High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides

Highlights

• Sensitive method for the determination of low-level quantities of nitrogen and phosphorous.

• A suitable method for quantification of nitrogen and phosphorous in any polysaccharide.

• Greater application in vaccines manufacturing and release.

Abstract

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods. However, available methods for quantifying nitrogen and phosphorus are not sensitive enough and laborious. We report a highly sensitive high-performance anion-exchange chromatography-conductivity detector (HPAEC-CD) method for quantifying nitrogen and phosphorus present in pneumococcal capsular polysaccharides. The method is reliable, robust and reproducible with no interference. The LOQ for nitrogen and phosphorus of 3.125 and 62.5 ng/mL, respectively, is highly critical for estimating low levels of total nitrogen and total phosphorus. We have implemented this method to quantify total nitrogen in Typhoid Vi polysaccharide and phosphorus in Haemophilus influenzae type-b polysaccharide. This method has greater application for quantification of nitrogen and phosphorus present in low concentrations in polysaccharide vaccines/biologicals.

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.