METHODS FOR QUANTIFICATION OF CARBOHYDRATES

Abstract

The present invention provides a colorimetric based method for quantifying carbohydrates in a given aqueous sample. The method provided by the invention uses 2-Phenoxyethanol as a novel reagent for quantifying carbohydrates in a given sample. The present invention is a rapid, sensitive, simple and direct method for carbohydrate quantification.

Claims

1. A method for quantification of carbohydrates in an aqueous sample, said method comprising the steps of: a. admixing sulphuric acid and 2-Phenoxyethanol with an aqueous sample containing carbohydrates to obtain a reaction mixture; b. incubating the reaction mixture to form a coloured complex; and c. measuring the absorbance of the coloured complex to quantify the amount of carbohydrate present in the aqueous sample.

2. The method as claimed in claim 1, wherein the absorbance of the coloured complex is measured at a wavelength ranging from about 490 nm to 510 nm.

3. The method as claimed in claim 1, wherein the quantity of carbohydrate in the sample is proportional to the measured absorbance of the coloured complex.

4. The method as claimed in claim 1, wherein the said carbohydrate is selected from a group comprising monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, their derivatives and combinations thereof

5. The method as claimed in claim 4, wherein the polysaccharide is selected from a group comprising Pneumococcal polysaccharides, Meningococcal polysaccharides, VI polysaccharide, O2 polysaccharide and the like.

6. The method as claimed in claim 1, wherein the volume of the sulphuric acid ranges from about 1 to about 3 times the volume of the aqueous sample.

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

8. The method as claimed in claim 1, wherein the reaction mixture is incubated at a temperature ranging from about 80° C. to 110° C. for the development of coloured complex.

9. The method as claimed in claim 1, wherein the reaction mixture is incubated for a duration ranging from about 1 minute to about 10 minutes for the development of the coloured complex.

10. A kit for quantifying carbohydrates in an aqueous sample essentially comprising sulphuric acid, 2-Phenoxyethanol and a combination thereof

11. Use of 2-Phenoxyethanol for quantification of carbohydrates in a sample.

12. Use of 2-Phenoxyethanol according to claim 11, wherein said carbohydrate is selected from a group comprising monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, their derivatives and combinations thereof

13. 2-Phenoxyethanol for use in quantification of carbohydrates in a sample.

14. 2-Phenoxyethanol for use in the quantification of carbohydrates in a sample according to claim 12, wherein said carbohydrate is selected from a group comprising monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, their derivatives and combinations thereof

Description

BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1 illustrates absorbance of Monosaccharides upon reacting with 2-PE (500 nm), anthrone (625 nm) and Phenol (490 nm) reagents;

[0025] FIG. 2 illustrates Absorbance of polysaccharides (Pneumococcal polysaccharides, Pullulan 800 and Starch) upon reacting with 2-PE (500 nm), anthrone (625 nm) and Phenol (490 nm) reagents; and

[0026] FIG. 3 illustrates absorbance of Pneumococcal polysaccharides as % recovery in presence of known impurities (2 and 5% w/v) present in purified polysaccharides.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to methods for estimation of carbohydrates using an aromatic ethanol such as 2-phenoxy-ethanol. More specifically, the present invention relates to methods for quantification of carbohydrates using 2-phenoxy-ethanol when carbohydrates are present in low concentration in pharmaceuticals, biopharmaceuticals like vaccines, protein formulations, peptide formulations and other biologicals.

[0028] In an embodiment , the present invention relates to the estimation of carbohydrates such as monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines and their derivatives and combination thereof in an aqueous sample.

[0029] In an embodiment of the present invention, monosaccharide includes, but not limited to, glucose, galactose, rhamnose, mannose, arabinose, xylose, fructose, ribose and their derivatives and combination thereof.

[0030] In an embodiment of the invention, disaccharide includes, but not limited to, sucrose, lactose, maltose, trehalose, cellobiose and their derivatives and combination thereof.

[0031] In an embodiment of the invention, uronic acid includes, but not limited to glucuronic acid, galacturonic acid, mannuronic acid and their derivatives and combination thereof.

[0032] In an embodiment of the invention, hexosamine includes, but not limited to, Fucosamine, Glucosamine, Galactosamine, Mannosamine, Pneumosamine, N-Acetyl L-fucosamine and N-Acetyl L-pneumosamine, N-Acetyl Glucosamine, N-Acetyl-Galactosamine N-Acetyl Mannosamine and their derivatives and combination thereof.

[0033] In one embodiment, the present invention relates to a method for quantification of polysaccharides such as Pneumococcal polysaccharides, Meningococcal polysaccharides, VI polysaccharide, O2 polysaccharide and other carbohydrates such as Cellulose, Starch, Chitin, Dextran and Pullulan and the like.

[0034] In an embodiment of the present invention, pneumococcal polysaccharides are prepared as disclosed in PCT Publication Number WO2016/174683 A1 and the structure thereof is deduced by .sup.1H NMR analysis. The purified capsular polysaccharides are found to be in compliance with commercially available specified composition of monosaccharides such as Glucose, Galactose, Rhamnose; Uronic Acids such as Glucuronic acid and Galacturonic Acid; Hexosamines such as N-Acetyl L-fucosamine and N-Acetyl L-pneumosamine, N-Acetyl Glucosamine, N-Acetyl-Galactosamine; O-Acetyl, Phosphorous and Nitrogen content.

[0035] The inventors of the present invention during their continuous efforts to develop high sensitive methods for the quantification of polysaccharides have developed a method for quantification of carbohydrates using aromatic alcohol such as 2-PE (2-Phenoxyethanol). The developed method provides higher specificity and sensitivity over other methods, such as quantification with anthrone or phenol, and contributes to the quantification of a broad range of carbohydrates, sugars and/or polysaccharides in a wide range of samples.

[0036] 2-PE reacts with the fural or 5-Hydroxymethylfurfural formed from acid hydrolysis of polysaccharide and has an absorbance maximum at 500 nm. Additionally, the colour development in this method is not impaired by uronic acid containing polysaccharides. Further, polysaccharide-protein conjugates can be quantified using the present invention with increased sensitivity over anthrone reagent. The polysaccharides isolated from bacteria (Streptococcus Pneumoniae,) sugars and their respective acids showed enhanced reactivity with 2-PE thus improving the method sensitivity. The method is simple, direct and can be used as a routine technique for any type of polysaccharide quantitation. Overall, the higher sensitivity of the methods of the present invention would contribute to the quantification of a broad range of carbohydrates, sugars and/or polysaccharides with higher sensitivity over other reported methods.

[0037] In an embodiment, the present invention provides a method for quantification of carbohydrates in an aqueous sample comprises the steps of : [0038] a. admixing sulphuric acid and 2-Phenoxyethanol with the aqueous sample to obtain a reaction mixture; [0039] b. incubating the reaction mixture to form a coloured complex; and [0040] c. measuring the absorbance of the coloured complex to quantify the amount of carbohydrate present in the aqueous sample. [0041] In another embodiment, the quantity of carbohydrates in the sample is proportional to the measured absorbance of the coloured complex.

[0042] In a more preferred embodiment of the invention, the carbohydrate sample is treated with concentrated sulphuric acid to obtain the reaction mixture.

[0043] In an embodiment of the invention, the volume of sulphuric acid added to the carbohydrate containing aqueous sample ranges from about 1 to 3 times the volume of the carbohydrate containing aqueous sample.

[0044] The term “about” as used herein contemplates a range of values for a given number of ±25% the magnitude of that number. In certain embodiments, the term “about” contemplates a range of values for a given number of ±20%, ±15%, ±10%, or ±5% the magnitude of that number.

[0045] In another embodiment of the invention, the volume of sulphuric acid added to the carbohydrate containing aqueous sample is twice the volume of the carbohydrate containing aqueous sample.

[0046] In an embodiment of the invention, the concentration of 2-Phenoxyethanol ranges from about 0.1% v/v to 2.5% v/v with respect to the reaction mixture

[0047] In an embodiment of the invention, the reaction mixture is incubated at a temperature ranging from about 80° C. to 110° C.

[0048] In another embodiment, the reaction mixture is incubated at a temperature of about 90° C.

[0049] In an embodiment of the invention, the reaction mixture is incubated for a time period ranging from about 1 minute to 10 minutes.

[0050] In another embodiment, the reaction mixture is incubated for a time period of about 5 minutes.

[0051] In an embodiment of the invention, the absorbance of the coloured complex is measured in a wavelength ranging from about 490 nm to 510 nm.

[0052] In another embodiment of the invention, the absorbance of the coloured complex is measured at a wavelength of 500 nm.

[0053] In an embodiment of the invention, the aqueous sample quantified is a pure carbohydrate sample.

[0054] In another embodiment of the invention, the aqueous sample quantified is an impure carbohydrates sample. The impurities include, but are not limited to, nucleic acids, proteins, lipids, residual reagents, excipients or a combination thereof.

[0055] It was found that the method of the present invention is very specific to carbohydrates in presence of different impurities and formulation excipients, indicating the specificity of the assay.

[0056] “Limit of Detection,” as that term is used herein, includes the lowest concentration at which one can identify a sample as containing a molecule of the substance of interest.

[0057] In an embodiment of the present invention, the Limit of Detection (LOD) is less than about 4 μg/mL.

[0058] “Limit of Quantification”, as used herein, refers to a point where measurements become quantitatively meaningful.

[0059] In an embodiment of the present invention, the Limit of Quantification (LOQ) is at least 6 μg/mL.

[0060] In another embodiment of the present invention, the Limit of Quantification (LOQ) is at least 8 μg/mL.

[0061] In an embodiment, the method of the present invention is used to quantify carbohydrates in a vaccine or a biopharmaceutical sample.

[0062] In an embodiment of the present invention, carbohydrate content in monoconjugate and multivalent Pneumococcal Conjugate Vaccine (PCV) drug product is quantified.

[0063] In another embodiment of the present invention, the total carbohydrate content was quantified for monoconjugate and multivalent PCV drug product wherein pneumococcal serotype such as 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 1 1A, 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. were prediluted to 4-100 μg/mL prior to being subjected for analysis.

[0064] The multivalent pneumococcal conjugate vaccine may 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, 30 valent pneumococcal vaccine composition.

[0065] In yet another embodiment , the method of the present invention is applied to quantify the polysaccharide in monovalent and multivalent vaccine products for interference and spike recovery evaluation.

[0066] In yet another embodiment, the multivalent Pneumococcal conjugate vaccine adsorbed with aluminium phosphate (equivalent to ˜70 μg/mL of polysaccharides) is subjected to total saccharide content analysis using methods of the present invention.

[0067] In an embodiment of the present invention, carbohydrate content in multivalent meningococcal conjugate drug product containing polysaccharides from Neisseria meningitidis serogroups A, C, W135, X and Y is quantified.

[0068] In an embodiment of the present invention, carbohydrate content in monovalent and bivalent typhoid conjugate drug product containing Vi polysaccharide and O2 polysaccahrides from Salmonella typhi and Salmonella paratyphi is quantified.

[0069] In an embodiment, this invention also includes a kit for quantifying carbohydrates in an aqueous sample.

[0070] In an embodiment, the kit for quantifying carbohydrates comprises of sulphuric acid, 2-Phenoxyethaol or a combination thereof.

[0071] In another embodiment, the invention provides the use of 2-Phenoxyethanol for quantification of carbohydrates in a sample.

[0072] In another embodiment, the invention provides the use of 2-Phenoxyethanol for quantification of carbohydrates in a sample, wherein said carbohydrate is selected from a group comprising monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, their derivatives and combinations thereof.

[0073] The following examples are provided to illustrate the invention and are merely for illustrative purpose only and should not be construed to limit the scope of the invention.

EXAMPLES

Example 1: Estimation of Monosaccharides and Polysaccharides by Three Different Methods as Under

a. 2-PE-Sulphuric Acid Assay.

[0074] Carbohydrates such as Glucose, Galactose, Rhamnose, Ribitol, Glycerol, Lactose, Mannose, Sucrose, Glucuronic acid, Galacturonic acid, N-Acetyl L-fucosamine and N-Acetyl L-pneumosamine, N-Acetyl Glucosamine, N-Acetyl-Galactosamine, and N-Acetyl Mannosamine,Pullulan 800, and Pneumococcal polysaccharides from serotypes 1, 3, 5, 6B, 9V, 14,19F, 22F, 23F were diluted to 20 μg in 250 μL MilliQ water, based on the dry weight and hydrolysed in the presence of concentrated H.sub.2SO.sub.4 to form monosaccharides or their derivatives which in hot acidic medium gets dehydrated to form hydroxymethyl furfural. These hydroxymethyl furfural structures then reacted with phenol reagent to form an orange-yellow coloured complex which gives a maximum absorbance at 500 nm.

[0075] The reaction of 2-phenoxyethanol with sugars in presence of H.sub.2SO.sub.4 (Based on Molisch's Test Reaction) is depicted in the following scheme:

##STR00001##

[0076] 250 μL (80 μg/mL) of the diluted monosaccharides and polysaccharides were dispensed in triplicates into clean and dry glass tubes and 250 μL of reagent/MilliQ water was taken in triplicates for blank correction. 10 μL of 98% of 2-PE and 500 μL of H.sub.2SO.sub.4 were added to all the tubes and vortexed gently. Tubes were incubated in a water bath at 90° C. for 5 minutes. Thereafter the tubes were cooled to room temperature and 250 μL were transferred into micro-plate and the absorbance was measured at 500 nm using a plate reader.

b. Phenol-Sulphuric Acid Assay.

[0077] All monosaccharides and polysaccharides were diluted to 20 μg in 165 μL MilliQ water, based on the dry weight and hydrolysed in the presence of 500 μL of concentrated H.sub.2SO.sub.4 to form monosaccharides or their derivatives which in hot acidic medium gets dehydrated to form hydroxymethyl furfural. These hydroxymethyl furfural structures then reacts with phenol reagent to form an orange-yellow coloured complex which gives a maximum absorbance at 490 nm.

[0078] Each standard of 0-20 μg was taken in a clean glass tube and 100 μL of 5% phenol reagent was added and thereafter incubated for 5 minutes at 90° C. in open tubes and 250 μL were transferred into microplate, followed by reading the absorbance using plate reader at 490 nm.

c. Anthrone-Sulphuric Acid Assay.

[0079] All monosaccharides and polysaccharides were diluted to 20 μg in 250 μL, based on the dry weight and hydrolysed in presence of H.sub.2SO.sub.4 to form monosaccharides or their derivatives which in hot acidic medium gets dehydrated to form hydroxymethyl furfural. These hydroxymethyl furfural structures then react with anthrone reagent to form a green coloured complex which gives a maximum absorbance at 625 nm. 250 μL (80 μg/mL) in triplicates were dispensed into a clean glass tube and 500 μL of anthrone reagent were added followed by incubation for 5 minutes at 90° C. and the absorbance was measured using plate reader at 625 nm.

[0080] In all above listed three methods, the final reaction volumes were ˜760 μL and 250 μL was taken for absorbance measurements in microplate.

TABLE-US-00001 TABLE 1 Carbohydrate Estimation Methods λ max S. No. Method Reagent (Absorbance) 1. 2-PE-Sulphuric Aromatic 500 nm Acid Assay Phenoxy Ethanol 2. Phenol-Sulphuric Aromatic Phenol 490 nm Acid Assay Component 3. Anthrone-Sulphuric Tricyclic Aromatic 625 nm Acid Assay Component

Results

Absorbance of Monosaccharides

[0081] The absorbance of monosaccharides upon reacting with 2-PE (500 nm), anthrone (625 nm) and phenol (490 nm) reagents were illustrated in FIG. 1.

[0082] In presence of H.sub.2SO.sub.4, all monosaccharides get hydrolysed to form hydroxymethylfurfural. These hydroxymethyl furfural structures when reacted with anthrone formed a green coloured complex having an absorbance maximum at 625 nm. Similarly, when hydroxymethyl furfural structures reacted with 2-PE gave an orange-yellow colour complex having absorbance maxima at 500 nm. The colour complex formed with Glucuronic acid and Galacturonic acid upon reacting with anthrone, has low absorbance (0.05 and 0.10 OD), whereas absorbance using 2-PE as a reagent the OD (Optical Density) units were 0.69 and 0.85 respectively. Hexoses, such as Glucose, Galactose and Mannose ODs were 1.55, 1.07 and 2.07 with 2-PE and with anthrone 0.93, 0.45 and 0.43 respectively. Similarly, for Rhamnose, i.e. methyl pentose, the OD with 2-PE was 1.02 and 0.62 with anthrone. The ODs for N-Acetylated amines (FucNAc; GlucNAc; GalNAc; ManNAc; PneuNAc) gave marginally different results with Anthrone or 2-PE reagents (FIG. 1).

Absorbance of Polysaccharides

[0083] The absorbance of polysaccharides (Pneumococcal polysaccharides, Pullulan 800 and Starch) upon reacting with 2-PE (500 nm), anthrone (625 nm) and phenol (490 nm) reagents were illustrated in FIG. 2.

[0084] The sulphuric acid used for polysaccharide hydrolysis results in the formation of the hydroxymethylfurfural of each sugar. These hydroxymethyl furfurals when reacted with anthrone gave a green coloured complex with an absorbance maxima at 625 nm. Similarly, when hydroxymethyl furfurals reacted with 2-PE gave an orange-yellow coloured complex with an absorbance maxima at 500 nm. The sulphuric acid hydrolysed pneumococcal polysaccharides when reacted with 2-PE the colour complex formed was higher than that of the colour complex generated with anthrone reagent (FIG. 2) thus increasing the sensitivity for quantification and improving the lower LOD (Limit of Detection) of the sugars. Similar sensitivity and reactivity were also observed for other polysaccharides such as pullulan 800 using 2-PE (FIG. 2).

Example 2: Polysaccharide Quantification in Mono-conjugate and Multivalent Adsorbed Vaccine

[0085] Different known concentrations of pneumococcal serotype 6B and 7F Poly-protein conjugates each of 4-100 μg/mL and multivalent conjugate vaccine samples were subjected for 2-PE analysis. The percent recovery of 6B and 7F polysaccharide or conjugates were between 90-110%, above their LOQ (Limit of Quantification) level (Table 2).

[0086] Similarly, the percentage recovery of total polysaccharides in pneumococcal multivalent adsorbed conjugate vaccine at different Al.sup.+3 concentrations (0.25, 0.5 and 1.0 mg/mL of AlPO.sub.4 gel) were 97, 101 and 99 respectively (Table 2), indicating no interference from aluminium phosphate in the drug product.

Interference from impurities and Validation

[0087] The most common impurities in purified polysaccharides are nucleic acids, proteins and residual reagents. The interference of these impurities at 2 and 5% w/v level against total polysaccharide concentration were assessed using 2-PE reagent (FIG. 3) reactivity. At 2% impurities level (which is the maximum allowed limit as per regulatory guidance) there was no effect on the reactivity, however there was ˜8% of less reactivity in presence of 5% impurities. Assay was validated for the specificity, accuracy, precision, spike recovery, limit of detection (LOD) and limit of quantification (LOQ) using polysaccharides and conjugates.

[0088] This method was further evaluated for polysaccharide quantification in vaccine conjugates. The polysaccharide concentration in 6B and 7F (Pneumo-CRM conjugates) were estimated at the entire standard range to check the assay LOD and LOQ (Table 2). Based on the % recovery of the polysaccharide or conjugates, the LOD of the assay was ≤4.0 μg/mL and LOQ of the assay was ≥8 μg/mL (Table 2).

[0089] The source of the biological material used in the present invention is as follows: [0090] 1. Streptococcus pneumoniae Serotype 6B obtained from Centers for Disease Control and Prevention (CDC) USA [0091] 2. Streptococcus pneumoniae Serotype 7F obtained from Centers for Disease Control and Prevention (CDC) USA
Further, Pneumo-CRM conjugates have been prepared using CRM197 protein isolated from Corynebacterium obtained from ATCC.

TABLE-US-00002 TABLE 2 Total polysaccharide content in 6 B, 7 F mono-conjugates and multivalent Pneumococcal conjugate vaccine by 2-PE method Expected Concentration % Recovery of Concentration by 2-PE method the total Ps Sample Details (μg/mL) (μg/mL) content 6 B Polysaccharide 4 2.8 69 8 7.4 92 16 14 90 24 24 101 32 34 107 40 40 99 100 100 100 6 B mono-conjugate 4 1 25 8 8 100 16 17 106 24 22 92 32 32 100 40 40 100 100 98 98 7 F Polysaccharide 4 0.1 3 8 7.2 90 16 15 94 24 26 108 32 35 110 40 44 110 100 97 97 7 F mono-conjugate 4 1.2 30 8 7.3 91 16 15 94 24 23 96 32 32 100 40 40 100 100 102 102 Multivalent vaccine with 70 68 97 0.25 mg/mL Al.sup.+3 Multivalent vaccine with 70 71 101 0.5 mg/mL Al.sup.+3 Multivalent vaccine with 70 69 99 1.0 mg/mL Al.sup.+3

Advantages of the Invention

[0092] 1. The method is simple and direct. [0093] 2. The method has a higher specificity and sensitivity over other methods known for quantification of carbohydrates. [0094] 3. The method can be used as a routine technique for quantifying a broad range of carbohydrates, sugars and/or polysaccharides in a wide range of samples. [0095] 4. The colour development in the developed method is not impaired by uronic acid containing polysaccharides. [0096] 5. The method of the present invention can accurately determine the amount of carbohydrates in presence of different impurities and formulation excipients, indicating the specificity of the assay.