Development and validation of a targeted LC-MS/MS quantitation method to monitor cell culture expression of tetanus neurotoxin during vaccine production.

The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20% mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools.

Purpose: Aluminum-based adjuvants including aluminum phosphate (AlPO) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO, such as particle size and chemical composition.

Identity, Structure and Compositional Analysis of Aluminum Phosphate Adsorbed Pediatric Quadrivalent and Pentavalent Vaccines.

Purpose: The goal of this study is to set an empirical baseline to map the structure-function relation of the antigens from the commercialized vaccine products.

Methods: To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM have been used.

Results: All antigens have shown wide range of hydrodynamic diameter from 7 nm to 182 nm.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding.

Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate.

HSV529 is a replication defective human herpes simplex virus (HSV)-2 viral vaccine candidate in clinical development. An engineered cell line is required to support production of HSV529 by transgenic expression of the HSV-1 transcription factors UL5 (HELI) and UL29 (DNBI). These 2 genes have been deleted from the vaccine candidate to ensure replication deficiency, and the transgene products are thus impurities that must be monitored in the final product.

Al and P NMR spectroscopy method development to quantify aluminum phosphate in adjuvanted vaccine formulations.

A novel qNMR method is described for the quantitative determination of total aluminum and phosphate in aluminum phosphate (AlPO) adjuvanted vaccine samples using solution Al and P nuclear magnetic resonance (NMR) spectroscopy. External standard calibrations of AlPO solutions established excellent linearity in the range of 15-40 × 10 M and additional studies determined the level of detection for both nuclei. A commercialized combination vaccine product (Quadracel®), along with several individual adsorbed antigen components used in the vaccine were employed as model systems for method development.

Tuberculosis vaccine candidate: Characterization of H4-IC31 formulation and H4 antigen conformation.

Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85 B and TB10.

Host cell protein testing strategy for hepatitis B antigen in Hexavalent vaccine - Towards a general testing strategy for recombinant vaccines.

Background: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine.

Characterization of Inherent Particles and Mechanism of Thermal Stress Induced Particle Formation in HSV-2 Viral Vaccine Candidate.

Background: Vaccine formulations may contain visible and/or subvisible particles, which can vary in both size and morphology. Extrinsic particles, which are particles not part of the product such as foreign contaminants, are generally considered undesirable and should be eliminated or controlled in injectable products. However, biological products, in particular vaccines, may also contain particles that are inherent to the product.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard.

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials.

Nuclear Magnetic Resonance (NMR) Study for the Detection and Quantitation of Cholesterol in HSV529 Therapeutic Vaccine Candidate.

This study describes the NMR-based method to determine the limit of quantitation (LOQ) and limit of detection (LOD) of cholesterol, a process-related impurity in the replication-deficient Herpes Simplex Virus (HSV) type 2 candidate vaccine HSV529. Three signature peaks from the 1D H NMR of a cholesterol reference spectrum were selected for the identification of cholesterol. The LOQ for a cholesterol working standard was found to be 1 μg/mL, and the LOD was found to be 0.

Differential Scanning Calorimetry - A Method for Assessing the Thermal Stability and Conformation of Protein Antigen.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural "fingerprint" that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; ∆H) of proteins.

Visible and subvisible particles in the BCG immunotherapeutic product Immucyst®.

Bacille Calmette-Guerin, BCG, is a live attenuated bovine tubercle bacillus used for the treatment of non-muscle invasive bladder cancer. In this study, an Electrical Sensing Zone (ESZ) method was developed to measure the particle count and the size of BCG immunotherapeutic (BCG IT), or ImmuCyst® product using a Coulter Counter Multisizer 4® instrument. The focus of this study was to establish a baseline for reconstituted lyophilized BCG IT product using visible and sub-visible particle concentration and size distribution as reportable values.

Investigation into the misincorporation of norleucine into a recombinant protein vaccine candidate.

A high level of norleucine misincorporation was detected in a recombinant methionine-rich protein vaccine candidate expressed in E. coli K12. An investigation was conducted to evaluate a simple remediation strategy to reduce norleucine misincorporation and to determine if the phenomenon was either (a) due to the depletion of methionine during fermentation, (b) a result of the cultivation environment, or (c) a strain-specific effect.

Structure-guided antigen engineering yields pneumolysin mutants suitable for vaccination against pneumococcal disease.

Pneumolysin (PLY) is a cholesterol-binding, pore-forming protein toxin. It is an important virulence factor of Streptococcus pneumoniae and a key vaccine target against pneumococcal disease. We report a systematic structure-driven approach that solves a long-standing problem for vaccine development in this field: detoxification of PLY with retention of its antigenic integrity.