[go: up one dir, main page]

WO1996025425A1 - Toxoide et toxine tetaniques purifies - Google Patents

Toxoide et toxine tetaniques purifies Download PDF

Info

Publication number
WO1996025425A1
WO1996025425A1 PCT/US1996/002173 US9602173W WO9625425A1 WO 1996025425 A1 WO1996025425 A1 WO 1996025425A1 US 9602173 W US9602173 W US 9602173W WO 9625425 A1 WO9625425 A1 WO 9625425A1
Authority
WO
WIPO (PCT)
Prior art keywords
tetanus
tetanus toxoid
toxin
toxoid
tetanus toxin
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.)
Ceased
Application number
PCT/US1996/002173
Other languages
English (en)
Inventor
Douglas J. Cecchini
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.)
Massachusetts Health Research Institute Inc
Original Assignee
Massachusetts Health Research Institute 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 Massachusetts Health Research Institute Inc filed Critical Massachusetts Health Research Institute Inc
Priority to AU49865/96A priority Critical patent/AU4986596A/en
Publication of WO1996025425A1 publication Critical patent/WO1996025425A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)

Definitions

  • the invention relates to tetanus toxin, tetanus toxoid, and methods for the purification thereof.
  • Tetanus toxoid is prepared and used worldwide for large-scale immunization against tetanus. Tetanus toxoid is used both singly and in combination with diphtheria and/or pertussis vaccines.
  • the parent protein, tetanus toxin is generally obtained in cultures of Clostridium tetani. Tetanus toxin is a protein of 150 kDa and consists of two subunits (100 kDa and 50 kDa) linked by a sulfide bond.
  • the toxin is detoxified with formaldehyde and most commonly purified from culture filtrates by a series of ammonium sulfate fractionation steps (see, e.g., Levin and Stone, J. Immunology 67:235-242, 1951).
  • the invention features, a method of purifying tetanus toxin or tetanus toxoid.
  • the method includes: (i) contacting the tetanus toxin or the tetanus toxoid with a hydrophobic moiety; and (ii) forming a complex between the tetanus toxin or the tetanus toxoid and the hydrophobic moiety, thereby providing for the purification of tetanus toxin or tetanus toxoid.
  • the method further includes a washing step, e.g.: the method further includes treating the hydrophobic moiety so as to free (e.g., release, elute or remove) a substance, other than tetanus toxin or tetanus toxoid, which has formed a complex with or has otherwise bound to or become associated with the hydrophobic moiety; the method further includes treating the complex between the hydrophobic moiety and the tetanus toxin or tetanus toxoid so as to free (e.g., release, elute or remove) a substance, other than tetanus toxin or tetanus toxoid, which has formed a complex with or has otherwise bound to or become associated with one or both of the hydrophobic moiety and the tetanus toxin or tetanus toxoid; the method further includes treating the hydrophobic moiety so as to free (e.g., release,
  • the washing step is sufficiently gentle that a biological activity (e.g., the antigenicity as measured, e.g., by the flocculation (LF) assay) of the complexed (or purified) tetanus toxin or tetanus toxoid is preserved.
  • a biological activity e.g., the antigenicity as measured, e.g., by the flocculation (LF) assay
  • LF flocculation
  • a substance other than tetanus toxin or tetanus toxoid is freed (e.g., released, eluted, or removed) from the hydrophobic moiety (or from the tetanus toxin or tetanus toxoid bound to the moiety) by contacting the substance with a washing agent, e.g., solution a of relatively high ionic strength, e.g. a high salt solution, e.g., a salt solution with a salt concentration of at least 1, more preferably 2, and most preferably more than 3 molar.
  • a washing agent e.g., solution a of relatively high ionic strength, e.g. a high salt solution, e.g., a salt solution with a salt concentration of at least 1, more preferably 2, and most preferably more than 3 molar.
  • the method fiirther includes an eluting step: e.g., the method further includes disrupting the complex between the tetanus toxin or tetanus toxoid and the hydrophobic moiety, e.g., by contacting the complex with an agent, e.g., an agent which disrupts or weakens hydrophobic interactions, e.g., an eluting solution.
  • the solution can be, e.g., a low salt solution, e.g., a solution of less than 0.5, more preferably less than 0.1 , more preferably 0.05, yet more preferably less than 0.015, molar salt.
  • the solution can include a detergent, e.g., an ionic or non-ionic detergent; an organic solvent, e.g., ethanol; a chaotropic agent, e.g., urea or guanidine hydrochloride.
  • a detergent e.g., an ionic or non-ionic detergent
  • an organic solvent e.g., ethanol
  • a chaotropic agent e.g., urea or guanidine hydrochloride.
  • the eluting step is sufficiently gentle that a biological activity (e.g., the antigenicity as measured, e.g., by the flocculation (LF) assay) of the complexed (or purified) tetanus toxin or tetanus toxoid is preserved.
  • a biological activity e.g., the antigenicity as measured, e.g., by the flocculation (LF) assay
  • eluting step results in a loss of less than 10, 20, 30, 40, or 50% of a biological activity.
  • the method further includes a wash step in which a substance other than tetanus toxin or tetanus toxoid is freed, e.g., released, eluted, or removed) from the hydrophobic moiety (or from the tetanus toxin or tetanus toxoid bound to the moiety) and a subsequent elution step in which tetanus toxin or tetanus toxoid is freed (e.g., released, eluted, or removed) from the hydrophobic moiety.
  • the wash step includes contacting the complex with a wash solution and the subsequent elution step includes contacting the complex with an elution solution having a greater ability to disrupt or weaken hydrophobic interactions than does the wash solution of the wash step, e.g., the subsequently applied elution solution has a lower salt concentration than does the wash solution.
  • the method further includes, prior to the purification step, supplying a preparation containing tetanus toxin or tetanus toxoid, e.g., a culture supernatant, a culture filtrate, or a cell-free supernatant, which includes the tetanus toxin or tetanus toxoid, for contacting with the hydrophobic moiety.
  • a preparation containing tetanus toxin or tetanus toxoid e.g., a culture supernatant, a culture filtrate, or a cell-free supernatant, which includes the tetanus toxin or tetanus toxoid, for contacting with the hydrophobic moiety.
  • the method further includes a subsequent purification step, e.g., the step of contacting the tetanus toxin or the tetanus toxoid that is obtained from the hydrophobic moiety with a charged moiety, e.g., an ion exchange moiety, e.g., DEAE linked to a substrate, to further purify the tetanus toxin or tetanus toxoid.
  • a subsequent purification step e.g., the step of contacting the tetanus toxin or the tetanus toxoid that is obtained from the hydrophobic moiety with a charged moiety, e.g., an ion exchange moiety, e.g., DEAE linked to a substrate, to further purify the tetanus toxin or tetanus toxoid.
  • the material from the hydrophobic purification is subjected to the subsequent purification without an interven
  • the hydrophobic moiety includes an aromatic hydrocarbon, e.g., a phenyl group, or a group with similar hydrophobicity.
  • the hydrophobic moiety is coupled to a substrate, e.g., an insoluble substrate, e.g., an insoluble polymer.
  • the insoluble polymer can be, e.g., a cross- linked agarose, e.g., sepharose, a cross-linked co-polymer of dextran and bisacrylamide, e.g., sephacryl, a cross-linked agarose, e.g., superose, or a cross-linked dextran, e.g., sephadex.
  • the invention features, a method of providing a purified preparation of tetanus toxin or tetanus toxoid.
  • the method includes: (i) supplying a preparation including the tetanus toxin or the tetanus toxoid; (ii) contacting the preparation with a hydrophobic moiety; (iii) forming a complex between the tetanus toxin or the tetanus toxoid and the hydrophobic moiety; (iv) (optionally) removing, eluting, freeing or releasing a substance other than the tetanus toxin or the tetanus toxoid which, e.g., has formed a complex with or has otherwise bound to or become associated with the one or both of the hydrophobic moiety and the tetanus toxin or tetanus toxoid; and (v) disrupting the complex between the t
  • the invention features, a tetanus toxin or tetanus toxoid preparation including tetanus toxin or tetanus toxoid complexed with a hydrophobic moiety, the preparation being substantially free of at least one bacterial cell product, e.g., a polypeptide, polysaccharide, amino acid, or lipid, which occurs naturally with the tetanus toxin or tetanus toxoid.
  • a tetanus toxin or tetanus toxoid preparation including tetanus toxin or tetanus toxoid complexed with a hydrophobic moiety, the preparation being substantially free of at least one bacterial cell product, e.g., a polypeptide, polysaccharide, amino acid, or lipid, which occurs naturally with the tetanus toxin or tetanus toxoid.
  • the invention features, a substantially purified preparation of tetanus toxin or tetanus toxoid made by a method described herein.
  • a substantially purified preparation of tetanus toxin or tetanus toxoid having one or more of the following properties: the preparation is at least 50, 60, 70, 80 or 90% monomeric tetanus toxin or tetanus toxoid (on e.g., a weight/weight basis); the preparation contains less than 3, 4, 5, 6, 8, 7, 8, 9, or 10% tetanus toxin or tetanus toxoid dimers (on e.g., a weight/weight basis); the preparation contains less than 0.4, 0.5, 1.0, 2.0, 3.0, 3.5, 4, or 5% higher order polymers of tetanus toxin or tetanus toxoid (where higher order means trimers or
  • the hydrophobic interaction-based purification method of the invention can be used to provide purified preparations of tetanus toxin or tetanus toxoid which have relatively low amounts of cross-linked tetanus toxin or tetanus toxoid.
  • the preparations are particularly well suited for the production of conjugated toxins which include a tetanus toxin or tetanus toxoid conjugated to a second vaccine component, e.g., a bacterial component (e.g., a bacterial protein or polysaccharide).
  • the invention features a method of making a conjugate vaccine having a tetanus toxin or tetanus toxoid moiety conjugated to a second vaccine component.
  • the method includes: (i) providing a purified tetanus toxin or tetanus toxoid made by a method of the invention; (ii) contacting the purified tetanus toxin or tetanus toxoid moiety with the second vaccine component; and (iii) conjugating the tetanus toxin or tetanus toxoid moiety with the second vaccine component to provide a conjugate vaccine.
  • a complex between a hydrophobic moiety and a tetanus toxin or tetanus toxoid refers to a complex characterization by a chemical interaction and preferably to a non-covalent interaction, e.g., a hydrophobic interaction.
  • a purified preparation of tetanus toxin or tetanus toxoid is one which is at least 40, 50, 60, 80, 90, 95, more preferably at least 98, more preferably at least 99, and most preferably at least 99.5 % (weight/weight) tetanus toxin or tetanus toxoid.
  • tetanus toxin or tetanus toxoid preparations of the invention have one or more of the following properties: the preparation is at least 50, 60, 70, 80 or 90% monomeric tetanus toxin or tetanus toxoid (on e.g., a weight/weight basis); the preparation contains less than 3, 5, 6, 7, 8, 9, or 10% tetanus toxin or tetanus toxoid dimers (on e.g., a weight/weight basis); the preparation contains less than 0.4, 0.5, 1.0, 2.0, 3.0, 3.5, 4.0 or 5% higher order polymers of tetanus toxin or tetanus toxoid (where higher order means trimers or higher)(on e.g., a weight/weight basis).
  • Methods of the invention provide a single chromatographic step which both purifies and concentrates tetanus toxoid or tetanus toxin.
  • the recovery of tetanus toxoid is similar to or better than the recovery obtained with the standard ammonium sulfate process.
  • a typical ammonium sulfate procedure includes three precipitation steps followed by dialysis).
  • Methods of the invention provide rapid purification: the entire process can be completed in a 24 hour period with proper column geometry and size.
  • the purity of the tetanus toxoid purified by methods of the invention can be higher than that obtained by the standard ammonium sulfate process.
  • Methods of the invention minimize contamination of the product with the higher molecular weight polymers of tetanus toxoid that are often generated during the formalinization of tetanus toxin. This results in better lot-to-lot consistency and results in a product which is well suited for the production of conjugate toxins.
  • Purified tetanus toxoid can be eluted from the hydrophobic column in a low salt buffer.
  • the material can be further purified by application of ion exchange chromatography (e.g., DEAE-Sepharose) without a prior desalting step.
  • ion exchange chromatography e.g., DEAE-Sepharose
  • the in vivo and in vitro potency of phenyl sepharose purified tetanus toxoid is similar to or better than tetanus toxoid purified by multistep ammonium sulfate processes.
  • phenyl sepharose purified tetanus toxoid is a desirable for use in human vaccines.
  • the methods of the invention can be scaled up with relative ease. For example, 100 L of crude tetanus toxoid (50 Lf/ml) can be purified on only 2 L of phenyl sepharose. The purified tetanus toxoid would be obtained in a total volume of 2-3 L.
  • Fig. 1 is a typical chromatogram for the elution of tetanus toxoid from phenyl sepharose 100 ml column.
  • Fig. 2 is a set of chromatograms of the analysis of tetanus toxoid purified on a TSK- 3000SW size exclusion HPLC column (A) and of tetanus toxoid purified by the ammonium sulfate method (B).
  • hydrophobic groups in aqueous solution are promoted by the presence of water structuring salts, and at high ionic strengths, hydrophobic residues on the surface of a protein associate strongly with other hydrophobic species. At relatively high ionic strengths, protein precipitation is observed. However, at intermediate ionic strengths, proteins may be adsorbed from solution onto hydrophobic surfaces. This adsorption is often reversible, and elution can often be achieved by simply lowering the ionic strength. The strength of adsorption is determined by a number of factors. These include the inherent hydrophobicity of the protein, the hydrophobicity of the adsorbent, temperature, and the concentration and type of electrolyte used to promote salting-out.
  • Water structuring salts are normally used for promoting protein adsorption.
  • Relatively hydrophobic proteins will, in general, require mildly hydrophobic ligands such as butyl groups (otherwise elution problems are encountered), whereas very hydrophilic proteins will, in general, require relatively hydrophobic ligands such as decyl groups (otherwise excessive salt concentrations may be required for binding).
  • the following references provide general guidance in this field: Hydrophobic Interaction Chromatography on non-charged agarose derivatives, J. Rosengren et al., (1980), Biochim. Biophys. Ada 412. 51 ; The Hydrophobic Effect, C. Tanford (1980), John Wiley & Sons, New York; Hydrophobic Interactions, A.
  • hydrophobic moieties can be used to purify tetanus toxoid or toxin.
  • the use of phenyl sepharose, as a hydrophobic interaction matrix, is described herein.
  • the hydrophobic moiety is other than a hydrophobic moiety on a tetanus toxin or toxoid molecule.
  • hydrophobic moieties can be used in methods of the invention.
  • Hydrophobic moieties suitable for use in the method of the invention can be moieties with a degree of hydrophobicity similar to that of the phenyl group used herein, or moieties of higher or lower hydrophobicity than that of the phenyl group.
  • Relative hydrophobicity can be determined by any of several methods known in the art (Hansch, ed., "Comprehensive Medicinal Chemistry", Vol. 4, Pergamon Press, Oxford, 1990), and can be used to guide the choice of potential hydrophobic moieties for use in the invention.
  • the partition coefficient, P between 1 -octanol and water has been used as a reference for measuring the hydrophobicity of a compound.
  • Hydrophobicity can be expressed as log P, the logarithm of the partition coefficient (Hansch et al., Nature 194, 178 (1962); Fujita et al., J. Am. Chem. Soc. 86, 5175 (1964)).
  • Preferred hydrophobic moieties include those with log P greater than 0, or preferably between 0 and about 10, more preferably in the range of about 0 to about 5, even more preferably with log P in the range of about 1 to about 4, and still more preferably between 2 and 3.
  • Suitable hydrophobic moieties may include aromatic hydrocarbons, substituted aromatic hydrocarbons, polyaromatic hydrocarbons, aromatic or non-aromatic heterocycles, cycloalkyl, alkyl, alkenyl, or alkynyl groups, or derivatives thereof.
  • alkyl refers to saturated aliphatic groups having one to thirty carbon atoms, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • Such hydrocarbon moieties may be substituted on one or more carbons.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to six carbon atoms. Representative of such alkyl groups are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopental, and the like.
  • middle alkyl as used herein means an alkyl group, as defined above, but having from seven to twelve carbon atoms.
  • higher alkyl as used herein means an alkyl group, as defined above, but having from thirteen to thirty carbon atoms.
  • aromatic as used herein includes 4-, 5- and 6-member single-ring aromatic groups which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • the aromatic ring may be substituted at one or more ring positions.
  • the aromatic ring may further be fused to one or more other aromatic or non-aromatic rings, forming a polycyclic ring system.
  • Hydrophobic interaction chromatography is a broadly applicable technique and is not necessarily limited to extremely hydrophobic biomolecules.
  • a column is filled with a suitable substrate (e.g., a cross-linked agarose such as sepharose derivatized with a hydrocarbon containing moiety) and equilibrated under conditions favoring hydrophobic binding (e.g., high ionic strength).
  • a suitable substrate e.g., a cross-linked agarose such as sepharose derivatized with a hydrocarbon containing moiety
  • hydrophobic binding e.g., high ionic strength
  • Hydrophobic moieties coupled to supports and suitable for use in hydrophobic interaction chromatography are known to those skilled in the art and are commercially available.
  • Typical materials include polystyrenedivinylbenzene beads which have been surface modified by the attachment of C3 or C4 functional groups (e.g., BioChrom HydrocellTM, Rainin); methyl or t-butyl derivatized methacrylate (Macro-Prep HIC Supports, BioRad); phenyl, butyl, hexyl, octyl, or decyl derivatized cross-linked agarose beads (e.g., PIKSITM H System, American International Chemical).
  • a candidate moiety is appropriate for use in the method of the invention by coupling a moiety to a substrate, e.g., agarose based sepharose or superose, acrylamide based sephacryl, or dextran based sephadex, or other suitable chromatographic support; making a column from the substrate-bound-moiety; and applying crude tetanus toxin or toxoid to the column.
  • the column can than be washed under various conditions to remove contaminants and tetanus toxin or toxoid eluted by use of stepwise or gradient elution, e.g., by eluting with a solution having a descending salt concentration gradient.
  • the product obtained can than be analyzed, e.g., by methods described herein. All fractions should be collected and individual fractions assayed for absorbance at 280 nm. Each fraction should be assayed for toxoid activity, e.g., by the methods described herein to determine the yield and purity of the effluent, thereby determining if the candidate moiety is suitable for use in the purification column.
  • One skilled in the art will be able to vary wash and elution buffers to optimize these for each particular candidate moiety tested in the method of the invention.
  • the entire hydrophobic purification procedure described herein can be carried out at room temperature or at +5°C.
  • the phenyl sepharose (phenyl sepharose CL-4B, Pharmacia, Piscataway, NJ) gel is thoroughly washed with distilled water to remove all traces of the ethanol storage solution.
  • a column is then poured and packed using distilled water as the mobile phase.
  • the column can be further cleaned and depyrogenated by washing with two to three bed volumes of 0.5 M sodium hydroxide.
  • the column is washed with water until the pH of effluent is less than 10.
  • the column is equilibrated with 0.02 M phosphate buffer (pH 7.2) containing 0.5 M ammonium sulfate.
  • Tetanus toxin is produced in anerobic cultures of Clostridium tetani. The cultures are sterilized and detoxified with formaldehyde. The cultures are clarified by filtration giving the crude tetanus toxoid solution, see e.g., Latham et al., 1962, Applied Microbiology 10:146 and W.H.O. Manual for the Production and Control of Vaccines: Tetanus Toxoid (BLG/UNDP/77.2 Rev.I.) Crystalline ammonium sulfate is dissolved in culture filtrate until the final concentration of ammonium sulfate is 0.5 M.
  • the solution is applied to the phenyl sepharose column at the linear flow rate of 20 cm/hr.
  • a culture filtrate containing approximately 150 ⁇ g tetanus toxoid/ml (50 Lf/ml) it is suitable to load 50-70 ml of solution per ml of column bed volume.
  • the column is washed with 5 bed volumes of 3 M sodium chloride.
  • the tetanus toxoid is eluted with 0.002 M phosphate buffer (pH 7.2) containing 0.015 M sodium chloride.
  • the column effluent is monitored by UV detection at 280 nm and the tetanus toxoid peak (as determined by absorption at 280 nm) is collected (Fig. 1).
  • the pooled peak fractions are generally filtered through a sterilizing filter to yield the final product.
  • the phenyl sepharose column generally turns dark brown after use.
  • the column can be cleaned and regenerated by washing with 40% ethanol until all the color is removed.
  • the column should be flushed with 2 bed volumes of 20% ethanol for storage.
  • PURIFICATION OF TETANUS TOXOID BY A STANDARD AMMONIUM SULFATE METHOD The following ammonium sulfate precipitation-based method can be used to produce preparation of tetanus toxoid.
  • Proteins are salted out at 60% SAS by adding 0.37 Kg AS per liter of crude toxoid solution. The solution is stirred to dissolve the AS and allowed to stand at 5°C several hours or overnight to permit flotation of the proteins. The proteins are collected by drawing off and discarding the underlying clear liquor using a syphon tube previously inserted. It may be necessary to filter or centrifuge the last liter or so of solution.
  • the precipitate is dissolved in distilled water to l/20 tn the original volume.
  • AS content is raised to 23% SAS by adding saturated AS solution.
  • the solution is then centrifuged and the precipitate is discarded.
  • the volume of the supernate is measured and the AS concentration brought to 60% SAS with saturated AS solution. The solution is stirred for 30 minutes and allowed to stand overnight at +5°C.
  • the precipitate is collected by centrifugation and the supernatant discarded.
  • the precipitate is dissolved in saline to 1/80* original volume. (A magnetic stirrer is a convenient aid in dissolving these precipitates.)
  • the solution is dialyzed until free of sulfate. (The presence of sulfate is assessed by adding barium chloride to a sample of the toxoid dialysate and detecting the formation of a barium sulfate precipitate.)
  • the product is filtered through a 0.2 ⁇ m sterilizing filter.
  • the molecular weight distribution of the purified tetanus toxoid can be determined by high performance size exclusion chromatography using Tosohaas (Montgomeryville, PA) TSK-3000SW column (Fig. 2).
  • the mobile phase is phosphate buffered saline (PBS) at a flow rate of 1 ml per minute.
  • the wavelength of detection is 280 nm.
  • the potency of purified tetanus toxoid can be tested as described in the NIH Minimum Requirements for Tetanus Toxoid.
  • In vitro antigenicity can be determined by the flocculation (Lf) assay with a standard antitoxin against tetanus toxin. The in vitro antigenicity is generally expressed as Lf units per mg of protein nitrogen. The in vivo potency is generally expressed as antitoxin units per ml (AU/ml) of guinea pig sera.
  • tetanus toxoid should meet the safety, purity and immunogenic potency specifications described in the NIH Minimum Requirements for tetanus toxoid (U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Maryland, 4th Revision, December 15, 1952).
  • EXAMPLE 1 A typical chromatogram for the elution of tetanus toxoid is shown in Fig. 1. In this example, 6 liters of crude tetanus toxoid were loaded in a 100 ml (5 cm X 5 cm) phenyl sepharose column.
  • the amount of tetanus toxoid recovered after chromatography ranges from 60-80% (Table 1). This is determined from the number of Lf units recovered in the product compared to the total Lf in the starting crude culture filtrate.
  • the in vitro potency of phenyl sepharose purified tetanus toxoid ranges from 1500-2700 Lf/mgN (Table 1).
  • EXAMPLE 2 The in vivo potency of purified tetanus toxoid in guinea pig is similar to tetanus toxoid purified by the ammonium sulfate method. For example, both materials behaved comparably (5-6 AU/ml) when tested as components of acellular pertussis vaccines (Table 3). The NIH Minimum Requirement for Tetanus toxoid in this assay is 2 AU/ml.
  • Group of 8 animals were immunized with 0.75 ml of vaccine containing the following antigens absorbed to aluminum phosphate: 37.5 ⁇ g of pertussis toxoid; 4.5 ⁇ g of FHA; 7.5 Lf of tetanus toxoid and 15 Lf of diphtheria toxoid.
  • Sera were collected at 4 and 6 weeks after immunization. Contains phenyl sepharose purified tetanus toxoid. Contains ammonium sulfate purified tetanus toxoid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Préparation de toxine ou toxoïde tétanique purifié(e) et leurs procédés de préparation.
PCT/US1996/002173 1995-02-16 1996-02-16 Toxoide et toxine tetaniques purifies Ceased WO1996025425A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49865/96A AU4986596A (en) 1995-02-16 1996-02-16 Purified tetanus toxoid and toxin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38958295A 1995-02-16 1995-02-16
US08/389,582 1995-02-16

Publications (1)

Publication Number Publication Date
WO1996025425A1 true WO1996025425A1 (fr) 1996-08-22

Family

ID=23538865

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/002173 Ceased WO1996025425A1 (fr) 1995-02-16 1996-02-16 Toxoide et toxine tetaniques purifies

Country Status (2)

Country Link
AU (1) AU4986596A (fr)
WO (1) WO1996025425A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063794A1 (fr) * 2003-12-30 2005-07-14 Bharat Biotech International Limited Procede de preparation et de purification de proteines de recombinaison
CN104327171A (zh) * 2014-09-30 2015-02-04 成都欧林生物科技股份有限公司 一种层析纯化法生产破伤风类毒素原液的方法
WO2018027126A1 (fr) 2016-08-05 2018-02-08 Sanofi Pasteur, Inc. Composition de conjugué polysaccharide pneumococcique multivalent-protéine
WO2018027123A1 (fr) 2016-08-05 2018-02-08 Sanofi Pasteur, Inc. Composition d'un conjugué polysaccharide-protéine pneumococcique multivalent
WO2021021729A1 (fr) 2019-07-31 2021-02-04 Sanofi Pasteur Inc. Compositions de conjugués de polysaccharide-protéine pneumococcique multivalents leurs méthodes d'utilisation
US11333642B2 (en) 2016-10-25 2022-05-17 Regeneran Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
US11369896B2 (en) 2016-08-16 2022-06-28 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11884698B2 (en) 2018-07-02 2024-01-30 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
US12042538B2 (en) 2017-09-19 2024-07-23 Regeneron Pharmaceuticals, Inc. Methods of reducing particle formation and compositions formed thereby
US12070502B2 (en) 2014-10-09 2024-08-27 Regeneron Pharmaceuticals, Inc. Process for reducing subvisible particles in a pharmaceutical formulation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000098A (en) * 1974-08-16 1976-12-28 Palo Alto Medical Research Foundation Separation of proteins by hydrophobic adsorption

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000098A (en) * 1974-08-16 1976-12-28 Palo Alto Medical Research Foundation Separation of proteins by hydrophobic adsorption

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOXICON, Vol. 30, No. 1, 1992, HOGY et al., "Increase of Permeability of Synaptosomes and Liposomes by the Heavy Chain of Tetanus Toxin", pages 63-76. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063794A1 (fr) * 2003-12-30 2005-07-14 Bharat Biotech International Limited Procede de preparation et de purification de proteines de recombinaison
CN104327171A (zh) * 2014-09-30 2015-02-04 成都欧林生物科技股份有限公司 一种层析纯化法生产破伤风类毒素原液的方法
CN104327171B (zh) * 2014-09-30 2017-08-25 成都欧林生物科技股份有限公司 一种层析纯化法生产破伤风类毒素原液的方法
US12070502B2 (en) 2014-10-09 2024-08-27 Regeneron Pharmaceuticals, Inc. Process for reducing subvisible particles in a pharmaceutical formulation
WO2018027126A1 (fr) 2016-08-05 2018-02-08 Sanofi Pasteur, Inc. Composition de conjugué polysaccharide pneumococcique multivalent-protéine
WO2018027123A1 (fr) 2016-08-05 2018-02-08 Sanofi Pasteur, Inc. Composition d'un conjugué polysaccharide-protéine pneumococcique multivalent
US11850535B2 (en) 2016-08-16 2023-12-26 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11369896B2 (en) 2016-08-16 2022-06-28 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11571636B2 (en) 2016-08-16 2023-02-07 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11333642B2 (en) 2016-10-25 2022-05-17 Regeneran Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
US11680930B2 (en) 2016-10-25 2023-06-20 Regeneron Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
US12042538B2 (en) 2017-09-19 2024-07-23 Regeneron Pharmaceuticals, Inc. Methods of reducing particle formation and compositions formed thereby
US11884698B2 (en) 2018-07-02 2024-01-30 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
WO2021021729A1 (fr) 2019-07-31 2021-02-04 Sanofi Pasteur Inc. Compositions de conjugués de polysaccharide-protéine pneumococcique multivalents leurs méthodes d'utilisation

Also Published As

Publication number Publication date
AU4986596A (en) 1996-09-04

Similar Documents

Publication Publication Date Title
JP2562192B2 (ja) 血清アルブミンの純化方法
US4568488A (en) Reverse immunoaffinity chromatography purification method
Irons et al. Isolation of the lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis by affinity chromatography
US4075193A (en) Process for producing intravenous immune globulin
CN105153297B (zh) 一种从Cohn组分Ⅳ沉淀中分离纯化α2-巨球蛋白的方法
IE44507B1 (en) Cationic material capable of reversibly fixing biological macromolecules
EP0140386B1 (fr) Méthode de purification de LPF-HA
JP3768485B2 (ja) 血清アルブミンの精製方法
WO1996025425A1 (fr) Toxoide et toxine tetaniques purifies
Ryan et al. Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-heme-octapeptide conjugates
EP0423938B1 (fr) Milieu contenant un ligand pour la séparation chromatographique, son procédé de préparation et son utilisation pour isoler des molécules synthétiques ou naturelles de mélanges de fluides
Klingenberg et al. [36] Isolation of functional membrane proteins related to or identical with the ADP, ATP carrier of mitochondria
CA1114293A (fr) Purification des hemagglutinines de b. pertussis
CN114805504B (zh) 一种低聚合破伤风类毒素制备方法
EP0391974B1 (fr) Procédé pour la purification des facteurs coagulants du sang dépendants de la vitamine K
JPH09506256A (ja) メンブレンクロマトグラフィーによるビタミンｋ依存性蛋白の精製
Colbeau et al. Purification, molecular properties and localization in the membrane of the hydrogenase of Rhodopseudomonas capsulata
US5445958A (en) Process for purifying blood clotting factors
Miccio et al. Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono-and poly-clonal antibodies
Bassett Large scale preparation of wheat germ agglutinin
CN1234729C (zh) 一种蛋白质交联剂及其交联方法
JPH09504532A (ja) クロマトグラフィー法による第８因子を含むウィルス不活性化分画の製造方法
JPWO1992014832A1 (ja) ヒトｂｃｄｆの精製法
CN109678932B (zh) 一种IgG抗体亲和的小分子肽及其应用
Lundblad Isolation of fucose-rich glycopeptides from normal urine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase