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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
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/644—Coagulation factor IXa (3.4.21.22)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21022—Coagulation factor IXa (3.4.21.22)

Definitions

• the invention relates to a method for purifying Factor B from human blood plasma.
• Complement plays a vital role in the defense of the body against infectious agents and in the inflammatory process. It represents an auxiliary system of immunity, in particular of the humoral response and the innate response.
• the complement comprises a set of approximately 30 plasma and sometimes membrane proteins, synthesized essentially by the liver and macrophages and which can be activated by proteolytic cascades. It includes both plasma proteins, many different cell surface receptors, some present on inflammatory cells and others on immune system cells, as well as regulating membrane proteins that protect host cells from autoattacking. . Plasma complement proteins function either as enzymes, as binding proteins, or as regulators (inhibitors or activators).
• Factor complement is a component of the alternative pathway of complement activation. It circulates in the blood as a protein composed of a single polypeptide chain of 93 kDa molecular weight. Upon activation of the alternate pathway, Factor B is cleaved by the resulting Complement Factor D into an inactive Ba (30 kDa) chain and the Bb (63 kDa) catalytic subunit.
• the active subunit Bb is a serine protease that associates with C3b to form the C3 converting C3 convertase of the alternative pathway (C3bBb).
• the Bb subunit is involved in the proliferation of pre-activated B lymphocytes, while the Ba chain inhibits their proliferation.
• Factor B preparations would be useful for the treatment of Factor B deficiencies, but for the time being no such preparation is commercially available. Summary of the invention
• the invention provides a method of purifying Factor B, comprising the steps of:
• step (ii) subjecting the fraction obtained in step (i) to heparin affinity chromatography
• step (iii) subjecting the fraction enriched in Factor B obtained in step (ii) to cation exchange chromatography;
• the method may further comprise at least one viral inactivation treatment, preferably by the action of solvent and detergent, and / or at least one viral elimination treatment, preferably by nanofiltration.
• the subject of the invention is also a process for obtaining a Factor B preparation for therapeutic use, which comprises
• the invention also relates to the preparation of Factor B obtainable by this method.
• Preferably said preparation is in freeze-dried form.
• the method of the invention has many advantages: it is industrializable, allows to purify Factor B from various production intermediates, and with a high degree of purity.
• FIG. 1 shows a diagram showing the steps of the purification process followed in Example 1. Detailed description of the invention
• Vector B any protein having the amino acid sequence of human native Factor B.
• the term “Factor B” further includes naturally occurring allelic variations and / or naturally occurring Factor B isoforms, and any form or degree of glycosylation or other post-translational modification. Also included are homologues or derivatives of Factor B which exhibit the same or higher biological activity with respect to the activity of the wild-type form and / or which have a sequence identity of at least 80%, preferably at least 85%. %, more preferably at least 90%.
• Factor B activity includes the ability to activate C3 convertase.
• Factor B activity can be measured in various ways, well known to those skilled in the art.
• a chromatography consists of contacting the Factor B in solution
• the starting material used is the supernatant of a cryoprecipitate of blood plasma optionally mixed with a plasma fraction not retained on anion exchange chromatography, DEAE-Sephadex type. It is also possible to use the supernatant resulting from an ethanolic precipitation. An unrestrained plasma fraction can also be used on anion exchange chromatography.
• Heparin-type affinity chromatography uses a matrix or a support, which is most often an agarose gel, on which heparin or heparin derivatives or mimetics are grafted.
• heparin-type ligands mention may be made especially of the following ligands: chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, or synthetic oligomers, for example cellubiosis sulfate ester (Matrex TM Cellufine Sulfate), or therapeutic analogs of heparin, for example sulfated oligosaccharides.
• the pH is adjusted to a value of 6, for example by equilibrating the column with a 20 mM sodium phosphate buffer solution, with an osmolality of 60 ⁇ 5 mOsm / kg, and a pH of 6.0 ⁇ 0.1.
• Example 1 An example of a particular protocol is presented in Example 1.
• a cation exchange chromatography uses a matrix, most often of the agarose or polymeric type, on which are grafted ligands such as carboxymethyl (CM), sulfopropyl (SP) or methyl sulfonate (S).
• Cation exchange supports are commercially available, for example CM-Sepharose, SP-Sepharose and S-Sepharose (GE Healthcare), Toyopearl CM-650, Toyopearl SP-650 (Tosoh Biosciences), Fractogel EMD carriers.
• SP sepharose FF is used.
• An example of a particular protocol is presented in Example 1.
• Factor B can under certain conditions bind to anion exchange resins, and be desorbed by high ionic buffer systems.
• Anion exchange chromatography uses a matrix, most often of the agarose or polymeric type, on which are grafted ligands such as diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) or quaternary ammonium (Q).
• DEAE diethylaminoethyl
• Q quaternary aminoethyl
• Q quaternary ammonium
• Anion exchange supports are commercially available, for example DEAE-Sepharose, Q-Sepharose (GE Healthcare), Toyopearl DEAE-650, Toyopearl Super Q-650 (Tosoh Biosciences), Fractogel EMD DEAE, Fractogel EMD TMAE (Merck KGaA), Macro-Prep DEAE Support, Macro-Prep High Q Support (BioRad), DEAE HyperD, Q HyperD, DEAE Trisacryl, DEAE Sperodex (Pall garlic).
• Q sepharose FF is used.
• An example of a particular protocol is presented in Example 1.
• Factor B is eluted by increasing the ionic strength of the equilibration buffer of anion exchange chromatography.
• the pH of the fraction of blood plasma containing Factor B and obtained in step (i) is adjusted to be in the range of pH 5.5 to pH 6.5 and preferably to be equal to pH 6.0.
• the pH of the fraction eluted from step (ii) and then diluted before step (iii) is adjusted to be in the range from pH 5.5 to pH 7.5.
• the pH of the eluted fraction of step (iii) and then diluted before step (iv) is adjusted to be in the range of 5.5 to 7.5.
• the purification of the factor B comprises the steps of:
• step (iii) (a) subjecting the fraction obtained in step (ii) to cation exchange chromatography, for example on SP-Sepharose;
• step (iv) subjecting the fraction enriched in Factor B obtained in step (iii) (a) or (b) to anion exchange chromatography, for example on Q-Sepharose.
• the process In order to obtain a preparation for therapeutic use, the process generally continues with formulation, concentration and then filtration steps of the Factor B concentrate.
• the method of manufacturing a Factor B preparation according to the invention may comprise the following steps:
• the chromatography of step (ii) is the only heparin-type affinity chromatography, the method comprising no additional step of heparin-type affinity chromatography.
• the method of the invention contains no chromatography other than those provided in steps (ii) to (iv) defined above.
• process of the invention may optionally comprise additional steps, for example other anion or cation exchange chromatographies, but also optionally one or more hydrophobic interaction chromatographies.
• Factor B can, under certain conditions, bind to hydrophobic interaction chromatography resins, and be desorbed by low ionic strength buffer systems.
• Such a chromatography uses a matrix, most often of agarose or polymeric type, on which are grafted ligands such as phenyl, octyl, butyl, methyl, or hexylamine, phenylpropylamine, 4-mercapto-ethyl-pyridine.
• grafted ligands such as phenyl, octyl, butyl, methyl, or hexylamine, phenylpropylamine, 4-mercapto-ethyl-pyridine.
• Supports for this type of chromatography are commercially available, for example phenyl sepharose, octyl sepharose, butyl sepharose and Capto TM carriers.
• the process of the invention may also comprise a chromatography of hydroxyapatite type. Indeed, Factor B can be purified using calcium phosphate, fluoroapatite and hydroxyapatite composites as the solid phase.
• Bio-Gel Hydroxyapatite HT Bio-Rad
• Ceramic Fluoroapatite Ceramic Fluoroapatite
• Ceramic Hydroxyapatite Bio-Rad
• HA Ultrogel Pall
• the method of the invention may also comprise immunoaffinity chromatography, which uses, for example, antibodies or aptamers immobilized on a chromatography matrix.
• the matrices are generally pre-activated resins, for example by epoxy (Sepharose 6B1 EAH Sepharose 4B, Amino Sepharose 6 FF, 6-AKS Sepharose 4FF, Toyopearl AF Epoxy, Toyopearl AF Amino, Toyopearl AF Tresyl).
• the unbound impurities are washed using suitable buffer systems (citrate buffer, phosphate buffer, or HEPES, for example), and the Factor B is then eluted using, for example, high concentrations of chaotropic salts (thiocyanate type, lithium bromide). ), buffer systems with low pH (glycine pH 2-3) or high pH (TRIS pH 9).
• the method of the invention may also include other treatments, such as delipidation.
• Delipidation consists in eliminating, preferably upstream or as soon as possible in the process, lipid impurities from the protein solutions by means of, for example, precipitation or adsorption.
• Suitable precipitating agents include, for example, polyethylene glycols (PEG) or ammonium sulfate, while silica powders ("fumed silica", Aerosil 200, Aerosil 380), dextran sulfates or fluorocarbons (Freon-13) are suitable adsorbents.
• proteolytic impurities ie enzymes, for example proteases or glycosidases, which would be able to cleave the Factor B molecule into inactive truncated forms.
• This elimination or inhibition is preferably carried out as soon as possible, preferably further upstream of the purification process.
• a filter which retains a part of the proteolytic impurities (Sartoclear® filter type).
• protease inhibitors may be, for example, benzamidine, 4-aminobenzamidine, benzamidine hydrochloride and its derivatives, lysine and its derivatives, 6-aminohexanoic acid ( ⁇ -aminocaproic acid) and its derivatives, trans-4-aminomethylcyclohexanecarboxylic acid (acid tranexamic) and its derivatives, 4- (2-aminoethyl) benzenesulfonylfluoride (AEBSF) and its derivatives, (4-Amidino-Phenyl) -methane-Sulfonyl Fluoride (APMSF) and its derivatives, 3,4-dichloroisocoumarin (DCI) and its derivatives, acetyl-leucyl-leucyl-arginal (Leupeptin) and its derivatives, aprotinin and its derivatives, trypsin inhibitor of soya, and its derivatives, ⁇
• proteolytic impurities can be inhibited by adding one or more protease inhibitors of the above-mentioned group, particularly antithrombin II and C1 inhibitor, and then eliminating the enzyme / inhibitor complex at the same time. subsequent purification steps.
• immobilized dyes such as Cibacron Blue F3GA or its derivatives, other triazine dyes such as Procion Red HE-3B and its derivatives, or Procion Green H-4G and its derivatives, Reactive Red 120 and its derivatives, Reactive Green 19 and its derivatives, Reactive Yellow 86 and its derivatives, Reactive Orange 14 and its derivatives, immobilized on agarose matrices or polymeric matrix.
• the preparation of Factor B useful in the invention has generally undergone at least one step of removing or inactivating at least one infectious agent.
• Infectious agents include viruses and NCTAs (unconventional transmissible agents) such as prions.
• Viral inactivation often involves treatment with chemicals, for example solvent, detergent and / or heat, for example by pasteurization.
• Nanofiltration is also useful for removing an infectious agent.
• the process comprises at least a solvent and detergent treatment, and a nanofiltration.
• Pasteurization refers to methods exposing liquid Factor B compositions at a temperature of 60 ° C for at least 10h.
• the treatment with solvent and / or detergent comprises in particular the treatment with tri-n-butyl phosphate TnBP and / or a detergent which is selected from Triton X-100, Tween (preferably Tween 80) and sodium cholate.
• the treatment is generally continued at 25 ° C for at least 6 hours.
• Nanofiltration generally refers to the filtration of a Factor B solution through a filter with a pore size of less than 80 nm.
• Available filters include Planova TM 75nm, Planova TM 35nm, Planova TM 20nm or Planova TM 15nm, BioEX (Asahi corporation), Ultipor DV 50 or DV 20 (Pall Corporation), Virosart CPV (Sartorius), Viresolve NFR or NFP (Millipore).
• nanofiltration is carried out after step (iv) of anion exchange chromatography.
• the purified Factor B fraction is filtered on a filter sequence with a pore size of 20 nm and 15 nm.
• the obtained Factor B preparation is formulated with suitable excipients and stabilizers. They may be diluents, agents, cryoprotectants, lyoprotectants, etc.
• sugars sucrose, trehalose, glucose, lactose, etc.
• polyols mannitol, sorbitol
• amino acids glycine, arginine, histidine, alanine
• Polysorbate surfactants Teween 20 or Tween 80 for example
• polyoxamer for example poloxamer 188
• polyethylene glycol can also be added.
• Antioxidants eg methionine, monothioglycerol, glutathione, citric acid, ascorbic acid, sodium metabisulfite, and sodium sulfite
• Antioxidants eg methionine, monothioglycerol, glutathione, citric acid, ascorbic acid, sodium metabisulfite, and sodium sulfite
• Buffer substances may be further used, for example in the form of carbonate, phosphate, citrate, acetate, borate, trimethamine [(2-amino-2-hydroxymethyl-1, 3-propanediol), TRIS], glycine and lysine.
• the liquid formulation of Factor B can desiccate if necessary, to obtain a solid form.
• Desiccation is a method of removing water at a high stage. It is a dehydration to remove as much water as possible. This phenomenon can be natural or forced. This desiccation can be carried out using freeze-drying, atomization and cryoatomization techniques.
• the preferred mode of obtaining the solid form of the composition for pharmaceutical use according to the invention is lyophilization.
• Lyophilization methods are well known to those skilled in the art, see for example Wang et al, Lyophilization and Development of Solid Protein Pesticides, International Journal of Pharmaceutics, Vol 203, p 1-60, 2000.
• the degree of humidity is less than or equal to 3% by weight, preferably less than or equal to 2.5%, preferably less than or equal to 2%, preferably less than or equal to 1.5%.
• the solid composition may be dissolved in water for injection (WFI) or in a reconstitution solvent to provide a therapeutic formulation.
• WFI water for injection
• a Factor B concentrate is prepared by following the steps shown in the attached figure.
• Cryosorbant is prepared by thawing fresh frozen plasma at a temperature between 1 ° C and 6 ° C (cryoprecipitation). After centrifugation, the cryoprecipitate is recovered to produce the fibrinogen, von Willebrand factor and factor VIII concentrates. The supernatant is collected, part of which is subjected to a purification step on DEAE-Sephadex in order to fix the vitamin k dependent factors such as protein C, Factor VII and Factor IX. The fraction not retained on DEAE-Sephadex is mixed with the remaining cryosurnant to form Solution A. The concentration of Factor B in the fraction is about 100-150 ⁇ g mL (for a reported plasma concentration of 200 ⁇ g / mL).
• Solution A is adjusted to a pH between 5.5 and 6.5 and preferably 6.0.
• the adjusted fraction is then subjected to chromatography on a Heparin Sepharose FF column or other chromatographic medium on which is grafted a heparin ligand. Most plasma proteins are found not delayed in the chromatography filtrate.
• Factor B is eluted by increasing the ionic strength of the column equilibration buffer.
• the eluted fraction is diluted and then chromatographed on a strong cationic exchange column of SP Sepharose type FF or equivalent. After washing the gel, the weakly adsorbed proteins on the gel are eluted by increasing the ionic strength of the column equilibration buffer.
• Factor B is eluted by further increasing the ionic strength of the column equilibration buffer.
• the eluted fraction is then subjected to a viral inactivation step by detergent solvent treatment (Polysorbate 80 and TnBP) effective on the enveloped viruses.
• the fraction is then diluted, adjusted to a pH of 6.0 (test 1) or 6.5 (test 2) and then subjected to chromatography on a strong anionic exchange column type Q Sepharose FF or equivalent equilibrated at pH 6, 0 or 6.5.
• Factor B is eluted by increasing the ionic strength of the column equilibration buffer.
• the fraction of Factor B obtained at this stage is of very high purity and can be subjected to a viral elimination step by nanofiltration, then concentrated and formulated by ultrafiltration.
• Factor B assay is carried out conventionally by immunoenzymatic method (ELISA) with commercial reagents (Diagnostica Stago). Briefly, the Factor B to be assayed is captured by a human anti-Factor B antibody immobilized on a solid phase. The fixed Factor B is then recognized by a peroxidase immunoconjugate. The level of bound peroxidase is measured by its activity on the ortho-phenylenediamine substrate in the presence of hydrogen peroxide. The intensity of the staining, after stopping the reaction with a strong acid, is a function of the amount of Factor B initially present in the sample. Factor B thus assayed is called "Antigen Factor B" or "Ag".

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