Method for determining the hexosamine content in pneumococcal polysaccharide sample solution

Abstract

The present invention relates to a method for determining the content of hexosamine in a pneumococcal polysaccharide sample solution. In quantitatively evaluating whether the hexosamine content of a produced pneumococcal polysaccharide undiluted solution sample is suitable for use in a vaccine, the method according to one aspect provides more accurate validation compared to the hexosamine content test method specified in the European Pharmacopoeia, and thus can be effectively utilized for quality control of pneumococcal polysaccharide vaccines or polysaccharide-protein conjugate vaccines containing hexosamine.

Description

Method for determining the content of hexosamine in a pneumococcal polysaccharide sample solution

[0001] A method for determining the content of hexosamine in a pneumococcal polysaccharide sample solution.

[0002] Streptococcus pneumoniae is a Gram-positive, hemolytic streptococcus, and is a major cause of meningitis, pneumonia, and serious invasive infections in infants, children, and the elderly worldwide. More than 1.6 million people die each year from pneumococcus-related illnesses, with a particularly high incidence of invasive infections in immunocompromised children under 5 years of age and in the elderly over 65 years of age. Streptococcus pneumoniae is classified into over 90 serotypes based on the structural and immunological characteristics of the capsular polysaccharide, the main virulence factor surrounding the outer (cell membrane) surface of the Streptococcus pneumoniae. Some serotypes have been reported to cause invasive diseases. A total of 37 serotypes were identified from invasive pneumococci collected between 1996 and 2008, and vaccines such as polysaccharide vaccines and polysaccharide-protein conjugate vaccines were manufactured using the capsular polysaccharides of these serotypes of pneumococci.

[0003] Meanwhile, the accuracy of vaccine quality assessment methods directly affects vaccine safety, and the European Pharmacopoeia (EP) presents a method for quantitatively evaluating the content of monosaccharides such as hexosamine in pneumococcal polysaccharide vaccines. However, the hexosamine content test method for polysaccharide vaccines presented in the European Pharmacopoeia (EP) has been raised as a problem in that it does not meet the hexosamine content compliance criteria for each serotype presented in the EP and WHO guidelines even when tested using commercial standard samples. Accordingly, the need for an evaluation method that can more accurately test the hexosamine content is greatly increasing.

[0004] [Prior Art Literature]

[0005] [Non-patent literature]

[0006] (Non-patent Document 1) EUROPEAN PHARMACOPOEIA 11.0, 2.5.20. Hexosamines in polysaccharide vaccine, 2022

[0007] One aspect provides a method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, comprising the steps of: (a) treating a series of concentrations of glucosamine standard solutions according to a predetermined method, measuring absorbance to create a standard curve; and (b) treating a polysaccharide sample solution according to a predetermined method, measuring absorbance to calculate the hexosamine content of the polysaccharide sample solution according to the standard curve obtained in step (a); wherein the predetermined method comprises: adding a hydrochloric acid solution to each test tube containing the standard solution or the polysaccharide sample solution to hydrolyze; adding a sodium carbonate solution to neutralize; adding an acetylacetone solution to react; adding ethanol to precipitate; adding a DMAB (dimethylaminobenzaldehyde) solution as a color developer for derivatization to dissolve the precipitate; and measuring absorbance.

[0008] One aspect provides a method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, comprising the steps of: (a) treating a series of concentrations of glucosamine standard solutions according to a predetermined method, measuring absorbance to create a standard curve; and (b) treating a polysaccharide sample solution according to a predetermined method, measuring absorbance to calculate the hexosamine content of the polysaccharide sample solution according to the standard curve obtained in step (a); wherein the predetermined method comprises: adding a hydrochloric acid solution to each test tube containing the standard solution or the polysaccharide sample solution to hydrolyze; adding a sodium carbonate solution to neutralize; adding an acetylacetone solution to react; adding ethanol to precipitate; adding a DMAB (dimethylaminobenzaldehyde) solution as a color developer for derivatization to dissolve the precipitate; and measuring absorbance.

[0009] The term “hexosamine” refers to a molecule with the formula C6H2 created by adding an amino group to hexose. 13 It refers to amino sugar NO5, and is a concept that includes galactosamine, glucosamine, fructosamine, and mannosamine.

[0010] The term “standard solution” refers to a solution used as a substance of known concentration to draw a calibration curve when quantitatively analyzing the concentration of an analyte using spectrophotometry. According to one aspect, it was confirmed that the hexosamine content compliance criteria presented in the EP and WHO guidelines were satisfied in all serotypes of commercial standard products regardless of whether the standard substance, glucosamine, was acetylated (Experimental Example 2.3). In one specific example, the glucosamine standard solution may be an N-Acetyl D-Glucosamine solution or a D-(+)-Glucosamine Hydrochloride solution. In one specific example, the series of concentrations falls within the range of 0-250 μg / mL, and the number of the series of concentrations may be at least 8. For example, the above series of concentrations may be 0, 25, 62.5, 125, 150, 175, 200, 225 μg / mL, but is not limited thereto, and sequential concentrations within the above range may be selected and used.

[0011] The term “polysaccharide sample solution” may be used interchangeably with the term “test solution” and refers to a test sample solution containing an analyte to be analyzed, the concentration of which is to be measured. For example, the method may be conducted using a purified pneumococcal capsular polysaccharide stock solution or a diluted solution thereof as a polysaccharide sample solution to determine whether the capsular polysaccharide stock solution obtained through cell lysis, isolation, and purification processes from a culture medium of a strain producing a specific serotype of pneumococcus is suitable for use in a vaccine. In one specific example, the polysaccharide sample solution may be a solution obtained by diluting the sample 10-fold to 15-fold. If the dilution factor of the polysaccharide sample solution is too large, exceeding the above range, or too small, falling below the above range, the absorbance of the sample may fall outside the absorbance range of the standard solution depending on the type of pneumococcal serotype, and therefore the assay method may not be suitable for use with all pneumococcal serotypes.

[0012] In one specific example, the serotype of the pneumococcus may be any serotype containing hexosamine, and specifically, may be any one selected from the group consisting of 4, 5, 9N, 9V, 10A, 12F, 14, 15B, 19A, and 19F.

[0013]

[0014] The above predetermined method is described in detail below.

[0015] Hydrolysis step:

[0016] This is a step of adding a hydrochloric acid solution, which is a hydrolysis buffer, to each test tube containing a glucosamine standard solution prepared at a series of concentrations or a prepared polysaccharide sample solution to react, which is a process for hydrolyzing and exposing specific residues in the sugar.

[0017] In one specific example, the concentration of the hydrochloric acid solution may be 12.0±0.5 M, for example, 12.0±0.4 M, 12.0±0.3 M, 12.0±0.2 M, 12.0±0.1 M, 12.0±0.05 M, or 12.0±0.01 M. If the concentration of the hydrochloric acid solution is too high, exceeding the above range, severe evaporation of hydrochloric acid may occur during the reaction at room temperature, making normal hydrolysis reaction difficult, and during the reaction at low temperature, not only the specific residue to be targeted but also other residues may be exposed, and the polysaccharide itself may be decomposed, making normal polysaccharide assay impossible. In addition, if the concentration of the hydrochloric acid solution is too low, below the above range, the residues of the polysaccharide are not sufficiently exposed to be able to bind to the reagent for color development, so that the intensity of the reaction is weak at the stage of adding a color development reagent such as DMAB later, making normal polysaccharide assay impossible.

[0018] In one specific example, the volume ratio of the hydrochloric acid solution to the standard solution or polysaccharide sample solution may be 2:0.5 to 1.5, for example, 2:0.5 to 1.3, 2:0.5 to 1.1, 2:0.5 to 1.0, 2:0.7 to 1.5, 2:0.7 to 1.3, 2:0.7 to 1.1, 2:0.7 to 1.1, 2:0.9 to 1.5, 2:0.9 to 1.3, 2:0.9 to 1.1, 2:0.9 to 1.0, 2:1.0 to 1.5, or 2:1.0 to 1.3. If the volume of hydrochloric acid solution added is too large or too low, outside the above range, the concentration of the hydrochloric acid solution at the start of hydrolysis may be too high or too low, causing the problems described above.

[0019] In one specific example, the hydrolysis step may be carried out for 4 to 6 hours, for example, 4 to 5 hours, or 5 to 6 hours. If the hydrolysis reaction time is too long, exceeding the above range, the hydrolysis may proceed too much and the polysaccharide may be damaged, which may cause the assay method to not be performed normally. If the hydrolysis reaction time is too short, which is less than the above range, the hydrolysis may not proceed sufficiently, which may cause less exposure of residues within the polysaccharide, which may cause the assay method to not be performed normally. The hydrolysis may be carried out by heating, and may be carried out at a temperature of 100°C or higher, for example, 100°C to 150°C, 100°C to 120°C, or 130°C to 150°C.

[0020] In one specific example, the test tube may be made of glass, and since thermal conductivity may vary depending on the test tube material, the reaction temperature and reaction time may be based on a test tube made of glass.

[0021] After the above hydrolysis reaction, a step of cooling each solution to room temperature may be performed.

[0022] Hydrolyzate neutralization titration steps:

[0023] This is a process of neutralizing the acidic solution in each test tube by adding a base. Specifically, this is a step of neutralizing by adding a sodium carbonate (Na2CO3) solution to each test tube in which the hydrolysis reaction was performed, and then reacting by adding an acetylacetone solution so that the color change in the subsequent color development reaction using a DMAB solution becomes more distinct.

[0024] In one specific example, the concentration of the sodium carbonate solution may be 2.0±1.0 M, for example, 2.0±0.9 M, 2.0±0.8 M, 2.0±0.7 M, 2.0±0.6 M, 2.0±0.5 M, 2.0±0.4 M, 2.0±0.3 M, 2.0±0.2 M, 2.0±0.1 M, 2.0±0.05 M, or 2.0±0.01 M.

[0025] In one specific example, the volume ratio of the sodium carbonate solution to the total volume of the solution contained in each test tube after the hydrolysis reaction may be 3:3.5 to 4.5, for example, 3:3.5 to 4.3, 3:3.5 to 4.1, 3:3.5 to 4.0, 3:3.7 to 4.5, 3:3.7 to 4.3, 3:3.7 to 4.1, 3:3.7 to 4.0, 3:3.9 to 4.5, 3:3.9 to 4.3, 3:3.9 to 4.1, 3:3.9 to 4.0, 3:4.0 to 4.5, or 3:4.0 to 4.3.

[0026] In one specific example, the concentration of the acetylacetone solution may be 0.2±0.1 M, for example, 0.2±0.09 M, 0.2±0.08 M, 0.2±0.07 M, 0.2±0.06 M, 0.2±0.05 M, 0.2±0.04 M, 0.2±0.03 M, 0.2±0.02 M, 0.2 to 0.3 M, or 0.216 M.

[0027] In one specific example, the volume ratio of the acetylacetone solution to the total volume of the solution in each test tube after addition of the sodium carbonate solution may be 7:4.5 to 5.5, for example, 7:4.5 to 5.3, 7:4.5 to 5.1, 7:4.5 to 5.0, 7:4.7 to 5.5, 7:4.7 to 5.3, 7:4.7 to 5.1, 7:4.7 to 5.0, 7:4.9 to 5.5, 7:4.9 to 5.3, 7:4.9 to 5.1, 7:4.9 to 5.0, 7:5.0 to 5.5, or 7:5.0 to 5.3.

[0028] In one specific example, the step of reacting by adding the acetylacetone solution may be reacting at a temperature of 100°C or higher, for example, 100°C to 150°C, 100°C to 120°C, or 130°C to 150°C, for 20 to 40 minutes, for example, 20 to 35 minutes, 20 to 30 minutes, 20 to 25 minutes, 25 to 40 minutes, 25 to 35 minutes, 25 to 30 minutes, 30 to 40 minutes, 30 to 35 minutes, or 35 to 40 minutes.

[0029] After adding the above acetylacetone solution and reacting, a step of cooling each solution to room temperature can be performed.

[0030] Color development steps:

[0031] This is a process in which ethanol is added to each neutralized solution to precipitate polysaccharides that have sufficiently exposed reagent sites to react with the reagent through hydrolysis in the previous step, then a solution of DMAB (dimethylaminobenzaldehyde), a coloring agent, is added to dissolve the precipitate and measure the absorbance.

[0032] In one specific example, the volume ratio of the ethanol to the total volume of the solution in each test tube after the addition of the acetylacetone solution may be 1.2:0.5 to 1.5, for example, 1.2:0.5 to 1.5, 1.2:0.5 to 1.3, 1.2:0.5 to 1.1, 1.2:0.5 to 1.0, 1.2:0.7 to 1.5, 1.2:0.7 to 1.3, 1.2:0.7 to 1.1, 1.2:0.7 to 1.1, 1.2:0.9 to 1.5, 1.2:0.9 to 1.3, 1.2:0.9 to 1.1, 1.2:0.9 to 1.0, 1.2:1.0 to 1.5, or 1.2:1.0 It may be 1.3. If the volume of the added ethanol solution is too small, below the above range, precipitation may not occur sufficiently, making normal reaction difficult.

[0033] In one specific example, the concentration of the DMAB solution may be 0.2±0.1 M, for example, 0.2±0.09 M, 0.2±0.08 M, 0.2±0.07 M, 0.2±0.06 M, 0.2±0.05 M, 0.2±0.04 M, 0.2±0.03 M, 0.2 to 0.3 M, or 0.223 M. If the concentration of the DMAB solution is too large, exceeding the above range, economic feasibility may be reduced, and if it is too small, below the above range, the color development reaction may not sufficiently occur, making measurement impossible.

[0034] In one specific example, the volume ratio of the DMAB solution to the total volume of the solution contained in each test tube after the addition of the ethanol may be 2.2:0.1 to 1.0, for example, 2.2:0.1 to 0.8, 2.2:0.1 to 0.5, 2.2:0.3 to 1.0, 2.2:0.3 to 0.8, 2.2:0.3 to 0.5, 2.2:0.5 to 1.0, or 2.2:0.5 to 0.8. If the volume of the DMAB solution added is too large, exceeding the above range, the concentration of the polysaccharide sample solution may be excessively diluted, resulting in inaccurate absorbance measurement results, and if it is too small, below the above range, the color reaction may not sufficiently occur, making measurement impossible.

[0035] In one specific example, the step of dissolving the precipitate by adding the DMAB solution may be to add the DMAB solution, vortex for 10 to 30 seconds, and then allow to stand at room temperature for 5 to 15 minutes, for example, 5 to 10 minutes, 8 minutes, 7 minutes, or 5 minutes. If the standing time is too long, exceeding the above range, the color development step may require the inspector to continuously observe the color change with the naked eye, which may reduce the convenience of the inspection, and the sample itself may decompose, making it difficult to measure the accurate absorbance. If the standing time is too short, less than the above range, a sufficient color development reaction may not occur, making it difficult to measure the absorbance. The above method has the advantage of improving the convenience of the inspection because it is possible to measure the absorbance with only a short standing time.

[0036] The above absorbance measurement may be performed by measuring the absorbance at an absorption wavelength of 530 nm using a UV-vis spectrophotometer for each test tube solution that has been left to stand for a certain period of time.

[0037]

[0038] In the case of the hexosamine content test method (EP method) presented in the European Pharmacopoeia, it was confirmed that the commercial standard sample did not satisfy the hexosamine content acceptance criteria for each serotype presented in the EP and WHO guidelines despite changes in various test conditions such as changing the hydrolysis concentration (8 M, 10 M, 12 M HCl), hydrolysis time (1, 2, 3, 4 hours), material of the analytical test tube (PP Tube, Glass Tube), color reaction method (Inverting, Vortexing), and whether the standard substance was acetylated (Experimental Example 1). In contrast, in the case of the hexosamine content test method of the present invention, it was confirmed that it stably exceeded the hexosamine content acceptance criteria presented in the EP and WHO guidelines for all serotypes of the commercial standard regardless of whether the standard substance was acetylated (Experimental Example 2), and thus, it can be usefully used in the production of a safe pneumococcal polysaccharide vaccine due to its excellent accuracy of assay performance.

[0039] The method according to the aspect of the invention can be used to quantitatively evaluate whether the hexosamine content of a pneumococcal polysaccharide stock sample produced is suitable for use in a vaccine, and can be used for quality control of a pneumococcal polysaccharide vaccine or a polysaccharide-protein conjugate vaccine of a serotype containing hexosamine, as it allows for more accurate testing than the hexosamine content test method presented in the European Pharmacopoeia.

[0040] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0041]

[0042] Experimental Example 1. EP Method and Modification Test

[0043] To assess the suitability of the quality of the produced pneumococcal polysaccharide stock samples for use in vaccines, the assay performance of the hexosamine content test method for polysaccharide vaccines presented in the European Pharmacopoeia (hereinafter referred to as the European Pharmacopoeia (EP) Method) and a method with modified test conditions based on this method were evaluated. Specifically, the commercial standard ATCC samples and the company's own samples were used to assess whether the samples met the hexosamine content standards for each serotype presented in the EP and WHO guidelines.

[0044]

[0045] 1.1 Hexosamine content test method according to EP method (comparative example)

[0046] 1.1.1 Preparation of D-(+)-Glucosamine Hydrochloride standard solution

[0047] Weigh 0.6 g of D-(+)-Glucosamine Hydrochloride and add it to 50 mL of distilled water. Vortex to dissolve it sufficiently and then dilute it to prepare a 10 mg / mL D-(+)-Glucosamine Stock Solution. Place 2.5 mL of 10 mg / mL D-(+)-Glucosamine Stock Solution into a 50 mL Cornical Tube, adjust the final volume to 50 mL with distilled water, and vortex to mix thoroughly to prepare a 500 μg / mL D-(+)-Glucosamine standard solution. The 500 μg / mL D-(+)-Glucosamine standard solution was added to the tube to obtain the standard solution concentrations as shown in Table 1 below and diluted to prepare the standard solution and blank.

[0048] ClassificationConcentration (μg / mL)500 μg / mL D-(+)-Glucosamine Standard Solution (μL)Tri-distilled Water (μL)Total Volume (μL)Blank0010001000STD 11252507501000STD 22505005001000STD 33757502501000STD 4500100001000

[0049] 1.1.2 Preparation of test solution

[0050] Table 2 below shows the compliance criteria (% content ratio) for hexosamine content by serotype in EP Volume 1. Vaccine Section Table 0966-1 (Percentage Contents of Components of Monovalent Bulk Polysaccharides). Referring to the % content ratio, the sample was appropriately diluted to ensure that the hexosamine concentration was within the standard solution concentration range of 125 to 500 μg / mL. The diluted sample was prepared in a tube so that the final volume was 1 mL.

[0051] Serotype% content ratio Serotype% content ratio 4≥ 4012F≥ 255≥ 2014≥ 209N≥ 2815B≥ 159V≥ 1319A≥ 1210A≥ 1219F≥ 12.5

[0052] Specifically, appropriate dilution was performed considering the minimum and maximum dilution factors of the sample by serotype. However, if the hexosamine content of the diluted sample was outside the concentration range of the standard solution, additional dilution was performed without being limited to the minimum and maximum dilution factors. If the dilution factor of the sample was 10 times or more, serial dilution was performed. The maximum and minimum dilution factors of the sample were calculated according to the following formula:

[0053] Maximum dilution factor = Dry weight value by serotype (mg / mL) x % content ratio by serotype / 125 μg / mL

[0054] Minimum dilution factor = Dry weight value by serotype (mg / mL) x % content ratio by serotype / 500 μg / mL

[0055] 1.1.3 Hydrolysis step

[0056] Polysaccharide hydrolysis was performed by adding hydrolysis buffer to the blank, D-(+)-Glucosamine standard solutions by concentration, and test solutions prepared in 1.1.1 and 1.1.2 above. Specifically, 1 mL of 8 M HCl solution was added to a tube containing 1 mL each of the blank, D-(+)-Glucosamine standard solutions by concentration, and test solutions prepared in 1.1.1 and 1.1.2 above, and then vortexing. The tube was capped, reacted for 1 hour in a water bath set to 90 to 99 °C, and then sufficiently cooled to room temperature for 15 minutes.

[0057] Here, the 8M HCl solution was prepared by adding 131.53 mL of 37% HCl to a 250 mL glass bottle, adding distilled water to make the final volume 200 mL, and stirring sufficiently using a magnetic stirrer.

[0058] 1.1.4 Hydrolyzate neutralization titration step

[0059] Each solution, which had become acidic due to hydrolysis, was titrated by adding a base. Specifically, 0.05 mL of thymolphthalein solution (5 g thymolphthalein / 1 L EtOH) was added to each tube cooled to room temperature and vortexed. Then, 200 g / L NaOH solution was slowly added until the colors of the blank, standard solution, and test solution changed from colorless to blue, and then 1 M HCl solution was added and titrated again until the blue color disappeared and the solution became colorless. Triple-distilled water was added so that the final volume of each tube became 10 mL and vortexed.

[0060] Of the 10 mL of the neutralized Blank, standard solution, and test solution, 1 mL was aliquoted into a new tube, and 1 mL of acetylacetone solution (1 volume acetylacetone / 50 volume Na2CO3) was added each time, followed by sufficient vortexing. The tube was capped and reacted in a water bath set to 90°C for 45 minutes, then cooled sufficiently at room temperature for 15 minutes.

[0061] Here, the Thymolphthalein solution was prepared by weighing 0.05 g of Thymolphthalein, adding it to 10 mL of Ethanol, and vortexing to sufficiently dissolve it. The 200 g / L NaOH solution was prepared by weighing 40 g of Sodium Hydroxide in a 250 mL Glass Bottle, adding distilled water to make a final volume of 200 mL, and stirring sufficiently using a Magnetic Stirrer. The Na2CO3 solution was prepared by weighing 2.65 g of Na2CO3, adding it to 50 mL of distilled water, and vortexing to sufficiently dissolve it. The Acetylacetone solution was prepared by adding 0.5 mL of Acetylacetone to 25 mL of Na2CO3 solution, and then vortexing to sufficiently mix it.

[0062] 1.1.5 Color development stage

[0063] 2.5 mL of ethanol was added to each tube that had cooled to room temperature, vortexed sufficiently, and confirmed that a precipitate had formed. 1 mL of DMAB (dimethylaminobenzaldehyde) solution (0.8 g dimethylaminobenzaldehyde / 15 mL EtOH + 15 mL HCl) was added and inverted to dissolve all the precipitate. 4.5 mL of ethanol was added so that the final volume was 10 mL, and inverted. After standing for 90 minutes at room temperature in a light-shielded environment, the absorbance was measured at a wavelength of 530 nm using a UV-Vis Spectrophotometer. A calibration curve was created using the measured absorbance values ​​of the standard solution, and the hexosamine content was quantified from the absorbance values ​​of the sample solution measured based on this.

[0064] Here, a DMAB solution of 0.179 M concentration was used, which was prepared by weighing 0.8 g of 4-(Dimetylamino)-benzaldehyde and dissolving it sufficiently in 15 mL of ethanol and 15 mL of 37% HCl by vortexing.

[0065] 1.2 Evaluation of hydrolysis concentration and time in EP method

[0066] In order to confirm whether the concentration conditions and reaction time conditions of HCL in the hydrolysis step in the EP method of 1.1 above are factors affecting the measurement of hexosamine content, serotypes whose suitability judgment may change due to a change in the test method were used as the serotypes to be investigated, and the hexosamine content of each serotype sample was measured under the hydrolysis conditions of 8 M, 10 M, and 12 M HCl for 1, 2, and 3 hours, and the results are shown in Table 3 below. In the case of serotypes 4 and 12F, a tendency for a lower hexosamine content was confirmed in the ATCC sample, which is a commercial standard, during the pre-test compared to the results of the company's polysaccharide stock solution, and therefore the serotype was considered the worst case and included as a control substance and then tested.

[0067] Here, the 10M HCl solution was prepared by adding 164.41 mL of 37% HCl to a 250 mL glass bottle, adding distilled water to make a final volume of 200 mL, and stirring sufficiently using a magnetic stirrer. The 12M HCl solution was prepared by using a 37% HCl stock solution.

[0068] SerotypeLot No.8M HCl10M HCl12M HCl standard (%)1 hr2 hr3 hr1 hr2 hr3 hr1 hr2 hr3 hr4A04042201C303429353234373332≥ 40ATCC23262626242631292712FCTAA1242282627242525303128≥ 25ATCC21212221222124232419AA96042301F131415121515161718≥ 1219FCTAA9242121113131415141617≥ 12.5

[0069] As a result, as shown in Table 3 above, it was confirmed that serotypes 19A and 19F could be used in vaccines because they exceeded the assay standard when heated in 12M HC for 3 hours. However, even though all serotypes were used in the assay method as commercial standards, it was confirmed that serotypes 4 and 12F did not exceed the assay standard, and thus the normal assay method was not performed.

[0070] 1.3 Evaluation according to test tube material in EP method

[0071] Since the water bath temperature of the test method 1.1 above is high, over 90 ℃, a comparative experiment was conducted according to the test tube material (PP (Polypropylene) Tube vs. Glass Tube) to confirm whether the thermal conductivity of the test tube is a factor affecting the measurement of the hexosamine content. The hexosamine content of serotype 4 was measured under hydrolysis conditions of 8 M, 10 M, and 12 M HCl for 4 hours, and the results are shown in Table 4 below. In this test, a material comparison test was conducted by fixing the time to 4 hours to confirm whether the test tube remained intact in appearance even when the hydrolysis reaction time was longer than the 3-hour condition in Table 6, and at the same time, whether the hexosamine content increased after 3 hours.

[0072] SerotypeLot No.Tube8M10M12MBased (%)4A04042201CPolypropylene313335≥40Glass393636

[0073] As a result, as shown in Table 4 above, it was confirmed that the use of a Glass Tube resulted in a higher hexosamine content than the use of a PP Tube. Since some differences in interactions between the sample and the test tube and in thermal conductivity were expected to occur, it was confirmed that the test tube needed to be changed to a Glass Tube, and therefore subsequent experiments were conducted using a Glass Tube.

[0074] 1.4 Evaluation according to the dissolution method of the sediment in the EP Method

[0075] In the test method of 1.1 above, DMAB solution is added to the precipitate formed when ethanol is added after the second water bath reaction, and the color reaction is confirmed by inverting and dissolving. In order to confirm whether there is a difference in the degree of color development depending on the method of dissolving the precipitate, a vortexing method was added and compared. Both inverting and vortexing in the color development step were performed for 10 to 30 seconds, and the hexosamine content of serotype 4, 9N, 12F, 19A, and 19F samples was measured under 8 M HCl, 1 hour hydrolysis conditions, and the results are shown in Table 5 below.

[0076] Based on SerotypeLot No.InvertingVortexing (%)4A04042201C3236≥ 409NCTAA9145J3031≥ 2812FCTAA12422727≥ 2519AA96042301F1112≥ 1219FCTAA974021212≥ 12.5

[0077] As a result, as shown in Table 5 above, all serotypes showed equal or higher hexosamine contents when vortexing was performed compared to inverting, confirming that vortexing was effective in more reliably dissolving the precipitate. In addition, although the inverting method was previously used to prevent damage to the polysaccharide, it was confirmed that the vortexing method could effectively dissolve the precipitate without damaging the polysaccharide when suspended. Therefore, subsequent experiments were conducted using the vortexing method when dissolving the precipitate in the color development step.

[0078] 1.5 Evaluation of acetylation of standard materials in the EP method

[0079] As confirmed in 1.2-1.4 above, despite several modifications based on the EP Method, only changing the test conditions and test tube material did not result in a result that stably exceeded the hexosamine content standards presented in the EP and WHO guidelines, even for the commercially available ATCC standard samples. Table 6 below shows the ratio of acetylated and non-acetylated hexosamines by serotype. Based on the fact that the commonality among the serotypes for which the existing EP hexosamine analysis method was not sufficient in the batch results was that the ratio of acetylated hexosamine was relatively high (Table 6), we attempted to confirm the quantitative results according to the acetylation status of the standard material.

[0080] HexosamineSerotype459N9V10A12F1415B19A19FGlucos-amineAc * 0010001100-0233021111Galactos-amineAc1000410000-1001112300Mannos-amineAc1011010011-0 000000000Fucos-amineAc1100010000-0000000000Pneumos-amineAc0100000000-0000000000Ratio of Acetylated to Non-acetylated Hexosamine3 / 42 / 42 / 51 / 51 / 53 / 61 / 41 / 51 / 21 / 20.750.500.400.200.200.500.250.200.500.50

[0081] (* Ac: Acetylated)

[0082] Specifically, unlike the non-acetylated conventional hexosamine (D-(+)-Glucosamine Hydrochloride, Chemical Formula 1), a comparative test was performed using acetylated hexosamine (N-Acetyl D-Glucosamine, Chemical Formula 2) as a standard according to the acetylation of the standard. The hydrolysis time was performed under the extreme conditions (minimum 1 hour, maximum 4 hours) among the conditions tested previously, and the experiment was conducted by adding DMAB solution and vortexing to develop a color reaction according to the result of 1.4. The results of measuring the hexosamine content using the N-Acetyl D-Glucosamine standard in the EP Method are shown in Table 7 below. When an acetylated standard is used, the molecular weight of the standard increases compared to the conventional standard, so the quantitative result also increases arithmetically by simply the difference in molecular weight. Therefore, in order to confirm only the improvement effect on reactivity according to acetylation, the measured content result was multiplied by the molecular weight ratio between the standard substances (179.17 / 221.21, approximately 81%), and the recalculated results so that the molecular weight of the acetyl group was canceled are also shown in Table 7 below. Meanwhile, in the case of the HCl molecule of the following chemical formula 1, since it is a salt bound to the glucosamine molecule, which is the target of quantification, it is ionized when dissolved and the corresponding molecular weight (36.46) is excluded from the standard solution concentration, so the molecule was not considered when comparing the structures between the two standard substances. Here, the N-acetyl D-Glucosamine standard solution was prepared by dissolving 500 mg of N-acetyl D-Glucosamine in 50 mL of triple-distilled water to make a stock of 10 mg / mL, and then diluting this 20-fold to prepare a standard solution with a concentration of 500 μg / mL and used.

[0083] [Chemical Formula 1]

[0084]

[0085] D-(+)-Glucosamine Hydrochloride (MW: 179.17)

[0086] [Chemical Formula 2]

[0087]

[0088] N-Acetyl D-Glucosamine (MW: 221.21)

[0089] Based on SerotypeLot No.8M HCl (%)1 hr4 hrRecalculated1 hr4 hr4A04042201C47453836≥ 40ATCC434635379NCTAA9145J41423334≥ 2812FCTAA124242353428≥ 25ATCC3232262619AA96042301F16191315≥ 1219FCTAA9740215201216≥ 12.5

[0090] As a result, as shown in Table 7 above, when comparing the results of Table 5 above with the recalculated values, the improvement due to the change in the standard material was minimal, and even with the ATCC sample standard, which is a commercial standard, it still did not stably exceed the hexosamine content standards presented in the EP and WHO guidelines.

[0091]

[0092] Experimental Example 2. SK Method and Modification Test

[0093] Despite various modifications, the existing EP method confirmed that even commercially available standards did not meet the hexosamine content standards presented in the EP and WHO guidelines for the serotypes under investigation. Therefore, a hexosamine content assay method (hereinafter referred to as the SK method) as described in 2.1 below was established to evaluate whether commercial standards met the hexosamine content standards for polysaccharide vaccines presented in the EP and WHO guidelines for the serotypes under investigation.

[0094] 2.1 Hexosamine content test method according to SK method (Example)

[0095] 2.1.1 Preparation of N-Acetyl D-Glucosamine standard solution

[0096] Weigh 125 mg of N-Acetyl D-Glucosamine in a 500 mL beaker, add 500 mL of triple-distilled water, and mix thoroughly using a magnetic stirrer to prepare a 250 μg / mL N-Acetyl D-Glucosamine standard solution. The 250 μg / mL N-Acetyl D-Glucosamine standard solution was placed in a glass tube and diluted to obtain the standard solution concentrations shown in Table 8 below, thereby preparing a standard solution and a blank.

[0097] ClassificationConcentration (μg / mL)250 μg / mL N-Acetyl D-Glucosamine Standard Solution (μL)Tri-distilled water (μL)Total Volume (μL)Blank00200200STD 12520180200STD 262.550150200STD 3125100100200STD 415012080200STD 517514060200STD 620016040200STD 722518020200

[0098] 2.1.2 Preparation of test solution

[0099] The test solution was appropriately diluted as shown in Table 9 below and only a final volume of 0.2 mL was placed in a glass tube to prepare it.

[0100] Dilution ratio sample (μL) Distilled water (μL) Total volume (μL) 1 / 10605406001 / 15405606001 / 2030570600

[0101] 2.1.3 Hydrolysis step

[0102] Hydrolysis was performed by adding a hydrolysis buffer to the blank, N-Acetyl D-Glucosamine standard solutions by concentration, and test solutions prepared in 2.1.1 and 2.1.2 above. Specifically, 0.1 mL of 12 M HCl solution was added to a glass tube containing 0.2 mL each of the blank, N-Acetyl D-Glucosamine standard solutions by concentration, and test solutions prepared in 2.1.1 and 2.1.2 above, and then vortexing. The glass tube was closed with its lid, reacted in a water bath set to 100 °C for 4 hours, and then sufficiently cooled at room temperature for 15 minutes.

[0103] Here, 12M HCl solution was used as 37% HCl stock solution.

[0104] 2.1.4 Hydrolyzate neutralization titration step

[0105] Each solution, which had become acidic due to hydrolysis, was neutralized by adding a base. Specifically, 0.4 mL of a 2 M Na2CO3 solution was added to each glass tube cooled to room temperature, and the resulting carbon dioxide gas was carefully vortexed. Next, 0.5 mL of an acetylacetone solution was added to each glass tube and vortexed. The glass tubes were capped and incubated in a water bath set to 100°C for 20 minutes, after which they were sufficiently cooled to room temperature for 15 minutes.

[0106] Here, the 2M Na2CO3 solution was prepared by weighing 53 g of Na2CO3 in a 250 mL bottle, adding 250 mL of distilled water, and stirring sufficiently using a magnetic stirrer. The acetylacetone solution was prepared by adding 0.4 mL of acetylacetone to 15 mL of 2M Na2CO3 solution, adding distilled water to adjust the final volume to 20 mL, and then vortexing to mix sufficiently.

[0107] 2.1.5 Color development stage

[0108] 1 mL of ethanol was added to each glass tube that had cooled to room temperature, vortexed sufficiently, and confirmed that a precipitate had formed. 0.5 mL of DMAB (dimethylaminobenzaldehyde) solution (Ehrlich's Reagent) was added to each glass tube, and vortexed carefully to remove the generated carbon dioxide gas. After allowing to stand at room temperature for 5 minutes, the absorbance was measured at a wavelength of 530 nm using a UV-Vis Spectrophotometer. A calibration curve was created using the measured absorbance values ​​of the standard solution, and the hexosamine content was quantified from the absorbance values ​​of the sample solution measured based on this.

[0109] Here, DMAB solution (Ehrlich's Reagent) was used, which was prepared by weighing 1 g of p-Dimethylaminobenzaldehyde, adding 15 mL of 95% ethanol, mixing thoroughly by vortexing, adding an additional 15 mL of 37% HCl, and mixing thoroughly by vortexing. A DMAB solution with a concentration of 0.223 M was used.

[0110] 2.2 Evaluation of hexosamine content analysis by SK method according to sample dilution factor

[0111] The SK method using the acetylated standard material of 2.1 above was used to analyze the same serotype as in Experimental Example 1. The results of analyzing the hexosamine content according to the sample dilution ratio using the SK method are shown in Table 10 below. Cases where the sample absorbance according to the dilution ratio was outside the standard solution absorbance range (STD Range Out) were marked with *.

[0112] Based on SerotypeLot No.2 289VCTAA924224*25262425≥ 13ATCC22222119*19*10ACTAA104217*19191920≥ 1212FCTAA124237*41383637≥ 25ATCC31*3232293014CTAA1441B29*35353636≥ 2015B210708T414*22*242427≥ 1519AA96042301F26*27272525*≥ 12ATCC2122222320*19FCTAA9740224*26282525≥ 12.5ATCC2122202120*

[0113] In order to confirm the results satisfying the standard solution absorbance range, experiments were conducted according to the dilution ratio, and as shown in Table 10 above, when the dilution ratio was outside the range of 10X to 15X, there were some serotypes for which the standard absorbance could not be measured, and thus, there were cases in which this assay method was not performed evenly for all serotypes. Therefore, it was confirmed that the dilution ratio range that can be commonly used in the hexosamine test method is 10X to 15X.

[0114] The results of three replicates with the confirmed dilution factor are shown in Table 11 below. When hexosamine content was analyzed using acetylated standards, the SK method satisfied the hexosamine content standards presented in the EP and WHO guidelines for both the commercially available ATCC standard sample and the purified pneumococcal polysaccharide stock sample. Furthermore, as shown in Table 7, recalculated values ​​that offset the effect of increased molecular weight also satisfied the standards for all samples.

[0115] Based on SerotypeLot No.1st2nd3rdAverage%CV (%)4A04042201C585357565≥ 40ATCC57474951105E19P344413838394≥ 209NCTAA9145J484747471≥ 289VCTAA9242262625262≥ 1310ACTAA1042192020203≥ 1212FCTAA1242383436366≥ 25ATCC322732301014CTAA1441B353436353≥ 2015B210708T4242526254≥ 1519AA96042301F272327269≥ 1219FCTAA97402282529278≥ 12.5

[0116] 2.3 Evaluation of acetylation status of standard materials in the SK Method

[0117] In order to confirm whether there was a difference according to the standard material in the SK method, an additional experiment was conducted using D-(+)-Glucosamine Hydrochloride, which is the standard material of the existing EP method. In order to set the reaction conditions before repeating the test, the conditions of 1 hour and 4 hours of hydrolysis with 12 M HCl, 5 minutes and 90 minutes of standing after adding DMAB solution were compared and analyzed, and the results are shown in Table 12 below. Here, the D-(+)-Glucosamine Hydrochloride standard solution was prepared by dissolving 150 mg of D-(+)-Glucosamine Hydrochloride in 500 mL of triple-distilled water, adding 250 μg / mL D-(+)-Glucosamine standard solution to a standard solution concentration as shown in Table 8, and diluting the standard solution and blank in a glass tube.

[0118] SerotypeLot No.Hydrolysis 1 hrHydrolysis 4 hr basis (%)5 min90 min5 min90 min4A04042201C51525454≥ 409NCTAA9145J38394141≥ 2819AA96042301F13142121≥ 1219FCTAA9740215162424≥ 12.5

[0119] The results of the experiment repeated three times for 10 serotypes under the conditions of 4 hours of hydrolysis, which was the method that measured the highest hexosamine content in Table 12 above, and 5 minutes of standing after adding DMAB solution are shown in Table 13 below.

[0120] Based on SerotypeLot No.1st2nd3rdAverage%CV (%)4A04042201C544551489≥ 40ATCC4247454565E19P344383736373≥ 209NCTAA9145J414039403≥ 289VCTAA9242222121213≥ 1310ACTAA1042171715167≥ 1212FCTAA1242333034327≥ 25ATCC313126291014CTAA1441B292928292≥ 2015B210708T4181919193≥ 1519AA96042301F212220215≥ 1219FCTAA97402242323233≥ 12.5

[0121] As a result, as shown in Table 13 above, when the test method itself used for measuring hexosamine content was applied as the SK method, it was confirmed that the hexosamine content standards presented in the EP and WHO guidelines were stably exceeded in all serotypes, including the ATCC sample, which is a commercial standard, without any difference according to the type of standard material.

Claims

1. (a) a step of processing a series of glucosamine standard solutions of a predetermined concentration according to a predetermined method, measuring absorbance, and creating a standard curve; and (b) a method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, comprising the steps of: (a) processing a polysaccharide sample solution according to a predetermined method, measuring absorbance, and calculating the hexosamine content of the polysaccharide sample solution according to the standard curve obtained in step (a); The above predetermined method is, A step of hydrolyzing by adding hydrochloric acid solution to each test tube containing the above standard solution or polysaccharide sample solution; A step of neutralizing by adding sodium carbonate solution; Step of reacting by adding acetylacetone solution; Step of precipitating by adding ethanol; A step of dissolving the precipitate by adding a DMAB (dimethylaminobenzaldehyde) solution, which is a coloring agent, for derivatization; and A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, comprising the step of measuring absorbance.

2. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the glucosamine standard solution is an N-Acetyl D-Glucosamine solution or a D-(+)-Glucosamine Hydrochloride solution.

3. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the series of concentrations falls within a range of 0-250 μg / mL, and the number of the series of concentrations is at least 8.

4. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the concentration of the hydrochloric acid solution is 12.0±0.5 M.

5. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the hydrolyzing step is performed for 4 to 6 hours.

6. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, wherein the volume ratio of the hydrochloric acid solution to the standard solution or polysaccharide sample solution in claim 1 is 2:0.5 to 1.

5.

7. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the concentration of the DMAB solution is 0.2±0.1 M.

8. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution, wherein the step of dissolving the precipitate by adding the DMAB solution according to claim 1 comprises adding the DMAB solution, vortexing the solution, and then allowing it to stand for 5 to 15 minutes.

9. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the test tube is made of glass.

10. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the polysaccharide sample solution is a solution in which the sample is diluted 10 to 15 times.

11. A method for determining the hexosamine content of a pneumococcal polysaccharide sample solution according to claim 1, wherein the serotype of the pneumococcus is any one selected from the group consisting of 4, 5, 9N, 9V, 10A, 12F, 14, 15B, 19A, and 19F.

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