Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use

Abstract

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM₁₉₇), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine's critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.

Keywords: Neisseria meningitidis; acetylation; adjuvant; chromatography; glycoconjugates; immunogenicity; meningitis; nuclear magnetic resonance spectroscopy; polysaccharide; serum bactericidal.

Figure 1

Percentage O-acetylation levels of purified capsular polysaccharide from meningococcal groups A, C, Y and W manufactured for Phase 1, 2 and 3 clinical and consistency lots of NmCV-5. ¹H-NMR spectroscopy was performed at NIBSC to determine the mol O-acetyl/mol repeating unit. The dotted lines indicate the lower limits according to WHO Guidelines for meningococcal polysaccharides [24] and conjugates [25,26].

Figure 2

Molecular size chromatograms of representative lots of group A, C, Y, W and X polysaccharides (A) and bulk conjugates (B) manufactured for Phase 3 trials with NmCV-5. A TSK 6000 + 5000_PWXL column series was used with a PBS (pH 7.4) buffer. Vo and Vt markers eluted at 46.2 min and 99.2 min, respectively. Data were obtained at NIBSC.

Figure 3

Free saccharide content of bulk conjugates from meningococcal groups A, C, Y, W and X manufactured for Phase 1, 2 and 3 clinical and consistency lots of NmCV-5. Free saccharide was separated from conjugated saccharide using DOC-precipitation at SIIPL (♦) or ultrafiltration at NIBSC (●, ◯). Open circles denote lower limits of quantification. Saccharide concentrations were determined by HPAEC-PAD using polysaccharide standards.

Figure 4

Free saccharide content of stability samples of NmCV-5 Phase 3 clinical and consistency lots. Samples were stored at 25 °C for up to 6 months (A) or at 40 °C for up to 4 weeks (B). Free non-conjugated saccharide was separated from conjugated saccharide using DOC-precipitation. Saccharide concentrations of groups A (♦), C (▲), Y (◯), W (●) and X (■) were determined by HPAEC-PAD using polysaccharide standards. Data were obtained by SIIPL and are from representative clinical consistency lots.

Figure 5

Molecular size distribution of monovalent bulk conjugates for Phase 3 lots of NmCV-5 stored at −20 °C for up to 24 months. Molecular size distributions of serogroups A (♦), C (▲), Y (◯), W (●) and X (☐) were determined using TSK 6000 + 5000_PWXL column series in PBS (pH 7.4). Data were obtained at SIIPL and are from representative clinical consistency lots.

Figure 6

Immunogenicity of NmCV-5 in BALB/c mice (EIGHT per group). Post dose-2 and post dose-3 polysaccharide-specific IgG from a pentavalent R&D lot were administered either with (■) or without (♦) aluminum phosphate adjuvant. The geometric mean of serotype-specific IgG concentrations in units/mL, were determined by ELISA. Error bars represent 95% confidence intervals. The study was performed at NIBSC.

Figure 7

Immunogenicity study in New Zealand White rabbits (eight/formulation) of NmCV-5 Phase 1 (A,B), and Phase 3 (C,D) clinical lots. Total IgG (A,C) and rabbit complement-dependent serum bactericidal activity titers (B,D) to meningococcal serogroups A, C, Y, W, and X in serum samples collected at three timepoints (Days 0, 28, and 35) were determined using the bead-based ELISA and rSBA assay. The immunogenicity of Phase 1 vaccine lots were tested as adjuvanted (■) and non-adjuvanted (♦) formulations (A,B). Phase 3 clinical consistency lots (n = 3). Lot 1 (♦), Lot2 (■), Lot3 (▲) were non-adjuvanted based upon data from the Phase 1 and 2 clinical trials [20,22] (C,D). Results are expressed as Geometric Mean Titers. Error bars represent 95% confidence intervals. Statistical significance between the adjvuanted and non-adjuvanted groups is described in the text. The study was performed at SIIPL.