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

Cervical intramedullary schwannoma: a case report and review of the literature

Intramedullary schwannomas unrelated with neurofibromatosis are uncommon tumors, but if correctly diagnosed and properly treated they may have a good prognosis