• 专利附录
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    • 专利摘要:
    The present invention provides methods of determining the endotoxin content of an aluminum salt for use in medicine, comprising the following steps: a. mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b. separating the aluminum salt from the endotoxin; and c. measuring the amount of endotoxin.
    公开号:BE1022256B1
    申请号:E2014/0418
    申请日:2014-06-05
    公开日:2016-03-04
    发明作者:Clèmentine Mascaux
    申请人:Glaxosmithkline Biologicals S.A.；
    IPC主号:
    专利说明:

    NEW PROCESS
    Field of the invention
    The present invention provides a method for determining the endotoxin content in an aluminum salt preparation for use in medicine, particularly for use as an adjuvant in vaccines.
    Context of the invention
    An analysis (LAL) of pyrogens or endotoxins is required for parenteral drugs. Conventional endotoxin assays can not be used to test the endotoxin content of aluminum salts. The endotoxin control added to the product is not recovered because of its interaction property with the aluminum salt. Validity criteria for assays require recovery and the ability to measure control endotoxin in bulk aluminum concentrations. Given this interaction, it is generally considered that conventional tests for endotoxins are unsuitable for endotoxin analysis in aluminum salt adjuvants. The only suitable test for endotoxin determination is the rabbit pyrogen test which involves the injection of aluminum salt into rabbits. Improved processes are needed. Summary of the invention
    The present invention provides methods for accurately quantifying endotoxin in an aluminum salt preparation. The invention provides methods for the determination of the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the steps of: ) mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b) the separation of aluminum salt from endotoxin; and c) measuring the amount of endotoxin. The invention further provides methods of making a desorption buffer for use in a method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use in as a vaccine adjuvant), comprising the steps of: a) mixing a base as defined herein with a metal chelating agent as defined herein and water; optionally b) stirring the mixture (in particular for a time equal to or greater than 30 seconds, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes) ; and optionally c) incubating the mixture for a time equal to or greater than 5, 10, 15, 20, 25, or 30 minutes.
    detailed description
    The present invention provides a method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the following steps: mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b) the separation of aluminum salt from endotoxin; and c) measuring the amount of endotoxin.
    The present invention provides a method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the following steps: mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b) centrifugation of the mixture, thereby separating the aluminum salt from the endotoxin; and c) measuring the endotoxin content of the supernatant.
    The present invention further provides a method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the steps of: a) mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b) centrifugation of the mixture, thereby separating the aluminum salt from the endotoxin; c) transferring the supernatant into a pyrogen-free receptacle; and d) measuring the endotoxin content of said supernatant.
    The term "endotoxin" is used herein to mean that defined in European Pharmacopoeia 5.1.10, i.e., this endotoxin is considered to be lipopolysaccharide (LPS) and / or lipo-oligosaccharide (LOS) derivatives. of Gram-negative bacteria.
    After the mixing step and before the separation / centrifugation step, the aluminum salt preparation and the desorption buffer can be incubated to ensure that any endotoxin bound to the aluminum salt is desorbed. Therefore, there are provided processes of the invention wherein the aluminum salt and desorption buffer mixture is incubated for a time greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. In a particular embodiment, the incubation period is less than 96 or 72 hours, i.e. the incubation period is between 1 and 96 hours, 5 and 72 hours, 10 and 48 hours , 15 and 36 hours, or between 20 and 28 hours. In a particular embodiment, the mixture is not stirred during the incubation period.
    The aluminum salt preparation and the desorption buffer are suitably mixed in a ratio of 1/9 to 9/1, for example 2/8, 3/7, 4/6, 5/5, 6/4 , 7/2 or 8/2. In particular embodiments, the aluminum salt preparation and the desorption buffer are mixed in a ratio of 3/7, 4/6, 5/5, 6/4, 7/3, especially 4/6. , 5/5 and 6/4.
    The methods of the invention can be carried out at any temperature that allows the desorption of endotoxin from the aluminum salt. Suitably, there are provided methods of the invention wherein said incubation is carried out at a temperature between 10 ° C and 70 ° C, for example between 20 ° C and 65 ° C, 20 ° C and 60 ° C, 15 ° C and 30 ° C, between 17 ° C and 28 ° C, between 19 ° C and 26 ° C, between 21 ° C and 23 ° C, or at about room temperature (about 22 ° C).
    The methods of the invention may comprise a step of stirring the mixture of the aluminum salt and the desorption buffer to ensure that the desorption buffer is mixed with the aluminum composition. Suitably, there are provided processes of the invention in which the mixture of the aluminum salt and the desorption buffer is stirred after step a) for more than 30 seconds, 40 seconds, 50 seconds, 1 minute, 1 minute 30 seconds or 2 minutes.
    Any buffer that facilitates the desorption of endotoxin can be used in the methods of the invention. Desorption buffers suitable for use in the present methods can be determined by exposing an aluminum salt candidate buffer comprising a known amount of endotoxin (e.g. 20 EU / ml), incubating for approximately 24 hours + / - 4 hours at room temperature, centrifugation at 10,000 rpm +/- 1000 rpm for 10 minutes +/- 1 min and then performing an LAL test on the supernatant. A buffer which desorbs between 50 and 200% of the endotoxin is considered a suitable desorption buffer.
    Methods of the invention are provided wherein said desorption buffer comprises a salt and a metal chelating agent. Suitable salts are well known to those skilled in the art and include, but are not limited to, Na 2 HPO 4. Na 2 HPO 4 is generally used in an amount of between 15% and 25%, for example about 18%, for example 17.8%. %, 0.224% EDTA, pH 8.0.
    In some embodiments, particularly wherein the methods of the invention are used to determine the endotoxin content of an aluminum hydroxide, the desorption buffer may comprise another salt, for example, NaCl. When NaCl is used, the desorption buffer generally comprises between 5 and 250 mM NaCl.
    Suitable metal chelating agents are well known to those skilled in the art and include, but are not limited to, EDTA (ethylene diamine tetraacetic acid). EDTA is generally used in an amount between 0.2 and 0.3%, for example about 0.22%, for example 0.224%.
    The desorption buffer can be at any pH that facilitates the desorption of endotoxin from the aluminum salt. In one embodiment, the desorption buffer of the invention has a pH greater than 5.5, particularly when the desorption buffer is used to desorb endotoxin from aluminum phosphate. There is provided a process of the invention wherein said desorption buffer is at a pH between about pH 7 and pH 9. In a particular embodiment, there is provided a desorption buffer which is at a pH of about pH 8 (for example at a pH between 7.5 and 8.5).
    In one embodiment, the desorption buffer of the invention has a pH greater than pH 9.5, particularly when the desorption buffer is used to desorb endotoxin from aluminum hydroxide. There is provided a process of the invention wherein said desorption buffer is at a pH between about pH 9.5 and pH 10.5. In a particular embodiment, there is provided a desorption buffer which is at a pH of about pH 10. In a particular embodiment, the pH is below pH 10.5.
    The desorption buffer may be at any temperature that prevents the precipitation of one or more of the buffer components, particularly when stored concentrated, for example concentrated between 1.5 and 2 times. Suitably, there is provided a process of the invention wherein said desorption buffer is stored at a temperature between about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C.
    The desorption buffer is ideally free of endotoxin, and methods of the invention are suitably provided in which the desorption buffer is substantially free of endotoxin, i.e., no endotoxin can be present. detected using any technique known to those skilled in the art. The endotoxin is desorbed from the aluminum salt and suitably separated to allow assay of the endotoxin content. The separation can be carried out using any method known to those skilled in the art. Suitably, methods of the invention are provided in which the mixture is centrifuged at a force required to pellet the aluminum salt and maintain the endotoxin in the supernatant. In particular, there are provided processes of the invention in which the mixture of the desorption buffer and the aluminum salt is centrifuged at a rate between 1000 and 1500 x g, for example approximately 1118 x g.
    The desorbed endotoxin content can be measured according to any test available to those skilled in the art for the measurement of endotoxin. In particular, there are provided methods of the invention wherein the endotoxin content is measured. In particular, there are provided methods in the invention wherein the endotoxin content is quantified. Processes available and known to those skilled in the art include, but are not limited to, LAL (horseshoe amoebocyte lysate) assay, monocyte activation assay or the EndoLISA method. In particular, the endotoxin is quantified in the methods of the invention by the LAL test (in particular by a chromogenic kinetic process).
    The methods of the invention can be used to determine the endotoxin content of any aluminum salt for use as an adjuvant in a vaccine. Suitable aluminum salt adjuvants are well known to those skilled in the art and include, but are not limited to, aluminum phosphate, aluminum hydroxide or a combination thereof. Aluminum salt adjuvants include, but are not limited to, Rehydragel ™ HS, Alhydrogel ™ 85, Rehydragel ™ PM, Rehydragel ™ AB, Rehydragel ™ HPA,
    Rehydragel ™ LV, Alhydrogel ™ or one of their combinations. In particular, the methods of the invention are used to determine the endotoxin content in Adjuphos, Rehydragel ™ HS (3% aluminum hydroxide in water [General Chemical]) or Alhydrogel ™ 85 (Brenntag BioSector [ Denmark]).
    In particular, the methods of the invention are used to determine the endotoxin content of aluminum salts which have a protein adsorption capacity of between 2.5 and 3.5, 2.6 and 3.4, 2.7 and 3.3 or 2.9 and 3.2, 2.5 and 3.7, 2.6 and 3.6, 2.7 and 3.5, or 2.8 and 3.4 protein (BSA) / ml of aluminum salt. In a particular embodiment of the invention, the methods of the invention are used to determine the endotoxin content of an aluminum salt with the protein adsorption capacity between 2.9 and 3.2 mg of BSA / mg of aluminum salt. The protein absorption capacity of the aluminum salt can be measured by any means known to those skilled in the art. In a particular embodiment of the invention, the protein adsorption capacity of the aluminum salt is measured using the method as described in Example 1 of WO 2012/136823 (which uses BSA). or its variations.
    The methods of the invention are used to determine the endotoxin content of aluminum salts described herein (i.e. having the protein adsorption capacity described herein) which have a crystal size between 2.8. and 5.7 nm measured by X-ray diffraction, for example 2.9 to 5.6 nm, 2.8 to 3.5 nm, 2.9 to 3.4 nm or 3.4 to 5.6 nm or 3, 3 and 5.7 nm measured by X-ray diffraction. X-ray diffraction is well known to those skilled in the art. In a particular embodiment of the invention, the crystal size is measured using the method described in Example 1 of WO 2012/136823 or its variations.
    The methods of the invention are particularly useful in quality control tests. The term "quality control" is used here to mean quality control (QC) or a set of procedures to ensure that a manufactured product or service adheres to a defined set of quality or quality criteria. meets the requirements of the manufacturer. Therefore, the methods of the invention are suitable for use in a quality control test.
    In a particular embodiment, the methods of the invention described herein are used to test the aluminum salt preparation prior to formulation of the aluminum salt with other components of the vaccine. In another embodiment, the methods of the invention described herein are used to test the aluminum salt preparation after formulating the aluminum salt with the other vaccine components (e.g., particular antigens). In another embodiment, the methods of the invention described herein are used to test the aluminum salt preparation for the endotoxin content both before and after the formulation of the aluminum salt preparation with the others. vaccine components (e.g., antigens, particularly those described herein).
    The methods of the invention may involve: extracting a sample from a bulk volume of aluminum salt; sample analysis by the methods defined herein; and then, if the sample passes the test, adsorbing one or more antigens (particularly those described herein) on said aluminum salt; and optionally combining said aluminum salt with one or more other antigens (particularly those described herein).
    The aluminum salts of the invention may be formulated with antigens before and / or after the endotoxin content assay.
    Antigens that can be formulated with the aluminum salt of the invention include, but are not limited to, tetanus toxoid (TT), diphtheria toxoid (DT), hepatitis B surface antigen , inactivated poliovirus (IPV), pertactin, with filamentous haemagglutinin (FHA), pertussis toxoid and / or Haemophilus influenzae type B (Hib) polysaccharide [in particular capsular saccharide polyribosyl-ribitol-phosphate ( PRP) of H. influenzae type B), human papillomavirus (e.g., pseudoviral particles from HPV 6, 11, 16, 18 or one of their combinations), polysaccharides derived from Streptococcus pneumoniae conjugated to a transport, for example TT, DT and / or CRM197.
    Tetanus toxoids (TT) and methods for their preparation are well known in the art. TTs can be produced by purifying the toxin from a Clostridium tetani culture followed by chemical detoxification, but it is alternatively manufactured by purifying a recombinant, or genetically detoxified, analog of the toxin (e.g. as described in EP 209281). A preferred method of detoxification is as follows. After fermentation, the broth is filtered through a 0.1 to 0.3 μm filter in the presence of Diatomite as a filter aid. The harvest is clarified through a 0.22 μm filter, concentrated and diafiltered on 30 kD flat sheet membranes against 10 volumes of phosphate buffer (20 mM - pH 7.3). The diafiltered toxin is then detoxified for 4 weeks at 37 ° C under the following conditions: 20 mM formaldehyde - 3 mM lysine - 100 mM potassium phosphate - initial pH 7.3 - 500 Lf / ml. The resulting toxoid is purified by fractionation with ammonium sulfate, concentrated and diafiltered (30 kD) against water for injection to remove ammonium sulfate. NaCl is added to a final concentration of 0.9%, the pH is adjusted to 7.3 and the purified toxoid is sterilized by filtration.
    Any tetanus toxoid can be used. "Tetanus toxoid" may include immunogenic fragments of the full-length protein (for example, fragment C - see EP 478602). The tetanus toxoid of the invention is generally adsorbed on an aluminum salt. In a particular embodiment of the invention, the aluminum salt is aluminum hydroxide. In another embodiment, the tetanus toxoid of the invention may be adsorbed on an aluminum salt such as aluminum phosphate. In another embodiment, the tetanus toxoid can be adsorbed on a mixture of both aluminum hydroxide and aluminum phosphate.
    Protein adsorption methods comprising tetanus toxoids on aluminum salts are well known to those skilled in the art (for example, the preparation of vaccines is generally described in Vaccine Design "The subunit and adjuvant approach" ( eds Powell MF & Newman MJ) (1995) Plenum Press New York).
    Diphtheria toxoids (DT) and their methods of preparation are well documented. Any suitable diphtheria toxoid can be formulated with the aluminum salt described here after or before the endotoxin assay. For example, DT may be produced by purification of the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, but it is alternatively manufactured by purification of a recombinant, or genetically detoxified analog of the toxin ( for example, CRM197, or other mutants as described in US 4,709,017, US 5,843,711, US 5,601,827, and US 5,917,017). A preferred method of detoxification is as follows. After fermentation, the diphtheria toxoid is harvested by TFF on a 0.45 μιη filter, clarified through a 0.22 μm filter, concentrated and diafiltered on 10 kD flat-leaf membranes against 10 volumes of phosphate buffer ( 20 mM - pH 7.2). The diafiltered toxin is then detoxified for 6 weeks at 37 ° C under the following conditions: 50 mM formaldehyde - 25 mM lysine - 50 mM potassium phosphate - initial pH 7.2 - 300 Lf / ml. The resulting toxoid is purified by fractionation with ammonium sulfate, concentrated and diafiltered (30 kD) against water for injection to remove ammonium sulfate. NaCl is added to a final concentration of 0.9%, the pH is adjusted to 7.3 and the purified diphtheria toxoid is sterilized by filtration.
    Inactivated poliomyelitis vaccine (IPV) may include Type 1 or Type 2 API or Type 3 API, or Type 1 and 2 API, or Type 1 and 3 API or Type 1 API 2 and 3, or IPV types 1, 2 and 3.
    Inactivated poliovirus (IPV) preparation methods are well known in the art. In one embodiment, the IPV should include types 1, 2 and 3 as is common in the vaccine art, and it may be the Salk polio vaccine that is inactivated with formaldehyde (see, for example, Sutter et al., 2000, Pediatr Clin, North Am 47: 287, Zimmerman & Spann 1999, Am Fam Physician 59: 113, Salk et al., 1954, Official Monthly Publication of the American Public Health Association 44 (5). ): 563 Hennesen, 1981, Develop Biol Standard 47: 139, Budowsky, 1991, Adv Virus Res 39: 255). Alternatively, IPV can be made using Sabin strains (Sabin-IPV, Kersten et al (1999), Vaccine 17: 2059).
    In one embodiment, the IPV is not adsorbed (for example, before mixing with other components). In another embodiment, the IPV component (s) of the invention may be adsorbed to an aluminum salt such as aluminum hydroxide (for example, before or after mixing with other components). In another embodiment, the IPV component (s) of the invention can be adsorbed onto an aluminum salt such as aluminum phosphate. In another embodiment, the IPV component (s) of the invention can be adsorbed onto a mixture of both aluminum hydroxide and aluminum phosphate. In case of adsorption, one or more IPV components may be adsorbed separately or together as a mixture. In another embodiment, the IPV is adsorbed on a salt / aluminum particle as described herein.
    Pertactin (the 69 kDa pertussis antigen) is an outer membrane protein that is heat stable and can be prepared by methods known in the art (see EP 0162639). Pertactin is optionally adsorbed on a particle of aluminum salt. In one embodiment of the invention, pertactin is adsorbed on aluminum hydroxide. In a particular embodiment of the invention, pertactin is adsorbed on an aluminum salt as described herein. Filamentous haemagglutinin (FHA) can be prepared in methods well known in the art (see the methods described and referenced in WO 1990/013313 (US 7,479,283)). FHA is optionally adsorbed on a particle of aluminum salt. In one embodiment of the invention, FHA is adsorbed on aluminum hydroxide. In a particular embodiment of the invention, FHA is adsorbed on an aluminum salt as described herein.
    Processes for producing pertussis toxoid are well known to those skilled in the art. The pertussis toxin can be detoxified by a well-known method of formaldehyde treatment or by means of mutations (PT derivative). Residue substitutions within the Si subunit of the protein have been found to result in a protein that retains the immunological and protective properties of the pertussis toxin, but with reduced toxicity or no toxicity (EP 322533) . The detoxifying mutations discussed in the claims of EP322533 are examples of the detoxified PT mutants of the present invention. The pertussis toxoid is optionally adsorbed on a particle of aluminum salt. In one embodiment of the invention, the pertussis toxoid is adsorbed on aluminum hydroxide. In a particular embodiment of the invention, the pertussis toxoid is adsorbed on an aluminum salt as described herein.
    The capsular saccharide polyribosyl ribitol phosphate (PRP) of Haemophilus influenzae type b can be conjugated to a transport protein. Saccharide is a polymer of ribose, ribitol and phosphate. The Hib antigen may be optionally adsorbed on aluminum phosphate as described in WO 97/00697, or it may be non-adsorbed as described in WO 02/00249 or it may not have undergone specific adsorption treatment.
    By "non-adsorbed on an aluminum adjuvant salt" ("non-adsorbed") antigen herein, it is meant for example that an express or dedicated adsorption step for the antigen on aluminum salt of Fresh adjuvant is not involved in the formulation process of the composition.
    Hib can be conjugated to any carrier that can provide at least one T-helper epitope, and can be tetanus toxoid, diphtheria toxoid, CRM-197 (diphtheria toxin mutants), or H protein D. influenzae not typable (EP 0594610).
    In another embodiment, there is provided a method of the invention further comprising the step of formulating the immunogenic composition with a hepatitis B surface antigen.
    The preparation of hepatitis B surface antigen (HBsAg) is well documented. See, for example, Hartford et al., 1983, Develop. Biol. Standard 54: 125; Gregg et al., 1987, Biotechnology 5: 479; EP 0226846; EP 0299108. It can be prepared as follows. One method involves the purification of the antigen in a particulate form from the plasma of chronic hepatitis B carriers, since large amounts of HBsAg are synthesized in the liver and released into the bloodstream during infection with the hepatitis B virus. HBV.
    Another method involves the expression of the protein by recombinant DNA methods. HBsAg can be prepared by expression in yeast Saccharomyces cerevisiae, Pichia, insect cells (e.g., Hi5) or mammalian cells. HBsAg can be inserted into a plasmid, and its expression from the plasmid can be controlled by a promoter such as the "GAPDH" promoter (from the glyceraldehyde-3-phosphate dehydrogenase gene). The yeast can be grown in a synthetic medium. HBsAg can then be purified by a process involving such steps. precipitation, ion exchange chromatography, and ultrafiltration. After purification, HBsAg can be dialyzed (eg, with cysteine). HBsAg can be used in particulate form.
    As used herein, the term "hepatitis B surface antigen" or "HBsAg" includes any HBsAg antigen or fragment thereof displaying the antigenicity of the HBV surface antigen. It should be understood that in addition to the 226 amino acid sequence of the S HBs HBs antigen (see Tiollais et al., 1985, Nature 317: 489 and references therein), the HBs Ag as described herein can, if desired, contain all or part of the pre-S sequence as described in the above references and in EP 0278940. In particular, the HBs Ag may comprise a polypeptide comprising a sequence of amino acids comprising residues 133 to 145 followed by residues 175 to 400 of the L protein of HBsAg relative to the open reading frame on a serotype ad hepatitis B virus (this polypeptide is called L *; EP 0414374). HBsAg within the scope of the invention may also comprise the preS1-preS2-S polypeptide described in EP 0198474 (Endotronics) or its analogs such as those described in EP 0304578 (McCormick and Jones). HBsAg as used herein may also refer to mutants, for example the "escape mutant" described in WO 91/14703 or EP 0511855A1, especially HBsAg in which the acid substitution amine at position 145 is an arginine from a glycine. HBsAg may be in the form of particles. The particles may comprise, for example, protein S alone or they may be composite particles, for example L *, S) where L * is as defined above and S indicates protein S of HBsAg. Said particle is advantageously in the form in which it is expressed in yeast.
    In one embodiment, HBsAg is the antigen used in Engerix B ™ (GlaxoSmithKline Biologicals S.A.), which is further described in WO 93/24148. The hepatitis B surface antigen may be optionally adsorbed on an aluminum salt, in particular aluminum phosphate, which may be carried out before mixing with the other components (described in WO 93/24148). ). The hepatitis B component should be substantially free of thiomersal (the method of preparing HBsAg without thiomersal has been published previously in EP 1307473).
    The present invention further provides methods of making a desorption buffer for use in a method for determining the endotoxin of an aluminum salt comprising the steps of: a) mixing one or more salts as defined herein, a metal chelating agent as defined herein and water; optionally b) stirring the mixture (in particular for a time equal to or greater than 30 seconds, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes) ; and optionally c) incubating the mixture for a time equal to or greater than 5, 10, 15, 20, 25, or 30 minutes.
    The present invention further provides methods of making a desorption buffer for use in a method for determining the endotoxin of an aluminum salt comprising the steps of: a) mixing a salt, d a metal and water chelating agent; optionally b) stirring the combined mixture at incubation periods at 37 ° C +/- 1 ° C (to dissolve); and optionally c) adding an acid or a base to set the pH; d) the addition of water to reach the final volume of the preparation; optionally e) storage at 37 ° C +/- 1 ° C.
    Steps a) and b) can be performed more than once, there are provided methods of making a desorption buffer as defined herein wherein steps b) and / or c) are repeated 1 or 2 times or more.
    Methods for making a desorption buffer as defined in which the water is substantially free of endotoxin are provided.
    There are provided methods of making a desorption buffer as defined wherein the water of step a) is preheated to about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C (± 1 ° C).
    There are provided methods of making a desorption buffer as defined in which the pH is adjusted between 7 and 9, in particular about pH 8. Any acid can be used to adjust this pH while not affecting the capacity. desorption buffer to desorb endotoxin from the aluminum salt. In particular, HCl can be used to adjust the pH in the manufacture of desorption buffers described herein. In particular, the pH is adjusted when the buffer is at about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C (± 1 ° C). Any suitable base or acid may be used to adjust this pH while not affecting the ability of the desorption buffer to desorb endotoxin from the aluminum salt.
    Methods are provided for making a desorption buffer as defined further comprising step d) storing the desorption buffer at about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C C and 38 ° C, or about 37 ° C (± 1 ° C).
    The terms "comprising", "include" and "includes" herein are intended by the inventors as possibly substitutable by the terms "consisting of", "consist of" and "consists of", respectively, in all cases .
    The term "about" used here is meant to mean the amount ± 10%.
    Examples 1. Preparation of the desorption buffer
    In order to make 100 ml of desorption buffer, 17.8 g of Na2HPO4, 0.224 g of EDTA and 90 ml of pyrogen-free water (if possible preheated in an incubator at 37 ° C) were placed in a Nunc pyrogen-free container. 250 ml.
    The salt and EDTA were dissolved by shaking for 15 minutes on a stirring table, heating for 30 minutes in an incubator at 37 ° C. ± 1 ° C. and repeating stirring and heating until dissolution occurred. total.
    To adjust the pH, 3 ml of 1N HCl was added, the pH was measured and adjusted to pH 8.0 with 1N HCl if necessary. The volume is completed to 100 ml with pyrogen-free water. The absence of endotoxin was determined by LAL analysis at a dilution of 1/100. The desorption buffer was stored at 37 ° C ± 1 ° C. 2. Determination of endotoxin
    In order to determine the endotoxin content of an aluminum sample (4 ml), desorption buffer (6 ml) was added to the aluminum salt sample. The mixture was stirred for 1 minute and then incubated at room temperature for 24 hours.
    After incubation, the mixture is stirred slowly by inverting the tube 5 times. A sample (1.5 ml) is taken and transferred to a 2 ml Eppendorf tube. The mixture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was then transferred to a pyrogen-free tube. The endotoxin content was then quantified using the LAL assay by the kinetic kinetic method at a 1:50 dilution.
    权利要求:
    Claims (28)
    [1]
    A method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the steps of: a. mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b. separation of aluminum salt from endotoxin; and c. measuring the amount of endotoxin.
    [2]
    A method for determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the steps of: a. mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b. centrifugation of the mixture, thereby separating the aluminum salt from the endotoxin; and c. measuring the endotoxin content of the supernatant.
    [3]
    A method of determining the endotoxin content of an aluminum salt preparation for use in medicine (particularly for use as an adjuvant in a vaccine), comprising the steps of: a. mixing the aluminum salt with a desorption buffer, thereby desorbing any endotoxin from the aluminum salt; b. centrifugation of the mixture, thereby separating the aluminum salt from the endotoxin; vs. transferring the supernatant into a pyrogen-free receptacle; and D. measuring the endotoxin content of said supernatant.
    [4]
    A process according to any one of the preceding claims wherein the aluminum salt preparation and the desorption buffer are mixed in a ratio of 1/9 to 9/1, for example 2/8, 3/7, 4/6, 5/5, 6/4, 7/2 or 8/2 (in particular between 3/7, 4/6, 5/5, 6/4 7/3, in particular 4/6, 5 / 5 and 6/4).
    [5]
    5. A process according to any one of the preceding claims, wherein the mixture is incubated (in particular without stirring) after mixing and before separation (in particular centrifugation) (in particular for a time greater than 1, 2, 3 , 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, in particular less than 96 or 72 hours, for example the incubation period is between 1 and 96 hours, 5 and 72 hours, 10 and 48 hours, 15 and 36 hours, or between 20 and 28 hours).
    [6]
    The method of claim 5 wherein said incubation (particularly for use in methods of determining aluminum phosphate endotoxin) is performed at room temperature (about 22 ° C).
    [7]
    The method according to claim 5, wherein said incubation (particularly for use in methods of determining aluminum hydroxide endotoxin) is carried out at a temperature above 30 ° C, 35 ° C, 40 ° C. ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C.
    [8]
    The method of any one of the preceding claims, wherein the mixture is stirred after step a) for more than 30 seconds, 40 seconds, 50 seconds, 1 minute, 1 minute 30 seconds or 2 minutes.
    [9]
    The process according to any one of the preceding claims, wherein said desorption buffer comprises a salt (in particular Na2HPO4 [in particular between 15 and 20%]) and a metal chelating agent (in particular EDTA [in particular between 0.2 and 0.3%]).
    [10]
    A process according to any one of the preceding claims (particularly that for use in methods of determining aluminum hydroxide endotoxin), wherein the desorption buffer comprises another salt (in particular NaCl ).
    [11]
    The method according to any one of the preceding claims, wherein said desorption buffer (particularly that for use in methods of determining aluminum phosphate endotoxin) has a pH equal to or greater than pH 5, 5, for example between about pH 7 and pH 9.
    [12]
    The process according to any one of the preceding claims, wherein said desorption buffer (particularly that for use in methods of determining aluminum hydroxide endotoxin) has a pH equal to or greater than pH 9. , 5, for example between about pH 9.5 and pH 10.5.
    [13]
    A process according to any one of the preceding claims, wherein said desorption buffer is at a temperature between about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C.
    [14]
    The process according to any one of the preceding claims, wherein said desorption buffer is at a temperature above 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C or 65 ° C. vs.
    [15]
    The method of any of the preceding claims, wherein the desorption buffer is substantially free of endotoxin and / or is sterile.
    [16]
    16. A process according to any one of the preceding claims, wherein the mixture is centrifuged at a rate between 100 and 1500 x g.
    [17]
    17. A method according to any one of the preceding claims wherein the endotoxin content is measured / quantified by the LAL test (limulus amoebocyte lysate).
    [18]
    18. A process according to any one of the preceding claims, wherein the aluminum salt is aluminum phosphate, aluminum hydroxide or a combination thereof.
    [19]
    19. Quality control test comprising the process according to any one of claims 1 to 18.
    [20]
    A method of making a desorption buffer for use in a method for determining the endotoxin of an aluminum salt comprising the steps of: a. mixing a base (especially Na2HPO4), a metal chelating agent (especially EDTA) and water; possibly b. stirring the mixture (especially for a time equal to or greater than 30 seconds, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes); and possibly c. incubating the mixture for a time equal to or greater than 5, 10, 15, 20, 25 or 30 minutes.
    [21]
    A method of making a desorption buffer for use in a method for determining endotoxin of an aluminum salt comprising the steps of: a. mixing a salt, a metal chelator and water; possibly b. stirring the combined mixture at incubation periods at 37 ° C +/- 1 ° C (to dissolve); possibly c. addition of an acid or a base to fix the pH; possibly d. the addition of water to reach the final volume of the preparation; and possibly e. storage at 37 ° C +/- 1 ° C.
    [22]
    The method of claim 20, wherein steps b) and / or c) are repeated 1 or 2 times or more.
    [23]
    23. The method of any one of claims 20 to 22, wherein the water is substantially free of endotoxin.
    [24]
    24. A process according to any one of claims 20 to 23, wherein the water of step a) is preheated to about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C. ° C, or about 37 ° C (± 1 ° C).
    [25]
    25. A process according to any one of claims 20 to 24, wherein the pH is adjusted between 7 and 9, in particular about pH 8 or between 9.5 and 10.5.
    [26]
    The method of claim 25, wherein the pH is adjusted when the buffer is at a temperature of about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C (± 1 ° C).
    [27]
    The method of any one of claims 20 to 26, further comprising step d) storing the desorption buffer at about 30 ° C and 45 ° C, 35 ° C and 40 ° C, 36 ° C and 38 ° C, or about 37 ° C (± 1 ° C).
    [28]
    28. The method of claim 17 wherein the endotoxin content is measured / quantified by the kinetic chromogenic method.
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    同族专利:
    公开号 | 公开日
    JP2016520842A|2016-07-14|
    CA2913025A1|2014-12-11|
    GB201310151D0|2013-07-24|
    US20160131652A1|2016-05-12|
    ES2775209T3|2020-07-24|
    US10060928B2|2018-08-28|
    CN105247369A|2016-01-13|
    EP3004886A1|2016-04-13|
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    BR112015030346A2|2017-07-25|
    WO2014195387A1|2014-12-11|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    GB201310151A|GB201310151D0|2013-06-07|2013-06-07|Novel method|
    GB13101514|2013-06-07|
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