Optimizing Virus Prefiltration for Biopharmaceutical Manufacturing

Virus filters are single-use devices that use a size-based separation process. In virus filters, contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filters are an essential component of the overall virus clearance strategy. Sections 1 and 2 of this dissertation provide an introduction and extensive review of monoclonal antibody (mAb) process development, where virus filtration is pivotal. In section 3, prefiltration studies were performed with an industrially relevant IgG1 type mAb using adsorptive and size-exclusion-based prefilters with different mechanisms of action. This mAb has an isoelectric point range of 7.1 to 8.0 and a molecular weight (MW) of 148 kDa. Decoupled prefiltration and virus filtration studies were conducted. We attempted to elute bound species from the membrane to identify them. Permeate fractions from the prefilters were introduced as feed fractions to a Planova BioEX (Asahi Kasei Medical, Tokyo, Japan) commercial virus filter for flux decay studies. Prefiltration and virus filtration studies were performed at different pH and ionic strength buffer conditions. By adjusting buffer conditions, and choosing prefilters with an appropriate mechanism of action, increased selectivity for foulant capture resulting in improved flux behavior during virus filtration could be achieved. Extensive characterization was also performed for the various filtration fractions to determine molecular species that increase fouling propensity in the virus filter and the efficacy of the different prefilters at removing these species. In section 4, prefiltration and flux decay studies on a Viresolve Pro (MilliporeSigma, Billerica, MA) as well as the Planova BioEX virus filter was performed with another industrially relevant mAb with an isoelectric point range of 5.95 - 6.55. The impact of excipients on mAb fouling behavior was determined. The impact of buffer pH was also evaluated with one pH condition below the isoelectric point (pI) of the mAb and another pH condition above the mAb pI. Decoupled prefiltration was performed to evaluate the impact of different types of prefilters on the filterability of this mAb. The pharmaceutical analysis system PA800 plus (SCIEX, Redwood City, CA) was also used to characterize the various mAb fractions from prefiltration and virus filtration. Dynamic light scattering (particle size analysis), size exclusion chromatography, SDS PAGE, capillary electrophoresis, and MALDI mass spectrometry were used for characterization. In section 5, a new technique of fractionating close molecular weight biomolecules was evaluated for virus clearance. The technique is known as internally staged ultrafiltration (ISUF), where layers of ultrafiltration membranes operate in stages to fractionate biomolecules based on differences in isoelectric points. The membranes of interest were the Pall Omega PES 300 kDa molecular weight cutoff (MWCO) flat sheet membrane, Pall Omega PES 100 kDa MWCO membrane, Millipore Ultracel 100kDa MWCO, and the Millipore Ultracel 30kDa MWCO. Virus clearance studies were performed using internally staged ultrafiltration membranes in skin and backing configurations. Section 6 is an overall conclusion for this work showing major findings and identifying areas for future study

Peptoid and Antibody-based GFP Sensors

In this work, we have made and characterized a pair of immunobiosensors for detecting the green fluorescent protein (GFP) in an aqueous matrix. An anti-GFP antibody-based biosensor was assembled to detect GFP, while a novel peptoid (N-substituted oligomers of glycine designated as IOS-1) biosensor was also assembled for GFP detection. A quartz crystal microbalance (QCM) gold sensor was used as the supporting substrate for self-assembly of the immunobiosensors. Gravimetric measurements of the QCM gold sensor during immunobiosensor construction and operation were available in real-time using a QCM instrument. X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and Fluorescence microscopy were used to characterize the immunobiosensors. Dose-dependent calibration curves were developed to contrast the performance of the peptoid immunobiosensor and the antibody-based immunobiosensor. The sensitivity of the biosensors shows that the peptoid could detect GFP at 8 nM, unlike the antibody immunobiosensor, which starts to measurably detect GFP at 40 nM. IOS-1 peptoid immunobiosensor had more adsorption capacity for GFP than the antibody-based immunobiosensor and could be reused through multiple adsorption/ desorption cycles. The peptoid immunobiosensor had a binding constant of 2.197 x 10(7) M(-1) with GFP

A versatile platform for three-dimensional dynamic suspension culture applications

In the last decades, the rapid upgrading in cell biological knowledge has bumped the interest in using cell-based therapeutic approaches as well as cell-based model systems for the treatment of diseases. Given the rapid translation towards cell-based clinical treatments and the consequent increasing demand of cell sources, three-dimensional (3D) suspension cultures have demonstrated to be an advantageous alternative to monolayer techniques for large scale expansion of cells and for the generation of three-dimensional model systems in a scale-up perspective. In this scenario, a versatile bioreactor platform suitable for 3D dynamic suspension cell culture under tuneable shear stress conditions is developed and preliminarily tested in two different biotechnological applications. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits a laminar hydrodynamics, enabling dynamic cell suspension in an environment favourable to mass transport. Technically, the bioreactor is conceived to produce dynamic suspension cell culture under tuneable shear stress conditions without the use of moving components (from ultralow to moderate shear stress). A multiphysics computational modelling strategy is applied for the development and optimization of the suspension bioreactor platform. The in silico modelling is used to support the design and optimization phase of the bioreactor platform, providing a comprehensive analysis of its operating principles, also supporting the development/optimization of culture protocols directly in silico, and thus minimizing preliminary laboratory tests. After the technical assessment of the functionality of the device and a massive number of in silico simulations for its characterization, the bioreactor platform has been employed for two preliminary experimental applications, in order to determine the suitability of the device for culturing human cells under dynamic suspension. In detail, the bioreactor platform has been used to culture lung cancer cells for spheroid formation (Calu-3 cell line) under ultralow shear stress conditions, and for human induced pluripotent stem cell (hiPSC) dynamic suspension culture. The use of the bioreactor platform for the formation of cancer cell spheroids under low shear stress conditions confirms the suitability of the device for its use as dynamic suspension bioreactor. In fact, compared to static cell suspension, after 5 days of dynamic suspension culture the bioreactor platform preserves morphological features, promotes intercellular connection, increases the number of cycling cells, and reduces double strand DNA damage. Calu-3 cells form functional 3D spheroids characterized by more functional adherence junctions between cells. Moreover, the computational model has been used as a tool for assisting the setup of the experimental framework with the extraction of the fluid dynamic features establishing inside the bioreactor culture chamber. As second proof of concept application, the bioreactor platform has been tested for the dynamic suspension of hiPSCs. Starting from the ‘a priori’ knowledge gained by the development of the in silico culture protocol, the agglomeration of human induced pluripotent stem cells has been modulated by means of the combination of moderate intermittent shear stress and free-fall transport within the bioreactor culture chamber. The inoculation of single cells suspensions inside the bioreactor chamber promotes cell-cell interaction and consequently the formation of human induced pluripotent stem cell aggregates. In conclusion, the impeller-free functioning principle characterizing the proposed bioreactor platform demonstrates to be promising for human cell dynamic suspension culture. In the future, this bioreactor platform will be further optimized for the realization of impeller-free dynamic suspension bioreactors dedicated and optimized to specific applications in stem cell and cancer cell culture

Differential Electrostatic Interaction Patterns in SARS-CoV-1 and SARS-CoV-2 variants: A Molecular Dynamics Simulation Study

The SARS-related coronavirus (SARS-rCoV) is a highly contagious virus that has raised significant worldwide health concerns. It caused outbreaks in 2002-2003 and more recently in 2019-2020 with SARS-CoV-2. SARS-CoV-2 is responsible for the COVID-19 pandemic, which has resulted in a significant global impact on health and the economy. The spike protein of the virus plays a critical role in its infectivity and transmission, and the receptor-binding domain (RBD) within the spike protein is of particular interest, as it is responsible for binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. In this study, we used Molecular dynamics (MD) simulations to investigate the electrostatic interaction patterns in the active and inactive models of SARS-CoV-1, SARS-CoV-2, and several variants of SARS-CoV-2, including the Alpha, Beta, Delta, and Epsilon variants. MD simulations are a computational method that allows us to model the motion of atoms and molecules over time, providing insights into the structure and behavior of biological molecules. The findings indicate differential electrostatic interaction patterns between the RBD of SARS-CoV-1 and SARS-CoV-2 spike protein. The RBD of SARS-CoV-2 exhibited a slower conformational pattern, which could influence higher stability, potentially affecting its binding affinity with the ACE2 receptor. Additionally, the Delta variant demonstrated significant differences in electrostatic interactions compared to the original SARS-CoV-2 strain, particularly in the N-terminal domain (NTD) and RBD regions. These findings suggest that Delta variant mutations could affect the RBD’s binding affinity to the ACE2 receptor, impacting transmission and virulence. Overall, this study highlights electrostatic interaction patterns in SARS-CoV-1, SARS-CoV-2, and variants, with implications for the development of long-term effective vaccines and therapeutics. Understanding the spike protein’s molecular basis may enable designing more effective treatments and strategies to prevent the spread of these viruses

Comparison of symmetrical hemodialysis catheters using computational fluid dynamics

Purpose: Symmetric-tip dialysis catheters have become alternative devices because of low access recirculation and ease of tip positioning. Flow characteristics of three symmetric catheters were compared based on computational fluid dynamics (CFD) as they relate to catheter function. Materials And Methods: In Palindrome, GlidePath, and VectorFloW catheters, a computational fluid dynamics based approach was used to assess W regions of flow separation, which are prone to thrombus development; (ii) shear-induced platelet activation potency; (iii) recirculation; and (iv) venous outflow deflection. A steady-state, laminar flow model simulated: catheter tip position within the superior vena cava. Catheter performance was investigated at high hemodialysis flow rate (400 mL/min). Blood was assumed as a Newtonian fluid. Results: Wide regions of flow separation downstream of the Palindrome side slot and close to the distal tip were observed in forward and reversed line configurations. Geometric asymmetry of the distal guide wire aperture of the GlidePath catheter produced the highest levels of inverted velocity flow when run in reversed configuration. The lowest mean shear-induced platelet activation was exhibited by GlidePath and VectorFloW catheters; the Palindrome catheter exhibited 152% higher overall platelet activation potency. All catheters were associated with a recirculation close to zero; the helically contoured lumens of the VectorFlow catheter produced the greatest amount of deflection of venous flow away from the arterial lumen. Conclusions: The VectorFlow catheter produced less shear-induced platelet activation than the Palindrome catheter and less flow separation than the Palindrome and GlidePath catheters irrespective of line configuration These findings have,potential implications for differences in thrombogenic risk during clinical performance of these catheters

Differential Electrostatic Interaction Patterns in SARS-CoV-1 and SARS-CoV-2 variants: A Molecular Dynamics Simulation Study

The SARS-related coronavirus (SARS-rCoV) is a highly contagious virus that has raised significant worldwide health concerns. It caused outbreaks in 2002-2003 and more recently in 2019-2020 with SARS-CoV-2. SARS-CoV-2 is responsible for the COVID-19 pandemic, which has resulted in a significant global impact on health and the economy. The spike protein of the virus plays a critical role in its infectivity and transmission, and the receptor-binding domain (RBD) within the spike protein is of particular interest, as it is responsible for binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. In this study, we used Molecular dynamics (MD) simulations to investigate the electrostatic interaction patterns in the active and inactive models of SARS-CoV-1, SARS-CoV-2, and several variants of SARS-CoV-2, including the Alpha, Beta, Delta, and Epsilon variants. MD simulations are a computational method that allows us to model the motion of atoms and molecules over time, providing insights into the structure and behavior of biological molecules. The findings indicate differential electrostatic interaction patterns between the RBD of SARS-CoV-1 and SARS-CoV-2 spike protein. The RBD of SARS-CoV-2 exhibited a slower conformational pattern, which could influence higher stability, potentially affecting its binding affinity with the ACE2 receptor. Additionally, the Delta variant demonstrated significant differences in electrostatic interactions compared to the original SARS-CoV-2 strain, particularly in the N-terminal domain (NTD) and RBD regions. These findings suggest that Delta variant mutations could affect the RBD’s binding affinity to the ACE2 receptor, impacting transmission and virulence. Overall, this study highlights electrostatic interaction patterns in SARS-CoV-1, SARS-CoV-2, and variants, with implications for the development of long-term effective vaccines and therapeutics. Understanding the spike protein’s molecular basis may enable designing more effective treatments and strategies to prevent the spread of these viruses

Impact analysis of palm oil mill effluent on the aerobic bacterial density and ammonium oxidizers in a dumpsite in Anyigba, Kogi State

The effects of palm oil mill effluent (POME) on the total aerobic bacterial populations and ammonium oxidizers in the soil were assessed. This was done by culturing soil samples from an effluent dumpsitefor the total aerobic bacterial counts and ammonium oxidizers. Results showed that the total aerobic bacterial populations in the POME soil samples (9.6 x 108 ± 0.3 at 20oC, 1.64 x 109 ± 0.2 at 30oC and 1.07x 109 ± 0.4 at 40oC) were significantly higher (P 0.05) than the counts for the non-POME soil samples (4.5 x 108 ± 0.3 at 20oC, 7.6 x 108 ± 0.3 at 30oC and 5.9 x 108 ± 0.3 at 40oC). In addition, ammonium oxidizers were isolated from the non-POME soil samples but not from the POME soil samples

Nigeria’s 2005 Bank Recapitalization: An Evaluation of Effects and Social Consequences

This study critically evaluated the 2005 recapitalization of banks in Nigeria in terms of the positive fallouts and the unanticipated consequences. It was necessary to look at the performance of banks, the recapitalization, while at same time evaluating the human resources and other developmental challenges that grew out of the recapitalization effort. This critical evaluation required the formulation of some testable hypotheses to confirm the merit of the recapitalization or  the absence of same. Tests of the differences of means were also applied to determine if there is any significant relationship between the pre and post recapitalization of banks and Nigerians economic growth. The results obtained confirmed that the 2005 recapitalization effort actually improved the performance of banks and also positively impacted the economy as a whole. The second hypothesis which tried to prove that there is no significant relationship in the pre and post performance of banks after the recapitalization. Our results tend affirm that the  standard of living of the population were negatively affected mostly because of number of banks staff  who had to be sacrificed and    thrown into unemployment to achieve, what some banks experts in the system call “ paper profits”. The extended family system practiced in the country means that the negative consequences of the recapitalization was far reaching than expected, with the result that most families are still licking their “wounds” occasioned by the 2005 recapitalization. Banks recapitalization is not adequate to strengthen and enhance the banking system to face the global challenges. The implication of this study is that government should formulate policies that aim at contribute to the growth of economy and improving standard of living. Keywords: Banks recapitalization, banks performance, economy, employment, standard of living