EP0576589A4 - Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers - Google Patents
Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymersInfo
- Publication number
- EP0576589A4 EP0576589A4 EP19920909326 EP92909326A EP0576589A4 EP 0576589 A4 EP0576589 A4 EP 0576589A4 EP 19920909326 EP19920909326 EP 19920909326 EP 92909326 A EP92909326 A EP 92909326A EP 0576589 A4 EP0576589 A4 EP 0576589A4
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- EP
- European Patent Office
- Prior art keywords
- macromolecular conjugate
- group
- glycopolypeptide
- polymer
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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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
- the present invention relates to biologicall active macromolecular conjugates, in particular, t conjugates of biologically active polypeptides an glycopolypeptides with water-soluble polymers.
- polypeptides wit water-soluble polymers such as polyethylene glycol (PEG)
- PEG polyethylene glycol
- the coupling of peptides an polypeptides to PEG and similar water-soluble polymers is disclosed by U.S. Patent No. 4,179,337 to Davis et al.
- Davis et al. discloses that physiologically active polypeptides modified with PEG exhibit dramatically reduced immunogenicity and antigenicity.
- the PEG-protein conjugates when injected into a living organism, have been shown to remain in the bloodstream considerably longer than the corresponding native proteins. Accordingly, a number of PEG-conjugated therapeutic proteins were developed exhibiting reduced immunogenicity and antigenicity and longer clearance times, while retaining a substantial portion of the protein's physiological activity.
- covalent attachment of the polymer is effected by reacting PEG-succinimide derivatives with amino groups on the exterior of protein molecules.
- the amino groups of many proteins are moieties responsible for polypeptide activity that can be readily inactivated as a result of such modification.
- the conjugation of such proteins is not desirable, because it results in the reduction of physiological activity.
- Other proteins may have only a small number of available amino groups, and consequently very few polymer anchoring sites. As a result, many proteins of interest cannot be conjugated with PEG in this manner.
- U.S. Patent No. 4,179,337 discloses the reaction of an amino-PEG derivative with l-ethyl-3-(3-dimethylamino-propyl) carbodiimide(EDC)- activated carboxylic acid groups of trypsin and other proteins.
- the selectivity of this reaction is rather poor because the reactivity of amino-PEG is similar to that of the lysyl residues of proteins, with both the amino-PEG and protein amino groups competing to react with the activated carboxylic acid groups. This results in intermolecular as well as intramolecular crosslinking and a loss of protein activity.
- Mater., 17, 208-9 (1990) also disclose the use of a norleucine spacer in PEG-succinimide derivatives covalently bonded to protein amino groups, noting that the use of such an unnatural amino acid helps in the characterization of the adduct because a single amino acid analysis would give both protein concentration and number of polymer chains bound to the amino groups.
- each single norleucine residue acid represents a polymer chain bound to an exterior amino grou .
- water-soluble polymers can be conjugated with biologically active polypeptides and glycopolypeptides utilizing acyl hydrazine derivatives of the water-soluble polymers.
- the acyl hydrazine derivatives of the water-soluble polymers covalently link to either the oxidized carbohydrate residues of the glycopolypeptides or the reactive carbonyl or activated carboxylic acid groups o peptide moieties of polypeptides or glycopolypeptides
- This invention extends the realm of water-solubl polymer-peptide conjugation to those polypeptide an glycopolypeptide materials that could not have bee modified heretofore by conventional methods
- pK a about 3 acyl hydrazine containing polymers of this inventio possess higher reactivity than the amino groups o polypeptides (pK a about 10.5), therefore minimizing an in most cases eliminating the competing reactions o these
- biologically active macromolecular conjugate is provide of a biologically active polypeptide or glycopolypeptid and one or more water-soluble polymer molecule covalently bonded thereto at a reactive carbonyl o carboxylic acid group of a peptide moiety on th polypeptide or glycopolypeptide by a linkage containin a hydrazide or hydrazone functional group.
- the linkag is formed by reacting an acyl hydrazine derivative o the water-soluble polymer with a polypeptide o glycopolypeptide having an activated carboxylic aci group or a reactive carbonyl group generated thereon.
- the present invention also provides biologically active macromolecular conjugate of biologically active glycopolypeptide and one or mor water-soluble polymer molecules covalently bonde thereto at an oxidized carbohydrate moiety of th glycopolypeptide by a linkage containing a hydrazide o hydrazone functional group bound to the polymer via short peptide sequence.
- the oxidation of th carbohydrate moiety produces reactive aldehydes.
- Th hydrazone linkage is formed by reacting an acy hydrazine derivative of the water-soluble polyme containing the peptide sequence with these aldehyde groups.
- the hydrazone can be further stabilized by reduction to a very stable alkyl hydrazine derivative.
- the peptide sequence influences the lability of the linkage to proteolytic enzymes and also allows convenient characterization of the polymer conjugates by amino acid analysis of their hydrolysates. By using state-of-the-art techniques of amino acid analysis, the quantity of peptide sequences, and consequently the degree of conjugation, can be determined for picomolar concentrations of the conjugate.
- the peptide sequences also be utilized with the polypeptide conjugates of the present invention to bind the linkages containing a hydrazide or hydrazone functional group to the water-soluble polymer.
- FIG. 1 is a GF-HPLC chromatogram comparison of mPEG-beta-alanine-bovine serum albumin conjugate to native bovine serum albumin.
- FIG. 2 is a GF-HPLC chro atogram comparison of mPEG-beta-alanine-ovalbumin conjugate to native ovalbumin.
- FIG. 3 is a GF-HPLC chromatogram comparison of PEG-beta-alanine-IgG, conjugated via oxidized carbohydrate moieties, to native IgG.
- FIG. 4 is a GF-HPLC chromatogram comparison of PEG-beta-alanine-rhG-CSF, conjugated via carboxylic acid groups of rhG-CSF, to native rhG-CSF. Best Mode of Carrying Out the Invention
- the macromolecules of the present invention are biologically active polypeptides or glycopolypeptides having one or more water-soluble polymer molecules covalently bonded thereto.
- biologically active is used consistently with the meaning commonly understood to those of ordinary skill in the polypeptide and glycopolypeptide art, which meaning is not limited to physiologically or pharmacologically activities of the polypeptides or glycopolypeptides in the therapeutic sense.
- physiologically active polypeptides such as enzymes, the water-soluble polymer conjugates of which have therapeutic applications, are also able to catalyze reactions in organic solvents.
- therapeutic uses exist for water-soluble polymer conjugates of proteins such as concanavalin A, immunoglobulins, and the like, the polymer conjugates of these proteins are also useful as laboratory diagnostic tools.
- Enzymes of interest for both biological applications in general and therapeutic applications in particular include the oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases disclosed by U.S. Patent No. 4,179,337, the disclosure of which is hereby incorporated herein by reference thereto.
- examples of specific enzymes of interest include asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase and bilirubin oxidase.
- Carbohydrate-specific enzymes are also of interest—for example, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase, glucuronidase, etc.
- proteins of general biological or therapeutic interest include, but are not limited to, Factor VIII and polypeptide hormones such as insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothamic releasing factors, antidiuretic hormones and prolactin.
- glycopolypeptides of interest include, but are not limited to, immunoglobulins, chorionic gonadotrophin, follicle-stimulating hormone, thyroid-stimulating hormone, ovalbumin, bovine serum albumin (BSA) , lectins, tissue plasminogen activator, numerous enzymes and glycosilated interleukins, interferons and colony stimulating factors.
- Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD and fragments thereof.
- glycopolypeptides such as the interleukins, interferons and colony stimulating factors also exist in non-glycosilated form, usually the result of preparation by recombinant protein techniques.
- the structure of such versions may not contain carbohydrate moieties.
- the non-glycosilated versions are still capable of conjugation at reactive carbonyl or carboxylic acid groups of the peptide moieties.
- allergen proteins and glycoproteins having reduced allerginicity when conjugated with water-soluble polymers and consequently suitable for use as tolerance inducers include those allergens disclosed by Dreborg et al., Crit. Rev. Therap. Drug Carrier Syst.. discussed above, the teachings of which are hereby incorporated herein by reference thereto.
- allergens disclosed by this article are Ragweed Antigen E, honey bee venom, mite allergen, and the like.
- the water-soluble polymers suitable for attachment to the polypeptides and glycopolypeptide include polyalkylene oxides, polyoxyethylenated polyols, polyacrylamides, polyvinyl pyrrolidone, polyvinyl alcohol, dextran, and other carbohydrate-based polymers.
- the polymer must be soluble in water at room temperature.
- Polyalkylene oxide homopolymers meeting this requirement are polyethylene glycol (PEG) and copolymers thereof.
- Block copolymers of PEG with polypropylene glycol or polypropylene oxide are also suitable for use with the present invention, provided that the degree of block copolymerization is not so great as to render the polymer insoluble in water at room temperature.
- polyoxyethylenated polyols examples include polyoxyethylenated glycerols, polyoxyethylenate sorbitols, polyoxyethylenated glucoses, and the like.
- the molecular weight of the polymer is no critical, and will depend mainly upon the end use of particular polymer conjugate. Those of ordinary skil in the art are capable of determining molecular weigh ranges suitable for their end use applications. I general, the useful range of molecular weight is number average molecular weight between about 600 an about 100,000 daltons, and preferably betwee about 2,000 and about 20,000 daltons.
- One or more polymer units can be attache covalently to the polypeptide or glycopolypeptide b reacting an acyl hydrazine derivative of the polyme with a polypeptide or glycopolypeptide having a reactiv carbonyl group or an activated peptide carboxylic aci group.
- th reactive carbonyl group is defined as being either ketone or aldehyde group, excluding othe carboxyl-containing groups such as amides.
- Aldehyd groups are preferred, because they are more reactiv than ketones.
- Carbonyl groups can be generated on saccharide units of glycopolypeptides, for example, by oxidizing vicinal diols of carbohydrate moieties of glycopolypeptides with excess periodate or enzymatically e.g. by use of galactose oxidase.
- the polymer acyl hydrazine reacts with the reactive carbonyl group on the polypeptide or glycopolypeptide to form a hydrazone linkage between the polymer and the polypeptide or glycopolypeptide.
- the hydrazone can be reduced to a more stable alkyl hydrazide by using for example NaBH 4 or NaCNBH 3 .
- the activated peptide carboxylic acid group can be derived either from a C-terminus carboxylic acid group or a carboxylic acid group of aspartic or glutamic acid residues.
- the polymer acyl hydrazine reacts with the activated peptide carboxylic acid group to form a diacylhydrazine linkage between the polymer and the polypeptide or glycopolypeptide.
- Such leaving groups include, but are not limited to, imidazolyl, triazolyl, N-hydroxysuccin- imidyl, N-hydroxynorbornenedicarboximidyl and phenolic leaving groups, and are substituted onto the peptide carboxylic acid group by reacting the polypeptide or glycopolypeptide in the presence of an activating reagent with the corresponding imidazole, triazole , N-hydroxysuccinimide, N-hydroxynorbornene dicarboximide and phenolic compounds.
- Suitable activating reagents are also well-known and disclosed by the above-cited Bodanszky, Principles of Peptide Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
- Examples of such activating reagents include, but are not limited to, water-soluble carbodiimides such as ethyl dimethyla ino-propyl carbodiimide (EDC) and 3-[2-morpholinyl-(4)-ethyl] carbodiimide, p-toluene sulfonate, 5-substituted isoxazolium salts, such a Woodward's Reagent K, and the like.
- acyl hydrazine polymer derivatives of th present invention will have the general structure (I) :
- R is one of the above-disclosed water-solubl polymers
- Z is 0, NH, S or a lower alkyl grou containing up to ten carbon atoms
- X is a termina group on the polymer.
- X can be a hydroxyl group, i which case the polymer has two labile groups per polyme moiety capable of reacting to form a derivative that can be covalently linked with a polypeptide or glycopolypeptide.
- X can therefore also be a group into which the terminal hydroxyl group may be converted, including the reactive derivatives of the prior art disclosed in U.S. Patent Nos.
- heterobi ⁇ functional polymers can be prepared by methods known to those skilled in the art, including the methods disclosed by the present specification with reference to the preparation of acyl hydrazine derivatives, as well as the methods disclosed by Zalipsky and Barany, Polym. Prepr.. 27(1.. 1 (1986) and Zalipsky and Barany, J. Bioact. Compat. Polym.. 5 , 227 (1990), the disclosures of which are hereby incorporated herein by reference thereto.
- X is a functional group useful for covalently linking the polymer with a second polypeptide or glycopolypeptide
- X can be a solid support or a small molecule such as a drug, or an acyl hydrazide derivative of the formula (II) :
- the selectivity of the acyl hydrazines for the reactive carbonyl or activated carboxylic acid groups over the peptide amino group prevents intermolecular crosslinking between peptide amino groups and the reactive carbonyl groups and activated carboxylic acid groups, limiting occurrences of such crosslinking to instances when bifunctional polymer derivatives are employed.
- X can also represent an antibody or solid support covalently coupled to the polymer by methods known to those skilled in the art.
- solid supports covalently coupled to water-soluble polymers and methods of coupling water-soluble polymers to solid supports are disclosed in Published European Patent Application No. 295,073, the disclosure of which is hereby incorporated herein by reference thereto.
- the acyl hydrazine derivative is prepared by reacting, for example, the terminal -OH group of methoxylated PEG (mPEG-OH) with phosgene to form mPEG-chloroformate as described in U.S. Patent Appln.
- a more preferred form of the present inventio uses polymer hydrazides of the general formula (III) :
- AA represents an amino acid or a peptide sequence.
- AA can be a peptide sequence of any of the common amino acids, or at least one amino acid residue. In the case of AA being one amino acid residue, it is preferable that it is a residue that does not appear naturally in proteins. Examples of such unusual residues include, but are not limited to, alpha- or gamma- amino butyric acid, norleucine, homoserine, beta-alanine, epsilon-caproic acid, and the like.
- the linkage is a urethane linkage, which is very stable at ambient temperature in a variety of buffers, even at extreme pH's, but is readily split under conditions normally used for protein hydrolysis, thus allowing determination of amino acid components of AA by amino acid analysis.
- the peptide sequence can serve two roles. First, it can provide for convenient characterization of the modified protein by quatitation of the sequence by amino acid analysis. In this instance, the peptide sequence preferably is as short as possible and preferably contains unusual amino acid residues. For characterization of the modified protein, the peptide sequence most preferably contains but one amino acid.
- AA can also contain a labeled amino acid residue (chromophore, fluorophore, or radioisotope containing) , or an amino acid that could be easily labeled (e.g. tyrosine can be iodinated) .
- a labeled amino acid residue chromophore, fluorophore, or radioisotope containing
- an amino acid that could be easily labeled e.g. tyrosine can be iodinated
- the peptide sequence can optimize the lability of the covalent linkage between the water-soluble polymer and the polypeptide to proteolytic enzymes.
- the peptide sequence is preferably as long as possible and preferably contains natural amino acid residues.
- the polymer conjugates can be used to deliver physiologically active polypeptides or glycopolypeptides to specific sites, such as cancer cells having elevated concentrations of certain proteolytic enzymes to which the peptide sequence is labile.
- the length and sequence of the peptide in this second instance can be fine-tuned depending on the system of use and specificity of the target enzyme. Usually, three to seven amino acid residues would be required. Using modern techniques of peptide chemistry such short peptide sequences can be readily assembled.
- X can also contain a second peptide sequence residue.
- X is an acyl hydrazine derivative, X would have the general formula
- the acyl hydrazine polymer derivativ containing a peptide sequence can be synthesized b first preparing the polymeric chloroformate as describe above.
- the polymeric chloroformate is then reacted wit the peptide or an amino acid derivative in a solvent i which the polymeric chloroformate is soluble, such a ethylene chloride.
- the peptide or amino acid i preferably in the form of the ester of the C-terminu acid group, more preferably methyl or ethyl esters.
- This reaction is also operative under mil conditions and typically runs to completion at roo temperature and * the resulting product can be readil converted to a hydrazide by hydrazinolysis.
- the acy hydrazine polymer derivative containing a peptid sequence is then recovered and purified by conventiona methods, such as repeated precipitation of the polymer product.
- the acyl hydrazine polyme derivative containing a peptide sequence or an amin acid can be prepared by reacting the peptide sequenc with a succinimidyl carbonate active ester of th polymer, as disclosed by the above-mentioned Zalipsky, U.S. Patent Appln. No. 340,928 or by directly reactin isocyanate derivatives of an amino acid with th terminal hydroxyl group of the polymer as disclosed b Zalipsky et al.. Int. J Peptide Protein Res.. 30. 740 (1987) , the disclosures of both of which are hereby incorporated herein by reference thereto.
- Either of the above polymer-polypeptide derivatives can be readily converted to a hydrazide by the hydrazinolysis method disclosed above to yield an acyl hydrazine.
- the preparation of peptide sequences is essentially conventional and disclosed by the above-cited Bodanszky, Principles of Peptide Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
- the hydrazone can be reduced to the more stable alkyl hydrazide by reacting the hydrazone with, for example, NaBH 4 or NaCNBH 3 .
- R3-C-OH e.g., EDC R 3 -C-R 4
- R again represents the above-described water-soluble polymers, and Z is the same as described above for Formulae I-IV.
- R 3 represents a polypeptide containing aspartic acid, glutamic acid or a C-terminus carboxylic acid residues.
- R 4 represents one of the above-described leaving groups substituted on the peptide carboxylic acid when the carboxylic acid group is activated as described above.
- the conjugation of a polypeptide or glycopolypeptide with a water-soluble polymer first involves either oxidizing carbohydrate moieties of the glycopolypeptide or activating carboxylic acid groups of peptide moieties of the polypeptides or glycopolypeptides.
- the carbohydrate moieties can be oxidized by reacting the glycopolypeptide in aqueous solution with sodium periodate or enzymatically usin galactose oxidase or combination of neuraminidase an galactose oxidase as disclosed by Solomon et al., J. Chromatographv. 510. 321-9 (1990) .
- the reaction runs rapidly to completion at room temperature.
- the reaction medium is preferably buffered, depending upon the requirements of the polypeptide or glycopolypeptide.
- the oxidized glycopolypeptide is then recovered and separated from the excess periodate by column chro atography.
- Carboxylic acid groups of peptide moieties can be activated by reacting the polypeptide or glycopolypeptide with an activating reagent such as a water-soluble carbodimide such as EDC.
- the reactants are contacted in an aqueous reaction medium at a pH between about 3.0 and 8.0, and preferably about 5.0, which medium may be buffered to maintain the pH. This reaction is taking place under mild conditions (typically 4 to 37 C) that are tolerated well by most proteins.
- Polypeptides or glycopolypeptides having peptide units on which reactive carbonyl groups have been generated may be directly reacted with the acyl hydrazine polymer derivatives in an aqueous reaction medium.
- This reaction medium may also be buffered, depending upon the pH requirements of the polypeptide or glycopolypeptide and the optimum pH for the reaction, which pH is generally between about 5.0 and about 7.0 and preferably about 6.0.
- the optimum reaction media pH for the stability of particular polypeptides or glycopolypeptides and for reaction efficiency, and the buffer in which this can be achieved is readily determined within the above ranges by those of ordinary skill in the art without undue experimentation.
- the operativeness of the within reactions under mild conditions is defined as meaning that the preferred temperature range is between about 4 and about 37 X C.
- the reactions will run somewhat faster to completion at higher temperatures, with the proviso that the temperature of the reaction medium cannot exceed the temperature at which the polypeptides or glycopolypeptides begin to denature.
- polypeptides and glycopolypeptides will require reaction with the polymer acyl hydrazine derivatives at reduced temperatures to minimize loss of activity and/or prevent denaturing.
- the reduced temperature required by particular polypeptides and glycopolypeptides is preferably no lower than 4 ⁇ C and in no event should this temperature be lower than 0 C. The reaction will still take place, although longer reaction times may be necessary.
- the polypeptide or glycopolypeptide is reacted in aqueous solution with a quantity of the acyl hydrazine polymer derivative in excess of the desired degree of conjugation. This reaction also proceeds under mild conditions, typically at 4 to 37 X C.
- the reaction medium may be optionally buffered, depending upon the requirements of the polypeptide or the glycopolypeptide, and the optimum pH at which the reaction takes place.
- the conjugated product is recovered and purified by diafiltration, column chromatography or the like.
- the degree of polymer conjugation of the polypeptide or glycopolypeptide can then be determined by amino acid analysis.
- acyl hydrazine polymer derivatives of the present invention possess the optimum balance of reactivity and selectivity so that polymer conjugates can be formed with non-amino functional groups of polypeptides and glycopolypeptides with virtually no competition between the acyl hydrazines and the peptid amino groups for the non-amino functional groups.
- crosslinking is prevented and the activity of th polypeptide or glycopolypeptide is preserved.
- Methoxy-PEG (mPEG) is available fro Union Carbide.
- the solvents used, as well as beta-alanine ethyl ester HCL, hydrazine, P2°5' EDC , N-hydroxy-5-norbornene-2,3-dicarboximide (HONb) , NaCNBH 3 and NaI0 4 are available from Aldrich Chemicals of Milwaukee, Wisconsin. Chymotrypsin was obtained from Worthington Chemical. BSA, ovalbumin and human immunoglobulin G (IgG) are available from Sigma Chemical of St. Louis, Missouri. G-CSF was obtained from Amgen of Thousand Oaks, California.
- EXAMPLE 1 SYNTHESIS OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE: mPEG (MW n 5,000, 100 g, 20 mmol) was dissolved in toluene (250 mL) and azeotropically dried for two hours under reflux. The solution was brought to 25 ⁇ C, diluted with methylene chloride (50 mL) and then treated with phosgene (30 mL of 20 percent toluene solution, 56 mmol) overnight. The solvents and the excess of phosgene were removed by rotary evaporation under vacuum.
- the mPEG-beta-alanine ethyl ester (62 g, 12 mmol) was dissolved in pyridine (120 L) and treated with hydrazine (12 mL, 0.375 mole) under reflux for six hours. The solution was rotary evaporated to dryness and the residue crystallized twice from isopropanol and dried in vacuo over P 2 0 5 . The yield was 60 g (97%) .
- TNBS gave 0.2 mmol/g (103% of theoretical) .
- the beta-alanine content of the polymer was 0.205 mmol/g (105% of theoretical) as determined by amino acid analysis of a completely hydrolysed (6N HC1, 110 C, 24 h) aliquot of the product.
- Example 2 The same conjugation protocol as Example 2 was employed, in the presence of HONb (28.7 mg, 0.16 mmol).
- the PEG-chymotrypsin obtained had an average 2.7 molecules of mPEG per molecule of protein, based on quantitation of beta-alanine by amino acid analysis. This demonstrates that the conjugation process is only slightly enhanced by the presence of HONb.
- EXAMPLE 4 COUPLING OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE TO EDC-ACTIVATED CARBOXYL GROUPS OF BSA: A solution of BSA (20 mg) and a mPEG-beta-alanine hydrazide derivative of Example 1 (800 mg, 0.16 mmol) in 50 mM NaCl (10 mL) was treated with EDC (15 mg, 0.078 mmol) overnight at pH 5.0, 25 C as in Example 2. Excess reagents were removed by extensive diafiltration of the reaction solution at 4 ⁇ C against phosphate buffer (50 mM, pH 7.7).
- phosphate buffer 50 mM, pH 7.7
- the content of beta-alanine in the conjugate corresponded to 8.1 residues of mPEG per molecule of BSA.
- a GF-HPLC comparison of the PEG-conjugate to native BSA was performed with a BIOSEP SEC 4000 column, the results of which are depicted in FIG. 1.
- the elution conditions were 10% (vol/vol) methanol/40 mM phosphate buffer.
- FIG. 1 depicts good homogeneity of the PEG-conjugate 1, with a substantially increased molecular weight as compared to the native BSA 2.
- Ovalbumin (20 mg, 4.4 x 10 ⁇ 7 mole) dissolved in Phosphate Buffered Saline (PBS) buffer, pH 6.0 (1.8 mL) was treated with NaI0 4 (0.2 mL of 200 mM aqueous solution) . The reaction was allowed to proceed in the dark at 4 ⁇ C. After one hour, the oxidized glycoprotein was separated from the excess of periodate by passing the reaction solution through a 12 mL Sephadex G-25 column equilibrated with acetate buffer to pH 5.0. Additional samples were prepared and the procedure was repeated equilibrating the column with PBS buffer at pH 6.0 and phosphate buffer at pH 7.0. This resulted in three separate reaction mixtures having different buffering systems.
- PBS Phosphate Buffered Saline
- Example 1 To each mixture was added the mPEG-beta-alanine-hydrazide derivative of Example 1 (150 mg, 2.9 x 10 ""5 mole). Each of the three reaction mixtures was divided into two equal portions and NaCNBH 3 (0.3 mL of 6.6 mg/mL solution, 3.15 x 10 ⁇ 5 mole) was added to one portion of each. The reactions were allowed to proceed overnight at 4 C. Each solution was diafiltered using phosphate buffer pH 7.7 until all the unreacted reagents were removed. The conjugates in the solutions to which the NaCNBH 3 was added formed
- FIG. 2 Depicted in FIG. 2 is the GF-HPLC analysis using a TSK G 4000SW column and a 10% (vol/vol) methanol/40 mM phosphate buffer pH 7.5 mobile phase, which showed good homogeneity of the mPEG-ovalbumin conjugate 3, and a substantially increased molecular weight as compared to the native ovalbumin 4.
- FIG. 3 depicts good homogeneity of the PEG-conjugate 5, with a substantially increased molecular weight as compared to the native IgG 6.
- the amount of beta-alanine was determined by amino acid analysis of a hydrolyzed (6 N HCl, 110 C, 24 h) aliquot of the PEG-IgG conjugate to correspond to six residues of mPEG per protein molecule.
- EXAMPLE 7 ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE TO THE CARBOHYDRATE MOIETY OF IMMUNOGLOBULIN G WITHOUT REMOVAL OF EXCESS PERIODATE:
- EXAMPLE 8 ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE TO CARBODIIMIDE- ACTIVATED CARBOXYL GROUPS OF G-CSF: The mPEG-beta-alanine-hydrazide of Example 1
- the average number of mPEG residues in the PEG-G-CSF was 5.8, as determined by measuring the amount of beta-alanine in an hydrolyzed (6 N HCl, 110 C, 24 h) aliquot of the conjugate.
- TNBS assay confirmed that both native and PEG-modified G-CSF-1 had the same number of amino groups, indicating that the EDC activated carboxylic acid groups of the protein did not react with amino groups of the protein.
- the preparation of mPEG-G-CSF gave four separate bands on SDS-PAGE (PhastGel-, Homogenous 12.5, Pharmacia) in the range from 29,000 to 67,000 daltons.
- the present invention is applicable to the production of polymers conjugated with various biologically active and pharmaceutically active compounds representing a novel form of drug delivery.
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Abstract
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US67269691A | 1991-03-18 | 1991-03-18 | |
US672696 | 1991-03-18 |
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EP0576589A1 EP0576589A1 (fr) | 1994-01-05 |
EP0576589A4 true EP0576589A4 (en) | 1994-07-27 |
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EP19920909326 Withdrawn EP0576589A4 (en) | 1991-03-18 | 1992-03-12 | Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers |
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EP (1) | EP0576589A4 (fr) |
JP (1) | JPH06506217A (fr) |
AU (1) | AU1676992A (fr) |
CA (1) | CA2101918A1 (fr) |
WO (1) | WO1992016555A1 (fr) |
Families Citing this family (154)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ244778A (en) * | 1991-10-21 | 1994-03-25 | Ortho Pharma Corp | Peg imidates and protein derivatives thereof |
US5169627A (en) * | 1991-10-28 | 1992-12-08 | Mount Sinai School Of Medicine Of The City University Of New York | Oral pharmaceutical composition containing a polyethylene glycol-immunoglobulin G conjugate for reconstitution of secretory immunity and method of reconstituting secretory immunity |
NZ250375A (en) * | 1992-12-09 | 1995-07-26 | Ortho Pharma Corp | Peg hydrazone and peg oxime linkage forming reagents and protein derivatives |
NO934477L (no) * | 1992-12-09 | 1994-06-10 | Ortho Pharma Corp | PEG hydrazon- og PEG oksim-bindingdannende reagenser og proteinderivater derav |
AU6029594A (en) * | 1993-01-15 | 1994-08-15 | Enzon, Inc. | Factor viii - polymeric conjugates |
US5581476A (en) * | 1993-01-28 | 1996-12-03 | Amgen Inc. | Computer-based methods and articles of manufacture for preparing G-CSF analogs |
US5516703A (en) * | 1993-08-20 | 1996-05-14 | The University Of Utah | Coating of hydrophobic surfaces to render them protein resistant while permitting covalent attachment of specific ligands |
WO1994028024A1 (fr) * | 1993-06-01 | 1994-12-08 | Enzon, Inc. | Polymere modifie aux glucides presentant une activite erythropoïetique |
AU7113594A (en) * | 1993-06-21 | 1995-01-17 | Enzon, Inc. | Site specific synthesis of conjugated peptides |
US6284503B1 (en) | 1993-08-20 | 2001-09-04 | University Of Utah Research Foundation | Composition and method for regulating the adhesion of cells and biomolecules to hydrophobic surfaces |
US5951974A (en) * | 1993-11-10 | 1999-09-14 | Enzon, Inc. | Interferon polymer conjugates |
AU691225B2 (en) * | 1993-11-10 | 1998-05-14 | Schering Corporation | Improved interferon polymer conjugates |
US5446090A (en) * | 1993-11-12 | 1995-08-29 | Shearwater Polymers, Inc. | Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules |
US5629384A (en) * | 1994-05-17 | 1997-05-13 | Consiglio Nazionale Delle Ricerche | Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces |
US5738846A (en) * | 1994-11-10 | 1998-04-14 | Enzon, Inc. | Interferon polymer conjugates and process for preparing the same |
SE9503380D0 (sv) * | 1995-09-29 | 1995-09-29 | Pharmacia Ab | Protein derivatives |
TW555765B (en) | 1996-07-09 | 2003-10-01 | Amgen Inc | Low molecular weight soluble tumor necrosis factor type-I and type-II proteins |
AU778790B2 (en) * | 1996-08-02 | 2004-12-23 | Ortho-Mcneil Pharmaceutical, Inc. | Polypeptides having a single covalently bound n-terminal water-soluble polymer |
AU3908597A (en) * | 1996-08-02 | 1998-02-25 | Ortho-Mcneil Pharmaceutical, Inc. | Polypeptides having a single covalently bound n-terminal water-soluble polymer |
EP0981548A4 (fr) | 1997-04-30 | 2005-11-23 | Enzon Inc | Proteines a chaine unique fixant les antigenes capables de glycosylation, production et utilisations de ces dernieres |
JP3688111B2 (ja) * | 1998-03-13 | 2005-08-24 | 科学技術振興事業団 | 樹脂固定化ヒドラジドとその誘導体並びにピラゾロン類の固相合成法 |
US6333396B1 (en) | 1998-10-20 | 2001-12-25 | Enzon, Inc. | Method for targeted delivery of nucleic acids |
AU772074B2 (en) * | 1999-04-28 | 2004-04-08 | Vectramed, Inc. | Enzymatically activated polymeric drug conjugates |
US6673361B1 (en) | 1999-05-19 | 2004-01-06 | Nof Corporation | Polymer, in vivo degradable material, and use |
EP1757701A1 (fr) | 1999-12-24 | 2007-02-28 | Genentech, Inc. | Procédés et compositions pour la prolongation de la demi-vie d'élimination des composés bioactifs |
WO2001045746A2 (fr) | 1999-12-24 | 2001-06-28 | Genentech, Inc. | Methodes et compositions permettant de prolonger les demi-vies d'elimination de composes bioactifs |
AU782580B2 (en) | 2000-01-10 | 2005-08-11 | Maxygen, Inc. | G-CSF conjugates |
DK1257295T3 (da) | 2000-02-11 | 2009-08-10 | Bayer Healthcare Llc | Faktor VII eller VIIA-lignende molekyler |
US20030211094A1 (en) | 2001-06-26 | 2003-11-13 | Nelsestuen Gary L. | High molecular weight derivatives of vitamin k-dependent polypeptides |
US7723296B2 (en) | 2001-01-18 | 2010-05-25 | Genzyme Corporation | Methods for introducing mannose-6-phosphate and other oligosaccharides onto glycoproteins and its application thereof |
WO2002074806A2 (fr) | 2001-02-27 | 2002-09-26 | Maxygen Aps | Nouvelles molecules de type interferon beta |
US7214660B2 (en) | 2001-10-10 | 2007-05-08 | Neose Technologies, Inc. | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US7173003B2 (en) | 2001-10-10 | 2007-02-06 | Neose Technologies, Inc. | Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF |
BR0214451A (pt) * | 2001-11-20 | 2006-05-30 | Pharmacia Corp | conjugados do hormÈnio do crescimento humano quimicamente modificado |
DK1517710T3 (da) | 2002-06-21 | 2011-07-18 | Novo Nordisk Healthcare Ag | Pegylerede faktor VII-glycoformer |
US7459435B2 (en) | 2002-08-29 | 2008-12-02 | Hoffmann-La Roche Inc. | Treatment of disturbances of iron distribution |
US7459436B2 (en) | 2002-11-22 | 2008-12-02 | Hoffmann-La Roche Inc. | Treatment of disturbances of iron distribution |
RS20050502A (en) | 2002-12-26 | 2007-08-03 | Mountain View Pharmaceuticals Inc., | Polymer conjugates of interferon- beta with enhanced biological potency |
WO2004061094A1 (fr) | 2002-12-30 | 2004-07-22 | Gryphon Therapeutics, Inc. | Composes thioesters et selenoesters hydrosolubles et leurs procedes de production et d'utilisation |
CA2788505C (fr) | 2003-02-26 | 2018-09-04 | Nektar Therapeutics | Conjugues de groupe fonctionnel a facteur polymerique viii |
CA2530725A1 (fr) * | 2003-03-28 | 2004-10-07 | Biopolymed Inc. | Materiau biologiquement actif conjugue avec un polymere biocompatible avec un complexe 1:1, technique de preparation de celui-ci et composition pharmaceutique comprenant celui-ci |
MXPA05010773A (es) | 2003-04-09 | 2005-12-12 | Neose Technologies Inc | Metodos de glicopegilacion y proteinas/peptidos producidos por los metodos. |
ES2340494T3 (es) | 2003-04-15 | 2010-06-04 | Glaxosmithkline Llc | Mutantes de sustitucion de il-18 humana y sus conjugados. |
EP1628686A2 (fr) | 2003-05-12 | 2006-03-01 | Affymax, Inc. | Fragment espaceur de peptides modifies par du poly (ethylene glycol) |
NZ543935A (en) | 2003-05-12 | 2008-06-30 | Affymax Inc | Peptides that bind to the erythropoietin receptor |
RS51130B (sr) | 2003-05-12 | 2010-10-31 | Affymax Inc. | Novi peptidi koji se vezuju za eritropoietinski receptor |
WO2005012484A2 (fr) | 2003-07-25 | 2005-02-10 | Neose Technologies, Inc. | Conjugues anticorps-toxines |
EP1653996A2 (fr) * | 2003-08-08 | 2006-05-10 | Novo Nordisk Health Care AG | Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique |
EP1673387B1 (fr) | 2003-10-10 | 2010-09-15 | Novo Nordisk A/S | Derives de il-21 |
EP2641611A3 (fr) | 2003-10-17 | 2013-12-18 | Novo Nordisk A/S | Thérapie combinée |
US20080305992A1 (en) | 2003-11-24 | 2008-12-11 | Neose Technologies, Inc. | Glycopegylated erythropoietin |
JP2007514673A (ja) | 2003-12-19 | 2007-06-07 | エフ.ホフマン−ラ ロシュ アーゲー | 慢性炎症性腸疾患の際の鉄分布障害の処置におけるエリスロポエチンの使用 |
CA2553040A1 (fr) | 2004-02-02 | 2005-08-18 | Ambrx, Inc. | Polypeptides a faisceau a quatre helices (4hb) humains modifies, et leur utilisation |
MXPA06014307A (es) | 2004-06-08 | 2007-03-12 | Alza Corp | Preparacion de conjugados macromoleculares por reaccion de condensacion de cuatro componentes. |
CN102603895B (zh) | 2004-06-18 | 2016-09-28 | Ambrx公司 | 新颖抗原结合多肽和其用途 |
EP2258399A3 (fr) | 2004-07-08 | 2011-08-10 | Elan Pharmaceuticals Inc. | Antagonistes multivalents de l'antigène VLA-4 comprenant des polymères |
JP5948627B2 (ja) | 2004-10-29 | 2016-07-20 | レイショファーム ゲーエムベーハー | 線維芽細胞成長因子(fgf)のリモデリングと糖質ペグ化 |
KR101252835B1 (ko) | 2004-12-22 | 2013-04-10 | 암브룩스, 인코포레이티드 | 아미노아실-tRNA 합성효소의 조성물 및 그것의 용도 |
WO2006073846A2 (fr) | 2004-12-22 | 2006-07-13 | Ambrx, Inc. | Procedes pour l'expression et la purification d'hormone de croissance humaine recombinante |
NZ555386A (en) | 2004-12-22 | 2011-01-28 | Ambrx Inc | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
KR20070090023A (ko) | 2004-12-22 | 2007-09-04 | 암브룩스, 인코포레이티드 | 변형 인간 성장 호르몬 |
EP1858543B1 (fr) | 2005-01-10 | 2013-11-27 | BioGeneriX AG | Facteur de stimulation de colonie de granulocytes glycopegylé |
JP2008534640A (ja) | 2005-04-05 | 2008-08-28 | イステイチユート・デイ・リチエルケ・デイ・ビオロジア・モレコラーレ・ピ・アンジエレツテイ・エツセ・ピー・アー | タンパク質の機能部位またはエピトープの遮蔽方法 |
EP1877099B1 (fr) | 2005-04-06 | 2012-09-19 | Genzyme Corporation | Conjugués thérapeutiques comprenant une enzyme lysosomale, l'acide polysialique et un agent de ciblage |
EP2386571B1 (fr) | 2005-04-08 | 2016-06-01 | ratiopharm GmbH | Compositions et méthodes utilisées pour la préparation de mutants par glycosylation de l'hormone de croissance humaine résistant à la protéase |
CN101247821B (zh) | 2005-06-03 | 2013-01-23 | Ambrx公司 | 经改良人类干扰素分子和其用途 |
US7550433B2 (en) | 2005-06-03 | 2009-06-23 | Affymax, Inc. | Erythropoietin receptor peptide formulations and uses |
US8324159B2 (en) | 2005-06-03 | 2012-12-04 | Affymax, Inc. | Erythropoietin receptor peptide formulations and uses |
EP1893632B1 (fr) | 2005-06-17 | 2015-08-12 | Novo Nordisk Health Care AG | Reduction et derivation séléctives de proteines facteur vii comprenant au moins une cysteine non native |
US20070105755A1 (en) | 2005-10-26 | 2007-05-10 | Neose Technologies, Inc. | One pot desialylation and glycopegylation of therapeutic peptides |
WO2007056191A2 (fr) | 2005-11-03 | 2007-05-18 | Neose Technologies, Inc. | Purification de sucre de nucleotide en utilisant des membranes |
KR20080079643A (ko) | 2005-11-16 | 2008-09-01 | 암브룩스, 인코포레이티드 | 비-천연 아미노산을 포함하는 방법 및 조성물 |
AU2007245190B2 (en) | 2006-03-31 | 2011-07-21 | Takeda Pharmaceutical Company Limited | Pegylated factor VIII |
US7982010B2 (en) | 2006-03-31 | 2011-07-19 | Baxter International Inc. | Factor VIII polymer conjugates |
US7985839B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US7645860B2 (en) | 2006-03-31 | 2010-01-12 | Baxter Healthcare S.A. | Factor VIII polymer conjugates |
JP2009537609A (ja) | 2006-05-24 | 2009-10-29 | ノボ ノルディスク ヘルス ケア アーゲー | 延長されたfixアナログ及び誘導体 |
US9187532B2 (en) | 2006-07-21 | 2015-11-17 | Novo Nordisk A/S | Glycosylation of peptides via O-linked glycosylation sequences |
JP5570809B2 (ja) | 2006-09-01 | 2014-08-13 | ノボ ノルディスク ヘルス ケア アーゲー | 修飾タンパク質 |
KR20090051227A (ko) | 2006-09-08 | 2009-05-21 | 암브룩스, 인코포레이티드 | 척추동물 세포를 위한 하이브리드 서프레서 tRNA |
EP2069396B1 (fr) | 2006-09-08 | 2015-10-21 | Ambrx, Inc. | Polypeptide plasmatique humain modifie ou squelettes de fc et leurs utilisations |
PT2064333E (pt) | 2006-09-08 | 2014-06-09 | Ambrx Inc | Supressor híbrido arnt para células de vertebrados |
US7985783B2 (en) | 2006-09-21 | 2011-07-26 | The Regents Of The University Of California | Aldehyde tags, uses thereof in site-specific protein modification |
JP2010505874A (ja) | 2006-10-03 | 2010-02-25 | ノヴォ ノルディスク アー/エス | ポリペプチドコンジュゲートの精製方法 |
US8637007B2 (en) | 2006-12-15 | 2014-01-28 | Baxter International Inc. | Factor VIIa-polysialic acid conjugate having prolonged in vivo half-life |
LT2457920T (lt) | 2007-01-18 | 2018-02-12 | Genzyme Corporation | Oligosacharidai, apimantys aminooksigrupę, ir jų konjugatai |
US8106154B2 (en) | 2007-01-31 | 2012-01-31 | Affymax, Inc. | Nitrogen-based linkers for attaching modifying groups to polypeptides and other macromolecules |
CN104163864B (zh) | 2007-03-30 | 2017-08-01 | Ambrx公司 | 经修饰fgf‑21多肽和其用途 |
KR20150064246A (ko) | 2007-04-03 | 2015-06-10 | 바이오제너릭스 게엠베하 | 글리코페길화 g―csf를 이용하는 치료 방법 |
AU2008247815B2 (en) | 2007-05-02 | 2012-09-06 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
CA2690611C (fr) | 2007-06-12 | 2015-12-08 | Novo Nordisk A/S | Procede ameliore pour la production de sucres de nucleotide |
MX338336B (es) | 2007-11-20 | 2016-04-07 | Ambrx Inc | Polipeptidos de insulina modificados y sus usos. |
CN101939443B (zh) | 2008-02-08 | 2014-01-29 | Ambrx公司 | 经修饰瘦素多肽和其用途 |
WO2009108806A1 (fr) | 2008-02-27 | 2009-09-03 | Novo Nordisk A/S | Molécules de facteur viii conjuguées |
PT2318029T (pt) | 2008-07-23 | 2018-01-10 | Ambrx Inc | Polipéptidos de g-csf bovino modificados e suas utilizações |
EP2342223B1 (fr) | 2008-09-26 | 2017-04-26 | Ambrx, Inc. | Polypeptides d érythropoïétine animale modifiés et leurs utilisations |
ES2660000T3 (es) | 2008-09-26 | 2018-03-20 | Ambrx, Inc. | Vacunas y microorganismos dependientes de la replicación de aminoácidos no naturales |
ITRM20080551A1 (it) * | 2008-10-15 | 2010-04-16 | Univ Catania | Derivati anfifilici del poliossietilenglicole (peg), procedimento di preparazione e loro usi nella preparazione di sistemi farmaceutici. |
KR20100052730A (ko) * | 2008-11-11 | 2010-05-20 | 한국유니온제약 주식회사 | 생체적합성 고분자와 결합한 신규한 에리스로포이에틴 접합체 |
CA2747230C (fr) | 2008-12-16 | 2019-06-11 | Genzyme Corporation | Conjugues oligosaccharide-proteine |
WO2010080720A2 (fr) * | 2009-01-12 | 2010-07-15 | Nektar Therapeutics | Conjugués d'une fraction enzymatique lysosomale et polymère soluble dans l'eau |
US8809501B2 (en) | 2009-07-27 | 2014-08-19 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
ES2856055T3 (es) * | 2009-07-27 | 2021-09-27 | Baxalta GmbH | Glicopolisialilación de proteínas diferentes de las proteínas de coagulación de la sangre |
US8642737B2 (en) | 2010-07-26 | 2014-02-04 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
CA2769326A1 (fr) | 2009-07-27 | 2011-02-10 | Baxter International Inc. | Conjugues de proteine de coagulation sanguine |
ES2590679T3 (es) * | 2009-07-27 | 2016-11-23 | Lipoxen Technologies Limited | Glicopolisialilación de proteínas diferentes a proteínas de coagulación de la sangre |
WO2011068853A2 (fr) * | 2009-12-01 | 2011-06-09 | Boston Medical Center Corporation | Traitement de maladie médiée par ige |
US20120283171A1 (en) | 2009-12-21 | 2012-11-08 | Ambrx, Inc. | Modified bovine somatotropin polypeptides and their uses |
SG181769A1 (en) | 2009-12-21 | 2012-07-30 | Ambrx Inc | Modified porcine somatotropin polypeptides and their uses |
WO2011107591A1 (fr) | 2010-03-05 | 2011-09-09 | Rigshospitalet | Molécules chimériques inhibitrices d'activation du complément |
JP2013528374A (ja) | 2010-05-10 | 2013-07-11 | パーシード セラピューティクス リミテッド ライアビリティ カンパニー | Vla4のポリペプチド阻害剤 |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
PL2605789T3 (pl) | 2010-08-17 | 2019-11-29 | Ambrx Inc | Zmodyfikowane polipeptydy relaksyny i ich zastosowania |
AR083006A1 (es) | 2010-09-23 | 2013-01-23 | Lilly Co Eli | Formulaciones para el factor estimulante de colonias de granulocitos (g-csf) bovino y variantes de las mismas |
KR102025442B1 (ko) | 2010-12-22 | 2019-09-25 | 박스알타 인코퍼레이티드 | 단백질에 수용성 지방산 유도체를 접합하기 위한 물질 및 방법 |
KR20140054009A (ko) | 2011-07-01 | 2014-05-08 | 바이엘 인텔렉쳐 프로퍼티 게엠베하 | 릴랙신 융합 폴리펩타이드 및 그의 용도 |
PT2717917T (pt) | 2011-07-05 | 2016-07-27 | Bioasis Technologies Inc | Conjugados de anticorpos p97 |
WO2013138730A1 (fr) * | 2012-03-16 | 2013-09-19 | Enzon Pharmaceuticals, Inc. | Conjugués polymères d'inhibiteurs de la c1-estérase |
SI2859017T1 (sl) | 2012-06-08 | 2019-05-31 | Sutro Biopharma, Inc. | Protitelesa, ki vsebujejo specifične nenaravne aminokislinske ostanke, postopki njihove priprave in postopki njihove uporabe |
WO2014004639A1 (fr) | 2012-06-26 | 2014-01-03 | Sutro Biopharma, Inc. | Protéines de fc modifiées, comprenant des résidus d'acides aminés non naturels spécifiques de site, leurs conjugués, leur préparation et leurs procédés d'utilisation |
ES2647082T3 (es) | 2012-07-31 | 2017-12-19 | Bioasis Technologies Inc | Proteínas desfosforiladas de la enfermedad de depósito lisosómico y métodos de uso de las mismas |
ES2907763T3 (es) | 2012-08-31 | 2022-04-26 | Sutro Biopharma Inc | Aminoácidos modificados que comprenden un grupo azido |
WO2014074218A1 (fr) | 2012-11-12 | 2014-05-15 | Redwood Bioscience, Inc. | Composés et procédés pour produire un conjugué |
EP2920148B1 (fr) | 2012-11-16 | 2019-06-12 | The Regents of the University of California | Ligature pictet-spengler pour la modification chimique de protéines |
US9310374B2 (en) | 2012-11-16 | 2016-04-12 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
CA2906003C (fr) | 2013-03-13 | 2021-07-06 | Bioasis Technologies Inc. | Fragments de p97 et leurs utilisations |
EA023360B1 (ru) * | 2013-03-28 | 2016-05-31 | Илья Александрович МАРКОВ | Линейный ацилазидный пегилирующий агент, способ его получения и способ получения пегилированного интерферона |
EA023323B1 (ru) * | 2013-03-28 | 2016-05-31 | Илья Александрович МАРКОВ | Разветвленный ацилазидный пегилирующий агент, способ его получения и способ получения пегилированного интерферона |
EA021643B1 (ru) * | 2013-03-28 | 2015-07-30 | Илья Александрович МАРКОВ | Монопегилированный интерферон-альфа линейной структуры и фармацевтическая композиция для приготовления лекарственного средства, обладающего активностью интерферона-альфа |
EA021610B1 (ru) * | 2013-03-28 | 2015-07-30 | Илья Александрович МАРКОВ | Жидкое противовирусное лекарственное средство |
EA022617B1 (ru) * | 2013-03-28 | 2016-02-29 | Илья Александрович МАРКОВ | Монопегилированный интерферон-альфа разветвленной структуры и фармацевтическая композиция для приготовления лекарственного средства, обладающего активностью интерферона-альфа |
EP3019522B1 (fr) | 2013-07-10 | 2017-12-13 | Sutro Biopharma, Inc. | Anticorps comprenant plusieurs résidus d'acides aminés non naturels site-spécifiques, des procédés permettant leur préparation et leurs méthodes d'utilisation |
EP3038657A2 (fr) | 2013-08-28 | 2016-07-06 | Bioasis Technologies Inc. | Conjugués comportant des régions fc modifiées pour cibler le snc et méthodes pour les utiliser |
WO2015054658A1 (fr) | 2013-10-11 | 2015-04-16 | Sutro Biopharma, Inc. | Acides aminés modifiés comprenant des groupes fonctionnels de tétrazine, procédés de préparation et procédés d'utilisation associés |
JP6745218B2 (ja) | 2013-11-27 | 2020-08-26 | レッドウッド バイオサイエンス, インコーポレイテッド | ヒドラジニル−ピロロ化合物及び複合体を生成するための方法 |
KR20240024362A (ko) | 2014-10-24 | 2024-02-23 | 브리스톨-마이어스 스큅 컴퍼니 | 변형된 fgf-21 폴리펩티드 및 그의 용도 |
WO2016201448A2 (fr) * | 2015-06-11 | 2016-12-15 | Prolong Pharmaceuticals, LLC | Facteur de stimulation des colonies de granulocytes pégylés (gcsf) |
JP6823055B2 (ja) | 2015-06-15 | 2021-01-27 | アンジオケム インコーポレーテッド | 軟髄膜癌腫症の治療方法 |
SG10201912034UA (en) | 2017-02-08 | 2020-02-27 | Bristol Myers Squibb Co | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
EP3648774A4 (fr) | 2017-07-07 | 2021-04-21 | Symic IP, LLC | Bioconjugués synthétiques |
US11492493B2 (en) | 2017-12-26 | 2022-11-08 | Becton, Dickinson And Company | Deep ultraviolet-excitable water-solvated polymeric dyes |
WO2019191482A1 (fr) | 2018-03-30 | 2019-10-03 | Becton, Dickinson And Company | Colorants polymères hydrosolubles portant des chromophores latéraux |
EP3813867A1 (fr) | 2018-07-22 | 2021-05-05 | Bioasis Technologies Inc. | Traitement de métastases lymphatiques |
CA3111576A1 (fr) | 2018-09-11 | 2020-03-19 | Ambrx, Inc. | Conjugues polypeptidiques d'interleukine-2 et leurs utilisations |
AU2019361206A1 (en) | 2018-10-19 | 2021-06-03 | Ambrx, Inc. | Interleukin-10 polypeptide conjugates, dimers thereof, and their uses |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2530247A1 (de) * | 1974-07-11 | 1976-01-29 | Yeda Res & Dev | Wasserunloesliche proteinpraeparate und deren herstellung |
EP0014263A2 (fr) * | 1979-01-12 | 1980-08-20 | Bayer Ag | Procédé d'amélioration de la solubilité d'agents biologiquement actifs dans l'eau et dans les alcools aliphatiques inférieurs ainsi que composés à solubilité améliorée |
WO1990013540A1 (fr) * | 1989-04-19 | 1990-11-15 | Enzon, Inc. | Carbonates actifs d'oxydes de polyalkylene pour la modification de polypeptides |
DD287950A5 (de) * | 1989-09-15 | 1991-03-14 | Adw Zi F. Molekularbiologie,De | Verfahren zur kovalenten bindung von biologisch aktiven verbindungen an substituierte polyoxyalkylenglykole und ihre monoalkoxyderivate |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5399384A (en) * | 1977-02-04 | 1978-08-30 | Toyo Tire & Rubber Co Ltd | Novel process for immobilization of enzyme |
US4970300A (en) * | 1985-02-01 | 1990-11-13 | New York University | Modified factor VIII |
US4766106A (en) * | 1985-06-26 | 1988-08-23 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
US4847325A (en) * | 1988-01-20 | 1989-07-11 | Cetus Corporation | Conjugation of polymer to colony stimulating factor-1 |
-
1992
- 1992-03-12 JP JP4508914A patent/JPH06506217A/ja active Pending
- 1992-03-12 EP EP19920909326 patent/EP0576589A4/en not_active Withdrawn
- 1992-03-12 AU AU16769/92A patent/AU1676992A/en not_active Abandoned
- 1992-03-12 CA CA002101918A patent/CA2101918A1/fr not_active Abandoned
- 1992-03-12 WO PCT/US1992/002047 patent/WO1992016555A1/fr not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2530247A1 (de) * | 1974-07-11 | 1976-01-29 | Yeda Res & Dev | Wasserunloesliche proteinpraeparate und deren herstellung |
EP0014263A2 (fr) * | 1979-01-12 | 1980-08-20 | Bayer Ag | Procédé d'amélioration de la solubilité d'agents biologiquement actifs dans l'eau et dans les alcools aliphatiques inférieurs ainsi que composés à solubilité améliorée |
WO1990013540A1 (fr) * | 1989-04-19 | 1990-11-15 | Enzon, Inc. | Carbonates actifs d'oxydes de polyalkylene pour la modification de polypeptides |
DD287950A5 (de) * | 1989-09-15 | 1991-03-14 | Adw Zi F. Molekularbiologie,De | Verfahren zur kovalenten bindung von biologisch aktiven verbindungen an substituierte polyoxyalkylenglykole und ihre monoalkoxyderivate |
Non-Patent Citations (2)
Title |
---|
CHEMICAL ABSTRACTS, vol. 115, no. 19, 11 November 1991, Columbus, Ohio, US; abstract no. 202770e * |
See also references of WO9216555A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH06506217A (ja) | 1994-07-14 |
EP0576589A1 (fr) | 1994-01-05 |
WO1992016555A1 (fr) | 1992-10-01 |
CA2101918A1 (fr) | 1992-09-19 |
AU1676992A (en) | 1992-10-21 |
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