Glycopolysialylation of non-blood coagulation proteins

Abstract

A water soluble polymer, in particular polysialic acid (PSA) or a modified PSA (mPSA), is conjugated to an oxidized carbohydrate moiety of a glycoprotein other than a blood coagulation protein or to a ganglioside or drug delivery system by contacting the oxidized carbohydrate moiety with the water soluble polymer, wherein said water soluble polymer contains an aminooxy group and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the water soluble polymer or wherein said water soluble polymer contains a hydrazide group and a hydrazone linkage is formed between the oxidized carbohydrate moiety and the hydrazide group on the water soluble polymer. Conjugates of aminooxy- or hydrazide-water soluble polymer, such as PSA and mPSA, are thus obtained in which the PSA or mPSA is attached via a carbohydrate moiety.

Claims

1. A conjugated glycoprotein, obtained by conjugating a polysialic acid (PSA) or a modified PSA (mPSA) to an oxidized carbohydrate moiety of a glycoprotein other than a blood coagulation protein, comprising a carbohydrate group, comprising contacting the oxidized carbohydrate moiety with PSA or mPSA under conditions that allow conjugation, thereby forming the conjugated glycoprotein, wherein said PSA or mPSA contains an aminooxy group, and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the PSA or mPSA.

2. The conjugated glycoprotein of claim 1, wherein the conjugated glycoprotein comprises: (a) the glycoprotein other than a blood coagulation protein and (b) at least one aminooxy-PSA or aminooxy-mPSA bound to the glycoprotein of (a), wherein said aminooxy-PSA or aminooxy-mPSA is attached to the glycoprotein via one or more carbohydrate moieties.

Description

FIGURES

(1) FIG. 1 shows the synthesis of the water soluble di-aminoxy linkers 3-oxa-pentane-1,5-dioxyamine and 3,6,9-trioxa-undecane-1,11-dioxyamine.

(2) FIG. 2 shows the preparation of aminooxy-PSA.

DETAILED DESCRIPTION OF THE INVENTION

(3) The pharmacological and immunological properties of carbohydrate-containing compounds, such as glycoproteins other than blood coagulations proteins can be improved by chemical modification and conjugation with water soluble polymer, in particular PEG or PSA or mPSA. The properties of the resulting conjugates generally strongly depend on the structure and the size of the polymer. Thus, polymers with a defined and narrow size distribution are usually preferred. PSA and mPSA, used in specific examples, can be purified in such a manner that results in a final PSA preparation with a narrow size distribution.

Glycoproteins

(4) As described herein, glycoproteins other than blood coagulation proteins including, but not limited to cytokines such as interleukins, alpha-, beta-, and gamma-interferons, colony stimulating factors including granulocyte colony stimulating factors, fibroblast growth factors, platelet derived growth factors, phospholipase-activating protein (PUP), insulin, plant proteins such as lectins and ricins, tumor necrosis factors and related alleles, soluble forms of tumor necrosis factor receptors, interleukin receptors and soluble forms of interleukin receptors, growth factors, tissue growth factors, transforming growth factors such as TGFs or TGFs and epidermal growth factors, hormones, somatomedins, pigmentary hormones, hypothalamic releasing factors, antidiuretic hormones, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, and immunoglobulins such as IgG, IgE, IgM, IgA, and IgD, erythropoietin (EPO), blood factors other than blood coagulation proteins, galactosidases, -galactosidases, -galactosidases, DNAses, fetuin, fragments thereof, and any fusion proteins comprising any of the above mentioned proteins or fragments thereof together with therapeutic glycoproteins in general are contemplated by the invention.

(5) As used herein biologically active derivative or biologically active variant includes any derivative or variant of a molecule having substantially the same functional and/or biological properties of said molecule, such as binding properties, and/or the same structural basis, such as a peptidic backbone or a basic polymeric unit.

(6) An analog, variant or derivative is a compound substantially similar in structure and having the same biological activity, albeit in certain instances to a differing degree, to a naturally-occurring molecule. For example, a polypeptide variant refers to a polypeptide sharing substantially similar structure and having the same biological activity as a reference polypeptide. Variants or analogs differ in the composition of their amino acid sequences compared to the naturally-occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or one or more internal regions of the naturally-occurring polypeptide sequence (e.g., fragments), (ii) insertion or addition of one or more amino acids at one or more termini (typically an addition or fusion) of the polypeptide and/or one or more internal regions (typically an insertion) of the naturally-occurring polypeptide sequence or (iii) substitution of one or more amino acids for other amino acids in the naturally-occurring polypeptide sequence. By way of example, a derivative refers to a polypeptide sharing the same or substantially similar structure as a reference polypeptide that has been modified, e.g., chemically.

(7) Variant or analog polypeptides include insertion variants, wherein one or more amino acid residues are added to a protein amino acid sequence of the invention. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the protein amino acid sequence. Insertion variants, with additional residues at either or both termini, include for example, fusion proteins and proteins including amino acid tags or other amino acid labels. In one aspect, the protein molecule optionally contains an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.

(8) In deletion variants, one or more amino acid residues in a protein or polypeptide as described herein are removed. Deletions can be effected at one or both termini of the protein or polypeptide, and/or with removal of one or more residues within the protein amino acid sequence. Deletion variants, therefore, include fragments of a protein or polypeptide sequence.

(9) In substitution variants, one or more amino acid residues of a protein or polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art. Alternatively, the invention embraces substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp. 71-77] and are set out immediately below.

(10) TABLE-US-00001 CONSERVATIVE SUBSTITUTIONS SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C. Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T Y B. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic) K R H Negatively charged (acidic) D E

(11) Alternatively, exemplary conservative substitutions are set out immediately below.

(12) TABLE-US-00002 CONSERVATIVE SUBSTITUTIONS II EXEMPLARY ORIGINAL RESIDUE SUBSTITUTION Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

Gangliosides

(13) In embodiments, of the invention, gangliosides are conjugated to water soluble polymers, e.g. PEG or PSA or mPSA. Gangliosides are known to provide cells with distinguishing surface markers that can serve in cellular recognition and cell-to-cell communication. They are useful as therapeutic agents.

(14) Conjugates of the invention may comprise a ganglioside and a water soluble polymer, in which the ganglioside comprises a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids linked on the sugar chain. Gangliosides can be classified according to how many sialic acid units are present on the molecule. Examples of gangliosides are GM1, GM2 and GM3 (monosialo-gangliosides), GD1a, GD1b, GD2 and GD3 (disialo-gangliosides), GT1b (trisialo-ganglioside) and GQ1 (tetrasialo-ganglioside).

(15) For use in the present invention, preferred gangliosides comprise a ceramide linked to glucose, which is linked to a first galactose, which is linked to N-acetylgalactosamine, which is linked to a second galactose. This second galactose can be linked to one sialic acid. The first galactose can be linked to one, two, three or four sialic acids. Sialic acids may be linked either as monomers (one on each of the galactose molecules), or as oligosialic acids (2-4 sialic acids) to the first galactose.

(16) Where administered therapeutic gangliosides need to circulate in the blood for long periods. So that their action on target tissues is more effective, gangliosides can be polysialylated, for example, by the method of the invention.

Drug Delivery Systems

(17) In further embodiments, of the invention, drug delivery systems are conjugated to a water soluble polymer, e.g. PEG or PSA or mPSA. In general, a drug delivery system (DDS) is any molecular or particulate entity which can control the fate and effect of drugs associated with the entity. DDSs can be separated into two general types. The first type comprises macromolecules (MDDSs), for instance antibodies, neoglycoproteins as well as synthetic polymers, such as poly(hydroxypropylmethacrylamide), polylysine and polymerised alkyl cyanoacrylates. The association of drugs with various types of macromolecular carriers, including monoclonal antibodies to target the drug to the desired sites is described for instance by Gregoriadis in Nature 265, 407-411 (1977). The second type is particulate DDSs (PDDSs), which comprises for instance nanospheres or microspheres, which comprise biodegradable materials such as albumin or semibiodegradable materials such as dextran and alkylcyanoacrylate polymers, or vesicles formed of nonionic surfactants or liposomesfor details of which see for example Gregoriadis in NIPS, 4, 146-151 (1989).

(18) Drugs can either be covalently linked to, or passively entrapped into, the DDS. For instance, PDDS comprising surfactant vesicles or liposomes may entrap hydrophilic or hydrophobic pharmaceutically active compounds by being formed of an appropriate combination of layers of surfactant or lipid molecules. Pharmaceutically active compounds are usually covalently linked to MDDSs, by a bond which may or may not be lysed in the body, for instance before or after the active compound performs its function.

(19) Many of the MDDSs have an intrinsic (e.g. antibodies) or acquired (e.g. neoglycoproteins) ability to be recognised by target cells or tissues through receptors on the latter's surface. Typically, such DDSs are taken up specifically by the target upon injection. Specific uptake is, however, limited with the bulk of the DDSs being taken up by other, irrelevant (to therapy) tissues. The reason for this is that antibodies and other DDS proteins (regardless of their specificity for the target) must be, like other proteins, catabolised at the end of their biological life.

(20) Synthetic polymers used in the macromolecular type MDDSs are for instance poly(hydroxypropylmethacrylamide) polylysine and polymerised alkyl cyanoacrylates. These may be catabolised in the reticuloendothelial system (RES) or other tissues by appropriate lysosomal enzymes. It would be desirable to reduce the rate of catabolism of such biodegradable macromolecular type DDS by some means, for instance by reducing uptake of the DDS by the RES or other tissues, or by reducing degradation by lysosomal enzymes once taken up by the RES.

(21) Particulate DDSs (PDDSs) are, as a rule, removed from the circulation by the RES. Because of their propensity for the RES, PDDSs are often used for the delivery of drugs to these tissues. It is often desirable however, that PDDSs are directed to tissues other than those of the RES. To achieve this goal, one must block or delay RES interception of PDDSs.

(22) DDSs for use in the invention may not initially contain glycons. An option is to add or otherwise incorporate a glycon into the DDS structure. Examples of such cases are liposomes incorporating a mannosylated or a galactosylated lipid. These glycoliposomes will target actives to tissues which express a mannose or galactose receptor respectively.

(23) Where DDSs need to circulate in the blood for long periods so that e.g. uptake by target tissues is more effective (as with hepatic parenchymal cells), they are advantageously polysialylated by the methods of the invention.

Administration

(24) In one embodiment a conjugated compound of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.

(25) To administer compositions comprising a conjugated compound of the present invention to human or test animals, in one aspect, the compositions comprise one or more pharmaceutically acceptable carriers. The terms pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and cleavage products, and in addition do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.

(26) As used herein, effective amount includes a dose suitable for treating a mammal having a clinically defined disorder.

(27) The compositions may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well. Generally, compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.

(28) Single or multiple administrations of the compositions can be carried out with the dose levels and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dosage will depend on the type of disease to be treated, as described above, the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.

(29) The present invention also relates to a pharmaceutical composition comprising an effective amount of a conjugated compound or protein as defined herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient. The pharmaceutical composition can be used for treating clinically-defined disorders. The pharmaceutical composition of the invention may be a solution or a lyophilized product. Solutions of the pharmaceutical composition may be subjected to any suitable lyophilization process.

(30) As an additional aspect, the invention includes kits which comprise a composition of the invention packaged in a manner which facilitates its use for administration to subjects. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising a conjugated protein), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. In one embodiment, the kit contains a first container having a composition comprising a conjugated protein and a second container having a physiologically acceptable reconstitution solution for the composition in the first container. In one aspect, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration. Preferably, the kit contains a label that describes use of the therapeutic protein or peptide composition.

(31) In one embodiment, the derivative retains the full functional activity of native therapeutic compounds, and provides an extended half-life in vivo, as compared to native therapeutic compounds. In another embodiment, the derivative retains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%) biological activity relative to native compound.

Sialic Acid and PSA

(32) As used herein, sialic acid moieties includes sialic acid monomers or polymers (polysaccharides) which are soluble in an aqueous solution or suspension and have little or no negative impact, such as side effects, to mammals upon administration of the PSA-protein conjugate in a pharmaceutically effective amount. PSA and mPSA are characterized, in one aspect, as having 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. In certain aspects, different sialic acid units are combined in a chain.

(33) In one embodiment of the invention, the sialic acid portion of the PSA or mPSA compound is highly hydrophilic, and in another embodiment the entire compound is highly hydrophilic. Hydrophilicity is conferred primarily by the pendant carboxyl groups of the sialic acid units, as well as the hydroxyl groups. The saccharide unit may contain other functional groups, such as, amine, hydroxyl or sulphate groups, or combinations thereof. These groups may be present on naturally-occurring saccharide compounds, or introduced into derivative polysaccharide compounds. The PSA and mPSA used in the methods and conjugates of the invention may be further characterized as described above in the Background of the Invention.

(34) The naturally occurring polymer PSA is available as a polydisperse preparation showing a broad size distribution (e.g. Sigma C-5762) and high polydispersity (PD). Because the polysaccharides are usually produced in bacteria carrying the inherent risk of copurifying endotoxins, the purification of long sialic acid polymer chains may raise the probability of increased endotoxin content. Short PSA molecules with 1-4 sialic acid units can also be synthetically prepared (Kang S H et al., Chem Commun. 2000; 227-8; Ress D K and Linhardt R J, Current Organic Synthesis. 2004; 1:31-46), thus minimizing the risk of high endotoxin levels. However PSA preparations with a narrow size distribution and low polydispersity, which are also endotoxin-free, can now be manufactured. Polysaccharide compounds of particular use for the invention are, in one aspect, those produced by bacteria. Some of these naturally-occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compounds are substantially free of terminal galactose units.

(35) In various embodiments, the compound is linked to or associated with the PSA or mPSA compound in stoichiometric amounts (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:7, 1:8, 1:9, or 1:10, etc.). In various embodiments, 1-6, 7-12 or 13-20 PSA and/or mPSA units are linked to the compound. In still other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more PSA and/or mPSA units are linked to the compound.

(36) Optionally, the compound is modified to introduce glycosylation sites (i.e., sites other than the native glycosylation sites). Such modification may be accomplished using standard molecular biological techniques known in the art. Moreover, the compound, prior to conjugation via one or more carbohydrate moieties, may be glycosylated in vivo or in vitro.

Aminooxy Linkage

(37) In one embodiment of the invention, the reaction of hydroxylamine or hydroxylamine derivatives with aldehydes (e.g., on a carbohydrate moiety following oxidation by sodium periodate) to form an oxime group is applied to the preparation of conjugates of compound. For example, a glycoprotein is first oxidized with a oxidizing agent such as sodium periodate (NaIO.sub.4) (Rothfus J A et Smith E L., J Biol Chem 1963, 238, 1402-10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246, 1889-94). The periodate oxidation of e.g. glycoproteins is based on the classical Malaprade reaction described in 1928, the oxidation of vicinal diols with periodate to form an active aldehyde group (Malaprade L., Analytical application, Bull Soc Chim France, 1928, 43, 683-96). Additional examples for such an oxidizing agent are lead tetraacetate (Pb(OAc).sub.4), manganese acetate (MnO(Ac).sub.3), cobalt acetate (Co(OAc).sub.2), thallium acetate (TlOAc), cerium sulfate (Ce(SO.sub.4).sub.2) (U.S. Pat. No. 4,367,309) or potassium perruthenate (KRuO.sub.4) (Marko et al., J Am Chem Soc 1997, 119, 12661-2), By oxidizing agent a mild oxidizing compound which is capable of oxidizing vicinal diols in carbohydrates, thereby generating active aldehyde groups under physiological reaction conditions is meant.

(38) The second step is the coupling of the polymer containing an aminooxy group to the oxidized carbohydrate moiety to form an oxime linkage. In one embodiment of the invention, this step can be carried out in the presence of catalytic amounts of the nucleophilic catalyst aniline or aniline derivatives (Dirksen A et Dawson P E, Bioconjugate Chem. 2008; Zeng Y et al., Nature Methods 2009; 6:207-9). The aniline catalysis dramatically accelerates the oxime ligation allowing the use of very low concentrations of the reagents. In another embodiment of the invention the oxime linkage is stabilized by reduction with NaCNBH.sub.3 to form an alkoxyamine linkage.

(39) In one embodiment of the invention, the reaction steps to conjugate PSA or mPSA to a protein are carried out separately and sequentially (i.e., starting materials (e.g., protein, polymer, etc), reagents (e.g., oxidizing agents, aniline, etc) and reaction products (e.g., oxidized carbohydrate on a protein, activated aminooxy polymer, etc) are separated between individual reaction steps).

(40) Additional information on aminooxy technology can be found in the following references, each of which is incorporated in their entireties: EP 1681303A1 (HASylated erythropoietin); WO 2005/014024 (conjugates of a polymer and a protein linked by an oxime linking group); WO96/40662 (aminooxy-containing linker compounds and their application in conjugates); WO 2008/025856 (Modified proteins); Peri F et al., Tetrahedron 1998, 54, 12269-78; Kubler-Kielb J and Pozsgay V., J Org Chem 2005, 70, 6887-90; Lees A et al., Vaccine 2006, 24(6), 716-29; and Heredia K L et al., Macromoecules 2007, 40(14), 4772-9.

(41) Advantages of the invention include high recovery of conjugate, high retention of activity of the conjugated glycoprotein compared to unconjugated protein and high conjugation efficiency.

(42) The invention is now illustrated with reference to the following examples. Examples 1-3, 9 and 11-27 illustrate specific embodiments of the invention. Examples 4-8 and 10 are included as reference examples for their relevance to preparation of corresponding conjugates of the invention.

EXAMPLES

Example 1

Preparation of the homobifunctional linker NH.SUB.2.[OCH.SUB.2.CH.SUB.2.].SUB.2.ONH.SUB.2

(43) The homobifunctional linker NH.sub.2[OCH.sub.2CH.sub.2].sub.2ONH.sub.2

(44) ##STR00005##
(3-oxa-pentane-1,5-dioxyamine) containing two active aminooxy groups was synthesized according to Boturyn et al. (Tetrahedron 1997; 53:5485-92) in a two step organic reaction employing a modified Gabriel-Synthesis of primary amines. In the first step, one molecule of 2,2-chlorodiethylether was reacted with two molecules of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in dimethylformamide (DMF). The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol, Except where otherwise specified, this is referred to as the diaminooxy linker in examples below.

Example 2

Preparation of the homobifunctional linker NH.SUB.2.[OCH.SUB.2.CH.SUB.2.].SUB.4.ONH.SUB.2

(45) The homobifunctional linker NH.sub.2[OCH.sub.2CH.sub.2].sub.4ONH.sub.2

(46) ##STR00006##
(3,6,9-trioxa-undecane-1,11-dioxyamine) containing two active aminooxy groups was synthesized according to Boturyn et al. (Tetrahedron 1997; 53:5485-92) in a two step organic reaction employing a modified Gabriel-Synthesis of primary amines. In the first step one molecule of Bis-(2-(2-chloroethoxy)-ethyl)-ether was reacted with two molecules of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol.

Example 3

Preparation of Aminooxy-PSA

(47) 500 mg of oxidized PSA (MW=18.8 kD) obtained from the Serum Institute of India (Pune, India) was dissolved in 8 ml 50 mM sodium acetate buffer, pH 5.5. Next, 100 mg 3-oxa-pentane-1,5-dioxyamine was added. After shaking for 2 hrs at room temperature, 44 mg sodium cyanoborohydride was added. After shaking for another 4 hrs at 4 C., the reaction mix was loaded into a Slide-A-Lyzer (Pierce, Rockford, Ill.) dialysis cassette (3.5 kD membrane, regenerated cellulose) and dialyzed against PBS pH 7.2 for 4 days. The product was frozen at 80 C. The preparation of the aminooxy-PSA according to this procedure is illustrated in FIG. 2.

Example 4

Coupling of Aminooxy-PSA to rFIX and Purification of the Conjugate

(48) To 12.6 mg rFIX, dissolved in 6.3 ml 50 mM sodium acetate buffer, pH 6.0, 289 l of an aqueous sodium periodate solution (10 mM) was added. The mixture was shaken in the dark for 1 h at 4 C. and quenched for 15 min at room temperature by the addition of 6.5 l 1M glycerol. Low molecular weight contaminates were removed by ultrafiltration/diafiltration (UF/DF) employing Vivaspin (Sartorius, Goettingen, Germany) concentrators (30 kD membrane, regenerated cellulose). Next, 43 mg aminooxy-PSA was added to the UF/DF retentate and the mixture was shaken for 18 hrs at 4 C. The excess PSA reagent was removed by hydrophobic interaction chromatography (HIC). The conductivity of the cooled reaction mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HIC column (1.62.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted within 2.4 column volumes (CV) with 50 mM HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5 ml/min. The preparation was analytically characterized by measuring total protein (BCA) and FIX chromogenic activity. For the PSA-rFIX conjugate a specific activity of 80.2 IU/mg protein was determined (56.4% in comparison to native rFIX). The results are summarized in Table 1.

(49) TABLE-US-00003 TABLE 1 Specific BCA FIX:chrom Specific Activity Activity Item [mg/ml] [IU/ml] [IU FIX:Chrom/mg BCA [%] rFIX 8.58 1221 142.3 100 PSA-rFIX 1.15 92.2 80.2 56.4

Example 5

Coupling of Aminooxy-PSA to rFIX in the Presence of Aniline as Nucleophilic Catalyst

(50) To 3.0 mg rFIX, dissolved in 1.4 ml 50 mM sodium acetate buffer, pH 6.0, 14.1 l of an aqueous sodium periodate solution (10 mM) was added. The mixture was shaken in the dark for 1 h at 4 C. and quenched for 15 min at room temperature by the addition of 1.5 l M glycerol. Low molecular weight contaminates were removed by means of size exclusion chromatography (SEC) employing PD-10 desalting columns (GE Healthcare, Fairfield, Conn.). 1.2 mg oxidized rFIX, dissolved in 1.33 ml 50 mM sodium acetate buffer, pH 6.0 was mixed with 70 l of aniline (200 mM aqueous stock solution) and shaken for 45 min at room temperature. Next, 4.0 mg aminooxy-PSA was added and the mixture was shaken for 2 hrs at room temperature and another 16 hrs at 4 C. Samples were drawn after 1 h, after 2 hrs and at the end of the reaction after 18 hrs. Next, excess PSA reagent and free rFIX were removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HIC column (1.62.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted with a linear gradient to 50 mM HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 in 20 CV with at a flow rate of 5 ml/min.

Example 6

Coupling of Aminooxy-PSA to rFIX and Reduction with NaCNBH.SUB.3

(51) To 10.5 mg rFIX, dissolved in 5.25 ml 50 mM sodium acetate buffer, pH 6.0, 530 of an aqueous sodium periodate solution (10 mM) was added. The mixture was shaken in the dark for 1 h at 4 C. and quenched for 15 min at room temperature by the addition of 5.3 l 1M glycerol. Low molecular weight contaminates were removed by means of UF/DF employing Vivaspin (Sartorius, Goettingen, Germany) concentrators (30 kD membrane, regenerated cellulose). Next, 35.9 mg aminooxy-PSA was added to the UF/DF retentate and the mixture was shaken for 2 hrs at room temperature. Then 541 of aqueous sodium cyanoborohydride solution (5M) was added and the reaction was allowed to proceed for another 16 hrs. Then the excess PSA reagent was removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF HIC (GE Healthcare, Fairfield, Conn.) column (1.62.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted within 2.4 CV with 50 mM HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5 ml/min.

Example 7

Coupling of Aminooxy-PSA (Linker: NH.SUB.2.[OCH.SUB.2.CH.SUB.2.].SUB.4.ONH.SUB.2.) to rFIX and Purification of the Conjugate

(52) To 5.6 mg rFIX, dissolved in 2.8 ml 50 mM sodium acetate buffer, pH 6.0, 102 l of an aqueous solution of sodium periodate (10 mM) was added. The mixture was shaken in the dark for 1 h at 4 C. and quenched for 15 min at room temperature by the addition of 2.9 l of 1M glycerol. Low molecular weight contaminates were removed by means of UF/DF employing Vivaspin (Sartorius, Goettingen, Germany) concentrators (30 kD membrane, regenerated cellulose). Then 19 mg aminooxy-PSA was added to the UF/DF retentate and the mixture was shaken for 18 hrs at 4 C. The excess PSA reagent was removed by means of HIC. The conductivity of the cooled reaction mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HIC column (1.62.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted within 2.4 CV with 50 mM HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5 ml/min.

Example 8

Coupling of Aminooxy-PSA to rFVIII

(53) To 11 mg rFVIII, dissolved in 11 ml Hepes buffer pH 6 (50 mM Hepes, 5 mM CaCl.sub.2, 150 mM NaCl, 0.01% Tween) 57 l 10 mM sodium periodate was added. The mixture was shaken in the dark for 30 min at 4 C. and quenched for 30 min at 4 C. by the addition of 107 l of an aqueous 1M glycerol solution. Then 19.8 mg aminooxy-PSA (18.8 kD) was added and the mixture was shaken over night at 4 C. The ionic strength was increased by adding a buffer containing 8M ammonium acetate (8M ammonium acetate, 50 mM Hepes, 5 mM CaCl.sub.2, 350 mM NaCl, 0.01% Tween 80, pH 6.9) to get a final concentration of 2.5M ammonium acetate. Next, the reaction mixture was loaded on a HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) column which was equilibrated with equilibration buffer (2.5M ammonium acetate, 50 mM Hepes, 5 mM CaCl.sub.2, 350 mM NaCl, 0.01% Tween 80, pH 6.9). The product was eluted with elution buffer (50 mM Hepes, 5 mM CaCl.sub.2, 0.01% Tween 80, pH 7.4), and the eluate was concentrated by centrifugal filtration using Vivaspin (Sartorius, Goettingen, Germany) devices with 30,000 MWCO.

Example 9

Preparation of the Homobifunctional Linker NH.SUB.2.[OCH.SUB.2.CH.SUB.2.].SUB.6.ONH.SUB.2

(54) The homobifunctional linker NH.sub.2[OCH.sub.2CH.sub.2].sub.6ONH.sub.2

(55) ##STR00007##
(3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine) containing two active aminooxy groups was synthesized according to Boturyn et al. (Tetrahedron 1997; 53:5485-92) in a two step organic reaction employing a modified Gabriel-Synthesis of primary amines. In the first step one molecule of hexaethylene glycol dichloride was reacted with two molecules of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis in ethanol.

Example 10

Polysialylation of rFIX Employing a Maleimido/Aminooxy Linker System

(56) A. Preparation of the Modification Reagent

(57) An Aminooxy-PSA reagent is prepared by use of a maleimido/aminooxy linker system (Toyokuni et al., Bioconjugate Chem 2003; 14, 1253-9). PSA-SH (20 kD) containing a free terminal SHgroup is prepared using a two step procedure: a) Preparation of PSA-NH.sub.2 by reductive amination of oxidized PSA with NH.sub.4Cl according to WO05016973A1 and b) introduction of a sulfhydryl group by reaction of the terminal primary amino group with 2-iminothiolane (Traut's reagent/Pierce, Rockford, Ill.) as described in U.S. Pat. No. 7,645,860. PSA-SH is coupled to the maleimido-group of the linker at pH 7.5 in PBSbuffer using a 10-fold molar excess of the linker and a PSA-SH concentration of 50 mg/ml. The reaction mixture is incubated for 2 hours under gentle shaking at room temperature. Then the excess linker reagent is removed and the aminooxy-PSA is buffer exchanged into oxidation buffer (50 mM sodium phosphate, pH 6.0) by diafiltration. The buffer is exchanged 25 times employing a Pellicon XL5 kD regenerated cellulose membrane (Millipore, Billerica, Mass.).

(58) B. Modification of rFIX after Prior Oxidation with NaIO.sub.4

(59) rFIX is oxidized in 50 mM sodium phosphate buffer, pH 6.0 employing 100 M sodium periodate in the buffer. The mixture was shaken in the dark for 1 h at 4 C. and quenched for 15 min at room temperature by the addition of glycerol to a final concentration of 5 mM. Low molecular weight contaminates were removed by means of size exclusion chromatography (SEC) employing PD-10 desalting columns (GE Healthcare, Fairfield, Conn.). Oxidized rFIX is then spiked with aniline to obtain a final concentration of 10 mM and mixed with the aminooxy-PSA reagent to achieve a 5 fold molar excess of PSA. The reaction mixture was incubated for 2 hours under gentle shaking in the dark at room temperature.

(60) C. Purification of the Conjugates

(61) The excess of PSA reagent and free rFIX is removed by means of HIC. The conductivity of the reaction mixture is raised to 180 mS/cm and loaded onto a column filled with 48 ml Butyl-Sepharose FF (GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 3 M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequently the conjugate is eluted with a linear gradient of 60% elution buffer (50 mM Hepes, 6.7 mM calcium chloride, pH 7.4) in 40 CV. Finally the PSA-rFIX containing fractions are collected and subjected to UF/DF by use of a 30 kD membrane made of regenerated cellulose (Millipore). The preparation is analytically characterized by measuring total protein (BCA) and FIX chromogenic activity. For the PSA-rFIX conjugates prepared with both variants a specific activity of >50% in comparison to native rFIX was determined.

Example 11

Preparation of Aminooxy-PSA Reagent

(62) An aminooxyPSA reagent was prepared according to Example 3. The final product was diafiltrated against buffer, pH 7.2 (50 mM Hepes) using a 5 kD membrane (regenerated cellulose, Millipore), frozen at 80 C. and lyophilized. After lyophilization the reagent was dissolved in the appropriate volume of water and used for preparation of PSA-protein conjugates via carbohydrate modification.

Example 12

Detailed Synthesis of the Aminooxy-PSA Reagent

(63) 3-oxa-pentane-1,5 dioxyamine was synthesized according to Botyryn et al (Tetrahedron 1997; 53:5485-92) in a two step organic synthesis as outlined in Example 1.

(64) Step 1:

(65) To a solution of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide (59.0 g; 1.00 eq) in 700 ml anhydrous N,N-dimethylformamide anhydrous K.sub.2CO.sub.3 (45.51 g; 1.00 eq) and 2,2-dichlorodiethylether (15.84 ml; 0.41 eq) were added. The reaction mixture was stirred for 22 hours at 50 C. The mixture was evaporated to dryness under reduced pressure. The residue was suspended in 2 L dichloromethane and extracted two times with saturated aqueous NaCl-solution (each 1 L). The Dichloromethane layer was dried over Na.sub.2SO.sub.4 and then evaporated to dryness under reduced pressure and dried in high vacuum to give 64.5 g of 3-oxapentane-1,5-dioxy-endo-2,3-dicarboxydiimidenorbornene as a white-yellow solid (intermediate 1).

(66) Step 2:

(67) To a solution of intermediate 1 (64.25 g; 1.00 eq) in 800 ml anhydrous Ethanol, 31.0 ml Hydrazine hydrate (4.26 eq) were added. The reaction mixture was then refluxed for 2 hours. The mixture was concentrated to the half of the starting volume by evaporating the solvent under reduced pressure. The occurring precipitate was filtered off. The remaining ethanol layer was evaporated to dryness under reduced pressure. The residue containing the crude product 3-oxa-pentane-1,5-dioxyamine was dried in vacuum to yield 46.3 g. The crude product was further purified by column chromatography (Silicagel 60; isocratic elution with Dichloromethane/Methanol mixture, 9+1) to yield 11.7 g of the pure final product 3-oxa-pentane-1,5-dioxyamine.

Example 13

Preparation of Aminooxy-PSA Polymer

(68) 1.3 g of oxidized colominic acid (23 kDa) was dissolved in 18 ml of 50 mM sodium acetate pH 5.50.02. 20 fold molar excess of 1,11-diamino-3,6,9-trioxaundecane (also referred to as 3,6,9-trioxa-undecane-1,11-dioxyamine) was dissolved in minimum amount of 50 mM sodium acetate (pH 5.50.02) and was added to the PSA solution. The final colominic acid concentration was 62.5 mg/ml. This reaction mixture was incubated for 20.1 hr at 221.0 C. on a gentle mixer (22 oscillations per minute). After this, 0.65 ml of 160 mg/ml NaCNBH.sub.3 solution was added to the above reaction mixture so as to make the final concentration of 5.00 mg/ml. This was incubated for 3.00.20 hours at 4.01.0 C. on a shaker (22 oscillations per minute) in a endotoxin free air tight container with enough headspace for mixing. For the purification, the sample was diluted with 2 mM triethanolamine, pH 8.00.02 to make final colominic acid concentration of 20 mg/ml. The reaction mixture was desalted to remove excess of 1,11-diamino-3,6,9-trioxaundecane, NaCNBH.sub.3 and byproducts of the reaction. This was followed by desalting on a Sephadex G25 column using 20 mM triethanolamine buffer (pH 8.00.02). The pH of the desalted sample was adjusted to pH 7.8-8.0 and was ultrafiltered/diafiltered with 20 mM TEA pH 8.0 once and 2 mM triethanolamine (TEA) pH 8.0 twice. The sample was freeze dried and stored at 80 C.

(69) Alternatively, purification was done in presence of high salt during desalting and ultrafiltration/diafiltration (UF/DF) steps. Anion exchange chromatography in high salt was also used to make highly pure aminooxy-PSA. Different molecular weights of aminooxy-PSA were synthesized.

Example 14

Coupling of Diaminooxy (3,6,9-trioxa-undecane-1,11-dioxyamine)PSA to -Galactosidase

(70) For oxidation of -Galactosidase (-Gal), different concentrations of NaIO.sub.4 (ranging from 0.157 mM to 2 mM) were used. 0.5 mg of -Gal was oxidized under acidic pH of 5.75 at 4 C. for 30 minutes in the dark. Oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized -Gal with diaminooxy PSA polymer (22 kDa). The final concentration of polymer in the reaction mixture was 1.25 mM whereas the concentration of -Gal ranged from 0.125 mg/ml to 0.76 mg/ml. All the reactions were done at pH5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. and samples were collected at time intervals of 1, 2 and 24 hours. Conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and this was also confirmed by western blotting.

(71) Based on the best reactions conditions, 1.9 mg of -Gal was oxidized with 1.5 mM of NaIO.sub.4 for 30 minutes at 4 C. and then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized -Gal with diaminooxy PSA polymer. The final concentrations of polymer and protein in the reaction mixture were 1.25 mM and 0.76 mg/ml respectively. The final pH of the reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 2 hours. Purified and unpurified conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and this was confirmed by western blotting using anti-PSA antibody. The in vitro activity PSA-Gal conjugates were comparable to native protein using All in one Gal assay kit (Pierce). Further, the overall process was scaled up to 3 fold.

Example 15

Coupling of Diaminooxy-PSA to Fetuin

(72) Fetuin and was oxidized with 10 mM NaIO.sub.4 for 60 minutes at 4 C. in the dark and the oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 10 mM. The conjugation reaction was carried out using the oxidized Fetuin with diaminooxy PSA polymer (23 kDa). The final concentration of polymer in the reaction mixture was 2.5 mM at pH 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The final protein concentration in the reaction was 0.714 mg/ml and the reaction was carried out at 4 C. for 2 hours. These conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugates in SDS PAGE and this was also confirmed by western blotting.

(73) For a scale up reaction, 5 mg Fetuin was oxidized with 10 mM NaIO.sub.4 for 60 minutes at 4 C. in dark and then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 10 mM. The conjugation reaction was carried out using the oxidized Fetuin with diaminooxy PSA polymer (23 kDa). The final concentration of polymer in the reaction mixture was 2.5 mM at pH of 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. and sample was collected after 2 hours. Purified and unpurified conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and this was also confirmed by western blotting.

Example 16

Coupling of Diaminooxy-PSA to Fetuin with Aniline to Act as a Nucleophilic Catalyst

(74) 0.2 mg of Fetuin was oxidized with 10 mM NaIO.sub.4 for 30 minutes at 4 C. in dark and then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized Fetuin with diaminooxy PSA polymer (23 kDa). The final concentration of polymer in the reaction mixture was 1.25 mM. The final pH of reaction mixture was 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The final protein concentration in the reaction was 0.125 mg/ml. 84.21 l of 200 mM aniline solution was added to the 1.6 ml of reaction mixture. The reaction was carried out at 4 C. overnight.

Example 17

Coupling of Diaminooxy-PSA to Erythropoietin (EPO)

(75) 0.2 mg of EPO was oxidized with 10 mM of NaIO.sub.4 for 30 minutes at 4 C. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized EPO with diaminooxy polymer of 23 kDa. The final concentration of polymer in the reaction mixture was 1.25 mM. The final concentration of EPO in the reaction mixture was 0.125 mg/ml. The final pH of the reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 24 hours. Unpurified conjugate was characterized using SDS PAGE. A shift in the band was seen for the conjugate in SDS PAGE.

Example 18

Coupling of Diaminooxy-PSA to EPO with Aniline to Act as a Nucleophilic Catalyst

(76) 0.2 mg of EPO was oxidized with 10 mM NaIO.sub.4 for 30 minutes at 4 C. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized EPO with diaminooxy PSA polymer (22 kDa). The final concentration of polymer in the reaction mixture was 1.25 mM. The final pH of the reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The final protein concentration in the reaction was 0.125 mg/ml. 84.21 l of 200 mM aniline solution was added to the 1.6 ml of reaction mixture. The reaction was carried out at 4 C. for overnight. The conjugates were characterized using SDS PAGE. A shift in the band was seen in the conjugates. No adverse effect of aniline was observed on activity of the conjugates.

Example 19

Coupling of Diaminooxy-PSA to DNAse

(77) For glycopolysialylation of DNAse, bovine pancreas DNAse was used for conjugation reaction. This source of DNAse was supplied as lyophilized powder, which was stored at 20 C. Prior to the reaction, this lyophilized powder was dissolved in sodium acetate buffer (pH 5.75). The polymer used for glycopolysialylation had a weight in the range of 10 kDa to 22 kDa. For oxidation of glycon moiety of DNAse, NaIO.sub.4 was used as oxidizing agent to a final concentration of 1 mM. DNAse was oxidized at acidic pH of 5.75 at 4 C. for 30 minutes. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 2 mM. After oxidation was complete, the conjugation reaction was carried out by addition of diaminooxy PSA polymer to a final concentration of 1.25 mM. NaCNBH.sub.3 was added to the reaction mixture to a final concentration of 50 mM or 3.17 mg/ml and the polysialylation of the DNAse was preformed 4.01.0 C. for at least 2 hours. The reactions were stopped with 25 molar excess of Tris over polymer. The conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and positing result was obtained from western blotting. Activity was measured as 95% (compared with the less than 50% observed in comparable conjugates made using aldehyde linker chemistry).

Example 20

Coupling of Diaminooxy (3 Oxa-Pentane-1,5-Dioxyamine Linker)-PSA to -Galactosidase

(78) For oxidation of -Galactosidase, NaIO.sub.4 was used at a concentration of 2 mM. 3 mg of -Galactosidase was oxidized at acidic pH of 5.75 at 4 C. for 30 minutes then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 2 mM. The conjugation reaction was carried out using the oxidized -Galactosidase with diaminooxy PSA polymer (23 kDa). The final concentration of polymer in the reaction mixture was 1.5 mM. The final concentration of -Galactosidase in reaction mixture was 0.867 mg/ml. The final pH of reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 2 hours. Conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and positing result was obtained from western blotting.

Example 21

Preparation of Hydrazine-Colominic Acid

(79) We used the following protocol to prepare a PSA-hydrazide (colominic acid-hydrazide) using adipic acid dihydrazide. Analogous methods are used to make other PSA-hydrazides.

(80) 1 Dissolve 1 g of activated colominic acid in 10 ml of 20 mM sodium acetate pH 5.50.02. Final colominic acid concentration should be 62.5 mg/ml

(81) 2 Dissolve 25-fold molar excess (over oxidized colominic acid CAO) of adipic acid dihydrazide (MW=174.2 gms) in minimum amount of 20 mM sodium acetate (pH 5.50.02) and add to solution from 1.

(82) $\begin{array}{c}\mathrm{Amount}\mathrm{of}\mathrm{adipic}\mathrm{acid}\mathrm{dihydrazide}\mathrm{to}\mathrm{be}\mathrm{added}=\frac{\mathrm{Weight}\mathrm{of}\mathrm{CAO}\mathrm{in}\mathrm{grams}25\mathrm{MW}\mathrm{of}\mathrm{adipic}\mathrm{acid}\mathrm{dihydrazide}\mathrm{in}\mathrm{gms}}{\mathrm{MW}\mathrm{of}\mathrm{CAO}\mathrm{in}\mathrm{Daltons}}\\ =\frac{125174.2}{15{10}^3}\\ =0.290g\end{array}$
4 After adding adipic acid dihydrazide solution, make up the volume of colominic acid with sodium acetate to a final concentration of 62.5 mg/ml. Therefore total reaction volume is 16 ml.
5 Incubate the reaction mixture for 210.1 hr at 22.01.0 C. on shaker (22 oscillations per minute).
6 Prepare concentrated NaCNBH.sub.3 solution (165 mg/ml) and add 0.5 ml to solution from 1 so that the final concentration of this becomes 5.0 mg/ml in the final reaction mixture. Incubate the reaction mixture for 3.010.20 hours at 4.01.0 C. on shaker (22 oscillations per minute).
7 Keep the reaction mixture in endotoxin-free, air tight container with excess 50 ml of headspace for proper mixing (there should be enough space so that reaction mixture should not touch the cap of container).
8 After 3 hours reaction at 4 C., dilute the sample with 2 mM triethanolamine (make the volume up to 50 ml), at pH 8.00.02 to make final colominic acid concentration to 20 mg/ml.
9 Desalt the reaction mixture to remove excess of untreated adipic acid dihydrazide, NaCNBH.sub.3 etc from the polymer. This can be done by GPC (using XK 50 Sephadex G-25 medium matrix; 1.8 mg of CA/ml matrix; 35 cm bed height; Column volume=687 ml) by observing UV 224 nm and conductivity. Desalting is carried out with 20 mM triethanolamine (pH 8.00.02) buffer.
10 After desalting, colominic acid-hydrazide is subjected to 1 cycle of ultrafiltration, 1 cycle of diafiltration using 20 mM TEA, pH 8.00.02 and at least 3 cycles of diafiltration using 2 mM TEA, pH 8.00.02. This can be done using 3 kDa vivaflow cassettes.
11 Adjust the pH of desalted sample to pH 7.8-8.0. Optionally, freeze-dry the sample and consecutively keep it for secondary drying to remove excess of moisture.

Example 22

Coupling of Hydrazide-PSA to Erythropoietin

(83) For oxidation of erythropoietin (EPO), NaIO.sub.4 was used at a concentration of 10 mM. EPO (1 mg) was oxidized at pH 5.75 at 4 C. for 30 minutes then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized EPO with hydrazide-PSA polymer. The molecular weight of the hydrazide-PSA used for conjugation was 24.34 kDa. The final concentration of hydrazide-PSA in the reaction mixture was 1.25 mM. The final concentration of EPO in the reaction mixture was 0.125 mg/ml. The final pH of the reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 24 hours. Conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and a positive result was obtained from western blotting.

Example 23

Coupling of Hydrazide-PSA to -Galactosidase

(84) -Galactosidase (0.5 to 4.5 mg) was oxidized with 0.625 to 2 mM of NaIO.sub.4 for 30 minutes at 4 C. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized -galactosidase with hydrazide-PSA ranging from 24.34 kDa to 27.9 kDa. The final concentration of hydrazide-PSA in the reaction mixture was 1.25 mM. The final concentration of -galactosidase in the reaction mixture was in a range from 0.125 mg/ml to 0.76 mg/ml. The final pH of reaction mixture should be around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. and samples were collected at 1, 2 and 24 hours. Purified and unpurified conjugate was characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and a positive result was obtained from western blotting. Activity was measured as 84%.

Example 24

Coupling of Hydrazide-PSA to Fetuin

(85) Fetuin (0.25 mg) was oxidized with NaIO4 (5 or 10 mM) for 30 or 60 minutes at 4 C. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 or 10 mM as appropriate to match the concentration of NaIO.sub.4 used for oxidation. The conjugation reactions were carried out using the oxidized Fetuin with adipic acid dihydrazide-PSA polymer. The final concentration of the polymer in the reaction mixture was between 1.25 and 2.5 mM. The final pH of reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 1 hour to 4 hours. The conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SD S PAGE for each set of reaction conditions and a positive result was obtained from western blotting.

(86) A scaled-up reaction for 5 mg Fetuin followed by purification of the resulting conjugate was carried out. 5 mg Fetuin was oxidized with 10 mM NaIO.sub.4 for 60 minutes at 4 C. and then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 10 mM. The conjugation reaction was carried out using the oxidized Fetuin with adipic acid dihydrazide-PSA polymer. The final concentration of polymer in the reaction mixture was 2.5 mM. The final pH of reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. and samples were collected at 2 hours. Purified and unpurified conjugate was characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and a positive result was obtained from western blotting.

Example 25

Coupling of Hydrazide-PSA to DNAse

(87) DNAse was oxidized with NaIO4 to a final concentration ranging from 0.2 mM to 2 mM for 30 minutes at 4 C. The oxidation reaction was stopped by adding NaHSO.sub.3 to a final concentration of between 2 and 5 mM depending upon the concentration of NaIO.sub.4 used for oxidation. Glycopolysialylation of oxidized DNAse was carried out by addition of hydrazide-PSA polymer to a final concentration of 1.25 mM to the oxidized DNAse. Sodium cyanoborohydride was added to the reaction mixture to a final concentration of 50 mM or 3.17 mg/ml and the glycopolysialylation of the DNAse was performed at 4 C. for a time period ranging from 1 hour to 2 hours. The reactions were stopped with 25-fold molar excess of Tris over polymer. The conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugates in SDS PAGE and a positive result was obtained from western blotting. The activity was measured as 49%.

Example 26

PEGylation of -Galactosidase Using Aminooxy Linker (3-Oxa-Pentane-1,5-Dioxyamine)

(88) -Galactosidase (1 mg) was oxidized with 1.5 mM of NaIO.sub.4 for 30 minutes at 4 C. The oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 1.5 mM. The conjugation reaction was carried out using the oxidized -Galactosidase with diaminooxy-PEG polymer (20 kDa). The final concentration of the polymer in the reaction mixture was 1.25 mM. The final concentration of -Galactosidase in the reaction mixture was 1 mg/ml. The final pH of reaction mixture should be around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4 C. for 2 hours. Unpurified conjugate was characterized using SDS PAGE and a shift in the band was seen for the conjugate in SDS PAGE. The activity was measured as 59%.

Example 27

PEGylation of Erythropoietin Using Aminooxy Linker

(89) Erythropoietin (EPO; 0.2 mg) was oxidised with 5 or 10 mM NaIO.sub.4 in 50 mM sodium acetate at pH 5.75 for 45 minutes at 4 C. and then oxidation was stopped by adding NaHSO.sub.3 to a final concentration of 5 or 10 mM (to match the concentration of NaIO.sub.4 used for oxidisation). The conjugation reaction was carried out using the oxidised EPO with diaminooxy PEG polymer (20 kDa). The final concentration of the polymer in the reaction mixture was 1.5 mM. The final pH of reaction mixture should be around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The final protein concentration in the reaction was 0.4 mg/ml. The conjugation reaction was carried out overnight at 4 C.

(90) The invention thus provides conjugates of compounds other than blood coagulation proteins with water soluble polymers, in particular PSA and mPSA.