Further Insights into the Measurement of Free Polysaccharide in Meningococcal Conjugate Vaccines

Abstract

Objectives: The purpose of this study was to further characterize the ultrafiltration (UF) method for determining free saccharide levels in glycoconjugate vaccines and compare it with other methods used for the determination of free saccharide levels in meningococcal glycoconjugate vaccines. Methods: We performed experiments on both meningococcal glycoconjugates and capsular polysaccharides, and compared UF, deoxycholate (DOC) precipitation, and solid-phase extraction (SPE) methods. Meningococcal capsular polysaccharides from groups A (MenA), C (MenC), and W (MenW) were depolymerized and characterized using SEC-MALS (size-exclusion chromatography with multi-angle laser light scattering) to determine the molecular weight and hydrodynamic size and then subjected to UF. The free saccharide content was quantified using HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Results: The characterization of size-reduced group C polysaccharide revealed weight-average molecular mass (Mw) ranging from 22,200 g/mol to 287,300 g/mol and hydrodynamic radii of 3.7 to 19.5 nm. Pore size studies confirmed that polysaccharides with diameters up to 15 nm filtered through the 100 kDa cellulose membrane. The smallest PS fragment tested (22,200 g/mol, 7.4 nm diameter) was partially recovered from the 30 kDa membrane. For MenC-CRM₁₉₇, DOC yielded the lowest free saccharide content (<1%), UF gave moderate results (7-8%), and SPE showed the highest and most variable values (up to 15%). For MenA- and MenW-CRM₁₉₇, UF and DOC consistently provided low free saccharide levels (<2% and 3-11%, respectively). Conclusions: The upper limits on the size of free group C meningococcal polysaccharides that can be ultrafiltered were assessed. Differences in the relative amount of free saccharide were observed between various methods used to control meningococcal conjugate vaccines.

Keywords: CRM; HPAEC; HPLC; Neisseria meningitidis; filtration; glycoconjugate; meningitis; meningococcal; stability.

Figure 1

SEC chromatograms of the purified MenC fractions after depolymerization and desalting. MenC full-length PS (blue) was subjected to hydrolysis for 15 (red), 30 (green), and 90 min (purple). The injections were performed using a Tosoh TSK PW_XL guard column in series with Tosoh TSKgel G6000 + 5000 PW_XL HPLC columns with PBS “A” as the mobile phase. The void volume (V₀) and total column volume (V_t) were determined using salmon DNA (Sigma Aldrich) and tyrosine (Sigma Aldrich), measured at 24.6 and 49.9 min, respectively. The absorbance of the polysaccharides was measured at 214 nm (indicated on the y-axis), which detects their amine, carbonyl, and carboxyl groups. Peak elution times are displayed for each.

Figure 2

Size characterization of ultrafiltration membranes using meningococcal group C polysaccharides. (a) Recovery of sized MenC polysaccharides from Microcon-10, -30 kDa, and -100 kDa ultrafiltration membranes. Percent filtrate recovery values are listed on each bar. For two samples run with the 10 kDa membrane, n.d., no data were recovered. (b) Effect of PS diameter on PS recovery. Data points are from purified PS samples: full-length (blue), 15 min (red), 30 min (green), and 90 min (purple) hydrolysates. The recovery of PS from ultrafiltration membranes with the measured diameters from this study and NMWCO values: 10 kDa (squares), 30 kDa (circles), and 100 kDa (diamonds) membranes are shown.