WO1994005332A2 - Glycolation of glycosylated macromolecules - Google Patents
Glycolation of glycosylated macromolecules Download PDFInfo
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- WO1994005332A2 WO1994005332A2 PCT/US1993/008196 US9308196W WO9405332A2 WO 1994005332 A2 WO1994005332 A2 WO 1994005332A2 US 9308196 W US9308196 W US 9308196W WO 9405332 A2 WO9405332 A2 WO 9405332A2
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- macromolecule
- glycosylated
- glycolated
- glycol
- polypeptide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
Definitions
- polypeptides in circulatory systems for the purpose of engendering a particular physiological response
- insulin which is used in the treatment of diabetes.
- Another group of polypeptides to which great therapeutic potential has been attributed are various enzymes.
- a principal factor which has severely limited the use in therapeutics of polypeptides is that most of these com ⁇ pounds elicit an immunogenic response in body fluids, evidenced by changes in the composition of the circulatory system, i.e., the production of antibodies to the polypeptides. This effect has one or both of two secondary consequences: first, neutralization of the polypeptides by the antibodies thus produced; second, and more seriously, the development of an allergic response.
- Neutralization of polypeptides by antibodies is be- lieved to be responsible for the rather low residence time of insulin in the human circulatory system; hence, persons afflicted with diabetes are forced to inject themselves fairly frequently with fresh doses of insulin.
- parenterally administered enzymes not only is there the problem of neutralization of the pclypep.tide and the subsequent negation of its physiological activity, but also the extremely undesirable elicitation of an allergic reaction.
- a limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is their potential for eliciting such immune response in the circulatory system.
- This immune response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), j. ciin. Endocr..31. 679-688, W. Moore (178), . ciin. Endroc ⁇ 'noi. Hetab., 51, 691-697.
- the antibody production may decrease or eliminate the desired bio ⁇ logical function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
- polypeptides include the modification of proteins with substantially straight- chain polymers such as polyethylene (PEG) or polypropylene glycol (PPG) .
- PEG polyethylene
- PPG polypropylene glycol
- U.S. Patent No. 4,055,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides.
- U.S. Patent No. 4,088,538 discloses a reversibly soluble, enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
- U.S. Patent No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group, and/or at least one aromatic hydroxy group (described in column 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in column 2, lines 42-66).
- U.S. Patent No. 4,496,689 discloses a covalently attached complex of ⁇ -1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
- U.S. Patent No. 3,788,948 discloses use of organic cyanate compounds to bind proteins to polymers.
- U.S. Patent No. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
- JP 57-92435 published November 26, 1982, discloses modified polypeptides, where all or part of the amino groups are substituted with a polyethoxyl moiety.
- DE 2312615 published September 27, 1973, discloses conju ⁇ gating of polymers to compounds containing hydroxy or amino groups.
- EP 147,761 discloses a covalent conjugate of ⁇ -1- proteinase inhibitor and a water-soluble polymer, where the polymer may be polyethylene glycol.
- EP 154,316 published September 11, 1985, discloses and claims chemically modified lymphokines, such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
- U.S. Patent No. 4,414,147 describes rendering inter- feron less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethylene succinic anhydride) .
- glycol couples most likely through an amino group on the protein, but also discloses an embodiment where the terminal hydroxy group of the glycol is converted to an amino group, e.g., with a sulfonating agent or a halogenating agent, and the resultant halide or tosylate is coupled with a carboxyl group of the polypeptide by known methods.
- a sulfonating agent or a halogenating agent e.g., a sulfonating agent or a halogenating agent
- the resultant halide or tosylate is coupled with a carboxyl group of the polypeptide by known methods.
- One aspect of the invention is a process for the glycolation of a glycosylated macromolecule, comprising activating a polyalkylene glycol, reacting the activated polyalkylene glycol with a diamino compound, whereby the activated polyalkylene glycol is coupled to the diamino compound through one of its amino groups, 93/08196
- the invention preferably comprises a process for the
- PEGylation of a glycosylated macromolecule comprising:
- n is preferably about 2-500.
- x is preferably about 1-20.
- Macromolecules usable in the invention include virtually any bioactive macromolecule bearing glyco ⁇ sylations (regardless of how bonded, e.g., covalently, etc.) or which can be glycosylated, e.g., polypeptides and/or proteins, nucleic acids, lipids, or carbohydrates.
- Preferred peptides include those comprising an antigen binding region, a cytokine, a receptor, an antithrom- botic, a growth factor, or an angiohypotensive reagent.
- the polypeptide is an immunoglobulin, an interferon, a receptor tyrosine kinase, a thrombomodulin, a transforming growth factor, an endothelin, or an analog of the above.
- One especially preferred protein is the monoclonal antibody TAb-250 or its chimeric analog BACh 250 ("BACh-250" or "C-erb-B2") . See Molecular Oncology as a Basis for New Strateties in Cancer Therapy: Efficacy of an Anti-c-erbB-2 Mouse/Human Chimeric Antibody Alone and in Combination with cis-
- CDDP Diammedichloroplatinum
- oxidoreductases such as Urate:oxygen- oxidoreductase, Hydrogen-peroxide:hydrogen-peroxide oxidoreductase, Cholesterol-reduced-NADP:oxygen oxidoreductase (20-3-hydroxylating) ; transferases, such as UDP glucuronate glucuronyl-transferase (acceptor unspecific) , UDP glucose: ⁇ -D-Galactose-1-phosphate; hydrolases, such as Mucopeptide N-acetyl- muramyl-hydrolase, Trypsin, L-asparagine aminohydrolase; lyases, such as Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase; isomerases, such as D-Xylose ketol-isomerase; and ligases, such as L-Citrulline
- peptide hormones examples include insulin, ACTH, Glycagon, Somatostatin, Somatotropin, Thymosis, Parathyroid hormone, Pigmentary hormones,
- Macromolecules which have no sugars may be glyco ⁇ sylated by means which are well known in the art, e.g. , as disclosed in Creighton, proteins, W.H. Freeman & Co., New York, 1983. Virtually any sugar which is reactive to oxidation is suitable. Examples include galactose, man- nose, glucose, N-acetylglucosamine, N-acetylgalactos- amine, sialic acids, fucose, and/or xylose.
- the length of the carbohydrate chains of the sugar may vary widely, i.e., poly- and oligosaccharides may be used. Normally, the glycosylation will be that which is indigenous to the species producing the macromolecule, e.g. , mammalian.
- the glycol is preferably polyethylene glycol, poly- propylene glycol, or a mixture thereof, as well as a mixed polyethylene-polypropylene glycol.
- Polyethylene glycols (PEG's) are most preferred, especially ono- methoxyethylene glycol.
- Preferred polyethylene glycols have the formula CH 3 0-(CH 2 CH 2 0) n -H, wherein n is 2-500, more preferably 20-400.
- the diamino compound preferably has the formula H 2 N- R-NH 2 , wherein R is an organic moiety.
- R may be, for example, a C ⁇ -aliphatic or C 3.20 -cycloaliphatic moiety or a C 5 . 20 -aryl moiety.
- Aliphatic moieties include straight- or branched-chain or cyclic alkyl, alkenyl, dienyl, and alkynyl groups.
- Preferred aliphatic moieties are C 2 . 12 - alkyl groups.
- Preferred aryl moieties are heterocyclic, i.e., containing one or more O, S, or N atoms, and aromatic, e.g., phenyl groups.
- Activation of the glycol with the addition of the diamino compound preferably occurs as in Veronese et al. , Applied Biochemistry-Biotechnology, Vol. 11, pp. 141-152 (1985) .
- the glycol is activated by reaction with 2,4,5- trichlorophenyl-chloroformate or p-nitrophenylchloro- formate and triethylamine to yield a glycolphenylcar- bonate, which is then reacted with the diamino compound in high excess so that only one amino group of the diamino compound reacts with the activated glycol.
- the molar ratio of diamino compound to activated glycol is at least 2:1, and more preferably at least
- the reaction proceeds rapidly for a time prefer ⁇ ably from about 1 minute to 2 hours, more preferably from about 5 minutes to an hour.
- temperatures from 4°C to 100°C will be utilized, preferably between 10° and 60°, more preferably near ambient, i.e., room temperature.
- the glycol may be activated as in
- oxidation is accomplished by reaction of the macromole ⁇ cule with sodium periodate (NaI0 ) in a preferred molar ratio of sugar to NaIO of 1000:1 to 10:1.
- the concentra ⁇ tion of NaI0 4 is preferably 10-1000 mM.
- the reaction may preferably proceed at temperatures of 0°C to 50°C, and preferably for 1 minute to 4 hours, more preferably about 30 minutes to 2 hours, most preferably about 30 minutes. Adjustment of the reaction time may be made to control the amount of glycols per protein, since longer incuba ⁇ tion results in greater oxidation of the sugars and, accordingly, more points on the macromolecule available for glycol attachment.
- the macromolecule is coupled with the amino group-bearing glycol, by simple mixing, at temperatures preferably of about 4°C to 100°C, and for times preferably from about 1 minute to 5 hours, more typically between 3 minutes and 1 hour, and more preferably between 5 minutes and 30 minutes. Isolation, if desired, and work-up for biological applications is conventional, as disclosed in, e.g., U.S. Patent No. 4,179,337 or as described below.
- the macromolecules may be used as diagnostic reagents, therapeutic reagents, test samples, etc. , as known in the art and dependent on their disclosed biological utilities.
- the triethylammonium chloride is then filtered off using a sintered glass funnel. 200 ml of ethyl ether is added, and the solution is left to crystallize overnight at 4°C. The product is filtered, washed with ether to remove all of the yellow color, and recrystallized from acetonitrile-ether. The yield is 75%. The product is then assayed spectrophotometrically by the release of p-nitrophenol by e-amino-n-caproic acid (ACA) .
- ACA e-amino-n-caproic acid
- the purity of the product is further verified spectrophotometrically.
- TAb-250 5 mg is dialyzed extensively into 50 iruM sodium borate buffer pH 8.3. A lower pH is used in order to ensure that only the very reactive epsilon amino groups of lysine are PEGylated.
- To the 2 ml dialyzed sample 3 mg of the activated mPEG is added, a 5 molar excess. Every 30 minutes, 5 ⁇ l is removed and mixed with 5 ⁇ l of 25 iruM ACA. Immediate ⁇ ly, 3 mg of activated mPEG is added and incubated at room temperature with shaking for a further 30 minutes.
- the reaction is stopped after 2 hours; final molar excess is 20-fold, by loading the sample on a NAP 25 (Pharmacia) desalting column and eluting it with 50 i ⁇ M NaP0 4 buffer, pH 6.8.
- the desalted sample is loaded on Superose 6 column (1 x 30 cm BioRad Econocolumn ® ) and eluted with 50 mM NaPO ⁇ buffer, pH 6.8.
- Four resultant peaks from the Superose column and the 30-minute time point samples are assayed by SDS-PAGE.
- the Superose peaks are further assayed by Radial Immuno-diffusion (RID) from Tago Immuno, Inc. for the quantitation of mouse IgG lr more specifically a kappa light chain. See Table 1.
- RID Radial Immuno-diffusion
- Tago Standards IgG 5.5 0.16/mg/mL Tago Immuno, Inc. (Cone, on vial 0.156 mg/mt )
- Immunoglobulin G contains approximately 3% carbohydrate by weight linked to the F c region of the protein.
- mPEG- ⁇ -p-nitrophenyl 0.5 g is slowly added to 5 mL of 50 mM Na-borate buffer, pH 9.0, containing 44.25 mg (100 mmoles) of 1, 4-aminobutane.
- the reaction is incu ⁇ bated at room temperature with shaking for 3 hours.
- the reaction is stopped by passing it through an NAP 25 desalting column and eluted with water and dialyzed into milli-Q H 2 0.
- the dialyzed material is lypophylized and weighed.
- 1,4-diaminobutane is in high excess to guard against reacting both amino groups.
- Immunoglobulin G., (IgG.,) contains approximately 3% carbohydrate by weight linked to the F c region of the protein.
- Coupling Buffer 0.05 M sodium acetate
- TAb-250 0.5 mg is buffer exchanged into the coupling buffer using an NAP-10 (Pharmacia) desalting column.
- NAP-10 Pulacia
- the solution is mixed gently, and the sealed reaction vial is shielded from light and incubated at room temperature for 30 minutes.
- To stop the reaction the sample is passed through a NAP-10 desalting column and is equilibrated with wash buffer. The column is eluted with the conjugation buffer.
- oxidized TAb-250 is added 5 mg of PEG- ⁇ -butamine.
- the reaction vial is overlayed with nitrogen and is tumbled gently overnight at 4°C.
- the molar ratio of TAb-250 to PEG- ⁇ -butamine is 1:100.
- the sample is then loaded following optional reduction of the TAb-250 onto the same Superose 6 column.
- the IgG peaks are pooled and are concentrated on an amicon stirred cell concentrator.
Abstract
A process is provided for coupling glycols to macromolecules through glycosylations on those macromolecules, rather than through amino or carboxyl groups on the macromolecule backbone. This produces macromolecules having decreased immunogenic response, and maintained activity. The present process for glycolation of a glycosylated macromolecule comprises activating a polyalkylene glycol; reacting the activated polyalkylene glycol with a diamino compound, whereby the activated polyalkylene glycol is coupled to the diamino compound through one of its amino groups; oxidizing the macromolecule to activate at least one glycosyl group therein; and reacting the polyalkylene glycol coupled to the diamino compound with the oxidized glycosyl group in the macromolecule. The result is a glycolated glycosylated macromolecule, wherein a glycol is bonded to the macromolecule through its glycosylations.
Description
GLYCOLATION OF GLYCOSYLATED MACROMOLECULES
Background of the Invention
The use of polypeptides in circulatory systems for the purpose of engendering a particular physiological response is well known in the medicinal arts. Among the best known polypeptides utilized for this purpose is insulin, which is used in the treatment of diabetes. Another group of polypeptides to which great therapeutic potential has been attributed are various enzymes. A principal factor which has severely limited the use in therapeutics of polypeptides is that most of these com¬ pounds elicit an immunogenic response in body fluids, evidenced by changes in the composition of the circulatory system, i.e., the production of antibodies to the polypeptides. This effect has one or both of two secondary consequences: first, neutralization of the polypeptides by the antibodies thus produced; second, and more seriously, the development of an allergic response. Neutralization of polypeptides by antibodies is be- lieved to be responsible for the rather low residence time of insulin in the human circulatory system; hence, persons afflicted with diabetes are forced to inject themselves fairly frequently with fresh doses of insulin. In the case of parenterally administered enzymes, not only is there the problem of neutralization of the pclypep.tide and the subsequent negation of its physiological activity, but also the extremely undesirable elicitation of an allergic reaction.
Overall, a limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is
their potential for eliciting such immune response in the circulatory system. This immune response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), j. ciin. Endocr..31. 679-688, W. Moore (178), . ciin. Endrocπ'noi. Hetab., 51, 691-697. The antibody production may decrease or eliminate the desired bio¬ logical function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
The problems set forth hereinabove are well recog¬ nized, and various approaches have been taken in attempts to solve them. The attachment of enzymes to insoluble supports has been the subject of a great deal of work. Reviews dealing with this subject will be found in Silman and Katchalski, Ann. Rev. Biochem., 35, 387 (1966), and Goldstein, Fermentation Advances, Academic Press, New York (1969) , page 391. This approach, however, while of academic interest, does not provide injectable long-life polypeptides. Another approach which has been taken to provide polypeptides of lengthened in vivo life has been the microencapsulation of enzymes which has been discussed in numerous articles by Chang and coworkers, namely, science,
14 6 , 52 4 ( 1964 ) ; Trans. Am. Soc , 12 , 13 ( 1966 ) ; Nature , 2 18 , 2 4 3 (1968) ; Can. J. Physiol. Pharmacol., 45, 705 (1967) . A further approach has been the heat stabilization of enzymes by attaching carboxy ethylcellulose to an enzyme such as Trypsin (Mitz and Su maria, Nature, 198, 576 (1961), and the attachment of proteases to hydrophilic carriers (Brummer et al., Eur. J. Bioche . , 25, 129 (1972) . These approaches, however, do not provide polypeptides in a soluble form, which form is the most desirable for injection and dosage control of injectable materials. Yet a further approach has been the attachment of synthetic polymers to polypeptidal proteins. A review of this work is found in Sela, "Advances in Immunology," 5, 30 (1966), Academic Press,
New York. In this work, it has been shown that while homopolymers of amino acids are nearly all non-immuno- genic, when these polymers are attached to immunogenic proteins, the immunogenic activity is not masked, and antibodies are produced in test circulatory systems. For example, while polyglycine itself is non-immunogenic, when attached to a protein, that conjugated protein becomes a hapten. Similarly, while dextran itself is slightly immunogenic, when coupled to insulin, the insulin-dextran-coupled material is believed to become substantially immunogenic.
Other modifications of polypeptides include the modification of proteins with substantially straight- chain polymers such as polyethylene (PEG) or polypropylene glycol (PPG) .
For example, U.S. Patent No. 4,055,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides. U.S. Patent No. 4,088,538 discloses a reversibly soluble, enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
U.S. Patent No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group, and/or at least one aromatic hydroxy group (described in column 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in column 2, lines 42-66).
U.S. Patent No. 4,496,689 discloses a covalently attached complex of α-1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
Abuchowski et al., J. Biol. Chem. 252(11), p. 357k, disclose covalent attachment to an amino group of bovine serum albumin of methoxypolyethylene glycols.
U.S. Patent No. 3,619,371 discloses a polymeric matrix having a biologically active substance chemically bound thereto.
U.S. Patent No. 3,788,948 discloses use of organic cyanate compounds to bind proteins to polymers.
U.S. Patent No. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
JP 57-92435, published November 26, 1982, discloses modified polypeptides, where all or part of the amino groups are substituted with a polyethoxyl moiety. DE 2312615, published September 27, 1973, discloses conju¬ gating of polymers to compounds containing hydroxy or amino groups. EP 147,761 discloses a covalent conjugate of α-1- proteinase inhibitor and a water-soluble polymer, where the polymer may be polyethylene glycol.
EP 154,316, published September 11, 1985, discloses and claims chemically modified lymphokines, such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
U.S. Patent No. 4,414,147 describes rendering inter- feron less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethylene succinic anhydride) .
PCT WO 87/00056, published January 15, 1987, dis¬ closes conjugation of PEG and polyoxyethylated polyols to such proteins as interferon-3, interleukin-2, and immuno- toxins. Davis et al., U.S. Patent No. 4,179,337, provide peptides and polypeptides coupled to polymers which are substantially non-immunogenic. In the process of Davis et al., a substantially straight-chain polymer is modi¬ fied, suitably at one end thereof, either by the alte- ration of the terminal group or by the addition thereto of a coupling group having activity vis-a-vis polypeptide and reacting said activated polymer with the polypeptide.
Davis et al. indicate that the glycol couples most likely through an amino group on the protein, but also discloses an embodiment where the terminal hydroxy group of the glycol is converted to an amino group, e.g., with a sulfonating agent or a halogenating agent, and the resultant halide or tosylate is coupled with a carboxyl group of the polypeptide by known methods. Although the reduction of biological activity is less than where amino groups on the protein are the coupling sides, the activity is still reduced.
Summary of the Invention
It is an object of the present invention to provide a method for reducing the immunogenicity of biologically active macromolecules, while maintaining their activity. Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art.
These objects have been satisfied by providing a process for coupling glycols to macromolecules through glycosylations on those macromolecules, instead of through amino or carboxyl groups on the macromolecule backbone itself, and by providing macromolecules having unexpectedly decreased immunogenic response and surprisingly maintained activity. Molecules in accordance with the invention, in addition, exhibit increased biological half-life, due to steric blocking of clearance receptors; increased solubility of hydrophobic molecules in an aqueous environment, due to the addition of lipophilic moieties; and increased resistance to proteolysis, due to steric hindrance.
One aspect of the invention, therefore, is a process for the glycolation of a glycosylated macromolecule, comprising activating a polyalkylene glycol, reacting the activated polyalkylene glycol with a diamino compound, whereby the activated polyalkylene glycol is coupled to the diamino compound through one of its amino groups,
93/08196
oxidizing the macromolecule to activate at least one glycosyl group therein, and reacting the polyalkylene glycol coupled to the diamino compound with the oxidized glycosyl group in the macromolecule. Specifically, the invention preferably comprises a process for the
PEGylation of a glycosylated macromolecule comprising:
(a) reacting a polyethylene glycol of the formula CH30-(CH2CH20)n-H with o-nitrophenylchloroformate and triethylamine to produce a nitro compound of the formula CH30-(CH2CH20)n-COO-Ph-N02,
(b) reacting the nitro compound with a diamino- alkane of the formula H2N-(CH2)χ-NH2 to produce an amino compound of the formula CH30-(CH2CH20)n-CO-NH-(CH2)χ-NH2,
(c) oxidizing sugar groups on the macromolecule to produce a macromolecule with an oxidized sugar residue, and
(d) reacting the amino compound with the activated macromolecule to produce a PEGylated molecule. The preferred molecular weight of the polyethylene glycol is up to about 24,000; accordingly, n is preferably about 2-500. In the diaminoalkane, x is preferably about 1-20. The result of this preferred process is a PEGylated glycosylated macromolecule, wherein PEG is bonded to the macromolecule through its glycosylations, specifically, of the formula PEG-OCO-NH-alkylene-N=CH-macromolecule. Macromolecules usable in the invention include virtually any bioactive macromolecule bearing glyco¬ sylations (regardless of how bonded, e.g., covalently, etc.) or which can be glycosylated, e.g., polypeptides and/or proteins, nucleic acids, lipids, or carbohydrates. Preferred peptides include those comprising an antigen binding region, a cytokine, a receptor, an antithrom- botic, a growth factor, or an angiohypotensive reagent. More preferably, the polypeptide is an immunoglobulin, an interferon, a receptor tyrosine kinase, a thrombomodulin, a transforming growth factor, an endothelin, or an analog of the above. One especially preferred protein is the
monoclonal antibody TAb-250 or its chimeric analog BACh 250 ("BACh-250" or "C-erb-B2") . See Molecular Oncology as a Basis for New Strateties in Cancer Therapy: Efficacy of an Anti-c-erbB-2 Mouse/Human Chimeric Antibody Alone and in Combination with cis-
Diammedichloroplatinum (CDDP) , Langton et al., Proceedings of the 2nd Joint Meeting of the American Association for Cancer Research and the Japenese Cancer Association. The procedures of the present invention are applic¬ able to enzymes and peptide hormones. Examples of enzymes which can be used are: oxidoreductases, such as Urate:oxygen- oxidoreductase, Hydrogen-peroxide:hydrogen-peroxide oxidoreductase, Cholesterol-reduced-NADP:oxygen oxidoreductase (20-3-hydroxylating) ; transferases, such as UDP glucuronate glucuronyl-transferase (acceptor unspecific) , UDP glucose:α-D-Galactose-1-phosphate; hydrolases, such as Mucopeptide N-acetyl- muramyl-hydrolase, Trypsin, L-asparagine aminohydrolase; lyases, such as Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase; isomerases, such as D-Xylose ketol-isomerase; and ligases, such as L-Citrulline and L-aspartate ligase.
Examples of peptide hormones that can be used are insulin, ACTH, Glycagon, Somatostatin, Somatotropin, Thymosis, Parathyroid hormone, Pigmentary hormones,
Somatomedin, Erythropoietin, Luteinizing hormone, Chorionic Conadotropin, Hypothalmic-releasing factors, Antidiuretic hormones, Thyroid-stimulating hormone, and Prolactin. ' Macromolecules which have no sugars may be glyco¬ sylated by means which are well known in the art, e.g. , as disclosed in Creighton, proteins, W.H. Freeman & Co., New
York, 1983. Virtually any sugar which is reactive to oxidation is suitable. Examples include galactose, man- nose, glucose, N-acetylglucosamine, N-acetylgalactos- amine, sialic acids, fucose, and/or xylose. The length of the carbohydrate chains of the sugar may vary widely, i.e., poly- and oligosaccharides may be used. Normally, the glycosylation will be that which is indigenous to the species producing the macromolecule, e.g. , mammalian.
The glycol is preferably polyethylene glycol, poly- propylene glycol, or a mixture thereof, as well as a mixed polyethylene-polypropylene glycol. Polyethylene glycols (PEG's) are most preferred, especially ono- methoxyethylene glycol. Preferred polyethylene glycols have the formula CH30-(CH2CH20)n-H, wherein n is 2-500, more preferably 20-400.
The diamino compound preferably has the formula H2N- R-NH2, wherein R is an organic moiety. R may be, for example, a C^^-aliphatic or C3.20-cycloaliphatic moiety or a C5.20-aryl moiety. Aliphatic moieties include straight- or branched-chain or cyclic alkyl, alkenyl, dienyl, and alkynyl groups. Preferred aliphatic moieties are C2.12- alkyl groups. Preferred aryl moieties are heterocyclic, i.e., containing one or more O, S, or N atoms, and aromatic, e.g., phenyl groups. Activation of the glycol with the addition of the diamino compound preferably occurs as in Veronese et al. , Applied Biochemistry-Biotechnology, Vol. 11, pp. 141-152 (1985) . The glycol is activated by reaction with 2,4,5- trichlorophenyl-chloroformate or p-nitrophenylchloro- formate and triethylamine to yield a glycolphenylcar- bonate, which is then reacted with the diamino compound in high excess so that only one amino group of the diamino compound reacts with the activated glycol. Pre¬ ferably, the molar ratio of diamino compound to activated glycol is at least 2:1, and more preferably at least
10:1. The reaction proceeds rapidly for a time prefer¬ ably from about 1 minute to 2 hours, more preferably from
about 5 minutes to an hour. Typically temperatures from 4°C to 100°C will be utilized, preferably between 10° and 60°, more preferably near ambient, i.e., room temperature. Alternatively, the glycol may be activated as in
Davis et al., U.S. Patent No. 4,179,337, by reacting the glycol at its terminal hydroxyl group either with a sulfonating agent, such as toluene chloride, or with a halogenating agent, such as triphenyl phosphine in carbon tetrachloride or triphenyl phosphine with a suitable N- halosuccimide. The thus-produced halide or tosylate is then treated with sodium azide and reduced with lithium aluminum hydride to give the corresponding terminal amino compound. The activated, amino group-containing glycol is then reacted with the macromolecule, on which a carbohydrate, i.e., a sugar moiety, has been oxidized. Preferably, oxidation is accomplished by reaction of the macromole¬ cule with sodium periodate (NaI0 ) in a preferred molar ratio of sugar to NaIO of 1000:1 to 10:1. The concentra¬ tion of NaI04 is preferably 10-1000 mM. The reaction may preferably proceed at temperatures of 0°C to 50°C, and preferably for 1 minute to 4 hours, more preferably about 30 minutes to 2 hours, most preferably about 30 minutes. Adjustment of the reaction time may be made to control the amount of glycols per protein, since longer incuba¬ tion results in greater oxidation of the sugars and, accordingly, more points on the macromolecule available for glycol attachment. Care should be taken to avoid over-glycolation, resulting in stearic hinderance of the macromolecule, reducing efficient reaction with its in¬ tended target. One of ordinary skill in the art could easily, with only routine experimentation, optimize this portion of the process for an intended use of the macromolecule.
Following oxidation of the sugars, the macromolecule is coupled with the amino group-bearing glycol, by simple
mixing, at temperatures preferably of about 4°C to 100°C, and for times preferably from about 1 minute to 5 hours, more typically between 3 minutes and 1 hour, and more preferably between 5 minutes and 30 minutes. Isolation, if desired, and work-up for biological applications is conventional, as disclosed in, e.g., U.S. Patent No. 4,179,337 or as described below. The macromolecules may be used as diagnostic reagents, therapeutic reagents, test samples, etc. , as known in the art and dependent on their disclosed biological utilities.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the disclosure in any way whatsoever.
In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
The entire disclosure of all applications, patents and publications, cited above and below, are hereby incorporated by reference.
11 EXAMPLES
1. Activation of Methoxy-PEG (mPEG)
Two grams of mPEG (0.1 mM, final concentration), 15 Kd, is dissolved in 20 ml of acetonitrile with 0.24 g of o-nitrophenylchloroformate (1.2 mM) and 33 μl of triethylamine (1.2 mM) and stirred for 24 hours at room temperature.
Chemical Reaction
P. NO - 2
II
CH ;0-(CH2CH20)r-H + CI-C-0- (CH3CH2)3N
The triethylammonium chloride is then filtered off using a sintered glass funnel. 200 ml of ethyl ether is added, and the solution is left to crystallize overnight at 4°C. The product is filtered, washed with ether to remove all of the yellow color, and recrystallized from acetonitrile-ether. The yield is 75%. The product is then assayed spectrophotometrically by the release of p-nitrophenol by e-amino-n-caproic acid (ACA) .
The purity of the product is further verified spectrophotometrically.
2. PEGylation of TAb-250 Through Lysine Groups Protocol
5 mg of TAb-250 (0.0316 μmoles) is dialyzed extensively into 50 iruM sodium borate buffer pH 8.3. A lower pH is used in order to ensure that only the very reactive epsilon amino groups of lysine are PEGylated.
To the 2 ml dialyzed sample 3 mg of the activated mPEG is added, a 5 molar excess. Every 30 minutes, 5 μl is removed and mixed with 5 μl of 25 iruM ACA. Immediate¬ ly, 3 mg of activated mPEG is added and incubated at room temperature with shaking for a further 30 minutes. The reaction is stopped after 2 hours; final molar excess is 20-fold, by loading the sample on a NAP 25 (Pharmacia) desalting column and eluting it with 50 iαM NaP04 buffer, pH 6.8. The desalted sample is loaded on Superose 6 column (1 x 30 cm BioRad Econocolumn®) and eluted with 50 mM NaPO^ buffer, pH 6.8. Four resultant peaks from the Superose column and the 30-minute time point samples are assayed by SDS-PAGE. The Superose peaks are further assayed by Radial Immuno-diffusion (RID) from Tago Immuno, Inc. for the quantitation of mouse IgGlr more specifically a kappa light chain. See Table 1.
Half of the pooled superose b sample is PEGylated as described above; however, this time, activated PEG was in lOOx molar excess and incubated for 3 hours. Half of this sample is allowed to incubate overnight before being stopped.
All samples were processed and assayed as reported above.
Chemistry:
0
Est. Binding
Sample Diameter μg/mL Efficiency %
PEG-μ-butamine 11.6 Out of range 100 TAb 250
PEG-μ-TAb 250 5.6 25 16.7 lOOx excess
TAb 250 in 6.8 150 100 borate pH 8 . 3
PEG-μ-TAb 250 7.7 129 100 2 Ox (peaks 1+2 )
Oxidized TAb 250 5.3 51.3 100
Tago Standards IgG 5.5 0.16/mg/mL Tago Immuno, Inc. (Cone, on vial = 0.156 mg/mt )
3. PEGylation of TAb-250 Through the Carbohydrate Moieties
Immunoglobulin G (IgG,) contains approximately 3% carbohydrate by weight linked to the Fc region of the protein.
Protocols
(a) Making the amino derivative of mPEG-μ-pNP
0.5 g of mPEG-μ-p-nitrophenyl is slowly added to 5 mL of 50 mM Na-borate buffer, pH 9.0, containing 44.25 mg (100 mmoles) of 1, 4-aminobutane. The reaction is incu¬ bated at room temperature with shaking for 3 hours. The reaction is stopped by passing it through an NAP 25 desalting column and eluted with water and dialyzed into milli-Q H20.
The dialyzed material is lypophylized and weighed.
Total yield of PEG-μ-butamine = 0.573 g.
Chemistry
NO.
PEG-C- (6 + H2N-( CH2)4-NH:
0
II PEG-C-NH-( CH2 ) 4 -NH2
( P EG-/z-bu lorni ne )
During the reaction, 1,4-diaminobutane is in high excess to guard against reacting both amino groups.
(b) Oxidation of TAb-250
Immunoglobulin G., (IgG.,) contains approximately 3% carbohydrate by weight linked to the Fc region of the protein.
Coupling Buffer: 0.05 M sodium acetate
0.1 M sodium chloride, pH 5.0 Wash Buffer: 0.1 M sodium acetate
0.5 M sodium chloride, pH 3.5 Storage buffer: 0.05 M sodium phosphate, pH 6.8
0.5 mg of TAb-250 is buffer exchanged into the coupling buffer using an NAP-10 (Pharmacia) desalting column. To the TAb-250 solution is added 0.1 L of freshly prepared 100 mM sodium m-periodate (NaI0 ) . The solution is mixed gently, and the sealed reaction vial is shielded from light and incubated at room temperature for 30 minutes. To stop the reaction, the sample is passed through a NAP-10 desalting column and is equilibrated with wash buffer. The column is eluted with the conjugation buffer.
(c) Coupling of oxidized TAb-250 to ,PEG-μ-butamine
To the desalted, oxidized TAb-250 is added 5 mg of PEG-μ-butamine. The reaction vial is overlayed with nitrogen and is tumbled gently overnight at 4°C. The
molar ratio of TAb-250 to PEG-μ-butamine is 1:100. The sample is then loaded following optional reduction of the TAb-250 onto the same Superose 6 column. The IgG peaks are pooled and are concentrated on an amicon stirred cell concentrator.
All the pegylated TAb 250 species were further analyzed by SDS-PAGE so as to estimate their molecular weights.
Discussion From the SDS-PAGE gel analysis, it can be seen that the experiments have generated species of a higher molecular weight than TAb-250. Table 1 demonstrates that lOOx molar excess sample has lost most of its binding capacity, as expected. It would indicate that all of the lysine binding sites are saturated, thus hindering binding. The 2Ox molar PEG excess and the carbohydrate conjugated samples all appear to retain all of their binding capacity.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Claims
1. A glycolated, glycosylated macromolecule, wherein a glycol is bonded to the macromolecule through a glycosylation moiety.
2. A glycolated, glycosylated macromolecule according to claim 1, wherein the glycol is a polyalkylene glycol.
3. A glycolated, glycosylated macromolecule according to claim 2, wherein the glycol is a polyethylene glycol.
4. A glycolated, glycosylated macromolecule according to claim 1 of the formula: glycol-diamine-macromolecule, wherein the diamine is bonded to the macromolecule through a carbohydrate moiety thereof, thereby forming a Schiff base linkage.
5. A glycolated, glycosylated macromolecule according to claim 1 of the formula: glycol-OCO-NH-alkylene-N=CH-macromolecule, wherein "alkylene" has 1 to 20 carbon atoms.
6. A glycolated, glycosylated macromolecule according to claim 1, which is a pharmacologically active
I compound.
7. A glycolated, glycosylated macromolecule according to claim 1, wherein said macromolecule comprises a nucleic acid, a polypeptide, or a lipid.
8. A glycolated, glycosylated macromolecule according to claim 7, wherein the polypeptide is a protein.
9. A glycolated, glycosylated macromolecule according to claim 8, wherein the protein is TAb-250 or BACh-250.
10. A glycolated, glycosylated macromolecule according to claim 1, having essentially undiminished bioactive half-life in a host and a reduced immunogenic side effect, an increased aqueous solubility, an increased resistance to proteolytic digestion, or a decreased affinity for formulation polymers, as compared to said glycosylated macromolecule which is not glycolated.
11. A glycolated, glycosylated macromolecule according to claim 1, having increased bioactive half- life compared to a glycosylated macromolecule which is not glycolated.
12. A glycolated, glycosylated macromolecule according to claim 1, having essentially undiminished bioactive half-life in a host, as compared to said glycosylated macromolecule which is glycolated through amino or carboxyl groups not on the glycosyl portion of the macromolecule.
13.' A glycolated, glycosylated macromolecule according to claim 7, wherein said macromolecule is a polypeptide which comprises an antigen binding region.
14. A glycolated, glycosylated macromolecule according to claim 7, wherein said macromolecule is a polypeptide which is a cytokine, a receptor, an anti- thrombotic, a growth factor, or an angiohypotensive reagent.
15. A glycolated, glycosylated macromolecule according to claim 7, wherein said polypeptide is an immunoglobulin, an interferon, a receptor tyrosine kinase, a thrombomodulin, a transforming growth factor, or an endothelin.
16. A process for the reduction of immunogenic side effect, for increasing aqueous solubility, for increasing resistance to proteolytic digestion, or for increasing the half-life of a bioactive glycosylated macromolecule upon administration to a host, comprising glycolating said macromolecule by bonding glycol to a carbohydrate moiety thereof.
17. A process according to claim 16, wherein the glycol is bonded to the macromolecule by a Schiff base linkage.
18. A process according to claim 16, wherein the macromolecule is a polypeptide.
19. A process according to claim 18, wherein the polypeptide is a protein.
20. A process according to claim 19, wherein the protein is TAb-250 or BACh-250.
21. A process for the glycolation of a glycosylated macromolecule, comprising activating a polyalkylene glycol, reacting the activated polyalkylene glycol with a diamino compound whereby the activated polyalkylene glycol is coupled to the diamino compound through one of its amino groups, oxidizing the macromolecule to activate at least one glycosyl group therein, and reacting the polyalkylene glycol coupled to the diamino compound with the oxidized glycosyl group in the macromolecule.
22. A method of claim 21, wherein said activating of polyalkylene glycol produces a glycol-nitrophenyl carbonate intermediate.
23. A process according to claim 21, wherein said diamino compound has the formula H2N-R-NH2, wherein R is an organic moiety.
24. A process according to claim 23, wherein R is an aliphatic hydrocarbon having from 2 to 20 carbon atoms or an aromatic hydrocarbon having from 5 to 20 carbon atoms.
25. A process according to claim 24, wherein R is an aliphatic hydrocarbon having from 5-12 carbon atoms.
26. A process according to claim 21 for the PEGylation of a glycosylated macromolecule comprising:
(a) reacting a polyethylene glycol of the formula
CH30-(CH2CH20)n-H with o-nitrophenylchloroformate and triethylamine to produce a nitro compound of the formula
CH30-(CH2CH20)n-C00-Ph-N02,
(b) reacting the nitro compound with a diaminoalkane of the formula
H2N-(CH2)χ-NH2 to produce an amino compoui'id of the formula CH30-(CH2CH20)n-C0-NH-(CH2)X~NH2, (c) oxidizing sugar groups on the macromolecule to produce a macromolecule with an oxidized sugar residue, and
(d) reacting the amino compound with the macromolecule to produce a PEGylated molecule, wherein n is 2-500 and x is 1-20.
27. A process according to claim 26, wherein n is 20-400 and x is 2-12.
28. A process according to claim 21, wherein the macromolecule is a nucleic acid, a polypeptide, or a lipid.
29. A process according to claim 21, wherein the macromolecule is a pharmacologically active compound.
30. A glycolated, glycosylated macromolecule produced by a process of claim 16.
31. A glycolated, glycosylated macromolecule produced by a process of claim 21.
32. A PEGylated, glycosylated macromolecule produced by a process of claim 26.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU50981/93A AU5098193A (en) | 1992-09-01 | 1993-09-01 | Glycolation of glycosylated macromolecules |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US93777992A | 1992-09-01 | 1992-09-01 | |
US07/937,779 | 1992-09-01 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1994005332A2 true WO1994005332A2 (en) | 1994-03-17 |
WO1994005332A3 WO1994005332A3 (en) | 1994-04-14 |
Family
ID=25470393
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/008196 WO1994005332A2 (en) | 1992-09-01 | 1993-09-01 | Glycolation of glycosylated macromolecules |
Country Status (2)
Country | Link |
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AU (1) | AU5098193A (en) |
WO (1) | WO1994005332A2 (en) |
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Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989006546A1 (en) * | 1988-01-20 | 1989-07-27 | Cetus Corporation | Conjugation of polymer to colony stimulating factor-1 |
EP0442724A2 (en) * | 1990-02-13 | 1991-08-21 | Kirin-Amgen, Inc. | Modified hIL-6 |
WO1993012220A1 (en) * | 1991-12-12 | 1993-06-24 | Berlex Laboratories, Inc. | RECOMBINANT AND CHIMERIC ANTIBODIES TO c-erbB-2 |
-
1993
- 1993-09-01 WO PCT/US1993/008196 patent/WO1994005332A2/en active Application Filing
- 1993-09-01 AU AU50981/93A patent/AU5098193A/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989006546A1 (en) * | 1988-01-20 | 1989-07-27 | Cetus Corporation | Conjugation of polymer to colony stimulating factor-1 |
EP0442724A2 (en) * | 1990-02-13 | 1991-08-21 | Kirin-Amgen, Inc. | Modified hIL-6 |
WO1993012220A1 (en) * | 1991-12-12 | 1993-06-24 | Berlex Laboratories, Inc. | RECOMBINANT AND CHIMERIC ANTIBODIES TO c-erbB-2 |
Non-Patent Citations (1)
Title |
---|
CHEMICAL ABSTRACTS, vol. 103, no. 11, 16 September 1985, Columbus, Ohio, US; abstract no. 84021, cited in the application & APPL. BIOCHEM. BIOTECHNOL., vol.11, no.2, 1985 pages 141 - 152 VERONESE F. ET AL. 'SURFACE MOFIFICATION OF PROTEINS. ACTIVATION OF MONOMETHOXY-POLYETHYLENE GLYCOLS BY PHENYLCHLOROFORMATES AND MODIFICATION OF RIBONUCLEASE AND SUPEROXIDE DISMUTASE.' * |
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US7214660B2 (en) | 2001-10-10 | 2007-05-08 | Neose Technologies, Inc. | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US7226903B2 (en) | 2001-10-10 | 2007-06-05 | Neose Technologies, Inc. | Interferon beta: remodeling and glycoconjugation of interferon beta |
US7265084B2 (en) | 2001-10-10 | 2007-09-04 | Neose Technologies, Inc. | Glycopegylation methods and proteins/peptides produced by the methods |
US7265085B2 (en) | 2001-10-10 | 2007-09-04 | Neose Technologies, Inc. | Glycoconjugation methods and proteins/peptides produced by the methods |
US7276475B2 (en) | 2001-10-10 | 2007-10-02 | Neose Technologies, Inc. | Remodeling and glycoconjugation of peptides |
US7297511B2 (en) | 2001-10-10 | 2007-11-20 | Neose Technologies, Inc. | Interferon alpha: remodeling and glycoconjugation of interferon alpha |
EP2322229A2 (en) | 2001-10-10 | 2011-05-18 | Novo Nordisk A/S | Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF) |
EP2305314A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of antibodies |
US7416858B2 (en) | 2001-10-10 | 2008-08-26 | Neose Technologies, Inc. | Pharmaceutical compositions of glycoconjugates |
US7439043B2 (en) | 2001-10-10 | 2008-10-21 | Neose Technologies, Inc. | Galactosyl nucleotide sugars |
US7138371B2 (en) | 2001-10-10 | 2006-11-21 | Neose Technologies, Inc | Remodeling and glycoconjugation of peptides |
EP2305312A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of follicle-stimulating hormone (FSH) |
EP2042196A2 (en) | 2001-10-10 | 2009-04-01 | Neose Technologies, Inc. | Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF) |
EP2305311A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Glycoconjugation of peptides |
EP1578771A2 (en) * | 2001-10-10 | 2005-09-28 | Neose Technologies, Inc. | Remodeling and glycoconjugation of peptides |
EP2305313A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of interferon-alpha (IFNa) |
JP2009108087A (en) * | 2001-10-10 | 2009-05-21 | Neose Technologies Inc | Remodeling and glycoconjugation of peptide |
EP2298354A2 (en) | 2001-10-10 | 2011-03-23 | BioGeneriX AG | Remodelling and glycoconjugation of interferon-beta |
EP1578771A4 (en) * | 2001-10-10 | 2007-02-21 | Neose Technologies Inc | Remodeling and glycoconjugation of peptides |
JP2016037503A (en) * | 2001-10-10 | 2016-03-22 | ノボ ノルデイスク エイ エス | Remodeling and glycoconjugation of peptides |
EP2279754A2 (en) | 2001-10-10 | 2011-02-02 | BioGeneriX AG | Remodelling and glycoconjugation of human growth hormone (hGH) |
EP2279753A3 (en) * | 2001-10-10 | 2011-10-12 | Novo Nordisk A/S | Remodeling and glycoconjugation of peptides |
US7696163B2 (en) | 2001-10-10 | 2010-04-13 | Novo Nordisk A/S | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
JP2005521635A (en) * | 2001-10-10 | 2005-07-21 | ネオス・テクノロジーズ・インコーポレーテツド | Peptide remodeling and glycoconjugation |
EP2279755A2 (en) | 2001-10-10 | 2011-02-02 | BioGeneriX AG | Remodelling and glycoconjugation of Fibroblast Growth Factor (FGF) |
US7473680B2 (en) | 2001-11-28 | 2009-01-06 | Neose Technologies, Inc. | Remodeling and glycoconjugation of peptides |
US7157277B2 (en) | 2001-11-28 | 2007-01-02 | Neose Technologies, Inc. | Factor VIII remodeling and glycoconjugation of Factor VIII |
US7541328B2 (en) | 2002-03-06 | 2009-06-02 | Fresenius Kabi Deutschland Gmbh | Coupling proteins to a modified polysaccharide |
US8618266B2 (en) * | 2002-09-11 | 2013-12-31 | Fresenius Kabi Deutschland Gmbh | Hasylated polypeptides |
JP2006522738A (en) * | 2002-10-09 | 2006-10-05 | ネオス テクノロジーズ インコーポレイテッド | Erythropoietin: Remodeling and glycoconjugation of erythropoietin |
US7204980B2 (en) | 2002-11-18 | 2007-04-17 | Phoenix Pharmacologics, Inc. | Methods for inhibiting viral replication in vivo |
US7803777B2 (en) | 2003-03-14 | 2010-09-28 | Biogenerix Ag | Branched water-soluble polymers and their conjugates |
US8247381B2 (en) | 2003-03-14 | 2012-08-21 | Biogenerix Ag | Branched water-soluble polymers and their conjugates |
EP2055189A1 (en) | 2003-04-09 | 2009-05-06 | Neose Technologies, Inc. | Glycopegylation methods and proteins/peptides produced by the methods |
US8853161B2 (en) | 2003-04-09 | 2014-10-07 | Novo Nordisk A/S | Glycopegylation methods and proteins/peptides produced by the methods |
US7691603B2 (en) | 2003-04-09 | 2010-04-06 | Novo Nordisk A/S | Intracellular formation of peptide conjugates |
EP2338333A2 (en) | 2003-04-09 | 2011-06-29 | BioGeneriX AG | Glycopegylation methods and proteins/peptides produced by the methods |
US8791070B2 (en) | 2003-04-09 | 2014-07-29 | Novo Nordisk A/S | Glycopegylated factor IX |
US8063015B2 (en) | 2003-04-09 | 2011-11-22 | Novo Nordisk A/S | Glycopegylation methods and proteins/peptides produced by the methods |
US9005625B2 (en) | 2003-07-25 | 2015-04-14 | Novo Nordisk A/S | Antibody toxin conjugates |
US8916360B2 (en) | 2003-11-24 | 2014-12-23 | Novo Nordisk A/S | Glycopegylated erythropoietin |
US7405198B2 (en) | 2003-11-24 | 2008-07-29 | Neose Technologies, Inc. | Glycopegylated erythropoietin |
WO2005056760A2 (en) * | 2003-12-03 | 2005-06-23 | Neose Technologies, Inc. | Glycopegylated follicle stimulating hormone |
WO2005056760A3 (en) * | 2003-12-03 | 2005-08-18 | Neose Technologies Inc | Glycopegylated follicle stimulating hormone |
US8361961B2 (en) | 2004-01-08 | 2013-01-29 | Biogenerix Ag | O-linked glycosylation of peptides |
US9023992B2 (en) | 2004-05-04 | 2015-05-05 | Novo Nordisk Healthcare Ag | Hydrophobic interaction chromatography purification of factor VII polypeptides |
US10844110B2 (en) | 2004-05-04 | 2020-11-24 | Novo Nordisk Healthcare Ag | O-linked glycoforms of polypeptides and method to manufacture them |
US8791066B2 (en) | 2004-07-13 | 2014-07-29 | Novo Nordisk A/S | Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1] |
US8268967B2 (en) | 2004-09-10 | 2012-09-18 | Novo Nordisk A/S | Glycopegylated interferon α |
US9200049B2 (en) | 2004-10-29 | 2015-12-01 | Novo Nordisk A/S | Remodeling and glycopegylation of fibroblast growth factor (FGF) |
US10874714B2 (en) | 2004-10-29 | 2020-12-29 | 89Bio Ltd. | Method of treating fibroblast growth factor 21 (FGF-21) deficiency |
US9029331B2 (en) | 2005-01-10 | 2015-05-12 | Novo Nordisk A/S | Glycopegylated granulocyte colony stimulating factor |
EP2514757A2 (en) | 2005-01-10 | 2012-10-24 | BioGeneriX AG | Glycopegylated granulocyte colony stimulating factor |
EP2386571A2 (en) | 2005-04-08 | 2011-11-16 | BioGeneriX AG | Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants |
EP2975135A1 (en) | 2005-05-25 | 2016-01-20 | Novo Nordisk A/S | Glycopegylated factor IX |
US8911967B2 (en) | 2005-08-19 | 2014-12-16 | Novo Nordisk A/S | One pot desialylation and glycopegylation of therapeutic peptides |
US8841439B2 (en) | 2005-11-03 | 2014-09-23 | Novo Nordisk A/S | Nucleotide sugar purification using membranes |
US7645860B2 (en) | 2006-03-31 | 2010-01-12 | Baxter Healthcare S.A. | Factor VIII polymer conjugates |
US8071728B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US7985839B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071726B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8003760B2 (en) | 2006-03-31 | 2011-08-23 | Baxter International Inc. | Factor VIII polymer conjugates |
US8067543B2 (en) | 2006-03-31 | 2011-11-29 | Baxter International Inc. | Factor VIII polymer conjugates |
US7982010B2 (en) | 2006-03-31 | 2011-07-19 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071724B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071727B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8053561B2 (en) | 2006-03-31 | 2011-11-08 | Baxter International Inc. | Pegylated factor VIII |
US8071725B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US7985838B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US11020458B2 (en) | 2006-03-31 | 2021-06-01 | Takeda Pharmaceutical Company Limited | Factor VIII polymer conjugates |
US8637007B2 (en) | 2006-12-15 | 2014-01-28 | Baxter International Inc. | Factor VIIa-polysialic acid conjugate having prolonged in vivo half-life |
US9050304B2 (en) | 2007-04-03 | 2015-06-09 | Ratiopharm Gmbh | Methods of treatment using glycopegylated G-CSF |
US9493499B2 (en) | 2007-06-12 | 2016-11-15 | Novo Nordisk A/S | Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography |
DE202008017456U1 (en) | 2007-08-27 | 2009-08-27 | Biogenerix Ag | Liquid formulation of G-CSF conjugates |
EP2578235A2 (en) | 2007-08-27 | 2013-04-10 | BioGeneriX AG | Liquid formulation of G-CSF conjugate |
US8546328B2 (en) | 2007-08-27 | 2013-10-01 | Biogenerix Ag | Liquid formulation of G-CSF conjugate |
EP2933264A2 (en) | 2008-01-22 | 2015-10-21 | Araim Pharmaceuticals, Inc. | Tissue protective peptides and peptide analogs for preventing and treating diseases and disorders associated with tissue damage |
US9150848B2 (en) | 2008-02-27 | 2015-10-06 | Novo Nordisk A/S | Conjugated factor VIII molecules |
EP2672260A1 (en) | 2008-05-13 | 2013-12-11 | Advanced Liquid Logic, Inc. | Droplet actuator devices, systems and methods |
EP2672259A1 (en) | 2008-05-13 | 2013-12-11 | Advanced Liquid Logic, Inc. | Droplet actuator devices, systems and methods |
US9795683B2 (en) | 2009-07-27 | 2017-10-24 | Lipoxen Technologies Limited | Glycopolysialylation of non-blood coagulation proteins |
US9492555B2 (en) | 2009-07-27 | 2016-11-15 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US8809501B2 (en) | 2009-07-27 | 2014-08-19 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
US9731024B2 (en) | 2009-07-27 | 2017-08-15 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US11040109B2 (en) | 2009-07-27 | 2021-06-22 | Takeda Pharmaceutical Company Limited | Blood coagulation protein conjugates |
US10350301B2 (en) | 2009-07-27 | 2019-07-16 | Baxalta Incorporated | Blood coagulation protein conjugates |
US10414793B2 (en) | 2009-07-27 | 2019-09-17 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US10576160B2 (en) | 2009-07-27 | 2020-03-03 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US10772968B2 (en) | 2009-07-27 | 2020-09-15 | Lipoxen Technologies Limited | Glycopolysialylation of non-blood coagulation proteins |
US11564992B2 (en) | 2009-07-27 | 2023-01-31 | Takeda Pharmaceutical Company Limited | Nucleophilic catalysts for oxime linkage |
US8637640B2 (en) | 2009-07-27 | 2014-01-28 | Baxter International Inc. | Blood coagulation protein conjugates |
US8642737B2 (en) | 2010-07-26 | 2014-02-04 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
US8945897B2 (en) | 2010-07-26 | 2015-02-03 | Baxter International Inc. | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
WO2013156488A2 (en) | 2012-04-16 | 2013-10-24 | Leverton Licence Holdings Limited | Optimised subcutaneous therapeutic agents |
US11351112B2 (en) | 2012-04-16 | 2022-06-07 | Cantab Biopharmaceuticals Patents Limited | Optimised subcutaneous therapeutic agents |
US10864253B2 (en) | 2016-04-29 | 2020-12-15 | Araim Pharmaceuticals, Inc. | Tissue protective peptides for preventing and treating diseases and disorders associated with tissue damage |
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