring Inst. Mitt., 93, 6380.[Medline]
Perkins, S.J., Smith, K.F., Amatayakul, S., Ashford, D., Rademacher, T.W., Dwek, R.A., Lachmann, P.J., and Harrison, R.A. (1990) Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering. J. Mol. Biol., 214, 751763.[ISI][Medline]
Punt, P.J., van Biezen, N., Conesa, A., Albers, A., Mangnus, J., and van den Hondel, C. (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol., 20, 200206.[CrossRef][ISI][Medline]
Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M., Hanai, M., and Shitara, K. (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem., 278, 34663473.
Swartz, J.R. (2001) Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol., 12, 195201.[CrossRef][ISI][Medline]
Vijay, I.K. (1998) Developmental and hormonal regulation of protein N-glycosylation in the mammary gland. J. Mammary Gland Biol. Neoplasia, 3, 325336.[CrossRef][ISI][Medline]
Werner, R.G. (1998) Innovative and economic potential of mammalian cell culture. Arzneimittelforschung, 48, 423426.[ISI][Medline]
|