Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particle were all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics.

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.

Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development.

Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.

Use of a noninfectious surrogate to predict minute virus of mice removal during nanofiltration.

Viruses can arise during the manufacture of biopharmaceuticals through contamination or endogenous expression of viral sequences. Regulatory agencies require "viral clearance" validation studies for each biopharmaceutical prior to approval. These studies aim to demonstrate the ability of the manufacturing process at removing or inactivating virus and are conducted by challenging scaled-down manufacturing steps with a "spike" of live virus. Due to the infectious nature of these live viruses, "spiking studies" are typically conducted in specialized biological safety level-2 facilities. The costs and logistics associated with these studies limit viral clearance analysis during process development and characterization. In this study, a noninfectious Minute Virus of Mice-Mock Virus Particle (MVM-MVP) was generated for use as an economical small virus spiking surrogate. An immunoglobin G containing solution was spiked with live MVM or MVM-MVP and processed through Planova nanofiltration units. Flux decay data was collected and particle reduction values were calculated from TCID50 and Immuno-qPCR analysis. The data indicated comparable filtration performance and particle reduction between infectious MVM and noninfectious surrogate, MVM-MVP. This proof of concept study suggests the feasibility of utilizing MVPs for predictive size-based viral clearance assessments during process development and characterization as an alternative to homologous infectious virus.

Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications.

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Investigations into the fouling mechanism of parvovirus filters during filtration of freeze-thawed mAb drug substance solutions.

Filtration to remove viruses is one of the single most expensive steps in the production of mAb drug products. Therefore, virus filtration steps should be fully optimized, and any decline in flow rates warrants investigation into the causes of such membrane fouling. In the current study, it was found that freezing and thawing of a mAb bulk drug solution caused a substantial decrease in viral filter membrane flow rate. Freezing and thawing also caused formation of aggregates and particles across a broad size range, including particles that could be detected by microflow imaging (≥1 µm in size). However, removal of these particles offered little protection against flow rate decline during viral filtration. Further investigation revealed that trace amounts of aggregates (ca. 10⁻6 of the total mass of protein in solution) approximately 20-40 nm in size were primarily responsible for the observed membrane fouling.