Validation and implementation of Planova™ BioEX virus filters in the manufacture of a new liquid intravenous immunoglobulin in China.

There is a continuous need to improve the viral safety of plasma products, and we here report the development and optimization of a manufacturing-scale virus removal nanofiltration step for intravenous immunoglobulin (IVIG) using the recently introduced Planova™ BioEX filter. IVIG throughput was examined for various operating parameters: transmembrane pressure, temperature, protein concentration, and prefiltration methods. The developed procedure was based on filtering undiluted process solution (50.0 g/l IVIG) under constant transmembrane pressure filtration at 294 kPa and 25 °C following prefiltration with a 0.1 µm MILLEX VV filter. The recovery of IgG was approximately 98%, and no substantial changes in biochemical characteristics were observed before and after nanofiltration in scaled-up production. A viral clearance validation study with parvovirus under worst-case conditions performed at the National Institutes for Food and Drug Control of China (NIFDC) showed PPV logarithmic reduction value (LRV) > 4. Improved viral safety of IVIG can be assured by implementing a Planova BioEX nanofiltration step to ensure effective parvovirus clearance under conditions providing excellent protein recovery and no detectable impact on product biochemical properties. This plasma-derived IVIG product is the first to be certified for parvovirus safety by the NIFDC in China.

Particle-based analysis elucidates the real retention capacities of virus filters and enables optimal virus clearance study design with evaluation systems of diverse virological characteristics.

In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number-based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co-spiking of empty and genome-containing particles resulted in a decrease in the virus removal performance proportional to the co-spike ratio. This suggests that empty particles are captured in the same way as genome-containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log10 difference in particle-to-infectivity ratios, there was a 20-fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity.

Direct visualization of virus removal process in hollow fiber membrane using an optical microscope.

Virus removal filters developed for the decontamination of small viruses from biotherapeutic products are widely used in basic research and critical step for drug production due to their long-established quality and robust performance. A variety of imaging techniques have been employed to elucidate the mechanism(s) by which viruses are effectively captured by filter membranes, but they are limited to 'static' imaging. Here, we propose a novel method for detailed monitoring of 'dynamic process' of virus capture; specifically, direct examination of biomolecules during filtration under an ultra-stable optical microscope. Samples were fluorescently labeled and infused into a single hollow fiber membrane comprising cuprammonium regenerated-cellulose (Planova 20N). While proteins were able to pass through the membrane, virus-like particles (VLP) accumulated stably in a defined region of the membrane. After injecting the small amount of sample into the fiber membrane, the real-time process of trapping VLP in the membrane was quantified beyond the diffraction limit. The method presented here serves as a preliminary basis for determining optimum filtration conditions, and provides new insights into the structure of novel fiber membranes.

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles.

Virus filtration with nanometer size exclusion membranes ("nanofiltration") is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18-26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.

Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance.

Membrane fouling commonly occurs in all filter types during virus filtration in protein-based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose-based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post-adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose-based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379-386, 2018.

Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal.

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova™ BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0-49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294-1302, 2017.

Effects of varying virus-spiking conditions on a virus-removal filter Planova™ 20N in a virus validation study of antibody solutions.

We aimed to investigate the effect of virus-spiking conditions on the filter performance (flux, flux decay, and parvovirus reduction) of the small virus filter Planova™ 20N. We used three kinds of porcine parvovirus (PPV) stocks: serum, serum-free, and purified. The flux profile with PPV spiking was similar to that without spiking for normal load filtration of about 250-300 L/m(2) . High volume (3 vol %) of serum-free PPV and 1 vol % serum PPV reduced the flux to some extent for high-load filtration (over 10 h, ca., 500 L/m(2) , 5 mg/mL IgG solution). Log reduction value (LRV) of PPV was maintained at a high level (>5) over the filtration volume. Flux for Planova™ 20N was only minimally affected by the use of different virus stocks for spiking. Transmission electron microphotography showed that the distribution of PPV particles captured inside the membrane wall was reached until the -60% thickness of the membrane, showing that the membrane of Planova™ 20N has a thick effective layer for virus removal. These results provided evidence for the robustness of the filter performance of Planova™ 20N, showing that it was not easily affected by virus spiking conditions and that it has a large capacity for high-load conditions.

Effect of antibody solution conditions on filter performance for virus removal filter Planova 20N.

We investigated the effect of antibody solution conditions (ionic strength, pH, IgG concentration, buffer composition, and aggregate level (dimer content)) on filter performance for a virus removal filtration process using the Planova 20N, a virus removal filter. Ionic strength and pH affected the filter flux. A consistent high flux was maintained at an ionic strength greater than 10 mM and at pH 4-8 under a typical buffer composition (sodium chloride, citrate, acetate, and phosphate). Optimum IgG concentration was 10-20 mg/mL allowing for high throughput (kg/m(2) of IgG). Dimer content negligibly affected the flux level. Under high throughput conditions, virus spiking did not affect flux whereas a parvovirus logarithmic reduction value greater than 5 was maintained. From the results of zeta potential analyses for IgG and the membrane, we considered that electrostatic interactions between antibodies and the membrane affect filter performance (flux level and throughput). These results indicate that the Planova 20N filter is applicable for a wide range of solution conditions typically used in antibody processing.