Patent Grant
11969512
The present invention relates to a method for producing a protein composition and a protein composition.
Proteins for use in medical applications and biochemical applications need to be sterilized before use. Sterilization is achieved by ethylene oxide gas sterilization, filtration sterilization, or radiosterilization. Radiosterilization is advantageous in terms of the absence of toxic residues (ethylene oxide gas) after sterilization as well as cost and validation.
Radiosterilization of proteins unfortunately causes changes such as degradation and denaturation. Such changes in proteins are induced by reactions of active radicals (hydroxy radicals, oxygen radicals) generated by radiation with the proteins.
Conventionally, the following methods have been employed in order to suppress changes in proteins induced by active radicals: a method in which proteins are irradiated with radiation under cooling conditions; a method in which water is removed from a radiation irradiation target; and a method in which a radical scavenger against active radicals is added (Patent Literatures 1 and 2).
Yet, the method in which proteins are irradiated with radiation under cooling conditions is not very effective, and the production cost for cooling is high. In addition, when sterilizing a highly hydrophilic protein or a protein that undergoes denaturation by complete removal of water, it is difficult to remove water from a radiation irradiation target. Further, in the case where a radical scavenger is used to prevent changes in proteins induced by active radicals, the radical scavenger needs to be added in a large amount, which unfortunately impairs physiological and physicochemical functions of the proteins.
The present invention aims to provide a method for producing a protein composition, the method being capable of suppressing changes such as degradation and denaturation of a protein which occur when a highly hydrophilic protein or a protein that undergoes denaturation by complete removal of water is irradiated with radiation.
As a result of extensive studies, the present inventors arrived at the present invention. Specifically, the present invention is directed to a method for producing a protein composition containing a protein (A), a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A), the method including a sterilization step of radiosterilizing an unsterilized protein composition, wherein the unsterilized protein composition contains the protein (A), the radical scavenger (RS), and the hydrogen-bond-formable compound (HC), the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond, the unsterilized protein composition has a water content of 0 to 30% by weight based on the weight of the unsterilized protein composition, a weight ratio of the radical scavenger (RS) to the protein (A) in the unsterilized protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1, and a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the unsterilized protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.5.
The present invention also relates to a protein composition containing a protein (A), wherein the protein composition further contains a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A), the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond, a weight ratio of the radical scavenger (RS) to the protein (A) in the protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50, and the protein composition has been radiosterilized.
According to the production method of the present invention, the addition amount of the radical scavenger (RS) can be reduced by effective scavenging of radicals generated when a highly hydrophilic protein or a protein that undergoes denaturation by complete removal of water is irradiated with radiation. Thus, physiological and physicochemical functions of the protein before being irradiated with radiation are maintained in a protein composition produced by the production method of the present invention.
In the method for producing a protein composition of the present invention, an unsterilized protein composition to be sterilized contains a protein (A), a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A).
The term “unsterilized protein composition” as used in the method for producing a protein composition of the present invention refers to a protein composition containing the protein (A), the radical scavenger (RS), and the hydrogen-bond-formable compound (HC) before being subjected to a sterilization step of the method for producing a protein composition of the present invention.
Examples of the protein (A) include animal-derived proteins, plant-derived proteins, microorganism-derived proteins, and recombinant proteins.
Examples of animal-derived proteins include protein formulations, enzymes, antibodies, coagulation factors, and extracellular matrices.
Examples of plant-derived proteins include enzymes and extracellular matrices.
Examples of microorganism-derived proteins include enzymes and extracellular matrices.
Examples of recombinant proteins include protein formulations and vaccines.
Examples of protein formulations include interferon α, interferon 13, interleukin 1 to 12, growth hormone, erythropoietin, insulin, granulocyte-colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), natriuretic peptide, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, and calcitonin.
Examples of vaccines include hepatitis A vaccine, hepatitis B vaccine, and hepatitis C vaccine.
Examples of enzymes include hydrolases, isomerases, oxidoreductases, transferases, synthases, and lyases.
Examples of hydrolases include protease, serine protease, amylase, lipase, cellulase, and glucoamylase.
Examples of isomerases include glucose isomerase.
Examples of oxidoreductases include peroxidase.
Examples of transferases include acyltransferase and sulfotransferase.
Examples of synthases include fatty acid synthase, phosphate synthase, and citrate synthase.
Examples of lyases include pectin lyase.
Examples of antibodies include IgD, IgE, IgG, IgA, and IgM.
Examples of coagulation factors include fibrinogen, fibrin, prothrombin, thrombin, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XII, and factor XIII.
Examples of extracellular matrices include collagen, fibronectin, laminin, and elastin.
The protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups.
Preferred among these are hydroxyl, amide, amino, and carboxyl groups in terms of the hydrogen bond distance between the protein (A) and the hydrogen-bond-formable compound (HC).
The protein (A) containing at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups preferably contains an amino acid having the at least one functional group.
Examples of amino acids having a sulfide group include methionine and cysteine.
Examples of amide groups include an amide group formed by a peptide bond between amino acids contained in the protein (A).
Examples of amino acids having a hydroxyl group include serine, threonine, and tyrosine.
Examples of amino acids having an amino group include arginine, asparagine, glutamine, histidine, lysine, and tryptophan.
Examples of amino acids having a carboxyl group include aspartic acid and glutamic acid.
In terms of stability of the protein (A), the protein (A) is preferably one containing methionine, cysteine, serine, threonine, tyrosine, arginine, asparagine, glutamine, histidine, lysine, tryptophan, aspartic acid, and glutamic acid, more preferably one containing methionine, serine, threonine, tyrosine, arginine, asparagine, glutamine, histidine, lysine, tryptophan, aspartic acid, and glutamic acid.
In the method for producing a protein composition of the present invention, the protein (A) preferably contains a repetitive sequence (X) in terms of stability of the protein.
In the method for producing a protein composition of the present invention, the repetitive sequence (X) preferably includes any one amino acid sequence (a1) selected from the group consisting of GAGAGS (SEQ ID NO:1), RGD, YIGSR (SEQ ID NO:2), GVGVP (SEQ ID NO3), PGVGV (SEQ ID NO:4), VPGVG (SEQ ID NO:5), GVPGV (SEQ ID NO:6), VGVPG (SEQ ID NO:7), GPP, GAP, GAHGPAGPK (SEQ ID NO:8), GAA, VAAGY (SEQ ID NO:9), GAGAGAS (SEQ ID NO:10), LGPLGP (SEQ ID NO:11), GAHGPAGPK (SEQ ID NO:12), GAPGPAGPPGSRGDPGPP (SEQ ID NO:13), GAQGPAGPG (SEQ ID NO:14), GAPGAPGSQGAPGLQ (SEQ ID NO:15), GAPGTPGPQGLPGSP (SEQ ID NO:16), GAAVTGRGDSPASAAGY (SEQ ID NO:17), and GAAPGASIKVAVSAGPSAGY (SEQ ID NO:18). The repetitive sequence (X) may include one or two or more of these amino acid sequences (a1).
In terms of stability of the protein, preferred among these are GAGAGS (SEQ ID NO:1), GAA, VAAGY (SEQ ID NO:9), and GAGAGAS (SEQ ID NO:10).
In the method for producing a protein composition of the present invention, in terms of stability of the protein, the repetitive sequence (X) includes preferably 2 to 200, more preferably 15 to 150, particularly preferably 30 to 120 units of GAGAGS (SEQ ID NO:1).
In the method for producing a protein composition of the present invention, in terms of stability of the protein, the repetitive sequence (X) preferably includes a sequence (Y) consisting of 2 to 200 repeats of an amino acid sequence (a2) that is one of GVGVP (SEQ ID NO:3), PGVGV (SEQ ID NO:4), VPGVG (SEQ ID NO:5), GVPGV (SEQ ID NO:6), VGVPG (SEQ ID NO7), GPP, GAP, or GAHGPAGPK (SEQ ID NO:8) and/or a sequence (Y1) in which 1 to 100 amino acids in the sequence (Y) are replaced by lysine (K) or arginine (R).
In terms of stability of the protein, the sequence (Y) is particularly preferably a sequence consisting of 2 to 200 repeats of GVGVP (SEQ ID NO:3), PGVGV (SEQ ID NO:4), VPGVG (SEQ ID NO:5), GVPGV (SEQ ID NO:6), or VGVPG (SEQ ID NO:7).
In addition, the repetitive sequence (X) may include one or two or more of these sequences (Y) and (Y1).
In the method for producing a protein composition of the present invention, a ratio of a number of units of GAGAGS (SEQ ID NO:1) to a total number of the amino acid sequences (a2) and amino acid sequences (a2′) described below in one molecule of the protein (A) [(the number of units of GAGAGS (SEQ ID NO:1)):(the total number of the amino acid sequences (a2) and (a2′))] is preferably [1:2] to [1:20], more preferably [1:10] to [1:5], in terms of stability of the protein,
amino acid sequence (a2′): an amino acid sequence in which one to five amino acids in the amino acid sequence (a2) are replaced by lysine (K) or arginine (R).
In the method for producing a protein composition of the present invention, in terms of bioactivity of the protein, the repetitive sequence (X) preferably includes a sequence (Y2) consisting of 1 to 50 repeats of an amino acid sequence (a3) that is one of GAAVTGRGDSPASAAGY (SEQ ID NO:17) or GAAPGASIKVAVSAGPSAGY (SEQ ID NO18).
In addition, the repetitive sequence (X) may include one or two or more of these sequences (Y2).
In the method for producing a protein composition of the present invention, the protein (A) may contain amino acids immediately upstream and downstream of the repetitive sequence (X) and between the repetitive sequences (X). In terms of solubility (solubility particularly in water) of the protein (A) and gelation time, the number of amino acids immediately upstream and downstream of the repetitive sequence (X) and between the repetitive sequences (X) is preferably 1 to 100, more preferably 5 to 40, particularly preferably 10 to 35.
Examples of the amino acids immediately upstream and downstream of the repetitive sequence (X) and between the repetitive sequences (X) include β-galactosidase-derived sequences and purified tags (e.g., 6xHis tag, V5 tag, Xpress tag, AUl tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag 09™, CruzTag22™ CruzTag41™, Glu-Glu tag, Ha.11 tag, KT3 tag, maltose binding protein, HQ tag, Myc tag, HA tag, and FLAG tag).
Some of preferred examples of the protein (A) in the method for producing a protein composition of the present invention are listed below.
(1) Examples of proteins in which the repetitive sequence (X) consists of GAGAGS (SEQ ID NO:1) and the sequence (Y1) include the followings:
In terms of stability of the protein, the protein (A) is preferably SELP OK or SELP 8K, more preferably SELP 8K, among the above examples.
(2) Examples of proteins in which the repetitive sequence (X) consists of GAGAGS (SEQ ID NO:1) and the sequence (Y2) include the followings:
In terms of stability of the protein (A), the amino acid composition of the protein (A) is preferably such that the number of proline (P) residues accounts for 1 to 50%, the number of serine (S) residues accounts for 1 to 50%, and the number of valine (V) residues accounts for 1 to 50%, based on the total number of amino acids in the protein (A). More preferably, the number of proline (P) residues accounts for 1 to 20%, the number of serine (S) residues accounts for 1 to 20%, and the number of valine (V) residues accounts for 1 to 30%.
In terms of solubility of the protein (A), the protein (A) content of the protein composition is preferably 50% by weight or less, more preferably 10% by weight or less, based on the weight of the protein composition.
In the method for producing a protein composition of the present invention, the protein (A) preferably has a molecular mass of 15 to 200 kDa as determined by SDS-polyacrylamide gel electrophoresis.
The molecular mass of the protein (A) is determined by a method in which test samples are isolated by SDS-polyacrylamide gel electrophoresis and the migration distance of each test sample is compared with that of the standard substance.
In the method for producing a protein composition of the present invention, in terms of stability of the protein (A), the radical scavenger (RS) in the protein composition is preferably at least one selected from the group consisting of oxygen-containing conjugated structures and nitrogen-containing conjugated structures.
In the method for producing a protein composition of the present invention, the radical scavenger (RS) preferably has a radical scavenging ability against diphenylpicrylhydrazyl radicals (DPPH radicals) of 0.01 to 90 mg Trolox eq/mg.
The DPPH radical scavenging ability can be measured by the method described in “Shokuhin kinosei hyoka manyuarushu dainishu (the second collection of food functionality evaluation manuals)”, DPPH radical scavenging activity evaluation method, Tomoyuki Oki, (2008) pp. 71-78, and can be evaluated as trolox equivalent.
In addition, the radical scavenging ability against peroxy radicals, hydroxy radicals, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, oxygen radicals, and alkyl radicals can be measured by a method such as superoxide dismutase assay (SOD assay), ABTS radical scavenging ability measurement method, potential antioxidant (PAO) measurement method, or EPR spin trapping method to set the radical scavenging ability to 0.01 mg Trolox eq/mg or more.
Examples of the radical scavenger (RS) include organic acids (e.g., ascorbic acid, erythorbic acid, uric acid, gallic acid, glutathione, phenolic acid, ellagic acid, and chlorogenic acid), glutathione, edaravone, polyphenols (e.g., flavonoid, phenolic acid, ellagic acid, lignan, curcumin, and coumarin), and phenolic compounds (e.g., vanillin, pyrogallol, dibutylhydroxytoluene, and butylhydroxyanisole).
In particular, in terms of miscibility between the protein (A) and the radical scavenger (RS), radical scavenging ability, and safety, the radical scavenger (RS) is preferably ascorbic acid, edaravone, vanillin, gallic acid, glutathione, or chlorogenic acid, more preferably ascorbic acid or edaravone.
In terms of solubility, the radical scavenger (RS) content of the unsterilized protein composition is preferably 40% by weight or less, more preferably 30% by weight or less, based on the weight of the unsterilized protein composition.
In the method for producing a protein composition of the present invention, the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups.
In terms of the hydrogen bond distance between the protein (A) and the hydrogen-bond-formable compound (HC), the hydrogen-bond-formable compound (HC) preferably contains at least one functional group selected from the group consisting of carboxyl, hydroxyl, and amino groups, among the above examples.
Examples of the hydrogen-bond-formable compound (HC) include amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, and their derivatives) and peptides (e.g., aspartame, vasopressin, glucagon, and selectin).
In terms of the hydrogen bond distance and miscibility between the protein (A) and the hydrogen-bond-formable compound (HC), the hydrogen-bond-formable compound (HC) is preferably tryptophan, tyrosine, or histidine, more preferably tryptophan, among the above examples.
In terms of solubility, the hydrogen-bond-formable compound (HC) content of the unsterilized protein composition is preferably 25% by weight or less, more preferably 10% by weight or less, based on the weight of the unsterilized protein composition.
In the method for producing a protein composition of the present invention, the water content of the unsterilized protein composition is 0 to 30% by weight based on the weight of the unsterilized protein composition. In terms of stability of the protein, the water content is preferably 0.01 to 30% by weight, more preferably 0.01 to 15% by weight.
In the method for producing a protein composition of the present invention, the unsterilized protein composition may be dried before the sterilization step.
Examples of the method of drying the unsterilized protein composition include freeze-drying and heat-drying.
The water content of the unsterilized protein composition can be measured as described below.
<Method for Measuring the Water Content of the Unsterilized Protein Composition>
An amount of 50 to 100 mg of the unsterilized protein composition is weighed in a glass vial. The weighed amount (Ws0), and the weight of the glass vial (Wb0) are recorded. A dryer is set to 100° C., and the glass vial containing the unsterilized protein composition is placed in the dryer when the temperature reaches 100° C. (The glass vial is uncapped.) Two hours later, the glass vial containing the unsterilized protein composition is taken out from the dryer, and is left to cool to room temperature in a desiccator. After cooling, the glass vial is capped, and the weight (W) is measured. Then, the water content of the unsterilized protein composition is calculated from the following formula (1).
(Ws0+Wb0−W)/Ws0×100=Water content (% by weight) of the unsterilized protein composition) (1)
In the method for producing a protein composition of the present invention, a weight ratio of the radical scavenger (RS) to the protein (A) in the unsterilized protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0. In terms of maintenance of physiological and physicochemical functions of the protein (A), the weight ratio is preferably 0.01 to 0.1, more preferably 0.01 to 0.05.
In the method for producing a protein composition of the present invention, a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the unsterilized protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50. In terms of protection of protein properties, the molar ratio is preferably 0.01 to 0.50, more preferably 0.01 to 0.10, still more preferably 0.01 to 0.05.
If the molar ratio exceeds 1.0, disadvantages resulting from the radical scavenger (RS) (e.g., changes in physiological and physicochemical functions, changes in pH environment, increased costs, inflammation, and carcinogenesis) will increase.
If the molar ratio is less than 0.01, the protein (A) will be denatured to a greater degree.
The “functional groups in the protein (A)” used for the calculation of the molar ratio refers to the functional groups in the protein (A) which form hydrogen bonds with the functional groups in the hydrogen-bond-formable compound (HC). The “functional groups in the hydrogen-bond-formable compound (HC)” used for the calculation of the molar ratio refers to the functional groups in the hydrogen-bond-formable compound (HC) which forms hydrogen bonds with the functional groups in the protein (A).
In the method for producing a protein composition of the present invention, as described above, the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups.
In addition, in the method for producing a protein composition of the present invention, the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond.
It is particularly preferred that the functional groups in the hydrogen-bond-formable compound (HC) can form hydrogen bonds with the functional groups in the amino acids in the protein (A) that is easily denatured when irradiated with radiation. The presence of the hydrogen-bond-formable compound (HC) in the unsterilized protein composition facilitates prevention of denaturation of the protein (A) in the sterilization step.
Examples of the amino acids in the protein (A) that is easily denatured when irradiated with radiation include serine and aspartic acid.
The functional groups of the protein (A) may be on side chains of the amino acids in the protein (A).
In the case where these functional groups are on the side chains of the amino acids, the functional groups on the side chains of the amino acids in the protein (A) are capable of binding to the functional groups of the hydrogen-bond-formable compound (HC) via first hydrogen bonds. In terms of radical scavenging properties, each first hydrogen bond has a distance of preferably 1.3 to 1.9 A, more preferably 1.7 to 1.8 Å.
In addition, an amide group, which is one of the functional groups in the protein (A) and which is formed by a peptide bond, is also in the protein (A).
The amide group formed by a peptide bond in the protein (A) is capable of binding to the hydrogen-bond-formable compound (HC) via a second hydrogen bond. In terms of radical scavenging properties, the second hydrogen bond has a distance of preferably 1.3 to 1.9 Å, more preferably 1.7 to 1.8 Å.
The hydrogen bond distance is calculated using simulation software (Gaussian: Gaussian, Inc.).
When calculating the hydrogen bond distance by simulation software (Gaussian), an amino acid having a predetermined functional group is selected as a model compound for the compound having the predetermined functional group in the protein (A). Then, the hydrogen bond distance between the functional group in the amino acid and the functional group in the hydrogen-bond-formable compound (HC) can be determined by calculation.
The hydrogen bond distance between the functional group in the amino acid and the functional group in the hydrogen-bond-formable compound (HC) refers to the hydrogen bond distance that is optimized such that the energy between these functional groups is minimized.
In the method for producing a protein composition of the present invention, in the case where the first hydrogen bond has a distance of 1.3 to 1.9 Å and the second hydrogen bond has a distance of 1.3 to 1.9 Å, these distances are shorter than the typical hydrogen bond distance (2.4 to 3.3 Å).
Thus, the presence of the hydrogen-bond-formable compound (HC) in the unsterilized protein composition can suppress denaturation of the protein (A) by radiation in the sterilization step. This is presumably because the hydrogen-bond-formable compound (HC) fulfills the role of facilitating transfer of radicals from the protein (A) to the radical scavenger (RS) through hydrogen bonds. Due to this synergistic effect, it is possible to drastically reduce the addition amount of the radical scavenger (RS) (the amount is reduced to about one hundredth), compared to the case where an attempt is made to suppress denaturation only by the radical scavenger (RS). This reduces disadvantages (e.g., changes in physiological and physicochemical functions, changes in pH environment, increased costs, inflammation induced by a protein to be produced, and carcinogenic properties of a protein to be produced) resulting from the addition of a large amount of the radical scavenger (RS) to the unsterilized protein composition.
In addition, in the method for producing a protein composition of the present invention, the hydrogen-bond-formable compound (HC) preferably binds to the protein (A) only via hydrogen bonds.
In the case where a compound that forms a covalent bond with the protein (A) is used instead of the hydrogen-bond-formable compound (HC), such a compound can also exhibit an equivalent function (role of facilitating transfer radicals from the protein (A) to the radical scavenger (RS)) to that of the hydrogen-bond-formable compound (HC); however, formation of such a covalent bond unfortunately changes physical and physiological functions of the protein (A). In contrast, in the case where the hydrogen-bond-formable compound (HC) binds to the protein (A) only via hydrogen bonds without forming covalent bonds, the above described disadvantages (e.g., changes in physiological and physicochemical functions and changes in safety) are prevented.
As a method for adjusting the hydrogen bond distance between at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups in the protein (A) and the functional group in the hydrogen-bond-formable compound (HC), for example, various functional groups, proteins, and hydrogen-bond-formable compounds (HC) are selected such that the hydrogen bond distance between at least one functional group selected from the group consisting of hydroxyl, amino, and carboxyl groups in the protein (A) and the functional group in the hydrogen-bond-formable compound (HC) is within the numerical range of 1.3 to 1.9 Å.
In other words, in the method for producing a protein composition of the present invention, the hydrogen-bond-formable compounds (HC) are preferably selected according to the type and other properties of the protein (A).
The hydrogen-bond-formable compounds (HC) may be screened by a method such as screening (1) or screening (2) described below.
<Screening (1)>
First, the protein (A) is irradiated with radiation to sterilize the protein (A). Subsequently, an amino acid (a) modified in the protein (A) irradiated with radiation is identified. Subsequently, the hydrogen-bond-formable compounds (HC) may be screened with the above-described simulation software or the like, using the amino acid (a) before modification as a model compound for the compound having a functional group on a side chain of an amino acid in the protein (A).
The modified amino acid (α) can be identified by LC-MSMS analysis. The LC-MSMS analysis conditions are preferably the same as those for “LC-MSMS measurement” (described later) for the calculation of the modification rate of the protein (A) using LC-MSMS.
The compounds (HC) capable of forming the first hydrogen bond can be screened by the method described here.
<Screening (2)>
The hydrogen-bond-formable compounds (HC) may be screened with the above-described simulation software or the like, using alanylalanine as a model compound for the compound having an amide group formed by a peptide bond in the protein (A).
The compounds (HC) capable of forming the second hydrogen bond can be screened by the method described here.
In the method for producing a protein composition of the present invention, the unsterilized protein composition may contain optional additives, in addition to the protein (A), the radical scavenger (RS), and the hydrogen-bond-formable compound (HC).
Examples of optional additives include antioxidants, antiseptics, stabilizers, solubilizing agents, and buffer components.
The method for producing a protein composition of the present invention includes the sterilization step of radiosterilizing the unsterilized protein composition.
The method for producing a protein composition of the present invention may also include, before the sterilization step, an unsterilized protein composition preparation step of preparing an unsterilized protein composition, a freeze-drying step of freeze-drying the unsterilized protein composition, and a packing step of packing the unsterilized protein composition, as described below.
(Unsterilized protein composition preparation step) Examples of methods for preparing the unsterilized protein composition include a method in which the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and the protein (A) are dissolved in water at room temperature. Any sterile water may be used. Examples of methods for sterilizing water include a method for filtering water through a microfiltration membrane having a pore diameter of 0.2 μm or less, a method for filtering water through an ultrafiltration membrane, a method for filtering water through a reverse osmosis membrane, and a method for heat-sterilizing water in an autoclave at 121° C. for 20 minutes to obtain ion-exchanged water.
(Freeze-Drying Step)
Examples of methods for freeze-drying the unsterilized protein composition include a method in which the temperature is lowered to about −30° C. to −35° C. to freeze the unsterilized protein composition. Then, the pressure is reduced to a vacuum state and the temperature is raised to about −10° C. to −20° C. (a temperature at which the equilibrium water vapor pressure is equal to or higher than the degree of vacuum) to sublime water.
Facility conditions are listed below, as an example.
In the method for producing a protein composition of the present invention, before the sterilization step, the unsterilized protein composition may be packed to be shielded from the outside.
Examples of methods for packing include a vacuum packing method.
(Sterilization Step)
In the method for producing a protein composition of the present invention, examples of methods for radiosterilizing the unsterilized protein composition in the sterilization step include methods such as γ-ray sterilization and electron beam sterilization under the following conditions.
In addition, in the method for producing a protein composition of the present invention, the protein composition is preferably sterilized to SAL 10−6 in accordance with JIS T 0806-2:2010 or ISO 11137-2:2006 in the sterilization step.
In addition, in the method for producing a protein composition of the present invention, the sterilization step may be performed only once or several times.
According to the method for producing a protein composition of the present invention, it is possible to produce a protein composition having a low denaturation rate. In particular, “the denaturation rate calculated based on HPLC measurement” and “the denaturation rate of the produced protein composition calculated based on LC-MSMS measurement”, which are measured by the following methods, can be reduced.
As used herein, the term “denaturation rate of the produced protein composition calculated based on HPLC measurement” refers to a numerical value calculated from the following formula (2), wherein M is the peak height of the unsterilized protein composition and N is the peak height of the produced protein composition, as determined by HPLC measurement of the unsterilized protein composition and the produced protein composition under the conditions described below.
Denaturation rate (%)=[1−(N/M)]×100 (2)
(HPLC Measurement)
The unsterilized protein composition or the produced protein composition is dissolved in deionized water (1 mL) to obtain a solution containing 1 mg of the protein (A), and the solution is filtered through a 0.45-μm filter to obtain a test sample.
The test sample is measured by HPLC (Shimadzu Corporation) under the following conditions.
Generally, proteins in a protein composition become hydrophilic when the protein composition is radiosterilized. Thus, the peak in HPLC analysis of the radiosterilized protein composition is broad and low. Conversely, if the percentage of denatured proteins is low, the peak in HPLC analysis is sharp and is not likely to be low.
As used herein, the term “denaturation rate of the produced protein composition calculated based on LC-MSMS measurement” refers to a numerical value calculated from the following formula (3), wherein On is the concentration of each amino acid in the unsterilized protein composition, Pn is the concentration of each amino acid in the produced protein composition, and Q is the number of types of amino acids measured, as determined by LC-MSMS measurement of the unsterilized protein composition and the produced protein composition under the conditions described below.
Denaturation rate (%)=1/Q×Σ[1−(Pn/On)]×100 (3)
(LC-MSMS Measurement)
The unsterilized protein composition or the produced protein composition is added to 6N hydrochloric acid (200 μL) to obtain a solution containing 1 mg of the protein (A), followed by degassing. The solution is degassed until foam is no longer formed, and then the protein (A) in the solution is hydrolyzed under vacuum sealed conditions at 110° C. for 22 hours. After hydrolysis, the solution is diluted in deionized water to 800 μL. The diluted solution is filtered through a 0.45-μm filter to obtain a test sample.
The test sample is measured by LC-MSMS (Shimadzu Corporation) under the following conditions.
The amino acid composition is determined from the elution time and the molecular weight of the standard sample (standard amino acid mixture: type H).
The method for producing a protein composition of the present invention is a method for producing a protein composition for use in medical applications and biochemical applications. The method is used to prevent radiosterilization-induced changes in proteins such as degradation and denaturation.
A protein composition produced by the method for producing a protein composition of the present invention is also encompassed by the present invention.
Specifically, the present invention is directed to a protein composition containing a protein (A), wherein the protein composition further contains a radical scavenger (RS), and at least one hydrogen-bond-formable compound (HC) selected from the group consisting of amino acids, peptides, and proteins other than the protein (A), the protein (A) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the hydrogen-bond-formable compound (HC) contains at least one functional group selected from the group consisting of sulfide, amide, hydroxyl, amino, and carboxyl groups, the at least one functional group in the protein (A) is capable of binding to the at least one functional group in the hydrogen-bond-formable compound (HC) via a hydrogen bond, a weight ratio of the radical scavenger (RS) to the protein (A) in the protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, a molar ratio of a total molar number of functional groups in the hydrogen-bond-formable compound (HC) to a total molar number of functional groups in the protein (A) in the protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50, and the protein composition has been radiosterilized.
In the protein composition of the present invention, the weight ratio of the radical scavenger (RS) to the protein (A) in the protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, preferably 0.01 to 0.1, more preferably 0.01 to 0.05.
As described above, the production of the protein composition of the present invention involves radiosterilization. When the weight ratio of the radical scavenger (RS) to the protein (A) in the protein composition [radical scavenger (RS)/protein (A)] is 0.01 to 1.0, radiosterilization is less likely to produce a denatured form of the protein (A), which facilitates maintenance of physiological and physicochemical functions of the protein (A).
In the protein composition of the present invention, the molar ratio of the total molar number of functional groups in the hydrogen-bond-formable compound (HC) to the total molar number of functional groups in the protein (A) in the protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50, preferably 0.01 to 0.10, more preferably 0.01 to 0.05.
As described above, the production of the protein composition of the present invention involves radiosterilization.
When the molar ratio of the total molar number of functional groups in the hydrogen-bond-formable compound (HC) to the total molar number of functional groups in the protein (A) in the protein composition [the total molar number of functional groups in the hydrogen-bond-formable compound (HC)/the total molar number of functional groups in the protein (A)] is 0.01 to 0.50, disadvantages resulting from the radical scavenger (RS) (e.g., changes in physiological and physicochemical functions, changes in pH environment, increased costs, inflammation, and carcinogenesis) are reduced. In addition, when radiosterilization is performed, the protein (A) is less likely to be denatured. Therefore, physiological and physicochemical functions of the protein (A) can be maintained.
The protein composition of the present invention has been radiosterilized.
Thus, use of the protein composition of the present invention in medical applications and biochemical applications can suitably prevent contamination.
The protein composition of the present invention has been preferably radiosterilized at 25 to 27 kGy.
The protein composition of the present invention has been more preferably sterilized to SAL 10−6 in accordance with JIS T 0806-2:2010 or ISO 11137-2:2006. The protein composition of the present invention that has been sterilized as described above is also usable as a medicinal product.
In the protein composition of the present invention, the protein composition preferably has a water content of 0 to 30% by weight, more preferably 0.01 to 30% by weight, still more preferably 0.01 to 15% by weight, based on the weight of the protein composition.
When the water content of the protein composition is 0 to 30% by weight based on the weight of the protein composition, the protein (A) in the protein composition has higher stability.
The protein composition of the present invention is preferably packed, more preferably vacuum-packed.
The protein composition when packed is shielded from the outside, and is thus less susceptible to contamination.
The present invention is described in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto.
(Provision of Radical Scavenger (RS))
Various radical scavengers (RS) shown in Table 1 were provided. Table 1 shows the radical scavenging ability and the structure of each radical scavenger.
(Screening of Hydrogen-Bond-Formable Compounds (HC))
First, as the protein (A), each of SELP 8K (sequence (22)), ProNectin F (sequence (30)), ProNectin L (sequence (32)), HRP-conjugated rabbit antibody (sequence (33)), glucose oxidase (sequence (34)), and bovine serum albumin (sequence (35)) was dissolved in water and, and these aqueous solutions were each freeze-dried, followed by vacuum-packing in a nitrogen atmosphere. Each vacuum-packed protein (A) was irradiated with electron beam at 25 kGy at −20° C.
Subsequently, the amino acid composition of the protein (A) before electron beam irradiation and the amino acid composition of the protein (A) after electron beam irradiation were measured by the following method to identify an amino acid modified by the electron beam irradiation.
<Evaluation: Amino Acid Analysis>
Each protein (1 mg) was individually added to 6N hydrochloric acid (200 μL), followed by degassing. The solution was degassed until foam was no longer formed, and then the protein (A) in the solution was hydrolyzed under vacuum sealed conditions at 110° C. for 22 hours. After hydrolysis, the solution was diluted in deionized water to 800 μL. The diluted solution was filtered through a 0.45-μm filter to obtain a test sample. Table 3 shows the results.
Analysis was performed by LC-MSMS (Shimadzu Corporation) under the following conditions.
The amino acid composition was determined from the elution time and the molecular weight of the standard sample (standard amino acid mixture: type H).
The amino acid in the protein (A) modified by the electron beam irradiation was serine.
Next, the compounds (HC) capable of forming a hydrogen bond with a hydroxyl group on the side chain of serine were screened with simulation software (Gaussian).
In addition, the compounds (HC) capable of forming a hydrogen bond with an amide group in the peptide bond of alanylalanine were screened.
As a result of screening, tryptophan, tyrosine, and histidine were identified as the hydrogen-bond-formable compounds (HC).
A hydroxyl group on the side chain of serine is considered to form a hydrogen bond (first hydrogen bond) with a carboxyl group on the side chain of tryptophan, a hydroxyl group on the side chain of tyrosine, or a carboxyl group on the side chain of histidine. Table 2 shows the distance (A) of each first hydrogen bond.
An amide group in the peptide bond of alanylalanine is considered to form a hydrogen bond (second hydrogen bond) with a carboxyl group on the side chain of tryptophan, a hydroxyl group on the side chain of tyrosine, or a carboxyl group on the side chain of histidine. Table 2 shows the distance (A) of each second hydrogen bond.
Radiosterilization of Protein Compositions Each Containing SELP 8K
SELP 8K as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 3 to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of SELP 8K for each example.
Subsequently, these unsterilized protein composition were freeze-dried and vacuum-packed in a nitrogen atmosphere. Each unsterilized protein had a water content of 8% by weight. The vacuum-packed unsterilized protein compositions were irradiated with electron beam at 25 kGy at −20° C. to produce protein compositions according to Examples 1 to 32.
Radiosterilization of Protein Compositions Each Containing ProNectin F
ProNectin F as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 4 to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of ProNectin F for each example.
Subsequently, these unsterilized protein composition were freeze-dried and vacuum-packed in a nitrogen atmosphere. Each unsterilized protein had a water content of 5% by weight. The vacuum-packed unsterilized protein compositions were irradiated with electron beam at 25 kGy at −20° C. to produce protein compositions according to Examples 33 to 65.
Radiosterilization of Protein Compositions Each Containing ProNectin L
ProNectin L as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 5 to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of ProNectin L for each example.
Subsequently, these unsterilized protein composition were freeze-dried and vacuum-packed in a nitrogen atmosphere. Each unsterilized protein had a water content of 4% by weight. The vacuum-packed unsterilized protein compositions were irradiated with electron beam at 25 kGy at −20° C. to produce protein compositions according to Examples 66 to 97.
Radiosterilization of a Protein Composition Containing HRP-Conjugated Rabbit Antibody
HRP-conjugated rabbit antibody as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 6 to obtain an aqueous solution of an unsterilized protein composition containing 0.1% by weight of HRP-conjugated rabbit antibody.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 7% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Example 98.
Radiosterilization of a Protein Composition Containing Glucose Oxidase
Glucose oxidase as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 6 to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of glucose oxidase.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 6% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Example 99.
Radiosterilization of a Protein Composition Containing Bovine Serum Albumin
Bovine serum albumin as the protein (A), the hydrogen-bond-formable compound (HC), the radical scavenger (RS), and water were mixed according to the weight ratio and the molar ratio described in Table 6 to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of bovine serum albumin.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 5% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Example 100.
Radiosterilization of a Protein Composition Containing SELP 8K
SELP 8K as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of SELP 8K.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 8% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 1.
Radiosterilization of a Protein Composition Containing ProNectin F
ProNectin F as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of ProNectin F.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 5% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 2.
Radiosterilization of a Protein Composition Containing ProNectin L
ProNectin L as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of ProNectin L.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 4% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 3.
Radiosterilization of a Protein Composition Containing HRP-Conjugated Rabbit Antibody
HRP-conjugated rabbit antibody as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 0.1% by weight of HRP-conjugated rabbit antibody.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 7% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 4.
Radiosterilization of a Protein Composition Containing Glucose Oxidase
Glucose oxidase as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of glucose oxidase.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 6% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 5.
Radiosterilization of a Protein Composition Containing Bovine Serum Albumin
Bovine serum albumin as the protein (A) and water were mixed to obtain an aqueous solution of an unsterilized protein composition containing 2.4% by weight of bovine serum albumin.
Subsequently, the unsterilized protein composition was freeze-dried and vacuum-packed in a nitrogen atmosphere. The unsterilized protein had a water content of 5% by weight. The vacuum-packed unsterilized protein composition was irradiated with electron beam at 25 kGy at −20° C. to produce a protein composition according to Comparative Example 6.
Radiosterilization of a Protein Composition Containing SELP 8K
A protein composition according to Comparative Example 7 was produced as in Comparative Example 1, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin F
A protein composition according to Comparative Example 8 was produced as in Comparative Example 2, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin L
A protein composition according to Comparative Example 9 was produced as in Comparative Example 3, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing HRP-Conjugated Rabbit Antibody
A protein composition according to Comparative Example 10 was produced as in Comparative Example 4, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing Glucose Oxidase
A protein composition according to Comparative Example 11 was produced as in Comparative Example 5, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing Bovine Serum Albumin
A protein composition according to Comparative Example 12 was produced as in Comparative Example 6, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing SELP 8K
A protein composition according to Comparative Example 13 was produced as in Comparative Example 1, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin F
A protein composition according to Comparative Example 14 was produced as in Comparative Example 2, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin L
A protein composition according to Comparative Example 15 was produced as in Comparative Example 3, except that ascorbic acid as the radical scavenger was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing SELP 8K
A protein composition according to Comparative Example 16 was produced as in Comparative Example 1, except that tryptophan as the hydrogen-bond-formable compound (HC) was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin F
A protein composition according to Comparative Example 17 was produced as in Comparative Example 2, except that tryptophan as the hydrogen-bond-formable compound (HC) was added in the proportion shown in Table 7.
Radiosterilization of a Protein Composition Containing ProNectin L
A protein composition according to Comparative Example 18 was produced as in Comparative Example 3, except that tryptophan as the hydrogen-bond-formable compound (HC) was added in the proportion shown in Table 7.
<Evaluation of the Denaturation Rate Calculated Based on HPLC Measurement>
(HPLC Measurement)
The unsterilized protein composition prepared for the production of the protein composition in each example and the protein composition according to each example were separately dissolved in deionized water (1 mL) to obtain solutions each containing 1 mg of the protein (A), and these solutions were filtered through a 0.45-μm filter to obtain test samples. Likewise, test samples of the unsterilized protein compositions prepared for the production of the protein compositions in the comparative examples and the protein compositions according to the comparative examples were prepared.
The test samples were measured by HPLC (Shimadzu Corporation) under the following conditions.
The denaturation rate calculated based on HPLC measurement was determined from the following formula (1), wherein M is the peak height of the unsterilized protein composition and N is the peak height of the produced protein composition as measured under the above conditions. Tables 3 to 7 show the results.
Denaturation rate (%)={1−(N/M)}×100 (1)
<Evaluation of the Denaturation Rate Calculated Based on LC-MSMS Measurement>
(Lc-Msms Measurement)
The unsterilized protein composition prepared for the production of the protein composition in each example and the protein composition according to each example were separately added to 6N hydrochloric acid (200 μL) to obtain solutions each containing 1 mg of the protein (A), followed by degassing. Each of these solutions was degassed until foam was no longer formed, and then the protein (A) in each solution was hydrolyzed under vacuum sealed conditions at 110° C. for 22 hours. After hydrolysis, each solution was diluted in deionized water to 800 μL. Each diluted solution was filtered through a 0.45-μm filter to obtain a test sample. Likewise, test samples of the unsterilized protein compositions prepared for the production of the protein compositions in the comparative examples and the protein compositions according to the comparative examples were prepared.
The test samples were analyzed by LC-MSMS (Shimadzu Corporation) under the following conditions.
The amino acid composition was determined from the elution time and the molecular weight of the standard sample (standard amino acid mixture: type H).
(Calculation of Denaturation Rate Calculated Based on LC-MSMS Measurement)
The denaturation rate calculated based on LC-MSMS measurement was determined from the following formula (2), wherein On is the concentration of each amino acid in the unsterilized protein composition, Pn is the concentration of each amino acid in the produced protein composition, and Q is the number of types of amino acids measured, which were determined under the above conditions. Tables 3 to 7 show the results.
Denaturation rate (%)=1/Q×Σ[1−(Pn/On)]×100 (2)
The results in Tables 3 to 7 show that the denaturation rate of the protein (A) is lower in each of the protein compositions obtained by the production method of the present invention than that in the protein compositions according to Comparative Examples 1 to 18.
The method for producing a protein composition of the present invention is excellent in allowing a protein composition to maintain physiological and physicochemical functions of a protein when the protein composition is subjected to radiosterilization. Thus, the present invention is effective as a method for producing a protein composition.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015-049302 | Mar 2015 | JP | national |
| Number | Name | Date | Kind |
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| 20020045567 | Cappello | Apr 2002 | A1 |
| 20030059338 | Mann et al. | Mar 2003 | A1 |
| 20100029542 | Jezek | Feb 2010 | A1 |
| 20140194370 | Cappello | Jul 2014 | A1 |
| Number | Date | Country |
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| 2000-514778 | Nov 2000 | JP |
| 2003-527210 | Sep 2003 | JP |
| 2010-514747 | May 2010 | JP |
| 2012-521776 | Sep 2012 | JP |
| 9717435 | May 1997 | WO |
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| 02103029 | Dec 2002 | WO |
| 03026704 | Apr 2003 | WO |
| 2008081166 | Jul 2008 | WO |
| 2010112576 | Oct 2010 | WO |
| 2016196662 | Dec 2016 | WO |
| WO-2016196662 | Dec 2016 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20210290790 A1 | Sep 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15556765 | US | |
| Child | 17336672 | US |
