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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8114—Kunitz type inhibitors
  • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
  • C12Y111/01—Peroxidases (1.11.1)
  • C12Y111/01006—Catalase (1.11.1.6)

Definitions

• the present invention relates to methods for derivatising proteins, in which a denaturant is included in the reaction mixture, to achieve high degrees of substitution.
• the method is of particular applicability to derivatisation methods involving reaction of an aldehyde reagent with epsilon amino groups of non-terminal lysyl units of proteins.
• Novel derivatised protein compounds have high degrees of substitution of polysialic acid chains.
• a new method in which a protein having at least two derivatisable pendant groups (being side chains of amino acyl units) is reacted with a derivatising reagent in aqueous solution to provide a protein derivative, and is characterised in that the derivatisation reaction is carried out in the presence of an effective denaturing concentration of a denaturant.
• the degree of substitution of the product at said pendant groups is at least two equivalent groups per mole of protein.
• the denaturant may be a chaotropic ion, such as I " or
• SCN “ a large anion derived from a strong acid, such as CIO 4 " or CCI 3 COO " , an organic solvent, urea or a derivative such as a guanidine compound.
• a strong acid such as CIO 4 " or CCI 3 COO "
• an organic solvent such as a guanidine compound.
• urea such as a guanidine compound.
• it is an amphiphilic compound, more preferably an anionic amphiphile.
• the anionic amphiphile is preferably a sulphate mono ester of 0 an alcohol having 8 to 24 carbon atoms.
• it is added to the reaction mixture in the form of an alkali metal salt.
• the amphiphile is a sodium or potassium C 8 . 24 alkyl sulphate, most preferably sodium dodecyl sulphate.
• amphiphile is generally present at a concentration 5 in the range 0.0001 to 0.01 M, most preferably in the range 0.0005 to 0.005M, most preferably about 0.001 M.
• the starting protein has at least 2, more preferably at least 5, for instance 10 or more derivatisable groups, all of the same nature.
• the groups may be hydroxyl groups, thiol groups, carboxylic acid groups or, o preferably primary amine groups.
• Most preferably the derivatisable groups are side chains of lysyl units.
• the protein therefore, has at least 2, more preferably at least 5, for instance 10 or more lysine units in the backbone.
• at least 2 reactive groups are derivatised more preferably at least 5 reactive groups are derivatised, that is the degree of derivatisation of the protein is at least 5.
• the derivatising reagent is a compound having a reactive group suitable for reacting with protein in aqueous solution, optionally in the presence of coupling compounds.
• Coupling compounds and activation chemistries are described in, for instance, "Methods in Enzymology” 135B (Immobilised Enzymes and Cells), 1987, Mosbach ed, Academic Press Inc., New York and in Nucci, M.L. et al. Adv. Drug Delivery Reviews 6, 133-151 (1991 ).
• the reagent is, for instance, a compound used to confer stability, reduce immunogenicity or increase circulation time or solubility of a protein, or to target the protein. It may be an oligomeric or polymeric compound, such as an oligo- or polysaccharide, poly (hydroxyalkyl(alk)acrylamide or -(alk)acrylates), polyvinyl alcohol or polyalkylene glycol, eg polyethylene glycol.
• the derivatising reagent may, for instance, be an activated polyethylene glycol (monofunctional), such as tresyl-PEG described in WO- A-9004606 or a succinimidyl succinate ester of PEG. These compounds may react with hydroxyl and thiol groups as well as amine groups.
• the present invention is of particular utility where the derivatising reagent to be reacted with primary amine pendant groups is an aldehyde compound.
• the aldehyde compound is a derivative of a saccharide or polysaccharide, for instance produced by the controlled oxidation of an alcohol.
• the aldehyde is generated in a preliminary step which may be a first step in the process of the invention, in which a saccharide or polysaccharide is reacted under controlled oxidation conditions, for instance using sodium periodate, in aqueous reaction.
• the saccharide or polysaccharide is sialic acid, or a derivative thereof, most preferably a polysaccharide having a terminal sialic acid group, and most preferably is a polysialic acid, that is a polysaccharide comprising at least 5 sialic acid units joined to one another through 2 ⁇ 8 or 2 -> 9 linkages.
• a suitable polysialic acid has a weight average molecular weight in the range 2 to 2000 kDa, preferably in the range 5 to 50 kDa.
• the polysialic acid is derived from a bacterial source, for instance being, or being derived from, polysaccharide B of E. coli Kl, N. meningitidis, Moraxella liquefaciens or Pasteurella aeruginosa or K92 polysaccharide of E. coli K92 strain. It is most preferably colominic acid from E. coli K1.
• proteins having at least 5 pendant polysialic acid chains are new. According to a further aspect of the invention there is provided a new protein having at least 5 pendant polysialic acid chains, each having at least 5 sialic acid units joined to one another.
• a sialic acid reagent is, for instance, reacted with sodium periodate under controlled oxidation conditions to form a terminal aldehyde at the C 7 atom.
• the oxidation conditions preferably involve sodium periodate at a concentration of around 0.1 M, being used in excess to derivatise a solution of polysialic acid.
• Reaction conditions preferably involve reaction at room temperature for 5 to 60 minutes. In order to deactivate excess periodate, conventional means such as reaction with ethylene glycol, are used.
• the derivatisation step of the method of the invention is preferably carried out with the protein in the aqueous reaction mixture at a temperature in the range 0 to 60°C, preferably 10 to 45°C, for instance at a raised temperature in the range 30 to 40°C.
• the protein is preferably present at a concentration in the range 0.1 to 100 g/l, preferably in the range 1 to 20 g/l.
• the reaction mixture may contain other ingredients such as dissolved inorganic salts, for instance to buffer the solution of a suitable pH.
• the protein which is derivatised in the invention may be, for instance, a therapeutically active compound.
• the derivatisation reaction may, for instance, be for controlling the hydro- or lipo-philicity of the protein, for instance to adjust its solubility in liquid media, especially to increase its hydrophilicity and solubility in aqueous media.
• the derivatisation reaction may be, as in our earlier publication WO-A-92/22331 , for increasing the circulation time, decreasing the immunogenicity and/or increasing the stability on storage, in vitro or in vivo, of a therapeutic compound.
• Derivatisation with poly(ethylene glycol) increases hydrophilicity of the protein; which may increase aqueous solubility or availability, increase circulation time or decrease the immunogenicity of the protein.
• the presence of the denaturant compound increases the degree of derivatisation, thereby enhancing the improvement in solubility, circulation time and/or immunogenicity or the increase in stability of the protein. It has been found that this increase in degree of derivatisation may be carried out without adversely affecting the activity of the protein, for instance its enzyme activity. Thus, in contrast to the findings of Visser ef al (op. cit.) the presence of the anionic amphiphile does not irreversibly deactivate the protein, indeed, as shown below, it inhibits deactivation.
• the derivatisation may alternatively be for providing active or passive targeting by attachment of polymers or binding ligands.
• cytokines such as interleukins, for instance IL-2, IL-6 or IL-1 , interferons, tumour necrosis factor (TNF), growth factors, peptide hormones, such as insulin, as well as enzymes for instance for use in enzyme therapy, as well as immuno- globulins, and aprotinin.
• the protein may be a carrier or adjuvant and the derivatising reaction conjugates pharmaceutically active, or diagnostically useful ligands to the protein to optimise the delivery of the useful ligand to a target issue.
• Figure 1 is a reaction scheme for the derivatisation of polysialic acid
• Figure 2 shows results of Example 1 ;
• Figure 3 shows results of Example 1 ; and Figure 4 shows the results of Example 6.
• catalase is used as a model protein.
• Catalase is a tetrameric haemprotein that catalyses the degradation of hydrogen peroxide to Oxygen and water.
• the reaction can be used to determine enzyme activity.
• the reaction is a first order reaction, whereby the amount of peroxide substrate decomposed is directly proportional to the concentration of both substrate and enzyme. Provided the concentration of substrate is constant between experiments, any difference in decomposition rate will therefore be a function of enzyme activity present.
• the drop in absorbance hydrogen peroxide at 240 ⁇ m from 0.450 to 0.400, corresponding to the decomposition of 3.45 ⁇ moles of hydrogen peroxide in a cuvette having a reaction volume of 3 ml is determined.
• Catalase concentration is determined spectrophotometrically.
• Catalase, insulin, IgG and aprotinin were radiolabelled using the conventional methods using 125 l, usually the chloramine-T method. Radiolabelled protein in the circulation was determined using techniques described in Fernandes, A.I. et al, Biochem. Biophys. Acta (1997) 1341 , 26-34.
• Colominic Acid is a derivative of E. coli K1 , having an average molecular weight of around 10kDa.
• the polysaccharide consists substantially only of 2-8 linked sialic acid units.
• the activation of (oxidation) colominic acid is carried out as follows. A 0.1M aqueous solution of sodium periodate is formed. 1 ml of the sodium periodate solution is mixed with 10 mg colominic acid in the dark, and the reaction mixture stirred for 15 minutes at room temperature and pressure (around 20°C, 1 bar). The reaction is terminated by addition of 2 ml ethyleneglycol, followed by stirring for 30 minutes under the same conditions. Subsequently the mixture is extensively dialysed at 40°C against a 0.01 % by weight ammonium carbonate buffer. The dialysate is freeze- dried overnight, then refrigerated until further use. The reaction scheme is shown in Figure 1.
• Sialic acid is determined by forming a 0.5 ml sample (having a concentration in the range 4 to 40 mg/ml sialic acid) in aqueous solution, added to this 0.5 ml resorcinol reagent. The mixture is boiled in a water bath for 30 minutes in sealed tubes. The mixture is cooled for 20-30 minutes and its absorbance read at 570 nm against control mixtures of appropriate buffer (i.e. of the type and concentration in which the original sample is presented) and reagent. Determination of Poly(ethylene glycol)
• the level of pegylation was estimated by the assay of PEG using ammonium ferrithiocyanate (A. Nag, G. Micra and C ghost, Anal. Biochem. 237:224-231 , 1996).
• the present example illustrates the effect of reaction time in the activated colominic acid - catalase derivatisation reaction, by assessing the catalase activity after recovery of the derivatised enzyme.
• the derivatisation was carried out using 24 mg of catalase with 50 mg of activated colominic acid, in the presence of 20 mg sodium cyanoborohydride in 5ml potassium hydrogen phosphate buffer. The reactants are stirred for a period up to 48 hours at 35 to 40°C.
• solid SDS is dissolved in the phosphate buffer to provide a final concentration of 1 x 10 '3 M SDS.
• reaction times of zero hours i.e. as quickly as possible after the reaction mixture is made up
• 6 hours, 12 hours, 24 hours and 48 hours the reaction is stopped by addition of 70% ammonium sulphate solution to precipitate out protein.
• the precipitated mixture is cooled on ice and stirred for one hour, then centrifuged at 3500 rpm for 45 minutes. The supernatant is discarded and the pellet washed with saturated ammonium sulphate solution, spun again for ten minutes at the same speed, and the supernatant discarded.
• the pellet is redissolved in 5 ml phosphate buffered saline.
• the resultant solution is dialysed extensively at -4°C against four changes of phosphate buffered saline.
• the solution is then passed down a Sephadex (trade mark) G-100 column and peaks collected and assayed for catalase and colominic acid content.
• Figure 2 indicates the conjugation ratio (degree of substitution) of colominic acid with catalase in the presence and absence of SDS.
• the results show that the presence of SDS increases the maximum conjugation ratio by a factor of about 3.
• the maximum level of derivatisation in the presence of SDS appears to be around 8 moles colominic acid per mole catalase.
• the derivatised catalase compounds were also tested against native catalase for their enzyme activity. The results are shown in Figure 3.
• the results for catalase alone in Figure 3 show the effect of subjecting catalase to the reaction conditions but without the addition of activated colominic acid. This shows that catalase activity is lost under those conditions, but that the loss of activity is inhibited by polysialylation. The inhibitor is greater when the polysialylation takes place in the presence of SDS. 0
• the derivatised catalase products were assessed for their catalase activity using the general test described above. The times taken for the enzymes to reduce the absorbance from 0.45 to 0.40 are shown in table 1 as compared to native catalase. 0
• the derivatised protein is recovered using the same general technique as in example 1 , but using centrifugation at 9000 rpm.
• the column used for isolation is Sephadex G-50.
• Table 2 shows the conjugation yields, that is the degree of derivatisation, with and without SDS, for the four proteins.
• Aprotinin has 4 lysyl units having derivatisable amine groups and 2 terminal amino groups available for derivatisation by aldehyde reagents.
• the labelled insulin, aprotinin and IgG, in their native forms, and derivatised with colominic acid (polysialic acid PSA) in the presence and absence of SDS are administered to mice to determine the rate of clearance from the circulation.
• the in vivo tests are carried out using the general technique described in example 1 of WO-A-92/22331 , by injection of protein or derivatised protein in the dose shown in table 3.
• the animals were bled from the tail vein immediately before and, immediately after, at 30 minutes, 1 hour, 4 hours, 6 hours, 12 hours, 24 and 48 hours after injection to determine the level of 1 5 l label remaining in the circulation. From the logarithmic curve of percent initial radioactivity against time following injection, the area under the curve is determined. The results for the various proteins are shown in table 3.
• the derivatisation method according to Example 1 was repeated using IgG in place of catalase. 5M urea or 1 x 10 "3 M SDS were used. The reaction in individual aliquots of reaction mixture was stopped after 6, 12, 24 and 48 hours. The degree of derivatisation was determined after assessing the level of protein using the Bradford technique described above and the level of sialic acid using the resorcinol method described above. The results are shown in Figure 4.
• IgG as used in the above examples was subjected to derivatisation with oxidised colominic acid (CA) in the absence and presence of 10 "3 M SDS under the general conditions used in Example 4.
• CA oxidised colominic acid
• ssPEG monomethoxy poly(ethlene glycol) succinimidyl succinate
• IgG in 0.2M phosphate buffer, pH 7.2 was allowed to react with a 50-fold molar excess of methoxypolyethylene glycol succinimidyl succinate (ss-PEG) at room temperature for 10 min. The reaction was stopped by addition of 5-fold molar excess ⁇ -amino caproic acid over ssPEG. The resulting PEG-lgG was purified and separated by gel permeation chromatography (SG-100; 0.2M phosphate buffer). For the reaction in the presence of SDS, the IgG was first contacted with 10 "3 M SDS in the 0.2M phosphate buffer at raised temperature for 12 hours. Otherwise the reaction and recovery was the same. The degree of derivatisation of pegylated and polysialyated IgG's was determined using the methods described above. The results are shown in table 4.
• the presence of SDS increases the level of derivatisation for a PEG reagent as well as for a polysialic acid reagent.
• the PEG reagent gives a higher degree of substitution than the colominic acid reagent.

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  • 2001-05-14 AU AU2001258536A patent/AU2001258536A1/en not_active Abandoned
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