Methods for carbohydrate quantification

Abstract

The present invention provides a colorimetric method for quantifying carbohydrates in a given aqueous sample. The method uses 2-phenoxyethanol as a novel reagent for quantifying carbohydrates in a given sample. The present invention provides a rapid, sensitive, simple, and direct method for quantifying carbohydrates.

Description

Technical Field

[0001] The present invention relates to a method for estimating carbohydrates. More particularly, the present invention relates to a method for quantifying carbohydrates using aromatic alcohols when the carbohydrates are present at low concentrations in pharmaceuticals, biopharmaceuticals, and biologicals. Background Art

[0002] Quantification of carbohydrate or polysaccharide content is a fundamental analytical procedure in the development of foods and beverages, nutritional supplements, agricultural products, pharmaceutical products, and vaccines. Polysaccharides are typically quantified by biochemical analysis using anthrone reagents and glucose as standards. Other biochemical methods using reagents such as orcinol or phenol-sulfuric acid with colorimetric reactions are also primarily used for polysaccharide estimation. To improve the sensitivity of colorimetric methods, several modifications to these methods (including the concentrations of anthrone reagent and H2SO4, heating time, and temperature) have been previously reported. These modifications aim to enhance the color development of the anthrone-furfural complex, which improves the optical density signal and thus increases the quantitative sensitivity of the test. On the other hand, these methods for analyzing polysaccharides containing mixtures of uronic acids (glucuronic acid and galacturonic acid) and hexosamines (fucosamine, glucosamine, galactosamine, mannosamine, and pneumosamine) exhibit low color development. These methods do not produce optimal color complexes and have low sensitivity, making them less suitable for evaluating these carbohydrate / polysaccharide species.

[0003] During carbohydrate quantification in the presence of sulfuric acid, hexoses and pentoses are hydrolyzed and converted to 5-hydroxymethylfurfural and furfural, respectively. These molecules, resulting from acid hydrolysis, then react with anthrone or phenol to form a color complex, the optical density (OD) of which is then measured at a specific wavelength. The absorption maximum of the anthrone-furfural complex is at 625 nm, while the absorption maximum of the phenol-furfural complex is at 490 nm. The absorbance intensity in both methods varies depending on the composition of the polysaccharide, as different polysaccharides contain different sugars, such as hexoses, pentoses, uronic acid, hexosamine, pentosamine, glycolides, glycoproteins, and nucleic acids, in varying proportions.

[0004] In conjugate vaccines, polysaccharides are bound to carrier proteins, and controlling the amount of each polysaccharide within the prescribed dose is crucial to maintaining vaccine quality and efficacy. Accurately quantifying polysaccharides at the microgram level is crucial for vaccine development. Polysaccharides are complex mixtures of hexoses, pentoses, uronic acids, and hexosamines.

[0005] Various biochemical methods have been reported to quantify the total polysaccharide content of pneumococcal serotypes or their components (methylpentoses, uronic acids, hexosamines, O-acetyl groups, phosphorus, and nitrogen). Most of these use acid hydrolysis to release monosaccharides, which are quantified using high-performance anion exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD), or acid hydrolysis combined with derivatization followed by quantification using gas chromatography coupled to a mass selective detector (GC-MSD).

[0006] Another method reported for the quantification of hexosamine-containing pneumococcal polysaccharides is based on acid hydrolysis, re-N-acetylation, and then labeling of the hexosamine with 2-aminobenzamide by reductive amination, followed by reversed-phase HPLC using a fluorescence detector. Although these methods are sensitive and accurate, they are time-consuming and laborious.

[0007] Indian Patent Application No. 5856 / DELNP / 2009 discloses the quantification of polysaccharides using sulfuric acid and anthrone reagents or sulfuric acid and tetraborate reagents using an automated colorimetric test.

[0008] EP 2290366 A1 discloses the analysis of saccharide vaccines without disturbing the monosaccharides and any other saccharide material from the composition, including a process for quantifying monosaccharides (sialic acid, galactose and glucose) by acid hydrolysis of Neisseria meningitidis serogroups C, W135 and Y using trifluoroacetic acid (TFA).

[0009] Roman Dreywood (Qualitative Testing of Carbohydrate Materials: August 1946), discloses the qualitative analysis of carbohydrate materials using a solution of anthrone in concentrated sulfuric acid, which gives the carbohydrate material a permanent green color.

[0010] Philippe et al. (Vaccine 20 (2002) 2474-2484) disclosed the quantitative assay of pneumococcal polysaccharides and conjugates using HPAEC-PAD, wherein pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TEA), methanolysis followed by TFA hydrolysis, and hydrofluoric acid followed by TFA hydrolysis.

[0011] Vincent et al. (Anal. Chem. 2010, 82, 1786-1792), disclose automation of anthrone assay for determining carbohydrate concentrations, wherein polysaccharide samples and standards are heated in a concentrated mixture of anthrone in sulfuric acid at an absorbance of 625 nm.

[0012] Verdnica et al. (Analytical Biochemistry 421 (2012) 250-255) disclosed the use of phenol-sulfuric acid, HPSEC (10 mM Na2HP04, 0.15 M NaCl, pH 7.5), competitive ELISA (o-phenylenediamine dihydrochloride in phosphate-citrate buffer, pH 5.0, and 0.05% hydrogen peroxide, and the reaction was stopped with 4.5 M H2SO4, absorbance of 492 nm) and sandwich ELISA methods to quantify the capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples, among which sandwich ELISA was found to be the most reproducible and sensitive method and the least susceptible to interference.

[0013] Earl Zablackis et al. (Vaccine Analysis: Strategies, Principles, and Control pp 271-299, 2015) disclose bacterial polysaccharide vaccines and their analytical prospects, wherein bacterial polysaccharides are quantified by a colorimetric method in which the polysaccharide is heated with a sulfuric acid / boric acid solution to release monosaccharides (uronic acids), then cooled, a carbazole / ethanol solution is added, and the absorbance is read at 530 nm. The polysaccharide (hexosamine) is digested with HCl, cooled, then reacted with acetylacetone / sodium carbonate, then with Ehrlich's reagent, and the absorbance is read at 530 nm.

[0014] The above-mentioned prior art discloses various methods for quantifying polysaccharides. However, these methods have various drawbacks, and therefore, there is a need to develop new methods to enhance color development and thereby increase sensitivity. The enhanced sensitivity can provide appropriate carbohydrate estimation when carbohydrates are present at low concentrations in any sample, including but not limited to pharmaceuticals, biopharmaceuticals, biological products, cosmetics, environmental matrices, foods, forensic samples, industrial chemicals, and nutritional supplements. Summary of the Invention

[0015] Purpose of the Invention

[0016] The main purpose of the present invention is to develop a simple, direct and more sensitive polysaccharide quantification method.

[0017] Another object of the present invention is to develop a simple and direct method for the quantification of polysaccharides when they are present at low concentrations in samples such as pharmaceuticals, biopharmaceuticals, biological products, cosmetics, environmental matrices, foods, forensic samples, industrial chemicals and nutritional products. SUMMARY OF THE INVENTION

[0019] In one aspect, the present invention relates to a method for quantifying carbohydrates (saccharides) using aromatic alcohols, such as 2-phenoxyethanol (2-PE).

[0020] The present invention provides a method for quantifying carbohydrates in an aqueous sample based on colorimetry, the method comprising the following steps:

[0021] a. mixing sulfuric acid and 2-phenoxyethanol with an aqueous sample containing a carbohydrate to obtain a reaction mixture;

[0022] b. incubating the reaction mixture to form a colored complex; and

[0023] c. Measure the absorbance of the colored complex to quantify the amount of carbohydrate present in the aqueous sample.

[0024] In one aspect, the present invention relates to methods for estimating carbohydrates, such as monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines and derivatives thereof, and combinations thereof.

[0025] In yet another aspect, the present invention also provides a kit for quantifying carbohydrates in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The absorbance of monosaccharides reacting with 2-PE (500 nm), anthrone (625 nm), and phenol (490 nm) reagents is shown;

[0027] Figure 2 shows the absorbance of polysaccharides (pneumococcal polysaccharide, pullulan 800, and starch) reacted with 2-PE (500 nm), anthrone (625 nm), and phenol (490 nm) reagents; and

[0028] Figure 3 Shown are the absorbance of pneumococcal polysaccharides expressed as % recovery in the presence of known impurities (2 and 5% w / v) in the purified polysaccharide. DETAILED DESCRIPTION

[0029] The present invention relates to a method for estimating carbohydrates using aromatic alcohols (e.g., 2-phenoxyethanol). More specifically, the present invention relates to a method for quantifying carbohydrates using 2-phenoxyethanol when carbohydrates are present at low concentrations in pharmaceuticals, biopharmaceuticals, such as vaccines, protein preparations, peptide preparations, and other biological products.

[0030] In one embodiment, the present invention relates to the estimation of carbohydrates, such as monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines and derivatives thereof, and combinations thereof, in aqueous samples.

[0031] In one embodiment of the present invention, monosaccharides include, but are not limited to, glucose, galactose, rhamnose, mannose, arabinose, xylose, fructose, ribose, and derivatives thereof, and combinations thereof.

[0032] In one embodiment of the present invention, disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, and derivatives thereof, and combinations thereof.

[0033] In one embodiment of the present invention, uronic acid includes but is not limited to glucuronic acid, galacturonic acid, mannuronic acid, and derivatives thereof, and combinations thereof.

[0034] In one embodiment of the present invention, hexosamines include, but are not limited to, fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine, N-acetyl L-fucosamine and N-acetyl L-pneumosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, and derivatives thereof, and combinations thereof.

[0035] In one embodiment, the present invention relates to a method for quantifying polysaccharides (eg, pneumococcal polysaccharides, meningococcal polysaccharides, VI polysaccharides, O2 polysaccharides) and other carbohydrates (eg, cellulose, starch, chitin, dextran, pullulan, etc.).

[0036] In one embodiment of the present invention, pneumococcal polysaccharide is prepared as described in PCT Publication No. WO2016 / 174683A1 and is prepared by 1 H NMR analysis revealed its structure. The purified capsular polysaccharide met the commercially available specifications for monosaccharide composition, including glucose, galactose, and rhamnose; uronic acids, such as glucuronic acid and galacturonic acid; hexosamines, such as N-acetyl L-fucosamine, N-acetyl L-pneumosamine, N-acetylglucosamine, and N-acetyl-galactosamine; and O-acetyl, phosphorus, and nitrogen content.

[0037] In their ongoing efforts to develop highly sensitive methods for polysaccharide quantification, the present inventors have developed a method for quantifying carbohydrates using aromatic alcohols, such as 2-PE (2-phenoxyethanol). The developed method offers higher specificity and sensitivity than other methods, such as those using anthrone or phenol, and facilitates the quantification of a wide range of carbohydrates, sugars, and / or polysaccharides in a wide range of samples.

[0038] 2-PE reacts with furfural or 5-hydroxymethylfurfural formed by acid hydrolysis of polysaccharides, and has an absorbance maximum at 500nm. In addition, the color development in this method is not affected by polysaccharides containing uronic acid. In addition, the present invention can be used to quantify polysaccharide-protein conjugates and has increased sensitivity compared to anthrone reagents. The reactivity of polysaccharides isolated from bacteria (Streptococcus Pneumoniae), sugars and their respective acids with 2-PE is enhanced, thereby improving the sensitivity of the method. This method is simple and direct and can be used as a conventional technique for the quantification of any type of polysaccharide. In general, the higher sensitivity of the method of the present invention will help to quantify a wide range of carbohydrates, sugars and / or polysaccharides with higher sensitivity than other reporting methods.

[0039] In one embodiment, the present invention provides a method for quantifying carbohydrates in an aqueous sample, comprising the following steps:

[0040] a. mixing sulfuric acid and 2-phenoxyethanol with the aqueous sample to obtain a reaction mixture;

[0041] b. incubating the reaction mixture to form a colored complex; and

[0042] c. Measure the absorbance of the colored complex to quantify the amount of carbohydrate present in the aqueous sample.

[0043] In another embodiment, the amount of carbohydrate in the sample is proportional to the measured absorbance of the colored complex.

[0044] In a more preferred embodiment of the present invention, the carbohydrate sample is treated with concentrated sulfuric acid to obtain a reaction mixture.

[0045] In one embodiment of the present invention, the volume of sulfuric acid added to the carbohydrate-containing aqueous sample is about 1 to 3 times the volume of the carbohydrate-containing aqueous sample.

[0046] As used herein, the term "about" contemplates a numerical range of ±25% of the magnitude of a given number. In certain embodiments, the term "about" contemplates a numerical range of ±20%, ±15%, ±10%, or ±5% of the magnitude of a given number.

[0047] In another embodiment of the present invention, the volume of sulfuric acid added to the aqueous carbohydrate-containing sample is twice the volume of the aqueous carbohydrate-containing sample.

[0048] In one embodiment of the present invention, the concentration of 2-phenoxyethanol ranges from about 0.1% v / v to 2.5% v / v relative to the reaction mixture.

[0049] In one embodiment of the invention, the reaction mixture is incubated at a temperature in the range of about 80°C to 110°C.

[0050] In another embodiment, the reaction mixture is incubated at a temperature of about 90°C.

[0051] In one embodiment of the invention, the reaction mixture is incubated for a period of about 1 minute to 10 minutes.

[0052] In another embodiment, the reaction mixture is incubated for a period of about 5 minutes.

[0053] In one embodiment of the invention, the absorbance of the colored complex is measured in the wavelength range of about 490 nm to 510 nm.

[0054] In another embodiment of the present invention, the absorbance of the colored complex is measured at a wavelength of 500 nm.

[0055] In one embodiment of the present invention, the aqueous sample to be quantified is a pure carbohydrate sample.

[0056] In another embodiment of the present invention, the quantified aqueous sample is an impure carbohydrate sample. Impurities include, but are not limited to, nucleic acids, proteins, lipids, residual reagents, excipients, or a combination thereof.

[0057] The method of the present invention was found to be very specific for carbohydrates in the presence of different impurities and formulation excipients, demonstrating the specificity of the assay.

[0058] As used herein, the term "limit of detection" includes the lowest concentration at which a sample can be considered to contain molecules of the substance of interest.

[0059] In one embodiment of the invention, the limit of detection (LOD) is less than about 4 μg / mL.

[0060] As used herein, "limit of quantitation" refers to the point at which a measurement has quantitative significance.

[0061] In one embodiment of the invention, the limit of quantitation (LOQ) is at least 6 μg / mL.

[0062] In another embodiment of the invention, the limit of quantitation (LOQ) is at least 8 μg / mL.

[0063] In one embodiment, the methods of the present invention are used to quantify carbohydrates in vaccine or biopharmaceutical samples.

[0064] In one embodiment of the invention, the carbohydrate content of monoconjugate and multivalent pneumococcal conjugate vaccine (PCV) drug products is quantified.

[0065] In another embodiment of the invention, the total carbohydrate content of monoconjugate and multivalent PCV drug products is quantified, wherein pneumococcal serotypes such as 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39, and 45 are pre-diluted to 4-100 μg / mL prior to analysis.

[0066] The multivalent pneumococcal conjugate vaccine can be a 10-valent, 12-valent, 13-valent, 14-valent, 15-valent, 17-valent, 18-valent, 19-valent, 20-valent, 22-valent, 23-valent, 24-valent, 25-valent, 27-valent, 28-valent, 29-valent, or 30-valent pneumococcal vaccine composition.

[0067] In yet another embodiment, the method of the invention is used to quantify polysaccharides in monovalent and multivalent vaccine products for interference and spike recovery assessment.

[0068] In another embodiment, a polyvalent pneumococcal conjugate vaccine adsorbed with aluminum phosphate (equivalent to approximately 70 μg / mL polysaccharide) was analyzed for total sugar content using the method of the present invention.

[0069] In one embodiment of the invention, the carbohydrate content of a multivalent meningococcal conjugate drug product containing polysaccharides from serogroups A, C, W135, X and Y of Neisseria meningitidis is quantified.

[0070] In one embodiment of the present invention, the carbohydrate content of monovalent and bivalent typhoid conjugate drug products containing Vi polysaccharide and O2 polysaccharide from Salmonella typhi and Salmonella paratyphi is quantified.

[0071] In one embodiment, the present invention also includes a kit for quantifying carbohydrates in an aqueous sample.

[0072] In one embodiment, the kit for quantifying carbohydrates comprises sulfuric acid, 2-phenoxyethanol, or a combination thereof.

[0073] In another embodiment, the present invention provides the use of 2-phenoxyethanol for the quantification of carbohydrates in a sample.

[0074] In another embodiment, the present invention provides use of 2-phenoxyethanol for quantifying carbohydrates in a sample, wherein the carbohydrates are selected from the group consisting of monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, derivatives thereof, and combinations thereof.

[0075] The following examples are provided to illustrate the present invention and are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0076] Example

[0077] Example 1: Estimation of monosaccharides and polysaccharides by the following three different methods.

[0078] a.2-PE-sulfuric acid determination method.

[0079] Sugars such as glucose, galactose, rhamnose, ribitol, glycerol, lactose, mannose, sucrose, glucuronic acid, galacturonic acid, N-acetyl L-fucosamine and N-acetyl L-pneumosamine, N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine, pullulan 800, and pneumococcal polysaccharides from serotypes 1, 3, 5, 6B, 9V, 14, 19F, 22F, 23F were diluted to 20 μg (dry weight basis) in 250 μL of MilliQ water and hydrolyzed in the presence of concentrated H2SO4 to form monosaccharides or their derivatives, which were dehydrated in hot acidic medium to form hydroxymethylfurfural. These hydroxymethylfurfural structures then reacted with phenolic reagents to form orange-yellow complexes that produced an absorbance maximum at 500 nm.

[0080] The reaction of 2-phenoxyethanol with sugar in the presence of H2SO4 (based on the Molisch test reaction) is shown in the following scheme:

[0081]

[0082] 250 μL (80 μg / mL) of diluted monosaccharides and polysaccharides were dispensed into clean, dry glass tubes in triplicate, and 250 μL of reagent / MilliQ water were taken in triplicate for blank correction. 10 μL of 98% 2-PE and 500 μL of H2SO4 were added to all tubes and vortexed gently. The tubes were incubated in a 90°C water bath for 5 minutes. Subsequently, the tubes were cooled to room temperature, 250 μL were transferred to a microplate, and the absorbance was measured at 500 nm using a microplate reader.

[0083] b. Phenol-sulfuric acid assay.

[0084] All monosaccharides and polysaccharides were diluted to 20 μg in 165 μL of MilliQ water based on dry weight and hydrolyzed in the presence of 500 μL of concentrated H2SO4 to form monosaccharides or their derivatives, which were dehydrated in hot acidic medium to form hydroxymethylfurfural. These hydroxymethylfurfural structures then reacted with phenolic reagents to form orange-yellow complexes that produced an absorbance maximum at 490 nm.

[0085] 0-20 μg of each standard was placed in a clean glass tube, 100 μL of 5% phenol reagent was added, and then incubated at 90 °C in an open tube for 5 minutes, 250 μL was transferred to a microplate, and the absorbance was read at 490 nm using a microplate reader.

[0086] c. Anthrone-sulfuric acid determination method.

[0087] All monosaccharides and polysaccharides were diluted to 20 μg in 250 μL based on dry weight and hydrolyzed in the presence of H2SO4 to form monosaccharides or their derivatives, which were dehydrated in hot acidic media to form hydroxymethylfurfural. These hydroxymethylfurfural structures then reacted with an anthrone reagent to form a green complex that produced an absorbance maximum at 625 nm. 250 μL (80 μg / mL) was dispensed in triplicate into clean glass tubes, 500 μL of anthrone reagent was added, and then incubated at 90°C for 5 minutes. The absorbance was measured at 625 nm using a microplate reader.

[0088] In all three methods described above, the final reaction volume was -760 μL and 250 μL was taken for absorbance measurement in a microplate.

[0089] Table 1: Carbohydrate Estimation Methods

[0090] Sample number method Reagents λmax (absorbance) 1. 2-PE-sulfuric acid determination method Aromatic phenoxyethanol 500nm 2. Phenol-sulfuric acid assay Aromatic phenol components 490nm 3. Anthrone-sulfuric acid determination method Three-ring aromatic components 625nm

[0091] result:

[0092] Absorbance of monosaccharides

[0093] The absorbance of monosaccharide when reacting with 2-PE (500 nm), anthrone (625 nm) and phenol (490 nm) reagents is as follows: Figure 1 shown.

[0094] In the presence of H2SO4, all monosaccharides hydrolyze to form hydroxymethylfurfural. These hydroxymethylfurfural structures, when reacted with anthrone, form a green complex with an absorbance maximum at 625 nm. Similarly, when the hydroxymethylfurfural structures react with 2-PE, an orange-yellow complex is produced with an absorbance maximum at 500 nm. The colored complexes formed by the reaction of glucuronic acid and galacturonic acid with anthrone have lower absorbances (0.05 and 0.10 OD), while the absorbance OD (optical density) units using 2-PE are 0.69 and 0.85, respectively. Hexoses such as glucose, galactose, and mannose have ODs of 1.55, 1.07, and 2.07, respectively, with 2-PE, and 0.93, 0.45, and 0.43, respectively, with anthrone. Similarly, rhamnose, a methyl pentose, has an OD of 1.02 with 2-PE and 0.62 with anthrone. The OD results of N-acetylated amines (FucNAc; GlucNAc; GalNAc; ManNAc; PneuNAc) were slightly different from those of anthrone or 2-PE reagents ( Figure 1 ).

[0095] Absorbance of polysaccharides

[0096] The absorbance of polysaccharides (pneumococcal polysaccharide, pullulan 800 and starch) reacting with 2-PE (500 nm), anthrone (625 nm) and phenol (490 nm) reagents is as follows: Figure 2 shown.

[0097] The sulfuric acid used for polysaccharide hydrolysis causes each sugar to form hydroxymethylfurfural. When these hydroxymethylfurfurals react with anthrone, a green colored complex is produced with an absorbance maximum at 625 nm. Similarly, when hydroxymethylfurfural reacts with 2-PE, an orange-yellow colored complex is produced with an absorbance maximum at 500 nm. When sulfuric acid-hydrolyzed pneumococcal polysaccharides react with 2-PE, the colored complex formed is higher than the colored complex produced using anthrone reagent ( Figure 2 ), thereby increasing the quantitative sensitivity and improving the lower limit of detection (LOD) of sugars. Similar sensitivity and reactivity were also observed for other polysaccharides such as pullulan 800 using 2-PE ( Figure 2 ).

[0098] Example 2: Quantification of polysaccharides in monoconjugate and multivalent adsorbed vaccines.

[0099] 2-PE analysis was performed on various known concentrations of pneumococcal serotype 6B and 7F polyprotein conjugates (4-100 μg / mL each) and multivalent conjugate vaccine samples. The percent recoveries of 6B and 7F polysaccharides or conjugates ranged from 90-110%, exceeding their LOQ (limit of quantitation) levels (Table 2).

[0100] Similarly, at different Al +3 The percentage recoveries of total polysaccharide in the pneumococcal polyvalent adsorbent conjugate vaccine were 97, 101, and 99 at the following concentrations (0.25, 0.5, and 1.0 mg / mL of AlPO4 gel), respectively (Table 2), indicating that there was no interference from aluminum phosphate in the drug product.

[0101] Impurity interference and verification

[0102] The most common impurities in purified polysaccharides are nucleic acids, proteins, and residual reagents. Figure 3 ) Reactivity was evaluated at 2 and 5% w / v levels for the interference of these impurities on the total polysaccharide concentration. At a 2% impurity level (the maximum allowable limit according to regulatory guidelines), there was no effect on reactivity, but in the presence of 5% impurity, reactivity decreased by approximately 8%. The assay was validated using polysaccharides and conjugates for specificity, accuracy, precision, spike recovery, limit of detection (LOD), and limit of quantification (LOQ).

[0103] This method was further evaluated for polysaccharide quantification in vaccine conjugates. Polysaccharide concentrations in 6B and 7F (lung-CRM conjugates) were estimated across the standard range to examine the test LOD and LOQ (Table 2). Based on the percent recovery of the polysaccharide or conjugate, the test LOD was ≤4.0 μg / mL, and the test LOQ was ≥8 μg / mL (Table 2).

[0104] The sources of the biological materials used in the present invention are as follows:

[0105] 1. Streptococcus pneumoniae serotype 6B was obtained from the Centers for Disease Control and Prevention (CDC) in the United States.

[0106] 2. Streptococcus pneumoniae serotype 7F was obtained from the Centers for Disease Control and Prevention (CDC)

[0107] In addition, a lung-CRM conjugate was prepared using CRM 197 protein isolated from Corynebacterium obtained from ATCC.

[0108] Table 2: Total polysaccharide content in 6B, 7F monoconjugates and multivalent pneumococcal conjugate vaccines by 2-PE method

[0109]

[0110]

[0111] Advantages of the present invention

[0112] 1. The method is simple and direct.

[0113] 2. Compared with other known carbohydrate quantification methods, this method has higher specificity and sensitivity.

[0114] 3. This method can be used as a routine technique to quantify a wide range of carbohydrates, sugars and / or polysaccharides in a wide range of samples.

[0115] 4. The color development in the developed method is not affected by polysaccharides containing uronic acid.

[0116] 5. The method of the present invention can accurately determine the amount of carbohydrates in the presence of different impurities and formulation excipients, demonstrating the specificity of the assay.

Claims

1. A method for quantifying carbohydrates in an aqueous sample, the method comprising the following steps: a. mixing sulfuric acid and 2-phenoxyethanol with an aqueous sample containing a carbohydrate to obtain a reaction mixture; b. incubating the reaction mixture to form a colored complex; and c. Measuring the absorbance of the colored complex to quantify the amount of carbohydrate present in the aqueous sample.

2. The method of claim 1, wherein the absorbance of the colored complex is measured at a wavelength in the range of 490 nm to 510 nm.

3. The method of claim 1, wherein the amount of carbohydrate in the aqueous sample is proportional to the absorbance measured by the colored complex.

4. The method of claim 1, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, uronic acid, hexosamine, derivatives thereof, and combinations thereof.

5. The method of claim 4, wherein the polysaccharide is selected from the group consisting of pneumococcal polysaccharide, meningococcal polysaccharide, VI polysaccharide and O2 polysaccharide.

6. The method of claim 1, wherein the volume of the sulfuric acid ranges from 1 to 3 times the volume of the aqueous sample.

7. The method of claim 1, wherein the concentration of 2-phenoxyethanol in the reaction mixture ranges from 0.1% v / v to 2.5% v / v.

8. The method of claim 1, wherein the reaction mixture is incubated at a temperature in the range of 80°C to 110°C to develop the colored complex.

9. The method of claim 1, wherein the reaction mixture is incubated for a period of time ranging from 1 minute to 10 minutes to develop the colored complex.

10. The method of any one of claims 1 to 9, wherein the aqueous sample comprises monovalent or multivalent saccharide conjugates.

11. The method according to claim 10, wherein: The glycoconjugate comprises glycoconjugates derived from one or more Streptococcus pneumoniae ( Streptococcus pneumoniae ) serotype, wherein the one or more S. pneumoniae serotypes are selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and 45.

12. A kit for quantifying carbohydrates in an aqueous sample, comprising sulfuric acid and 2-phenoxyethanol.

13. Use of 2-phenoxyethanol in a method for quantifying carbohydrates in an aqueous sample, wherein the method comprises the following steps: a. mixing sulfuric acid and 2-phenoxyethanol with an aqueous sample containing a carbohydrate to obtain a reaction mixture; b. incubating the reaction mixture to form a colored complex; and c. Measuring the absorbance of the colored complex to quantify the amount of carbohydrate present in the aqueous sample.

14. The use according to claim 13, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, uronic acid, hexosamine, derivatives thereof and combinations thereof.

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