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2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification
Journal Pre-proof 2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification Burki Rajendar, Ravikumar Mulagalapati, M.V.N Janardhan Reddy, Sumapriya Patri, Yellepeddi K. Karthik, Ramesh V. Matur PII:
S0003-2697(19)31176-5
DOI:
https://doi.org/10.1016/j.ab.2020.113624
Reference:
YABIO 113624
To appear in:
Analytical Biochemistry
Received Date: 27 November 2019 Revised Date:
22 January 2020
Accepted Date: 10 February 2020
Please cite this article as: B. Rajendar, R. Mulagalapati, M.V.N.J. Reddy, S. Patri, Y.K. Karthik, R.V. Matur, 2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification, Analytical Biochemistry (2020), doi: https://doi.org/10.1016/j.ab.2020.113624. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Authors statement B R, R M and M V N J R designed the experiments, reviewed the data and wrote the manuscript S P and Y K K executed the experiments R V M is conceptualized the work and reviewed the manuscript All authors discussed the results and contributed to the final manuscript
1
2-Phenoxyethanol: A Novel Reagent for Improved Sensitivity of carbohydrate quantification
Burki Rajendar,* Ravikumar Mulagalapati, M V N Janardhan Reddy, Sumapriya Patri, Yellepeddi K Karthik, Ramesh V. Matur,*
Research & Development, Biological E Limited, Shameerpet, Hyderabad 500078, India
*
To whom correspondence should be addressed
CORRESPONDING AUTHOR FOOTNOTE Dr. Ramesh V. Matur Research & Development, Biological E Limited, Plot No.1, Phase II, SP Biotech Park, Genome Valley, Shameerpet, Hyderabad 500078, India Tel: +91-40-6738-8227, E-mail: [email protected] Dr. Burki Rajendar Tel: +91-40-6738-8229, E-mail: [email protected] Keywords: Anthrone assay, 2-Phenoxyethanol, Polysaccharide quantification, Carbohydrate analysis Abbrevations used: 2-PE, 2-Phenoxyethanol, AlPO4, Aluminium Phosphate, OD, Optical Density, H2SO4, Sulphuric Acid
ABSTRACT
Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulfuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm. Though anthrone reacts well with polysaccharides containing hexoses (such as glucose and galactose) and rhamnose, it is less reactive with uronic acids (such as glucuronic acid and galacturonic acid) and hexosamines (such as fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). Here, we report a novel reagent, 2-Phenoxyethanol, which reacts with furfural or hydroxymethyl furfural resulting in higher absorptivity. This method is rapid, sensitive, simple and direct, and can be used for quantitative determination of any type of carbohydrate that contains neutral sugars and uronic acids. For these saccharides, the sensitivity of the assay using 2-Phenoxyethanol (2-PE) is twice over anthrone method. Uronic acids show improved sensitivity using 2-PE over Phenol and it is more than twice with glucuronic acid. 2-PE reagent method has greater application for quantification of carbohydrates when present in low concentration
in
vaccines/biologicals.
1. Introduction Quantification of carbohydrate/polysaccharide content is an essential analytical procedure in food and beverages, nutraceuticals, agricultural products, medicinal products and vaccine pharmaceuticals. Polysaccharides are quantified by colorimetric assay using anthrone reagent and glucose as standard.1 Other reagents such as orcinol, or phenol sulphuric acid with colorimetric reaction are also largely been used for polysaccharide estimation (Table 1).1-5 Several modifications of the anthrone method (concentration of anthrone reagent and H2SO4, heating time and temperature, etc.) were reported earlier for improving the sensitivity of the method.6 These modifications were aimed at enhancement of the color development of the anthrone-furfural complex, which improves the optical density signal, thereby enhancing quantification sensitivity of the assay. On the other hand, the color development is still low with these methods for analyzing polysaccharides containing mixtures of uronic acids (glucoronic acid and galacturonic acid) and hexosamines (fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). These methods do not generate optimum color complexes and suffer from low sensitivity making
them less applicable for estimating such carbohydrates/polysaccharides, especially for polysaccharide based vaccines. During carbohydrate quantification in presence of sulphuric acid, hexoses and pentoses are hydrolyzed and converted to 5-Hydroxymethyl furfural and furfural respectively. These molecules generated by acid hydrolysis are then reacted with anthrone or phenol to form color complex, which is then measured for optical density at a specific wavelength. The absorption maxima for the Anthrone-Furfural complex is at 625 nm7 and that of the phenol-furfural complex is at 490 nm.4 As mentioned earlier, the color intensity at the maximum absorbance wavelength in both these methods varies with the composition of the polysaccharide, as different polysaccharides contain different sugars such as hexoses, pentoses, uronic acids, hexosamines, and pentosamines etc. , and in different proportions. Therefore, there exists a critical need for further optimization of existing methods to enhance the color development and thereby enhancing the sensitivity through development of novel methods. Thus the increased signal intensity helps to estimate the smallest amount of polysaccharide present in the sample. For pneumococcal conjugate vaccines, serotype specific polysaccharides are conjugated to a carrier protein and therefore controlling each serotype polysaccharide amount in the prescribed dose is critical for maintaining the quality and efficacy of the vaccine. Accurate quantification of pneumococcal polysaccharides at microgram level is very essential in developing these vaccines. Pneumococcal polysaccharides are comprised of complex mixtures of hexoses, pentoses, uronic acids and hexosamines.8 Several investigators have recognized the importance of making these colorimetric assays more sensitive, high-throughput by scaling down to microplates. Two common colorimetric assay reagents used for this purpose are anthrone and phenol-sulfuric acid. We noticed that the reactivities of anthrone/sulphuric acid or phenol/sulphuric acid with polysaccharides containing uronic acids and hexosamines were very weak and could not be quantitated by these two methods. For quantification of the total polysaccharide content of pneumococcal serotypes or their components (methyl pentoses,9-10 uronic Acids,11-13 hexosamines,14 O-acetyl,15 phosphorous and nitrogen) various biochemical methods were reported. Majority of them use acid hydrolysis to release the monosaccha3
rides, which are then quantified using high-performance anion-exchange chromatography (HPAEC)16-17 with pulsed amperometric detection (PAD), or acid hydrolysis along with derivatization followed by quantification of monosaccharides using Gas chromatography connected to a mass selective detector (GC-MSD).18 Another method reported for quantification of pneumococcal polysaccharides containing hexosamines involves acid hydrolysis, re-N-acetylation followed by labeling the hexosamines with 2aminobenzamide by reductive amination and RP-HPLC with fluorescence detector.19 Although these methods are sensitive and offer the required accuracy they are laborious, time consuming and may not be amenable for Quality Control scientists. Here we report a novel method that utilizes an aromatic ethanol component, 2-Phenoxyethanol (Figure 1), which reacts the same way as Anthrone or Phenol with significant enhancement in color development. This reaction is based on the Molisch’s Test (Figure 2). The 2-Phenoxyethanol reacts with the furfural or 5-Hydroxymethylfurfural formed from acid hydrolysis of polysaccharide resulting in the formation of a color complex with an absorbance maximum at 500 nm. We also demonstrated that this colorimetric assay is suitable for components containing uronic acids unlike for the anthrone assay. 2. Materials and methods 2.1 Chemicals and Reagents All pneumococcal polysaccharides used were prepared in-house and confirmed their structure by 1H NMR analysis. Suitable purification steps were employed to remove the endotoxins, protein and nucleic acid impurities. The final purified capsular polysaccharides were found to be in compliance with specified composition20 for methyl pentoses, uronic acids, hexosamines, O-acetyl, phosphorous and nitrogen contents. N-acetyl L-fucosamine and N-acetyl L-pneumosamine were purchased from Omicron Biochemicals, Inc. USA. Pullulan 800, rhamnose, glucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine, glucuronic acid, galacturonic acid, and all other reagents and buffer components were purchased from Sigma-Aldrich, India. 2.2 2-PE-Sulphuric Acid Assay
4
All monosaccharides and polysaccharides were diluted to 80 µg per mL based on the dry weight. Dispensed 250 µL (80 µg/mL) containing 20 µg monosaccharides and/or polysaccharides in triplicates in to clean and dry glass tubes. Each standard of 0-20 µg in 250 µL was taken in a clean glass tube. Water 250 µL was taken as blank in triplicates. Added 10 µL of 98% 2-Phenoxyethanol (2-PE) and 500 µL of H2SO4 to all the tubes and vortexed gently. Incubated the tubes in a water bath at 90°C for 5 min. Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 500 nm using plate reader. 2.3 Phenol-Sulphuric Acid Assay5 Dispensed 165 µL containing 20 µg monosaccharides and/or polysaccharides in to clean and dry glass tubes. Each standard of 0-20 µg in 165 µL was taken in a clean glass tube as the standard along with water blank. Added 500 µL of H2SO4 and 100 µL of 5% phenol reagent to the sample/ standard/blank tubes and incubated for 5 mins at 90˚C Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 490 nm using plate reader. 2.4 Anthrone-Sulphuric Acid Assay1 Dispensed 250 µL containing 20 µg monosaccharides and/or polysaccharides in to clean and dry glass tubes. Each standard of 0-20 µg in 250 µL was taken in a clean glass tube as the standard along with water blank. Added 500 µL of anthrone reagent (0.2% w/v anthrone in concentrated H2SO4) and incubated for 5 mins at 90˚C. Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 625 nm using plate reader. In all above listed three methods the final reaction volumes were ~750 to 765 µL and 250 uL was transferred into microplate for absorbance measurements. 2.5 Sample preparation of mono-conjugates and PCV drug product Serotypes 6B, 7F monovalent bulk conjugates were pre-diluted to 4-100 µg/mL and subjected for total saccharide content analysis using 2-PE as test reagent. Similarly, the multivalent Pneumococcal conju-
5
gate vaccine adsorbed onto aluminum phosphate (equivalent to ~70 µg/mL of polysaccharides) was subjected to total saccharide content analysis using 2-PE as test reagent. 3. Results 3.1 Reactivity of Monosaccharides The relative absorbance spectra of the color complex formed by the reaction of glucose and glucuronic acid with H2SO4 followed by the reaction with 2-PE, anthrone and phenol are presented in Figure 3A and 3B. In presence of H2SO4, glucose reacts with anthrone resulting in the formation of a green colored complex having an absorbance maximum at 625-630 nm (Figure 3A).7 Similarly when these furfural structures react with 2-PE, they give an orange yellow color complex having absorbance maxima at 500 nm (Figure 3) and with phenol, they produce yellow color complex with an absorbance maxima at 490 nm (Figure 3). The absorption spectrum of the color complex formed by glucuronic acid standard with 2-PE and phenol is similar to that of glucose. However, the reactivity of anthrone reagent with the furfural derivative of glucuronic acid appears to be poor with no sharp absorption maxima as seen for phenol or 2-PE. To facilitate appropriate comparisons between all 3 assays (2-PE, anthrone and phenol sulfuric acid), the total volume for each reaction procedure was adjusted to 750 to 765 uL. Figure 4 provides comparison of the 3 colorimetric assays for neutral sugars (Figure 4A: glucose, galactose, rhamnose, lactose, mannose and sucrose), hexuronic acids (Figure 4B: glucuronic acid, galacturonic acid), N-acetyl sugar amines (N-acetyl fucosamine, N acetyl pneumosamine, N acetyl galactosamine, N acetyl mannosamine), ribitol and glycerol (Figure 4C). Overall the absorbance values for all neutral sugars as well as hexuronic acids are relatively higher for the 2-PE based assay compared to that of anthrone and phenol assays (Figure 4A and B). However, N-acetyl sugar amines, ribitol and glycerol are not sensitive to any of the reagents tested, although 2-PE appears to be slightly better in some cases (Figure 4C). Linearity plots (Supporting information; Figure S1) were generated for the sugars, which showed higher and moderate reactivity. As shown in Table 4, the LOD and LOQ was determined from the linearity pots. 3.2 Reactivity of Pneumococcal Polysaccharides 6
Reactivity of 2-PE with pneumococcal polysaccharides were compared (Figure 5). Similar to the results observed for the monosaccharides, the reactivity of all polysaccharides are relatively higher with 2-PE reagent compared to that of phenol and anthrone. Between anthrone and phenol reagents, the reactivity of phenol with polysaccharides is slightly better resulting in higher sensitivity (Figure 5). The absorption spectra for all the polysaccharide hydrolysates are shown in Supporting information, Figure S2. Each serotype of pneumococcal polysaccharide has unique composition of monosaccharides such as hexoses, pentoses, uronic acids, hexosamines etc depending on the serotype. Although some polysaccharides8 such as Serotype 5, 14, 19F and 22F contain N-acetyl glucosamine, N-acetyl galactosamine, N-acetyl mannosamine, N-acetyl fucosamine and N-acetyl pneumosamine which demonstrated low reactivity (Figure 4C), these polysaccharides still react well with 2-PE due to the presence of other reacting sugars (hexose, pentose, hexuronic acid) (Figure 5 and Table 2) enabling to quantify with improved sensitivity over other methods. 3.3 Polysaccharide quantification in mono-conjugate and multivalent adsorbed vaccine drug product The newly developed 2-PE method was applied not only for the vaccine intermediates containing purified serotype specific polysaccharides, but also for those polysaccharides conjugated to a carrier protein as the monovalent conjugate drug substance as well as the drug product containing a mixture of monovalent conjugate drug substances including AlPO4. The known concentration of various serotype pneumococcal polysaccharides and different known concentrations of serotype 6B & 7F Poly-protein conjugates ranging from 4 to 100 µg/mL were used to determine the measured concentration in order to establish a linear range in which the % recovery is within the accepted level 90-110%. Furthermore, multivalent conjugate vaccine samples containing mixture of 14 different pneumococcal serotype conjugates at a known polysaccharide concentration of 70 µg/mL were subjected to analysis by 2-PE assay. The recovery was between 90-110 %, at the linearity range of 8-100 µg/mL (Table 3). Similarly, the % recovery of total polysaccharides in pneumococcal multivalent adsorbed conjugate vaccine at different Al+3 concentrations (0.25, 0.5 and 1.0 mg/mL of AlPO4 gel) were 97, 101 and 99 7
respectively (Table 3), indicating no interference with aluminum phosphate in the drug product. In addition, the presence of carrier protein (closer to the equal amount of the polysaccharide content) in the monovalent conjugates and the polyvalent drug product doesn’t interfere with the polysaccharide quantitation. 3.4 Interference from impurities and Validation The most common impurities in purified polysaccharides are nucleic acids and residual reagents. The interference of these impurities at 2 and 5% w/v against total polysaccharide concentration were assessed with 2-PE reagent (Figure 6). At 2% impurity level (which is maximum allowed limit as per regulatory guidance) there was no or negligible effect on the recovery. However, when impurities at 5%, there was a marginal effect on the total polysaccharide recovery (Figure 6). We further evaluated this method for polysaccharide quantification in Polysaccharide-CRM197 conjugates. The polysaccharide concentration in serotype 6B and 7F (Pneumococcal polysaccharide-CRM conjugates) were estimated at entire standard range to determine the assay linearity (Table 3). The assay is linear from 8 to 100µg /mL (Table 3) based on the % recovery of the polysaccharide or conjugates. 4. Discussion Anthrone1 and other sugar reacting agents such as phenol sulphuric acid show less reactivity with uronic acids and hexosamines present in the polysaccharides. In this paper, we have demonstrated the utility of 2-PE reagent to provide substantially higher sensitivity for saccharides containing neutral sugars and uronic acid containing saccharides. Production and quality control of vaccines such as pneumococcal conjugate vaccines are highly complex and take about a year from start to finish. In general, the manufacturing process requires fermentation and purification of individual serotype specific polysaccharides to high level of purity with least amount of residual impurities such as nucleic acids, proteins, endotoxin, as well as residuals from the purification components. Once these serotype specific polysaccharides are purified and quality controlled, then they are subjected to the activation process prior to the conjugation to a carrier protein. Following the conjugation to a carrier protein, these individual conjugated polysaccharides are further puri8
fied using a variety of chromatographic and/or other purification methodologies and further quality controlled to release each serotype specific conjugate as Drug Substance. The final step of the manufacturing process involves formulation of all 14 different serotype specific polysaccharide conjugates based on the polysaccharide content, with appropriate excipients including the adjuvant such as aluminum phosphate. In each and every step of the production process, the determination of saccharide content becomes very crucial starting from polysaccharide purification process all the way to the final drug product as in-process, release and stability parameters. Therefore, we needed a very sensitive assay for the determination of polysaccharide content we developed this sensitive 2-PE based assay. The method that we developed is very useful in quality control as little or no interference with carrier protein and aluminum adjuvant present in polysaccharide-protein conjugate vaccine. Furthermore, the presence of residual amounts of protein, nucleic acids and sodium deoxycholate is shown to have negligible effect on the assay values. This assay was developed using test tubes, the methodology can also be adopted to automated microtiter plate based high-throughput assay.7 5. Conclusions We reported a novel method for polysaccharide quantification using 2-PE by colorimetry. We demonstrated that different monosaccharides (hexoses, uronic acids and hexosamines), whole polysaccharides, polysaccharide-protein conjugates can be quantified using 2-PE as a reagent with increased sensitivity over anthrone as well as the phenol reagent. The polysaccharides isolated from bacteria (S. pneumoniae, A. pullulans) 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 polysaccharide quantitation.
Acknowledgements We thank the Scientific Advisory Board and Management of Biological E Limited for their active support and encouragement. 9
References 1.
Morris, D. L., Quantitative Determination of Carbohydrates With Dreywood's Anthrone
Reagent. Science 1948, 107 (2775), 254-5. 2.
Trevelyan, W. E.; Forrest, R. S.; Harrison, J. S., Determination of yeast carbohydrates with the
anthrone reagent. Nature 1952, 170 (4328), 626-7. 3.
Cuesta, G.; Suarez, N.; Bessio, M. I.; Ferreira, F.; Massaldi, H., Quantitative determination of
pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method. J Microbiol Methods 2003, 52 (1), 69-73. 4.
DuBois, M. G., K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F., Colorimetric Method for
Determination of Sugars and Related Substances. Anal Chem 1956, 28 (3), 350. 5.
Masuko, T.; Minami, A.; Iwasaki, N.; Majima, T.; Nishimura, S.; Lee, Y. C., Carbohydrate
analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 2005, 339 (1), 69-72. 6.
Loewus, F. A., Improvement in Anthrone Method for Determination of Carbohydrates. Anal
Chem 1952, 24 (1), 219-219. 7.
Turula, V. E., Jr.; Gore, T.; Singh, S.; Arumugham, R. G., Automation of the anthrone assay for
carbohydrate concentration determinations. Anal Chem 2010, 82 (5), 1786-92. 8.
Geno, K. A.; Gilbert, G. L.; Song, J. Y.; Skovsted, I. C.; Klugman, K. P.; Jones, C.; Konradsen,
H. B.; Nahm, M. H., Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev 2015, 28 (3), 871-99. 9.
Dische, Z.; Shettles, L. B., A specific color reaction of methylpentoses and a spectrophotometric
micromethod for their determination. J Biol Chem 1948, 175 (2), 595-603. 10.
Dische, Z.; Shettles, L. B., A new spectrophotometric test for the detection of methylpentose. J
Biol Chem 1951, 192 (2), 579-82. 11.
Bitter, T.; Muir, H. M., A modified uronic acid carbazole reaction. Anal Biochem 1962, 4, 330-4.
12.
Blumenkrantz, N.; Asboe-Hansen, G., New method for quantitative determination of uronic
acids. Anal Biochem 1973, 54 (2), 484-9. 13.
De Ruiter, G. A.; Schols, H. A.; Voragen, A. G.; Rombouts, F. M., Carbohydrate analysis of
water-soluble uronic
acid-containing polysaccharides with
high-performance
anion-exchange
chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. Anal Biochem 1992, 207 (1), 176-85. 14.
Blumenkrantz, N.; Asboe-Hansen, G., An assay for total hexosamine and a differential assay for
glucosamine and galactosamine. Clin Biochem 1976, 9 (6), 269-74. 15.
Kao, G.; Tsai, C. M., Quantification of O-acetyl, N-acetyl and phosphate groups and
determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance 10
anion-exchange chromatography with conductivity detection (HPAEC-CD). Vaccine 2004, 22 (3-4), 335-44. 16.
Lee, Y. C., High-performance anion-exchange chromatography for carbohydrate analysis. Anal
Biochem 1990, 189 (2), 151-62. 17.
Talaga, P.; Vialle, S.; Moreau, M., Development of a high-performance anion-exchange
chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine 2002, 20 (19-20), 2474-84. 18.
Kim, J. S.; Laskowich, E. R.; Arumugham, R. G.; Kaiser, R. E.; MacMichael, G. J.,
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Canaán-Haden, L.; Cremata, J.; Chang, J.; Valdés, Y.; Cardoso, F.; Bencomo, V. V., High-
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Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate
vaccines. World Health Organization, 2009 (Technical Report Series, No. 977), Annex 3., 91-151.
11
Anthrone
Phenol
2-Phenoxyethanol
Figure 1. Structure of Anthrone, Phenol and 2-Phenoxyethanol
HO
HO O OH
O
O HO HO
H3O+ O
HO OH Glucose
OH
HO -3H 2 O (H 2SO4)
O
2-phenoxyethanH 1-ol O
HO
(O)H 3O+ -H +, -2e (H 2SO 4)
O
5-(hydroxymethyl)furan2-carbaldehyde O
HO
HO
O
O
HO Yellow orange dye wavelength max 500 nm
Figure 2. Reaction of 2-Phenoxyethanol with Sugars in presence of H2SO4 (Based on Molisch’s Test Reaction)
12
Figure 3. Absorption spectra of (A) Glucose and (B) Glucoronic acid upon reacting with 2-PE, Anthrone and Phenol reagents. Inside the figures the λmax is mentioned to indicate the maximum absorbance value obtained at a given wavelength.
13
A) 2-Phenoxyethanol
2.5
Anthrone
Phenol
Absorbance
2.0
1.5
1.0
0.5
0.0 Glucose
Galactose
Rhamnose
Lactose
Mannose
Sucrose
B) 1.0
2-Phenoxyethanol
Anthrone
Phenol
Absorbance
0.8
0.6
0.4
0.2
0.0 Glucuronic acid
Galacturonic acid
14
C) 0.30
2-Phenoxyethanol
Anthrone
Phenol
0.25
Absorbance
0.20 0.15 0.10 0.05 0.00
Figure 4. Absorbance of Monosaccharides upon reacting with 2-PE (500 nm), anthrone (625 nm) and Phenol (490 nm) reagents. (A) Absorbance with Neutral sugars (B) Absorbance with Hexuronic acids and (C) Absorbance with Low-reacting sugars (N-acetyl sugar amines, glycerol and ribitol). All the sugars are of same concentration in all three methods and processed, followed by measuring the absorbance.
15
2.0
2-Phenoxyethanol
Anthrone
Phenol
Absorbance
1.6
1.2
0.8
0.4
0.0 1
3
5
6B
9V
14
19F
22F
23F
Pullulan 800
Polysaccharide
Figure 5. Absorbance of polysaccharides (Pneumococcal polysaccharides and Pullulan 800) upon reacting with 2-PE (500 nm), Anthrone (625 nm) and Phenol (490 nm) reagents. All the polysaccharides are of same concentration in all three methods and processed, followed by measuring the absorbance.
16
2% impurity
5% impurity
100.0
%recovery
80.0
60.0
40.0
20.0
0.0 Protein
Nucleic acids
Na DOC
Impurities
Figure 6. Absorbance of Pneumococcal polysaccharides as % recovery in presence of known impurities (2 and 5% w/v) present in purified polysaccharides. 80 µg/mL of polysaccharide was spiked with 2% and 5% w/v of known impurities (Protein, Nucleic acids and NaDOC). Against the native polysaccharide absorbance (considered as 100%), %recovery of the absorbance with impurities presence was plotted.
Method Anthrone Phenol sulphuric Acid
2-Phenoxyethanol
Application Reagent λmax (Reference) To determine total Carbohydrate Concen- Tricyclic aromatic ke625nm tration (Dreywood; 1946 & Morris; 1948) tone To determine Simple sugars, Oligosaccharides and their derivatives including meth- Aromatic Phenol Com490nm ylated pentose groups (Michel Dubois et ponent al; 1956) To accurately estimate the saccharide conAromatic Phenoxy ethatent in Pneumococcal Polysaccharides and 500nm nol other carbohydrates
Table 1. Carbohydrate estimation methods. 17
Polysaccharide Neutral sugar
Uronic acids
Serotype 1
Two Galacturonic acids
Hexosamines
Other sugars AATGalp
Serotype 3
Glucose
Glucuronic acid
Serotype 5
Glucose
Glucuronic acd
Serotype 6B
Galactose, Glucose, Rhamnose
Serotype 9V
Galactose, Two Glucose
Serotype 14
Glucose, Two Galactose
GlcNAc
Serotype 19F
Glucose, Rhamnose
ManNAc
Serotype 22F
Galactose, Two Glucose, Glucuronic acid Two Rhamnose
Serotype 23F
Galactose, Glucose, Two Rhamnose
Pullulan800
Three Glucose (maltotriose)
FucNAc, PneuNAc
Sugp Ribitol
Glucuronic acid
ManNAc
Glycerol
Table 2. Composition of polysaccharides used in the analysis using Anthrone, Phenol and 2-PE assay. GlcNAc: N-acetyl glucosamine, GalNAc: N-acetyl galacosamine, ManNAc: N-acetyl mannosamine, FucNAc: N-acetyl fucosamine and PneuNAc: N-acetyl pneumosamine, AATGalp: 2-acetamido-4amino-2,4,6-trideoxy-D-galactose, SugP: 2-acetamido-2,6-dideoxy-D-xylo-heos-4-ulose.
18
Sample Details
6B Polysaccharide
6B mono-conjugate
7F Polysaccharide
7F mono-conjugate
Multivalent vaccine with 0.25 mg/mL Al+3 Multivalent vaccine with 0.5 mg/mL Al+3 Multivalent vaccine with 1.0 mg/mL Al+3
4 8 16 24 32 40 100 4 8 16 24 32 40 100 4 8 16 24 32 40 100 4 8 16 24 32 40 100
Concentration by 2-PE method (µg/mL) 2.8 7.4 14 24 34 40 100 1 8 17 22 32 40 98 0.1 7.2 15 26 35 44 97 1.2 7.3 15 23 32 40 102
% Recovery of the total Ps content 69 92 90 101 107 99 100 25 100 106 92 100 100 98 3 90 94 108 110 110 97 30 91 94 96 100 100 102
70
68
97
70
71
101
70
69
99
Expected Concentration (µg/mL)
19
Table 3. Total polysaccharide content in serotypes 6B, 7F polysaccharides and their mono-conjugates including multivalent Pneumococcal conjugate vaccine by 2-PE method. %recovery was calculated against the expected total concentration and obtained concentration.
Sugar
LOD (µg)
LOQ (µg)
Glucose
0.25
0.76
Galactose
0.36
1.10
Rhamnose
0.38
1.15
Lactose
0.26
0.78
Mannose
0.19
0.57
Sucrose
0.22
0.66
Glucuronic acid
0.57
1.72
Galacturonic acid
0.46
1.41
N-Acetyl-Mannosamine
1.91
5.78
Table 4. Limit of detection (LOD) and Limit of quantification values of individual sugars using 2-PE method.
20
Highlights: •
2-Phenoxyethanol reagent is more sensitive than Anthrone reagent for carbohydrate quantification
•
2-Phenoxyethanol reactivity is high for uronic acids sugars
•
A suitable method for quantification of any type of carbohydrate/polysaccharides
•
Greater application in Vaccines and Biologicals
1
S0003-2697(19)31176-5
DOI:
https://doi.org/10.1016/j.ab.2020.113624
Reference:
YABIO 113624
To appear in:
Analytical Biochemistry
Received Date: 27 November 2019 Revised Date:
22 January 2020
Accepted Date: 10 February 2020
Please cite this article as: B. Rajendar, R. Mulagalapati, M.V.N.J. Reddy, S. Patri, Y.K. Karthik, R.V. Matur, 2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification, Analytical Biochemistry (2020), doi: https://doi.org/10.1016/j.ab.2020.113624. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Authors statement B R, R M and M V N J R designed the experiments, reviewed the data and wrote the manuscript S P and Y K K executed the experiments R V M is conceptualized the work and reviewed the manuscript All authors discussed the results and contributed to the final manuscript
1
2-Phenoxyethanol: A Novel Reagent for Improved Sensitivity of carbohydrate quantification
Burki Rajendar,* Ravikumar Mulagalapati, M V N Janardhan Reddy, Sumapriya Patri, Yellepeddi K Karthik, Ramesh V. Matur,*
Research & Development, Biological E Limited, Shameerpet, Hyderabad 500078, India
*
To whom correspondence should be addressed
CORRESPONDING AUTHOR FOOTNOTE Dr. Ramesh V. Matur Research & Development, Biological E Limited, Plot No.1, Phase II, SP Biotech Park, Genome Valley, Shameerpet, Hyderabad 500078, India Tel: +91-40-6738-8227, E-mail: [email protected] Dr. Burki Rajendar Tel: +91-40-6738-8229, E-mail: [email protected] Keywords: Anthrone assay, 2-Phenoxyethanol, Polysaccharide quantification, Carbohydrate analysis Abbrevations used: 2-PE, 2-Phenoxyethanol, AlPO4, Aluminium Phosphate, OD, Optical Density, H2SO4, Sulphuric Acid
ABSTRACT
Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulfuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm. Though anthrone reacts well with polysaccharides containing hexoses (such as glucose and galactose) and rhamnose, it is less reactive with uronic acids (such as glucuronic acid and galacturonic acid) and hexosamines (such as fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). Here, we report a novel reagent, 2-Phenoxyethanol, which reacts with furfural or hydroxymethyl furfural resulting in higher absorptivity. This method is rapid, sensitive, simple and direct, and can be used for quantitative determination of any type of carbohydrate that contains neutral sugars and uronic acids. For these saccharides, the sensitivity of the assay using 2-Phenoxyethanol (2-PE) is twice over anthrone method. Uronic acids show improved sensitivity using 2-PE over Phenol and it is more than twice with glucuronic acid. 2-PE reagent method has greater application for quantification of carbohydrates when present in low concentration
in
vaccines/biologicals.
1. Introduction Quantification of carbohydrate/polysaccharide content is an essential analytical procedure in food and beverages, nutraceuticals, agricultural products, medicinal products and vaccine pharmaceuticals. Polysaccharides are quantified by colorimetric assay using anthrone reagent and glucose as standard.1 Other reagents such as orcinol, or phenol sulphuric acid with colorimetric reaction are also largely been used for polysaccharide estimation (Table 1).1-5 Several modifications of the anthrone method (concentration of anthrone reagent and H2SO4, heating time and temperature, etc.) were reported earlier for improving the sensitivity of the method.6 These modifications were aimed at enhancement of the color development of the anthrone-furfural complex, which improves the optical density signal, thereby enhancing quantification sensitivity of the assay. On the other hand, the color development is still low with these methods for analyzing polysaccharides containing mixtures of uronic acids (glucoronic acid and galacturonic acid) and hexosamines (fucosamine, glucosamine, galactosamine, mannosamine, pneumosamine). These methods do not generate optimum color complexes and suffer from low sensitivity making
them less applicable for estimating such carbohydrates/polysaccharides, especially for polysaccharide based vaccines. During carbohydrate quantification in presence of sulphuric acid, hexoses and pentoses are hydrolyzed and converted to 5-Hydroxymethyl furfural and furfural respectively. These molecules generated by acid hydrolysis are then reacted with anthrone or phenol to form color complex, which is then measured for optical density at a specific wavelength. The absorption maxima for the Anthrone-Furfural complex is at 625 nm7 and that of the phenol-furfural complex is at 490 nm.4 As mentioned earlier, the color intensity at the maximum absorbance wavelength in both these methods varies with the composition of the polysaccharide, as different polysaccharides contain different sugars such as hexoses, pentoses, uronic acids, hexosamines, and pentosamines etc. , and in different proportions. Therefore, there exists a critical need for further optimization of existing methods to enhance the color development and thereby enhancing the sensitivity through development of novel methods. Thus the increased signal intensity helps to estimate the smallest amount of polysaccharide present in the sample. For pneumococcal conjugate vaccines, serotype specific polysaccharides are conjugated to a carrier protein and therefore controlling each serotype polysaccharide amount in the prescribed dose is critical for maintaining the quality and efficacy of the vaccine. Accurate quantification of pneumococcal polysaccharides at microgram level is very essential in developing these vaccines. Pneumococcal polysaccharides are comprised of complex mixtures of hexoses, pentoses, uronic acids and hexosamines.8 Several investigators have recognized the importance of making these colorimetric assays more sensitive, high-throughput by scaling down to microplates. Two common colorimetric assay reagents used for this purpose are anthrone and phenol-sulfuric acid. We noticed that the reactivities of anthrone/sulphuric acid or phenol/sulphuric acid with polysaccharides containing uronic acids and hexosamines were very weak and could not be quantitated by these two methods. For quantification of the total polysaccharide content of pneumococcal serotypes or their components (methyl pentoses,9-10 uronic Acids,11-13 hexosamines,14 O-acetyl,15 phosphorous and nitrogen) various biochemical methods were reported. Majority of them use acid hydrolysis to release the monosaccha3
rides, which are then quantified using high-performance anion-exchange chromatography (HPAEC)16-17 with pulsed amperometric detection (PAD), or acid hydrolysis along with derivatization followed by quantification of monosaccharides using Gas chromatography connected to a mass selective detector (GC-MSD).18 Another method reported for quantification of pneumococcal polysaccharides containing hexosamines involves acid hydrolysis, re-N-acetylation followed by labeling the hexosamines with 2aminobenzamide by reductive amination and RP-HPLC with fluorescence detector.19 Although these methods are sensitive and offer the required accuracy they are laborious, time consuming and may not be amenable for Quality Control scientists. Here we report a novel method that utilizes an aromatic ethanol component, 2-Phenoxyethanol (Figure 1), which reacts the same way as Anthrone or Phenol with significant enhancement in color development. This reaction is based on the Molisch’s Test (Figure 2). The 2-Phenoxyethanol reacts with the furfural or 5-Hydroxymethylfurfural formed from acid hydrolysis of polysaccharide resulting in the formation of a color complex with an absorbance maximum at 500 nm. We also demonstrated that this colorimetric assay is suitable for components containing uronic acids unlike for the anthrone assay. 2. Materials and methods 2.1 Chemicals and Reagents All pneumococcal polysaccharides used were prepared in-house and confirmed their structure by 1H NMR analysis. Suitable purification steps were employed to remove the endotoxins, protein and nucleic acid impurities. The final purified capsular polysaccharides were found to be in compliance with specified composition20 for methyl pentoses, uronic acids, hexosamines, O-acetyl, phosphorous and nitrogen contents. N-acetyl L-fucosamine and N-acetyl L-pneumosamine were purchased from Omicron Biochemicals, Inc. USA. Pullulan 800, rhamnose, glucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine, glucuronic acid, galacturonic acid, and all other reagents and buffer components were purchased from Sigma-Aldrich, India. 2.2 2-PE-Sulphuric Acid Assay
4
All monosaccharides and polysaccharides were diluted to 80 µg per mL based on the dry weight. Dispensed 250 µL (80 µg/mL) containing 20 µg monosaccharides and/or polysaccharides in triplicates in to clean and dry glass tubes. Each standard of 0-20 µg in 250 µL was taken in a clean glass tube. Water 250 µL was taken as blank in triplicates. Added 10 µL of 98% 2-Phenoxyethanol (2-PE) and 500 µL of H2SO4 to all the tubes and vortexed gently. Incubated the tubes in a water bath at 90°C for 5 min. Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 500 nm using plate reader. 2.3 Phenol-Sulphuric Acid Assay5 Dispensed 165 µL containing 20 µg monosaccharides and/or polysaccharides in to clean and dry glass tubes. Each standard of 0-20 µg in 165 µL was taken in a clean glass tube as the standard along with water blank. Added 500 µL of H2SO4 and 100 µL of 5% phenol reagent to the sample/ standard/blank tubes and incubated for 5 mins at 90˚C Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 490 nm using plate reader. 2.4 Anthrone-Sulphuric Acid Assay1 Dispensed 250 µL containing 20 µg monosaccharides and/or polysaccharides in to clean and dry glass tubes. Each standard of 0-20 µg in 250 µL was taken in a clean glass tube as the standard along with water blank. Added 500 µL of anthrone reagent (0.2% w/v anthrone in concentrated H2SO4) and incubated for 5 mins at 90˚C. Cooled the tubes to room temperature, transferred 250 µL into microplate and measured the absorbance at 625 nm using plate reader. In all above listed three methods the final reaction volumes were ~750 to 765 µL and 250 uL was transferred into microplate for absorbance measurements. 2.5 Sample preparation of mono-conjugates and PCV drug product Serotypes 6B, 7F monovalent bulk conjugates were pre-diluted to 4-100 µg/mL and subjected for total saccharide content analysis using 2-PE as test reagent. Similarly, the multivalent Pneumococcal conju-
5
gate vaccine adsorbed onto aluminum phosphate (equivalent to ~70 µg/mL of polysaccharides) was subjected to total saccharide content analysis using 2-PE as test reagent. 3. Results 3.1 Reactivity of Monosaccharides The relative absorbance spectra of the color complex formed by the reaction of glucose and glucuronic acid with H2SO4 followed by the reaction with 2-PE, anthrone and phenol are presented in Figure 3A and 3B. In presence of H2SO4, glucose reacts with anthrone resulting in the formation of a green colored complex having an absorbance maximum at 625-630 nm (Figure 3A).7 Similarly when these furfural structures react with 2-PE, they give an orange yellow color complex having absorbance maxima at 500 nm (Figure 3) and with phenol, they produce yellow color complex with an absorbance maxima at 490 nm (Figure 3). The absorption spectrum of the color complex formed by glucuronic acid standard with 2-PE and phenol is similar to that of glucose. However, the reactivity of anthrone reagent with the furfural derivative of glucuronic acid appears to be poor with no sharp absorption maxima as seen for phenol or 2-PE. To facilitate appropriate comparisons between all 3 assays (2-PE, anthrone and phenol sulfuric acid), the total volume for each reaction procedure was adjusted to 750 to 765 uL. Figure 4 provides comparison of the 3 colorimetric assays for neutral sugars (Figure 4A: glucose, galactose, rhamnose, lactose, mannose and sucrose), hexuronic acids (Figure 4B: glucuronic acid, galacturonic acid), N-acetyl sugar amines (N-acetyl fucosamine, N acetyl pneumosamine, N acetyl galactosamine, N acetyl mannosamine), ribitol and glycerol (Figure 4C). Overall the absorbance values for all neutral sugars as well as hexuronic acids are relatively higher for the 2-PE based assay compared to that of anthrone and phenol assays (Figure 4A and B). However, N-acetyl sugar amines, ribitol and glycerol are not sensitive to any of the reagents tested, although 2-PE appears to be slightly better in some cases (Figure 4C). Linearity plots (Supporting information; Figure S1) were generated for the sugars, which showed higher and moderate reactivity. As shown in Table 4, the LOD and LOQ was determined from the linearity pots. 3.2 Reactivity of Pneumococcal Polysaccharides 6
Reactivity of 2-PE with pneumococcal polysaccharides were compared (Figure 5). Similar to the results observed for the monosaccharides, the reactivity of all polysaccharides are relatively higher with 2-PE reagent compared to that of phenol and anthrone. Between anthrone and phenol reagents, the reactivity of phenol with polysaccharides is slightly better resulting in higher sensitivity (Figure 5). The absorption spectra for all the polysaccharide hydrolysates are shown in Supporting information, Figure S2. Each serotype of pneumococcal polysaccharide has unique composition of monosaccharides such as hexoses, pentoses, uronic acids, hexosamines etc depending on the serotype. Although some polysaccharides8 such as Serotype 5, 14, 19F and 22F contain N-acetyl glucosamine, N-acetyl galactosamine, N-acetyl mannosamine, N-acetyl fucosamine and N-acetyl pneumosamine which demonstrated low reactivity (Figure 4C), these polysaccharides still react well with 2-PE due to the presence of other reacting sugars (hexose, pentose, hexuronic acid) (Figure 5 and Table 2) enabling to quantify with improved sensitivity over other methods. 3.3 Polysaccharide quantification in mono-conjugate and multivalent adsorbed vaccine drug product The newly developed 2-PE method was applied not only for the vaccine intermediates containing purified serotype specific polysaccharides, but also for those polysaccharides conjugated to a carrier protein as the monovalent conjugate drug substance as well as the drug product containing a mixture of monovalent conjugate drug substances including AlPO4. The known concentration of various serotype pneumococcal polysaccharides and different known concentrations of serotype 6B & 7F Poly-protein conjugates ranging from 4 to 100 µg/mL were used to determine the measured concentration in order to establish a linear range in which the % recovery is within the accepted level 90-110%. Furthermore, multivalent conjugate vaccine samples containing mixture of 14 different pneumococcal serotype conjugates at a known polysaccharide concentration of 70 µg/mL were subjected to analysis by 2-PE assay. The recovery was between 90-110 %, at the linearity range of 8-100 µg/mL (Table 3). Similarly, the % recovery of total polysaccharides in pneumococcal multivalent adsorbed conjugate vaccine at different Al+3 concentrations (0.25, 0.5 and 1.0 mg/mL of AlPO4 gel) were 97, 101 and 99 7
respectively (Table 3), indicating no interference with aluminum phosphate in the drug product. In addition, the presence of carrier protein (closer to the equal amount of the polysaccharide content) in the monovalent conjugates and the polyvalent drug product doesn’t interfere with the polysaccharide quantitation. 3.4 Interference from impurities and Validation The most common impurities in purified polysaccharides are nucleic acids and residual reagents. The interference of these impurities at 2 and 5% w/v against total polysaccharide concentration were assessed with 2-PE reagent (Figure 6). At 2% impurity level (which is maximum allowed limit as per regulatory guidance) there was no or negligible effect on the recovery. However, when impurities at 5%, there was a marginal effect on the total polysaccharide recovery (Figure 6). We further evaluated this method for polysaccharide quantification in Polysaccharide-CRM197 conjugates. The polysaccharide concentration in serotype 6B and 7F (Pneumococcal polysaccharide-CRM conjugates) were estimated at entire standard range to determine the assay linearity (Table 3). The assay is linear from 8 to 100µg /mL (Table 3) based on the % recovery of the polysaccharide or conjugates. 4. Discussion Anthrone1 and other sugar reacting agents such as phenol sulphuric acid show less reactivity with uronic acids and hexosamines present in the polysaccharides. In this paper, we have demonstrated the utility of 2-PE reagent to provide substantially higher sensitivity for saccharides containing neutral sugars and uronic acid containing saccharides. Production and quality control of vaccines such as pneumococcal conjugate vaccines are highly complex and take about a year from start to finish. In general, the manufacturing process requires fermentation and purification of individual serotype specific polysaccharides to high level of purity with least amount of residual impurities such as nucleic acids, proteins, endotoxin, as well as residuals from the purification components. Once these serotype specific polysaccharides are purified and quality controlled, then they are subjected to the activation process prior to the conjugation to a carrier protein. Following the conjugation to a carrier protein, these individual conjugated polysaccharides are further puri8
fied using a variety of chromatographic and/or other purification methodologies and further quality controlled to release each serotype specific conjugate as Drug Substance. The final step of the manufacturing process involves formulation of all 14 different serotype specific polysaccharide conjugates based on the polysaccharide content, with appropriate excipients including the adjuvant such as aluminum phosphate. In each and every step of the production process, the determination of saccharide content becomes very crucial starting from polysaccharide purification process all the way to the final drug product as in-process, release and stability parameters. Therefore, we needed a very sensitive assay for the determination of polysaccharide content we developed this sensitive 2-PE based assay. The method that we developed is very useful in quality control as little or no interference with carrier protein and aluminum adjuvant present in polysaccharide-protein conjugate vaccine. Furthermore, the presence of residual amounts of protein, nucleic acids and sodium deoxycholate is shown to have negligible effect on the assay values. This assay was developed using test tubes, the methodology can also be adopted to automated microtiter plate based high-throughput assay.7 5. Conclusions We reported a novel method for polysaccharide quantification using 2-PE by colorimetry. We demonstrated that different monosaccharides (hexoses, uronic acids and hexosamines), whole polysaccharides, polysaccharide-protein conjugates can be quantified using 2-PE as a reagent with increased sensitivity over anthrone as well as the phenol reagent. The polysaccharides isolated from bacteria (S. pneumoniae, A. pullulans) 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 polysaccharide quantitation.
Acknowledgements We thank the Scientific Advisory Board and Management of Biological E Limited for their active support and encouragement. 9
References 1.
Morris, D. L., Quantitative Determination of Carbohydrates With Dreywood's Anthrone
Reagent. Science 1948, 107 (2775), 254-5. 2.
Trevelyan, W. E.; Forrest, R. S.; Harrison, J. S., Determination of yeast carbohydrates with the
anthrone reagent. Nature 1952, 170 (4328), 626-7. 3.
Cuesta, G.; Suarez, N.; Bessio, M. I.; Ferreira, F.; Massaldi, H., Quantitative determination of
pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method. J Microbiol Methods 2003, 52 (1), 69-73. 4.
DuBois, M. G., K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F., Colorimetric Method for
Determination of Sugars and Related Substances. Anal Chem 1956, 28 (3), 350. 5.
Masuko, T.; Minami, A.; Iwasaki, N.; Majima, T.; Nishimura, S.; Lee, Y. C., Carbohydrate
analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 2005, 339 (1), 69-72. 6.
Loewus, F. A., Improvement in Anthrone Method for Determination of Carbohydrates. Anal
Chem 1952, 24 (1), 219-219. 7.
Turula, V. E., Jr.; Gore, T.; Singh, S.; Arumugham, R. G., Automation of the anthrone assay for
carbohydrate concentration determinations. Anal Chem 2010, 82 (5), 1786-92. 8.
Geno, K. A.; Gilbert, G. L.; Song, J. Y.; Skovsted, I. C.; Klugman, K. P.; Jones, C.; Konradsen,
H. B.; Nahm, M. H., Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev 2015, 28 (3), 871-99. 9.
Dische, Z.; Shettles, L. B., A specific color reaction of methylpentoses and a spectrophotometric
micromethod for their determination. J Biol Chem 1948, 175 (2), 595-603. 10.
Dische, Z.; Shettles, L. B., A new spectrophotometric test for the detection of methylpentose. J
Biol Chem 1951, 192 (2), 579-82. 11.
Bitter, T.; Muir, H. M., A modified uronic acid carbazole reaction. Anal Biochem 1962, 4, 330-4.
12.
Blumenkrantz, N.; Asboe-Hansen, G., New method for quantitative determination of uronic
acids. Anal Biochem 1973, 54 (2), 484-9. 13.
De Ruiter, G. A.; Schols, H. A.; Voragen, A. G.; Rombouts, F. M., Carbohydrate analysis of
water-soluble uronic
acid-containing polysaccharides with
high-performance
anion-exchange
chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. Anal Biochem 1992, 207 (1), 176-85. 14.
Blumenkrantz, N.; Asboe-Hansen, G., An assay for total hexosamine and a differential assay for
glucosamine and galactosamine. Clin Biochem 1976, 9 (6), 269-74. 15.
Kao, G.; Tsai, C. M., Quantification of O-acetyl, N-acetyl and phosphate groups and
determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance 10
anion-exchange chromatography with conductivity detection (HPAEC-CD). Vaccine 2004, 22 (3-4), 335-44. 16.
Lee, Y. C., High-performance anion-exchange chromatography for carbohydrate analysis. Anal
Biochem 1990, 189 (2), 151-62. 17.
Talaga, P.; Vialle, S.; Moreau, M., Development of a high-performance anion-exchange
chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine 2002, 20 (19-20), 2474-84. 18.
Kim, J. S.; Laskowich, E. R.; Arumugham, R. G.; Kaiser, R. E.; MacMichael, G. J.,
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11
Anthrone
Phenol
2-Phenoxyethanol
Figure 1. Structure of Anthrone, Phenol and 2-Phenoxyethanol
HO
HO O OH
O
O HO HO
H3O+ O
HO OH Glucose
OH
HO -3H 2 O (H 2SO4)
O
2-phenoxyethanH 1-ol O
HO
(O)H 3O+ -H +, -2e (H 2SO 4)
O
5-(hydroxymethyl)furan2-carbaldehyde O
HO
HO
O
O
HO Yellow orange dye wavelength max 500 nm
Figure 2. Reaction of 2-Phenoxyethanol with Sugars in presence of H2SO4 (Based on Molisch’s Test Reaction)
12
Figure 3. Absorption spectra of (A) Glucose and (B) Glucoronic acid upon reacting with 2-PE, Anthrone and Phenol reagents. Inside the figures the λmax is mentioned to indicate the maximum absorbance value obtained at a given wavelength.
13
A) 2-Phenoxyethanol
2.5
Anthrone
Phenol
Absorbance
2.0
1.5
1.0
0.5
0.0 Glucose
Galactose
Rhamnose
Lactose
Mannose
Sucrose
B) 1.0
2-Phenoxyethanol
Anthrone
Phenol
Absorbance
0.8
0.6
0.4
0.2
0.0 Glucuronic acid
Galacturonic acid
14
C) 0.30
2-Phenoxyethanol
Anthrone
Phenol
0.25
Absorbance
0.20 0.15 0.10 0.05 0.00
Figure 4. Absorbance of Monosaccharides upon reacting with 2-PE (500 nm), anthrone (625 nm) and Phenol (490 nm) reagents. (A) Absorbance with Neutral sugars (B) Absorbance with Hexuronic acids and (C) Absorbance with Low-reacting sugars (N-acetyl sugar amines, glycerol and ribitol). All the sugars are of same concentration in all three methods and processed, followed by measuring the absorbance.
15
2.0
2-Phenoxyethanol
Anthrone
Phenol
Absorbance
1.6
1.2
0.8
0.4
0.0 1
3
5
6B
9V
14
19F
22F
23F
Pullulan 800
Polysaccharide
Figure 5. Absorbance of polysaccharides (Pneumococcal polysaccharides and Pullulan 800) upon reacting with 2-PE (500 nm), Anthrone (625 nm) and Phenol (490 nm) reagents. All the polysaccharides are of same concentration in all three methods and processed, followed by measuring the absorbance.
16
2% impurity
5% impurity
100.0
%recovery
80.0
60.0
40.0
20.0
0.0 Protein
Nucleic acids
Na DOC
Impurities
Figure 6. Absorbance of Pneumococcal polysaccharides as % recovery in presence of known impurities (2 and 5% w/v) present in purified polysaccharides. 80 µg/mL of polysaccharide was spiked with 2% and 5% w/v of known impurities (Protein, Nucleic acids and NaDOC). Against the native polysaccharide absorbance (considered as 100%), %recovery of the absorbance with impurities presence was plotted.
Method Anthrone Phenol sulphuric Acid
2-Phenoxyethanol
Application Reagent λmax (Reference) To determine total Carbohydrate Concen- Tricyclic aromatic ke625nm tration (Dreywood; 1946 & Morris; 1948) tone To determine Simple sugars, Oligosaccharides and their derivatives including meth- Aromatic Phenol Com490nm ylated pentose groups (Michel Dubois et ponent al; 1956) To accurately estimate the saccharide conAromatic Phenoxy ethatent in Pneumococcal Polysaccharides and 500nm nol other carbohydrates
Table 1. Carbohydrate estimation methods. 17
Polysaccharide Neutral sugar
Uronic acids
Serotype 1
Two Galacturonic acids
Hexosamines
Other sugars AATGalp
Serotype 3
Glucose
Glucuronic acid
Serotype 5
Glucose
Glucuronic acd
Serotype 6B
Galactose, Glucose, Rhamnose
Serotype 9V
Galactose, Two Glucose
Serotype 14
Glucose, Two Galactose
GlcNAc
Serotype 19F
Glucose, Rhamnose
ManNAc
Serotype 22F
Galactose, Two Glucose, Glucuronic acid Two Rhamnose
Serotype 23F
Galactose, Glucose, Two Rhamnose
Pullulan800
Three Glucose (maltotriose)
FucNAc, PneuNAc
Sugp Ribitol
Glucuronic acid
ManNAc
Glycerol
Table 2. Composition of polysaccharides used in the analysis using Anthrone, Phenol and 2-PE assay. GlcNAc: N-acetyl glucosamine, GalNAc: N-acetyl galacosamine, ManNAc: N-acetyl mannosamine, FucNAc: N-acetyl fucosamine and PneuNAc: N-acetyl pneumosamine, AATGalp: 2-acetamido-4amino-2,4,6-trideoxy-D-galactose, SugP: 2-acetamido-2,6-dideoxy-D-xylo-heos-4-ulose.
18
Sample Details
6B Polysaccharide
6B mono-conjugate
7F Polysaccharide
7F mono-conjugate
Multivalent vaccine with 0.25 mg/mL Al+3 Multivalent vaccine with 0.5 mg/mL Al+3 Multivalent vaccine with 1.0 mg/mL Al+3
4 8 16 24 32 40 100 4 8 16 24 32 40 100 4 8 16 24 32 40 100 4 8 16 24 32 40 100
Concentration by 2-PE method (µg/mL) 2.8 7.4 14 24 34 40 100 1 8 17 22 32 40 98 0.1 7.2 15 26 35 44 97 1.2 7.3 15 23 32 40 102
% Recovery of the total Ps content 69 92 90 101 107 99 100 25 100 106 92 100 100 98 3 90 94 108 110 110 97 30 91 94 96 100 100 102
70
68
97
70
71
101
70
69
99
Expected Concentration (µg/mL)
19
Table 3. Total polysaccharide content in serotypes 6B, 7F polysaccharides and their mono-conjugates including multivalent Pneumococcal conjugate vaccine by 2-PE method. %recovery was calculated against the expected total concentration and obtained concentration.
Sugar
LOD (µg)
LOQ (µg)
Glucose
0.25
0.76
Galactose
0.36
1.10
Rhamnose
0.38
1.15
Lactose
0.26
0.78
Mannose
0.19
0.57
Sucrose
0.22
0.66
Glucuronic acid
0.57
1.72
Galacturonic acid
0.46
1.41
N-Acetyl-Mannosamine
1.91
5.78
Table 4. Limit of detection (LOD) and Limit of quantification values of individual sugars using 2-PE method.
20
Highlights: •
2-Phenoxyethanol reagent is more sensitive than Anthrone reagent for carbohydrate quantification
•
2-Phenoxyethanol reactivity is high for uronic acids sugars
•
A suitable method for quantification of any type of carbohydrate/polysaccharides
•
Greater application in Vaccines and Biologicals
1