Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology.

Impact of aluminum adjuvants on the stability of pneumococcal polysaccharide-protein conjugate vaccines.

Development of a vaccine drug product requires formulation optimization to ensure that the vaccine's effectiveness is preserved upon storage throughout the shelf-life of the product. Although aluminum adjuvants have been widely used in vaccine formulations to safely and effectively potentiate an immune response, careful attention must be directed towards ensuring that the type of aluminum adjuvant does not impact the stability of the antigenic composition. PCV15 is a polysaccharide-protein conjugate vaccine comprising the pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F), each individually conjugated to the protein carrier CRM197.

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode.

Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells.

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc.

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size, shape, and salt concentration.

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles.

Functional profiling of Covid 19 vaccine candidate by flow virometry.

Vaccine development is a complex process, starting with selection of a promising immunogen in the discovery phase, followed by process development in the preclinical phase, and later by clinical trials in tandem with process improvements and scale up. A large suite of analytical techniques is required to gain understanding of the vaccine candidate so that a relevant immunogen is selected and subsequently manufactured consistently throughout the lifespan of the product. For viral vaccines, successful immunogen production is contingent on its maintained antigenicity and/or infectivity, as well as the ability to characterize these qualities within the context of the process, formulation, and clinical performance.

Regeneration of Capto Core 700 resin through high throughput and laboratory scale studies and impact on production of a SARS-CoV-2 vaccine candidate.

During the development of a SARS-CoV-2 vaccine candidate, at the height of the COVID-19 pandemic, raw materials shortages, including chromatography resins, necessitated the determination of a cleaning in place (CIP) strategy for a multimodal core-shell resin both rapidly and efficiently. Here, the deployment of high throughput (HT) techniques to screen CIP conditions for cleaning Capto Core 700 resin exposed to clarified cell culture harvest (CCCH) of a SARS-CoV-2 vaccine candidate produced in Vero adherent cell culture are described. The best performing conditions, comprised of 30% n-propanol and ≥0.

Analytical technology development to monitor the stability of Polysaccharide-Protein conjugate vaccines.

The covalent attachment of a bacterial-derived capsular polysaccharide to protein is of critical importance in transforming the polysaccharide from an antigen with limited immunogenicity in infants and older adults to an antigen that can prevent potentially fatal disease. For a polysaccharide-protein conjugate vaccine (PCV) candidate to be successful, it must be sufficiently stable. Chemical breakage of carbohydrate bonds in the polysaccharide may result in the reduction of "conjugate dose" and could negatively impact immunogenicity and the ability of the vaccine to prime for memory responses.

Multi-attribute characterization of pneumococcal conjugate vaccine by Size-exclusion chromatography coupled with UV-MALS-RI detections.

Streptococcus pneumoniae bacterial infection can cause serious diseases. Among more than 90 known streptococcus pneumoniae serotypes, more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Initially, a 23-valent polysaccharide vaccines (PPSV) PNEUMOVAX®23, was developed to generate an antigen-specific immune response and prevent diseases caused by these pneumoniae serotypes.

real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need.

Protein Adsorption on Core-shell Particles: Comparison of Capto™ Core 400 and 700 Resins.

Structural and functional characteristics of the two core-shell resins Capto™ Core 400 and 700, which are useful for the flow-through purification of bioparticles such as viruses, viral vectors, and vaccines, are compared using bovine serum albumin (BSA) and thyroglobulin (Tg) as models for small and large protein contaminants. Both resins are agarose-based and contain an adsorbing core surrounded by an inert shell. Although shell thicknesses are comparable (3.

Immunogenicity of PCV24, an expanded pneumococcal conjugate vaccine, in adult monkeys and protection in mice.

Invasive pneumococcal disease (IPD) is responsible for serious illnesses such as bacteremia, sepsis, meningitis, and pneumonia in young children, older adults, and persons with immunocompromising conditions and often leads to death. Although the most recent pneumococcal conjugate vaccines (PCVs) have been designed to target serotypes identified as the primary causative agents of IPD, the epidemiological landscape continues to change stressing the need to develop new PCVs. We have developed an investigational 24-valent PCV (PCV24) including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F all conjugated to CRM197 and evaluated this vaccine in adult monkeys.

Immunogenicity Comparison of a Next Generation Pneumococcal Conjugate Vaccine in Animal Models and Human Infants.

Background: Evaluation of a pneumococcal conjugate vaccine (PCV) in an animal model provides an initial assessment of the performance of the vaccine prior to evaluation in humans. Cost, availability, study duration, cross-reactivity and applicability to humans are several factors which contribute to animal model selection. PCV15 is an investigational 15-valent PCV which includes capsular polysaccharides from pneumococcal serotypes (ST) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F all individually conjugated to cross-reactive material 197 (CRM197).

Safety and immunogenicity of 15-valent pneumococcal conjugate vaccine (PCV15) in healthy infants.

Background: Pediatric use of pneumococcal conjugate vaccines (PCV) has been associated with significant decrease in disease burden. However, disease caused by non-vaccine serotypes has increased. Safety and immunogenicity of 15-valent PCV (PCV15) containing serotypes included in 13-valent PCV (PCV13) plus serotypes 22F and 33F were evaluated in infants (NCT01215188).

Immunogenicity differences of a 15-valent pneumococcal polysaccharide conjugate vaccine (PCV15) based on vaccine dose, route of immunization and mouse strain.

Pneumococcal disease continues to be a medical need even with very effective vaccines on the market. Globally, there are extensive research efforts to improve serotype coverage with novel vaccines; therefore, conducting preclinical studies in different animal models becomes essential. The work presented herein focuses on evaluating a 15-valent pneumococcal conjugate vaccine (PCV15) in mice.

Large-scale, nonchromatographic purification of plasmid DNA.

A large-scale approach to the purification of plasmid DNA has been developed that overcomes many of the limitations of current chromatography-based processes. The process consists of a scaleable lysis using recombinant lysozyme and a rapid heating and cooling step followed by a selective precipitation with cetyltrimethylammonium bromide (CTAB). Calcium silicate batch adsorption is then utilized to remove residual genomic DNA, linear plasmid, open circular plasmid, endotoxin, detergents, and proteins.

Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation.

Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized.

Monitoring of RNA clearance in a novel plasmid DNA purification process.

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays.

Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities.

The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid.

Fractional precipitation of plasmid DNA from lysate by CTAB.

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins.