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Glycobiology vol. 16 no. 9 pp. 833–843, 2006 doi:10.

1093/glycob/cwl004 Advance Access publication on May 22, 2006

GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli

Shawn DeFrees3, Zhi-Guang Wang3, Ruye Xing3, Arthur E. Scott3, Jin Wang3, David Zopf1,3, Dominique L. Gouty4, Eric R. Sjoberg4, Krishnasamy Panneerselvam4, Els C.M. Brinkman-Van der Linden2,4, Robert J. Bayer4, Mads A. Tarp5, and Henrik Clausen5
Technologies, Inc., 102 Witmer Road Drive, Horsham, PA 19044; Neose Technologies, Inc., 6330 Nancy Ridge Road, Suites 102–103, San Diego, CA 92121; and 5Department of Medical Biochemistry and Genetics, Glycobiology, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark Received on February 8, 2006; revised on May 11, 2006; accepted on May 13, 2006
3 Neose 4

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-a2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use. Key words: G-CSF/glycosylation/GM-CSF/IFN-α2b/ PEGylation Introduction Modification of protein drugs by covalent attachment of polyethylene glycol (PEG) can improve physical and thermal stabilities, protect against degradation by enzymes, enhance solubility, prolong circulating half-life, and, in some cases, reduce immunogenicity of the polypeptide (Katre, 1993; Francis et al., 1998; Harris et al., 2001; Harris and Chess, 2003). The circulatory half-life of a parenterally administered therapeutic protein may be extended significantly by covalent attachment of one or more chains of PEG. For example,

1

To whom correspondence should be addressed; e-mail: dzopf@neose.com 2 Present address: Noordwijkerhout 2211 AR, Schaepmanlaan 19, The Netherlands

conjugation of 20 kDa PEG to the amino terminus of Escherichia coli-produced G-CSF (Filgrastim; Amgen, Thousand Oaks, CA) gives a product (Pegfilgrastim; Amgen) whose plasma half-life in humans is increased by a factor of ∼20 (Zamboni, 2003; Neupogen, 2005). PEGylation of proteins represents a significant manufacturing challenge in that coupling reactions between chemically activated PEG and free amino groups, even after extensive optimization, commonly result in multiple PEGylated isoforms with nonuniform chemical and pharmaceutical properties (Kinstler et al., 1996; Francis et al., 1998; Foser et al., 2003; Jolling et al., 2004). Drug activity typically relies upon contact of a properly folded active site of a protein agonist with the complementary region of its cognate receptor. In the past, considerable effort has gone into tailoring a PEG coupling strategy for each individual protein drug to avoid modification of its active site (Harris and Chess, 2003). Chemically activated PEGs that react with lysyl or histidyl residues have generated successful long-acting products from a subset of drug proteins, but reactivities of target substrates vary in response to local chemical environments (e.g., charge) created by neighboring amino acids at the surface of the folded polypeptide, and for many proteins, alternative approaches have proved necessary (Katre, 1993; Harris and Chess, 2003). These have included altering amino acid sequences by the introduction of an unpaired cysteine that can be selectively PEGylated (Yang et al., 2003), random mutagenesis and high-throughput screening to select bioactive muteins lacking lysyl groups in the region of the active site (Yoshioka et al., 2004), and the use of the enzyme transglutaminase to introduce alkyl PEG on to glutamine residues (Sato, 2002). We have designed a novel strategy for site-directed enzymatic PEGylation based on the finding that sialic acid covalently substituted with PEG at the 5′-amino position can be enzymatically transferred to glycan acceptors on glycoproteins. Several clinically important drugs such as granulocyte colony stimulating factor (G-CSF), interferonalpha2b (IFN-α2b), and granulocyte/macrophage colony stimulating factor (GM-CSF) are naturally O-glycosylated human glycoproteins but are manufactured by recombinant expression in E. coli as nonglycosylated polypeptides. In the work described here, we utilized a simple two-step process for site-directed PEGylation using enzymatic GalNAc O-glycosylation followed by enzymatic PEGylation of the introduced O-glycans (Figure 1). Escherichia coliexpressed G-CSF, IFN-α2b, and GM-CSF proteins were selectively GalNAc O-glycosylated at their natural O-glycosylation sites using specific recombinant human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) isoforms. The selection of the appropriate GalNAc-transferase

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© 2006 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Once incorporated. the GalNAc residue may serve as a direct acceptor for PEGylated sialic acid transferred by ST6GalNAc-I. Thus.S. Downloaded from glycob. GalNAc-T2. The enzyme reactions can be run separately with intermediate purification of the product or in a single vessel without intermediate purification steps. screening short 15–20mer peptide sequences derived from a protein of interest by in vitro glycosylation assays with GalNAc-T isoforms is predicted to provide reasonable assessment of GalNAc-Ts function with the protein. studies indicate that the primary sequence context is the major determining factor for substrate specificities (Hassan et al. chemically homogeneous GlycoPEGylated protein with extended plasma half-life. Monitoring reactions by matrix-assisted laser desorption ionization-time-offlight mass spectrometry (MALDI-TOF/MS) time-course analysis revealed that the glycosylation of G-CSF and IFN-α2b . We used short synthetic peptides covering natural O-glycosylation sites in G-CSF (Figure 2A). Enzymatic PEGylation of the introduced GalNAc O-glycan was achieved with sialyltransferases that can transfer sialic acid conjugated with PEG. Starting materials (left) are nonglycosylated recombinant polypeptides expressed in Escherichia coli. Once incorporated. for example. The process forms a biologically active. DeFrees et al. 2011 Fig. isoform was based on a simple screening assay of substrate specificities with short synthetic peptides covering O-glycan acceptor sites of interest. for example. Sequential in vitro enzyme-mediated reactions are employed to carry out site-directed O-glycosylation and subsequent PEGylation at the O-glycan site. coli-produced unglycosylated cytokines. Although distinct consensus 834 acceptor sequence motifs have not been elucidated. Results Selection strategy for GalNAc-T isoforms for site-directed O-glycosylation GalNAc-T isoforms have unique and partly overlapping acceptor substrate specificities. (B) Polypeptides such as GM-CSF may contain multiple closely positioned natural nonutilized O-glycosylation sites (orange) in which a serine (S) or threonine (T) residue may serve as an acceptor for selective addition of GalNAc ( ) by a polypeptide GalNAc transferase. natural nonutilized O-glycosylation sites (orange) in which a serine (S) or threonine (T) residue may serve as an acceptor for selective addition of GalNAc ( ) by a polypeptide GalNAc transferase.org by guest on April 3. GalNAc-T2. and GM-CSF (Figure 4A) to evaluate GalNAc-T isoforms suitable for site-directed GalNAc glycosylation of the E. IFN-α2b (Figure 3A). 1. the product of which creates an acceptor for transfer of PEGylated sialic acid (✧-PEG) by ST3Gal-1.oxfordjournals. 2000). (A) Polypeptides such as G-CSF and IFN-α2b produced in this way may contain a single. Depiction of GlycoPEGylation strategies.. the GalNAc residues may serve as an acceptor for transfer of galactose (❍) by Core1 GalT.

and/or Thr10) (Kaushansky et al. and GM-CSF recombinant proteins was obtained as predicted from the screening assay with the GalNAc-T2 isoform. (D) Survival of E. or G-CSF-PEG-20K. IFN-α2b. coli-produced (nonglycosylated) G-CSF. pharmacokinetics.org by guest on April 3. (B) MALDI-TOF/MS spectra of Escherichia coliproduced G-CSF before (top) and after (bottom) in vitro O-glycosylation with GalNAc-T2.. (C) SDS–PAGE analysis of purified preparations of E. 1992. Results are normalized at each time point to the pre-dose vehicle control value. G-CSF(GalNAc-SiaPEG-20K).GlycoPEGylation of recombinant therapeutic proteins Downloaded from glycob.oxfordjournals. and bioactivity of G-CSF(GalNAc-SiaPEG-20K). peptides were essentially complete. Forno et al. Analysis. Recombinant forms of these cytokines expressed in mammalian cells are similarly O-glycosylated. (A) MALDI-TOF/MS spectra of a synthetic peptide including amino acids 126–139 of G-CSF before (top) and after (bottom) O-glycosylation with GalNAc-T2. Concentrations of G-CSF and PEGylated variants were determined by ELISA (see Materials and Methods). (E) Peripheral blood white blood cell (WBC) response in mice following a single intravenous dose (250 mg/kg) of E. but when expressed in E. respectively). coli. while several isoforms including T2 were candidates for glycosylation of IFN-α2b (Figure 3A). while GM-CSF is O-glycosylated at multiple sites (Ser5. they are not. Among the six human recombinant GalNAc-Ts tested. 1987. coli-produced (nonglycosylated) G-CSF (Filgrastim). Lane 1 contains molecular weight standards labeled as kilodaltons. and G-CSF-PEG-20K (Pegfilgrastim) in plasma after intravenous bolus injection in rats. coliproduced G-CSF (lane 2) after in vitro O-glycosylation with GalNAc-T2 (lane 3) and after addition of SiaPEG-20K with ST6GalNAc-I (lane 4). Ser9.. Native G-CSF and IFN-α2b are O-glycosylated at a single site (Thr133 and Thr106. coli-expressed cytokines Site-specific GalNAc glycosylation of the reported natural O-glycosylation sites in G-CSF. 2011 Fig. while the glycosylation of the GM-CSF peptide did not go to completion with two and three residues of GalNAc incorporated. MALDI-TOF/ MS analysis suggested incorporation of a single-GalNAc 835 . 2004). GalNAc-T2 was identified as the only candidate isoform for glycosylation of G-CSF (Figure 2A) and GM-CSF (Figure 4A). 2. GlycoPEGylation of E. Ser7. G-CSF-GalNAc-SiaPEG-20K.

coli-produced IFN-α2b and IFN-α2b(GalNAcSiaPEG-20K) after intravenous bolus injection in rats. we used this feature of ST6GalNAc-I for efficient transfer of sialic acid-PEG from CMP-sialic acid-PEG-20K to the enzymatically introduced GalNAc residues in G-CSF(GalNAc) and IFN-α2b(GalNAc). representing 124LGMAPALQPT133QGAMPAFASAFQR146 plus GalNAc. 2011 Fig. Analysis. (B) MALDI-TOF/MS spectra of Escherichia coliproduced IFN-α2b before (top) and after (bottom) in vitro O-glycosylation with GalNAc-T2. representing 97ACVIQGVGVT106ETPLMKE113 plus GalNAc) was shown by MS/MS peptide sequencing to 836 be glycosylated at T106.27.. and bioactivity of IFN-α2b(GalNAc-SiaPEG-20K). coliproduced IFN-α2b (lane 2) after in vitro O-glycosylation with GalNAc-T2 (lane 3) and after addition of sialic acid-5-N-PEG-20K with ST6GalNAc-I (lane 4).oxfordjournals. Sialyltransferases including ST6GalNAc-I were initially found to transfer cytidine monophosphate (CMP)-sialic acid with 20 kDa PEG linked through the 5′-amino nitrogen of the sialic acid residue to simple acceptor substrates (C. (C) SDS–PAGE analysis of purified preparations of E. which was further confirmed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) analysis (Figures 2C and 3C). (E) Antiviral activity of IFN-α2b (nonglycosylated) and IFN-α2b(GalNAc-SiaPEG-20K) when MDBK cells were challenged with VSV. Here. 3. (A) MALDI-TOF/MS spectra of a synthetic peptide including amino acids 100–113 of IFN-α2b before (top) and after (bottom) O-glycosylation with GalNAc-T2. Downloaded from glycob. Ions for the corresponding unglycosylated peptides were not observed. GalNAc-T2 incorporated two GalNAc residues into GM-CSF at Ser7 and Ser9. et al.org by guest on April 3. residue into G-CSF and IFN-α2b (Figures 2B and 3B). manuscript in preparation). The reaction proceeded essentially to . pharmacokinetics. DeFrees et al. (D) Survival in circulation of E.69. Similarly. and a third site of low incorporation was not identified. Peptide mapping of a GluC plus trypsin digest of G-CSF(GalNAc) by liquid chromatography – mass spectrometry/mass spectrometrometry (LC-MS/MS) confirmed the presence of a doubly charged peptide ion 1282. a doubly charged ion from GalNAc-T2-glycosylated IFN-α2b (1018. in which GalNAc was shown by MS/MS peptide sequencing to be linked to T133 (data not shown).S. Lane 1 contains molecular weight standards labeled as kilodaltons. Bowe.

peak 1. 4.001% Tween-80. (B) MALDI-TOF/MS spectra of Escherichia coli-produced GM-CSF before (top) and after (bottom) in vitro O-glycosylation with GalNAc-T2. with trace incorporation after prolonged incubation into a third site which we have not identified. GlycoPEGylation of GM-CSF followed a modified strategy and did not result in similar homogenous glycosylation products (Figure 1B).oxfordjournals. a bulky PEG substituent at one site sterically hinders approach of the enzyme to an acceptor GalNAc linked to a nearby amino acid. peak 2.3 mL/min. 10 × 300 cm) eluted with phosphate-buffered saline. and lane 4. pH 5. protein markers. (D) SDS–PAGE analysis of fractions pooled from chromatography illustrated in (C): lane 1. A doubly charged ion from a peptide digest of the glycosylated GM-CSF protein product (1235. and two residues with traces of one and three residues were incorporated into the GM-CSF protein (Figure 4A and B). lane 3.GlycoPEGylation of recombinant therapeutic proteins Downloaded from glycob. 2011 Fig. GalNAc-T2 incorporated two to three GalNAc residues into the short synthetic peptide after prolonged incubation (22 h).0 plus 0.org by guest on April 3. Preliminary experiments showed that ST6GalNAc-I could add only one mole of SiaPEG-20K to GM-CSF(GalNAc)2. Numbers are m/z values followed in parentheses by the number of added GalNAc residues. and the final purified products appear as single bands by SDS–PAGE analysis (Figures 2C and 3C). (C) Purification of GM-CSF (GalNAc-Gal-SA-PEG-20K)2 (peak 2) from GM-CSF[GalNAc](GalNAc-Gal-SA-PEG-20K) (peak 3) and a trace component (peak 1) assumed to be GM-CSF(GalNAc-Gal-SA-PEG-20K)3 on Superdex 200 (Amersham. and Edman degradation was used to determine that the major residues substituted with O-GalNAc were Ser7 and Ser9 (data not shown).00. representing 4RS5PS7PS9T10QPWEHVNAIQE21 plus two GalNAc residues) was identified. lane 2. We hypothesized that addition of a sugar residue “spacer” to each GalNAc 837 . presumably because once added. (A) LC/EMS spectra of a synthetic peptide including amino acids 1–16 of GM-CSF before (top) and after (bottom) O-glycosylation with GalNAc-T2. Fractions were pooled as indicated. Thus. flow rate 0. the combined data from MS/MS peptide sequencing and chemical sequencing by Edman degradation indicate that in vitro O-glycosylation by GalNAc-T2 attaches GalNAc residues almost exclusively to Ser7 and Ser9. completion. Analysis of GM-CSF[GalNAc](GalNAc-Gal-SA-PEG-20K). peak 3.

8 pg/mL compared with 439 pg/mL for IFN-α2b (Figure 3E). G-CSF(GalNAc-SiaPEG20K). coli (Figure 3D). and 0. coli. 1999). 1985a. therefore. and G-CSF were 1. SDS–PAGE analysis shows that both the singly and doubly PEGylated products migrate as single bands with only a faint trace of doubly and triply PEGylated products. We. Studies in an animal model that has generally proved predictive of myelogenic responses to G-CSF in Downloaded from glycob. the same dose of unmodified E. 2011 838 .3 G-CSF (GalNAc-SiaPEG-20K) 2470 0. coli-produced G-CSF raised the WBC count only 2.74 h). The calculated area under the curve for G-CSF(GalNAc-SiaPEG-20K) is increased Table I. respectively.. G-CSF(PEG-20K). a response profile comparable with.. G-CSF(GalNAc-SiaPEG-20K) caused the peripheral blood white blood cell (WBC) count to increase to a maximum 5.org by guest on April 3. with return to baseline at 48 h.0896 7. 1999).71 41.761 26.6-fold above baseline after 48 h with return to baseline at 84 h. Bioactivity of G-CSF and IFN-a Comparative analysis of binding affinities of PEGylated and nonPEGylated G-CSF variants for the G-CSF receptor demonstrated that receptor-binding properties of the molecules were largely preserved: Ki values for G-CSF(GalNAc-SiaPEG-20K). Pharmacokinetic data for G-CSF.71 h). When combined in a single reaction mixture.718 50. Feasibility of the strategy was confirmed with two therapeutic proteins. and G-CSF(PEG-20K) (E. DeFrees et al. Rotondaro et al.oxfordjournals.225 2.1 × 104 U/μg.8 × 104. and ST3Gal-1 is known to transfer sialic acid in α-linkage to the 3-O-position of galactose linked β1-3 to GalNAc (Blixt et al. 1985b. G-CSF(GalNAcSiaPEG-20K). After chromatographic separation. Specific activities of the three compounds as stimulators of in vitro proliferation of NFS-60 murine myeloid leukemia cells were 1.4 nM. Steric maps constructed from X-ray crystal data show that the glycosylation sites of these cytokines are remote from their active sites (Radhakrishnan et al.2 49. which are currently in clinical use. and G-CSF(PEG-20K) in blood plasma following intravenous injection in rats G-CSF C0 (ng/mL) Ke (1/h) t1/2z (h) MRT (h) AUC0–t (ng*h/mL) AUC0–∞ (ng*h/mL) Cl (mL/h/kg) Vz (mL/kg) Vss (mL/kg) 2170 0. that of G-CSF(PEG-20K) (Figure 2E). a value comparable with the t1/2 for G-CSF(PEG-20K) (7. by uncertainties regarding metabolic turnover of the [125I]-labeled protein more than 18 h after intravenous administration. GlycoPEGylated IFN-α2b(GalNAc-SiaPEG-20K) also showed a significantly slower clearance rate compared with the unmodified IFN-α2b protein produced in E. followed by SiaPEG-20K using ST3Gal-1 proceeds under mild buffer conditions.S. whereas addition of a second SiaPEG-20K proceeds to only 75% (Figure 4C.6 G-CSF (PEG-20K) 2080 0. Discussion This study demonstrates an alternative strategy for sitedirected attachment of PEG chains by the enzymatic process termed GlycoPEGylation.74 13. coli-produced protein). however. 2004).406 1.71 1. Aritomi et al. This data indicate that the pharmacokinetic profile of G-CSF(GalNAc-SiaPEG-20K) is comparable with that of a chemically PEGylated G-CSF molecule in current clinical use and may provide a slightly improved prolonged systemic exposure following a single dose. PEGylation of the two O-linked GalNAc residues occupying adjacent amino acid residues on GM-CSF(GalNAc)2 was therefore achieved via a different pathway. Like the addition of SiaPEG-20K by ST6GalNAc-I.. might provide a more favorable substrate for the addition of two SiaPEG-20K residues and found that at least 75% of polypeptide molecules can be converted to the di-PEGylated form using this approach.2 28.984 3. Pharmacokinetics of GlycoPEGylated G-CSF and IFN-a Comparative analysis of plasma concentrations of G-CSF (unmodified E. but increased more than 5-fold over t1/2 for G-CSF (1. The strategy relies on enzymatic transfer of GalNAc to natural O-glycosylation sites that are not glycosylated in the recombinant proteins expressed in E. When injected as a single intravenous dose in mice. peak 1). 1996. which is comparable with the reported value for rhG-CSF of 105 U/μg (Neupogen.8-fold with respect to G-CSF(PEG-20K).2. 2002. glycosylation is not required for folding or receptor binding.4 49.78 14. and its selectivity for the O-linked GalNAc carbohydrate acceptor assures a well-defined product. Precise calculation of pharmacokinetic parameters for radiolabeled protein is compromised. 3. respectively. 3. and 9. lane 2 and Figure 4C..14. 2005).0 25.5 13-fold with respect to G-CSF and 1. or somewhat better than.2 26.0 41. employing two enzymes in a “one-pot” reaction: Core1 GalT is known to add galactose in β-linkage to the 3-O-position of GalNAc (Ju et al.4 65. coli-produced G-CSF with 20 kDa PEG chemically coupled to the amino terminus) at various time points following bolus intravenous administration in rats shows that the clearance of GlycoPEGylated G-CSF(GalNAc-SiaPEG-20K) similar to G-CSF(PEG-20K) was reduced more than 10-fold compared with G-CSF (Figure 2D and Table I).4 × 104. The terminal phase plasma half-life observed for G-CSF(GalNAc-SiaPEG20K) was 8. coli as nonglycosylated polypeptides (Bodo and Maurer-Fogy.8-fold at 24 h.78 h. respectively (Figure 4D).0790 8. hypothesized that the attachment of O-glycans extended with PEG to the natural O-glycosylation sites would not impair their biological activities. A trace of triply PEGylated product was detected in some preparations (Figure 4D. 2002). the addition of Gal using Core1 GalT. G-CSF and IFN-α2b. the enzymes act consecutively such that addition of the first SiaPEG-20K proceeds essentially to completion. peak 2). By contrast.. Since both G-CSF and IFNα2b proteins retain their respective biological activities when expressed in E. The ED50 of IFN-α2b(GalNAc-SiaPEG20K) in the Madin–Darby bovine kidney (MDBK)-vesicular stomatitis virus (VSV) assay was 16. Jeanneau et al.57 3778 3788 26..

. chST6GalNAc-I (GenBank accession No. The GalNAc-T family contains up to 20 isoforms that transfer GalNAc to serine and/or threonine residues (Hassan et al. although the products were more heterogeneous than the products for G-CSF and IFN-α2b. Since preliminary studies showed that the transfer of SiaPEG-20K by ST6GalNAc-I was incomplete. We have shown that this sialyltransferase can efficiently utilize a modified sugar nucleotide donor in which sialic acid is substituted at the 5′-amino position with a 20 kDa linear PEG chain. pilot scale galactosylation of N-glycans of human immunoglobulin (IgG) to create molecules with >98% G2 glycoform was performed using recombinant β4Gal-T1 in a reaction containing 1 kg of IgG-acceptor glycoprotein (Warnock et al. NJ) and washed with 5 column volume (CV) of buffer A (200 mM NaCl. MA. The applied screening strategy using a few synthetic peptides that include natural O-glycosylation sites from a protein of interest rapidly identifies one or more isoforms useful for O-glycosylation at the corresponding natural site in the complete protein. A slightly modified pathway for GlycoPEGylation of GM-CSF was developed. 2002). was used. In recent years. Gal. GalNAc-T6 (Bennett et al.... Introduction of O-linked GalNAc residues in proteins provides an acceptor site for several other glycosyltransferases that can add sialic acids. 2000). 2000). Enzyme was loaded onto an SP Sepharose FF cation-exchange column (Pharmacia.. MA.. 1989) and milk fucosyltransferase catalyzed transfer of fucose substituted at the C-6 position (Tsuboi et al. scaled-up production of recombinant glycosyltransferases overexpressed in mammalian or fungal host cell systems has provided soluble enzyme reagents suitable for industrial in vitro remodeling of glycan chains attached to pharmaceutically active glycoproteins. 2004)... and expressed in Sf9 cells. 25 mM 2-[4-morpholino]-ethane sulfonic acid [MES]. 2001) demonstrated that G-CSF(GalNAc-SiaPEG-20K) has a pharmacodynamic profile consistent with prolonged ability to stimulate proliferation and differentiation of stem cells to granulocytes. These results provide further support for the importance of the primary sequence context in acceptor substrate specificities of GalNAc-Ts (Hassan et al. we have employed the sialyltransferase ST6GalNAc-I.. an enzyme. AD-0021. IFN-α2b (GalNAc-SiaPEG-20K) exhibits antiviral titers comparable with the specific activities reported for chemically PEGylated interferons approved for human use (Youngster et al. 2002). the combined use of recombinant ST3Gal-III and FT-VI to link sialic acid and fucose. Several reports by others have previously demonstrated that glycosyltransferases may catalyze transfer of sugars with substituents.oxfordjournals. (Horsham.. 2003). their contribution to overall cost of manufacturing may be offset by the value of enhanced performance characteristics of the product.. As these enzymes are required in relatively small amounts relative to the glycoprotein protein drug (∼0. 2004).. Here.. These include sialyltransferase ST6Gal-1 catalyzed transfer of sialic acids substituted at the C-9 position (Gross et al. Bowe. C. La Jolla. and GalNAc-T3 (Wandall et al. Foser et al. 2002. was expressed in insect cells and purified to homogeneity. Filgrastim and Pegfilgrastim are products of Amgen (purchased from Besse Pharmaceutical Supply).. we demonstrate a novel approach for sitedirected enzymatic attachment of large PEG groups to the recombinant therapeutic proteins G-CSF. Similarly. 839 . 1998). 2005). et al.6sialyltransferase (ST6GalNAc-I) accession No. highly site-selective mechanism for PEGylation. 2000.. Canton. giving a chemically uniform product with wellpreserved bioactivity. GalNAc-T4 (Bennett et al. or GlcNAc monosaccharides. For example. Glycosyltransferase enzymes Soluble secreted human polypeptide GalNAc transferases were expressed in insect cells and purified as described previously for GalNAc-T1. Downloaded from glycob. 1995).org by guest on April 3. In conclusion. ITC-DTPAEuropium labeling kits were obtained from Perkin Elmer. Similarly. Catalog No. Viral stock was plaque-purified. respectively. 1999).1% on a mass basis). Transfer occurs exclusively to single-GalNAc residues introduced in G-CSF and IFNα2b... 2003). to create Slex epitopes at the termini of biantennary N-linked glycans on the human complement inhibitor sCR1 was carried out at the 10 g scale (Thomas et al. ST3Gal-1. Inc. 1997). and GalNAc-T11 (Schwientek et al. DeFrees et al. Soluble secreted chicken sialyltransferase. Wellesley.. and this study demonstrates that the unique substrate specificity of these enzymes can be used for site-directed enzymatic O-glycosylation of proteins. a substituent considerably larger than any previously described. PA. 2011 Materials and Methods Materials CMP-sialic acid with 20 kDa linear methoxypolyethylene glycol linked to the 5′-amino nitrogen of the sialic acid residue (CMP-SiaPEG-20K) was prepared at Neose Technologies. GM-CSF and IFN-α2b were purchased from Cell Sciences. galactosylation to form the core 1 structure Galβ1-3GalNAcα before transfer of SiaPEG20K with a different sialyltransferase. enabling the manufacture of long-acting protein drugs with greater structural homogeneity as compared with PEGylated proteins prepared by conventional chemical methods (Roberts et al. and GM-CSF. Selective addition of sialic acid-PEG to O-linked GalNAc on a protein provides a novel. which naturally transfers sialic acid from the sugar nucleotide donor CMP-sialic acid onto the 6-O-position of O-linked GalNAc (Kurosawa et al. Piscataway. The GlycoPEGylation strategy relies on two unique features of glycosyltransferases: the unique substrate specificities of polypeptide GalNAc-T isoforms and the ability of glycosyltransferases to use modified donor nucleotide substrates. GalNAc-T2. The product exhibited improved pharmacokinetic properties and a 10-fold increase in affinity for E-selectin.. Ten Hagen et al. IFN-α2b. thereby reducing clearance via receptor-mediated endocytosis (Kuwabara et al. A non-plaque-purified baculovirus stock of recombinant chicken CMP-sialic acid: N-acetylgalactosaminide α2. X74946).GlycoPEGylation of recombinant therapeutic proteins humans (Lord et al. 1990) and to decrease cognate receptor affinity.. GalNAc-T2 introduced two GalNAc residues in close proximity (Ser7 and Ser9). 2000). manuscript in preparation. CA). PEGylation of G-CSF has been proposed both to reduce renal clearance (Tanaka and Tokiwa. X74946 was a gift from Dr Jim Paulson (Scripps Institute. amplified.

25 mM Tris. The pooled product was concentrated as before and stored in the elution buffer at 4°C.5. pH 6.5 mU GalNAc-Ts in 25 μL reaction mixtures containing 0.8 mmol) UDP-Gal.2 mL of the same buffer containing 400 mU ST6GalNAc-I and 0.2 mg/200 μL) were further purified by HPLC on a Superdex 75 column (HR 10/30. 10 mM MnCl2. 25 mM MES buffer. coli was GalNAc glycosylated with GalNAc-T2 (40 mU) in a reaction mixture of 1 mL containing 9 mM UDP-GalNAc. and 0. pH 6. Amersham) eluted with 25 mM sodium acetate plus 0. plus 0. was purified by HPLC on SP Sepharose eluted with 25 mM NaOAc.05% polysorbate 80. 2011 IFN-a2b. 1 mg of GCSF(GalNAc) was incubated in a final volume of 1 mL containing 200 mU ST6GalNAc-I. GM-CSF.2. (GalNAc)G-CSF. This construct was transfected into Sf9 cells using the BaculoGold system (Invitrogen. was reconstituted into 1. Screening enzyme reactions were performed with 0. pH 6. and 4 mM MnCl2. Pooled fractions were concentrated with 5 kDa MWCO spin filter. The enzyme was loaded in 10 CV and washed to A280 baseline with 5 CV buffer A. pH 7. pH 7. and purified further using Superdex 200. A 1-mL aliquot of the reaction mixture was buffer exchanged into 150 mM NaCl. 25 mM MES. which was washed with buffer A (25 mM NaCl. and 0. and 0. coli-expressed cytokines G-CSF. Additional CMP-SiaPEG-20K (6 mg. Recombinant GM-CSF produced in E. 0. Carlsbad. concentrated.5 M ammonium sulfate and loaded onto a Phenyl 650C column equilibrated with 5 CV of buffer A (0. 20 mM sodium phosphate. Reaction products were quantified by scintillation counting after chromatography on Dowex 1X8 or analyzed by high-performance liquid chromatography (HPLC) and MALDI-TOF.2. 5 mM MgCl2. The product.0) to A280 baseline.5.. 100 μM UDP-[14C]-GalNAc (3200 cpm/nmol) (Amersham Pharmacia Biotech.4). Pooled fractions were concentrated as before and stored in the elution buffer at 4°C. was purified by HPLC size exclusion 840 chromatography on successive 10 × 300 mm Superdex 75 and Superdex 200 columns (Amersham) eluted at 1 mL/min with 0.25 mL 25 mM MES buffer containing 6 mg (9.S. G-CSF(GalNAcSiaPEG-20K). MALDI-TOF/MS analysis showed the reaction was complete at 24 h. The reaction mixture .0)..005% NaN3 at room temperature.3 mL aqueous solution containing 20 mM MES buffer. pH 7. 6 mg (0. The enzyme was eluted with 1.0 plus 0.2 mM UDP-GalNAc. FF 1 mL.3 μmol) was added. pH 4. Secreted enzyme was loaded onto a Q Sepharose FF column. 20 mM sodium phosphate. The active ST6GalNAc-I fraction pool from the SP Sepharose FF elution was adjusted to 0. 20 mM MES. The resulting mixture was slowly rotated at room temperature for 24 h. LD20186.4. The active fractions were pooled. and 100 mM MnCl2. and 0.005% NaN3.25 mM CMP-SiaPEG-20K. 1997). and aliquots (0. 5 mM MgCl2. 10 × 300 mm.3 μmol) CMP-SiaPEG-20K. IFN-α2b(GalNAc-SiaPEG-20K). 1. as described above. was purified by HPLC on SP Sepharose (HiTrap SP.6-sialyltransferase (ST6GalNAc-I) and its use for semipreparative synthesis of glycopeptides have been described (Kurosawa et al. and ∼250 μM peptide substrate (Wandall et al. Drosophila UDP-Gal: GalNAcα β1. 0. 0. Recombinant G-CSF (960 μg) produced in E. Core1 GalT-1.005% polysorbate 80.5 CV of TBS.0. The yield based on starting protein measured by absorbance at 280 nm was >95%. After concentration and buffer exchange into 25 mM NaOAc. followed by Superdex 75 eluted with 20 mM sodium phosphate. 50 mM Tris. coli (1 mg. Blixt et al. pH 7. 80 mU GalNAc-T2. 25 mM Tris.12 mL at 32°C with slow rotary mixing.25 mM MnCl2 at room temperature. 5 mM MnCl2. was subcloned in frame into pACGP67. Active fractions from the Phenyl 650C step were pooled and concentrated to 4 mL with a 10K MWCO Amicon Ultra and loaded onto a Superdex 200 Hiload 16/60 column that was equilibrated with 1.0). with gentle rocking at 32°C for 72 h. pH 7. 2002) accession No. pH 6.005% polysorbate 80. and 0. 0. The enzyme was eluted by a 20 CV gradient from 99% buffer A/ 1% buffer B (1 M NaCl. CA) and expressed in Sf9 cells according to the manufacturer’s instructions. The product. MALDI-TOF/MS analysis showed the reaction to be complete after 96 h. pH 6.3 galactosyltransferase (Core1 GalT) (Ju et al. 20 mM sodium phosphate.005% NaN3. IFN-α2b(GalNAc). Screening of GalNAc-transferase isoforms for site-directed O-glycosylation Semi-purified recombinant human GalNAc-Ts were tested for the capacity to transfer GalNAc to synthetic peptides derived from sequences covering potential O-glycan acceptor sites in proteins of interest. The enzyme was eluted with a 20 CV elution gradient from 0 to 100% buffer B (1 M NaCl.0). pH 7.. Amersham) eluted with 150 mM NaCl.0 plus 0.15 M NaCl. Glycosylation and PEGylation of E. DeFrees et al.05% NaN3 in 2. MALDI-TOF/ MS indicated that the formation of GM-CSF(GalNAc)2 was complete.2.42 mM CMP-SiaPEG-20K.5. Piscataway.5 [Tris-buffered saline (TBS)]. 25 mM Na-cacodylate.01% Tween-80) until material absorbing at 280 nm was no longer detectable. pH 4. and incubation continued at room temperature for an additional 24 h. pH 6. NJ).5 CV of 150 mM NaCl.org by guest on April 3. after 72 h. 5 mM MnCl2. 4 mM MnCl2.05% NaN3 using a Centricon 5 kDa MWCO filter spin cartridge. 0. 40 mU Core1 GalT-1.75 M ammonium sulfate.2. The resulting product.05% polysorbate 80.005% polysorbate 80. 0. 150 mM NaCl. pH 7. pH 4. 20 mM MES (pH 7. truncated at E48. and 6.1–0. coli was GalNAc-O-glycosylated with GalNAc-T2 (80 mU). 2002).069 μmol) was incubated in a 1. 120 mU ST3Gal-1. followed by concentration to a final volume of 1 mL using a Centricon filter (MWCO 5 kDa). The acceptor specificity of recombinant chicken CMP-NeuAc: GalNAcα α2.2. pH 6. After buffer exchange into 25 mM MES. as measured by a molecular weight increase of 406 KDa. GM-CSF(GalNAc)2 (1 mg) was reconstituted into 1. The enzyme was eluted with a 10 CV elution gradient from 0 to 100% buffer B (20 mM sodium phosphate. Downloaded from glycob. 1994.oxfordjournals. 3 mM UDP-GalNAc.005% polysorbate 80 at 1 mL/min for 10 min. 150 mM NaCl. and the mixture was incubated at 32°C with slow rotary mixing for 96 h. the product.5 M NaCl in the same buffer.01% Tween-80) to 100% buffer B.. Recombinant IFN-α2b (4 mg) produced in E.25% Triton X-100. 0. followed by gradient elution with 0–0.

dissolved in 30% aqueous acetonitrile. The antibody was labeled with ITC-DTPAEuropium according to the manufacturer’s instructions (Perkin Elmer).1% formic acid gradient at a flow rate of 3 μL/ min.5-dihydroxybenzoic acid. 1982). at a flow rate of 0. G-CSF competition binding assay A competition binding assay to estimate the affinity of G-CSF GlycoPEGylated variants to human G-CSF receptor was constructed using a truncated G-CSF receptor/IgG-Fc chimera immobilized on microtiter wells coated with protein A. The ELISA (LOD 0. PA).org/). followed by a linear gradient to 100% buffer B (2 M NaCl.org by guest on April 3. Samples containing [125I]-radiolabeled IFN-α2b or IFN-α2b(GalNAcSiaPEG-20K) (10 μCi. Viral inhibition assay Viral inhibition titers for IFN-α2b and derivatives were performed at PBL BioMedical Laboratory (Piscataway. Pharmacokinetic calculations Mean serum radioactivity or drug concentration–time data for each compound were analyzed using noncompartmental methods (Gibaldi and Perrier. and the peptides were eluted from the column by an ACN/0. Foster City.GlycoPEGylation of recombinant therapeutic proteins was concentrated to 1 mL. Matrix for peptides was 2. NIH reference Gxa01-901-535). St.005% polysorbate 80. G-CSF. All mass spectra were obtained in the positive mode. AF-214-NA) as a trapping antibody and europium-labeled murine antihuman G-CSF (R&D Catalog No. The AUC was estimated with the linear trapezoidal rule and extrapolated to time infinity (last concentration value/Ke). MAB214) as a detection antibody. MO). One microliter volume of the digest was injected. Details of the assay are given in Supplementary Data.5 mL in a 5000 MWCO centrifugal filter. Acknowledgments We thank Bruce Mico and Tom Stevenson for helpful insights regarding preparation of this manuscript and GloriaMay Machado for excellent technical assistance.3% trifluoroacetic acid. Pharmacokinetic studies Proteins were formulated in physiological saline. Funding for this work was provided to Henrik Clausen by the Danish Cancer Society and the Danish Medical Research Council. Samples were tested in duplicate against an international reference standard (human IFN-α2b. 20 μCi/μg) (Amersham) or unlabeled G-CSF. 25 mM NaOAc.3 mL/min. NJ) using MDBK cells challenged with VSV according to procedures described by Familletti and others (1981). and macrophage colony stimulating factor (M-CSF) (Nakoinz et al. and 0. Structural analysis of peptides and proteins MALDI-TOF mass spectrometry was performed on a Voyager-DE MALDI time-of-flight mass spectrometer (PerSeptive Biosystems.5 mL in a 5000 MWCO centrifugal filter. 0. and study protocols were reviewed by the Institutional Animal Care and Use Committees at Calvert Preclinical Services (Olyphant. pH 5.0 kV.0. buffer exchanged with buffer A (25 mM NaOAc.5 min was pooled and concentrated as before. 0. The terminal elimination halflife (t1/2) was calculated as 0. and concentrated to 2.0). For peptide mapping. The concentrated protein solution was further purified on an Amersham HiLoad Superdex 200 (10 × 300 mm) eluted with 0.oxfordjournals.005% polysorbate 80. G-CSF(GalNAc-SiaPEG-20K). Clearance (Cl) was estimated by dividing dose by AUC0–∞. protein samples were digested by GluC with or without trypsin overnight at 37°C and loaded on an LC-MS system (Finnigan LCQ-classic ion trap) with an electrospray ion source interfaced to a 15 cm × 300 μm id LC Packings PepMap reversed-phase capillary chromatography column. 1990) and are used in a cell proliferation assay described in Supplementary Data. The resulting solution was purified on an Amersham HiTrap SP Sepharose FF column (5 mL) with isocratic elution of 100% buffer A for 10 min. 2011 Supplementary Data Supplementary data are available at Glycobiology online (http://glycob. pH 6. G-CSF(GalNAc-SiaPEG-20K). The electrospray ion source was operated at 4. Data processing was carried out using GRAMS/386 software. pH 6. Concentration values at time zero (C0) for area under the concentration–time curve (AUC) estimates were obtained by fitting concentration– time profiles to a one-compartment model and using the y-intercept for the time zero values. The peak eluting at 25% buffer B was collected and concentrated to 1. All animal studies were conducted in compliance with the USDA Animal Welfare Act and the PHS Policy on Humane Care and Use of Laboratory Animals. or heparinized plasma was assayed by enzyme-linked immunosorbent assay (ELISA) to determine concentrations of G-CSF.. Downloaded from glycob. A peak containing the desired product eluting at 28.005% polysorbate 80.05% polysorbate 80.4 ng/mL) was performed utilizing goat anti-human G-CSF (R&D Catalog No.15 M NaCl plus 20 mM sodium phosphate. CA) equipped with delayed extraction. 25 g/L (Sigma.693/Ke. Funding 841 . Louis. and G-CSF(PEG-20K).5-dimethoxy-4-hydroxycinnamic acid (Sigma) 100 g/L dissolved in 30% acetonitrile in 0. The terminal elimination rate constant (Ke) was estimated by log-linear regression of plasma concentration values in the apparent terminal elimination phase.0) over 20 min at a flow rate of 3 mL/min. G-CSF-dependent NFS-60 cellular proliferation assay NFS-60 cells are responsive to interleukin-3. pH 6.5% mannitol and 0. The MALDI matrix for the proteins was 3. At timed intervals after injection.oxfordjournals. or G-CSF(PEG-20K) (3 μg protein/rat) were injected as a bolus via the tail vein in rats (five animals per time point).5 plus 2. whole blood samples were withdrawn via the jugular vein and assayed for radioactivity in a gamma counter to determine IFN-α2b or IFN-α2b(GalNAc-SiaPEG-20K). Concentrations of injected proteins were measured by HPLC by comparing eluted sample peaks monitored at A280 nm with a bovine serum albumin (BSA) standard.

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