Short communicationHigh-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides
Introduction
Pneumococcal vaccines protect from infection by serotypes most frequently associated with severe pneumococcal diseases [1], [2]. The pneumococcal capsular polysaccharides (PnPs) are used to manufacture conjugated and unconjugated vaccines. Based on the sugar composition and linkage of the glycosidic bond, they are classified into different serotypes. The pneumococcal polysaccharides are heteropolymers constituting sugar derivatives, such as uronic acids, hexosamines, methyl pentose and other functional groups are attached to the sugars, such as N-Acetyl and O-acetyl groups, nitrogen, and phosphorus.
The amines (hexosamines) are significant sources of nitrogen in the polysaccharides. The polysaccharides contain 0 to 7% phosphorus and 0 to 6% nitrogen [3], [4]. Not only the amines, but the protein impurity in the Ps also contributes to the total % nitrogen content. Similarly, the nucleic acid impurity in the Ps contributes to the total % phosphorus. As part of the Ps release, meeting the composition specification as per recommended guidelines [4] is essential in producing polysaccharide conjugate vaccines. The list of recommended biochemical methods for polysaccharide composition analysis are, uronic acid by Carbazole method [5], hexosamines by Elson Morgan method [6], methyl pentose by cysteine HCl method [7], O-acetyl groups by Hestrin method [8] or HPAEC-CD [9], nitrogen by Kjeldahl method [10], [11] and phosphorus by Chen method [12].
Except for nitrogen and phosphorus, methods recommended in pharmacopeia for the remaining components (uronic acids, hexosamines, methyl pentose, and o-acetyl) are suitable for polysaccharide release. Due to their poor sensitivity and recoveries, total nitrogen quantification by Kjeldahl and phosphorus by Chen methods are not ideal for analyzing pneumococcal polysaccharides. The assay execution must happen manually, so there is no scope to comply with the 21CFR Part 11 requirement. The equipment employed does not have any ability for system-generated reports.
Using a single method, we report a highly sensitive and accurate ion chromatography-based method to determine total nitrogen and phosphorus in different polysaccharides. Besides, as the method execution is fully automated and controlled by a software can comply with the 21CFR Part 11 compliance. We have demonstrated that this method can replace the nitrogen estimation by Kjeldahl and phosphorus estimation by Chen methods recommended in pharmacopeia for polysaccharide and conjugate vaccine analysis.
Section snippets
Materials and methods
Thermo Scientific ICS-5000 Plus (Thermo Fisher Scientific, Sunnyvale, CA, USA) system was used in this study. The system integrates an ion exchange chromatography system with a dual-piston pump, vacuum degas and AS-AP auto sampler with a six-port injection valve coupled with a 25 µL injection loop. In addition, the system is equipped with a column compartment with temperature control. The total nitrogen and total phosphorus in the form of nitrate and phosphate are detected by a conductivity
Method optimization and interference
Since all the polysaccharides were purified, they do not contain anion molecules like chloride, chlorate, nitrites, carbonate, sulfides and others, and hence there will be no potential anion interference in quantifying low levels of total phosphorus as phosphates (P) and total nitrogen as nitrates (N).
We subjected BSA and ribose-5-phosphate to alkaline persulfate digestion as shown in Scheme 1, and analyzed by HPAEC-CD method [15]. The %nitrogen and %phosphorous in both samples matched with
Conclusions
We reported a novel method for quantifying low amounts of nitrogen and phosphorus present in purified polysaccharides using HPAEC-CD. The reported method is very sensitive (∼0.001% w/w), based on the dry weight of polysaccharides with good resolution and peak profiles. The developed method also met the method validation parameters of linearity, specificity, accuracy, precision, and LOQ. This method can be applied in the manufacturing of polysaccharide vaccines. The method is simple, direct, and
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank the Scientific Advisory Board and Management of Biological E Limited for their active support and encouragement.
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