WO2021064748A1 - Methods for quantification of carbohydrates - Google Patents
Methods for quantification of carbohydrates Download PDFInfo
- Publication number
- WO2021064748A1 WO2021064748A1 PCT/IN2020/050841 IN2020050841W WO2021064748A1 WO 2021064748 A1 WO2021064748 A1 WO 2021064748A1 IN 2020050841 W IN2020050841 W IN 2020050841W WO 2021064748 A1 WO2021064748 A1 WO 2021064748A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polysaccharides
- carbohydrates
- quantification
- phenoxyethanol
- sample
- Prior art date
Links
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- 235000014633 carbohydrates Nutrition 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 66
- 238000011002 quantification Methods 0.000 title claims abstract description 42
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- 239000005017 polysaccharide Substances 0.000 claims description 80
- 238000002835 absorbance Methods 0.000 claims description 33
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- 238000011534 incubation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 238000006140 methanolysis reaction Methods 0.000 description 1
- 229940031346 monovalent vaccine Drugs 0.000 description 1
- 229940031348 multivalent vaccine Drugs 0.000 description 1
- 150000008278 pentosamines Chemical class 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 229940124733 pneumococcal vaccine Drugs 0.000 description 1
- 229940031937 polysaccharide vaccine Drugs 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Definitions
- the present invention relates to methods for the estimation of carbohydrates. More specifically, the present invention relates to methods for quantification of carbohydrates using aromatic ethanol when carbohydrates are present in low concentration in pharmaceuticals, biopharmaceuticals and biologicals.
- Quantification of carbohydrate or polysaccharide content is an essential analytical procedure in food and beverages, nutraceuticals, agricultural products, medicinal products and vaccine development.
- Polysaccharides are generally quantified by biochemical assay using anthrone reagent and glucose as standards.
- Other biochemical methods using reagents such as orcinol, or phenol sulphuric acid with colorimetric reaction are also largely been used for polysaccharide estimation.
- concentration of anthrone reagent and H2SO4, heating time and temperature, etc. were reported earlier for improving the sensitivity of the colorimetric method.
- the intensity of the absorbance in both these methods varies with the composition of the polysaccharide, as different polysaccharides contain different sugars such as Hexoses, Pentoses, Uronic acids, Hexosamines, Pentosamines, Glycolides, Glycoproteins and Nucleic Acids, etc., and in different proportions.
- polysaccharides are conjugated to a carrier protein and controlling each polysaccharide amount in the prescribed dose is critical for maintaining the quality and efficacy of the vaccine. Accurate quantification of polysaccharides at the microgram level is very essential in developing the vaccine.
- Polysaccharides are complex mixtures of Hexoses, Pentoses, Uranic acids and Hexosamines.
- Indian Patent Application No. 5856/DELNP/2009 discloses quantification of polysaccharide using automated colorimetric assay using sulphuric acid and anthrone reagent or sulphuric acid and tetraborate reagent.
- EP 2290366 Al discloses the analysis of saccharide vaccines without interference between the monosaccharides and from any other saccharide materials in the composition, comprising the process of quantifying monosaccharides (sialic acid, galactose and glucose) by acid hydrolysis using trifluoroacetic acid (TFA) for Neisseria meningitidis serogroups C, W135 and Y.
- monosaccharides sialic acid, galactose and glucose
- TFA trifluoroacetic acid
- Vincent et al. (Anal. Chem. 2010, 82, 1786-1792), discloses the automation of the anthrone assay for determination of carbohydrate concentration wherein the polysaccharide test samples and standards are heated in a concentrated mixture of anthrone in sulphuric acid at absorbance 625nm.
- Verdnica et al. discloses the quantification of capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples using phenol-sulfuric, HPSEC (10 mM Na 2 HP0 4 , 0.15 M NaCl, pH 7.5), competitive ELISA (o-phenylenediamine dihydrochloride in phosphate-citrate buffer, pH 5.0, and 0.05% hydrogen peroxide were added and the reaction was stopped with 4.5 M H2SO4, and the absorbance at 492 nm), and sandwich ELISA methods, wherein sandwich ELISA was found to be the method with the best reproducibility and sensitivity and the least susceptible to interferences.
- HPSEC mM Na 2 HP0 4 , 0.15 M NaCl, pH 7.5
- competitive ELISA o-phenylenediamine dihydrochloride in phosphate-citrate buffer, pH 5.0, and 0.05% hydrogen peroxide were added and the reaction was stopped with 4.5 M H2SO
- the above prior art discloses various method to quantify the polysaccharides.
- these methods suffer from various infirmities and hence, there is a need for development of new method which enhances the colour development and consequently, enhances the sensitivity.
- the enhanced sensitivity may provide proper estimation of the carbohydrates when present in low concentration in any sample, including, but not limited to, pharmaceuticals, biopharmaceuticals, biologicals, cosmetics, environmental matrices, food, forensic samples, industrial chemicals, and nutraceuticals.
- the invention relates to a method for quantification of carbohydrates using an aromatic ethanol, such as 2-Phenoxyethanol (2-PE).
- an aromatic ethanol such as 2-Phenoxyethanol (2-PE).
- the present invention provides a colorimetric based method for the quantification of carbohydrates in an aqueous sample, the method comprising the steps of: a. admixing sulphuric acid and 2-Phenoxyethanol with the 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.
- the present invention relates to a method for estimation of carbohydrates such as, monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines and their derivatives and combination thereof.
- the invention also provides a kit for the quantification of carbohydrates in a sample.
- Figure 1 illustrates absorbance of Monosaccharides upon reacting with 2-PE (500 nm), anthrone (625 nm) and Phenol (490 nm) reagents;
- Figure 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
- Figure 3 illustrates absorbance of Pneumococcal polysaccharides as % recovery in presence of known impurities (2 and 5% w/v) present in purified polysaccharides.
- 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.
- 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.
- monosaccharide includes, but not limited to, glucose, galactose, rhamnose, mannose, arabinose, xylose, fructose, ribose and their derivatives and combination thereof.
- disaccharide includes, but not limited to, sucrose, lactose, maltose, trehalose, cellobiose and their derivatives and combination thereof.
- uronic acid includes, but not limited to glucuronic acid, galacturonic acid, mannuronic acid and their derivatives and combination thereof.
- 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.
- the present invention relates to a method for quantification of polysaccharides such as Pneumococcal polysaccharides, Meningococcal polysaccharides, VI polysaccharide, 02 polysaccharide and other carbohydrates such as Cellulose, Starch, Chitin, Dextran and Pullulan and the like.
- polysaccharides such as Pneumococcal polysaccharides, Meningococcal polysaccharides, VI polysaccharide, 02 polysaccharide and other carbohydrates such as Cellulose, Starch, Chitin, Dextran and Pullulan and the like.
- pneumococcal polysaccharides are prepared as disclosed in PCT Publication Number WO2016/174683 A1 and the structure thereof is deduced by *H 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.
- monosaccharides such as Glucose, Galactose, Rhamnose
- Uronic Acids such as Glucuronic acid and Galacturonic Acid
- Hexosamines such as N-Acetyl L-fucosamine and
- 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).
- 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.
- 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.
- the present invention provides a method for quantification of carbohydrates in an aqueous sample comprises the steps of : a. admixing sulphuric acid and 2-Phenoxyethanol with the aqueous sample 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.
- the quantity of carbohydrates in the sample is proportional to the measured absorbance of the coloured complex.
- the carbohydrate sample is treated with concentrated sulphuric acid to obtain the reaction mixture.
- 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.
- 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.
- the volume of sulphuric acid added to the carbohydrate containing aqueous sample is twice the volume of the carbohydrate containing aqueous sample.
- the concentration of 2-Phenoxyethanol ranges from about 0.1% v/v to 2.5% v/v with respect to the reaction mixture
- the reaction mixture is incubated at a temperature ranging from about 80 ° C to 110 ° C.
- reaction mixture is incubated at a temperature of about
- the reaction mixture is incubated for a time period ranging from about 1 minute to 10 minutes.
- reaction mixture is incubated for a time period of about 5 minutes.
- the absorbance of the coloured complex is measured in a wavelength ranging from about 490 nm to 510 nm.
- the absorbance of the coloured complex is measured at a wavelength of 500nm.
- the aqueous sample quantified is a pure carbohydrate sample.
- 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.
- “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. In an embodiment of the present invention, the Limit of Detection (LOD) is less than about 4 ⁇ g/mL.
- “Limit of Quantification”, as used herein, refers to a point where measurements become quantitatively meaningful.
- the Limit of Quantification is at least 6 ⁇ g/mL.
- the Limit of Quantification is at least 8 ⁇ g/mL.
- the method of the present invention is used to quantify carbohydrates in a vaccine or a biopharmaceutical sample.
- carbohydrate content in monoconjugate and multivalent Pneumococcal Conjugate Vaccine (PCV) drug product is quantified.
- 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, 22R 23A, 23B, 23R 24B, 24F, 31 , 33F, 34, 35B, 35F, 38, 39 and 45. were prediluted to 4-100 ⁇ g/mL prior to being subjected for analysis.
- 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, 22R 23A, 23B, 23R 24B, 24F, 31
- 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.
- the method of the present invention is applied to quantify the polysaccharide in monovalent and multivalent vaccine products for interference and spike recovery evaluation.
- the multivalent Pneumococcal conjugate vaccine adsorbed with aluminium phosphate (equivalent to ⁇ 70 ⁇ g/mL of polysaccharides) is subjected to total saccharide content analysis using methods 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.
- carbohydrate content in monovalent and bivalent typhoid conjugate drug product containing Vi polysaccharide and 02 polysaccahrides from Salmonella typhi and Salmonella paratyphi is quantified.
- this invention also includes a kit for quantifying carbohydrates in an aqueous sample.
- the kit for quantifying carbohydrates comprises of sulphuric acid, 2-Phenoxyethaol or a combination thereof.
- the invention provides the use of 2-Phenoxyethanol for quantification of carbohydrates in a sample.
- 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.
- Example 1 Estimation of monosaccharides and polysaccharides by three different methods as under. a. 2-PE-Sulphuric Acid Assay.
- 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 ⁇ g in 250 ⁇ L MilliQ water, based on the dry weight and hydrolysed in the presence of concentrated H2SO4 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
- 250 ⁇ L (80 ⁇ g/mL) of the diluted monosaccharides and polysaccharides were dispensed in triplicates into clean and dry glass tubes and 250 ⁇ L of reagent/MilliQ water was taken in triplicates for blank correction.
- 10 ⁇ L of 98% of 2-PE and 500 ⁇ L of H2SO4 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 ⁇ L were transferred into micro-plate and the absorbance was measured at 500 nm using a plate reader.
- Phenol-Sulphuric Acid Assay were dispensed in triplicates into clean and dry glass tubes and 250 ⁇ L of reagent/MilliQ water was taken in triplicates for blank correction.
- 10 ⁇ L of 98% of 2-PE and 500 ⁇ L of H2SO4 were added to all the tubes and vortexed gently. Tubes were incuba
- the final reaction volumes were -760 ⁇ L and 250 ⁇ L was taken for absorbance measurements in microplate.
- 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.
- 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 ( Figure 3) reactivity.
- impurities level which is the maximum allowed limit as per regulatory guidance
- Assay was validated for the specificity, accuracy, precision, spike recovery, limit of detection (LOD) and limit of quantification (LOQ) using polysaccharides and conjugates. This method was further evaluated for polysaccharide quantification in vaccine conjugates.
- the polysaccharide concentration in 6B and 7F 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 ⁇ g/mL and LOQ of the assay was >8 ⁇ g/mL (Table 2).
- the source of the biological material used in the present invention is as follows:
- Streptococcus pneumoniae Serotype 6B obtained from Centers for Disease Control and Prevention (CDC) USA
- Streptococcus pneumoniae Serotype 7F obtained from Centers for Disease Control and Prevention (CDC) USA
- Pneumo-CRM conjugates have been prepared using CRM 197 protein isolated from Corynebacterium obtained from ATCC.
- Table 2 Total polysaccharide content in 6B, 7F mono-conjugates and multivalent Pneumococcal conjugate vaccine by 2-PE method
- the method is simple and direct.
- the method has a higher specificity and sensitivity over other methods known for quantification of carbohydrates.
- 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.
- the colour development in the developed method is not impaired by uranic acid containing polysaccharides. 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.
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US17/764,351 US20220390423A1 (en) | 2019-10-01 | 2020-09-30 | Methods for quantification of carbohydrates |
KR1020227014457A KR20220075374A (en) | 2019-10-01 | 2020-09-30 | Methods for Quantification of Carbohydrates |
CA3152573A CA3152573A1 (en) | 2019-10-01 | 2020-09-30 | Methods for quantification of carbohydrates |
EP20872366.8A EP4038369A4 (en) | 2019-10-01 | 2020-09-30 | Methods for quantification of carbohydrates |
CN202080067950.XA CN114450576A (en) | 2019-10-01 | 2020-09-30 | Method for carbohydrate quantification |
ZA2022/02670A ZA202202670B (en) | 2019-10-01 | 2022-03-04 | Methods for quantification of carbohydrates |
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UA23879U (en) * | 2007-01-29 | 2007-06-11 | Yurii Yuriiovych Malynovskyi | Process for preparation and quantification of polysaccharide complex from spotted hemlock |
EP2290366A1 (en) | 2004-03-17 | 2011-03-02 | Novartis Vaccines and Diagnostics S.r.l. | Analysis of saccharide vaccines without interference |
WO2016174683A1 (en) | 2015-04-28 | 2016-11-03 | Biological E Limited | Method for separation of protein and other impurities from microbial capsular polysaccharides |
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EP2290366A1 (en) | 2004-03-17 | 2011-03-02 | Novartis Vaccines and Diagnostics S.r.l. | Analysis of saccharide vaccines without interference |
UA23879U (en) * | 2007-01-29 | 2007-06-11 | Yurii Yuriiovych Malynovskyi | Process for preparation and quantification of polysaccharide complex from spotted hemlock |
WO2016174683A1 (en) | 2015-04-28 | 2016-11-03 | Biological E Limited | Method for separation of protein and other impurities from microbial capsular polysaccharides |
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S. SUZANNE NIELSEN: "Food Analysis Laboratory Manual", 26 November 2009, SPRINGER NEW YORK, NY, Boston, MA, ISBN: 978-1-4419-1463-7, article S. SUZANNE NIELSEN : "Phenol-sulfuric acid method for total carbohydrates.", pages: 47 - 53, XP009535344, DOI: 10.1007/978-1-4419-1463-7 * |
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