EP0576589A4 - Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers - Google Patents

Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers

Info

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
Authority
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.)
Withdrawn
Application number
EP19920909326
Other languages
English (en)
Other versions
EP0576589A1 (fr
Inventor
Samuel Zalipsky
Chyi Lee
Sunitha Menon-Rudolph
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzon Pharmaceuticals Inc
Original Assignee
Enzon Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Enzon Inc filed Critical Enzon Inc
Publication of EP0576589A1 publication Critical patent/EP0576589A1/fr
Publication of EP0576589A4 publication Critical patent/EP0576589A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Polyamides (AREA)
  • Polyethers (AREA)

Abstract

On décrit des conjugés macromoléculaires biologiquement actifs d'un polypeptide ou d'un glycopolypeptide biologiquement actif associés à une ou plusieurs molécules d'un polymère hydrosoluble liées de manière covalente, au niveau d'un groupe de carbonyle ou d'acide carboxylique réactif d'une fraction de peptide sur le polypeptide ou glycopolypeptide ou au niveau d'une fraction d'hydrate de carbone oxydé du glycopolypeptide, par une liaison contenant un groupement fonctionnelle d'hydrazide ou d'hydrazone. Ladite liaison comprend de préférence également un acide aminé ou une séquence peptidique.
EP19920909326 1991-03-18 1992-03-12 Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers Withdrawn EP0576589A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US67269691A 1991-03-18 1991-03-18
US672696 1991-03-18

Publications (2)

Publication Number Publication Date
EP0576589A1 EP0576589A1 (fr) 1994-01-05
EP0576589A4 true EP0576589A4 (en) 1994-07-27

Family

ID=24699627

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19920909326 Withdrawn EP0576589A4 (en) 1991-03-18 1992-03-12 Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers

Country Status (5)

Country Link
EP (1) EP0576589A4 (fr)
JP (1) JPH06506217A (fr)
AU (1) AU1676992A (fr)
CA (1) CA2101918A1 (fr)
WO (1) WO1992016555A1 (fr)

Families Citing this family (165)

* Cited by examiner, † Cited by third party
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
WO1995000162A1 (fr) * 1993-06-21 1995-01-05 Enzon, Inc. Synthese de peptides conjuges modifies, sur des sites specifiques
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
KR960705579A (ko) * 1993-11-10 1996-11-08 에릭에스. 딕커 개선된 인터페론 중합체 결합체(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
FR2733914B1 (fr) 1995-05-11 1997-08-01 Sanofi Sa Composition de liquide stable contenant de l'urate oxydase et composition lyophilisee pour sa preparation
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
EP1731174A3 (fr) * 1996-08-02 2007-01-17 Ortho-McNeil Pharmaceutical, Inc. Polypeptides possédant un polyéthylène glycol à N-terminal par liaison via hydrazone ou oxime
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
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
EP1176985A2 (fr) * 1999-04-28 2002-02-06 Vectramed, Inc. Conjugues de medicaments polymeres actives par voie enzymatique
WO2000071602A1 (fr) * 1999-05-19 2000-11-30 Nof Corporation Polymere, materiau degradable in vivo et utilisation
EP1757311B1 (fr) 1999-12-24 2009-02-11 Genentech, Inc. Méthodes et compositions pour la prolongation de la demi-période d'élimination de composés bioactifs
EP2180054A1 (fr) 1999-12-24 2010-04-28 Genentech, Inc. Procédés et compositions pour prolonger la demi-vie d'élimination de composés bioactifs
SK11672002A3 (sk) 2000-01-10 2002-12-03 Maxygen Holdings Ltd. Polypeptidový konjugát, spôsob jeho výroby, farmaceutický prostriedok s jeho obsahom a jeho použitie
DE60138364D1 (de) 2000-02-11 2009-05-28 Bayer Healthcare Llc Gerinnungsfaktor vii oder viia konjugate
US20030211094A1 (en) 2001-06-26 2003-11-13 Nelsestuen Gary L. High molecular weight derivatives of vitamin k-dependent polypeptides
US7118737B2 (en) 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
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
EP2080771A3 (fr) 2001-02-27 2010-01-06 Maxygen Aps Nouvelles molécules de type interféron 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
AP2004003050A0 (en) * 2001-11-20 2004-06-30 Pharmacia Corp Chemically modified human growth hormone conjugates
RU2362807C2 (ru) 2002-06-21 2009-07-27 Ново Нордиск Хелт Кэр Аг Конъюгат полипептида фактора vii, способ его получения, его применение и содержащая его фармацевтическая композиция
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
EA008866B1 (ru) 2002-12-26 2007-08-31 Маунтин Вью Фамэсьютикэлс, Инк. ПОЛИМЕРНЫЙ КОНЪЮГАТ МОДИФИКАЦИЙ ИНТЕРФЕРОНА-β, ФАРМАЦЕВТИЧЕСКИЕ ПРОДУКТЫ НА ЕГО ОСНОВЕ И СПОСОБ ИХ ПОЛУЧЕНИЯ
US8034900B2 (en) 2002-12-30 2011-10-11 Amylin Pharmaceuticals, Inc. Water-soluble thioester and selenoester compounds and methods for making and using the same
WO2004075923A2 (fr) 2003-02-26 2004-09-10 Nektar Therapeutics Al, Corporation Conjugues de groupe fonctionnel a facteur polymerique viii
AU2004224466B2 (en) * 2003-03-28 2008-01-03 Biopolymed Inc. Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same
EP2055189A1 (fr) 2003-04-09 2009-05-06 Neose Technologies, Inc. Méthode de glycopegylation et proteines/peptides produits au moyen de ces méthodes
ATE459647T1 (de) 2003-04-15 2010-03-15 Glaxosmithkline Llc Humane il-18 substitutionsmutanten und deren konjugate
WO2004100997A2 (fr) * 2003-05-12 2004-11-25 Affymax, Inc. Nouveau fragment espaceur de composes peptidiques modifies par du poly(ethylene glycol)
SI1625156T1 (sl) 2003-05-12 2013-02-28 Affymax, Inc. Peptidi, ki se veĹľejo k eritropoetinskemu receptorju
JP4266028B2 (ja) 2003-05-12 2009-05-20 アフィーマックス・インコーポレイテッド エリスロポエチン受容体に結合する新規ペプチド
WO2005012484A2 (fr) 2003-07-25 2005-02-10 Neose Technologies, Inc. Conjugues anticorps-toxines
EP1654004A2 (fr) * 2003-08-08 2006-05-10 Novo Nordisk A/S Synthese et utilisation de nouveaux polymeres ramifies structuraux bien definis comme agents de conjugaison de peptides
DE602004029173D1 (de) 2003-10-10 2010-10-28 Novo Nordisk As Il-21-derivate
EP2633866A3 (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
EP1696947B1 (fr) 2003-12-19 2014-02-26 F.Hoffmann-La Roche Ag UTILISATION DE L'ÉRYTROPOïÉTINE POUR TRAITER LES PERTURBATIONS DE LA DISTRIBUTION DE FER DANS LES MALADIES INTESTINALES INFLAMMATOIRES CHRONIQUES
EP2327724A3 (fr) 2004-02-02 2011-07-27 Ambrx, Inc. Polypeptides d'hormone de croissance humaine et leur utilisations
JP2008502788A (ja) 2004-06-08 2008-01-31 アルザ コーポレイション ４成分縮合反応による高分子コンジュゲートの調製
KR101699142B1 (ko) 2004-06-18 2017-01-23 암브룩스, 인코포레이티드 신규 항원-결합 폴리펩티드 및 이의 용도
TWI418346B (zh) 2004-07-08 2013-12-11 伊蘭製藥公司 包括聚合物部分之多價ｖｌａ-４拮抗劑
JP2008507280A (ja) 2004-07-21 2008-03-13 アンブレツクス・インコーポレイテツド 非天然コードアミノ酸を用いた生合成ポリペプチド
US20080176790A1 (en) 2004-10-29 2008-07-24 Defrees Shawn Remodeling and Glycopegylation of Fibroblast Growth Factor (Fgf)
JP2008525473A (ja) 2004-12-22 2008-07-17 アンブレツクス・インコーポレイテツド 修飾されたヒト成長ホルモン
GB2438760A (en) 2004-12-22 2007-12-05 Ambrx Inc Methods for expression and purification of recombinant human growth hormone
CN102732588B (zh) 2004-12-22 2015-01-07 Ambrx公司 氨酰基-tRNA合成酶的组合物及其用途
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
CA2602654A1 (fr) 2005-04-05 2006-10-12 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Procede permettant de proteger des sites fonctionnels ou des epitopes sur des proteines
EP1877099B1 (fr) 2005-04-06 2012-09-19 Genzyme Corporation Conjugués thérapeutiques comprenant une enzyme lysosomale, l'acide polysialique et un agent de ciblage
US20070154992A1 (en) 2005-04-08 2007-07-05 Neose Technologies, Inc. Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US8324159B2 (en) 2005-06-03 2012-12-04 Affymax, Inc. Erythropoietin receptor peptide formulations and uses
WO2006133089A2 (fr) 2005-06-03 2006-12-14 Ambrx, Inc. Molecules d'interferons humains ameliorees et utilisations de celles-ci
US7550433B2 (en) 2005-06-03 2009-06-23 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
MX2008002149A (es) 2005-08-18 2008-04-22 Ambrx Inc Composiciones de arnt y sus usos.
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
EP1951890A4 (fr) 2005-11-16 2009-06-24 Ambrx Inc Procedes et compositions comprenant des acides amines non naturels
CA2647314A1 (fr) 2006-03-31 2007-11-08 Baxter International Inc. Facteur viii pegyle
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
KR20090013816A (ko) 2006-05-24 2009-02-05 노보 노르디스크 헬스 케어 악티엔게젤샤프트 연장된 생체내 반감기를 갖는 인자 ⅸ 유사체
ES2516694T3 (es) 2006-07-21 2014-10-31 Ratiopharm Gmbh Glicosilación de péptidos a través de secuencias de glicosilación con unión en O
WO2008025856A2 (fr) 2006-09-01 2008-03-06 Novo Nordisk Health Care Ag Protéines modifiées
MX2009002460A (es) 2006-09-08 2009-03-20 Ambrx Inc Arnt supresor hibrido para celulas de vertebrados.
CN106008699A (zh) 2006-09-08 2016-10-12 Ambrx公司 经修饰的人类血浆多肽或Fc骨架和其用途
EP2064333B1 (fr) 2006-09-08 2014-02-26 Ambrx, Inc. Transcription d'arnt suppresseur dans des cellules de vertébrés
US7985783B2 (en) 2006-09-21 2011-07-26 The Regents Of The University Of California Aldehyde tags, uses thereof in site-specific protein modification
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
CA2670618C (fr) 2006-12-15 2016-10-04 Baxter International Inc. Conjugue du facteur viia et de l'acide (poly)sialique presentant une demi-vie in vivo prolongee
SI2457920T1 (en) 2007-01-18 2018-02-28 Genzyme Corporation Oligosaccharides containing the amino acid group and their conjugates
US8106154B2 (en) 2007-01-31 2012-01-31 Affymax, Inc. Nitrogen-based linkers for attaching modifying groups to polypeptides and other macromolecules
CA2682147C (fr) 2007-03-30 2017-08-08 Ambrx, Inc. Polypeptides fgf-21 modifies, et leurs utilisations
KR20100016160A (ko) 2007-04-03 2010-02-12 바이오제너릭스 에이지 글리코페길화 ｇ―ｃｓｆ를 이용하는 치료 방법
MX2009011870A (es) 2007-05-02 2009-11-12 Ambrx Inc Polipeptidos de interferon beta modificados y usos de los mismos.
WO2008154639A2 (fr) 2007-06-12 2008-12-18 Neose Technologies, Inc. Procédé amélioré pour la production de sucres de nucléotide
MX2010005317A (es) 2007-11-20 2010-06-02 Ambrx Inc Polipeptidos de insulina modificados y sus usos.
NZ586947A (en) 2008-02-08 2012-11-30 Ambrx Inc Modified leptin polypeptides and their uses
KR101582841B1 (ko) 2008-02-27 2016-01-11 노보 노르디스크 에이/에스 콘쥬게이트된 인자 ｖｉｉｉ 분자
PT2318029T (pt) 2008-07-23 2018-01-10 Ambrx Inc Polipéptidos de g-csf bovino modificados e suas utilizações
MX2011003272A (es) 2008-09-26 2011-04-28 Ambrx Inc Polipeptidos de eritropoyetina animal modificados y sus usos.
PE20110480A1 (es) 2008-09-26 2011-07-01 Ambrx Inc Microorganismos y vacunas dependientes de replicacion de aminoacidos 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
CA3040973A1 (fr) 2008-12-16 2010-07-01 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
CN106110311A (zh) 2009-07-27 2016-11-16 百深公司 凝血蛋白缀合物
US8809501B2 (en) 2009-07-27 2014-08-19 Baxter International Inc. Nucleophilic catalysts for oxime linkage
KR101912335B1 (ko) * 2009-07-27 2018-10-26 리폭센 테크놀로지즈 리미티드 비혈액 응고 단백질의 글리코폴리시알화
CN102497884A (zh) 2009-07-27 2012-06-13 巴克斯特国际公司 凝血蛋白缀合物
US8642737B2 (en) 2010-07-26 2014-02-04 Baxter International Inc. Nucleophilic catalysts for oxime linkage
WO2011068853A2 (fr) * 2009-12-01 2011-06-09 Boston Medical Center Corporation Traitement de maladie médiée par ige
JP2013515081A (ja) 2009-12-21 2013-05-02 アンブルックス，インコーポレイテッド 修飾されているブタのソマトトロピンポリペプチドおよびそれらの使用
NZ600361A (en) 2009-12-21 2014-06-27 Ambrx Inc Modified bovine somatotropin polypeptides and their uses
CA2791841C (fr) 2010-03-05 2023-01-03 Rigshospitalet Molecules chimeriques inhibitrices d'activation du complement
EP2569331A1 (fr) 2010-05-10 2013-03-20 Perseid Therapeutics LLC Polypeptides inhibiteurs de vla4
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
MA34521B1 (fr) 2010-08-17 2013-09-02 Ambrx Inc Polypeptides de relaxine modifiés et leurs utilisations
TWI480288B (zh) 2010-09-23 2015-04-11 Lilly Co Eli 牛顆粒細胞群落刺激因子及其變體之調配物
HUE049352T2 (hu) 2010-12-22 2020-09-28 Baxalta GmbH Anyagok és módszerek egy vízoldható zsírsavszármazéknak egy fehérjéhez való konjugálására
EP2726502A1 (fr) 2011-07-01 2014-05-07 Bayer Intellectual Property GmbH Polypeptides de fusion de relaxine et leurs utilisations
ES2582869T3 (es) 2011-07-05 2016-09-15 Bioasis Technologies Inc Conjugados de P97-anticuerpo
WO2013138730A1 (fr) * 2012-03-16 2013-09-19 Enzon Pharmaceuticals, Inc. Conjugués polymères d'inhibiteurs de la c1-estérase
PL2859017T3 (pl) 2012-06-08 2019-07-31 Sutro Biopharma, Inc. Przeciwciała zawierające swoiste dla miejsca, niewystępujące naturalnie reszty aminokwasowe, sposoby ich wytwarzania i sposoby ich zastosowania
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
HRP20220455T1 (hr) 2012-08-31 2022-05-27 Sutro Biopharma, Inc. Modificirane aminokiseline koje sadrže azid grupu
AU2013341711A1 (en) 2012-11-12 2015-05-21 Redwood Bioscience, Inc. Compounds and methods for producing a conjugate
CN104870423A (zh) 2012-11-16 2015-08-26 加利福尼亚大学董事会 用于蛋白质化学修饰的pictet-spengler连接反应
US9310374B2 (en) 2012-11-16 2016-04-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
JP6586412B2 (ja) 2013-03-13 2019-10-02 バイオアシス テクノロジーズ インコーポレイテッド ｐ９７のフラグメントおよびその使用
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
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
EA023323B1 (ru) * 2013-03-28 2016-05-31 Илья Александрович МАРКОВ Разветвленный ацилазидный пегилирующий агент, способ его получения и способ получения пегилированного интерферона
EA021610B1 (ru) * 2013-03-28 2015-07-30 Илья Александрович МАРКОВ Жидкое противовирусное лекарственное средство
EA023360B1 (ru) * 2013-03-28 2016-05-31 Илья Александрович МАРКОВ Линейный ацилазидный пегилирующий агент, способ его получения и способ получения пегилированного интерферона
ES2865473T3 (es) 2013-07-10 2021-10-15 Sutro Biopharma Inc Anticuerpos que comprenden múltiples residuos de aminoácidos no naturales sitio-específicos, métodos para su preparación y métodos de uso
US20150093399A1 (en) 2013-08-28 2015-04-02 Bioasis Technologies, Inc. Cns-targeted conjugates having modified fc regions and methods of use thereof
EP3055298B1 (fr) 2013-10-11 2020-04-29 Sutro Biopharma, Inc. Acides aminés modifiés comprenant des groupes fonctionnels de tétrazine, procédés de préparation et procédés d'utilisation associés
WO2015081282A1 (fr) 2013-11-27 2015-06-04 Redwood Bioscience, Inc. Composés hydrazinyl-pyrrolo et procédés de production d'un conjugué
SMT202100388T1 (it) 2014-10-24 2021-09-14 Bristol Myers Squibb Co Polipeptidi fgf-21 modificati e loro usi
JP2018519359A (ja) * 2015-06-11 2018-07-19 アンビオ ファーマシューティカルズ，エルエルシー Ｐｅｇ化顆粒球コロニー刺激因子（ｇｃｓｆ）
DK3307326T3 (da) 2015-06-15 2020-10-19 Angiochem Inc Fremgangsmåder til behandling af leptomeningeal karcinomatose
KR102783230B1 (ko) 2015-11-09 2025-03-19 알.피.쉐러 테크놀러지즈 엘엘씨 항-cd22 항체-메이탄신 콘쥬게이트 및 그것의 사용 방법
EP4610656A3 (fr) 2016-04-15 2025-11-19 Beckman Coulter, Inc. Macromolécules photoactives et leurs utilisations
KR20230159629A (ko) 2016-12-12 2023-11-21 벡톤 디킨슨 앤드 컴퍼니 수용성 중합체 염료
CN110637027B (zh) 2017-02-08 2024-08-30 百时美施贵宝公司 包含药代动力学增强子的修饰的松弛素多肽及其用途
WO2019010484A2 (fr) 2017-07-07 2019-01-10 Symic Ip, Llc Bioconjugués synthétiques
CN119286279A (zh) 2017-12-26 2025-01-10 贝克顿·迪金森公司 深紫外线可激发的水溶剂化聚合物染料
CN119220109A (zh) 2018-03-30 2024-12-31 贝克顿·迪金森公司 含侧基发色团的水溶性聚合染料
US20220162336A1 (en) 2018-07-22 2022-05-26 Bioasis Technologies, Inc. Treatment of lymphatic metastases
HRP20240016T1 (hr) 2018-09-11 2024-03-29 Ambrx, Inc. Konjugati polipeptida interleukina-2 i njihove uporabe
WO2020082057A1 (fr) 2018-10-19 2020-04-23 Ambrx, Inc. Conjugués polypeptidiques d'interleukine-10, dimères de ceux-ci et leurs utilisations
JP7695885B2 (ja) 2019-02-12 2025-06-19 アンブルックス，インコーポレイテッド 抗体－ｔｌｒアゴニストコンジュゲートを含有する組成物、その方法、及び使用
WO2021183832A1 (fr) 2020-03-11 2021-09-16 Ambrx, Inc. Conjugués polypeptidiques d'interleukine-2 et leurs procédés d'utilisation
US20210355468A1 (en) 2020-05-18 2021-11-18 Bioasis Technologies, Inc. Compositions and methods for treating lewy body dementia
US20210393787A1 (en) 2020-06-17 2021-12-23 Bioasis Technologies, Inc. Compositions and methods for treating frontotemporal dementia
EP4199968A1 (fr) 2020-08-20 2023-06-28 Ambrx, Inc. Conjugués anticorps-agonistes de tlr, procédés et utilisations de ceux-ci
US20250161471A1 (en) 2021-04-03 2025-05-22 Feng Tian Anti-her2 antibody-drug conjugates and uses thereof
EP4155349A1 (fr) 2021-09-24 2023-03-29 Becton, Dickinson and Company Colorants absorbants jaunes verts solubles dans l'eau
EP4547759A2 (fr) 2022-07-01 2025-05-07 Beckman Coulter, Inc. Nouveaux colorants fluorescents et polymères issus de dérivés de dihydrophenanthrène
WO2024044327A1 (fr) 2022-08-26 2024-02-29 Beckman Coulter, Inc. Monomères dhnt et colorants polymères ayant des propriétés photophysiques modifiées
CN120936679A (zh) 2023-03-17 2025-11-11 贝克曼库尔特有限公司 苯并噻吩并吡咯花菁染料
WO2025064842A1 (fr) 2023-09-21 2025-03-27 Beckman Coulter, Inc. Colorants pontés par dihydrophénanthrène (dhp) destinés à être utilisés en cytométrie en flux
WO2026043823A2 (fr) 2024-08-19 2026-02-26 Sutro Biopharma, Inc. Anticorps comprenant des résidus d'acides aminés non naturels spécifiques à un site, leurs procédés de préparation et leurs utilisations

Citations (4)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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
WO1992016555A1 (fr) 1992-10-01
JPH06506217A (ja) 1994-07-14
AU1676992A (en) 1992-10-21
EP0576589A1 (fr) 1994-01-05
CA2101918A1 (fr) 1992-09-19

Similar Documents

Publication Publication Date Title
WO1992016555A1 (fr) Conjuges de polypeptides et de glycopolypeptides associes a des polymeres, contenant de l'hydrazine
US5643575A (en) Non-antigenic branched polymer conjugates
US7511095B2 (en) Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith
US6566506B2 (en) Non-antigenic branched polymer conjugates
US5321095A (en) Azlactone activated polyalkylene oxides
US7419600B2 (en) Method for purifying a branched water-soluble polymer
WO1994028024A1 (fr) Polymere modifie aux glucides presentant une activite erythropoïetique
WO1996041813A2 (fr) Polymeres fonctionnalises destines a une ligation dirigee
JP2006321808A (ja) 化学的に修飾されたヒト成長ホルモンコンジュゲート
AU2002360257A1 (en) Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith
US20070117924A1 (en) Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same
EP0539167A2 (fr) Imidates de PEG et leurs dérivés protéiniques
EP0632082B1 (fr) Préparation de carbamates actifs de polyalkylène glycol et leur utilisation
Bonora et al. Reactive PEGs for protein conjugation

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930908

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MENON-RUDOLPH, SUNITHA

Inventor name: LEE, CHYI

Inventor name: ZALIPSKY, SAMUEL

A4 Supplementary search report drawn up and despatched

Effective date: 19940606

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE

18D Application deemed to be withdrawn

Effective date: 19971001