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

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.

Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens.

A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease.

Trumenba (bivalent rLP2086) is a vaccine licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B (NmB) in individuals 10-25 years of age in the USA. The vaccine is composed of two factor H binding protein (fHbp) variants that were recombinantly expressed in Escherichia coli as native lipoproteins: rLP2086-A05 and rLP2086-B01. The vaccine was shown to induce potent bactericidal antibodies against a broad range of NmB isolates expressing fHbp that were different in sequence from the fHbp vaccine antigens. Here, we describe the characterization of the vaccine antigens including the elucidation of their structure which is characterized by two distinct motifs, the polypeptide domain and the N-terminal lipid moiety. In the vaccine formulation, the lipoproteins self-associate to form micelles driven by the hydrophobicity of the lipids and limited by the size of the folded polypeptides. The micelles help to increase the structural stability of the lipoproteins in the absence of bacterial cell walls. Analysis of the lipoproteins in Toll-like receptor (TLR) activation assays revealed their TLR2 agonist activity. This activity was lost with removal of the O-linked fatty acids, similar to removal of all lipids, demonstrating that this moiety plays an adjuvant role in immune activation. The thorough understanding of the structure and function of each moiety of the lipoproteins, as well as their relationship, lays the foundation for identifying critical parameters to guide vaccine development and manufacture.

Preservative of choice for Prev(e)nar 13™ in a multi-dose formulation.

Development of a Prev(e)nar 13™ multi-dose vaccine, in support of vaccinating populations against pneumococcal disease, required the addition of a preservative to the vaccine formulation that met antimicrobial effectiveness tests based on the European Pharmacopoeia (EP) requirements, including deliberate multiple challenge studies and recommendation by the WHO Open Vial Policy. In this study, the antimicrobial effectiveness of several preservatives in Prev(e)nar 13™ formulations was evaluated. A Prev(e)nar 13™ formulation containing 2-Phenoxyethanol (2-PE) at a concentration of 5.0mg/dose was stable and met EP recommended criteria for antimicrobial effectiveness tests when the formulation was kept over a 30 month period. In contrast, a recommended dose of Thimerosal, as a comparator, or other preservatives did not meet EP antimicrobial effectiveness acceptance criteria. The rate of growth inhibition of Thimerosal compared to 2-PE on Staphylococcus aureus, a resilient organism in these tests, was significantly slower in single and multi-challenge studies. These results indicate that 2-PE provides a superior antimicrobial effectiveness over Thimerosal for this vaccine formulation.

High-throughput screening of stabilizers for respiratory syncytial virus: identification of stabilizers and their effects on the conformational thermostability of viral particles.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in children worldwide. Recombinant live attenuated viral preparations are one of the most promising strategies for vaccination but they typically possess poor thermostability. In this work, a library of compounds was screened and stabilizers were selected based on their ability to inhibit the aggregation of RSV perturbed at 56 degrees C. After screening and selection of excipients, the conformational stability of the RSV proteins was evaluated in the presence of potential stabilizers. The secondary and tertiary structures as well as aggregation/dissociation of RSV were monitored using circular dichroism and second derivative UV absorption spectroscopies and light scattering, respectively, as a function of temperature (10-90 degrees C). RSV membrane fluidity was also evaluated by generalized polarization of Laurdan fluorescence. Screening experiments showed that a variety of sugars, amino acids, polyols and polyanions inhibited the aggregation of viral particles. Conformational stability studies demonstrated that the addition of sugars and polyols stabilized RSV as indicated by a significant increase in the transition melting temperature (Tm) of both the secondary and tertiary structures as well as the gel to liquid crystalline membrane transition. These results should provide the basis for rational development of more physically stable formulations of live attenuated RSV vaccines.