US 2011 O150820A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0150820 A1 Van Gelder et al. (43) Pub. Date: Jun. 23, 2011

(54) METHODS FOR COVALENTLY ATTACHING Related U.S. Application Data A POLYMER TO A METHONNE RESIDUE IN AND PEPTIDES (60) Provisional application No. 61/136,332, filed on Aug. 28, 2008. (75) Inventors: Joel M. Van Gelder, Jerusalem (IL); Andrei Serban, Publication Classification Rishon-LeZion (IL); Susanna (51) Int. Cl. Tchilibon, Jerusalem (IL); Daphna A6II 47/48 (2006.01) Miron, Rechovot (IL) C08G 65/48 (2006.01) A6IP37/04 (2006.01) (73) Assignee: Insight Biopharmaceuticals Ltd., Rehovot (IL) (52) U.S. Cl...... 424/78.17:525/54.1: 525/418 (21) Appl. No.: 13/060,439 (57) ABSTRACT (22) PCT Fled: Aug. 27, 2009 Conjugates of polypeptides and a polymeric moiety Such as PEG covalently attached to the sulfur atom of a methionine (86) PCT NO.: PCT/ILO9AO0832 side chain are disclosed. Processes of preparing Such conju gates, including intermediates and reagents utilized therefore S371 (c)(1), are also disclosed. Further disclosed are therapeutic uses of (2), (4) Date: Feb. 24, 2011 these conjugates. Patent Application Publication Jun. 23, 2011 Sheet 1 of 23 US 2011/O150820 A1

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1a. US 2011/O 150820 A1 Jun. 23, 2011

METHODS FOR COVALENTLY ATTACHING linking, via covalent attachment, of one or more poly(ethyl A POLYMER TO A METHONNE RESIDUE ene glycol) (PEG) chains thereto. IN PROTEINS AND PEPTIDES 0008. The optimization of both the polymerization proce dure and purification process allowed the development of FIELD AND BACKGROUND OF THE PEGs with low polydispersity, spanning from 1.01, for PEG INVENTION below 5kDa molecular weight, to 1.1 for PEG with molecular 0001. The present invention relates to methods of attach weight as high as 50 kDa. ing a polymeric moiety to active proteins and peptides, and, 0009 Alcohol dehydrogenase can degrade low molecular more particularly, to methods of attaching polyethylene gly weight PEGs, and chain cleavage can be catalyzed by P450 col and/or related polymeric moieties to therapeutically active proteins and peptides so as to improve the pharmaco microsomal enzymes. PEG has been used for several years as logical performance thereof. an excipient in foods, cosmetics and pharmaceuticals and is 0002 Most therapeutic proteins are short-lived and have considered non-toxic and therefore FDA-approved for human often a short circulatory half-life in vivo, and therefore the use. The first common positive effect of PEGylation is an pharmaceutical uses thereof are critically limited by their in extended half-life in the bloodstream due to reduced kidney vivo and ex vivo instability and by their poor pharmacokinet filtration and clearance. Consequently, a PEGylated thera ics. This is particularly valid for non-glycosylated proteins of peutic requires reduced frequency of administration a molecular mass less than 50 kDa. with respect to a non-PEGylated protein. Moreover, PEG is a 0003. The short lifetime of proteins in vivo is attributed to highly suitable polymer for protein conjugation in the context several factors, including glomerular filtration in the kidney, of therapeutic purposes due to the lack of immunogenicity and proteolysis in the stomach, bloodstream and liver. More and antigenicity on its part. PEG has been shown to decrease over, proteins are typically characterized by poor absorption immunogenicity of the protein presumably by protecting the after oral ingestion, in particular due to their relatively high protein from being recognized as foreign antigen by the molecular mass and/or the lack of specific transport systems. owing to coverage or blockage of critical Proteins are easily degraded in oxidative and acidic environ protein's sites which are recognized epitopes, and by masking ments and therefore typically require intravenous administra specific sequence regions which are degraded by proteolytic tion (so as to avoid, e.g., degradation in the gastrointestinal enzymes. tract). (0010 Site-preferential PEGylation of proteins and 0004 Considering that therapeutic proteins are not polypeptides can be achieved by exploiting the different sur absorbed orally, prolonged maintenance of these therapeuti face accessibility of the protein's amino groups, as demon cally active drugs in the circulation is still a considerable strated for a truncated form of growth hormone-releasing challenge of great clinical importance, since proteins are fur hormone (hGRF1-29), thiol groups, as demonstrated for ther broken down in the blood system and liver by proteolytic beta (IFN-beta), carboxyl groups as demonstrated enzymes and are rapidly cleared from the circulation, and with truncated thrombomodulin mutant, hydroxyl groups as further have the tendency to evoke an immunological demonstrated with epidermal , and also demon response particularly when their sequence is not recognized strated with some protein's guanidine groups. However, site by the host’s immune system. preferred method is often inapplicable, or limited by low 0005. In addition, proteins are heat and humidity sensitive yield, in cases where, for example, the PEGylation is directed and therefore their maintenance requires costly care, complex at a buried or less accessible , and particularly and inconvenient modes of administration, and high-cost of when a high molecular weight PEG is required. Thus, other production and maintenance. The above disadvantages approaches are desired, and some have been designed. impede the use of proteins as efficient drugs and stimulate the 0011 While PEGylation chemistry of proteins through quest for means to alter some of the characteristics of proteins reactions with a variety of reactive amino acids has been so as to bestow robustness and stability thereto. thoroughly investigated, none of the presently known meth 0006. It has been shown that when a suitable polymer is odologies for protein PEGylation teach the attachment of conjugated to a protein, the conjugate polymer endows the PEG to the sulfur atom in the side chain of the amino acid aqueous solubility to the protein, masks potential epitope and residue methionine, albeit chemical modification thereof is proteolysis sites on the protein, and increases the molecular known for many years Glazer, AN, Annu. Rev. Biochem. weight and volume thereof. The attachment of a suitable 39, 101, 1970. This particular amino acid is the second least polymer to a protein has been shown to be efficacious in frequent amino acid in expressed having the single reducing the rate of clearance of the therapeutic protein drug tri-nucleotide codon AUG, and an observed frequency of from the physiological system, reducing renal clearance, 1.8% in vertebrates. Having a unique chemical feature among reducing proteolysis, reducing antigenicity, and increasing all amino acid side chains, thiomethoxy, and being relatively water solubility, while retaining a Substantial proportion of infrequent, this amino acid residue presents a lucrative oppor the biological activity of the protein. tunity for site-directed and discriminating PEGylation of pro 0007 Among all the polymers studied so far, polyethylene teins. glycol (PEG) emerged as an optimal candidate for being 0012. An example of a naturally occurring modification of conjugated to proteins and polypeptides and modification methionine side-chain is S-methylmethionine, also known as thereof due to its uncommon properties, discussed hereinbe “vitamin U”. This compound is produced in plants by the low, which are conferred on the protein-PEG conjugate. This enzyme methionine S-methyltransferase and was identified promising technology for improving pharmaceutical and as an anti-ulcer agent McRolly, R A. et al., J. Am. Chem. clinical properties of therapeutic proteins is known as PEGy Soc., 76, 115, 1954 and as potential agent against nephritic lation. Thus, the term PEGylation defines the modification of hyperlipidemia Seri, K. et al., Arzneim. Forsch. 29, 1517, a protein, peptide or non-peptide molecule by the chemical 1979. In addition, nature also makes extensive use of the US 2011/O 150820 A1 Jun. 23, 2011

S-adenosylmethionine Sulfonium salt as a biological methy tive moiety, the reactive moiety being selected capable of lating agent Grillo, MA and Colombatto, S. Amino Acids, reacting with the sulfur atom of the methylsulfanyl-ethyl 28, 357, 2005). side-chain. 0013 Introduction of a carboxymethyl group (carboxym 0021. According to some embodiments of the invention ethylation) into various side chains of amino acids such as the reactive moiety is selected from the group consisting of cysteine, lysine, histidine and methionine is currently used in amine, carboxyl, amide, acetamide, 2-halo-acetamide, (4-ha chemical modification reactions of proteins Gurd, F R N. lomethyl)-benzamide, benzyl-halide, hydrazine, hydrazide, Methods Enzymol. 1, 532, 1967. The alkylating agents used acetohydrazide, alkyl, haloalkyl, alkyl sulfonylhalide, alkyl in this type of reaction are typically haloacetates or haloac tosylate, alkyl triflate, allyl, haloallyl, allyl sulfonylhalide, etamides. This modification reaction is typically most effec allyl tosylate, allyl triflate, aryl, haloaryl heteroaryl, 4-(ha tive near or above the pH corresponding to the pKa of the lomethyl)benzyl, benzyl and halobenzyl, and any combina individual amino acid, namely for histidine at or above pH=5 tion thereof. and for lysine at or above pH-7. Only in the case of methion 0022. According to some embodiments of the invention ine, the thioether is available for reaction over the entire pH the reactive moiety comprises a leaving group Such as, but not range unless the side chain is in a masked State. Therefore, limited to, halide, acetate, tosylate, triflate, Sulfonate, azide, accessible methionine side-chain residues may be selectively hydroxy, thiohydroxy, alkoxy, cyanate, thiocyanate, nitro and alkylated at an acidic pH, namely pH-4. This reaction has cyano. been used for selective chemical modification of methionines 0023. According to some embodiments of the invention in several proteins see, for example, Gundlach, H.G. et al., J. the linking moiety further comprises a spacer. According to Biol. Chem. 234, 1754, 1959; Holeysovsky, V. and Lazdun Some embodiments of the invention the spacer is selected ski, M., Biochim. Biophys. Acta, 154, 457, 1968; Colma, RF, from the group consisting of a linear or branched, Saturated or J. Biol. Chem., 243, 2454, 1968; Naider, F. et al., Biochem unsaturated, Substituted or unsubstituted alkylene chain hav istry, 11, 3202, 1972: Cheng, K W., Biochem. J., 159, 79, ing 1-30 carbon atoms, and a linear or branched, Saturated or 1976: CZupryn, M.J. et al., J. Biol. Chem.,270,978, 1995; and unsaturated, Substituted or unsubstituted alkylene chain hav Blouin, C. et al., Biochem. Biol. 80, 197, 2002. ing 1-30 carbon atoms interrupted by one or more heteroa 0014. The carboxymethylation reaction has also been used tom, whereby the one or more heteroatom is selected from the for selective binding of methionyl-containing peptides and group consisting of oxygen, Sulfur, nitrogen, phosphor and/or proteins to polymeric resins having a haloacetamide linker silicon. Shechter, Y. et al., Biochemistry, 16, 1424, 1977). Other 0024. According to some embodiments of the invention alkylation agents. Such as benzyl bromide, have also been the polypeptide is selected from the group consisting of an used for selective modification of methionines in proteins interferon, a , a hormone, a growth factor, an enzyme, Such as fumarase Rogers, G. A. et al., J. Biol. Chem..., 251, a blood protein (factor), an antibody, an antigen, a viral pro 5711, 1976), CAMP factor and calmodulin Lang, S. et al., tein, a fusion protein, and any part or segment thereof. Anal. Biochem., 359, 253, 2006. 0025 Representative examples include adalimumab, 00.15 Methionine residues have also been labeled by alky adenosine deaminase, agallsidase-beta, alglucosidase-alpha, lation with dansylaziridine, as in the case of the calcium alpha-galactosidase, asparaginase, B-deleted domainFactor binding component of troponin Grabarek, Z. et al., J. Biol. VIII, bone morphogenetic protein-2 (BMP-2), bone morpho Chem. 258, 14098, 1983 and with epoxides under acidic genetic protein-7 (BMP-7), brain-derived neurotrophic factor conditions Alferiev, I Set al., Biomaterials, 22, 2501, 2001. (BDNF), cetuximab, chorionic gonadotropin (CG), dornase alpha, (EPO), , Factor IX, Factor SUMMARY OF THE INVENTION VIIa, Factor VIII, follicle stimulating hormone (FSH), gal sulfase, glial cell line derived neurotrophic factor (GDNF), 0016 While chemical modification of some proteins at the glucagon, granulocyte colony stimulating factor (G-CSF), side-chain of methionine has been reported to Some extent, granulocyte-macrophage colony stimulating factor (GM proteins which have been modified, at the side-chain of a CSF), growth hormone (GH), hemoglobin, heparanase, methionine residue found in their amino-acid sequence, by hyaluronidase, imiglucerase, infliximab, insulin-like growth moieties such as PEG, which improve the pharmacokinetic factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), inter profile and hence the therapeutic activities of the proteins, feron-alpha-2b (IFNalpha-2b), interferon-beta-1a (IFN beta have not been reported hitherto. 1a), interferon-beta-1b (IFN beta-1b), interferon-gamma-1b 0017. The present inventors have now designed and suc (IFN gamma-1b), (IL-11), interleukin (IL-2), cessfully practiced a novel method of PEGylating proteins via interleukin-1 (IL-1) receptor antagonist, interleukin-1 recep a side chain of a methionine residue within the protein. tor antagonist (IL-1 ra), keratinocyte growth factor (KGF), 0018 Thus, according to one aspect of embodiments of laronidase, luteinizing hormone (LH), megakaryocyte the present invention there is provided a conjugate which growth differentiation factor (MGDF), obesity protein (OB includes (a) a polypeptide having one or more methionine protein or leptin), (OPG), parathyroid hor residue(s), each methionine residue having a methylsulfanyl mone (PTH or 1-34 segment or PTH 1-34), palivizumab, ethyl side-chain; and (b) one or more polymer moieties being platelet-derived growth factor (PDGF). Protein C, rituximab, covalently attached to a sulfur atom of the methylsulfanyl (SCF), streptokinase, thrombin, thrombopoi ethyl side-chain of one or more the methionine residue(s). etin (TPO), thyrotropin (TSH), tissue plasminogen activator 0019. According to some embodiments of the invention (tPA), trastuzumab, binding protein the polymer moiety is covalently attached to the sulfur atom (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate via a linking moiety. oxidase. 0020. According to some embodiments of the invention 0026. According to some embodiments of the invention the linking moiety comprises one or more residues of a reac the polymer moiety is selected from the group consisting of a US 2011/O 150820 A1 Jun. 23, 2011

polyalkylene glycol, a (PEG), a poly (IFN gamma-1b), interleukin (IL-11), interleukin (IL-2), (lactic acid) (PLA), a polyester, a polyglycolide (PGA), a interleukin-1 (IL-1) receptor antagonist, interleukin-1 recep polycaprolactone (PCL), a polyamide, a polymethacryla tor antagonist (IL-1 ra), keratinocyte growth factor (KGF), mide, a polyvinyl alcohol, a polycarboxylate, a polyvinyl laronidase, luteinizing hormone (LH), megakaryocyte pyrrolidinone, a dextran, a cellulose, a chitosan, a hydroxy growth differentiation factor (MGDF), obesity protein (OB ethyl Starch (HES), polyamino acids Such as polyglutamic protein or leptin), osteoprotegerin (OPG), parathyroid hor acid and polyglycine and any copolymer thereof. mone (PTH or 1-34 segment or PTH 1-34), palivizumab, 0027. According to some embodiments of the invention platelet-derived growth factor (PDGF). Protein C, rituximab, the polymer moiety has an average molecular weight that stem cell factor (SCF), streptokinase, thrombin, thrombopoi ranges from about 1 kDa to about 100 kDa. etin (TPO), thyrotropin (TSH), tissue plasminogen activator 0028. According to some embodiments of the invention (tPA), trastuzumab, tumor necrosis factor binding protein the polymer moiety is a polyethylene glycol (PEG). Accord (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate ing to some embodiments of the invention the polyethylene oxidase. glycol has an average molecular weight that ranges from 4 0036. According to some embodiments of the invention kDa to 40 kDa. the first reactive moiety is selected capable of reacting with 0029. According to some embodiments of the invention the Sulfur atom, and can be selected from the group consisting the polypeptide has a characterizing biological activity and of amine, carboxyl, amide, acetamide, 2-halo-acetamide, the conjugate has a biological activity of a kind characterizing (4-halomethyl)-benzamide, benzyl-halide, hydrazine, the polypeptide. hydrazide, acetohydrazide, alkyl, haloalkyl, alkylsulfonylha 0030. According to some embodiments of the invention lide, alkyl tosylate, alkyl triflate, allyl, haloallyl, allyl sulfo the conjugate is soluble in Saline. nylhalide, allyl tosylate, allyl triflate, aryl, haloaryl het 0031. According to some embodiments of the invention eroaryl, 4-(halomethyl)benzyl, benzyl and halobenzyl, and the polypeptide has a characterizing three dimensional struc any combination thereof. ture and the conjugate has a three dimensional structure of a 0037 According to some embodiments of the invention kind characterizing the polypeptide. the first reactive moiety comprises one or more leaving 0032. According to some embodiments of the invention groups such as, but not limited to, halide, acetate, tosylate, the polypeptide has a characterizing three dimensional struc triflate, Sulfonate, azide, hydroxy, thiohydroxy, alkoxy, cyan ture in which one or more methionine residues is positioned at ate, thiocyanate, nitro and cyano. an outer surface of the three dimensional structure. 0038 According to some embodiments of the invention the second reactive moiety is selected from the group consist 0033 According to some embodiments of the invention ing of amine, carboxyl, amide, hydrazine, hydrazide, thiol, the polypeptide has a characterizing three dimensional struc hydroxyl and hydroxylamine, and any combination thereof. ture, in which one or more methionine residues is positioned 0039. According to some embodiments of the invention at an outer Surface of the three dimensional structure, and the the modifying moiety further comprises a spacer connecting polymer forms a stable chemical interaction with the outer the residue of the first reactive moiety and the second reactive Surface at physiological conditions. moiety. 0034. According to some embodiments of the invention 0040. According to some embodiments of the invention the polypeptide has a characterizing half life under physi the spacer is selected from the group consisting of methane ological conditions and the conjugate has a greater half life di-yl, ethane 1-yl-2-yl, propane 1-yl-3-yl, butane 1-yl-4-yl, under the physiological conditions. 1,4-benzene-diyl and 1,10-biphenyl-diyl. 0035. According to another aspect of embodiments of the 0041 According to some embodiments of the invention present invention there is provided a compound which com the compound further comprises a labeling moiety being prises (a) a polypeptide having one or more methionine resi covalently attached to the modifying moiety. dues, each methionine residue having a methylsulfanyl-ethyl 0042. According to some embodiments of the invention side-chain; and (b) one or more modifying moieties, each the labeling moiety is selected from the group consisting of a comprising a residue of a first reactive moiety and a second fluorescent moiety, a radioactive moiety, a magnetic moiety, a reactive moiety. The modifying moiety is covalently attached chromophore, a phosphorescent moiety and a heavy metal to a sulfur atom of the methylsulfanyl-ethyl side-chain of a cluster, and any combination thereof. methionine residue via the residue of the first reactive moiety, 0043. According to another aspect of embodiments of the and the polypeptide is selected from the group consisting of present invention there is provided a conjugate comprising adalimumab, adenosine deaminase, agallsidase-beta, alglu cosidase-alpha, alpha-galactosidase, asparaginase, B-deleted interferon-beta-1b and 30 kDa methoxy polyethylene glycol domain Factor VIII, bone morphogenetic protein-2 (BMP-2), N-ethyl-acetamide, being attached therebetween via a sulfur bone morphogenetic protein-7 (BMP-7), brain-derived neu atom of a methionine residue of the interferon-beta-1b, and rotrophic factor (BDNF), cetuximab, chorionic gonadotropin having a formula: (CG), dornase-alpha, erythropoietin (EPO), etanercept, Fac tor IX, Factor VIIa, Factor VIII, follicle stimulating hormone 30 kDa methoxy polyethylene glycol. (FSH), galsulfase, glial cell line derived neurotrophic factor (GDNF), glucagon, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth hormone (GH), hemoglobin, heparanase, hyaluronidase, imiglucerase, infliximab, insulin-like growth factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), inter interferon-beta-1b feron-alpha-2b (IFNalpha-2b), interferon-beta-1a (IFN beta 1a), interferon-beta-1b (IFN beta-1b), interferon-gamma-1b US 2011/O 150820 A1 Jun. 23, 2011

0044 According to another aspect of embodiments of the erythropoietin and 30 kDa methoxy polyethylene glycol present invention there is provided a conjugate comprising N-ethyl-(4-bromomethyl)-benzamide, being attached ther interferon-beta-1b and 30 kDa methoxy polyethylene glycol ebetween via a sulfur atom of a methionine residue of the N-ethyl-(4-bromomethyl)-benzamide, being attached ther erythropoietin, and having a formula: ebetween via a sulfur atom of a methionine residue of the interferon-beta-1b, and having a formula: S N-1NH S H 30 kDa methoxy erythropoietin O polyetnyolwethylene glyglycol. N-1-N30 kDa methoxy O olwethylene glycol. interferon-beta-1b polyetny gly 0049 According to another aspect of embodiments of the present invention there is provided a conjugate comprising 0045. According to another aspect of embodiments of the granulocyte colony-stimulating factor (G-CSF) and 30 kDa present invention there is provided a conjugate comprising methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben interferon-beta-1a and 30 kDa methoxy polyethylene glycol Zamide, being attached therebetween via a Sulfur atom of a N-ethyl-acetamide, being attached therebetween via a sulfur methionine residue of the G-CSF, and having a formula: atom of a methionine residue of the interferon-beta-1a, and having a formula:

S -- H 30 kDa methoxy polyethylene glycol. N-1-N30 kDa methoxy G-CSF O polyethylene glycol.

0050. According to another aspect of embodiments of the interferon-beta-1a present invention there is provided a conjugate comprising human growth hormone (h-GH) and 30 kDa methoxy poly 0046 According to another aspect of embodiments of the ethylene glycol N-ethyl-(4-bromomethyl)-benzamide, being present invention there is provided a conjugate comprising attached therebetween via a sulfur atom of a methionine interferon-alpha-2a and 30 kDa methoxy polyethylene glycol residue of the h-GH, and having a formula: N-ethyl-acetamide, being attached therebetween via a sulfur atom of a methionine residue of the interferon-alpha-2a, and having a formula: S H

30 kDa methoxy polyethylene glycol. N-1-N30 kDa methoxy -GH O polyetnyolwethylene glyglycol.

0051. According to another aspect of embodiments of the present invention there is provided a conjugate comprising interferon-alpha-2a. human follicle stimulating hormone (h-FSH) and 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben 0047 According to another aspect of embodiments of the Zamide, being attached therebetween via a Sulfur atom of a present invention there is provided a conjugate comprising methionine residue of the h-FSH, and having a formula: interferon-alpha-2a and 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide, being attached ther ebetween via a sulfur atom of a methionine residue of the interferon-alpha-2a, and having a formula: S -- H N-1-N30 kDa methoxy -FSH O polyetnyolwethylene glyglycol S H

N-1-N30 kDa methoxy 0.052 According to yet another aspect of embodiments of interferon-alpha-2a. O polyethylene glycol. the present invention there is provided a process of preparing a conjugate as described herein, which comprises a polypep tide having one or more methionine residues and one or more 0048. According to another aspect of embodiments of the polymer moieties attached to a Sulfur atom of a methylsulfa present invention there is provided a conjugate comprising nyl-ethyl side-chain of the methionine residues, the process is US 2011/O 150820 A1 Jun. 23, 2011

effected by reacting the polypeptide with a polymer having 0063. According to some embodiments of the invention one or more reactive moieties attached thereto under acidic the polyalkylene glycol-benzyl halide compound further conditions ranging from about pH of 2 to pH of 5. includes a spacer linking the benzyl halide moiety and the 0053 According to still another aspect of embodiments of linking moiety. the present invention there is provided a process of preparing 0064. Unless otherwise defined, all technical and scien the modified polypeptide compound described herein, the tific terms used herein have the same meaning as commonly process is effected by reacting the polypeptide with a modi understood by one of ordinary skill in the art to which this fying moiety having a first reactive moiety and a second invention belongs. Although methods and materials similar or reactive moiety under acidic conditions ranging from pH 2 to equivalent to those described herein can be used in the prac pH 5, whereby the first and second reactive moieties are tice or testing of the present invention, Suitable methods and selected such that a covalent bond is formed between the first materials are described below. In case of conflict, the patent reactive group and the Sulfur atom. specification, including definitions, will control. In addition, 0054 According to still another aspect of embodiments of the materials, methods, and examples are illustrative only and the present invention there is provided a process of preparing not intended to be limiting. a conjugate which includes (a) a polypeptide having one or 0065. The term “comprising means that other steps and more methionine residues; and (b) one or more polymer moi ingredients that do not affect the final result can be added. eties attached to a sulfur atom of a methylsulfanyl-ethyl side This term encompasses the terms "consisting of and “con chain of the methionine residues; the process is effected by sisting essentially of. reacting the polypeptide with a modifying moiety having a 0066. The phrase “consisting essentially of means that first reactive moiety and a second reactive moiety under the composition or method may include additional ingredi acidic conditions ranging from pH 2 to pH 5, the first and ents and/or steps, but only if the additional ingredients and/or second reactive moieties are selected Such that a covalent steps do not materially alter the basic and novel characteris bond is formed between the first reactive moiety and the tics of the claimed composition or method. Sulfur atom, to thereby obtain a polypeptide having the one or 0067. As used herein, the singular form “a” “an and more modifying moiety attached thereto; and reacting the “the include plural references unless the context clearly polypeptide having the one or more modifying moieties dictates otherwise. For example, the term “a compound” or attached thereto with a polymer having a third reactive moi “at least one compound may include a plurality of com ety, the third reactive moiety is selected capable of reacting pounds, including mixtures thereof. with the second reactive moiety in the modifying moiety. 0068 Throughout this disclosure, various aspects of this 0055 According to some embodiments of the invention invention can be presented in a range format. It should be the second and the third reactive moieties are each indepen understood that the description in range format is merely for dently selected from the group consisting of amine, carboxyl, convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accord amide, hydrazine, hydrazide, thiol, hydroxyl and hydroxy ingly, the description of a range should be considered to have lamine, and any combination thereof. specifically disclosed all the possible Subranges as well as 0056. According to an additional aspect of embodiments individual numerical values within that range. For example, of the present invention there is provided a pharmaceutical description of a range such as from 1 to 6 should be consid composition which comprises, as an active ingredient, a con ered to have specifically disclosed Subranges Such as from 1 jugate as described herein. to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 0057 According to still an additional aspect of embodi to 6 etc., as well as individual numbers within that range, for ments of the present invention there is provided a use of a example, 1, 2, 3, 4, 5, and 6. This applies regardless of the conjugate as described herein in the manufacture of a medi breadth of the range. Cament. 0069. Whenevera numerical range is indicated herein, it is 0058 According to still an additional aspect of embodi meant to include any cited numeral (fractional or integral) ments of the present invention there is provided a method of within the indicated range. The phrases “ranging/ranges treating a medical condition treatable by a polypeptidehaving between a first indicate number and a second indicate num at least one methionine residue, the method is effected by ber and “ranging/ranges from a first indicate number “to a administering to a Subject in need thereofan therapeutically second indicate number are used herein interchangeably and effective amount of a conjugate as described herein. are meant to include the first and second indicated numbers 0059. According to a further aspect of embodiments of the and all the fractional and integral numerals therebetween. present invention there is provided a compound which com prises a polyalkylene glycol moiety and a benzyl halide moi BRIEF DESCRIPTION OF THE DRAWINGS ety being covalently linked therebetween via a linking moi (0070. The invention is herein described, by way of ety. example only, with reference to the accompanying drawings. 0060 According to some embodiments of the invention With specific reference now to the drawings in detail, it is the polyalkylene glycol moiety is a polyethylene glycol stressed that the particulars shown are by way of example and (PEG). for purposes of illustrative discussion of the preferred 0061 According to some embodiments of the invention embodiments of the present invention only, and are presented the polyethylene glycol has an average molecular weight that in the cause of providing what is believed to be the most ranges from 4 kDa to 40 kDa. useful and readily understood description of the principles 0062 According to some embodiments of the invention and conceptual aspects of the invention. In this regard, no the linking moiety is selected from the group consisting of attempt is made to show structural details of the invention in amine, alkyl, aryl, heteroaryl, carboxyl, amide, hydrazine, more detail than is necessary for a fundamental understand hydrazide and any combination thereof. ing of the invention, the description taken with the drawings US 2011/O 150820 A1 Jun. 23, 2011

making apparent to those skilled in the art how the several N-ethyl-(4-bromomethyl)-benzamide (FIG. 7A) and recom forms of the invention may be embodied in practice. binant human interferon-beta-1b (FIG. 7B); (0071. In the drawings: (0079 FIGS. 8A-B present RP-HPLC chromatograms 0072 FIG. 1 presents a reverse-phase HPLC (RP-HPLC) showing the peptide maps of purified PEGylation product of chromatogram of the PEGylation reaction products of recom recombinant human interferon-beta-1b with 30 kDa methoxy binant human interferon-beta-1b and 30 kDa methoxy poly polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide ethylene glycol N-ethyl-2-iodo-acetamide, obtained using a photo-diode array set at 280 nm, an injection Volume of 40 ul, (FIG. 8A), and of recombinant human interferon-beta-1b total run-time of 70 minutes and 0.2% TFA in waterfacetoni (FIG. 8B), as obtained by applying peptide digestion using trile as a mobile phase, showing a peak having a retention Lys-C; time of 29.17 minutes and a peak corresponding to interferon 0080 FIGS. 9A-B present color images of a non-reducing beta-1b having a retention time of 33.84 minutes: SDS-PAGE separation gel in which the peptides which were 0073 FIGS. 2A-B present color images of a non-reducing formed after peptide Lys-C digestion of the purified PEGy SDS-PAGE gel slab in which the PEGylation reaction prod lation product of recombinant human interferon-beta-1b with uct of recombinant human interferon-beta-1b and 30 kDa 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom methoxy polyethylene glycol N-ethyl-2-iodo-acetamide (as ethyl)-benzamide were run and stained with Coomassie Blue isolated by RP-HPLC) and the starting materials of the (FIG.9A) and subsequently with iodine (FIG.9B), wherein PEGylation reaction were run and stained with Coomassie the collected fractions of peptide K3 and the new formed Blue (FIG. 2A) and subsequently with iodine (FIG. 2B), peptide with a retention time of 47.5 minutes were run in wherein an un-PEGylated sample of interferon-beta-1b was lanes 1 and 2 respectively; a 30 kDa methoxy polyethylene run in lane 1; the isolated peak by RP-HPLC was run in lane glycol N-ethyl-(4-bromomethyl)-benzamide sample was run 2; molecular weight markers were run in lane 3; and a series in lane 3 and molecular weight markers were run in lane 4, of samples of 30 kDa methoxy polyethylene glycol N-ethyl I0081 FIG. 10 presents a comparative plot of the concen 2-iodo-acetamide (the PEGylation reagent) at various con tration of interferon-beta-1b (INF-B1b) in plasma of rats as a centrations (0.078 ug, 0.3125 ug, 3.125 ug, 6.25 ug, 12.5ug function of time, following intravenous administration of 0.4 and 25 ug) which were run in lanes 4, 5, 6, 7, 8 and 9 mg/kg of 30 kDa PEG-Interferon beta-1b (in red) and respectively; BETAFERONR) (in green), showing the pharmacokinetic 0074 FIG. 3 presents a MALDI-TOF mass spectrogram profile of the two drugs and the remarkable longer pharma of the PEGylation reaction product of recombinant human cologic range of the PEGylated interferon according to some interferon-beta-1b with 30 kDa methoxy polyethylene glycol embodiments of the present invention: N-ethyl-2-iodo-acetamide, using a 2,4-dihydroxybenzoic I0082 FIGS. 11A-B present color images of a non-reduc acid matrix, showing a small peak with an average molecular ing SDS-PAGE gel slab in which the PEGylation reaction weight of about 50,000 Da corresponding to the molecular products of recombinant human interferon-beta-1a and 30 weight of a mono-PEGylated interferon-beta-1b conjugate: kDa methoxy polyethylene glycol N-ethyl-2-iodo-aceta 0075 FIG. 4 presents a RP-HPLC chromatogram of the mide, eluted on a Fractogel COO column, were run and PEGylation reaction product of recombinant human inter stained with Coomassie Blue (FIG. 11A) and subsequently feron-beta-1b and 30 kDa methoxy polyethylene glycol with iodine (FIG. 11B), wherein molecular weight markers N-ethyl-(4-bromomethyl)-benzamide, showing a peak hav were run in lane 1; an un-PEGylated sample of interferon ing a retention time of 30.27 minutes corresponding to meth beta-1a was run in lane 2; the eluted fraction was run in lane oxy polyethylene glycol N-ethyl-(4-bromomethyl)-benza 3; the unbound fraction washed with sodium acetate buffer mide, a second peak having a retention time of 38 minutes Solutions was run in lane 4, and the crude reaction mixture corresponding to the PEGylated protein, and a peak having a which was loaded on the column was run in lane 5; showing retention time of 48.44 minutes corresponding to recombi the PEGylated protein (marked with an arrow) and an nant human interferon-beta-1b: increase of about 50 kDa in the apparent molecular weight of 0076 FIGS.5A-B present color images of a non-reducing the PEGylated protein as compared to the un-PEGylated pro SDS-PAGE separation gel in which the PEGylation reaction tein; products of recombinant human interferon-beta-1b and 30 I0083 FIGS. 12A-B present color images of a non-reduc kDa methoxy polyethylene glycol N-ethyl-(4-bromom ing SDS-PAGE separation gel in which the PEGylation reac ethyl)-benzamide, after collection by RP-HPLC, were run tion products of recombinant human interferon-alpha-2a and and stained with Coomassie Blue (FIG.5A) and subsequently 30 kDa methoxy polyethylene glycol N-ethyl-2-iodo-aceta with iodine (FIG. 5B), wherein the collected fractions were mide, which were eluted on a Source 15-S column, were run run in lanes 1, 2 and 3; an un-PEGylated sample of recombi and stained with Coomassie Blue (FIG. 12A) and iodine nant human interferon-beta-1b was run in lane 4, a 30 kDa (FIG. 12B), wherein the fraction eluted with NaC1 solution methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben was run in lane 1; an un-PEGylated sample of interferon Zamide sample was run in lane 5 and molecular weight mark alpha-2a was run in lane 2, and molecular weight markers ers were run in lane 6: were run in lane 3: showing the PEGylated protein (marked 0077 FIG. 6 presents a MALDI-TOF mass spectrogram with an arrow), and an increase of about 50 kDa in the appar of the purified PEGylation product of recombinant human ent molecular weight of the PEGylated protein as compared interferon-beta-1b with 30 kDa methoxy polyethylene glycol to the un-PEGylated protein; N-ethyl-(4-bromomethyl)-benzamide, using a 2,4-dihy I0084 FIG. 13 presents a RP-HPLC chromatogram of the droxybenzoic acid matrix: PEGylation reaction products of recombinant human inter 0078 FIGS. 7A-B present ESI mass spectrograms of the feron-alpha-2a and 30 kDa methoxy polyethylene glycol purified PEGylation product of recombinant human inter N-ethyl-(4-bromomethyl)-benzamide wherein several feron-beta-1b with 30 kDa methoxy polyethylene glycol formed peaks are observed (retention times of 21.77, 23.78, US 2011/O 150820 A1 Jun. 23, 2011

25.05, 25.57 and 27.42 minutes) and recombinant human ylene glycol N-ethyl-(4-bromomethyl)-benzamide (FIG. interferon-alpha-2a has a retention time of 28.42 minutes; 20A) and recombinant human granulocyte colony Stimulat 0085 FIGS. 14A-B present color images of a non-reduc ing factor (FIG. 20B); ing SDS-PAGE gel slab in which the PEGylation reaction 0092 FIG. 21 presents a RP-HPLC chromatogram of the products of recombinant human interferon-alpha-2a and 30 PEGylation reaction products of recombinant human growth kDa methoxy polyethylene glycol N-ethyl-(4-bromom hormone and 30 kDa methoxy polyethylene glycol N-ethyl ethyl)-benzamide, after collection by RP-HPLC, were run (4-bromomethyl)-benzamide wherein a new formed peak is and stained with Coomassie Blue (FIG. 14A) and subse observed with a retention time of 46.65 minutes and recom quently with iodine (FIG. 14B), wherein the collected frac binant human growth hormone has a retention time of 52 tions were run in lanes 1,2,3 and 4; an un-PEGylated sample minutes; of recombinant human interferon-alpha-2a was run in lane 5, 0093 FIGS. 22A-B present color images of a non-reduc a 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom ing SDS-PAGE separation gel in which the PEGylation reac tion products of recombinant human growth hormone and 30 ethyl)-benzamide sample was run in lane 6 and molecular kDa methoxy polyethylene glycol N-ethyl-(4-bromom weight markers were run in lane 7: ethyl)-benzamide, after collection by RP-HPLC, were run I0086 FIG. 15 presents a RP-HPLC chromatogram of the and stained with Coomassie Blue (FIG. 22A) and subse PEGylation reaction products of recombinant human eryth quently with iodine (FIG. 22B), wherein the collected frac ropoietin and 30 kDa methoxy polyethylene glycol N-ethyl tions were run in lanes 1, 2, 3; an un-PEGylated sample of (4-bromomethyl)-benzamide wherein a new peak is observed recombinant human growth hormone was run in lane 4, a 30 with a retention time of 25.62 minutes and recombinant kDa methoxy polyethylene glycol N-ethyl-(4-bromom human erythropoietin has a retention time of 28.35 minutes: ethyl)-benzamide sample was run in lane 5 and molecular 0087 FIGS. 16A-B present color images of a non-reduc weight markers were run in lane 6; and ing SDS-PAGE separation gel in which the PEGylation reac 0094 FIGS. 23A-B present color images of a non-reduc tion products of recombinant human erythropoietin and 30 ing SDS-PAGE separation gel in which the PEGylation reac kDa methoxy polyethylene glycol N-ethyl-(4-bromom tion products of recombinant follicular stimulating hormone ethyl)-benzamide, after collection by RP-HPLC, were run (rh-FSH) and 30 kDa methoxy polyethylene glycol N-ethyl and stained with Coomassie Blue (FIG. 16A) and subse (4-bromomethyl)-benzamide without purification were run quently with iodine (FIG. 16B), wherein the collected frac and stained with Coomassie Blue (FIG. 23A) and subse tions were run in lanes 1 and 2; an un-PEGylated sample of quently with iodine (FIG. 23B), wherein the reaction mixture recombinant human erythropoietin was run in lane 3, a 30 after 17 hours was run in lane 1, the reaction mixture at time kDa methoxy polyethylene glycol N-ethyl-(4-bromom Zero was run in lane 2, an un-PEGylated sample of recom ethyl)-benzamide sample was run in lane 4 and molecular binant human follicle stimulating hormone was run in lane 3, weight markers were run in lane 5: a 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom I0088 FIGS. 17A-B present RP-HPLC chromatograms, ethyl)-benzamide sample was run in lane 4 and molecular comparing the peptide maps of purified PEGylation product weight markers were run in lane 5. of recombinant human erythropoietin (rh-EPO) with 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben DESCRIPTION OF THE PREFERRED Zamide (FIG. 17A), and of a un-PEGylated hr-EPO (FIG. EMBODIMENTS 17B), as obtained by applying peptide digestion using 0.095 The present invention, in some embodiments trypsin; thereof, relates to novel conjugates of proteins and of pro I0089 FIG. 18 presents a RP-HPLC chromatogram of the cesses of preparing same, and, more specifically, but not PEGylation reaction products of recombinant human granu exclusively, to conjugates of a protein and one or more poly locyte colony stimulating factor and 30 kDa methoxy poly mers which are attached to a side chain of one or more ethylene glycol N-ethyl-(4-bromomethyl)-benzamide methionine residues within the protein. Embodiments of the wherein PEGylated protein peaks are observed with a reten present invention also relate to pharmaceutical compositions tion time of 36.9, 48.2 and 49.4 minutes and recombinant containing these conjugates and to uses thereofas therapeutic human granulocyte colony stimulating factor has a retention agents for treating various medical conditions. The conju time of 53.5 minutes; gated proteins of the present embodiments are characterized 0090 FIGS. 19A-B present color images of a non-reduc by improved pharmacokinetic profile, which renders them ing SDS-PAGE separation gel in which the PEGylation reac highly Suitable for use in therapeutic applications. tion products of recombinant human granulocyte colony 0096. The principles and operation of the present inven stimulating factor and 30 kDa methoxy polyethylene glycol tion may be better understood with reference to the figures N-ethyl-(4-bromomethyl)-benzamide, after collection by and accompanying descriptions. RP-HPLC, were run and stained with Coomassie Blue (FIG. 0097. Before explaining at least one embodiment of the 19A) and subsequently with iodine (FIG. 19B), wherein the invention in detail, it is to be understood that the invention is collected fractions were run in lanes 1, 2, 3 and 4; an un not necessarily limited in its application to the details set forth PEGylated sample of recombinant human granulocyte in the following description or exemplified by the Examples. colony stimulating factor was run in lane 5, a 30kDa methoxy The invention is capable of other embodiments or of being polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide practiced or carried out in various ways. sample was run in lane 6 and molecular weight markers were 0098. As discussed hereinabove, therapeutic proteins are run in lane 7: prone to evoke an immunogenic response, are relatively water 0091 FIGS. 20A-B present ESI mass spectrograms of the insoluble, and generally suffer from a short and insufficient in purified PEGylation product of recombinant human granulo vivo half-life. The pharmacokinetics of the particular protein cyte colony stimulating factor with 30 kDa methoxy polyeth will govern both the efficacy and duration of therapeutic US 2011/O 150820 A1 Jun. 23, 2011

effect of the drug, and therefore it is important to reduce the rate of clearance of the protein so that prolonged action can be TABLE 1-continued achieved. 0099. One of the most effective approaches for accom Observed plishing a better to pharmacokinetic profile for pharmaceuti Amino Acids Codons Frequency cal proteins is effected by attaching a polymer, such as poly Serine UCU, UCA, UCC, UCG, AGU, 8.1% ethylene glycol (PEG), to the protein. The polymer improves Stop Codons UAA, UAG, UGA the aqueous solubility of the protein, masks potential epitope and proteolysis sites on the protein, and increases the molecu 0103) The relatively low abundance and the unique chem lar weight and volume thereof. The attachment of PEG to a protein, known as PEGylation, has been shown to be effica istry of its side chain renders methionine a highly suitable cious in reducing the rate of clearance of the therapeutic target for polymer attachment since it increases the probabil protein drug from the physiological system, reducing renal ity of a single and site-directed modification event versus a clearance, reducing proteolysis, reducing antigenicity, and multiple and/or random attachment points. increasing water solubility, while retaining a Substantial pro 0104 PEGylation of proteins via the side-chain of portion of the biological activity of the therapeutic protein. methionine has not been described in the art hitherto. 0100. As further discussed hereinabove, proteins may be 0105. Another example of a rare amino acid relevant in the PEGylated in a variety of methods, depending on the type, context of the present embodiments is selenomethionine. number and availability of particular functional groups on its Selenomethionine (Sel-met or Sem) is an amino acid contain surface which are suitable for PEGylation. ing selenium instead of the sulfur atom of methionine. The 0101 While conceiving the present invention, the present L-isomer of selenomethionine is a naturally occurring amino inventor have recognized that methionine may serve as a acid. In vivo, Sel-met is randomly incorporated instead of favorable PEGylation site, particularly in cases wherein site methionine in lower organisms, and its redox activity stems specific or otherwise preferential PEGylation is required due from its ability to deplete reactive species. As methionine, to its relative scarcity and to its unique chemical reactivity. Sel-met can undergo alkylation (Lang, S. et al., Anal. Bio 0102 Table 1 below presents the observed frequency of all chem., 342, 271-279, 2005; and Lang, S. et al., Anal. Bio naturally occurring amino acids in Vertebrates and clearly chem., 359, 253-258, 2006) and may therefore undergo show the relatively low abundance of methionine. As shown PEGylation according to the present embodiments. in the structural formula of methionine presented below, this amino acid has a unique thioether group in its side-chain, 0106 Hence, the present embodiments encompass pep which is characterized by a unique chemical reactivity with tides and proteins which have one or more methionine and/or respect to the reactivity of other available functional groups Selenomethionine residues which are either naturally, inten found in other amino acids in proteins. tionally, synthetically or genetically incorporated thereto. 0.107 The term “methionine', as used herein, therefore encompasses both methionine and selenomethionine. 0108. Thus, according to one aspect of the present inven tion there is provided a conjugate which comprises: SN 0109 (a) a polypeptide having one or more methionine NH residues in its amino acid sequence, each methionine residue L-Methionine is characterized by a methylsulfanyl-ethyl side-chain; and 0110 (b) one or more polymer moieties being covalently attached to the polypeptide via the sulfur atom of the meth ylsulfanyl-ethyl side-chain of the methionine residue(s). TABLE 1. 0111. The terms “polypeptide' and “protein', which are Observed used herein interchangeably, refer to a polymeric form of Amino Acids Codons Frequency amino acids of any length (e.g., of two or more, or 10 or more Tryptophan UGG 1.3% and more, or 100 or more amino acids), which can include Methionine AUG 1.8% coded and non-coded amino acids, chemically or biochemi Histidine CAU, CAC 2.9% cally modified or derivatized amino acids, and polypeptides Cysteine UGU, UGC 3.3% having modified peptide backbones. Polypeptides may be Tyrosine UAU, UAC 3.3% polymers of naturally occurring amino acid residues; non Glutamine CAA, CAG 3.7% Isoleucine AUU, AUA, AUC 3.8% naturally occurring amino acid residues. Such as, for example Phenylalanine UUU, UUC 4.0% N-Substituted glycine residues, amino acid Substitutes, and Arginine CGU, CGA, CGC, CGG, AGA, 4.2% the like; and both naturally occurring and non-naturally Asparagine AAU, AAC 4.4% occurring amino acid residues/substitutes. This term does not Proline CCU, CCA, CCC, CCG S.0% Glutamic Acid GAA, GAG 5.8% refer to or exclude post-translational modifications of the Aspartic Acid GAU, GAC 5.9% polypeptide, for example, glycosylations, acetylations, phos Threonine ACU, ACA, ACC, ACG 6.2% phorylations and the like. The term includes ribosomally or Valine GUU, GUA, GUC, GUG 6.8% synthetically made polypeptides, fusion proteins, including, Lysine AAA, AAG 7.2% Alanine GCU, GCA, GCC, GCG 7.4% but not limited to, fusion proteins with a heterologous amino Glycine GGU, GGA, GGC, GGG 7.4% acid sequence, fusions with heterologous and homologous Leucine CUU, CUA, CUC, CUG, UUA, 7.6% leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. In US 2011/O 150820 A1 Jun. 23, 2011

the context of the present invention, these terms refer to polypeptides and proteins which comprise at least one TABLE 2 methionine in their amino acid sequence. 0112. In general, the term “polypeptide', as used herein, Three-Letter One-letter refers to all the polypeptides which can be used in any of the Amino Acid Abbreviation Symbol embodiments of the present invention, and encompasses a Alanine Ala naturally occurring, a mutated, an altered, a genetically engi Arginine Arg Asparagine ASn neered, a synthetic, an extracted and/or a recombinant Aspartic acid Asp polypeptide. Cysteine Cys 0113. As used hereinthroughout, the term "amino acid” or Glutamine Gln Glutamic Acid Glu “amino acids” is understood to include the 20 genetically Glycine Gly coded or naturally occurring amino acids; those amino acids Histidine His often modified post-translationally in Vivo, including, for Isoleucine Iie example, hydroxyproline, phosphoserine and phosphothreo Leucine Leu Lysine Lys nine; and other unusual amino acids including, but not limited Methionine Met to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor Phenylalanine Phe valine, nor-leucine and ornithine. Furthermore, the term Proline Pro “amino acid' includes both D- and L-amino acids and other Serine Ser non-naturally occurring amino acids. Threonine Thr Tryptophan Trp 0114 Tables 2 and 3 below list the genetically encoded or Tyrosine Tyr naturally occurring amino acids (Table 2) and non-limiting Valine Wall examples of non-conventional/modified amino acids (Table 3).

TABLE 3

Non-conventional amino acid Code Non-conventional amino acid Code C-aminobutyric acid Abu L-N-methylalanine Nimala C-amino-C.-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane-carboxylate Cpro L-N-methylasparagine NmaSn aminoisobutyric acid Aib L-N-methylaspartic acid Nmasp aminonorbornyl-carboxylate Norb L-N-methylcysteine Nmcys Cyclohexylalanine Chexa L-N-methylglutamine Nmgin Cyclopentylalanine Cpen L-N-methylglutamic acid Nmglu D-alanine Dal L-N-methylhistidine Nmhis D-arginine Darg L-N-methylisoleucine Nmile D-aspartic acid Dasp L-N-methyleucine Nimleu D-cysteine Dcys L-N-methyllysine Nmlys D-glutamine Dglin L-N-methylmethionine Nmmet D-glutamic acid Dglu L-N-methylnorleucine Nmnle D-histidine Dhis L-N-methylnorvaline Nminva D-isoleucine Dile L-N-methylornithine Nmorn D-leucine Deu L-N-methylphenylalanine Nmphe D-lysine Dlys L-N-methylproline Nmpro D-methionine Dmet L-N-methylserine Nmser DL-ornithine DiLorn L-N-methylthreonine Nimthr D-phenylalanine Dphe L-N-methyltryptophan Nmtrp D-proline Dpro L-N-methyltyrosine Nmtyr D-serine Dser L-N-methylvaline Nmval D-threonine Dthr L-N-methylethylglycine Nmetg D-tryptophan Dtrp L-N-methyl-t-butylglycine Nmtbug D-tyrosine Dtyr L-norleucine Nle D-valine Dval L-norvaline Nwa D-O-methylalanine Dmala C.-methyl-aminoisobutyrate Maib D-O-methylarginine Dmarg C-methyl-y-aminobutyrate Mgabu D-O-methylasparagine DmaSn C.-methylcyclohexylalanine Mchexa D-O-methylaspartate Dmasp C.-methylcyclopentylalanine Mcpen D-O-methylcysteine Dmcys C.-methyl-C-napthylalanine Manap D-O-methylglutamine Dmgln C.-methylpenicillamine Mpen D-O-methylhistidine Dmhis N-(4-aminobutyl)glycine Nglu D-O-methylisoleucine Dmile N-(2-aminoethyl)glycine Naeg D-O-methylleucine Dmleu N-(3-aminopropyl)glycine Norn D-O-methyllysine Dmlys N-amino-C.-methylbutyrate Nmaabu D-O-methylmethionine Dmmet C-napthylalanine D-O-methylornithine Dmorn N-benzylglycine Nphe D-O-methylphenylalanine Dmphe N-(2-carbamylethyl)glycine Ngln D-O-methylproline Dmpro N-(carbamylmethyl)glycine Nasin D-O-methylserine Dmser N-(2-carboxyethyl)glycine Nglu D-O-methylthreonine Dmthr N-(carboxymethyl)glycine Nasp D-O-methyltryptophan Dmitrp N-cyclobutylglycine Nicbut D-O-methyltyrosine Dmity N-cycloheptylglycine Nchep US 2011/O 150820 A1 Jun. 23, 2011 10

TABLE 3-continued

Non-conventional amino acid Code Non-conventional amino acid Code D-O-methylvaline Dmval N-cyclohexylglycine Nchex D-O-methylalnine Dnimala N-cyclodecylglycine Nicdec D-O-methylarginine Dnmarg N-cyclododeclglycine Nicdod D-O-methylasparagine DnmaSn N-cyclooctylglycine Nicoct D-O-methylasparatate Dnmasp N-cyclopropylglycine Ncpro D-O-methylcysteine Dnmcys N-cycloundecylglycine Ncund D-N-methyleucine Dnmleu N-(2,2-diphenylethyl)glycine Nbhm D-N-methyllysine Dnmlys N-(3,3-diphenylpropyl)glycine Nbhe N-methylcyclohexylalanine Nmchexa N-(3-indolylyethyl)glycine Nhtrp D-N-methylornithine Dnmorn N-methyl-y-aminobutyrate Nmgabu N-methylglycine Nala D-N-methylmethionine Dnmmet N-methylaminoisobutyrate Nmaib N-methylcyclopentylalanine Nmcpen N-(1-methylpropyl)glycine Nile D-N-methylphenylalanine Dnmphe N-(2-methylpropyl)glycine Nile D-N-methylproline Dnimpro N-(2-methylpropyl)glycine Neu D-N-methylserine Dnmser D-N-methyltryptophan Dnmtrp D-N-methylserine Dnmser D-N-methyltyrosine Dnmtyr D-N-methylthreonine Dnmthr D-N-methylvaline Dnmval N-(1-methylethyl)glycine Nwa Y-aminobutyric acid Gabu N-methyla-napthylalanine Nmanap L-t-butylglycine Tbug N-methylpenicillamine Nmpen L-ethylglycine Etg N-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine Hphe N-(thiomethyl)glycine Ncys L-C.-methylarginine Marg penicillamine Pen L-C.-methylaspartate Masp L-C.-methylalanine Mala L-C.-methylcysteine Mcys L-C.-methylasparagine Masin L-C.-methylglutamine Mglin L-C.-methyl-t-butylglycine Mtbug L-C-methylhistidine Mhis L-methylethylglycine Metg L-C.-methylisoleucine Mile L-C.-methylglutamate Mglu D-N-methylglutamine Dnmgn L-C-methylhomo phenylalanine Mhphe D-N-methylglutamate Dnmglu N-(2-methylthioethyl)glycine Ninet D-N-methylhistidine Dnmhis N-(3-guanidinopropyl)glycine Narg D-N-methylisoleucine Dnmile N-(1-hydroxyethyl)glycine Nthr D-N-methyleucine Dnmleu N-(hydroxyethyl)glycine Nser D-N-methyllysine Dnmlys N-(imidazolylethyl)glycine Nhis N-methylcyclohexylalanine Nmchexa N-(3-indolylyethyl)glycine Nhtrp D-N-methylornithine Dnmorn N-methyl-y-aminobutyrate Nmgabu N-methylglycine Nala D-N-methylmethionine Dnmmet N-methylaminoisobutyrate Nmaib N-methylcyclopentylalanine Nmcpen N-(1-methylpropyl)glycine Nile D-N-methylphenylalanine Dnmphe N-(2-methylpropyl)glycine Neu D-N-methylproline Dnimpro D-N-methyltryptophan Dnmtrp D-N-methylserine Dnmser D-N-methyltyrosine Dnmtyr D-N-methylthreonine Dnmthr D-N-methylvaline Dnmval N-(1-methylethyl)glycine Nval Y-aminobutyric acid Gabu N-methyla-napthylalanine Nmanap L-t-butylglycine Tbug N-methylpenicillamine Nmpen L-ethylglycine Etg N-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine Hphe N-(thiomethyl)glycine Ncys L-C.-methylarginine Marg penicillamine Pen L-C.-methylaspartate Masp L-C.-methylalanine Mala L-C.-methylcysteine Mcys L-C.-methylasparagine Masin L-C.-methylglutamine Mglin L-C.-methyl-t-butylglycine Mtbug L-C-methylhistidine Mhis L-methylethylglycine Metg L-C.-methylisoleucine Mile L-C.-methylglutamate Mglu L-C.-methyleucine Meu L-C-methylhomophenylalanine Mhphe L-C.-methylmethionine Mmet N-(2-methylthioethyl)glycine Ninet L-C.-methylnorvaline Mnva L-C.-methyllysine Mlys L-C.-methylphenylalanine Mphe L-C.-methylmorleucine Minle L-C.-methylserine Se L-C-methylornithine Morn L-C-methylvaline Mtrp L-C.-methylproline Mpro L-C.-methyleucine Mval Nnbhm L-O-methylthreonine Mthr N-(N-(2,2-diphenylethyl)carbamylmethyl-glycine Nnbhm L-C.-methyltyrosine Mtyr -carboxy-1-(2,2-diphenyl ethylamino)cyclopropane Nmbc L-N-methylhomophenylalanine Nmhphe N-(N-(3,3-diphenylpropyl)carbamylmethyl(1)glycine Nnbhe DL-citruline Di Lctr

0115. As is well accepted in the art, the term “residue, as part of a polypeptide chain after the formation of the polypep used herein, describes a portion, and typically a major por- tide chain, is an amino acid residue. An amino acid residue is tion, of a molecular entity, Such as molecule or a part of a therefore that part of an amino acid which is present in a molecule Such as a group, which has underwent a chemical peptide sequence upon reaction of for example, an alpha reaction and is now covalently linked to another molecular amine group thereof with an alpha-carboxylic group of an entity. For example, the molecular entity can be anamino acid adjacentamino acid in the peptide sequence, to form a peptide molecule, and the portion of the amino acid which forms a amide bond and/or of an alpha-carboxylic acid group thereof US 2011/O 150820 A1 Jun. 23, 2011 with an alpha-amine group of an adjacent amino acid in the polymer under certain conditions and/or by using polymer peptide sequence, to form a peptide amide bond. conjugation reagents that are selected Suitable to this effect. 0116 For example, in the case of an alkyl halide, the term 0.124. The phrase “solvent-accessible surface', as used “residue' describes the alkyl part of the alkylhalide, which is herein, refers to the surface area of the polypeptide that is present in a molecule upon being Subjected to a nucleophilic accessible to the molecules of the solvent it is dissolved in. Substitution reaction, in which the halide serves as a leaving The solvent-accessible surface is oftentimes referred to as the group. Lee-Richards molecular surface Lee B. and Richards F M., 0117 The term “side-chain', as used herein with refer 1971, “The interpretation of protein structures: estimation of ence to amino acids, refers to a chemical group which is static accessibility”, J. Mol. Biol., 55(3), pp. 379-400. A attached to the a-carbon atom of an amino acid. The side functional group of an amino-acid residue which is posi chain is unique for each type of amino acid and does not take tioned at or near the solvent-accessible surface of a protein is part in forming the peptide bond which connects the amino more likely to be available for chemical modifications and acids in a polypeptide. For example, the side chain for glycine polymer conjugation, such as PEGylation. is hydrogen, for alanine it is methyl, for valine it is isopropyl 0.125. The protein, according to some embodiments of the and for methionine it is methylsulfanyl-ethyl. For the specific present invention, can be any protein which is administered side chains of all amino acids reference is made to A. L. exogenously into a subject, Such as a human. Exemplary Lehninger's text on Biochemistry (see, chapter 4). proteins which are relevant in the context of the present 0118. As used herein, the phrase "moiety” describes a part embodiments include, without limitation, therapeutic protein or a major part of a chemical entity or compound, which drugs and agents such as , , hormones, typically has certain functionality or distinguishing features. growth factors, blood proteins (blood factors), plasma-de 0119) Due to the beneficial features attributed to polypep rived proteins, urine-derived proteins, antibodies and anti tides by conjugating thereto a polymeric moiety that alters gens, enzymes, viral proteins and fusion proteins. their pharmacokinetic profile, delineated hereinabove, the I0126. As mentioned above, the polypeptide can consist of polypeptides in the conjugates presented herein can be thera a part of a larger protein having either a longer polypeptide peutic proteins or proteins which otherwise exhibit a benefi chain or more than one polypeptide chain. Hence, according cial pharmacological and/or diagnostic activity. to the present embodiments, the polypeptide can be any part 0120 In the context of the present embodiments, the or segment of a protein, e.g. antibody fragments, such as Fab, phrase “therapeutic protein’ describes a protein of any source Fv, and ScHv, and therefore any part or segment of a thera and origin, synthetic of naturally occurring, which has been peutic protein, provided that it has at least one methionine identified as having a beneficial therapeutic effect when residue in its sequence. administered exogenously to a subject. I0127 Depending on their specific therapeutic purpose, 0121 According to the present embodiments, the most therapeutic proteins for human consumption are of polypeptide has at least one methionine (or selenomethion human origins; hence, according to Some embodiments, the ine) residue in its sequence. The methionine (or selenom polypeptides described herein are recombinant human pro ethionine) can be naturally found in the polypeptide (as a teins. Other therapeutic proteins, particularly for vaccination result of natural processes and evolution), and can be entered and other immunotherapeutic purposes consist or include into the sequence by genetic engineering techniques, for proteins or segments thereof from pathogenic organisms and example, having codons for methionine inserted into the cor other sources. responding genetic code which is expressed to yield a given I0128 Representative examples of interferons which can polypeptide. According to the present embodiments, the be utilized in the context of the present embodiments include, methionine can be entered into the sequence of the polypep without limitation, interferon-alpha such as IFNA1, IFNA2, tide also by synthetic techniques which are known in the art IFNA4, IFNA5, IFNA6. IFNA7, IFNA8, IFNA10, IFNA13, for preparing relatively short polypeptides. IFNA14, IFNA16, IFNA17 and IFNA21, interferon-beta 0122 Hence, according to the some embodiments, the such as IFNB1 and IFNB3, interferon-lambda such as IFN polypeptide may have a methionine (or selenomethionine) w1 IFN-22 and IFN-23 also called IL29, IL28A and IL28B inserted into its amino-acid sequence artificially, either as an respectively, interferon-kappa, interferon-delta, interferon added amino-acid (insertion mutation), or as a replacement to epsilon, interferon-tau, interferon-omega and interferon-Zeta another amino-acid (replacement mutation). These polypep (limitin), tide or proteins are modified so as to have a methionine in I0129 Representative examples of viral proteins and other their amino-acid sequence, and are sometimes referred to as antigens which can be utilized in the context of the present genetically engineered mutant proteins and recombinant pro embodiments include, without limitation, HW proteins, teins. In the context of some embodiments of the present envelope protein, porcine transmissible gas invention, Some proteins and polypeptides which do not have troenteritis virus glycoprotein S, SIgA/G, sclv-bryodin 1, a methionine coded for in their native amino-acid sequence Norwalk virus capsid protein, rabies virus glycoprotein, are encompassed herein as being modified so as to have at rotavirus enterotoxin and enterotoxigenic, cholera toxin B or least one methionine residue present therein, and are still A2 subunit, diabetes autoantigen and Escherichia coli entero referred to in their original name. toxin. 0123. According to some embodiments, at least one of the 0.130 Representative examples of antibodies which can be methionine residues present in the polypeptide is accessible utilized in the context of the present embodiments include, to chemical conjugation to a polymer, Such as PEGylation, by without limitation, herpes simplex virus IgG, herpes simplex being positioned at. or close to, the solvent-accessible Surface virus LSC, rituximab, trastuzumab, cetuximab, palivizumab, of the fully formed and folded polypeptide. As discussed infliximab and adalimumab. herein, buried and otherwise less accessible methionine resi I0131 Representative examples of blood factors which can dues side-chains may still undergo chemical conjugation to a be utilized in the context of the present embodiments include, US 2011/O 150820 A1 Jun. 23, 2011 without limitation, alpha 2-antiplasmin, antithrombin III, 0.136 Exemplary therapeutically active proteins which are aprotinin, B-deleted domain Factor VIII, cancer procoagu suitable for use in the context of the present embodiments lant, Factor I (fibrinogen), Factor II (prothrombin), Factor Ha include, without limitation, adalimumab, adenosine deami (activated Factor II), Factor IX (Christmas factor), Factor V nase, agallsidase-beta, alglucosidase-alpha, alpha-galactosi (proaccelerin, labile factor), Factor VII (stable factor), Factor dase, asparaginase, B-deleted domain Factor VIII, bone mor VIIa, Factor VIII (antihemophilic factor), Factor X (Stuart phogenetic protein-2 (BMP-2), bone morphogenetic Prower factor), Factor XI (plasma thromboplastin anteced protein-7 (BMP-7), brain-derived neurotrophic factor ent), Factor XII (Hageman factor), Factor XIII (fibrin-stabi (BDNF), cetuximab, chorionic gonadotropin (CG), dornase lizing factor), fibronectin, heparin cofactor II, high molecular alpha, erythropoietin (EPO), etanercept, Factor IX, Factor weight kininogen (HMWK), plasminogen, plasminogen acti VIIa, Factor VIII, follicle stimulating hormone (FSH), gal sulfase, glial cell line derived neurotrophic factor (GDNF), vator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 glucagon, granulocyte colony stimulating factor (G-CSF), (PAI2), prekallikrein, protein C, protein S. protein Z. protein granulocyte-macrophage colony stimulating factor (GM Z-related protease inhibitor (ZPI), thrombin, Tissue factor CSF), growth hormone (GH), hemoglobin, heparanase, (formerly known as Factor III), tissue plasminogen activator hyaluronidase, imiglucerase, infliximab, insulin-like growth (tPA), urokinase and von Willebrand factor. factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), inter 0132 Representative examples of hormones which can be feron-alpha-2b (IFNalpha-2b), interferon-beta-1a (IFN beta utilized in the context of the present embodiments include 1a), interferon-beta-1b (IFN beta-1b), interferon-gamma-1b antimullerian hormone (AMH or mullerian inhibiting factor (IFN gamma-1b), interleukin (IL-11), interleukin (IL-2), or hormone), adiponectin (Acrp30), adrenocorticotropic hor interleukin-1 (IL-1) receptor antagonist, interleukin-1 recep mone (ACTH or corticotropin), angiotensinogen/angiotensin tor antagonist (IL-1 ra), keratinocyte growth factor (KGF), (AGT), antidiuretic hormone (ADH or vasopressin, arginine laronidase, luteinizing hormone (LH), megakaryocyte vasopressin), atrial-natriuretic peptide (ANP or atriopeptin), growth differentiation factor (MGDF), obesity protein (OB calcitonin (CT), cholecystokinin (CCK), chorionic gonadot protein or leptin), osteoprotegerin (OPG), parathyroid hor ropin (CG(, corticotropin-releasing hormone (CRH), eryth mone (PTH or 1-34 segment or PTH 1-34), palivizumab, ropoietin (EPO), follicle-stimulating hormone (FSH), gastrin platelet-derived growth factor (PDGF). Protein C, rituximab, (GRP), ghrelin, glucagon, glucagons, gonadotropin-releas stem cell factor (SCF), streptokinase, thrombin, thrombopoi ing hormone (GnRH), growth hormone (GH orhGH), growth etin (TPO), thyrotropin (TSH), tissue plasminogen activator hormone-releasing hormone (GHRH), human chorionic (tPA), trastuzumab, tumor necrosis factor binding protein gonadotropin (hCG), human placental lactogen (HPL), (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate inhibin, insulin (INS), insulin-like growth factor (IGF or oxidase. Somatomedin), leptin, luteinizing hormone (LH), melanocyte 0.137 In some embodiments, the therapeutically active stimulating hormone (MSH or C-MSH), neuropeptidey, oxy protein is interferon-alpha-2a (INF-C2a), interferon-beta-1a tocin, parathyroid hormone (PTH), prolactin (PRL), relaxin, (INF-B1a), interferon-beta-1b (INF-B1b), erythropoietin secretin, somatostatin (SRIF), , thyroid (EPO), granulocyte colony-stimulating factor (G-CSF), stimulating hormone (TSH), thyrotropin (TSH) and thyrotro human growth hormone (h-GH) or human follicle stimulating pin-releasing hormone (TRH). hormone (h-FSH). 0.133 Representative examples of growth factors and 0.138. The polymer moiety is attached to the protein in cytokines which can be utilized in the context of the present order to endow certain beneficial qualities to the conjugate embodiments include erythropoietin (EPO), thrombopoietin protein-polymer, and thus allow or improve the pharmacoki (TPO), granulocyte-macrophage colony stimulating factor netic characteristics of the protein. (GM-CSF), granulocyte colony stimulating factor (G-CSF), 0.139. The term “polymer as used herein encompasses insulin-like growth factor-1 (IGF-1), keratinocyte growth one or more of a polymer, a copolymer or a mixture thereof, factor (KGF), platelet-derived growth factor (PDGF), bone as well as linear or branched form thereof when applicable. morphogenetic protein-2 (BMP-2), bone morphogenetic pro 0140. Hence, the polymer, according to some embodi tein-7 (BMP-7), tumor necrosis factor-alpha (TNF-alpha), ments of the invention, is: interferon-alpha-2a (IFN-alpha-2a), interferon-alpha-2b 0141 i) capable of forming a covalent bond and a stable (IFN alpha-2b), interferon-beta-1a (IFN beta-1a), inter chemical interaction with the polypeptide either directly or feron-beta-1b (IFN beta-1b), interferon-gamma-1b (IFN via a linking moiety, as defined hereinbelow: gamma-1b), interleukin-1 (IL-1) receptor antagonist, inter 0.142 ii) non-toxic, namely, the polymer and/or its leukin (IL-2) and interleukin (IL-11). metabolites have no harmful effects to a biological system 0134 Representative examples of enzymes which can be upon administration; utilized in the context of the present embodiments include 0.143 iii) highly soluble in aqueous solutions, so as to human-secreted alkaline phosphatase, C.1-antitrypsin, endow a less Soluble moiety the capacity to dissolve in aque heparanase, alglucosidase-alpha, imiglucerase, laronidase, ous solutions; agallsidase-beta, galsulfase, hyaluronidase, alpha-galactosi 0144) iv) highly flexible, so as to have the capacity to dase, urate oxidase and human dornase-alpha, urokinase, assume a wide range of conformations so as to have low arginase, asparaginase, methioninase, histaminase, adenos immunogenicity and low rate of bio-degradation; and ine deaminase, catalase, Superoxide dismutase and streptoki 0145 V) of suitable mass and size, in order to endow a aSC. protein Sufficient mass and protection, as discussed herein 0135 Representative examples of fusion proteins which above. can be utilized in the context of the present embodiments 0146 These characteristics constitute some of the require include etanercept, alefaceptand r-IL-2 diphteria toxinfusion ments which render a polymer Suitable for conjugation with a protein. protein, and in particular a therapeutic protein. As discussed US 2011/O 150820 A1 Jun. 23, 2011 hereinabove, these requirements include endowing the pro determine properties Such as, for example, the hydration of a tein with the necessary solubility and bioavailability, protec polymer and thus its aqueous solubility, cross-section size tion from proteolysis, masking from the immune system and which influences the rate of clearance from a biological sys extended half-life within the biological system it is adminis tem, immunogenicity and rate of bio-degradation. tered to, and allowing the polymer to have favorable interac 0156 Due to the process of preparing polymers, it is tion with the protein. impractical to attempt to achieve a uniform sample of a poly 0147 Thus, in some embodiments, the polymer is selected mer in which all the molecules have the same length (which Such that when it is conjugated to a protein: corresponds to the molecular weight thereof). Therefore, 0148 a) the protein-polymer conjugate Substantially pre polymers are characterized by a molecular weight distribu serves the characterizing biological activity of the unconju tion and an average molecular weight. gated protein in physiological conditions; 0157. In the context of the present embodiments, the poly 0149 b) the protein-polymer conjugate is substantially mer is selected so as to have an optimal average molecular soluble in aqueous and physiological solutions such as Saline, weight which is Suitable for the protein to be conjugated to, even and particularly when the unconjugated protein is less and for the particular use of the conjugate. soluble in the same solutions; 0158. In some embodiments, the polymer moiety has an 0150 c) the characterizing three dimensional structure of average molecular weight which ranges from about 1 kDa to the unconjugated protein is substantially preserved when about 100 kDa. conjugated to the polymer under physiological conditions; 0159 Polyalkylene glycol is a general name which refers and to a family of polyether polymers which share the following 0151 d) the characterizing half-life of the protein-poly general formula: HO—(CH)n-O-m-CHOH, wherein n mer conjugate under physiological conditions is substantially represents the number of methylene groups present in each greater than the half-life of the unconjugated protein under monomer unit, and m represents the number of repeating similar conditions. monomer units, and therefore represents the size of the poly 0152. As discussed hereinabove, one of the rudimentary mer. For example, when n=2, the polymer is referred to as objectives of PEGylation of polypeptides is to allow a polyethylene glycol, and when n=3, the polymer is referred to polypeptide to exert its particular biologic activity when used, as polypropylene glycol. for example, as an exogenously administered drug. The con 0160. As discussed hereinabove, polyethylene glycol jugates described herein are therefore characterized by hav (PEG) is a highly suitable polymer for conjugation with pro ing a biological activity similar or identical to that of the teins and, hence, according to Some embodiments, the poly unconjugated polypeptide, and by retaining the level of that mer is PEG. In some embodiments, the PEG moiety has an activity at least to some significant extent, while being further average molecular weight that ranges from 4 kDa to 40 kDa. advantageously characterized, according to some embodi In some embodiments, the PEG moiety is a 30 kDa methoxy ments, by improved pharmacokinetic features such as Solu polyethylene glycol moiety. A methoxy polyethylene glycol bility, stability and bioavailability, and the like, which are moiety is a polyalkylene glycol residue that terminates with a expressed by, for example, extended half-life. methoxy ( OCH) group (instead of hydroxy). 0153. For example, a polymer-conjugated therapeutic pro 0.161 Derivatives of polyalkylene glycols are therefore tein may exhibit a considerably lower level of bioactivity as also contemplated. These include, for example, polyalkylene compared to the level of bioactivity of the non-conjugated glycols in which one of more of the methylene groups in the protein, e.g. retaining even less than 10% thereof (due to repeating monomer units is Substituted by, for example, an many possible factors such as hindrance the interaction with alkyl, a alkenyl, a cycloalkyl, an alkoxy, a thioalkoxy, a a receptor due to the polymer mass and motility), and still be halide, and more. beneficially used for therapeutic and diagnostic purposes. In 0162 According to some embodiments, the polypeptide Such cases the contribution in the extension of a protein's and the polymer moiety are covalently attached to each other half-life in a biologic system (e.g., the blood) may contribute at the Sulfur atom of a methionine side-chain via a linking to the efficacy and usefulness of a conjugated protein as a drug moiety. even more than preserving the original level of its bioactivity. 0163 As used herein, the phrase “linking moiety’ 0154 Exemplary polymers which are suitable for conju describes a chemical moiety or a group, as defined herein, gation with a polypeptide according to the present embodi which links the polymeric moiety and the polypeptides. The ments include, without limitation, a polyalkylene glycol, a linking moiety can thus be, for example, formed upon react polyethylene glycol (PEG), a poly(lactic acid) (PLA), a poly ing a reactive moiety within the polymer with the thioether ester, a polyglycolide (PGA), a polycaprolactone (PCL), a group at the side chain of a methionine. polyamide, a polymethacrylamide, a polyvinyl alcohol, a 0164. Thus, according to some embodiments of the polycarboxylate, a polyvinyl pyrrolidinone, a dextran, a cel present invention, the linking moiety includes at least one lulose, a chitosan, a hydroxyethyl starch (HES), polyamino residue of a reactive moiety, whereby the reactive moiety is acids such as polyglutamic acid and polyglycine and any selected capable of reacting with a Sulfur atom of a methion copolymer thereof. ine side-chain. 0155 The molecular weight of a polymer determines 0.165. As discussed in further details hereinbelow, in some many of its physical and biochemical properties. In the con embodiments of the invention, the conjugation reaction of the text of physical properties, the molecular weight of a polymer polymer moiety to the sulfur atom of the methionine side will determine properties such as, for example, the tempera chain is effected via an alkylation, or a methylation, reaction, tures for transitions from liquids to waxes to rubbers to solids, and according to some embodiments, it is a nucleophilic and mechanical properties such as stiffness, strength, vis Substitution of a leaving group on the reactive moiety of an coelasticity, toughness and Viscosity. In the context of bio alkylating agent by the Sulfur atom. Hence, the linking moi chemical properties, the molecular weight of a polymer will ety, according to some embodiments, comprises a residue, as US 2011/O 150820 A1 Jun. 23, 2011

defined herein, of a reactive moiety, whereby the residue is chain of aromatic rings forms the spacer moiety. An example formed upon an interaction of the reactive moiety with the of a spacer moiety made of two benzene rings is a 1.10 thioether group of the methionine residue biphenyl-diyl moiety. 0166 The phrase “reactive moiety', as used herein, 0173 As discussed hereinabove, the conjugation of a describes a chemical group that is capable of undergoing a polymer moiety to a polypeptide can be conducted under chemical reaction that typically leads to a bond formation. conditions that enable preferential conjugation at a Sulfur The bond, according to some embodiments, is a covalent atom of a methionine residue side-chain. Thus, according an bond. Chemical reactions that lead to a bond formation additional aspect of the present invention there is provided a include, for example, nucleophilic and electrophilic Substitu process of preparing the conjugate presented herein. The tions, nucleophilic and electrophilic addition reactions, alky process is effected by providing a polymer which has, or is lations, addition-elimination reactions, cycloaddition reac modified so as to have, at least one reactive moiety attached tions, rearrangement reactions and any other known organic thereto, and reacting this polymer with the polypeptide under reactions that involve a reactive group. acidic conditions, to thereby obtain the conjugate. The acidic 0167 For example, according to some embodiments, the conditions of the conjugation reaction are set to range from reactive moiety which is capable of reacting with the sulfur pH of 2 to pH of 5, or from pH of 3 to pH of 5, such that the atom of the side-chain of a methionine, comprises a leaving reactive moiety, which is selected capable of reacting with a group, as defined hereinbelow, and can be selected capable of Sulfur atom of a methionine side-chain, forms a covalent bond alkylating the Sulfur atom by means of a nucleophilic Substi with the sulfur. tution. 0.174. It should be noted that while reacting the polymer 0168 As discussed hereinabove, some cases wherein the with the polypeptide can be performed under various (e.g., methionine is not at, or close to, the solvent-accessible Sur acidic, basic or neutral) conditions, it can be conducted at a face of the polypeptide, the conjugation of the polymer pH range of from pH of 2 to pH of 5, so as to render the thereto can be effected by means of an “elongation arm’, or a reaction specific to methionine side-chains, while avoiding spacer, which can penetrate through the amino-acid residues reactions of the polymer with other functional groups found on the surface of the polypeptide and reach a buried or less in proteins, and particularly while avoiding conjugation of the accessible methionine, owing to a thin and flexible structure polymer to functional groups of the N-terminal amino acid, thereof. Thus, according to some embodiments of the present and of histidine, lysine and cysteine residues, as discussed invention, the linking moiety further comprises a spacer. hereinabove. 0169. For the purposes of this invention, the term “spacer' 0.175 Representative examples of a reactive moiety, as or 'spacer moiety' is intended to encompass any chemical defined hereinabove, and is also referred to hereinbelow as entity that covalently connects between two or more com the first reactive group, include, but are not limited to, amine, pounds, moieties or residues thereof. Such as a polypeptide carboxyl, amide, acetamide, 2-halo-acetamide, (4-halom and a polymer and/or a linking moiety. In the context of the ethyl)-benzamide, benzyl-halide, hydrazine, hydrazide, present embodiments, the spacer covalently connects acetohydrazide, alkyl, haloalkyl, alkyl sulfonylhalide, alkyl between two moieties or residues thereof, one is the first tosylate, alkyl triflate, allyl, haloallyl, allyl sulfonylhalide, reactive moiety which is attached to the sulfur atom in the allyl tosylate, allyl triflate, aryl, haloaryl heteroaryl, 4-(ha side-chain of a methionine residue in the polypeptide, and the lomethyl)benzyl, benzyl, halobenzyl, and any combination other is the second reactive moiety which is attached to the thereof. polymer moiety. Further in the context of the present embodi 0176). In some embodiments, the reactive moiety is amide, ments, such spacer moieties can be designed to facilitate, 2-halo-acetamide, (4-halomethyl)-benzamide, benzyl-ha modulate, regulate or otherwise influence the attachment of lide, haloalkyl, hydrazine, hydrazide or acetohydrazide. the polymer to a methionine side-chain on a polypeptide, 0177. As used herein, the term “amine” describes a particularly in cases where the methionine side-chain is Sub —NR'R" group where each of R and R" is independently stantially buried within the polypeptide chain or otherwise hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or het less accessible for chemical modifications. eroaryl, as these terms are defined herein. 0170 Spacer groups, as described herein, include, but are 0.178 As used herein, the term “alkyl describes an ali not limited to, a linear or branched, Saturated or unsaturated, phatic hydrocarbon including straight chain and branched Substituted or unsubstituted alkylene —(CH2)— chain hav chain groups. According to Some embodiments, the alkyl ing 1-30 carbon atoms (n is an integer ranging from 1 to 30), group has 1 to 20 carbon atoms, or 1-10 carbon atoms. When and a linear or branched, Saturated or unsaturated, Substituted ever a numerical range; e.g., "1-10'. is stated herein, it or unsubstituted alkylene chain having 1-30 carbon atoms implies that the group, in this case the alkyl group, may interrupted by at least one heteroatom, whereby the at least contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., one heteroatom is selected from the group consisting of oxy up to and including 10 carbon atoms. The alkyl can be Sub gen, Sulfur, nitrogen, phosphor and/or silicon. stituted or unsubstituted. When substituted, the substituent 0171 In some embodiments, the spacer moiety is a linear, can be, for example, an alkyl, an alkenyl, an alkynyl, a unsubstituted alkylene chain wherein n is 1, as in methane cycloalkyl, an aryl, a heteroaryl, a halide, a hydroxy, an di-yl; n is 2, as in ethane-1-yl-2-yl; n is 3, as in propane-1-yl alkoxy and a hydroxyalkyl as these terms are defined herein 3-yl; and n is 4, as in butane 1-yl-4-yl. below. The term “alkyl, as used herein, also encompasses 0172 Alternatively, the spacer can comprise one or more saturated or unsaturated hydrocarbon, hence this term further aryl or heteroaryl groups. An example of a spacer moiety is a encompasses alkenyl and alkynyl. 1,4-benzene-diyl moiety. If more than one aryls or heteroar 0179 The term “alkenyl' describes an unsaturated alkyl, yls are present, they can be linked to one another Such that a as defined herein, having at least two carbon atoms and at US 2011/O 150820 A1 Jun. 23, 2011

least one carbon-carbon double bond. The alkenyl may be pates. Hence, according to some embodiments, the reactive substituted or unsubstituted by one or more substituents, as moiety comprises an alkyl group and a leaving group. described hereinabove. 0194 Such a nucleophilic alkylation reaction typically 0180. The term “alkynyl', as defined herein, is an unsat produces a Sulfonium ion, namely, a positively charged Sulfur urated alkyl having at least two carbonatoms and at least one atom carrying three alkyl groups as Substituents (SR). Thus, carbon-carbon triple bond. The alkynyl may be substituted or the product of such a Sulfur-alkylation reaction is typically a unsubstituted by one or more substituents, as described here Sulfonium salt consisting of a Sulfonium ion (cation) and a inabove. counter ion (a counter-anion). The counter anion can be the 0181. The term "carboxyl', as used herein, refers to a leaving group itself, as it is obtained upon its release during —C(=O)—O—R', where R' is as defined herein. the nucleophilic reaction. Otherwise, the leaving group can 0182. The term “amide' describes a NR' C(=O)— be replaced by any other chemically compatible moiety. group, a NR' C(=O)—R" group or a —C(=O)—NR'R'" According to some embodiments, the counter ion in the group, wherein R is as defined herein and R" is as defined for obtained Sulfonium salt forms a pharmaceutically acceptable R". For example, an acetamide refers to a NR' C(=O)— Sulfonium salt. CR" group. 0.195 The phrase “pharmaceutically acceptable salt”, as 0183. As used herein, the term “hydrazine' describes a used herein, refers to a salt form of the conjugates presented - NR NR"R" group, wherein R is as defined herein and herein, having a positively charged Sulfonium ion and a phar R" and R" are as defined for R'. maceutically acceptable counter ion, which possesses prop 0184 The term “hydrazide', as used herein, refers to a erties such as absorption, distribution, metabolism, excretion —C(=O) NR NR"R" group wherein R', R" and R" are and toxicity that render it Suitable for use in as a pharmaceu each independently hydrogen, alkyl, cycloalkyl or aryl, as tical agent. these terms are defined herein. For example, acetohydrazide 0196. Exemplary pharmaceutically acceptable counter refers to a -CH C(=O) NR NR"R" group. anions include, without limitation, acetate, ascorbate, aspar 0185. As used herein, the terms “halo' and “halide', tate, benzenesulfonate, benzoate, besylate, camphorsul which are referred to herein interchangeably, describe an fonate, citrate, decanoate, esylate, fumarate, glutamate, atom of a halogen, that is fluorine, chlorine, bromine or glycollate, halide (fluoride, chloride, bromide and iodide), iodine, also referred to herein as fluoride, chloride, bromide hexanoate, isethionate, lactate, malate, maleate, methane and iodide. Sulfonate (mesylate), naphthalenesulfonate (napsylate), 0186 The term "haloalkyl describes an alkyl group as naphthylsulfonate, nitrate, octanoate, oleate, oxalate, pamo defined above, further substituted by one or more halide(s). ate, phosphate (orthophosphate), polystyrene Sulfonate, pro 0187. The term “alkoxy” refers to a OR group, were R' pionate, salicylate, Stearate. Succinate, Sulfate, tartrate and is as defined herein. toluenesulfonate (tosylate). In some embodiments, the phar 0188 The term “thioalkoxy' refers to a —SR' group, were maceutically acceptable counter-anion is a halide, namely R" is as defined herein. fluoride, chloride, bromide or iodide. (0189 The term “sulfonylhalide” refers to a S(=O)— 0.197 As used herein, the phrase “leaving group' X, wherein X is a halide. For example, an alkylsulfonylhalide describes a labile atom, group or chemical moiety that readily refers to a R—S(=O). X wherein X is a halide and R is undergoes detachment from an organic molecule during a an alkyl. chemical reaction, while the detachment is facilitated by the (0190. The term “allyl refers to a C–C–C group. relative stability of the leaving atom, group or moiety there 0191 The term “aryl' describes an all-carbon monocyclic upon. Typically, any group that is the conjugate base of a or fused-ring polycyclic (i.e., rings which share adjacent pairs strong acid can act as a leaving group. Representative of carbon atoms) groups having a completely conjugated examples of Suitable leaving groups according to the present pi-electron system. The aryl group may be substituted or embodiments therefore include, without limitation, halide, unsubstituted by one or more substituents, as described here acetate, tosylate, triflate, Sulfonate, azide, hydroxy, thiohy inabove. droxy, alkoxy, cyanate, thiocyanate, nitro and cyano. 0.192 The term "heteroaryl describes a monocyclic or 0.198. The term “acetate' refers to acetic acid anion. fused ring (i.e., rings which share an adjacent pair of atoms) (0199 The term “tosylate” refers to toluene-4-sulfonic acid group having in the ring(s) one or more atoms, such as, for anion. example, nitrogen, oxygen and Sulfur and, in addition, having 0200. The term “triflate' refers to trifluoro-methane a completely conjugated pi-electron system. Examples, with Sulfonic acid anion. out limitation, of heteroaryl groups include pyrrole, furane, 0201 The term “azide” refers to an N. thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, (0202) The terms “hydroxy” and “thiohydroxy” refer to the pyrimidine, quinoline, isoquinoline and purine. The het OH and SH anions respectively. eroaryl group may be substituted or unsubstituted by one or (0203 The term “cyanate” and “thiocyanate” refer to more substituents, as described hereinabove. Representative O—C—NIT and S—C—NIT anions respectively. examples are thiadiazole, pyridine, pyrrole, oxazole, indole, (0204. The term “nitro” refers to NO. purine and the like. (0205 The term “cyano” refers to IC=N. 0193 In some embodiments of the present invention, the 0206. In some embodiments, the leaving group is halide. conjugation reaction is effected via an alkylating reaction, 0207. In order to further facilitate the alkylation reaction, while utilizing the unique chemical activity of the thioether according to some embodiments, the reactive moiety further side chain of methionine. Without being bound by any par comprises an electron withdrawing activating group. There ticular theory, it is assumed that alkylation is effected via a fore, according to some embodiments of the present inven nucleophilic substitution reaction, in which the nucleophilic tion, the reactive moiety can comprise, for example, a short sulfur atom in the thioether side chain of methionine partici alkyl (e.g., methylene) Substituted by a halide as a leaving US 2011/O 150820 A1 Jun. 23, 2011 group and an activating electron withdrawing group. Such as methoxy polyethylene glycol N-ethyl-acetamide, being an amide, that renders the carbon atom more Susceptible to a attached therebetween via a sulfur atom of a methionine nucleophilic substitution by the sulfur atom of a methionine residue of the interferon-beta-1b (see, Example 2), having a side-chain. In another example, the reactive moiety can com formula: prise a short alkyl (e.g., methylene) which is substituted with a tosylate as a highly reactive leaving group, and an allyl as an activating electron withdrawing group. Alternatively, the 30 kDa methoxy polyethylene glycol. reactive moiety can comprise a short alkyl (e.g., methylene) which is substituted with a triflate as a highly reactive leaving group, and a benzyl as an activating electron withdrawing group. 0208. The reactive moiety can thus be a combination of interferon-beta-1bDr several moieties, and according to some embodiments, of a short alkyl, Substituted by a reactive leaving group, and fur ther linked to an activating electron withdrawing group. The reactive moiety can thus be a combination of for example, 0216 Similarly, another exemplary conjugate comprises haloalkyl, haloalkyl-amide, (4-halomethyl)-benzamide, interferon-beta-1b and 30 kDa methoxy polyethylene glycol halo-benzyl, halosulfonyllalykl, halotosylalkyl and the like N-ethyl-(4-bromomethyl)-benzamide, being attached ther with amide, allyl, aryland the like. ebetween via a sulfur atom of a methionine residue of the 0209. In some embodiments the reactive moiety is a interferon-beta-1b (see, Example 4), and having a formula: haloalkyl, a haloalkyl-amide, (4-halomethyl)-benzamide and/or halo-benzyl. 0210. In general, the conjugation reaction between the -- polypeptide and the polymer moiety can use commercially S H available PEG reagents, as demonstrated in the Examples N-1-N30 kDa methoxy section that follows. For example, the PEGylation of recom O olwethylene glycol. binant human interferon-beta-1b (rh-IFN-B1b) can be interferon-beta-1b polyetny gly effected with a commercially available 30kDa methoxy poly ethylene glycol N-ethyl-2-iodo-acetamide, as demonstrated in Example 2 hereinbelow, and illustrated in Scheme 1 below. 0217. Another exemplary conjugate comprises interferon The same PEG reagent was used in Examples 8 and 9 in the beta-1a and 30 kDa methoxy polyethylene glycol N-ethyl PEGylation of rh-IFN-B1a and rh-IFN-C2a respectively. acetamide, being attached therebetween via a Sulfur atom of 0211. The PEGylation reaction can further be achieved a methionine residue of the interferon-beta-1a (see, Example with other PEGylation reagents, such as, for example, 4-ha 8), and having a formula: lobenzyl (also referred to hereinas benzyl halide), and haloal lyl. 0212. Thus, in other embodiments, the PEG reagent, 30 30 kDa methoxy polyethylene glycol. kDa methoxy polyethylene glycol N-ethyl-(4-bromom ethyl)-benzamide, is prepared (see, Example 3 hereinbelow) and used in the PEGylation of rh-IFN-B1b and rh-IFN-C2a, EPO, rh-G-CSF; rh-GH and rh-FSH (see, Examples 4, 10, 11, 13, 14 and 15 respectively), as illustrated in Scheme 2 below. interferon-beta-1aDr 0213. The general concept which is shared by PEGylation reagents, according to Some embodiments of the invention, is the attachment of the PEG moiety to the polypeptide via a 0218. Another exemplary conjugate comprises interferon reactive moiety. The reactive moiety can be attached in one or alpha-2a and 30 kDa methoxy polyethylene glycol N-ethyl more steps, namely be first attached to one of the members of acetamide, being attached therebetween via a Sulfur atom of the conjugate, and then to the other, or to both of the members a methionine residue of the interferon-alpha-2a (see, of the conjugate in one reaction. Example 9), and having a formula: 0214 Hence, the exemplary reactive moieties in the PEGylation reagents methoxy polyethylene glycol N-ethyl 2-iodo-acetamide and methoxy polyethylene glycol N-ethyl 30 kDa methoxy polyethylene glycol. (4-bromomethyl)-benzamide, can be regarded as having two functional groups, wherein one functional group is already attached to the PEG, represented by the N-ethyl acetamide moiety in the case of methoxy polyethylene glycol N-ethyl 2-iodo-acetamide and in the case of methoxy polyethylene interferon-alpha-2a.Dr.- glycol N-ethyl-(4-bromomethyl)-benzamide, and another functional group used to react with the sulfur atom of the methionine residue in the polypeptide and forms a methion 0219. Another exemplary conjugate comprises interferon ine Sulfonium bond, such as the 2-iodo-acetamide or the alpha-2a and 30 kDa methoxy polyethylene glycol N-ethyl (bromomethyl)benzene, respectively. (4-bromomethyl)-benzamide, being attached therebetween 0215. An exemplary conjugate according to the present via a sulfur atom of a methionine residue of the interferon embodiments is a conjugate of interferon-beta-1b and 30 kDa alpha-2a (see, Example 10), and having a formula: US 2011/O 150820 A1 Jun. 23, 2011 17

0225. Another exemplary conjugate comprises human fol licle stimulating hormone (h-FSH) and 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide, S H being attached therebetween via a sulfur atom of a methion ine residue of the h-FSH (see, Example 15), and having a N-1N30 kDa methoxy formula: interferon-alpha-2a. O polyethylene glycol.

0220 Another exemplary conjugate comprises erythro S H poietin and 30 kDa methoxy polyethylene glycol N-ethyl-(4- bromomethyl)-benzamide, being attached therebetween via a N-1-N30 kDa methoxy sulfur atom of a methionine residue of the erythropoietin -FSH O polyetnyolwethylene glycol.gly (EPO, see. Example 11), and having a formula: 0226. As demonstrated in the Examples section that fol lows, a novel PEGylation reagent has been designed, pre St H pared and successfully practiced in methionine PEGylation. This reagent is based on a benzyl halide moiety, as a moiety N-1-N30 kDa methoxy that is highly susceptible to nucleophilic Substitution reac O polyethylene glycol. tions, linked to a polyethylene glycol moiety. erythropoietin 0227 Hence, according to another aspect of embodiments of the invention, there is provided a compound, or a reactive 0221) The PEGylated EPO conjugate demonstrates a polyalkylene glycol compound, which includes a polyalky unique feature of site-specific PEGylation, Stemming from lene glycol moiety and a benzyl halide moiety, being the fact that EPO contains a single (site-specific) and acces covalently linked therebetween via a linking moiety. 0228. According to some embodiments, the polyalkylene sible (reactive) methionine residue in its sequence. glycol moiety is a polyethylene glycol (PEG), and more spe 0222. In the general case, the peptide may have more than cifically, the reactive polyalkylene glycol compound com one methionine with varying reactivities, allowing situations prises a polyethylene glycol which has an average molecular where the peptide is PEGylated with more than one PEG weight that ranges from 4 kDa to 40 kDa. According to some moiety in a non-homogeneous distribution, affording a popu embodiments, the reactive polyalkylene glycol compound lation of more than one conjugate species which are distrib comprises a polyethylene glycol which has an average uted into groups according to the reactivity of the methionine molecular weight that ranges from 20 kDa to 40 kDa. In an residues in the peptide. exemplary embodiment, the PEG has a molecular weight of 0223. Another exemplary conjugate comprises granulo about 30 kDa. cyte colony-stimulating factor (G-CSF) and 30 kDa methoxy 0229. The linking moiety which covalently connects polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide, between the polyalkylene glycol moiety and the benzyl halide being attached therebetween via a sulfur atom of a methion moiety can be, for example, amine, alkyl, aryl, heteroaryl, ine residue of the G-CSF (see, Example 13), and having a carboxyl, amide, hydrazine, hydrazide and any combination formula: thereof. 0230. The linking moiety can further comprise a spacer moiety, as defined hereinabove, to facilitate the conjugation of the polyalkylene glycol moiety to the side-chains of less St H accessible methionine residues of a polypeptide. The spacer N-1-N therefore links between the benzyl halide moiety and the 30 kDa methoxy polyalkylene glycol moiety. G-CSF O polyethylene glycol. 0231. According to some embodiments, the spacer is a linear, Saturated, unsubstituted alkylene chain having 1-10 carbon atoms, and in some embodiments, the alkylene chain 0224. Another exemplary conjugate comprises human has 2-4 carbon atoms. In an exemplary embodiment, the growth hormone (h-GH) and 30 kDa methoxy polyethylene spacer is an ethylene (n=2). glycol N-ethyl-(4-bromomethyl)-benzamide, being attached 0232. It should be noted in this regard that similar PEGy therebetween via a sulfur atom of a methionine residue of the lation reagents, having other leaving groups instead of h-GH (see, Example 14), and having a formula: halides, are also contemplated. Hence, the compound described herein can include, for example, a benzylsulfonyl halide or a benzyl triflate instead of benzyl halide. 0233. As demonstrated in the Examples section that fol St H lows, 30 kDa methoxy polyethylene glycol N-ethyl-(4-bro / V ( ) ~N-1-N momethyl)-benzamide, a novel PEGylation reagent was suc 30 kDa methoxy cessfully prepared (see, Example 3) and utilized (see, O polyethylene glycol. -GH Examples 4, 10, 11, 13, 14 and 15 respectively). Thus, an efficient PEGylation of interferon-beta-1b, interferon-alpha 2a, EPO, GCSF, h-GH (human growth hormone) and h-FSH US 2011/O 150820 A1 Jun. 23, 2011

was afforded by using a 4-bromomethyl-N-(PEG)-benza 0241. The term “thiohydroxyl or “thiol, as used herein, mide reagent at acidic conditions. refers to an —SH group. 0234. As discussed herein, in cases where the methionine 0242. The term “hydroxylamine', as used herein, refers to is not at, or close to, the solvent-accessible Surface of the a —NR' OH group, wherein R is as defined herein. polypeptide, or in cases where the polymer moiety is bulky 0243 According to some embodiments, the modifying Such that the conjugation process thereof to the polypeptide moiety includes a hydrazine or a hydrazide as a second reac becomes inextricable, the conjugation process can be effected tive moiety. These second reactive moieties are useful due to via an intermediate compound wherein a modifying moiety is their reactivity towards amines under mild conditions, and the attached to the Sulfur atom of a methionine side-chain under widely available chemistry by which an amine can be intro favorable conditions, and thereafter the polymer moiety is duced to a polymer moiety. attached to the modifying moiety. Thus, according to another 0244 As discussed hereinabove, such a modified polypep aspect of embodiments of the invention, there is provided a tide compound can be used in cases where the methionine compound, or a modified polypeptide compound, which side-chain is less accessible and hence, the modifying moiety, includes: according to some embodiments, further comprises a spacer which connects the residue of the first reactive moiety and the 0235 (a) a polypeptide having at least one methionine second reactive moiety. By means of this spacer, a polypep residue, and tide which has a buried methionine side-chain (meaning not at 0236 (b) at least one modifying moiety which comprises or close to the solvent-accessible surface of the polypeptide) a residue of a first reactive moiety and a functional moiety, as can be conjugated to a polymer moiety, and even to a bulky these are defined hereinabove. polymer moiety. 0237. In this modified polypeptide compound, the modi 0245 Apart from being useful for conjugation with poly fying moiety is covalently attached to the sulfur atom of the mer moieties, a modified polypeptide, having a modifying methionine side-chain via a residue of a first reactive moiety, moiety attached to the sulfur atom thereof, may be utilized for which is selected capable of reacting with the sulfur atom. covalently attaching to the polypeptide, via its methionine 0238 According to some embodiments of this aspect, the side chain, an additional moiety Such as, for example, a label polypeptide is a therapeutically active polypeptide such as, ing moiety. Such modified and labeled polypeptide can be for example, agallsidase-beta, alglucosidase-alpha, alpha-ga used for various medical, analytical, imaging and diagnostic lactosidase, B-deleted domain Factor VIII, bone morphoge purposes. netic protein-2 (BMP-2), bone morphogenetic protein-7 0246. As used herein, the phrase “labeling moiety” refers (BMP-7), brain-derived neurotrophic factor (BDNF), cetux to a detectable moiety or a probe and includes, for example, imab, chorionic gonadotropin (CG), dornase-alpha, erythro fluorescent moieties, phosphorescent moieties, chro poietin (EPO), etanercept, Factor IX, Factor VIIa, Factor mophores, phosphorescent moieties, heavy metal clusters, VIII, follicle stimulating hormone (FSH), galsulfase, glial magnetic moieties and radioactive labeling moieties, as well cell line derived neurotrophic factor (GDNF), glucagon, as any other known detectable moieties and any combination granulocyte colony Stimulating factor (G-CSF), granulocyte thereof. macrophage colony Stimulating factor (GM-CSF), growth 0247. As used herein, the term "chromophore” refers to a hormone (GH), hemoglobin, heparanase, hyaluronidase, imi chemical moiety that, when attached to another molecule, glucerase, insulin-like growth factor-1 (IGF-1), interferon renders the latter colored and thus visible when various spec alpha-2a (IFNalpha-2a), interferon-alpha-2b (IFNalpha-2b). trophotometric measurements are applied. interferon-beta-1a (IFN beta-1a), interferon-beta-1b (IFN 0248. The phrase “fluorescent moiety” refers to a moiety beta-1b), interferon-gamma-1b (IFN gamma-1b), interleukin that emits at a specific wavelength during exposure to (IL-11), interleukin (IL-2), interleukin-1 (IL-1) receptor radiation from an external source. antagonist, interleukin-1 receptor antagonist (IL-1 ra), kerati 0249. The phrase “phosphorescent moiety” describes a nocyte growth factor (KGF), laronidase, luteinizing hormone moiety emitting light without appreciable heat or external (LH), megakaryocyte growth differentiation factor (MGDF), excitation as by slow oxidation of phosphorous. obesity protein (OB protein or leptin), osteoprotegerin 0250) A heavy metal cluster can be for example a cluster of (OPG), parathyroid hormone (PTH or 1-34 segment or PTH gold atoms used, for example, for labeling in electron micros 1-34), platelet-derived growth factor (PDGF). Protein C, rit copy techniques. uximab, stem cell factor (SCF), thrombin, thrombopoietin 0251 For example, by attempting to label a given thera (TPO), thyrotropin (TSH), tissue plasminogen activator peutically active polypeptide with a detectible moiety, (tPA), trastuzumab, tumor necrosis factor binding protein according to embodiments presented herein, one can verify (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate whether a methionine side-chain is accessible for a beneficial oxidase. conjugation with a polymer moiety. As presented in the 0239. The functional moiety, according to some embodi Examples section that follows, a therapeutically active ments, is selected so as to allow conjugation of other moieties polypeptide labeled with Luciferyellow (a well-known polar to the modified polypeptide, via the modifying moiety. tracer for neurons) as labeling moiety can be used to verify According to Some embodiments, the functional moiety is a whether a PEG moiety could be attached to a given polypep second reactive moiety. The second reactive moiety can be, tide (see, Example 16). for example, amine, carboxyl, amide, hydrazine, hydrazide, 0252. The modified polypeptide compound presented thiol, hydroxyl and hydroxylamine, and any combination herein can be prepared using conditions similar to those used thereof, including any moiety that enables covalent attach for the preparation of the polymer-polypeptide conjugate pre ment of a desired moiety. sented hereinabove. Thus, according to another aspect of the 0240. The term “hydroxyl, as used herein, refers to an present invention there is provided a process of preparing the —OH group. modified polypeptide compound described herein, which is US 2011/O 150820 A1 Jun. 23, 2011 effected by reacting a polypeptide with a modifying moiety which comprises a pharmaceutically acceptable carrier and, which has a first reactive moiety and a second reactive moiety as an active ingredient, a conjugate as presented herein, which under acidic conditions ranging from pH 2 to pH 5. includes: 0253) The first reactive moiety and the second reactive 0262 (a) a polypeptide, as defined and exemplified moiety are selected such that a covalent bond is selectively hereinabove, which has at least one methionine residue formed between the first reactive moiety and the sulfur atom, in its amino-acid sequence; and thereby obtaining the modified polypeptide compound. The 0263 (b) at least one polymer moiety being covalently particular selection of the first reactive moiety and the second attached to the Sulfur atom a methionine side-chains of reactive moiety should be considered since the desired effect at least one the methionine residues. is a reaction between the first reactive group and the protein, 0264. Accordingly, there is provided a use of a conjugate leaving the second reactive moiety unaffected and possibly as presented herein in the manufacture of a medicament. free for another reaction at different condition with, for example, a polymer moiety or a labeling moiety. This dis 0265. As used herein a “pharmaceutical composition' criminating reactivity with respect to the first reactive moiety refers to a preparation of the conjugates presented herein, versus the second reactive moiety can be achieved by select with other chemical components such as pharmaceutically ing two moieties which are reactive under different condi acceptable and Suitable carriers and excipients. The purpose tions, or by protecting the second reactive group with an of a pharmaceutical composition is to facilitate administra acid-proof protecting group Such that it will not be removed tion of a compound to an organism. during the reaction in which the reactive moiety forms a 0266. Hereinafter, the term “pharmaceutically acceptable covalent bond with the sulfur. carrier” refers to a carrier or a diluent that does not cause 0254 An example of such selective reactivity can be significant irritation to an organism and does not abrogate the achieved with an alkyl halide for one reactive group and an biological activity and properties of the administered com amide for the other reactive group. Similarly, a hydrazine and pound. Examples, without limitations, of carriers are: propy an acyl-halide can be used having discriminating reactivity. lene glycol, saline, emulsions and mixtures of organic Sol 0255 Hence, according to another aspect of embodiments vents with water, as well as solid (e.g., powdered) and of the invention there is provided a process of preparing a gaseous carriers. conjugate which comprises: 0267. Herein the term “excipient” refers to an inert sub 0256 a) a polypeptide having at least one methionine resi stance added to a pharmaceutical composition to further due; and facilitate administration of a compound. Examples, without 0257 b) at least one polymer moiety attached to a sulfur limitation, of excipients include calcium carbonate, calcium atom of a methylsulfanyl-ethyl side-chain of said at least one phosphate, various Sugars and types of starch, cellulose methionine residue, which is effected by reacting a polypep derivatives, gelatin, vegetable oils and polyethylene glycols. tide with a modifying moiety as presented hereinabove, under 0268 Techniques for formulation and administration of acidic conditions ranging from pH 2 to pH 5, and thereafter drugs may be found in “Remington's Pharmaceutical Sci reacting this modified polypeptide having at least one modi ences’ Mack Publishing Co., Easton, Pa., latest edition, fying moiety attached thereto with a polymer having a third which is incorporated herein by reference. reactive moiety which is selected capable of reacting with the 0269 Pharmaceutical compositions for use in accordance second reactive moiety of the modifying moiety, thereby with the present embodiments thus may be formulated in obtaining the conjugate. conventional manner using one or more pharmaceutically 0258. In other words, this two-step process is based on a acceptable carriers comprising excipients and auxiliaries, first step wherein a hetero-bifunctional moiety (a moiety hav which facilitate processing of the present conjugates into ing at least two different reactive moieties) is attached to a preparations which can be used pharmaceutically. Proper for polypeptide, thereby affording a modified polypeptide, and a mulation is dependent upon the route of administration cho second step wherein a polymer is attached to that moiety of sen. The dosage may vary depending upon the dosage form the modified polypeptide, thereby affording a polypeptide employed and the route of administration utilized. The exact polymer conjugate. formulation, route of administration and dosage can be cho 0259. According to some embodiments of the present sen by the individual physician in view of the patient's con invention, the third reactive moiety, which forms a part of the dition (see e.g., Finglet al., 1975, in “The Pharmacological polymer moiety, is selected capable of interacting with the Basis of Therapeutics' Ch. 1 p. 1). second reactive moiety, which forms a part of the modifying 0270. The pharmaceutical composition may be formu moiety, and covalently attach the polymer moiety thereto. lated for administration in either one or more of routes Exemplary Such (third) reactive groups include, but are not depending on whether local or systemic treatment or admin limited to, amine, carboxyl, amide, hydrazine, hydrazide, istration is of choice, and on the area to be treated. Adminis thiol, hydroxyl and hydroxylamine, and any combination tration may be done orally, by inhalation, or parenterally, for thereof. example by intravenous drip or intraperitoneal, Subcutane 0260. As discussed hereinabove, the conjugates presented ous, intramuscular or intravenous injection, or topically (in herein can be used in a variety of medical, diagnostic and cluding ophtalmically, vaginally, rectally, intranasally). other pharmaceutical and therapeutic purposes. In any of the 0271 Formulations for topical administration may purposes mentioned herein, the conjugates of the present include but are not limited to lotions, ointments, gels, creams, embodiments can be utilized either perse or, alternatively, as Suppositories, drops, liquids, sprays and powders. Conven a part of a pharmaceutical composition that further comprises tional pharmaceutical carriers, aqueous, powder or oily bases, a pharmaceutically acceptable carrier. thickeners and the like may be necessary or desirable. 0261 Thus, according to another aspect of the present 0272 Compositions for oral administration include pow invention, there is provided a pharmaceutical composition ders or granules, Suspensions or Solutions in water or non US 2011/0150820 A1 Jun. 23, 2011 20 aqueous media, Sachets, pills, caplets, capsules or tablets. used to accelerate recovery from neutropenia after chemo Thickeners, diluents, flavorings, dispersing aids, emulsifiers therapy; to increase the number of hematopoietic stem cells in or binders may be desirable. the blood of the donor before collection by leukapheresis for 0273 Formulations for parenteral administration may use in hematopoietic stem cell transplantation; and to treat include, but are not limited to, sterile solutions which may heart degeneration. also contain buffers, diluents and other suitable additives. 0282 Human growth hormones (GH) are known to have a Slow release compositions are envisaged for treatment. beneficial effect on many human diseases. A human GH 0274 The amount of a composition to be administered PEGylated according to the present embodiments, can there will, of course, be dependent on the subject being treated, the fore be used to treat diseases such as, for example, Turner severity of the affliction, the manner of administration, the syndrome, chronic renal failure, Prader-Willi syndrome, judgment of the prescribing physician, etc. intrauterine growth retardation, severe idiopathic short stat 0275 Compositions of the present embodiments may, if ure, AIDS, short bowel syndrome, remission of multiple scle desired, be presented in a pack or dispenser device, such as an rosis, aging in older adults, obesity, fibromyalgia, Crohn's FDA (the U.S. Food and Drug Administration) approved , disease and ulcerative colitis, and can also be used for other which may contain one or more unit dosage forms containing purposes such as bodybuilding or athletic enhancement. Non the active ingredient. The pack may, for example, comprise human growth hormones, modified according to the present metal or plastic foil, such as, but not limited to a blister pack embodiments, can be used to treat farm animals or to modify or a pressurized container (for inhalation). The pack or dis their productivity, such as to increase milk production in penser device may be accompanied by instructions for admin cattle. istration. The pack or dispenser may also be accompanied by (0283) Follicle-Stimulating Hormone (FSH) is involved in a notice associated with the containerina form prescribed by controlling the menstrual cycle and the production of eggs by a governmental agency regulating the manufacture, use or the ovaries. The amount of FSH varies throughout a woman's sale of pharmaceuticals, which notice is reflective of approval menstrual cycle and peaks just before ovulation. In men, FSH by the agency of the form of the compositions for human or is involved in controlling the production of sperm, and its Veterinary administration. Such notice, for example, may be level typically remains constant. Abnormally low level of of labeling approved by the U.S. Food and Drug Administra FSH can result in failure of gonadal function (hypogo tion for prescription drugs or of an approved product insert. nadism), which is typically manifested in males as failure in Compositions comprising a conjugate of the invention for production of normal numbers of sperm, and cessation of mulated in a compatible pharmaceutical carrier may also be reproductive cycles in females. Conditions which are associ prepared, placed in an appropriate container, and labeled for ated with abnormally low level of FSH include infertility, treatment of a medical condition which is associated with the polycystic ovarian syndrome (POS), POS combined with polypeptide which forms a part of the conjugate. obesity hirsutism and infertility, Kallmann syndrome, hypo 0276 According to further embodiments of the any of the thalamic suppression, hypopituitarism, hyperprolactinemia, compositions, methods and uses presented herein, the conju gonadotropin deficiency, and gonadal suppression. gates of the present invention can be combined with other (0284 Hence, according to another aspect of embodiments active ingredients. of the invention, there is provided a method of treating a (0277 As discussed hereinabove, PEGylation of polypep medical condition which is treatable by a polypeptide that at tide drugs can impart several significant pharmacological least one methionine residue. According to some embodi advantages over the unmodified form thereof, by increasing ments of the present invention, the method is effected by the molecular weight of the polypeptide and providing some administering to a subject in need thereof a therapeutically degree of protection thereto, which also improves the drug effective amount of one or more of the PEGylated conjugates, solubility, reduces dosage frequency without diminished effi as described hereinabove. cacy with potentially reduced toxicity, extends circulating (0285) As used herein, the terms “treating” and “treatment” life, increases drug stability and enhances protection from includes abrogating, substantially inhibiting, slowing or proteolytic degradation. The PEGylation process also opens reversing the progression of a condition, substantially ame other avenues, such as new delivery formats and dosing and liorating clinical or aesthetical symptoms of a condition or administration regimens. Substantially preventing the appearance of clinical or aes 0278 For example, interferons (e.g., as interferon beta-1a thetical symptoms of a condition. and interferon beta-1b) which are PEGylated via a methion 0286 As used herein, the phrase “therapeutically effective ine side chain, can be used to treat many diseases which are amount” describes an amount of the composite being admin treatable by these interferons, such as multiple sclerosis and istered which will relieve to some extent one or more of the the relapsing-remitting form of multiple sclerosis, respec symptoms of the condition being treated. tively. (0287. The method of treatment, according to some 0279 Interferons belonging to the interferon alpha-2 fam embodiments of the invention, may include the administra ily, which are PEGylated via a methionine side chain, can be tion of an additional therapeutically active agent. used to treat many diseases which are treatable by these (0288 Medical conditions which are treatable by polypep interferons, such as Kaposi's sarcoma, anogenital warts, tides that have at least one methionine residue include, for hepatitis B and C and anti-HIV. Some non-limiting examples, Kaposi's sarcoma, anogenital 0280 Erythropoietin (EPO), PEGylated via a methionine warts, hepatitis B and C. AIDS, anaemia, neurodegenerative side chain, can be used to treat many diseases such as diseases and chronic kidney diseases, cancer as adjuvant anaemia, neurodegenerative diseases and chronic kidney dis therapy, neutropenia after ; heart degeneration, eases, and as adjuvant therapy in the treatment of cancer. Turner syndrome, chronic renal failure, Prader-Willi syn (0281 Granulocyte colony-stimulating factor (G-CSF), drome, intrauterine growth retardation, severe idiopathic PEGylated according to the present embodiments, can be short stature, short bowel syndrome, remission of multiple US 2011/O 150820 A1 Jun. 23, 2011

Sclerosis, aging in older adults, obesity, fibromyalgia, 0304 MALDI-TOF and ESI mass spectrometry was per Crohn's disease and ulcerative colitis, infertility, polycystic formed at the Weizmann Institute, Israel ovarian syndrome (POS), POS combined with obesity hirsut ism and infertility, Kallmann syndrome, hypothalamic Sup Example 1 pression, hypopituitarism, hyperprolactinemia, gonadotropin deficiency and gonadal Suppression. PEGylation of Methionine Containing Proteins— 0289 Since the aforementioned polypeptides are in a form General Procedure of a PEGylated conjugate, the administration can be effected, 0305 PEGylation of recombinant and/or native proteins for example, orally, by intravenous injection, by Subcutane which exhibit at least one unmodified methionine side chain ous injection or topically. in their structure is performed with an exemplary PEG moiety 0290. It is appreciated that certain features of the inven Such as 30 kDa methoxy polyethylene glycol reagents as tion, which are, for clarity, described in the context of separate follows: embodiments, may also be provided in combination in a 0306 A solution of 2.5 umol of a methoxy polyethylene single embodiment. Conversely, various features of the glycol reagent in acetate buffer (pH 4) is added to a vial invention, which are, for brevity, described in the context of a containing a solution of 0.055 Limol protein dissolved in single embodiment, may also be provided separately or in any acetate buffer (pH 4). The reaction is stirred in the dark at 25° suitable subcombination or as suitable in any other described C. for a time period ranging from about 24 hours to about 200 embodiment of the invention. Certain features described in hours. the context of various embodiments are not to be considered 0307. The reaction mixture is thereafter diluted with the essential features of those embodiments, unless the embodi reaction buffer and loaded onto a chromatographic column ment is inoperative without those elements. which is pre-equilibrated with the reaction buffer, utilizing a FPLC system. The loaded columnis washed with the reaction 0291 Various embodiments and aspects of the present buffer and the unbound fraction is collected. The sample is invention as delineated hereinabove and as claimed in the eluded with an acidic, neutral oralkali solution or saline in the claims section below find experimental support in the follow reaction buffer solution and the fractions are collected into ing examples. tubes. 0308 Alternatively, the reaction mixture is purified by EXAMPLES preparative reverse-phase chromatography (RP-HPLC) by injecting the mixture into a Jupiter C4 column with acetoni 0292 Reference is now made to the following examples, trile-water and 0.2% TFA as mobile phase. The fraction con which together with the above descriptions; illustrate the taining the PEGylated protein is collected and concentrated invention in a non limiting fashion. by centrifugation of the media through a filter and by ultra filtration on Nanosep 3K (Pall). Materials and Methods 0309 An SDS-PAGE is run under non-reducing condi tions in order to avoid cleavage of the methionine Sulfonium 0293. The human recombinant protein samples inter bonds by mercaptoethanol and other harsh reagents as shown feron-beta-1b, interferon-beta-1a, G-CSF and FSH were previously Naider, F and Bohak, Z, Biochemistry, 11, 3208, obtained from InSight Biopharmaceuticals, Israel. 1972. The samples are subjected to non-reducing SDS 0294 Recombinant human erythropoietin (rh-EPO), mar PAGE 4-12% bis-tris gel with MOPS buffer. The separation gel is run for 50 minutes at 200 V, and thereafter the gel is keted as NeoRecormon R) was purchased from Hoffman exposed for 15 minutes with fixing solution and stained, after LaRoche (lot #MH69265) washing the gel for 1 hour with GelCode Blue. The separation 0295 Recombinant human interferon-alpha-2a (rh-INF gel is thereafter destained for 4 hours, and photographed. The C2a) was purchased from Amoytop Biotech, China. separation gel is incubated in a solution of barium chloride in 0296 Recombinant human growth hormone (rh-GH) water (5%), according to the procedure described in Basu et (cathicYT-202) was purchased from Prospec-Tany, Israel al., Bioconjug. Chem., 17, 618, 2006. After several rinses 0297 30 kDa Methoxy polyethylene glycol N-ethyl-2- with water, the gel is stained with iodine solution (0.1 N iodo-acetamide (cathé293) was purchased from Biovectra Titrisol, Merck) for 5 minutes followed by destaining with DCL, Canada. several replacements of the water. 0298 30 kDa Methoxy polyethylene glycol ethylamine (catio234) was purchased from Biovectra DCL, Canada Example 2 0299 Endoproteinase Lys-C (cat #P3428) was purchased from Sigma-Aldrich PEGylation of Recombinant Human Interferon-beta 0300 Empigen BB detergent (cat #45165) was purchased 1b (rh-IFN-B1b) with 30 kDa Methoxy Polyethylene from Sigma-Aldrich Glycol N-ethyl-2-iodo-acetamide 0301 Tosyl phenylalanyl chloromethyl ketone (TPCK) 0310 Interferon beta-1b, marketed as BETASERONR) by treated trypsin (cat #3740) was purchased from Worthington Berlex Corporation, is produced in modified E. coli strands Biochemical Corporation. and used to treat multiple Sclerosis typically by Subcutaneous 0302 HPLC was performed on an Agilent 1200 and a injection, and has been shown to slow the advance of the Waters 2695 instrument. affliction as well as reduce the frequency of attacks. 0303 Determination of protein concentration was per 0311. The PEGylation reaction was performed using formed on a Nanodrop ND1000 spectrophotometer PEG-iodoacetamide, as depicted in Scheme 1 below. US 2011/O 150820 A1 Jun. 23, 2011 22

2; molecular weight markers were run in lane 3; and a series of samples of 30 kDa methoxy polyethylene glycol N-ethyl Scheme 1 2-iodo-acetamide (the PEGylation reaction product) at vari / PEG ous concentrations (0.078 ug, 0.3125 ug, 3.125 ug, 6.25 ug, S 12.5 lug and 25 ug) which were run in lanes 4, 5, 6, 7, 8 and 9 Selective respectively. alkylation 0316. As can be seen in FIGS. 2a-b, interferon-beta-1b -- -e- appeared as a blue band having a molecular weight of about 18 kDa in the Coomassie blue stained gel (FIG.2a A, lane 1), Or acidic pH and was not stained in brown with iodine (FIG.2bB, lane 1). The PEG N-ethyl-2-iodo-acetamide reagent at different con methioning side-chain I centrations stained only with iodine and not by the Coomassie on a protein PEG-iodoacetamide blue, appeared as a brown band having an apparent molecular PEG weight of about 55 kDa (FIG.2bB, lanes 4, 5, 6, 7, 8 and 9). The sample collected by RP-HPLC exhibited a new species which is seen as a new band (marked with an arrow in FIGS. 2a A-bB, lane 2) having an apparent molecular weight of O NH about 70 kDa, a size which is the sum of the molecular weight of interferon-beta-1b and the molecular weight of the PEG moiety. This band was stained by both with Coomassie blue r and iodine. 0317. The fraction of the PEGylated interferon-beta-1b, I collected by RP-HPLC, was further analyzed by MALDI TOF mass spectrometry using a 2,4-dihydroxybenzoic acid matrix. FIG. 3 presents a MALDI-TOF mass spectrogram of the PEGylation reaction product of recombinant human inter feron-beta-1b with 30 kDa methoxy polyethylene glycol PEGylated protein N-ethyl-2-iodo-acetamide, using a 2,4-dihydroxybenzoic acid matrix, showing a small peak having an average molecu 0312. A solution of 75 mg (2.5 umol) 30 kDa methoxy lar weight of about 50,000 Da corresponding to the molecular polyethylene glycol N-ethyl-2-iodo-acetamide in 0.15 ml of weight of a mono-PEGylated interferon-beta-1b conjugate: phosphoric acid buffer solution (0.1 M. pH 2.52) was added to 0318. As can be seen in FIG. 3, this analysis revealed the a vial containing a solution of 0.975 mg (0.055umol) rh-IFN presence of a peak which corresponds to an average molecu B1b in 1 ml phosphoric acid buffer solution (0.1 M. pH 2.52), lar weight of about 50 kDa, corroborating that a mono-PE and the reaction was stirred in the dark at 25°C. for 1 week Gylated interferon-beta-1b was formed. Surprisingly, the (168 hours). main peak corresponds to an average molecular weight of 0313 Purification of the reaction mixture by preparative about 20kDa, corresponding to interferon-beta-1b which was reverse-phase chromatography was performed according to not visible in the SDS-PAGE. A possible explanation to this the general procedure presented hereinabove. Briefly, 60 ul of discrepancy may be that the methionine sulfonium bond of the mixture was injected into a Jupiter C4 column with aceto the PEGylated interferon-beta-1b severed during the mass nitrile-water and 0.2% TFA as mobile phase. The fraction of spectrometry experiment leading back to the starting mate the PEGylated protein was collected at a retention time of rial. A further analysis of this phenomenon is delineated 28.9 minutes. hereinafter (see, Example 4). 0314 FIG. 1 presents an reverse-phase HPLC (RP-HPLC) 0319. The fact that the PEGylation occurred at pH 2.5 is chromatogram of the PEGylation reaction products of recom indicative of the involvement of the thiomethoxy group of binant human interferon-beta-1b and 30 kDa methoxy poly methionine in the chemical modification, since methionine is ethylene glycol N-ethyl-2-iodo-acetamide, obtained using a the only amino acid that is chemically reactive in alkylation photo-diode array set at 280 nm, an injection Volume of 40 ul, reactions at this pH. total run-time of 70 minutes and 0.2% TFA in waterfacetoni trile as a mobile phase, showing a peak having a retention Example 3 time of 29.17 minutes and a peak corresponding to interferon Synthesis of 30 kDa Methoxy Polyethylene Glycol beta-1b having a retention time of 33.84 minutes. This frac N-ethyl-(4-bromomethyl)-benzamide tion was concentrated to 100 ul by speed-vac and by ultrafil tration on NanoSep 3K (Pall), and analyzed in a non-reducing 0320 Oxalyl chloride (14.4 mg., 0.114 mmol) and 1 drop SDS-PAGE. of DMF were added to a mixture of 4-(bromomethyl)benzoic 0315 FIGS. 2A-B present color images of a non-reducing acid (8.2 mg; 0.038 mmol) dissolved in 0.5 ml dry THF and SDS-PAGE gel slab in which the PEGylation reaction prod cooled to 0° C. The mixture was stirred for 2 hours and uct of recombinant human interferon-beta-1b and 30 kDa thereafter the solvent was evaporated under reduced pressure methoxy polyethylene glycol N-ethyl-2-iodo-acetamide (as to give a yellowish product. This product was dissolved in 1 isolated by RP-HPLC) and the starting materials of the ml of dry dioxane and added to a mixture of 30 kDa PEG PEGylation reaction were run and stained with Coomassie ethylamine (190 mg; 0.0063 mmol) and triethylamine (22.9 Blue (FIG. 2A) and subsequently with iodine (FIG. 2B), mg, 0.2 mmol) in 1.5 ml of dioxane. The resulting mixture wherein an un-PEGylated sample of interferon-beta-1b was was stirred for 16 hours at 25°C. and a white suspension was run in lane 1; the isolated peak by RP-HPLC was run in lane formed. After addition of 10 ml of dry ether the mixture was US 2011/O 150820 A1 Jun. 23, 2011

filtered and the white solid residue was triturated with dry 50 ul of the mixture into a Jupiter C4 column with acetoni ether and thereafter dried to afford 187.5 mg of product at an trile-water and 0.2%TFA as mobile phase. The fraction of the overall yield of 97%. PEGylated protein was collected at a retention time of 38 0321) The reactivity of the PEG-benzyl bromide for thiol minutes. groups found in cysteine side-chains and methylsulfanyl 0325 FIG. 4 presents an RP-HPLC chromatogram of the groups found in methionine side-chains, was evaluated at PEGylation reaction product of recombinant human inter about 90% by an indirect Ellman's test assay, performed feron-beta-1b and 30 kDa methoxy polyethylene glycol according to a published procedure Morpurgo and Veronese, N-ethyl-(4-bromomethyl)-benzamide, showing a peak hav Methods Mol. Biol. 288, 45-70, 2004). ing a retention time of 30.27 minutes corresponding to meth Example 4 oxy polyethylene glycol N-ethyl-(4-bromomethyl)-benza mide, a second peak having a retention time of 38 minutes PEGylation of Recombinant Human Interferon-beta corresponding to the PEGylated protein, and a peak having a 1b (rh-IFN-B1b) with 30 kDa Methoxy Polyethylene retention time of 48.44 minutes corresponding to recombi Glycol N-ethyl-(4-bromomethyl)-benzamide nant human interferon-beta-1b. 0322. In order to study the efficiency of other PEGylation 0326. The fractions were concentrated to 100 ul by speed reagents, the reaction presented hereinabove with PEG-io vac and were analyzed on two non-reducing SDS-PAGE gel doacetamide was performed using PEG-benzyl bromide as a slabs and color images of the obtained slabs are presented in PEGylation reagent, as depicted in Scheme 2 below. FIGS. SA-B. 0327 FIGS.5A-B present color images of a non-reducing SDS-PAGE separation gel in which the PEGylation reaction Scheme 2 products of recombinant human interferon-beta-1b and 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom ethyl)-benzamide, after collection by RP-HPLC, were run and stained with Coomassie Blue (FIG.5A) and subsequently with iodine (FIG. 5B), wherein the collected fractions were Selective O ". NH run in lanes 1, 2 and 3; an un-PEGylated sample of recombi benzylation -- -e- nant human interferon-beta-1b was run in lane 4, a 30 kDa acidic pH methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben Zamide sample was run in lane 5 and molecular weight mark ers were run in lane 6. 0328. As can be seen in FIGS. 5A-B, lane 4, interferon methioning side-chain beta-1b appeared as an 18 kDa band stained with Coomassie on a protein and not with iodine. The PEG N-ethyl-(4-bromomethyl)-ben Br Zamide reagent stained only with iodine and not by the Coo PEG-benzyl bromide massie blue, appeared as a brown band having an apparent molecular weight of about 55 kDa (FIG. 5B, lane 5). It can be ar observed that fraction 1 collected from RP-HPLC (retention O NH time of about 30 minutes) is mainly a di-PEGylated product since it appeared as a band having a molecular weight of about 120 kDa (which is the sum of interferon-beta-1b and twice the apparent molecular weight of PEG) in the Coo massie stained gel (FIG. 5A, lane 1), and was stained brown with iodine (FIG. 5b, lane 1). In addition, it can be seen that fraction 2 collected from RP-HPLC (retention time of about / 38 minutes) is mainly a mono-PEGylated product since it St appeared as a band having a molecular weight of about 70 kDa (which is the sum of interferon-beta-1b and the apparent Br molecular weight of PEG) in the Coomassie stained gel (FIG. 5A, lane 2), and was stained in brown with iodine (FIG. 5B, lane 2). Fraction 3 collected from RP-HPLC (retention time of about 48 minutes) is the non-reacted interferon-beta-1b (FIGS. 5a-b, lane 3) similarly to the interferon-beta-1b stan PEGylated protein dard (FIGS.5A-B, lane 4). These results indicate that a mono PEGylated interferon-beta-1b was formed by using a PEG 0323 Methoxy polyethylene glycol N-ethyl-(4-bromom N-ethyl-(4-bromomethyl)-benzamide reagent at acidic con ethyl)-benzamide (30 kDa, 27.7 mg, 0.92 umol) was added to ditions. a solution of rh-IFN-B1b (3.70 mg, 0.18 umol) in phosphoric 0329. The sample collected by RP-HPLC that was acid (0.77ml, 0.01 M. pH 2.5), and the reaction was incubated assigned as the PEGylated protein was analyzed by MALDI in a "head over tail shaker at 22°C. for 24 hours. Substantial TOF mass spectrometry. FIG. 6 presents a MALDI-TOF amounts of PEGylated product was formed in 24 hours, indi mass spectrogram of the purified PEGylation product of cating the high reactivity of the PEG-benzyl bromide reagent. recombinant human interferon-beta-1b with 30 kDa methoxy 0324 Purification of the reaction mixture by preparative polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide, reverse-phase chromatography was performed by injecting using a 2,4-dihydroxybenzoic acid matrix. US 2011/O 150820 A1 Jun. 23, 2011 24

0330. As can be seen in FIG. 6, a peak corresponding to 0332 The 30 kDa PEGylated interferon-beta-1b was also species of an average molecular weight of about 50 kDa was analyzed by the more accurate electrospray ionization (ESI) detected, as in the case of PEGylated interferon beta-1b pro mass spectrometry. FIGS. 7A-B present ESI mass spectro duced with 30 kDa PEG-iodoacetamide, corroborating that a grams of the purified PEGylation product of recombinant mono-PEGylated interferon-beta-1b was formed. Surpris human interferon-beta-1b with 30kDa methoxy polyethylene ingly, the main peak with an average molecular weight of glycol N-ethyl-(4-bromomethyl)-benzamide (FIG. 7A) and about 20 kDa, was identified as belonging to interferon-beta recombinant human interferon-beta-1b (FIG. 7B). 1b, a species which was not detectable in the SDS-PAGE. As 0333 As can be seen in FIGS. 7A-B, no molecular peak of already postulated in the case of PEGylated interferon beta a 30kDa PEG-interferon beta-1b conjugate was observed (about 50 kDa), as in the case of the MALDI-TOF mass 1b from PEG-iodoacetamide, a possible explanation to this spectrometry analysis, and the main peak was due to the discrepancy was that the relatively labile methionine sulfo Hoffman elimination described hereinabove, resulting in a nium bond of the PEGylated interferon-beta-1b was dis mass of 19,829 Da. rupted during the mass spectrometry experiment, leading 0334. The measured molecular mass of interferon beta-1b back to the starting material. Decomposition of proteins bear by ESI was 19,877 Da (see FIG. 7B). Therefore, the differ ing alkylated methionines has been reported to occur under ence in mass between decomposed 30kDa PEG-interferon mass spectrometry conditions Bykova, N V et al., Anal. beta-1b by Hoffman elimination and interferon beta-1b is Chem., 78, 1093-103, 2006. A likely reaction mechanism for exactly 48 Da, which is very specific to alkylated methionines decomposition of the Sulfonium salt in the source of the mass (see Scheme 3). spectrometer is the Hoffman elimination reaction. Derivati 0335) Since no other cases of decomposition of PEGylated zation of methionine followed by this decomposition yields a proteins have been described, both mass spectrometry tech net decrease of 48 Da from the mass of the starting protein niques indicate very strongly that the PEGylation atacidic pH (see Scheme 3 below). Careful analysis of the MW of the occurred via the methionines as was targeted. main peak in the MALDI-TOF spectrum shows that the MW of 30 kDa PEG-interferon beta-1b after decomposition is 51 Example 5 Da less than the theoretical MW value of interferon beta-1b (19,879 Da). This net decrease is similar to the MW of the Peptide Mapping of PEGylated Recombinant Human Hofmann elimination product (decrease by 48 Da) and may Interferon-beta-1b be in the inaccuracy range of the MALDI-TOF measurement. 0336. In order to study the effect of PEGylation and to 0331 Scheme 3 presents a typical methionine side-chain verify the PEGylation site along the polypeptide chain, modification with iodoacetamide undergoing collision-in PEGylated recombinant human interferon-beta-1b (rh-IFN duced dissociation (CID) fragmentation in mass spectrom B1b) was subjected to protease digestion, and the breakup etry by Hoffman elimination. products were compared to those obtain from the un-PEGy lated peptide by a method known as peptide mapping. 0337 Peptide mapping was performed according to Scheme 3 Johnson-Jackson, D et al. WO 08/020968. Recombinant O human-IFN-B1b was prepared at a concentration of 0.5 H O mg/ml in 2 mMaspartic acid. Empigen BB (30%, 1.3 ul) was N added to rh-IFN-B1b, and the pH was adjusted to 7 using -- I sus Tris-HCl pH 7 (1 M., 62.5 ul). The digest was started by NH2 addition of 1 Jul Lys-C (1 mg/ml), and the reaction mixture iodoacetamide was incubated at 37°C. with a “head-over-tail shaking for 4 hours. Thereafter, another aliquote of 1 Jul of Lys-C was added SN and sample was incubated for an additional 24 h. To quench a methionine the digest, 13 ul of 8 M guanidine HCl was added to the residue in a protein reaction mixture. 0338 Peptide fragments were resolved by HPLC chroma H N tography by injecting 50 ul of the mixture into a Jupiter C4 column with acetonitrile-water and 0.1% TFA as mobile

". -e-CID phase. 0339. In a control reaction in which the digested peptides O were reduced at the end of the incubation, 0.5ul of 1 MDTT S. was added to 90 ul of the reaction mixture, and incubation was -- N performed for 45 minutes at 37° C. methionine Sulfonium ion (0340 Peptide mapping of PEGylated rh-IFN-B1b was O performed simultaneously and in the same manner as peptide H O mapping of rh-IFN-B1b described above. (0341 FIGS. 8A-B present RP-HPLC chromatograms 1N -- HN --- S N showing the peptide maps of purified PEGylation product of recombinant human interferon-beta-1b with 30 kDa methoxy N polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide a protein missing 48 Da (FIG. 8A), and of recombinant human interferon-beta-1b (FIG. 8B), as obtained by applying peptide digestion using Lys-C. US 2011/O 150820 A1 Jun. 23, 2011

0342. As can be seen in FIG. 8B, the peptide map that was (0347 LC/MS/MS analysis of the content of the new peak obtained by Lys-C digestion of interferon beta-1b is similar to (FIG.9, lane 2) has shown that it contains molecular masses the published peptide map of the commercial interferon beta that correspond to peptides K3-48 Da (MW=1457.7) and 1b under similar conditions Lin, L S. Kunitani, M G and K9-48 Da (MW=879.5) and fragmentations of 44 Da which Hora, MS, Interferon-beta-1b (BETASERONR): A model are typical for the disrupted PEG chain. This is an additional for hydrophobic therapeutic proteins in Formulation, charac evidence that PEGylation of rh-IFN-?31b with 30 kDa meth terization and stability of protein drugs, Pearlman, R and oxy polyethylene glycol N-ethyl-(4-bromomethyl)-benza Wang, YJ (Eds.), Plenum Press, NY. pp. 275-301, 1996 and mide leads to the formation of only two mono-PEGylated Johnson-Jackson, D et al., WO 08/020968, which allows isoforms via methionines M117 and M36. assigning the peaks to the corresponding peptides. When peptide mapping of interferon beta-lb by Lys-C was per Example 6 formed under reducing conditions the retention time of the peptide containing the single S-S bond (K2-12) changed Antiviral Activity of 30kDa PEG-Interferon Beta-1b from 43.07 minutes to 43.52 minutes (K12) and a new peak VerSuS BETASERONOR) was formed with a retention time of 33.55 minutes corre 0348. The antiviral activity of the conjugate of 30 kDa sponding to K2 according to Johnson-Jackson, D et al., WO PEG-Interferon beta-1b, an exemplary PEGylated active 08/020968 (data not shown). cytokine according to Some embodiments of the present 0343 As can be seen in FIG. 8A, the peptide map of invention, was determined by the capacity of the PEGylated PEGylated interferon beta-1b, which is probably a mixture of cytokine to protect human amnion WISH cells against vesicu PEGylated isoforms, digested by Lys-C is similar to the pep lar stomatitis virus (VSV) induced cytopathic effects Rubin tide map of interferon beta-1b digested by Lys-C, but some stein, M. et al. J. Virol, 37, 755-758, 1981. differences are observed (see FIG. 8B). The intensity of (0349 Lyophilized 30 kDa PEG-Interferon-beta-1b conju mainly two methionine containing peptides, K9 and K3. gate was reconstituted in 2 ml formulation buffer (5 mM decreased in the digested PEGylated protein as compared to aspartic acid, 5% mannitol, pH 4) to afford a solution at a the corresponding peaks in interferon beta-1b digests. The concentration of 40 ug/ml. intensity of the third peptide that contains methionine (K5) 0350. The introduction of 30 kDa PEG-Interferon-beta-1b was the same in both peptide maps. In addition, in the case of to the WISH cells had a specific anti-viral activity of approxi the digested PEGylated protein a new peptide was formed mately 85% compared to that of BETASERONR, indicating having a retention time of 47.5 minutes. From comparison of that the PEGylation of the cytokine did not reduce its bioac both peptide maps it may be concluded that a selective PEGy tivity. This result is extraordinarily positive for a PEGylated lation occurred via methionine amino acids M117 and M36 cytokine as other known and commercially available PEGy and not via M62. These results are in agreement with the lated cytokines, such as PEGASYSR), are said to retain only surface accessibility of these methionines as observed in the 7% of the original bioactivity of the non-PEGylated cytokine crystal structure of interferon beta-1a Karpusas, M et al., Bailon, Pet al., Bioconj. Chem., 12, 195-202, 2001). Proc. Natl. Acad. Sci., 94, 11813, 1997 and with the observed chemical susceptibility of interferon beta-1a after Example 7 oxidation and alkylation Orru, Set al., Biol. Chem., 381, 7. 2000. Pharmacokinetic Profile of 30 kDa PEG-Interferon 0344) The peptide K3 and the new peptide, having a reten beta-1b tion time of 47.5 minutes, which were formed after the pep 0351. The pharmacokinetic profile of 30 kDa PEG-Inter tide Lys-C digestion of the purified PEGylation product of feron-beta-1b, an exemplary PEGylated active cytokine recombinant human interferon-beta-1b with 30 kDa methoxy according to some embodiments of the present invention, was polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide compared to that of a commercially available for of the cytok with Lys-C, were collected by RP-HPLC and subjected to ine, namely BETAFERONR). The study was performed by electrophoresis, and the results are shown in FIGS. 9A-B. monitoring the cytokine concentration in the plasma of rats 0345 FIGS. 9A-B present color images of a non-reducing following intravenous administration of the two drugs. SDS-PAGE separation gel in which the peptides which were 0352 Lyophilized 30 kDa PEG-Interferon-beta-1b was formed after peptide Lys-C digestion of the purified PEGy reconstituted in formulation buffer (5 mMaspartic acid, 5% lation product of recombinant human interferon-beta-1b with mannitol, pH4), affording a solution of 1 mg/ml of the PEGy 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom lated cytokine. BETAFERONR) (250 ug in each vial) was ethyl)-benzamide were run and stained with Coomassie Blue dissolved in water for injection to afford a solution of 1 (FIG.9A) and subsequently with iodine (FIG.9B), wherein mg/ml. The test compounds were administered intravenously the collected fractions of peptide K3 and the new formed through the cannula of three cannulated rats each at a single peptide with a retention time of 47.5 minutes were run in dose of 0.4 mg/kg. lanes 1 and 2 respectively; a 30 kDa methoxy polyethylene 0353 For both test compounds, the blood samples were glycol N-ethyl-(4-bromomethyl)-benzamide sample was run collected from the rats after 0.083, 0.25, 0.5, 1, 2, 4, 7 and 24 in lane 3 and molecular weight markers were run in lane 4. hours. In the case of 30 kDa PEG-Interferon-beta-1b, addi (0346. As can be seen from FIGS. 9A-B, peptide K3 (lane tional blood samples were collected after 48,72 and 96 hours. 1) may not be seen on the gel, as expected due to its low MW. The plasma was separated by centrifugation at 5000 rpm at 4 In contrast, the new peak (FIG.9, lane 2) appeared with a MW C. for 5 minutes, transferred to plastic vials, frozen on dry ice similar to the PEG reagent, as expected for a PEGylated and stored at -80° C. peptide. This band stained both with Coomassie and iodine. 0354 Analysis of samples was performed using Human This evidence indicates that the new peak that appeared in the IFN-B Elisakit (Fujirebio Inc., cat #KAC1201), according to peptide mapping with Lys-C is the newly PEGylated peptide. the protocol provided with the kit. The concentration of 30 US 2011/O 150820 A1 Jun. 23, 2011 26 kDa PEG-Interferon-beta-1b and BETAFERONR) in the 0361. As can be seen in FIGS. 11A-B, rh-IFN-B1a blood samples were calculated from their respective standard appeared as a blue band having a molecular weight of about CUWCS. 22 kDa in the Coomassie blue stained gel (FIG. 11A, lanes 2, 0355 FIG. 10 presents a comparative plot of the concen 3 and 5), whereas an un-PEGylated sample thereof was not tration of interferon-beta-1b (INF-B1b) in plasma of rats as a stained in brown. In addition to the 22 kDa band in lane 3, function of time, following intravenous administration of 0.4 wherein the eluted fraction was run, a new band appeared mg/kg of 30 kDa PEG-Interferon beta-1b (in red) and having an apparent molecular weight of about 75 kDa which BETAFERONR) (in green), showing the pharmacokinetic is the sum of interferon-beta-1a and the apparent molecular profile of the two drugs and the remarkable longer pharma weight of 30 kDa PEG. This band was stained both with cologic range of the PEGylated interferon according to some Coomassie and iodine Solution. embodiments of the present invention. 0356. As can be seen in FIG. 10, the concentration of 0362. These results clearly show that a mono-PEGylated BETAFERONR) in blood decreases very rapidly and 24 hours interferon-beta-1a was afforded. after administration no drug was detected (half-life is 1 hour). In sharp contrast, 24 hours after administration of the 30 kDa Example 9 PEG-Interferon-beta-1b, substantial amounts thereof were detected markedly in blood and even after 96 hours traceable PEGylation of Recombinant Human Interferon-al amounts (1.2 ng/ml) were still detected (half life is 10.2 pha-2a (rh-IFN-C2a) with 30 kDa Methoxy Polyeth hours). The 10-fold longer half-life of PEGylated INF-B1b as ylene Glycol N-ethyl-2-iodo-acetamide compared to BETAFERONR is similar to other PEG-INF B1b molecules that are PEGylated through sites other than 0363 Interferon alpha-2a, marketed as Roferon(R)-A by methionine sulfur Basu et al., Bioconjug. Chem., 17, 618, Hoffmann-Laroche, is produced in modified E. coli strands. 2006). The PEGylation reaction thereof was performed using PEG iodoacetamide, as depicted in Scheme 1 hereinabove. Example 8 0364. A solution of 23.2 mg (0.78 umol) 30 kDa methoxy PEGylation of Recombinant Human Interferon-beta polyethylene glycol N-ethyl-2-iodo-acetamide in 0.1 ml of 1a (rh-IFN-?31a) acetate buffer Solution (pH 4) was added to a vial containing a solution of 0.3 mg (0.015 umol) rh-IFN-C2a in 0.3 ml 0357 Recombinant human interferon beta-la (rh-IFN acetate buffer solution (pH 4), and the reaction was stirred in B1a), marketed as Avonex(R) by Biogen Idec and RebifR by the dark at 25°C. for 5 days (120 hours). Serono, is produced by mammalian cells and used to treat 0365. The reaction mixture (400 ul) was diluted with a multiple sclerosis (MS) typically by subcutaneous injection, solution of sodium acetate (1.6 ml, 20 mM, pH 4) and the and has been shown to have about a 30% to 35% reduction in mixture was loaded onto a Source 15-S column (5 mmx100 the rate of MS relapses, and to slow the progression of dis mm) which was pre-equilibrated with sodium acetate solu ability in MS patients. tion (20 mM, pH 4), utilizing an AKTA FPLC system. The 0358. A solution of 11.2 mg (0.38 umol) 30 kDa methoxy loaded column was washed with sodium acetate solution (60 polyethylene glycol N-ethyl-2-iodo-acetamide in 0.07 ml ml. 20 mM, pH 4), and the unbound fraction was collected. acetate buffer (50 mM, pH 4) was added to a vial containing The elution was done with a NaCl solution (0.5 M) in sodium a solution of 0.168 mg (0.0075umol) rh-IFN-B1a in 0.28 ml acetate solution (20 mM). acetate buffer (50 mM, pH 4). The reaction was stirred in the dark at 25°C. for 144 hours. 0366. The fractions were analyzed by SDS-PAGE and 0359. The reaction mixture (350 ul) was diluted with a color images are presented in FIGS. 12A-B. solution of sodium acetate (0.07 ml, 20 mM, pH 4) and the 0367 FIGS. 12A-B present color images of a non-reduc mixture was loaded onto a Fractogel COO column (0.5 ing SDS-PAGE separation gel in which the PEGylation reac cmx5 cm; by Merck) which was pre-equilibrated with tion products of recombinant human interferon-alpha-2a and sodium acetate solution (20 mM, pH 4), utilizing an AKTA 30 kDa methoxy polyethylene glycol N-ethyl-2-iodo-aceta FPLC system. The loaded column was washed first with mide, which were eluted on a Source 15-S column, were run sodium acetate solution (50 ml, 20 mM, pH 4) and second and stained with Coomassie Blue (FIG. 12A) and iodine with phosphate buffer with 10% propylene glycol (50 ml, 20 (FIG. 12B), wherein the fraction eluted with NaCl solution mM, pH 7), and the unbound fraction was collected. The was run in lane 1; an un-PEGylated sample of interferon elution was done with phosphate buffer with 10% propylene alpha-2a was run in lane 2, and molecular weight markers glycol and 1 MNaCl (20 mM, pH 7), and the fractions were were run in lane 3: showing the PEGylated protein (marked collected into tubes. with an arrow), and an increase of about 50 kDa in the appar 0360. The fractions were analyzed on one non-reducing ent molecular weight of the SDS-PAGE and color images of the obtained slab are pre 0368 PEGylated protein as compared to the un-PEGy sented in FIGS. 11A-B. The gel slab was run and thereafter lated protein. stained with Coomassie Blue (FIG. 11A), and thereafter the 0369. As can be seen in FIGS. 12A-B, interferon-alpha-2a same gel slab was stained with iodine (FIG. 11B). Molecular appeared as a blue band having a molecular weight of about weight markers were run in lane 1; an un-PEGylated sample 16 kDa in the Coomassie blue stained gel (FIG. 12A, lanes 1 of interferon-beta-1a was run in lane2; the eluted fraction was and 2), whereas an un-PEGylated sample thereof was not run in lane 3: the unbound fraction washed with sodium stained in brown with iodine (FIG. 12B, lane 2). The eluted acetate buffer Solutions was run in lane 4, and the crude sample exhibited a new species which is seen as a new band reaction mixture which was loaded on the column was run in (marked with an arrow in FIGS. 12A-B, lane 1) having an lane 5. apparent molecular weight of about 70 kDa, a size which is US 2011/O 150820 A1 Jun. 23, 2011 27 the sum of interferon-alpha-2a and PEG molecular weight. 1), and was stained in brown with iodine (FIG. 14B, lane 1). This band was stained both with Coomassie blue and iodine. In addition, it can be seen that fractions 2 and 3 collected from RP-HPLC are mainly mono-PEGylated products (mono-PE Example 10 Gylated isoforms) since they appeared as bands having a molecular weight of about 70 kDa (which is the sum of PEGylation of Recombinant Human Interferon-al interferon-beta-1b and the apparent molecular weight of pha-2a (rh-IFN-C2a) with 30 kDa Methoxy Polyeth PEG) in the Coomassie stained gel (FIG. 14A, lanes 2-3), and ylene Glycol N-ethyl-(4-bromomethyl)-benzamide was stained brown with iodine (FIG. 14B, lanes 2-3). Fraction 0370. In order to further study the efficiency of PEG 4 collected from RP-HPLC is the non-reacted interferon benzyl bromide as a PEGylation reagent, the PEGylation of alpha-2a (FIGS. 14A-B, lane 4) similarly to the interferon interferon alpha-2a, was conducted with PEG-benzyl bro alpha-2a standard (FIGS. 14A-B, lane 5). mide as depicted in Scheme 2 hereinabove. 0377 The results indicate that a mono-PEGylated inter 0371 Glacial acetic acid (0.5ul) was added to the solution feron-alpha-2a was formed by using a PEG N-ethyl-(4-bro of rh-IFN-C2a(0.5 ml) to reduce the solution pH to pH4. This momethyl)-benzamide reagent at acidic conditions. Solution was then concentrated to 125ul corresponding to a protein concentration of 4 mg/ml using Amicon Ultra-4 fil Example 11 ters. Protein concentration was determined by Bradford assay. 30 kDa methoxy polyethylene glycol N-ethyl-(4-bro PEGylation of Recombinant Human Erythropoietin momethyl)-benzamide (3.7 mg, 0.12 umol) was added to the (EPO) with 30 kDa Methoxy Polyethylene Glycol above solution of rh-IFN-C2a (0.48 mg, 0.025umol) and the N-ethyl-(4-bromomethyl)-benzamide reaction was incubated in a head over tail shaker at 25°C. for 24 hours. 0378 Recombinant human erythropoietin (rh-EPO), mar 0372 Purification of the reaction mixture by preparative keted as NeoRecormon R by Hoffman-Laroche and Procrit(R) reverse-phase chromatography: 20 Jul of the mixture were by Johnson and Johnson, is produced by mammalian cells and injected to a Jupiter C4 column with acetonitrile-water and is used for the treatment of anemia. 0.2% TFA as mobile phase. Three main peaks that were 0379 Two syringes of NeoRecormon containing 20,000 assigned to be the PEGylated protein were collected at reten IU each were used. The buffer was exchanged to 50 mM tion time between 20.4-27.2 minutes. These fractions were acetate buffer pH 4 and the protein was concentrated to 80 ul concentrated by speed-Vac and analyzed in a non-reducing using Amicon Ultra-4 filters. Protein concentration (3 mg/ml) SDS-PAGE. was determined using Nanodrop spectrophotometer. 30 kDa 0373 The products of the reaction between interferon Methoxy polyethylene glycol N-ethyl-(4-bromomethyl)- alpha-2a and 30 kDa methoxy polyethylene glycol N-ethyl benzamide (1.2 mg 0.040 umol) was added to the above (4-bromomethyl)-benzamide were collected by preparative solution of EPO (0.24 mg. 0.008 umol) and the reaction was reverse-phase chromatography. incubated in a head over tail shaker at 25°C. for 24 hours. 0374 FIG. 13 presents a RP-HPLC chromatogram of the 0380 Purification of the reaction mixture by preparative PEGylation reaction products of recombinant human inter reverse-phase chromatography was performed by loading 20 feron-alpha-2a and 30 kDa methoxy polyethylene glycol ul of the mixture on a Jupiter C4 column with acetonitrile N-ethyl-(4-bromomethyl)-benzamide wherein several water and 0.2% TFA as mobile phase. The main peak was formed peaks are observed (retention times of 21.77, 23.78, assigned to the fraction containing the PEGylated protein, 25.05, 25.57 and 27.42 minutes) and recombinant human which was collected at retention time between 25-29 minutes. interferon-alpha-2a has a retention time of 28.42 minutes. This fraction was concentrated by speed-Vac and analyzed in 0375 FIGS. 14A-B present color images of a non-reduc a non-reducing SDS-PAGE. ing SDS-PAGE separation gel in which the PEGylation reac (0381 FIG. 15 presents a RP-HPLC chromatogram of the tion products of recombinant human interferon-alpha-2a and PEGylation reaction products of recombinant human eryth 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom ropoietin and 30 kDa methoxy polyethylene glycol N-ethyl ethyl)-benzamide, after collection by RP-HPLC, were run (4-bromomethyl)-benzamide wherein a peak is observed and stained with Coomassie Blue (FIG. 14A) and subse with a retention time of 25.62 minutes and recombinant quently with iodine (FIG. 14B), wherein the collected frac human erythropoietin has a retention time of 28.35 minutes. tions were run in lanes 1,2,3 and 4; an un-PEGylated sample 0382. The products of the reaction between erythropoietin of recombinant human interferon-alpha-2a was run in lane 5, and 30 kDa methoxy polyethylene glycol N-ethyl-(4-bro a 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom momethyl)-benzamide were isolated using preparative ethyl)-benzamide sample was run in lane 6 and molecular reverse-phase chromatography. weight markers were run in lane 7. 0383 FIGS. 16A-B present color images of a non-reduc 0376. As can be seen in FIGS. 14A-B (lane 5), interferon ing SDS-PAGE separation gel in which the PEGylation reac alpha-2a appeared as an 18kDa band stained with Coomassie tion products of recombinant human erythropoietin and 30 and not with iodine. The PEG N-ethyl-(4-bromomethyl)-ben kDa methoxy polyethylene glycol N-ethyl-(4-bromom Zamide reagent stained only with iodine and not by the Coo ethyl)-benzamide, after collection by RP-HPLC, were run massie blue, appeared as a brown band having an apparent and stained with Coomassie Blue (FIG. 16A) and subse molecular weight of about 55 kDa (FIG. 14B, lane 6). It can quently with iodine (FIG. 16B), wherein the collected frac be observed that fraction 1 collected from RP-HPLC is tions were run in lanes 1 and 2; an un-PEGylated sample of mainly a di-PEGylated product since it appeared as a band recombinant human erythropoietin was run in lane 3, a 30 having a molecular weight of about 120 kDa (which is the kDa methoxy polyethylene glycol N-ethyl-(4-bromom Sum of interferon-alpha-2a and twice the apparent molecular ethyl)-benzamide sample was run in lane 4 and molecular weight of PEG) in the Coomassie stained gel (FIG. 14A, lane weight markers were run in lane 5: US 2011/O 150820 A1 Jun. 23, 2011 28

0384 As can be seen in FIGS. 16A-B (lane 3), EPO that this new peak contains fragmentations of 44 Da which is appeared as a 35kDa band stained with Coomassie and not typical fingerprint of the disrupted PEG chain. with iodine, and fraction 1 collected from RP-HPLC contains 0393. From comparison of both peptide maps it is con a mono-PEGylated product since it appeared as a band having cluded that a site-specific PEGylation occurred via methion a molecular weight of about 90 kDa (which is the sum of ine amino acid M54. erythropoietin and the apparent molecular weight of PEG) in the Coomassie stained gel (FIG.16A, lane 1), and was stained Example 13 in brown with iodine (FIG. 16B, lane 1). Fraction 2 collected PEGylation of Recombinant Human Granulocyte from RP-HPLC is the non-reacted erythropoietin (FIGS. Colony Stimulating Factor (rhG-CSF) with 30 kDa 15A-B, lane 2) similarly to the erythropoietin standard Methoxy Polyethylene Glycol N-ethyl-(4-bromom (FIGS. 16A-B, lane 3). ethyl)-benzamide 0385. These results indicate that a mono-PEGylated EPO was formed by using a PEG N-ethyl-(4-bromomethyl)-ben 0394 Granulocyte colony stimulating factor (G-CSF) Zamide reagent at acidic conditions. In this case PEGylation stimulates the production of white blood cells, and therefore may even be site-specific, since EPO has only one methion a recombinant form of G-CSF is used in oncology and hema 1C. tology. rh G-CSF is used with certain cancer patients in treat ment-resistant and/or metastatic breast cancer to prolong Sur Example 12 vival by accelerating recovery from neutropenia, allowing high-intensity chemotherapy regimens to be more Sustained Peptide Mapping of PEGylated rh-EPO against myelosuppression and unacceptably-low levels of 0386. In order to study the effect of PEGylation and to white blood cells. One of the most widely used recombinant verify the PEGylation site along the polypeptide chain, human G-CSFs is synthesized in E. coli and is called PEGylated rh-erythropoietin (EPO) was subjected to pro (NeupogenR), which is structurally slightly dif tease digestion, and the breakup products were compared to ferent from the structure of the natural glycoprotein in terms those obtain from the un-PEGylated peptide. of the post-translational saccharides modifications. Another 0387 Peptide mapping was performed according to form of recombinant human G-CSF is called , Moya, Get al. Biotecnologia Aplicada, 20, 214, 2003. 120 which is synthesized in Chinese Hamster Ovary cells (CHO ug of RECORMONR (30,000 IU/0.6 ml; an un-PEGylated cells), and since it is made in a mammalian cell expression form of rh-EPO by F. Hoffmann La Roche, Ltd.) were system, it is indistinguishable from the 174-amino acid natu concentrated and buffer exchanged to 100 mM Tris Acetate ral human G-CSF. There are no differences between buffer pH 8.5 to a volume of 50 ul. Protein concentration (0.24 Filgrastim and Lenograstim from the clinical or therapeutic mg/ml) was determined using NANODROPR) (by Thermo point of view. Fisher Scientific Inc.). Trypsin (2310 U/mg) treated with 0395 GCSF solution (0.3 mg/ml) was concentrated using tosyl phenylalanyl chloromethyl ketone (TPCK) was pre Amicon Ultra 4 filter-10 kDa cut-off to a final concentration pared at a concentration of 1 mg/ml, and 0.5 mg/ml was added of about 4 mg/ml in Acetate buffer (10 mM, pH 4) with 5% to 50 ul the RECORMONR preparation. Reaction was per mannitol. Final protein concentration was determined using formed at 37° C. for 18 hours under mild rotation. Nanodrop spectrophotometer. 30 kDa methoxy polyethylene 0388 Peptide fragments were resolved by HPLC chroma glycol N-ethyl-(4-bromomethyl)-benzamide (8 mg 0.26 tography by injecting 50 ul of the mixture into a Jupiter C4 umol) was added to the above solution of GCSF (1 mg, 0.073 column heated to 30° C. with acetonitrile-water and 0.1% umol) and the reaction was incubated in a head-over-tail TFA as mobile phase. shaker at 25°C. for 24 hours. 0389. Peptide mapping of PEGylated rh-EPO was per 0396 Purification of the reaction mixture by preparative formed simultaneously and in the same manner as peptide reverse-phase chromatography by loading 40 ul of the mix mapping of rh-EPO described above. ture on a Jupiter C4 column with acetonitrile-water and 0.2% 0390 FIGS. 17A-B present RP-HPLC chromatograms TFA as mobile phase. comparing the peptide maps of purified PEGylation product 0397 FIG. 18 presents a RP-HPLC chromatogram of the of rh-EPO with 30 kDa methoxy polyethylene glycol PEGylation reaction products of recombinant human granu N-ethyl-(4-bromomethyl)-benzamide (FIG. 17A), and of the locyte colony stimulating factor and 30 kDa methoxy poly un-PEGylated rh-EPO (FIG. 17B), as obtained by applying ethylene glycol N-ethyl-(4-bromomethyl)-benzamide peptide digestion using trypsin. wherein PEGylated protein peaks are observed with a reten 0391) As can be seen in FIG. 17B, the peptide map that tion time of 36.9, 48.2 and 49.4 minutes and recombinant was obtained by trypsin digestion of rh-EPO is similar to the human granulocyte colony stimulating factor has a retention published peptide maps of rh-EPO under similar conditions time of 53.5 minutes. These fractions were concentrated to Moya, Get al., Biotecnologia Aplicada, 20, 214, 2003 and about 2/3 volume by speed-vac, and to avoid aggregation dur Labrenz, S R et al., PDA. J. Pharm. Sci. Technol., 62,211, ing further concentration, buffer was exchange to 10 mM 2008, which allows assigning the peaks to the corresponding acetate buffer, containing 5% mannitol, using Amicon Ultra 4 peptides. filters, and samples from these fractions were analyzed in a 0392. As can further be seen in FIG. 17A, the peptide map non-reducing SDS-PAGE. of PEGylated rh-EPO digested by trypsin is similar to the 0398. The products from the reaction between GCSF and peptide map of the un-PEGylated rh-EPO digested by the 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromom same protease. One makeable difference stems from the ethyl)-benzamide were collected by preparative reverse methionine residue in the peptide, which has almost totally phase chromatography. disappeared from the digested PEGylated peptide map (FIG. 0399 FIG. 19A-B present color images of a non-reducing 17A) as compared to the corresponding peak in rh-EPO SDS-PAGE separation gel in which the PEGylation reaction digest map (FIG. 17B). In addition, in the case of the digested products of recombinant human granulocyte colony stimulat PEGylated protein, a new peptide was formed having a reten ing factor and 30 kDa methoxy polyethylene glycol N-ethyl tion time of 94 minutes. Analysis by LC/MS/MS has shown (4-bromomethyl)-benzamide, after collection by RP-HPLC, US 2011/O 150820 A1 Jun. 23, 2011 29 were run and stained with Coomassie Blue (FIG. 19A) and ul of the mixture on a Jupiter C4 column with acetonitrile subsequently with iodine (FIG. 19B), wherein the collected water and 0.2%TFA as mobile phase. The fraction which was fractions were run in lanes 1, 2, 3 and 4; an un-PEGylated identified as the PEGylated protein was collected at retention sample of recombinant human granulocyte colony Stimulat time between 43-48 minutes. This fraction was concentrated ing factor was run in lane 5, a 30 kDa methoxy polyethylene by speed-vac and analyzed in a non-reducing SDS-PAGE. glycol N-ethyl-(4-bromomethyl)-benzamide sample was run (0407. The products from the reaction between GH and 30 in lane 6 and molecular weight markers were run in lane 7. kDa methoxy polyethylene glycol N-ethyl-(4-bromom (0400. As can be seen in FIGS. 19A-B (lane 5), GCSF has ethyl)-benzamide were collected by preparative reverse an 18 kDa band stained with Coomassie and not with iodine. phase chromatography. The PEG N-ethyl-(4-bromomethyl)-benzamide reagent (0408 FIG. 21 presents a RP-HPLC chromatogram of the stained only with iodine and not by the Coomassie blue, PEGylation reaction products of recombinant human growth appeared as a brown band having an apparent molecular hormone and 30 kDa methoxy polyethylene glycol N-ethyl weight of about 55 kDa (FIG. 19B, lane 6). It can be observed (4-bromomethyl)-benzamide wherein a new formed peak is that fraction 1 collected from RP-HPLC is a di-PEGylated observed with a retention time of 46.65 minutes and recom product since it appeared as a band having a molecular weight binant human growth hormone has a retention time of 52 of about 120 kDa (which is the sum of GCSF and twice the minutes. apparent molecular weight of PEG) in the Coomassie stained 04.09 FIG.22A-B present color images of a non-reducing gel (FIG. 18A, lane 1), and was stained brown with iodine SDS-PAGE separation gel in which the PEGylation reaction (FIG. 19B, lane 1). Fractions 2 and 3 are mainly mono products of recombinant human growth hormone and 30 kDa PEGylated products (mono-PEGylated isoforms) since they methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben appeared as bands having a molecular weight of about 70 kDa Zamide, after collection by RP-HPLC, were run and stained (which is the sum of GCSF and the apparent molecular weight with Coomassie Blue (FIG. 22A) and subsequently with of PEG) in the Coomassie stained gel (FIG. 19A, lanes 2-3), iodine (FIG.22B), wherein the collected fractions were run in and was stained brown with iodine (FIG. 19B, lanes 2-3). lanes 1, 2,3; an un-PEGylated sample of recombinant human Fraction 4 collected from RP-HPLC is the non-reacted GCSF growth hormone was run in lane 4, a 30 kDa methoxy poly (FIGS. 19A-B, lane 4) similarly to the GCSF standard (FIGS. ethylene glycol N-ethyl-(4-bromomethyl)-benzamide 19A-B, lane 5). sample was run in lane 5 and molecular weight markers were 04.01. The 30kDa PEGylated GCSF was also analyzed by run in lane 6. electrospray ionization (ESI) mass spectrometry. FIGS. 0410. As can be seen in FIGS. 22A-B (lane 4), GH 20A-B present ESI mass spectrograms of the purified PEGy appeared as an 18 kDa band stained with Coomassie and not lation product of recombinant human GCSF with 30 kDa with iodine. The PEG N-ethyl-(4-bromomethyl)-benzamide methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben reagent stained only with iodine and not by the Coomassie Zamide (FIG. 20A) and recombinant human interferon-beta blue, appeared as a brown band having an apparent molecular 1b (FIG.20B). weight of about 55 kDa (FIG.22B, lane 5). It can be observed 0402. As can be seen in FIGS. 20A-B, no molecular peak that fraction 1 collected from RP-HPLC is mainly a mono of a 30 kDa PEG-GCSF conjugate was observed (about 50 PEGylated product since it appeared as bands having a kDa) and the main peak was due to the Hoffman elimination molecular weight of about 70 kDa (which is the sum of GH described hereinabove, resulting in a mass of 18,755 Da and the apparent molecular weight of PEG) in the Coomassie which is exactly 48 Daless than the molecular mass of GCSF. stained gel (FIG. 22A, lane 1), and was stained brown with 0403. The results indicate that a mono-PEGylated GCSF iodine (FIG. 22B, lane 1). Fraction 3 collected from RP was formed through a linkage with a methionine Sulfur by HPLC is the non-reacted GH (FIGS. 22A-B, lane 3) similarly using a PEG N-ethyl-(4-bromomethyl)-benzamide reagent at to the GH standard (FIGS. 22A-B, lane 3). acidic conditions. 0411. The results indicate that a mono-PEGylated GH was formed by using a PEG N-ethyl-(4-bromomethyl)-benza Example 14 mide reagent at acidic conditions. PEGylation of Growth Hormone (GH) with 30 kDa Methoxy Polyethylene Glycol N-ethyl-(4-bromom Example 15 ethyl)-benzamide PEGylation of Follicle Stimulating Hormone (FSH) 0404 Recombinant human Growth Hormone (rh-GH), with 30 kDa Methoxy Polyethylene Glycol N-ethyl marketed as Nutropin (Genentech), Humatrope (Lilly), (4-bromomethyl)-benzamide Genotropin (Pfizer), Norditropin (Novo), and Saizen (Merck Serono) is produced by E. coli and mammalian cells. GH is 0412 Recombinant human Follicle Stimulating Hormone used as replacement therapy in adults with GH deficiency of (rh-FSH), marketed as GONAL-fR (Merck Serono S.A.) and either childhood-onset (after completing growth phase) or PUREGONR) (Schering-Plough Corp.) is produced by mam adult-onset (usually as a result of an acquired pituitary malian cells. tumor). 0413 Buffer of 100 ul of rh-FSH solution was exchanged 04.05 rh-GH (1 mg) was reconstituted by adding water to 20 mM acetate buffer pH 4 using MICROCONR) 10K (250 ul) to obtain a protein concentration of 4 mg/ml acetic (Millipore) with 3 volume changes of 400 uL. Final protein acid (1 ul, 1:3) was added to the above solution (125 ul) concentration (2.17 mg/ml) was determined using NANO reducing the pH to 4. DROPR) spectrophotometer. 6 ul of a 20 mg/ml solution of 30 0406 30 kDa methoxy polyethylene glycol N-ethyl-(4- kDa methoxy polyethylene glycol N-ethyl-(4-bromom bromomethyl)-benzamide (6.73 mg, 0.22 Lumol) was added ethyl)-benzamide (0.12 mg, 0.004 umol) was added to 18.5ul to the above solution of GH (0.5 mg, 0.022 umol) and the of the above solution of rh-FSH (0.04 mg., 0.002 umol) and reaction was incubated in a head-over-tail shaker at 25°C. for the reaction was incubated in ahead-over-tail shaker at 25°C. 24 hours. Purification of the reaction mixture by preparative for 17 hours. The reaction mixture was analyzed without reverse-phase chromatography was performed by loading 15 further purification in a non-reducing SDS-PAGE. Another US 2011/O 150820 A1 Jun. 23, 2011 30 sample was prepared immediately before running a non-re ducing SDS-PAGE as time Zero reference. -continued 0414 FIGS. 23A-B present color images of a non-reduc ing SDS-PAGE separation gel in which the PEGylation reac O tion products of recombinant human follicle stimulating hor mone and 30 kDa methoxy polyethylene glycol N-ethyl-(4- HN --- bromomethyl)-benzamide were run and stained with Fluorescent r Coomassie Blue (FIG. 23A) and subsequently with iodine Selective (FIG.23B), wherein the reaction mixture after 17 hrs. was run Carboxymethylation O N O --- in lane 1; the reaction mixture at time Zero was run in lane 2: pH = 4 an un-PEGylated sample of recombinant human follicle stimulating hormone was run in lane 3, a 30 kDa methoxy polyethylene glycol N-ethyl-(4-bromomethyl)-benzamide sample was run in lane 4 and molecular weight markers were run in lane 5. KOS1 YSOK 0415. As can be seen in FIGS. 23A-B, rh-FSH appeared as an 35 kDa band stained with Coomassie and not with iodine Lucifer Yellow Iodoacetamide (lane 3). The PEG N-ethyl-(4-bromomethyl)-benzamide reagent stained only with iodine and not by the Coomassie H blue, appeared as a brown band having an apparent molecular N -- / weight of about 55 kDa (FIG. 23B, lane 4). It can be observed Lucifer Yellow 1. r S I that a new product was formed since a new band appeared in the reaction mixture after 17 hours (lane 1) which did not exist at time Zero (lane 2). The newly formed product is mono PEGylated rh-FSH since it appeared as bands having a molecular weight of about 90 kDa, which is the sum of the apparent molecular weights of rh-FSH and PEG, in the Coo massie stained gel (FIG.23A, lane 1), and was stained brown Fluorescent protein with iodine (FIG. 23B, lane 1). 0416) These results indicate that a mono-PEGylated rh FSH was formed by using only 2 equivalents of PEG N-ethyl (4-bromomethyl)-benzamide reagent at acidic conditions. Example 17 Example 16 Two-Steps PEGylation of Methionine Containing Proteins Attachment of a Fluorescent Probe to Methionine Containing Proteins 0419. A two-step PEGylation of recombinant and/or native proteins which exhibit at least one unmodified 0417. Lucifer yellow is a well-known polar tracer for neu methionine side chain in their structure can be performed in rons. Its iodoacetamide derivative has high water solubility cases wherein the methionine side-chain(s) is less accessible. and visible absorption and emission similar to those of Luci fer yellow. 0420. Thus, the protein is modified with a modifying moi ety having three basic components, a first and second reactive 0418. A methionine-containing protein is reacted with an moieties and a linking moiety which can be a spacer. The iodoacetamide derivative of Lucifer yellow, at an acidic pH, protein is modified at a methionine side-chain as illustrated in using the procedure described in Example 2 hereinabove, as Scheme 5 hereinbelow. depicted in Scheme 4 below.

Scheme 5 Scheme 4

methioning side-chain on a protein US 2011/O 150820 A1 Jun. 23, 2011 31

-continued -continued third Second PEG reactive reactive second moiety reactive moiety moiety

Her First step

first reactive first reactive leaving moiety group modifying moiety

second PEGylated protein reactive moiety 0423. In a typical example, a methionine residue is reacted leaving with a reactive derivative of an alkylacetylhydrazide to form group a methionine Sulfonium moiety having a substituent termina tion with an acetylhydrazide. The hydrazide group which becomes exposed to the surface is then selectively modified at first low pH with high MW PEG-aldehyde by reductive alkyla reactive moiety tion, as depicted in Scheme 7.

Scheme 7

S modified protein H -- Prot N 0421. This reaction is conducted under conditions that N n Prot favora formation of a covalent bond between the first reactive H O moiety and the Sulfur atom of the methionine side-chain, and O according to some embodiments, under acidic conditions. Selective Carboxymethylation 0422 The modified protein is then reacted with a PEG H2NN XL -e- moiety having a third reactive group which is capable of N pi pH = 4 forming a covalent bond with the second reactive group, as H illustrated in Scheme 6 hereinbelow. XL = reactive moiety; L = leaving group Scheme 6 HN1 "X n.1 S L Second O reactive moiety H

Prot N N n Prot third PEG reactive H O moiety -as first Second step Y reactive HN his1 X ns1+ L O

H --

Prot N N n Prot

modified protein H O US 2011/O 150820 A1 Jun. 23, 2011 32

cosidase-alpha, recombinant human imiglucerase, recombi -continued nant human laronidase, recombinant human agallsidase-beta, O recombinant human galsulfase, recombinant human hyalu

-e- ronidase, recombinant human alpha-galactosidase, recombi High MWPEG ~us H NaCNBHpH = 3 nant urate oxidase and recombinant human dornase-alpha. H 0430 Representative examples of antibodies which can be High MWPEG 1N1N1N N ny - s 1. L PEGylated according to the present embodiments include recombinant human rituximab, recombinant human trastu O Zumab and recombinant human cetuximab. | 0431 Representative examples of fusion proteins which Prot N can be PEGylated according to the present embodiments a Prot include etanercept, alefaceptand r-IL-2 diphteria toxinfusion H O protein. 0432 Although the invention has been described in con junction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be appar Example 18 ent to those skilled in the art. Accordingly, it is intended to 0424. Using the General procedure described herein embrace all Such alternatives, modifications and variations above, a wide variety of proteins, and particularly proteins of that fall within the spirit and broad scope of the appended therapeutic and pharmacological importance can be PEGy claims. lated owing to the fact that they have at least one methionine 0433 All publications, patents and patent applications residue within their sequence. mentioned in this specification are herein incorporated in 0425 Exemplary protein families which can be PEGy their entirety by reference into the specification, to the same lated using the abovementioned general procedure include extent as if each individual publication, patent or patent appli human blood factors, human hormones, human growth fac cation was specifically and individually indicated to be incor tors and cytokines, enzymes, antibodies and fusion proteins. porated herein by reference. In addition, citation or identifi 0426 Representative examples of human blood factors cation of any reference in this application shall not be which can be PEGylated according to the present embodi construed as an admission that Such reference is available as ments include recombinant human Factor VIII, recombinant prior art to the present invention. To the extent that section human B-deleted domain Factor VIII, recombinant human headings are used, they should not be construed as necessarily Factor VIIa, recombinant human Factor IX, recombinant limiting. human tissue plasminogen activator (TPA), recombinant 1-45. (canceled) human activated Protein Cand recombinant humanthrombin. 46. A conjugate comprising: 0427 Representative examples of human hormones (a) a polypeptide having at least one methionine residue, which can be PEGylated according to the present embodi each said methionine residue having a methylsulfanyl ments include recombinant human growth hormone (GH), ethyl side-chain; and recombinant human follicle stimulating hormone (FSH), (b) at least one polymer moiety being covalently attached recombinant human luteinizing hormone (LH), recombinant to a sulfur atom of said methylsulfanyl-ethyl side-chain human parathyroid hormone (PTH), recombinant human par of at least one said methionine residue. athyroid hormone (1-34) (PTH 1-34), recombinant human 47. The conjugate of claim 46, wherein said at least one chorionic gonadotropin (CG), recombinant human thyrotro polymer moiety is covalently attached to said Sulfur atom via pin (TSH) and recombinant human glucagons. a linking moiety. 0428 Representative examples of human growth factors 48. The conjugate of claim 47, wherein said linking moiety and cytokines which can be PEGylated according to the comprises at least one residue of a reactive moiety, said reac present embodiments include recombinant human erythro tive moiety being selected capable of reacting with said Sulfur poietin (EPO), recombinant human thrombopoietin (TPO), atom of said methylsulfanyl-ethyl side-chain and said residue recombinant human granulocyte-macrophage colony Stimu of said reactive moiety being formed upon said reacting. lating factor (GM-CSF), recombinant human granulocyte 49. The conjugate of claim 48, wherein said reactive moi colony stimulating factor (G-CSF), recombinant human insu ety is selected from the group consisting of amine, carboxyl, lin-like growth factor-1 (IGF-1), recombinant human kerati amide, acetamide, 2-halo-acetamide, (4-halomethyl)-benza nocyte growth factor (KGF), recombinant human platelet mide, benzyl-halide, hydrazine, hydrazide, acetohydrazide, derived growth factor (PDGF), recombinant human bone alkyl, haloalkyl, alkyl sulfonylhalide, alkyl tosylate, alkyl morphogenetic protein-2 (BMP-2), recombinant human bone triflate, allyl, haloallyl, allyl sulfonylhalide, allyl tosylate, morphogenetic protein-7 (BMP-7), recombinant human allyl triflate, aryl, haloarylheteroaryl, 4-(halomethyl)benzyl, tumor necrosis factor-alpha (TNF-alpha), recombinant benzyl and halobenzyl, and any combination thereof. human interferon-alpha-2a (IFN alpha-2a), recombinant 50. The conjugate of claim 48, wherein said reactive moi human interferon-alpha-2b (IFN alpha-2b), recombinant ety is selected from the group consisting of 2-halo-acetamide, human interferon-gamma-1b (IFN gamma-1b), recombi (4-halomethyl)-benzamide, benzyl-halide, haloalkyl, hydra nant human interleukin-1 (IL-1) receptor antagonist, recom Zine, hydrazide and acetohydrazide. binant human interleukin (IL-2) and recombinant human 51. The conjugate of claim 48, wherein said reactive moi interleukin (IL-11). ety comprises at least one leaving group selected from the 0429 Representative examples of enzymes which can be group consisting of halide, acetate, tosylate, triflate, Sul PEGylated according to the present embodiments include fonate, azide, hydroxy, thiohydroxy, alkoxy, cyanate, thiocy recombinant human heparanase, recombinant human alglu anate, nitro and cyano. US 2011/O 150820 A1 Jun. 23, 2011

52. The conjugate of claim 47, wherein said linking moiety attached therebetween via a sulfur atom of a methionine further comprises a spacer. residue of said interferon-beta-1b, and having a formula: 53. The conjugate of claim 52, wherein said spacer is selected from the group consisting of methane-di-yl, ethane 1-yl-2-yl, propane-1-yl-3-yl, butane-1-yl-4-yl, 1,4-benzene 30 kDa methoxy polyethylene glycol; diyl and 1,10-biphenyl-diyl. 54. The conjugate of claim 46, wherein said polypeptide is selected from the group consisting of an interferon, a cytok ine, a hormone, a growth factor, an enzyme, a blood protein (factor), an antibody, an antigen, a viral protein, a fusion interferon-beta-1bDr protein, and any part or segment thereof. 55. The conjugate of claim 46, wherein said polypeptide is selected from the group consisting of adalimumab, adenosine deaminase, agallsidase-beta, alglucosidase-alpha, alpha-ga a conjugate comprising interferon-beta-1b and 30 kDa lactosidase, asparaginase, B-deleted domainFactor VIII, methoxy polyethylene glycol N-ethyl-(4-bromom bone morphogenetic protein-2 (BMP-2), bone morphoge ethyl)-benzamide, being attached therebetween via a netic protein-7 (BMP-7), brain-derived neurotrophic factor Sulfur atom of a methionine residue of said interferon (BDNF), cetuximab, chorionic gonadotropin (CG), dornase beta-1b, and having a formula: alpha, erythropoietin (EPO), etanercept, Factor IX, Factor VIIa, Factor VIII, follicle stimulating hormone (FSH), gal sulfase, glial cell line derived neurotrophic factor (GDNF), glucagon, granulocyte colony stimulating factor (G-CSF), -- granulocyte-macrophage colony stimulating factor (GM S H CSF), a growth hormone (GH), hemoglobin, heparanase, N-1-N30 kDa methoxy hyaluronidase, imiglucerase, infliximab, insulin-like growth O olyethylene glycol; factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), inter interferon-beta-1b polyetny gly feron-alpha-2b (IFNalpha-2b), interferon-beta-1a (IFN beta 1a), interferon-beta-1b (IFN beta-1b), interferon-gamma-1b (IFN gamma-1b), interleukin (IL-11), interleukin (IL-2), a conjugate comprising interferon-beta-1a and 30 kDa interleukin-1 (IL-1) receptor antagonist, interleukin-1 recep methoxy polyethylene glycol N-ethyl-acetamide, being tor antagonist (IL-1 ra), keratinocyte growth factor (KGF), attached therebetween via a sulfur atom of a methionine laronidase, luteinizing hormone (LH), megakaryocyte residue of said interferon-beta-1a, and having a formula: growth differentiation factor (MGDF), obesity protein (OB protein or leptin), osteoprotegerin (OPG), parathyroid hor mone (PTH or 1-34 segment or PTH 1-34), palivizumab, 30 kDa methoxy polyethylene glycol; platelet-derived growth factor (PDGF). Protein C, rituximab, stem cell factor (SCF), streptokinase, thrombin, thrombopoi etin (TPO), thyrotropin (TSH), tissue plasminogen activator (tPA), trastuzumab, tumor necrosis factor binding protein (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate oxidase, and any part or segment thereof. interferon-beta-1a 56. The conjugate of claim 46, wherein said polypeptide is selected from the group consisting of interferon-alpha-2a (INF-C2a), interferon-beta-1a (INF-B1a), interferon-beta-1b a conjugate comprising interferon-alpha-2a and 30 kDa (INF-B1b), erythropoietin (EPO), granulocyte colony-stimu methoxy polyethylene glycol N-ethyl-acetamide, being lating factor (G-CSF) human growth hormone (h-GH). attached therebetween via a sulfur atom of a methionine 57. The conjugate of claim 46, wherein said polymer moi residue of said interferon-alpha-2a, and having a for ety is selected from the group consisting of a polyalkylene mula: glycol, a polyethylene glycol (PEG), a poly(lactic acid) (PLA), a polyester, a polyglycolide (PGA), a polycaprolac tone (PCL), a polyamide, a polymethacrylamide, a polyvinyl 30 kDa methoxy polyethylene glycol; alcohol, a polycarboxylate, a polyvinyl pyrrolidinone, a dex tran, a cellulose, a chitosan, a hydroxyethyl starch (HES), polyglutamic acid, polyglycine and any copolymer thereof. 58. The conjugate of claim 46, wherein said polymer moi ety is a polyethylene glycol (PEG). interferon-alpha-2a.)-r- 59. The conjugate of claim 58, wherein said polyethylene glycol has an average molecular weight that ranges from 4 kDa to 40 kDa. a conjugate comprising interferon-alpha-2a and 30 kDa 60. The conjugate of claim 46, being selected from the methoxy polyethylene glycol N-ethyl-(4-bromom group consisting of ethyl)-benzamide, being attached therebetween via a a conjugate comprising interferon-beta-1b and 30 kDa Sulfur atom of a methionine residue of said interferon methoxy polyethylene glycol N-ethyl-acetamide, being alpha-2a, and having a formula: US 2011/O 150820 A1 Jun. 23, 2011 34

S H S H

N-1-N30 kDa methoxy N-1-N30 kDa methoxy O olwethylene glycol. interferon-alpha-2a. O polyetnyolyethylene glycol;gly -FSH polyetny gly

61. A process of preparing the conjugate of claim 46, the a conjugate comprising erythropoietin and 30 kDa meth process comprising: oxy polyethylene glycol N-ethyl-(4-bromomethyl)-ben reacting said polypeptide with said polymer having at least one reactive moiety attached thereto, under acidic con Zamide, being attached therebetween via a Sulfur atom ditions ranging from about pH of 2 to pH of 5, said of a methionine residue of said erythropoietin, and hav reactive moiety being selected capable of reacting with ing a formula: said Sulfur atom in said methylsulfanyl-ethyl side-chain, thereby obtaining the conjugate. 62. A pharmaceutical composition comprising the conju gate of claim 46. St H 63. The pharmaceutical composition of claim 62, wherein N-1-N said polypeptide is selected from the group consisting of an 30 kDa methoxy interferon, a cytokine, a hormone, a growth factor, an enzyme, erythropoietin O polyetnyolyethylene glycol;gly a blood protein (factor), an antibody, an antigen, a viral pro tein, a fusion protein, and any part or segment thereof. 64. The pharmaceutical composition of claim 62, wherein said polypeptide is selected from the group consisting of a conjugate comprising granulocyte colony-stimulating adalimumab, adenosine deaminase, agallsidase-beta, alglu factor (G-CSF) and 30 kDa methoxy polyethylene gly cosidase-alpha, alpha-galactosidase, asparaginase, B-deleted col N-ethyl-(4-bromomethyl)-benzamide, being domainFactor VIII, bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), brain-derived neu attached therebetween via a sulfur atom of a methionine rotrophic factor (BDNF), cetuximab, chorionic gonadotropin residue of said G-CSF, and having a formula: (CG), dornase-alpha, erythropoietin (EPO), etanercept, Fac tor IX, Factor VIIa, Factor VIII, follicle stimulating hormone (FSH), galsulfase, glial cell line derived neurotrophic factor (GDNF), glucagon, granulocyte colony stimulating factor S -- H (G-CSF), granulocyte-macrophage colony stimulating factor N-1-N (GM-CSF), a growth hormone (GH), hemoglobin, hepara 30 kDa methoxy nase, hyaluronidase, imiglucerase, infliximab, insulin-like G-CSF O polyethylene glycol; growth factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), interferon-alpha-2b (IFN alpha-2b), interferon-beta-1a (IFN beta-1a), interferon-beta-1b (IFN beta-1b), -1b (IFN gamma-1b), interleukin (IL-11), interleukin a conjugate comprising human growth hormone (h-GH) (IL-2), interleukin-1 (IL-1) receptor antagonist, interleukin-1 and 30 kDa methoxy polyethylene glycol N-ethyl-(4- receptor antagonist (IL-1 ra), keratinocyte growth factor bromomethyl)-benzamide, being attached therebetween (KGF), laronidase, luteinizing hormone (LH), megakaryo via a sulfur atom of a methionine residue of said h-GH, cyte growth differentiation factor (MGDF), obesity protein and having a formula: (OB protein or leptin), osteoprotegerin (OPG), parathyroid hormone (PTH or 1-34 segment or PTH 1-34), palivizumab, platelet-derived growth factor (PDGF). Protein C, rituximab, stem cell factor (SCF), streptokinase, thrombin, thrombopoi S -- H etin (TPO), thyrotropin (TSH), tissue plasminogen activator (tPA), trastuzumab, tumor necrosis factor binding protein N-1-N30 kDa methoxy (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate -GH O polyetnyolyethylene glycol;gly oxidase, and any part or segment thereof. 65. The pharmaceutical composition of claim 62, wherein said polypeptide is selected from the group consisting of interferon-alpha-2a (INF-C2a), interferon-beta-1a (INF B1a), interferon-beta-1b (INF-B1b), erythropoietin (EPO), and granulocyte colony-stimulating factor (G-CSF) human a conjugate comprising human follicle stimulating hor growth hormone (h-GH). mone (h-FSH) and 30 kDa methoxy polyethylene glycol 66. The pharmaceutical composition of claim 62, wherein N-ethyl-(4-bromomethyl)-benzamide, being attached said polymer moiety is selected from the group consisting of therebetween via a sulfur atom of a methionine residue a polyalkylene glycol, a polyethylene glycol (PEG), a poly of said h-FSH, and having a formula: (lactic acid) (PLA), a polyester, a polyglycolide (PGA), a US 2011/O 150820 A1 Jun. 23, 2011

polycaprolactone (PCL), a polyamide, a polymethacryla 74. The method of claim 69, wherein said polymer moiety mide, a polyvinyl alcohol, a polycarboxylate, a polyvinyl is a polyethylene glycol (PEG). pyrrolidinone, a dextran, a cellulose, a chitosan, a hydroxy 75. A compound comprising: ethyl starch (HES), polyglutamic acid, polyglycine and any (a) a polypeptide having at least one methionine residue, copolymer thereof. each said methionine residue having a methylsulfanyl 67. The pharmaceutical composition of claim 62, wherein ethyl side-chain; and said polymer moiety is a polyethylene glycol (PEG). (b) at least one modifying moiety which comprises a resi 68. The pharmaceutical composition of claim 67, wherein due of a first reactive moiety and a second reactive said polyethylene glycol has an average molecular weight moiety, said modifying moiety being covalently that ranges from 4 kDa to 40 kDa. attached to a sulfur atom of said methylsulfanyl-ethyl 69. A method of treating a medical condition treatable by a side-chain of at least one said methionine residue via polypeptide having at least one methionine residue, the said residue of said first reactive moiety, method comprising administering to a subject in need thereof said polypeptide being selected from the group consisting antherapeutically effective amount of the conjugate of claim of adalimumab, adenosine deaminase, agallsidase-beta, 46. alglucosidase-alpha, alpha-galactosidase, asparaginase, 70. The method of claim 69, wherein said polypeptide is B-deleted domainFactor VIII, bone morphogenetic pro selected from the group consisting of an interferon, a cytok tein-2 (BMP-2), bone morphogenetic protein-7 (BMP ine, a hormone, a growth factor, an enzyme, a blood protein 7), brain-derived neurotrophic factor (BDNF), cetux (factor), an antibody, an antigen, a viral protein, a fusion imab, chorionic gonadotropin (CG), dornase-alpha, protein, and any part or segment thereof. erythropoietin (EPO), etanercept, Factor IX, Factor 71. The method of claim 69, wherein said polypeptide is VIIa, Factor VIII, follicle stimulating hormone (FSH), selected from the group consisting of adalimumab, adenosine galsulfase, glial cell line derived neurotrophic factor deaminase, agallsidase-beta, alglucosidase-alpha, alpha-ga (GDNF), glucagon, granulocyte colony Stimulating fac lactosidase, asparaginase, B-deleted domainFactor VIII, tor (G-CSF), granulocyte-macrophage colony stimulat bone morphogenetic protein-2 (BMP-2), bone morphoge ing factor (GM-CSF), growth hormone (GH), hemoglo netic protein-7 (BMP-7), brain-derived neurotrophic factor bin, heparanase, hyaluronidase, imiglucerase, (BDNF), cetuximab, chorionic gonadotropin (CG), dornase infliximab, insulin-like growth factor-1 (IGF-1), inter alpha, erythropoietin (EPO), etanercept, Factor IX, Factor feron-alpha-2a (IFN alpha-2a), interferon-alpha-2b VIIa, Factor VIII, follicle stimulating hormone (FSH), gal (IFN alpha-2b), interferon-beta-1a (IFN beta-1a), inter sulfase, glial cell line derived neurotrophic factor (GDNF), feron-beta-1b (IFN beta-1b), interferon-gamma-1b glucagon, granulocyte colony stimulating factor (G-CSF), (IFN gamma-1b), interleukin (IL-11), interleukin (IL granulocyte-macrophage colony stimulating factor (GM 2), interleukin-1 (IL-1) receptor antagonist, interleu CSF), a growth hormone (GH), hemoglobin, heparanase, kin-1 receptor antagonist (IL-1 ra), keratinocyte growth hyaluronidase, imiglucerase, infliximab, insulin-like growth factor (KGF), laronidase, luteinizing hormone (LH), factor-1 (IGF-1), interferon-alpha-2a (IFN alpha-2a), inter megakaryocyte growth differentiation factor (MGDF), feron-alpha-2b (IFNalpha-2b), interferon-beta-1a (IFN beta obesity protein (OB protein or leptin), osteoprotegerin 1a), interferon-beta-1b (IFN beta-1b), interferon-gamma-1b (OPG), parathyroid hormone (PTH or 1-34 segment or (IFN gamma-1b), interleukin (IL-11), interleukin (IL-2), PTH 1-34), palivizumab, platelet-derived growth factor interleukin-1 (IL-1) receptor antagonist, interleukin-1 recep (PDGF). Protein C, rituximab, stem cell factor (SCF), tor antagonist (IL-1 ra), keratinocyte growth factor (KGF), streptokinase, thrombin, thrombopoietin (TPO), thy laronidase, luteinizing hormone (LH), megakaryocyte rotropin (TSH), tissue plasminogen activator (tPA), tras growth differentiation factor (MGDF), obesity protein (OB tuZumab, tumor necrosis factor binding protein (TN protein or leptin), osteoprotegerin (OPG), parathyroid hor Fbp), tumor necrosis factor-alpha (TNF-alpha) and mone (PTH or 1-34 segment or PTH 1-34), palivizumab, urate-oxidase. platelet-derived growth factor (PDGF). Protein C, rituximab, 76. The compound of claim 75, wherein said polypeptide is stem cell factor (SCF), streptokinase, thrombin, thrombopoi selected from the group consisting of interferon-alpha-2a etin (TPO), thyrotropin (TSH), tissue plasminogen activator (INF-C2a), interferon-beta-1a (INF-B1a), interferon-beta-1b (tPA), trastuzumab, tumor necrosis factor binding protein (INF-B1b), erythropoietin (EPO), granulocyte colony-stimu (TNFbp), tumor necrosis factor-alpha (TNF-alpha)andurate lating factor (G-CSF), human growth hormone (h-GH) and oxidase, and any part or segment thereof. follicle stimulating hormone (FSH). 72. The method of claim 69, wherein said polypeptide is 77. The compound of claim 75, wherein said first reactive selected from the group consisting of interferon-alpha-2a moiety is capable of reacting with said Sulfur atom and is (INF-C2a), interferon-beta-1a (INF-B1a), interferon-beta-1b selected from the group consisting of amine, carboxyl, amide, (INF-B1b), erythropoietin (EPO), granulocyte colony-stimu acetamide, 2-halo-acetamide, (4-halomethyl)-benzamide, lating factor (G-CSF) human growth hormone (h-GH). benzyl-halide, hydrazine, hydrazide, acetohydrazide, alkyl, 73. The method of claim 69, wherein said polymer moiety haloalkyl, alkyl sulfonylhalide, alkyl tosylate, alkyl triflate, is selected from the group consisting of a polyalkylene glycol, allyl, haloallyl, allyl sulfonylhalide, allyl tosylate, allyl tri a polyethylene glycol (PEG), a poly(lactic acid) (PLA), a flate, aryl, haloarylheteroaryl, 4-(halomethyl)benzyl, benzyl polyester, a polyglycolide (PGA), a polycaprolactone (PCL), and halobenzyl, and any combination thereof. a polyamide, a polymethacrylamide, a polyvinyl alcohol, a 78. The compound of claim 75, wherein said second reac polycarboxylate, a polyvinyl pyrrolidinone, a dextran, a cel tive moiety is selected from the group consisting of amine, lulose, a chitosan, a hydroxyethyl starch (HES), polyglutamic carboxyl, amide, hydrazine, hydrazide, thiol, hydroxyl and acid, polyglycine and any copolymer thereof. hydroxylamine, and any combination thereof. US 2011/O 150820 A1 Jun. 23, 2011 36

79. The compound of claim 77, wherein said modifying (INF-C2a), interferon-beta-1a (INF-B1a), interferon-beta-1b moiety further comprises a spacer connecting said residue of (INF-B1b), erythropoietin (EPO), granulocyte colony-stimu said first reactive moiety and said second reactive moiety. lating factor (G-CSF), a growth hormone (GH) and follicle 80. The compound of claim 75, further comprising a label stimulating hormone (FSH). ing moiety being covalently attached to said modifying moi ety. 84. The process of claim 82, wherein said first reactive 81. A process of preparing the compound of claim 75, the moiety is selected from the group consisting of amine, car process comprising: boxyl, amide, acetamide, hydrazine, hydrazide, acetohy reacting said polypeptide with a modifying moiety having drazide, alkyl, haloalkyl, alkyl sulfonylhalide, alkyl tosylate, a first reactive moiety and a second reactive moiety alkyl triflate, allyl, haloallyl, allyl sulfonylhalide, allyl tosy under acidic conditions ranging from pH 2 to pH 5, said late, allyl triflate, aryl, haloaryl heteroaryl, 4-(halomethyl) first and second reactive moieties are selected Such that benzyl, 4-(halomethyl)benzamide, benzyl and halobenzyl, a covalent bond is formed between said first reactive and any combination thereof. group and said Sulfur atom, thereby obtaining the com pound. 85. The process of claim 82, wherein said second and said 82. A process of preparing a conjugate which comprises: third reactive moieties are each independently selected from a) a polypeptide having at least one methionine residue; the group consisting of amine, carboxyl, amide, hydrazine, and hydrazide, thiol, hydroxyl and hydroxylamine, and any com b) at least one polymer moiety attached to a Sulfur atom of bination thereof. a methylsulfanyl-ethyl side-chain of said at least one 86. The process of claim 82, wherein said polymer moiety methionine residue; the process comprising: is selected from the group consisting of a polyalkylene glycol, reacting said polypeptide with at least one modifying moi a polyethylene glycol (PEG), a poly(lactic acid) (PLA), a ety having a first reactive moiety and a second reactive polyester, a polyglycolide (PGA), a polycaprolactone (PCL), moiety under acidic conditions ranging from pH 2 to pH a polyamide, a polymethacrylamide, a polyvinyl alcohol, a 5, said first and second reactive moieties are selected polycarboxylate, a polyvinyl pyrrolidinone, a dextran, a cel such that a covalent bond is formed between said first lulose, a chitosan, a hydroxyethyl starch (HES), polyglutamic reactive moiety and said Sulfur atom, to thereby obtain a acid, polyglycine and any copolymer thereof. polypeptide having said at least one modifying moiety 87. The process of claim 82, wherein said polymer moiety attached thereto; and reacting said polypeptide having said at least one modify is a polyethylene glycol (PEG). ing moiety attached thereto with a polymer having a 88. A compound comprising a polyalkylene glycol moiety third reactive moiety, said third reactive moiety is and a benzyl halide moiety being covalently linked therebe Selected capable of reacting with said second reactive tween via a linking moiety, said polyalkylene glycol moiety moiety in said modifying moiety, thereby obtaining the having an average molecular weight that ranges from 20 kDa conjugate. to 40 kDa. 83. The process of claim 82, wherein said polypeptide is selected from the group consisting of interferon-alpha-2a