Continuous viral vaccine manufacturing and viral detection strategies

Viruses are responsible for many human diseases that lead to suffering and death. Acquired immunodeficiency syndrome (AIDS), influenza, and coronavirus disease 2019 (COVID-19) have claimed the lives of millions of people. Two methods have been created to reduce the suffering caused by these diseases. The first is the manufacturing of vaccines that prevent disease and the second is the development of virus detection tests that lead to treatment. The work summarized in this dissertation discusses the development of a continuous virus purification process using an aqueous two-phase system for use in vaccine manufacturing. Additionally, two novel virus detection methods were developed using graphene biosensors and a gold nanoparticle aggregation assay. This dissertation contains results obtained from a novel continuous viral particle purification technique. The biomanufacturing industry would greatly benefit by switching from traditional batch processes to continuous processing. The reduction in costs and increase in productivity of continuous unit operations are driving the desire for continuous biomanufacturing processes. Switching to continuous processing would eliminate the current downstream processing challenges of viral-based biotherapeutics including limited yield and throughput. The method developed here was a chromatography-free separation technique aimed to increase throughput, purity, and yield of viral particles. The technique utilized an aqueous two-phase system (ATPS) to partition viral particles to a different phase than contaminant proteins. The ATPS consisted of poly(ethylene glycol) (PEG) and sodium citrate to separate viral particles from contaminant proteins from cell culture media. The ATPS was scaled-up from 1 g batch systems and run continuously for porcine parvovirus (PPV) and human immunodeficiency virus-like particles (HIV VLPs). Results from the purification of both viruses showed \u3e70% recovery with a majority of the contaminating proteins and DNA being removed. This system has the potential to be further scaled-up for full-scale continuous biomanufacturing of vaccines. This dissertation also contains results from the development of two viral detection methods. The first detection method used a graphene ink-based paper biosensor to electrochemically detect the presence of biomolecules. These graphene biosensors were created with the goal of being lab-on-a-chip devices for rapid detection in low-resource areas without the need of expensive laboratory equipment. The biosensors initially showed promise for portable, inexpensive, and rapid detection of proteins via electrical resistance changes at the sensor surface, but ultimately could not be applied to virus particles. The second detection test utilized osmolyte-induced gold nanoparticle (AuNP)/virus complex aggregation to detect whole viral particles. The AuNP aggregation assay was previously developed to detect two model viruses, PPV and bovine viral diarrhea virus (BVDV), using one size of AuNPs. The AuNP assay was capable of nanomolar level detection of both viruses. Optimization of this previously established virus detection method was performed by utilizing various sizes of AuNPs to lower the limit of detection (LOD). Results from using other sizes of AuNPs showed no improvement to the LOD

Tie line framework to optimize non-enveloped virus recovery in aqueous two-phase systems.

Viral particle purification is a challenge due to the complexity of the broth, the particle size, and the need to maintain virus activity. Aqueous two-phase systems (ATPSs) are a viable alternative for the currently used and expensive downstream processes. This work investigated the purification of two non-enveloped viruses, porcine parvovirus (PPV), and human rhinovirus (HRV) at various ATPS tie lines. A polyethylene glycol (PEG) 12 kDa-citrate system at pH 7 was used to study the behavior of the partitioning on three different thermodynamic tie line lengths (TLLs). It was experimentally determined that increasing the TLL, and therefore increasing the hydrophobic and electrostatic driving forces within the ATPS, facilitated higher virus recoveries in the PEG-rich phase. A maximum of 79% recovery of infectious PPV was found at TLL 36 w/w% and tie line (TL) ratio 0.1. Increased loading of PPV was studied to observe the change in the partitioning behavior and similar trends were observed for all the TLs. Most contaminants remained in the citrate-rich phase at all the chosen TLLs, demonstrating purification of the virus from protein contaminants. Moderate DNA removal was also measured. Net neutral charged HRV was studied to demonstrate the effects of driving forces on neutrally charged viruses. HRV recovery trends remained similar to PPV on each TLL studied, but the values were lower than PPV. Recovery of viral particles in the PEG-rich phase of the PEG-citrate system utilized the difference in the surface hydrophobicity between virus and proteins and showed a direct dependence on the surface charge of each studied virus. The preferential partitioning of the relatively hydrophobic viral particles in the PEG-rich phase supports the hypothesis that both hydrophobic and electrostatic forces govern the purification of viruses in ATPS

Mannitol-induced gold nanoparticle aggregation for the ligand-free detection of viral particles

Traditional virus detection methods require ligands that bind to either viral capsid proteins or viral nucleic acids. Ligands are typically antibodies or oligonucleotides and they are expensive, have limited chemical stability, and can only detect one specific type of virus at a time. Here, the biochemical surface properties of viruses are exploited for ligand-free, nonspecific virus detection. It has been found that the osmolyte mannitol can preferentially aggregate virus, while leaving proteins in solution. This led to the development of a ligand-free detection of virus using gold nanoparticle (AuNP) aggregation. Porcine parvovirus (PPV) was incubated with AuNPs and aggregation of the PPV-AuNP complex with mannitol was detected by dynamic light scattering (DLS). The lowest detectable concentration of PPV was estimated to be 106 MTT50 per mL, which is lower than standard antibody assays. PPV was also detected when swabbed from a dry surface and in the presence of a protein solution matrix. The enveloped bovine viral diarrhea virus (BVDV) was also detected using mannitol-induced aggregation of BVDV-coated AuNPs. The lowest detectable concentration of BVDV was estimated to be 104 MTT50 per mL. This demonstrates that gold nanoparticle aggregation can detect virus without the use of specific ligands

Shielding effectiveness of carbon-filled polypropylene composites

© SAGE Publications. Adding conductive carbon fillers to insulating thermoplastic resins increases composite shielding effectiveness. In this study, varying amounts of three different carbons (carbon black, synthetic graphite particles, and carbon nanotubes) were added to polypropylene resin. The resulting single filler composites were tested for shielding effectiveness. The effects of single fillers and combinations of two different carbon fillers were studied via a factorial design. At the highest single filler loadings, the following shielding effectiveness results were obtained at 800 MHz: 23.4 dB for 10 wt% carbon black/polypropylene, 34.7 dB for 70 wt% synthetic graphite/polypropylene, and 45.9 dB for 15 wt% carbon nanotubes/polypropylene. The factorial results indicated that for the composites containing only single fillers, carbon nanotubes, carbon black, and synthetic graphite cause a statistically significant increase in composite shielding effectiveness. All composites containing combinations of two different fillers had a statistically significant effect which increased shielding effectiveness. The shielding effectiveness values for the 2.5 wt% carbon black/65 wt% synthetic graphite/polypropylene and 65 wt% synthetic graphite/6 wt% carbon nanotubes/polypropylene composites are \u3e 60 dB, which is higher than that of many metals

Physiochemical properties of enveloped viruses and arginine dictate inactivation

Background: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. Results: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. Conclusions: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane

Continuous purification of an enveloped and non-enveloped viral particle using an aqueous two-phase system

Meeting the increasing demand for vaccines throughout the world is key to decrease the spread of infectious diseases. The switch to a fully continuous vaccine manufacturing process would increase productivity and the supply of crucial vaccines. To aid in this switch, we have developed a novel, continuous downstream purification technique based on an aqueous two-phase system (ATPS). The system has the potential to be used as a platform system for viral product purification. A 12 kDa poly(ethylene glycol) (PEG) and trisodium citrate ATPS was able to purify porcine parvovirus (PPV) and human immunodeficiency virus type-1 group antigen virus-like particles (HIV VLPs) from cell supernatant. PPV was recovered in the PEG-rich phase at 90 ± 16% with a DNA removal of 96 ± 3% and ≥89% protein removal. The system was also able to recover 99 ± 2% of HIV VLPs in the PEG-rich phase with a 73 ± 1% DNA removal and high protein removal shown by SDS-PAGE. Continuous ATPS recovered virus at the same amount as batch recovery. This demonstrates that continuous ATPS can be scaled up and runrun continuously without a loss in purity or recovery. Mixing and settling time both played an important role in developing a continuous ATPS for viral particles

Osmolyte enhanced aqueous two-phase system for virus purification

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele