WO2023027044A1 - Procédé de réduction des endotoxines - Google Patents

Procédé de réduction des endotoxines Download PDF

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Publication number
WO2023027044A1
WO2023027044A1 PCT/JP2022/031625 JP2022031625W WO2023027044A1 WO 2023027044 A1 WO2023027044 A1 WO 2023027044A1 JP 2022031625 W JP2022031625 W JP 2022031625W WO 2023027044 A1 WO2023027044 A1 WO 2023027044A1
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endotoxin
liquid
magnesium carbonate
basic magnesium
recombinant
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PCT/JP2022/031625
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English (en)
Japanese (ja)
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史憲 鴻池
正克 西八條
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株式会社カネカ
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Publication of WO2023027044A1 publication Critical patent/WO2023027044A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium

Definitions

  • the present invention relates to a method that can effectively and simply reduce endotoxin in liquid.
  • Endotoxin also called lipopolysaccharide, endotoxin, LPS, pyrogen, etc.
  • Endotoxin is present in the outer membrane of Gram-negative bacteria, and has a structure composed of three parts: an O-antigen polysaccharide, a core polysaccharide, and a lipid portion called lipid A. have.
  • the sugar chain structure of such O-antigen polysaccharides varies greatly depending on the type of bacteria, and each bacterium induces a specific immune response.
  • Endotoxin causes no particular problems when taken orally, but when it enters the blood even in extremely small amounts, it causes fever, lethal shock, blood pressure drop, intravascular coagulation, activation of leukocytes, and the like.
  • transformed cells have been used to produce proteins and peptides, and such proteins and peptides are also used as active ingredients in pharmaceuticals.
  • Gram-negative bacteria such as Escherichia coli are also used as transformed cells, and endotoxin contamination is a concern.
  • endotoxins can cause problems even in minute amounts, so techniques for removing endotoxins are important.
  • Patent Document 1 discloses an endotoxin adsorbent containing polymyxin
  • Patent Document 2 discloses an endotoxin adsorbent containing polylysine
  • Patent Document 2 discloses a endotoxin adsorbent containing polylysine.
  • No. 3 discloses an endotoxin adsorption-removing agent containing aluminum silicate
  • Patent Document 4 discloses an endotoxin adsorbent such as hydroxyapatite: Ca 5 (PO 4 ) 3 (OH).
  • Patent Document 1 since polymyxin is relatively expensive, the manufacturing cost of the endotoxin adsorbent of Patent Document 1 is considered high.
  • cationic ligands such as polylysine in Patent Document 2 may adsorb not only endotoxin but also target proteins and peptides.
  • the aluminum silicate of Patent Document 3 can be said to be relatively inexpensive, but it is necessary to adjust the zeta potential to be positive. It may get lost.
  • the hydroxyapatite of Patent Document 4 also adsorbs antibodies and the like in addition to endotoxin.
  • Patent Document 5 discloses a method for purifying antibodies (Patent Document 5) and a method for purifying viruses (Patent Document 6) using inexpensive inorganic compounds.
  • Patent Document 7 discloses a method for purifying a protein, which comprises the steps of forming a solid containing cations and anions in a protein-containing medium and separating the solid.
  • an object of the present invention is to provide a method capable of effectively and simply reducing the amount of endotoxin in a liquid.
  • the present inventors have made intensive studies to solve the above problems. As a result, the present inventors have found that the amount of endotoxin can be reduced by bringing basic magnesium carbonate into contact with a substance to be purified that contains endotoxin as an impurity, and that the subsequent purification load of the target useful substance can be reduced, thereby completing the present invention. .
  • the present invention is shown below.
  • a method for reducing the amount of endotoxin in a liquid comprising: contacting a liquid containing 1 EU/mL or more of endotoxin with basic magnesium carbonate to adsorb at least part of the endotoxin onto the basic magnesium carbonate; A method comprising separating said basic magnesium carbonate from the liquid fraction of said liquid.
  • the liquid is a gram-negative bacterial culture, homogenate, or extract.
  • the Gram-negative bacterium is transformed.
  • the method according to any one of [1] to [3], wherein the liquid further contains a useful substance.
  • the useful substance is a recombinant protein.
  • the recombinant protein is a recombinant enzyme, a recombinant growth factor, a recombinant hormone, or a recombinant cytokine.
  • the recombinant protein is a recombinant antibody or a recombinant antibody-like molecule.
  • the amount of endotoxin in the liquid is reduced for the production of the useful substance.
  • a method for producing a useful substance comprising: contacting the liquid containing the useful substance and 1 EU/mL or more of endotoxin with basic magnesium carbonate to adsorb at least part of the endotoxin onto the basic magnesium carbonate; A method comprising separating said basic magnesium carbonate from the liquid fraction of said liquid.
  • the liquid is a culture medium, homogenate or extract of Gram-negative bacteria.
  • the Gram-negative bacterium is transformed.
  • the recombinant protein is a recombinant enzyme, a recombinant growth factor, a recombinant hormone, or a recombinant cytokine.
  • the recombinant protein is a recombinant antibody or a recombinant antibody-like molecule.
  • endotoxin can be adsorbed to basic magnesium carbonate, while target useful substances such as proteins and peptides are not adsorbed or hardly adsorbed, so that the amount of endotoxin can be reduced. It is possible to suppress the contamination of the useful substance of interest with endotoxin and to roughly purify the useful substance of interest.
  • basic magnesium carbonate is inexpensive, does not require any special treatment, and can adsorb endotoxin simply by contacting it with a liquid containing endotoxin. Therefore, the method of the present invention can be easily implemented at low cost. is possible.
  • basic magnesium carbonate can be industrially mass-produced in advance, the particle size and shape can be controlled, and the effect can be easily obtained with good reproducibility. Furthermore, since basic magnesium carbonate can be used in a relatively small amount for treatment, it hardly gives adverse effects such as denaturation to the intended useful substance, and can suppress the generation of water-insoluble magnesium salts in the latter stage. In addition, since basic magnesium carbonate can improve filtration efficiency as a filter aid, it can be separated from a liquid in a short period of time by filtration. Therefore, the present invention is industrially extremely useful as it can contribute to mass production of useful substances such as antibodies, for which demand is expected to increase in the future.
  • the present invention reduces the amount of endotoxin in the liquid by contacting basic magnesium carbonate with liquid containing endotoxin at a concentration that can cause problems, thereby adsorbing at least part of endotoxin to basic magnesium carbonate. It is about how to The method of the present invention will be described below by dividing it into steps, but the present invention is not limited to the following specific examples. For example, an embodiment in which two or more of the following individually described embodiments are combined is also included in the scope of the present invention.
  • a liquid containing endotoxin to be reduced is prepared. Implementation of this step is optional, and there is no need to perform this step when such a liquid has already been obtained.
  • the liquid may be an aqueous solution or suspension.
  • An aqueous solution refers to a solution in which all components are dissolved in water, which is a solvent, and substantially free of insoluble components
  • a suspension refers to a liquid that may contain solutes but also includes insoluble components.
  • insoluble components include insoluble components of cell-derived components such as cells, cell debris, aggregated proteins, and the like. The insoluble component may be dispersed or sedimented in the solution.
  • Endotoxin also called lipopolysaccharide, endotoxin, LPS, pyrogen, etc.
  • Endotoxin is present in the outer membrane of Gram-negative bacteria, and has a structure composed of three parts: an O-antigen polysaccharide, a core polysaccharide, and a lipid portion called lipid A. have.
  • This lipid A is buried in the outer membrane of Gram-negative bacteria, and the O-antigen polysaccharide extends from lipid A through the core polysaccharide.
  • Endotoxin is known as a pathogenic factor, is released by the death or destruction of Gram-negative bacteria, and lipid A excessively enhances the immune response, causing successive or simultaneous dysfunction of vital organs. Therefore, it is necessary to suppress contamination of the useful substance to be purified with endotoxin.
  • Endotoxins are generally contaminated from Gram-negative bacteria, the host, but there are also known cases of endotoxin contamination during the manufacturing process of useful substances.
  • the endotoxin concentration in the liquid is preferably 1 EU/mL or more. If the concentration is less than 1 EU/mL, there is a possibility that the amount of endotoxin can be reduced to a non-problematic level without using the present invention by purifying the intended useful substance.
  • the concentration is preferably 10 2 EU/mL or higher, more preferably 10 3 EU/mL or higher.
  • the upper limit of the concentration is not particularly limited, but can be, for example, 10 8 EU/mL.
  • the concentration is, for example, preferably 10 7 EU/mL or less or 10 6 EU/mL or less, more preferably 10 5 EU/mL or less.
  • the liquid to be treated with specific basic magnesium carbonate is not particularly limited as long as it contains endotoxin as an impurity, and may contain useful substances.
  • Such liquids include, for example, culture solutions, culture supernatants, cultured cells, cell lysates, or cell lysates suspended in water, extracts thereof, biological extracts, viruses or virus-like particles.
  • the cells may be virus-infected cells.
  • a liquid containing a virus or virus-like particles may be produced by infecting cultured cells or chicken fertilized eggs with the virus to produce viruses or virus-like particles in a culture solution or body fluid.
  • body fluids are fluids that fill between tissues, body cavities, tubes and circulatory systems spread throughout the body, or are secreted and excreted inside and outside the body of animals.
  • chorioallantoic fluid separated after inoculation of blood or virus strain into the allantoic cavity of chicken eggs and culturing may be used.
  • Liquids containing endotoxins include, for example, culture fluids of Gram-negative bacteria, their lysates, and extracts.
  • the lysate of Gram-negative bacteria refers to a suspension obtained by homogenizing a culture solution of Gram-negative bacteria using a homogenizer, a French press, or the like, or by ultrasonicating or freeze-thawing.
  • a solution may be obtained from the suspension by filtration, centrifugation, or the like.
  • lysozyme or a surfactant may be used in combination when disrupting bacteria.
  • the liquid may contain useful substances to be purified.
  • useful substances include proteins such as antibodies, antibody-like molecules, antibody-binding proteins, enzymes, growth factors, hormones, cytokines, and blood proteins; peptides containing active sites of proteins; gene therapy; Examples include viruses and virus-like particles that can be used.
  • the "protein” that can be purified in the present invention may be any protein that is considered useful for industrial use, and is a functional protein having a polypeptide structure and a secondary structure such as an ⁇ -helix or ⁇ -sheet structure.
  • a functional protein having a polypeptide structure and a secondary structure such as an ⁇ -helix or ⁇ -sheet structure.
  • those having a sugar chain those modified with sugars, those subjected to modifications such as phosphorylation and tyrosination, and those coordinated with metals.
  • those produced by genetic recombination techniques those whose functions have been improved, structures consisting only of functional sites, linkages of different functional sites, Includes concatenations of identical functional sites.
  • those intramolecularly crosslinked by disulfide bonds of intramolecular cysteine residues include proteins linked by modification, and proteins functionalized by chemical modification or addition of functional molecules.
  • Antibodies or antibody-like molecules are not particularly limited, and include polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, heavy-chain antibodies, multivalent antibodies, Fab, F(ab'), F(ab') 2 , Fc, Fc fusion protein, bispecific antibody, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc(Fv) 2 , disulfide-bonded Fv (sdFv) , Diabodies, antibody-like molecular target peptides (microantibodies), and the like.
  • these antibodies or antibody-like molecules may be immunoglobulins, Fc-containing proteins such as Fc fusion proteins having an Fc portion, and the above Fab, F(ab'), F(ab') 2 , Fc, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc (Fv) 2 , disulfide bond Fv (sdFv), single chain antibody, heavy chain antibody, multivalent antibody, bivalent Any of low-molecular-weight antibodies such as specific antibodies, diabodies, and antibody-like molecule-targeting peptides (microantibodies) can be preferably targeted.
  • Fc-containing proteins such as Fc fusion proteins having an Fc portion
  • Enzymes include, for example, lipase, protease, steroidogenic enzyme, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.
  • Growth factors include, for example, epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial cell proliferation factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast proliferation factor (FGF), hepatocyte growth factor (HGF).
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • TGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • VEGF vascular endothelial cell proliferation factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Hormones include, for example, insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin.
  • Cytokines include, for example, interleukins, interferons (IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factor (TNF).
  • Blood proteins include, for example, thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.
  • Antibody-binding protein is not particularly limited as long as it is a protein having specific binding ability to antibody, for example, protein A, protein G, protein L, Fc ⁇ receptor, these antibody-binding domains, and antibody or These variants that maintain or improve their ability to bind to antibody-like molecules are included.
  • non-enveloped viruses include adeno-associated virus, adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus, rotavirus. , coxsackievirus, sapovirus, norovirus, poliovirus, echovirus, coronavirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, and the like.
  • Adeno-associated viruses include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV -12, AAV-13, AAV-14, AAV-15 and AAV-16.
  • enveloped viruses include retrovirus, lentivirus, Sendai virus, herpes simplex virus, vaccinia virus, measles virus, baculovirus, influenza virus, and the like.
  • Virus-like particles are all or part of the viral coat protein that mainly constitutes the capsid, and since they do not contain nucleic acids, there is no concern about infection. can.
  • liquids containing endotoxins include culture fluids of Gram-negative bacteria, their crushed fluids, and extracts. Transformed cells of Gram-negative bacteria are also used for the production of recombinant proteins. Therefore, the liquid containing endotoxin may be the culture medium of the transformed Gram-negative bacterium, its lysate or extract, and the useful substance to be purified may be a recombinant protein.
  • Recombinant proteins include recombinants of the above useful substances, specifically one or more recombinant proteins selected from recombinant enzymes, recombinant growth factors, recombinant hormones, recombinant cytokines, recombinant antibodies, and recombinant antibody-like molecules. is mentioned.
  • the liquid may contain other impurities in addition to endotoxins and useful substances.
  • Other impurities include, but are not limited to, aggregated proteins, plasmids, medium components, nucleic acids such as plasmid DNA, and the like.
  • water is preferable as a liquid solvent, a buffer solution may be used.
  • Basic magnesium carbonate may adsorb impurities other than endotoxin in addition to endotoxin.
  • water-insoluble refers to the degree of dissolution within 30 minutes when magnesium compound powder is placed in purified water and strongly shaken for 30 seconds every 5 minutes at 20 ⁇ 5 ° C. Specifically, 1 g of magnesium compound The amount of purified water required to dissolve is 100 mL or more. The amount of purified water is preferably 1000 mL or more.
  • an alkali metal carbonate such as sodium carbonate or potassium carbonate
  • water-insoluble inorganic magnesium salts containing metal ions other than magnesium such as hydrotalcite: Mg 6 Al 2 CO 3 (OH) 16 4H 2 O, are useful for antibodies and the like.
  • the size of the basic magnesium carbonate may be appropriately adjusted, and for example, the average particle size can be 0.1 ⁇ m or more and 1000 ⁇ m or less. If the average particle size is 1000 ⁇ m or less, the specific surface area of basic magnesium carbonate is sufficiently large, and endotoxin can be adsorbed more efficiently. no energy required.
  • the average particle size is preferably 1 ⁇ m or more, more preferably 5 ⁇ m or more.
  • the average particle size is measured by a laser diffraction particle size distribution analyzer, and the average particle size may be measured on a volume basis, a weight basis, or a number basis, but the volume basis is preferable.
  • the specific surface area of the basic magnesium carbonate may be appropriately adjusted, but is preferably 1 m 2 /g or more and 1000 m 2 /g or less, for example.
  • the specific surface area is preferably 10 m 2 /g or more and 500 m 2 /g or less.
  • the specific surface area shall be measured by a nitrogen gas adsorption device using a dried basic magnesium carbonate sample.
  • the amount of basic magnesium carbonate to be used may be adjusted according to the amount of endotoxin in the liquid. For example, 0.01 g or more and 100 g or less of basic magnesium carbonate may be used per 100 mL of the liquid. The ratio is preferably 15 g/100 mL or less.
  • Basic magnesium carbonate can be used in an amount of 0.01 w/v% or more and 100 w/v% or less with respect to the liquid, and the ratio is preferably 1 w/v% or more and 15 w/v% or less.
  • a larger amount of basic magnesium carbonate may be used.
  • the method of contacting the liquid and basic magnesium carbonate may be selected as appropriate.
  • basic magnesium carbonate may be added to the liquid and shaken or stirred.
  • the temperature at that time may be room temperature, specifically, 0° C. or higher and 40° C. or lower.
  • the temperature is preferably 1° C. or higher, more preferably 10° C. or higher or 15° C. or higher, and preferably 30° C. or lower and more preferably 25° C. or lower.
  • the contact time can be 1 second or more and 10 hours or less.
  • the basic magnesium carbonate on which at least part of the endotoxin contained in the liquid is adsorbed is separated from the liquid component of the liquid.
  • the liquid component of the liquid refers to the remainder after removing the insoluble components and the endotoxin adsorbed on the basic magnesium carbonate from the liquid. Separation means is not particularly limited as long as it can separate the basic magnesium carbonate from the liquid component, and examples thereof include centrifugation and filtration.
  • the useful substances are mainly dispersed in the liquid, and all or part of the other impurities are mainly adsorbed on the basic magnesium carbonate.
  • part of the useful substances may be adsorbed by the basic magnesium carbonate and part of the impurities may be dissolved in the liquid, but at least the total amount of endotoxin in the liquid can be reduced, Useful substances are concentrated.
  • a column may be packed with basic magnesium carbonate and the liquid may be circulated to allow endotoxin to be adsorbed on the basic magnesium carbonate.
  • endotoxin adsorption and liquid separation from basic magnesium carbonate can be performed simultaneously.
  • the amount of basic magnesium carbonate packed in the column and the flow rate of the liquid are preferably adjusted within a range in which at least endotoxin is sufficiently adsorbed by basic magnesium carbonate.
  • steps 2 and 3 at least part of the endotoxin and relatively large insoluble components such as cells are separated and removed, and if useful substances are contained, it is preferable to further purify them by chromatography.
  • the amount of impurities or other impurities may be further reduced by common processing steps. The general processing steps are described below.
  • Step 4 Treatment Step with Activated Carbon
  • the liquid containing impurities is brought into contact with activated carbon.
  • impurities in the present disclosure refer not only to endotoxins but also to compounds other than intended useful substances.
  • This step may be performed before or after step 2 described above, or may be performed simultaneously using basic magnesium carbonate and activated carbon. However, implementation of this step is optional.
  • Activated carbon is made by burning charcoal, coconut shells, or the like to develop pores to make it porous, and has excellent adsorption performance.
  • the general specific surface area of activated carbon is about 800 m 2 /g or more and 2500 m 2 /g or less.
  • the average pore diameter of the activated carbon is not particularly limited, but is usually 0.1 nm or more and 20 nm or less, preferably 0.5 nm or more and 5.0 nm or less, more preferably 2.0 nm or more and 5.0 nm or less, and still more preferably It is 3.0 nm or more and 5.0 nm or less.
  • the average pore diameter of activated carbon can be calculated from a nitrogen adsorption isotherm curve using the BJH method.
  • the means of the purification method using activated carbon of the present invention is not particularly limited, but examples thereof include a batch method, a membrane treatment method, a column chromatography method, and the like, and an appropriate form of activated carbon is selected according to each method. be. If necessary, in the form of particles in which activated carbon is encapsulated in a porous polymer or gel, in the form of a membrane in which activated carbon is adsorbed, fixed or molded using a support agent such as polypropylene or cellulose or fibers, or in the form of a cartridge. You can also use
  • the amount of activated carbon used may be adjusted according to the concentration of impurities in the liquid to be treated, but for example, 0.5 g or more and 5 g or less of activated carbon may be used per 100 mL of liquid.
  • activated carbon may be added to the impurity-containing liquid and shaken or stirred, or the column may be filled with activated carbon, as in the case of basic magnesium carbonate.
  • this step 4 and the above step 2 are performed at the same time, a mixture of basic magnesium carbonate and activated carbon may be used.
  • the liquid and the activated carbon are separated.
  • the step 3 may be performed after performing the step 4 and the step 2 at the same time.
  • a liquid containing impurities is treated with a flocculant.
  • This step may be performed before or after the treatment step 2 with basic magnesium carbonate and/or the treatment step 4 with activated carbon, or the basic magnesium carbonate and/or activated carbon and flocculant may be used simultaneously. However, implementation of this step is optional.
  • the flocculants include caprylic acid, polyamines, divalent cations, polyetherimine, chitosan, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, poly(diallyldimethylammonium monochloride) (pDADMAC), and the like.
  • Divalent cations include, for example, Ca 2+ , Mg 2+ , Cu 2+ , Co 2+ , Mn 2+ , Ni 2+ , Be 2+ , Sr 2+ , Ba 2+ , Ra 2+ , Zn 2+ , Cd 2+ , Ag 2+ , Pd 2+ , Rh 2+ , and these divalent cations can be used in free form or as hydrochloride, sulfate, citrate and the like.
  • the amount of flocculant used may be adjusted depending on the concentration of impurities contained in the liquid to be treated.
  • the following flocculants may be used, and more preferably 0.1 w/v % or more and 1 w/v % or less of flocculants may be used.
  • the flocculant is caprylic acid, chitosan, polyethylene glycol, polyvinyl alcohol or polyvinylpyrrolidone
  • the floculant should be used in an amount of 0.01 w/v% or more and 10 w/v% or less, more preferably 1 w/v% or more and 5 w. /v% or less flocculant may be used.
  • the flocculant When the flocculant is pDADMAC, 0.01 w/v% or more and 1 w/v% or less of floculant may be used, and more preferably 0.1 w/v% or more and 0.5 w/v% or less of floculant is used. Just do it.
  • the flocculant When the flocculant is a divalent cation, it may be added in an amount such that the divalent cation concentration is 1 mM or more and 100 mM or less, more preferably 2 mM or more and 50 mM or less.
  • the floculant may be added to the impurity-containing liquid and shaken or stirred, or the column may be filled with the floculant, as in the case of basic magnesium carbonate.
  • this step 5 and the treatment step 2 with basic magnesium carbonate and/or the treatment step 4 with activated carbon are performed at the same time, a mixture of floculant and basic magnesium carbonate and/or activated carbon may be used.
  • the liquid and the flocculant are separated.
  • the step 3 may be performed after performing the step 5 and the steps 2 and 4 at the same time.
  • the liquid containing impurities is treated with an endonuclease.
  • This step may be performed before or after each of the above steps, or may be performed at the same time using one or more selected from basic magnesium carbonate, activated carbon and flocculant and an endonuclease in combination. may However, implementation of this step is optional.
  • An endonuclease is one of the enzymes that degrade DNA and has the function of degrading even the center of the base sequence.
  • commercially available products include Benzonase (manufactured by Millipore) and Kaneka Endonuclease (manufactured by Kaneka).
  • the amount of endonuclease used may be adjusted according to the concentration of DNA contained in the liquid, but is preferably 10 U/mL or more, more preferably 100 U/mL or more, for the liquid.
  • the upper limit is not particularly limited, but is preferably 10,000 U/mL or less.
  • the above processes can be suitably used not only before or after the general impurity reduction process such as membrane or column treatment described below, but
  • the above steps are performed before the column treatment, it is necessary to suppress the decrease in the adsorption capacity of the chromatography carrier, the decrease in the separation ability, the decrease in the treatment speed due to the increase in back pressure, and the decrease in the carrier life due to the decrease in washing and regeneration efficiency.
  • the membrane filtration process it can be expected to suppress the decrease in the carrier life due to the decrease in the processing capacity per unit membrane area, the increase in back pressure, the decrease in the processing speed, and the decrease in washing and regeneration efficiency. That is, it is also a preferred embodiment of the present invention to further subject the useful substance-containing liquid in which impurities have been reduced by the above steps to column treatment or membrane filtration treatment. , the method of the present invention can also be used.
  • Liquids containing useful substances can be purified by column treatment such as chromatography.
  • the chromatography to be used is not particularly limited as long as it is a method capable of recovering and purifying the useful substance of interest.
  • Anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, mixed mode chromatography. Chromatography, affinity chromatography, and the like may be mentioned, and these may be used alone or in combination.
  • the above steps, particularly steps 2 and 3 can be suitably used before or after the chromatography step.
  • the amount of solvent can be reduced to concentrate the useful substance, or the solvent can be replaced.
  • Impurities can be quantified at any stage by absorbance analysis, electrophoresis, HPLC, and commercially available assay kits.
  • fluorescence analysis using fluorescent reagents in addition to absorption analysis at the maximum absorption wavelength of nucleic acids, fluorescence analysis using fluorescent reagents, high performance liquid chromatography isotope dilution mass spectrometry (LC IDMS), inductively coupled plasma emission spectrometry (ICP OES) and Quantification by emission spectrometry such as inductively coupled plasma mass spectrometry (ICP MS), mass spectrometry, chromatographic analysis, or analytical methods that combine them, or methods such as gel electrophoresis, q-PCR, and next-generation DNA sequencing can be done.
  • LC IDMS high performance liquid chromatography isotope dilution mass spectrometry
  • ICP OES inductively coupled plasma emission spectrometry
  • Quantification by emission spectrometry such as inductively coupled plasma mass spectrometry (ICP MS), mass spect
  • host-derived proteins derived from CHO cells can be quantified using a CHO HCP ELISA kit (manufactured by Cygnus). If there is no commercially available assay kit for the desired contaminant protein, a desired detection system can be created by immunizing animals such as chickens with the contaminant protein. If some impurities other than useful substances can be removed before chromatography, the load on chromatography can be reduced and more efficient purification becomes possible.
  • Example 1 Removal of endotoxin from E. coli lysate E. E. coli W3110 was cultured with shaking at 30° C. for 40 hours in a medium having the composition shown in Table 1 below to obtain an E. coli culture solution.
  • the resulting Escherichia coli culture solution is ultrasonically disrupted, and basic magnesium carbonate (“Basic magnesium carbonate, light” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added at a rate of 1% by mass or 5% by mass, and stirred for 1 hour. bottom. Then, it was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected as a treated liquid.
  • Example 2 Removal of endotoxin from the periplasm The E. coli culture of Example 1 was centrifuged at 10,000 x G for 20 minutes and the supernatant discarded to obtain an E. coli pellet. 20 w/v% sucrose-30 mM Tris-HCl buffer (pH 7.1) containing 5 times the amount of 1 mM EDTA was added to the resulting E. coli pellet, and after thorough stirring, centrifugation was performed at 10,000 ⁇ G for 20 minutes. Separate and discard the supernatant.
  • Example 3 Endotoxin adsorption of E. coli lysate
  • the E. coli lysate prepared in the same manner as in Example 1 was added with 1% by mass of calcium carbonate (“calcium carbonate” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), calcium phosphate (“phosphate Calcium monohydrogen” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), synthetic magnesite ("Magthermo (R) " manufactured by Kojima Chemical Co., Ltd.), or basic magnesium carbonate (“basic magnesium carbonate, light” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ) was added and stirred for 1 hour.
  • calcium carbonate (“calcium carbonate” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
  • calcium phosphate phosphate Calcium monohydrogen” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • synthetic magnesite Magnetic magnesite
  • basic magnesium carbonate basic magnesium carbonate, light” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • Endotoxin removal rate (1-endotoxin concentration in treatment solution/endotoxin concentration before treatment) x 100 (%)
  • Example 4 Purification of Insulin Using Basic Magnesium Carbonate To E. coli lysate prepared in the same manner as in Example 1, the hormone insulin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1 mg/mL. After adding 1 w/v% or 5 w/v% of basic magnesium carbonate and stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to recover the supernatant to obtain a treated liquid. The obtained treated solution and the solution before treatment were subjected to SDS-PAGE, and the band intensity around 5.8 kDa was read with an imaging system ("Chemidoc TM Touch" manufactured by Bio-Rad). Insulin recovery was calculated by dividing the band intensity before treatment from the band intensity after treatment. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 5 shows the results.
  • Example 5 Purification of DNaseI Using Basic Magnesium Carbonate To E. coli lysate prepared in the same manner as in Example 1, an enzyme, DNaseI (manufactured by Nippon Gene) was added to 0.05 mg/mL. After adding 15 w/v% or 5 w/v% basic magnesium carbonate and stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to recover the supernatant to obtain a treated liquid. The obtained treated solution and the solution before treatment were subjected to SDS-PAGE, and the band intensity around 31 kDa was read with an imaging system ("Chemidoc TM Touch" manufactured by Bio-Rad). DNase I recovery was calculated by dividing the band intensity before treatment from the band intensity after treatment. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 6 shows the results.
  • Example 6 Purification of Fab Using Basic Magnesium Carbonate Fab, which is a partial antibody, was expressed in Escherichia coli (“E. Coli W3110”), and an E. coli lysate was prepared in the same manner as in Example 1. 1 w/v% or 5 w/v% of basic magnesium carbonate was added to the obtained E. coli lysate, and after stirring for 1 hour, the supernatant was recovered by centrifuging at 15,000 rpm for 5 minutes to obtain a treated liquid. rice field. The obtained treated solution and the solution before treatment were subjected to Western blotting, and the band intensity was read with an imaging system ("Chemidoc TM Touch" manufactured by Bio-Rad). Fab recovery was calculated by dividing the band intensity before treatment from the band intensity after treatment. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 7 shows the results.
  • Example 7 Purification of scFV Using Basic Magnesium Carbonate After expressing the partial antibody scFV in E. coli (“E. coli W3110”), an E. coli lysate was prepared in the same manner as in Example 1. 2 w/v % of basic magnesium carbonate was added to the obtained E. coli lysate, and after stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to collect the supernatant to obtain a treated liquid. The obtained treated solution and the solution before treatment were subjected to Western blotting, and the band intensity was read with an imaging system ("Chemidoc TM Touch" manufactured by Bio-Rad). scFV recovery was calculated by dividing the band intensity before treatment from the band intensity after treatment. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 8 shows the results.
  • Example 8 Purification of exonuclease using basic magnesium carbonate After exonuclease, which is a nucleic acid enzyme, was expressed in E. coli ("E.Coli W3110"), an E. coli lysate was prepared in the same manner as in Example 1. bottom. 1 w/v % of basic magnesium carbonate was added to the resulting E. coli lysate, and after stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to collect the supernatant to obtain a treated liquid.
  • E.Coli W3110 E. Coli W3110
  • Example 9 Purification of bromelain using basic magnesium carbonate After dissolving 100 mg of the enzyme bromelain in 1 mL of 25 mM Tris-HCl buffer (pH 7.5), endotoxin standard (E. E. coli O113) was added and dissolved. 1 wt % or 5 wt % of basic magnesium carbonate was added to the obtained solution, and after stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to recover the supernatant to obtain a treated liquid. The bromelain recovery rate was obtained by dividing UV280 nm of the solution before treatment from the absorbance of UV280 nm of the treatment solution. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 10 shows the results.
  • Example 10 Purification of Pepsin Using Basic Magnesium Carbonate 100 mg of the enzyme pepsin was dissolved in 1 mL of 25 mM Tris-HCl buffer (pH 7.5) and diluted with an endotoxin standard (E. coli O1133) was added and dissolved. 1 wt % or 5 wt % of basic magnesium carbonate was added to the obtained solution, and after stirring for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes to recover the supernatant to obtain a treated liquid. The pepsin recovery rate was obtained by dividing the UV280nm of the solution before treatment from the UV280nm absorbance of the treatment solution. Also, the endotoxin concentration was calculated in the same manner as in Example 3. Table 11 shows the results.

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Abstract

Le but de la présente invention est de fournir un procédé qui permet une réduction efficace et facile de la quantité d'endotoxines dans un liquide. Un procédé de réduction de la quantité d'endotoxines dans un liquide selon la présente invention est caractérisé en ce qu'il comprend : une étape de mise en contact d'un liquide contenant au moins 1 EU/mL d'endotoxines avec du carbonate de magnésium basique de sorte qu'au moins une partie des endotoxines soit absorbée par le carbonate de magnésium basique ; et une étape de séparation ultérieure du carbonate de magnésium basique de la fraction liquide du liquide.
PCT/JP2022/031625 2021-08-27 2022-08-22 Procédé de réduction des endotoxines WO2023027044A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020045290A1 (fr) * 2018-08-31 2020-03-05 株式会社カネカ Procédé de purification d'un anticorps ou d'une molécule de type anticorps
WO2021172217A1 (fr) * 2020-02-28 2021-09-02 株式会社カネカ Procédé de réduction d'acide nucléique et filtre d'adsorption

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020045290A1 (fr) * 2018-08-31 2020-03-05 株式会社カネカ Procédé de purification d'un anticorps ou d'une molécule de type anticorps
WO2021172217A1 (fr) * 2020-02-28 2021-09-02 株式会社カネカ Procédé de réduction d'acide nucléique et filtre d'adsorption

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