JP2018072339A - Protein quantification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of reliably and accurately quantifying protein in a specimen containing protein and a viscosity agent.SOLUTION: A protein quantification method defined in the compendial method is modified to replace a step of adding a 10 w/v% trichloroacetic acid solution to a sample prepared from a specimen in a same amount with a step of adding a trichloroacetic acid solution to the sample prepared from the specimen such that trichloroacetic acid concentration becomes 20.0-31.0 w/v%. Such a modification enables accurate and reliable protein quantification even when the specimen contains a viscosity agent.SELECTED DRAWING: None

Description

  The present invention relates to a method for quantifying protein. More specifically, the present invention relates to a method for quantifying a protein in a specimen containing a protein and a viscous agent with high accuracy and high accuracy.

  In recent years, biological preparations using vaccines, antibodies and the like have been used for the purpose of preventing and treating various diseases. In such a biopharmaceutical manufacturing process, various tests and tests are performed in accordance with the “Biological Formulation Standard” (Non-Patent Document 1) established by the Ministry of Health, Labor and Welfare, and strict quality control is performed. . On the other hand, from the viewpoints of efficacy, safety, and usability, the use of a wide variety of new additives and changes in dosage forms are progressing. The number of cases in which quality control is difficult by applying the existing method indicated as “general test method” is increasing.

  An important test item in the manufacturing process of a biologic is protein quantification. Conventionally, protein quantification has been performed in accordance with the “general test method protein quantification method” (hereinafter, official document method) disclosed in Non-Patent Document 1. The official method adopts a method for measuring protein in a specimen precipitated by heated trichloroacetic acid by the Raleigh method as a method for quantifying protein. Specifically, the following procedure is established: (1 ) Standard albumin for protein quantification is dissolved in water to make a standard diluted solution of 1 mg / mL, and this solution is used to make three or more different standard diluted solutions. (3) Take exactly 1 mL each of the sample and the standard diluent, add the same amount of 10 w / v% trichloroacetic acid solution, and then in a water bath for 15 minutes. Cool after heating. (4) Collect the precipitate by centrifugation, add 2 mL of 5 w / v% trichloroacetic acid solution to the precipitate, shake and centrifuge again. (5) Add 2.5 mL of alkaline copper reagent to the precipitate. Add and shake for 10 minutes (6) Add 2.5 mL of water and 0.5 mL of dilute forin reagent, and leave at 37 ° C. for 30 minutes, and then measure the absorbance of this solution at a wavelength of 750 nm using a spectrophotometer. (7) Obtain the protein mass in the sample from the calibration curve obtained from the measurement results of the standard dilution, and calculate the content in the sample. Separately, as a control, water is measured in the same manner, and the absorbance is measured and used for correction.

  The official method is considered to be capable of quantifying proteins in specimens with high accuracy and high accuracy, but the effects of various additives in biological products on the quantitative properties of proteins are not sufficient. It cannot be said that it has been studied.

Biologics Standards, Amended March 28, 2016 Ministry of Health, Labor and Welfare Notification No. 106, Internet <http://www.nih.go.jp/niid/images/qa/seibutuki/seibutsuki_japanese/20160328.pdf>

  The present inventor examined the quantitative property of the protein by the official method using a biological preparation containing a viscous agent having a known protein content. A gap was confirmed. That is, the present inventor has found a drawback of the official document method that the quantitative accuracy of the protein in the sample containing the protein and the viscous agent is low.

  An object of the present invention is to provide a method for quantifying a protein in a sample containing a protein and a viscous agent with high accuracy and high accuracy.

  The present inventor has intensively studied to solve the above-mentioned problems. As a result, in the official document method, the sample prepared from the specimen is subjected to the operation of adding the same amount of 10 w / v% trichloroacetic acid solution to the specimen prepared from the specimen. By changing the trichloroacetic acid solution so that the concentration of trichloroacetic acid is 20.0 to 31.0 w / v%, the protein can be obtained with high accuracy even if the specimen contains a viscous agent. It was found that it can be quantified with high accuracy. The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A method for quantifying protein in a specimen containing a protein and a viscous agent, comprising preparing a sample using the specimen,
Step 2 of adding and heating the trichloroacetic acid solution to the sample obtained in Step 1 so that the concentration of trichloroacetic acid is 20.0 to 31.0 w / v%
Step 3 for recovering the precipitate from the solution obtained in Step 2;
Adding a trichloroacetic acid solution and / or water to the precipitate obtained in step 3 and mixing the precipitate, and then measuring the protein mass in the precipitate obtained in step 4 by the Raleigh method; Step 5 for determining the protein mass in the specimen
A quantitative method comprising:
Item 2. Item 2. The quantification method according to Item 1, wherein the viscosity agent is a polyacrylic acid-based viscosity agent.
Item 3. Item 3. The method according to Item 1 or 2, wherein the viscosity agent is a carboxyvinyl polymer.
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the specimen is a biological preparation.
Item 5. Item 5. The quantification method according to any one of Items 1 to 4, wherein the protein is an antigenic protein of an influenza vaccine.
Item 6. Item 6. The quantification method according to any one of Items 1 to 5, wherein the trichloroacetic acid solution used in Step 4 is a solution containing 15 w / v% or less of trichloroacetic acid.
Item 7. Item 7. The quantification method according to any one of Items 1 to 6, wherein the step 5 includes the following steps 5-1 to 5-3:
Step 5-1, in which an alkaline copper test solution is added to the precipitate obtained in Step 4 to dissolve the precipitate,
After adding a diluted forin test solution to the solution obtained in Step 5-1, the absorbance was measured in Step 5-2, and the calibration curve prepared with the standard diluent was used to measure the solution. Step 5-3 for obtaining the protein mass in the specimen from the absorbance.

  The official method has a drawback that the quantitative accuracy of the protein in the sample containing the protein and the viscous agent is low, but according to the present invention, the disadvantage is overcome and the protein in the sample containing the protein and the viscous agent is removed. It becomes possible to quantify the protein with high accuracy and high accuracy. Therefore, according to the present invention, it becomes possible to perform strict quality control of biological preparations and the like containing proteins and viscous agents with high accuracy and high accuracy.

  In this specification, “accuracy” in the measurement of protein represents the degree of deviation between the quantitative value and true value (actual protein mass) of the protein in the sample, and “high accuracy” It means that the quantitative value of protein is close to the true value. In this specification, “accuracy” in protein measurement refers to the degree of variation in the quantitative value of protein in the sample, and “high accuracy” refers to the quantitative value of the protein in the sample. It means that there is little variation every time.

The quantification method of the present invention is a method for quantifying a protein in a sample containing a protein and a viscous agent, and includes the following steps 1 to 5:
Step 1 of preparing a sample using the specimen,
Step 2, in which a trichloroacetic acid solution is added and heated so that the concentration of trichloroacetic acid is 20.0 to 31.0 w / v% with respect to the sample obtained in Step 1;
Step 3 for recovering the precipitate from the solution obtained in Step 2;
Adding a trichloroacetic acid solution and / or water to the precipitate obtained in step 3 and mixing the precipitate, and then measuring the protein mass in the precipitate obtained in step 4 by the Raleigh method; Step 5 for determining the protein mass in the specimen.
Hereinafter, the quantitative method of the present invention will be described in detail.

[Sample]
In the quantification method of the present invention, a protein in a specimen containing a protein and a viscous agent is to be quantified. The official method has the disadvantage that the quantitative accuracy of the protein in the specimen containing the viscous agent is lowered, but the quantitative method of the present invention eliminates this disadvantage and increases the protein in the specimen containing the viscous agent. It is possible to quantify with high accuracy and accuracy.

  The specimen may contain any protein and a viscous agent, and any protein that requires quantitative determination of the protein may be used. Examples thereof include pharmaceuticals, preferably biological preparations. Further, the dosage form of the specimen is not particularly limited, and may be any of liquid, solid, semi-solid, and the like.

  The type of protein contained in the specimen (that is, the protein to be quantified) is not particularly limited, and examples thereof include proteins used for biological preparations and other pharmaceuticals. Specifically, proteins used for biological preparations include influenza vaccine, influenza HA vaccine, cell culture influenza vaccine (H5N1 strain), sedimentation influenza vaccine (H5N1 strain), sedimentation cell culture influenza vaccine (H5N1 strain). ), Emulsion cell culture influenza HA vaccine (H5N1 strain), dry tissue culture inactivated hepatitis A vaccine, dry attenuated live mumps vaccine, gas foal antitoxin (gas pelvic antitoxin), dry gas pelvic antitoxin (dried) Anti-toxin gas), inactivated rabies vaccine, dried tissue culture inactivated rabies vaccine, cholera vaccine, dried diphtheria equine antitoxin (dried diphtheria antitoxin), diphtheria toxoid, precipitated diphtheria toxoid, adult precipitated diphtheria toxoid diphtheria tetanus Combined toxoid, precipitated diphtheria tetanus mixed toxoid, varicella antigen, dry attenuated live varicella vaccine, tetravalent meningococcal vaccine (diphtheria toxoid conjugate), typhoid paratyphoid mixed vaccine, purified tuberculin, pruritus vaccine (rice seedling), dried moth Vaccine (Dried Koji Seedling), Cell Culture Koji Vaccine, Dried Cell Culture Koji Vaccine, Japanese Encephalitis Vaccine, Dried Japanese Encephalitis Vaccine, Dried Cell Culture Japanese Encephalitis Vaccine, Pneumococcal Vaccine, Precipitated 7-valent Streptococcus pneumoniae-binding vaccine Mutant diphtheria toxin conjugate), precipitated 10-valent pneumococcal conjugate vaccine (non-capsular Haemophilus influenzae protein D, tetanus toxoid, diphtheria toxoid conjugate), precipitated 13-valent pneumococcal conjugate vaccine (non-toxic mutant diphtheria toxin conjugate) ), Tetanus toxoid, sedimentary tetanus toki Soid, dried horseshoe antitoxin (dried horseskin antitoxin), precipitated hepatitis B vaccine, precipitated hepatitis B vaccine (derived from huGK-14 cells), recombinant precipitated hepatitis B vaccine (derived from yeast), recombinant precipitated type B Hepatitis vaccine (derived from Chinese hamster ovary cells), recombinant precipitated pre-S2 antigen / HBs antigen-containing hepatitis B vaccine (derived from yeast), dry BCG intravesical (Connaught strain), dry BCG intravesical (Japan strain) , Dry BCG vaccine, recombinant precipitated bivalent human papillomavirus-like particle vaccine (derived from nettle guava cells), recombinant precipitated tetravalent human papillomavirus-like particle vaccine (derived from yeast), oral attenuated live human rotavirus vaccine, pertussis vaccine , Sedimentation purified pertussis vaccine, pertussis diphtheria mixed vaccine, pertussis diphtheria tetanus mixed vaccine Precipitated purified pertussis diphtheria tetanus mixed vaccine, Precipitated purified pertussis diphtheria tetanus inactivated polio (Sabin strain) mixed vaccine, Precipitated purified pertussis diphtheria tetanus inactivated polio (soak vaccine) mixed vaccine, dry attenuated live shin Vaccine, Dry Hemophilus b type vaccine (tetanus toxoid conjugate), Precipitated Hemophilus b type vaccine (Non-toxic mutant diphtheria toxin conjugate), Typhoid vaccine, Dry botulinum antitoxin (Dry botulinum antitoxin), Oral live polio vaccine, Inactivated polio vaccine (soak vaccine), dry attenuated live hemp shin vaccine, dry attenuated live hemp shinpo mushroom-like mixed vaccine, dry attenuated raw hemp shin-style mixed vaccine, dry maize horse antitoxin (dry maize antitoxin), pentavalent oral attenuated live mouth virus vaccine Proteins used in vaccines such as Weil's disease autumn mixed vaccine; heated human plasma protein, human serum albumin, dried human fibrinogen, human blood coagulation factor VIII, human blood coagulation factor IX complex, human blood coagulation factor X Factor activation factor VII, human immunoglobulin, alkylated human immunoglobulin, sulfonated human immunoglobulin, pH4-treated acidic human immunoglobulin, pH4-treated acidic human immunoglobulin, plasmin-treated human immunoglobulin dry pepsin-treated human immunoglobulin, Examples include polyethylene glycol-treated human immunoglobulin, human antithrombin III, human activated protein C, and human haptoglobin. These proteins may be included alone in the specimen, or may be included in combination of two or more.

  Among these proteins, the subject of quantification of the present invention is preferably a protein derived from a microorganism, more preferably a protein derived from a virus, and particularly preferably a protein derived from an influenza virus. The microorganism may be present alive in the specimen, may be present in an attenuated state, and has been inactivated by an appropriately selected technique such as addition of formalin or ultraviolet irradiation. It may exist in a state. In addition, the microorganism may have a perfect shape in the specimen, or may have an incomplete shape, such as a structure modified or divided by an appropriately selected physical or chemical technique.

  Moreover, influenza vaccine is mentioned as a suitable example of the said sample. Among influenza vaccines, an influenza vaccine that preferably induces mucosal IgA, and more preferably an influenza vaccine that induces nasal mucosal IgA. In addition, influenza vaccines can be classified into three types according to their forms: live attenuated vaccines, inactivated whole particle vaccines, and inactivated split vaccines.

  The type of the viscosity agent contained in the specimen is not particularly limited. For example, as the viscosity agent used in the biological preparation, specifically, a polyacrylic acid viscosity agent such as carboxyvinyl polymer; methylcellulose Cellulose-based viscosity agents such as hydroxyethyl cellulose, hydroxypropylmethyl cellulose, and carboxymethyl cellulose; and vinyl-based viscosity agents such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl methyl ether. These viscous agents may be contained alone in the sample, or may be contained in combination of two or more.

  Among these viscous agents, a specimen containing a polyacrylic acid-based viscous agent, particularly a carboxyvinyl polymer, is suitable as an application target of the quantification method of the present invention.

  The ratio of the protein and the viscous agent contained in the specimen is not particularly limited. For example, the viscosity agent is 50 to 6000 parts by mass, preferably 400 to 2000 parts by mass, and more preferably 600 to 100 parts by mass per 100 parts by mass of the protein. 1000 parts by mass can be mentioned. For a sample containing protein and a viscous agent at such a ratio, a decrease in the quantitative accuracy is noticeable in the conventional official document method, but according to the quantitative method of the present invention, it is highly accurate and accurate. It is possible to quantify white matter.

  In addition to proteins and viscous agents, specimens support additives necessary for formulation (buffering agents, pH adjusters, tonicity agents, polyhydric alcohols, etc.), and immunity other than proteins Components (such as an adjuvant) may be included.

[Step 1]
In step 1, a sample is prepared using the specimen.

  If the sample is liquid or pasty and the expected range of protein mass contained in the sample is 1 to 500 μg / mL, preferably 10 to 300 μg / mL, more preferably 20 to 150 μg / mL, the sample Can be used as a sample. If the sample is solid, or the sample is liquid or pasty and the predicted protein mass contained in the sample exceeds the above-mentioned range, it can be predicted using a dilution solvent. What is necessary is just to dilute so that protein mass may become the range mentioned above. Further, when the sample is in a liquid or paste form and the predicted value of the protein mass contained in the sample is lower than the above-described range, it may be concentrated so that the predicted protein mass is in the above-mentioned range.

  In Step 1, when a sample is diluted to prepare a sample, it is desirable to use water as a solvent for dilution. In addition, since the dilution factor employed in step 1 is used when calculating the protein content, it is important to dilute the specimen accurately in step 1.

[Step 2]
In step 2, the trichloroacetic acid solution is added to the sample obtained in step 1 so that the concentration of trichloroacetic acid is 20.0 to 31.0 w / v% and heated. In the conventional official document method, the same amount of 10 w / v% trichloroacetic acid solution is added to the sample (that is, the trichloroacetic acid solution is added so that the concentration of trichloroacetic acid is 5.0 w / v%). However, in the quantification method of the present invention, it is possible to quantify the protein with high accuracy and high accuracy even when a viscous agent coexists by adopting the operating conditions specified in the step 2. become.

  In step 2, the trichloroacetic acid solution may be added to the sample so that the final concentration of trichloroacetic acid is 20.0 to 31.0 w / v%, but the protein mass quantitative accuracy is further improved. From the viewpoint of improving, the concentration of trichloroacetic acid after addition to the sample is preferably 23.1 to 31.0 w / v%, more preferably 23.1 w / v%.

  In step 2, the concentration of trichloroacetic acid in the trichloroacetic acid solution added to the sample is not particularly limited as long as the concentration after addition to the sample can satisfy the above-mentioned range. For example, 50 w / v% or more Is mentioned. From the viewpoint of further improving the quantitative accuracy of protein mass, the concentration of trichloroacetic acid in the trichloroacetic acid solution added to the sample is preferably 50 to 100 w / v%, more preferably 75 to 100 w / v%, particularly Preferably, 100 w / v% is mentioned.

  In the present invention, the “trichloroacetic acid solution” refers to a solution containing trichloroacetic acid, and the trichloroacetic acid solution having a concentration of trichloroacetic acid of 100 w / v% is a solute except for unavoidably mixed impurities. Refers to a solution consisting essentially of trichloroacetic acid. In the trichloroacetic acid solution, the solvent is usually water.

  Moreover, what is necessary is just to set suitably about the quantity of the said sample with which it uses for the process 2 according to the protein mass contained in the said sample, and it should just set to 0.1-1 mL normally. In addition, when the protein mass contained in the sample is small, the sample to be subjected to step 2 may be set to an appropriate amount of 1 mL or more.

  In step 2, a trichloroacetic acid solution is added to the sample obtained in step 1 so that trichloroacetic acid has a predetermined concentration, and the mixture is stirred as necessary, followed by heating. Specifically, the heating conditions are 15 ° C. or higher, preferably 60 to 100 ° C., more preferably 100 ° C., 5 to 60 minutes, preferably 10 to 30 minutes, and more preferably 15 minutes. It is done.

  The heating method in step 2 is not particularly limited, but it is desirable to heat in a water bath.

  Precipitation (aggregation) occurs due to heating in step 2.

[Step 3]
In step 3, the precipitate is recovered from the solution obtained in step 2. Prior to precipitation collection in Step 3, the solution obtained in Step 2 may be cooled to about room temperature (1 to 30 ° C.) as necessary.

  The method for recovering the precipitate in step 3 is not particularly limited and may be any solid-liquid separation method such as centrifugation or filtration, but preferably includes centrifugation.

  In Step 3, when the precipitate is collected by centrifugation, the specific conditions are not particularly limited, but for example, 1400 g or more is 10 minutes or more, more preferably 1400 g or more is 20 minutes or more, and further preferably 1500 to Examples include 20 to 60 minutes at 20000 g, and most preferably 20 minutes at 1800 to 12000 g. In addition, the temperature condition at the time of centrifugation is not particularly limited, and examples thereof include 5 to 35 ° C, preferably 15 to 25 ° C, and more preferably 25 ° C.

[Step 4]
In step 4, the trichloroacetic acid solution and / or water is added to and mixed with the precipitate obtained in step 3, and then the precipitate is recovered.

  In Step 4, the solution added to the precipitate obtained in Step 3 may be at least one of a trichloroacetic acid solution and water, and preferably a trichloroacetic acid solution.

  The concentration of trichloroacetic acid in the trichloroacetic acid solution used in step 4 is not particularly limited, but is usually 15 w / v% or less, preferably 2 to 10 w / w from the viewpoint of further improving the quantitative accuracy of protein mass. v%, more preferably 5 w / v%. In the trichloroacetic acid solution used in step 4, the balance other than trichloroacetic acid is water.

  The amount of trichloroacetic acid solution and / or water added in step 4 is not particularly limited. For example, the volume ratio is 0.5 to 10 times, preferably 1 based on the amount of the sample used in step 1 above. ˜5 times amount, more preferably 2 times amount.

  In Step 4, after adding and mixing a trichloroacetic acid solution and / or water to the precipitate obtained in Step 3, the precipitate is collected again. The mixing method is not particularly limited, but usually it may be mixed for about 3 to 5 seconds. The method for collecting the precipitate is the same as in the case of step 3 above.

[Step 5]
In step 5, the protein mass in the precipitate obtained in step 4 is measured by the Raleigh method to determine the protein mass in the sample.

The measurement of the protein in the step 5 may be performed by combining the biuret reaction and the oxidation reaction of the amino acid side chain by the Raleigh method, and the specific method thereof is not particularly limited, but the protein mass by the Raleigh method in the step 5 is measured. As a suitable example of the measurement, there is a method of performing the following steps 5-1 to 5-3.
Step 5-1, in which an alkaline copper test solution is added to the precipitate obtained in Step 4 to dissolve the precipitate,
After adding a diluted forin test solution to the solution obtained in Step 5-1, the absorbance was measured in Step 5-2, and the calibration curve prepared with the standard diluent was used to measure the solution. Step 5-3 for obtaining the protein mass in the specimen from the absorbance.
Hereinafter, the steps 5-1 to 5-3 will be described.

(Step 5-1)
The alkaline copper test solution used in the step 5-1 may be any alkaline solution that contains alkaline copper ions and can be used for the biuret reaction. The concentration is not particularly limited as long as it is within a range in which the precipitate obtained in Step 4 can be dissolved to cause a biuret reaction and a linear relationship is established between absorbance and protein concentration. As the copper test solution, one prepared by the following procedure can be used.
(Preparation procedure of alkaline copper test solution)
Dissolve 0.8 g of sodium hydroxide in water to make 100 mL, and dissolve 4 g of anhydrous sodium carbonate in this. This is A liquid. Mix 1 mL of 2 w / v% copper sulfate solution and 1 mL of 4 w / v% sodium tartrate solution. This is designated as B liquid. Mix 50 mL of solution A and 1 mL of solution B at the time of use to obtain an alkaline copper test solution.

  The amount of the alkaline copper reagent added in the step 5-1 is within a range in which the precipitate obtained in the step 4 can be dissolved to cause a biuret reaction and a linear relationship is established between the absorbance and the protein concentration. However, for example, when an alkaline copper test solution prepared by the above procedure is used, the volume ratio of the sample subjected to Step 1 is 0.1 to 10 times in terms of volume ratio based on the standard (1 time amount). The amount is preferably 1 to 5 times, more preferably 2.5 times.

  In Step 5-1, an alkaline copper test solution is added to the precipitate obtained in Step 4, and usually 5 minutes or longer, preferably 10 minutes or longer, more preferably 1 to 5 hours, at room temperature (15 to 25 ° C.). By allowing to stand, the precipitate obtained in the step 4 is dissolved and subjected to the step 5-2.

(Step 5-2)
The dilute forin test solution used in step 5-2 is a test solution prepared by adding water to a forin test solution (phenol test solution). The acid concentration in the diluted forin test solution is not particularly limited as long as the linear relationship is established between the absorbance and the protein concentration, but it is usually adjusted to 1 mol / L.

  In step 5-2, the amount of dilute forin test solution added is not particularly limited as long as the linear relationship is established between absorbance and protein concentration. For example, dilute forin sample solution with an acid concentration of 1 mol / L is used. Is used, the volume ratio of the sample subjected to Step 1 is 0.02 to 2 times, preferably 0.2 to 1 times, and more preferably 0.5, in terms of the volume ratio (standard amount). Double the amount.

  In step 5-2, water may be added as necessary. The amount added is not particularly limited as long as the linear relationship is established between the absorbance and the protein concentration. For example, the volume ratio based on the liquid amount of the sample subjected to the above step 1 as a reference (1 time amount) The amount is 0.1 to 10 times, preferably 1 to 5 times, and more preferably 2.5 times.

  In Step 5-2, after adding a diluted forin test solution and water as necessary to the solution obtained in Step 5-1, a temperature of 30 to 45 ° C., preferably 35 to 40 ° C., more preferably 37 ° C. Under the condition, after standing for 5 to 60 minutes, preferably 15 to 45 minutes, more preferably 30 minutes, the absorbance is measured using a spectrophotometer.

  In Step 5-2, when turbidity is observed after adding the diluted forin test solution and water as necessary to the solution obtained in Step 5-1, and leaving it to stand, centrifugation (for example, 1400 g) What is necessary is just to use the supernatant liquid isolate | separated by the above for 10 minutes for the measurement of a light absorbency.

  The absorbance measurement in step 5-2 is usually performed with the measurement wavelength set to 750 nm, but the measurement wavelength is 450 to 900 nm, preferably 700 to 900, as long as the measurement accuracy and accuracy are not adversely affected. You may change suitably in the range of 800 nm.

(Step 5-3)
The calibration curve used in Step 5-3 is obtained by performing Steps 1 to 4, Step 5-1, and Step 5-2 using a standard diluent with a known protein concentration. Can be created by deriving relationships.

  The type of standard diluent with a known protein concentration is not particularly limited. For example, a standard dilution solution containing 1 mg / mL of protein quantification albumin is prepared, and three or more different standard dilution solutions are used. Examples include standard dilutions adjusted to the concentration. In addition, the concentration of albumin for protein quantification in the standard diluted solution is specifically, the maximum concentration is in the range of about 100 to 500 μg / mL, and the minimum concentration is in the range of about 1 to 50 μg / mL. It should be set as follows.

  Further, in the calculation of the protein mass in the sample in step 5-3, for the absorbance value obtained in step 5-2, steps 1-4, step 5-1, and step 5-2 were performed for water. It is desirable to correct using the absorbance value obtained in this way. This makes it possible to quantify proteins with higher accuracy and accuracy.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Test example 1
1. Test material
1-1. Samples shown in Sample Table 1 were prepared.

1-2. Standard diluted solution Standard albumin for protein quantification defined in Non-Patent Document 1 was dissolved in water so as to be 1 mg / mL to prepare a standard diluted solution. Subsequently, the standard diluted solution was further diluted with water to prepare standard diluted solutions having albumin concentrations of 20, 50, 100, and 150 μg / mL.

1-3. Water was added to trichloroacetic acid solution trichloroacetic acid (Wako Pure Chemical Industries, Ltd.) to prepare 100 w / v% trichloroacetic acid solution, 5 w / v% trichloroacetic acid solution, and 10 w / v% trichloroacetic acid solution, respectively.

1-4. Alkaline copper test solution 0.8 g of sodium hydroxide was dissolved in water to make 100 mL, and 4 g of anhydrous sodium carbonate was dissolved therein to prepare solution A. Separately, 1 mL of 2 w / v% aqueous solution of copper sulfate and 1 mL of 4 w / v% aqueous solution of sodium tartrate were mixed to prepare solution B. 50 mL of A solution and 1 mL of B solution were mixed at the time of use to prepare an alkaline copper test solution.

1-5. Diluted forin test solution Forin test solution (phenol reagent, acid concentration 2 mol / L: Wako Pure Chemical Industries, Ltd.) 10 mL of water was added to prepare an acid concentration of 1 mol / L to prepare a diluted forin test solution. .

2. Protein quantification
2-1. Sample The specimen was used as a sample.

2-2. Formation of Precipitate by Addition of Trichloroacetic Acid Solution A trichloroacetic acid solution was added to 1 mL of a sample under the conditions shown in Table 2, and heated at 100 ° C. for 15 minutes in a water bath. Next, after cooling, centrifugation (1872 g, 20 minutes) was performed at 25 ° C., and the precipitate was collected.

2-3. Formation of precipitate by re-addition of trichloroacetic acid solution 2 mL of 5 w / v% trichloroacetic acid solution was added to the recovered precipitate, shaken and centrifuged at 25 ° C. (1872 g, 20 minutes) to recover the precipitate.

2-4. Measurement of protein by the Raleigh method 2.5 mL of alkaline copper test solution was added to the collected precipitate, shaken and allowed to stand at 25 ° C. for 5 hours to dissolve the precipitate. Next, 2.5 mL of water and 0.5 mL of dilute forin reagent solution were added and allowed to stand at 37 ° C. for 30 minutes, and then the absorbance of the solution at a wavelength of 750 nm was measured using a spectrophotometer.

2-5. Calculation of the protein mass in the sample The standard dilution solution was operated under the same conditions as described above to prepare a calibration curve showing the relationship between the protein mass and the absorbance. Next, the protein mass in the sample is calculated from the absorbance value obtained in the above “2-4. Measurement of protein by the Raleigh method” using a calibration curve, and further, from the dilution factor of the sample, The protein mass was determined. The absorbance was measured by operating under the same conditions as described above using water instead of the sample, and after correcting the absorbance of each sample and standard dilution with the absorbance, creating a calibration curve and The protein mass was calculated.

3. Test results The results obtained are shown in Table 2. In this test, the measurement was performed three times under the same conditions, and the average value of each protein mass in the sample under the same conditions was calculated. The results of conditions 1 and 2 are protein masses determined according to the official document law. As is clear from the comparison between conditions 1 and 2 and the comparison between conditions 12 and 13, in sample 1 not containing a viscosity agent (carboxyvinyl polymer), protein could be quantified with high accuracy by the official method. In the specimen 2 containing the viscosity agent (carboxyvinyl polymer), the quantitative accuracy of the protein was significantly lowered by the official method.

  On the other hand, the operation of adding the same amount of 10 w / v% trichloroacetic acid solution as that of the sample in the official document method is performed so that the concentration of trichloroacetic acid finally becomes 20.0 to 31.0 w / v%. When the sample is replaced with an operation of adding the sample to the sample (conditions 5 to 9, 14 and 15), the protein can be quantified with high accuracy and high accuracy even in a specimen containing a viscous agent (carboxyvinyl polymer). It was.

Test example 2
1. Test material
1-1. Samples shown in Sample Table 3 were prepared.

1-2. Standard diluent, trichloroacetic acid solution, alkaline copper test solution, and dilute folin test solution In the same manner as in Test Example 1, a standard dilute solution, trichloroacetic acid solution, alkaline copper test solution, and dilute folin test solution were prepared.

2. Protein quantification The sample was diluted with purified water so that the content of the influenza virus antigen-containing protein was about 100 μg / mL. Specifically, samples 6 diluted 3 times with purified water, samples 7 diluted 6 times with purified water, and samples 8 diluted 9 times with purified water were used as samples.

  Protein was quantified under the same conditions as in Test Example 1. In addition, the process of “2-2. Precipitation generation by addition of trichloroacetic acid solution” was performed under a condition in which 0.3 mL of trichloroacetic acid solution (trichloroacetic acid 100 w / v%) was added to 1 mL of the sample.

3. Test results The results obtained are shown in Table 4. In this test, the measurement was performed three times under the same conditions, and the average value of each protein mass in the sample under the same conditions was calculated. From this result, the operation of adding the same amount of 10 w / v% trichloroacetic acid solution as that of the sample in the official method, the trichloroacetic acid solution was adjusted so that the final concentration of trichloroacetic acid was 20.0 to 31.0 w / v%. By replacing with the operation of adding to the sample, protein can be quantified with high accuracy and high accuracy even if the specimen contains additives such as buffering agents and tonicity agents as well as viscosity agents (carboxyvinyl polymer). confirmed.

Test example 3
1. Test material
1-1. Sample 6 shown in Sample Table 3 was prepared.

2. Protein quantification
2-1. Sample The sample diluted with purified water so that the content of the influenza virus antigen-containing protein was about 100 μg / mL was used as a sample. Specifically, a sample 6 diluted 3 times with purified water was used as a sample.

2-2. Formation of precipitate by addition of trichloroacetic acid solution 0.06 mL of a trichloroacetic acid solution (trichloroacetic acid 100 w / v%) was added to 0.2 mL of the sample, and the mixture was heated at 100 ° C. for 15 minutes in a water bath. Next, after cooling, the mixture was centrifuged under the conditions shown in Table 5 to collect the precipitate.

2-3. Formation of precipitate by re-addition of trichloroacetic acid solution 2 mL of a 5 w / v% trichloroacetic acid solution was added to the recovered precipitate, shaken, and centrifuged under the conditions shown in Table 5 to recover the precipitate.

2-4. Measurement of protein by the Raleigh method 0.5 mL of alkaline copper test solution was added to the collected precipitate, shaken and allowed to stand at 25 ° C. for 5 hours to dissolve the precipitate. Subsequently, after adding 0.5 mL of water and 0.1 mL of dilute forin reagent solution and allowing to stand at 37 ° C. for 30 minutes, the absorbance of the solution at a wavelength of 750 nm was measured using a spectrophotometer.

2-5. Calculation of protein mass in sample A calibration curve was prepared and the protein mass in the sample was calculated under the same conditions as in Test Example 1.

3. Test results The results obtained are shown in Table 5. In this test, the measurement was performed three times under the same conditions, and the average value of each protein mass in the sample under the same conditions was calculated. From this result, it was confirmed that the protein could be quantified with high accuracy and high accuracy by the method of the present invention even when precipitation collection after addition of the trichloroacetic acid solution was performed under conditions of 3000 to 10000 g for 20 minutes.

Reference Test Example A protein was prepared under the same conditions as in Test Example 1 except that the sample shown in Table 6 was used, the trichloroacetic acid solution was added to 1 mL of the sample under the conditions shown in Table 7, and the number of measurements was one. Was quantified.

  The results obtained are shown in Table 7. From these results, it was confirmed that, even if arginine and glycerin were included in the sample, the quantitative accuracy of the protein was not affected by the official document method.

Claims (7)

A method for quantifying protein in a specimen containing a protein and a viscous agent, comprising preparing a sample using the specimen,
Step 2, in which a trichloroacetic acid solution is added and heated so that the concentration of trichloroacetic acid is 20.0 to 31.0 w / v% with respect to the sample obtained in Step 1;
Step 3 for recovering the precipitate from the solution obtained in Step 2;
Adding a trichloroacetic acid solution and / or water to the precipitate obtained in step 3 and mixing the precipitate, and then measuring the protein mass in the precipitate obtained in step 4 by the Raleigh method; Step 5 for determining the protein mass in the specimen
A quantitative method comprising:   The quantitative method according to claim 1, wherein the viscous agent is a polyacrylic acid-based viscous agent.   The method according to claim 1 or 2, wherein the viscosity agent is a carboxyvinyl polymer.   The quantification method according to claim 1, wherein the specimen is a biological preparation.   The quantification method according to any one of claims 1 to 4, wherein the protein is an antigenic protein of an influenza vaccine.   The quantification method according to any one of claims 1 to 5, wherein the trichloroacetic acid solution used in Step 4 is a solution containing 15 w / v% or less of trichloroacetic acid. The quantification method according to any one of claims 1 to 6, wherein the step 5 includes the following steps 5-1 to 5-3:
Step 5-1, in which an alkaline copper test solution is added to the precipitate obtained in Step 4 to dissolve the precipitate,
After adding a diluted forin test solution to the solution obtained in Step 5-1, the absorbance was measured in Step 5-2, and the calibration curve prepared with the standard diluent was used to measure the solution. Step 5-3 for obtaining the protein mass in the specimen from the absorbance.