ABSTRACT
Compared to batch operation, continuous bioprocessing can offer numerous advantages, including increased productivity, improved process control, reduced footprint, and increased flexibility. However, integration of traditional batch operations into a connected process can be challenging. In contrast to batch operations run at constant pressure or high flux, virus filtration in continuous processes may be operated at very low flux. This change in operating conditions may reduce the viral retention performance of the filter which has inhibited adoption of truly continuous virus filtration. To overcome this limitation, a novel approach is described that utilizes serial virus filtration, with a high area ratio between first to second stage filters, to achieve virus retention targets. In this study, virus filters were operated continuously (except for planned process interruptions) for 200 h in a serial configuration at a first to second stage filter area ratio of 13:1 and at a first stage flux of 5 L/m2/h. While the minute virus of mice (MVM) retention performance of the first stage filter was about 4 log reduction value (LRV), there was no virus detected in the second stage filtrate, translating to an MVM LRV across the filtration train of ≥6.7. The second stage filter was the dominant flow resistance at the start of the run but, as it was protected from foulants by the first stage filter, it suffered minimal fouling and the life of the filter train was controlled by the first stage. A theoretical case study projected that continuous virus filtration using serial configuration at high area ratio would have about 30% longer filter changeout time, 14% higher productivity, and virus retention nearly six LRV greater than single stage operation. The findings of this research are expected to provide valuable insights into optimizing virus filtration in continuous bioprocessing.
ABSTRACT
As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5-100 L/m2 /h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20-30 L/m2 /h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.
Subject(s)
Membranes, Artificial , Viruses , Humans , Filtration/methods , Immunoglobulin G , FoodABSTRACT
Bubble point tests are widely used for assessing the integrity of sterilizing-grade membrane cartridge filters. While many authors have considered the limitations of bubble point tests as applied to cartridge filters, the level of bacterial retention assurance provided by this test as conducted with automated integrity testers (AITs) has not, until now, been quantified. Contrary to the notion that filter leaks result in a depressed bubble point, it was shown that the bubble point as reported by AITs was insensitive to defect size up until the point where the AIT either determined a gross leak failure or was not able to return a valid result. For the three AITs used in this study, the minimum laser hole defect diameter in 10-inch (25.4 cm) sterilizing-grade cartridge filters that resulted in a failing bubble point test was between about 30 and 60 µm, depending on the filter type and test conditions. These defect sizes were associated with bacterial log reduction values in the 4.0 to 4.5 range. This study supports the generally recommended practice of pairing the bubble point test (which does confirm proper pore size rating) with a complementary gas-liquid diffusion test (better suited for detecting defects) to achieve a more comprehensive assessment of filter integrity.
ABSTRACT
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 ÏX174, 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 ÏX174 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.
Subject(s)
Filtration , Viruses , Animals , Kinetics , Leukemia Virus, Murine , Mammals , Mice , VirionABSTRACT
Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Immunoglobulin G/isolation & purification , Membranes, Artificial , Nanoparticles/chemistry , Viruses/chemistry , Antibodies, Monoclonal/chemistry , Humans , Immunoglobulin G/chemistryABSTRACT
Reliability of retention performance is of paramount importance for membrane filters designed for sterile and virus filtration. To achieve dependable retention, an integrity test can be applied to ensure the absence of oversize pores or defects that can compromise the retention capability of the filter. Probably the most commonly applied nondestructive integrity test for membrane filters is the gas-liquid diffusion test, with air and water often used as the gas-liquid pair. However, the sensitivity of the air-water diffusion test is limited by the fact that the diffusive flow rate for an integral membrane can span a range that is large compared to the flow contributed by a defect. A novel nondestructive air-water integrity test for microporous and nanoporous membranes is introduced here that provides improved test sensitivity by measuring the gas composition in addition to gas flow rate. Oxygen permeates through water faster than does nitrogen, so with air as the challenge gas and water as the wetting fluid, the permeate stream will be enriched in oxygen. The permeate oxygen concentration is predictable, accurately measurable, and within a narrow and repeatable range for an integral membrane. A leak through the membrane will result in a deviation from the integral permeate concentration, signaling a defect. Compared to the conventional air-water diffusion test, this air binary gas (i.e., O2 and N2) test in which the permeate gas composition is measured (in addition to the diffusive flow rate) has a superior signal-to-noise ratio and was demonstrated to provide a significantly higher level of retention assurance for both sterilizing grade and virus filters. Because air and water are used as the gas-liquid pair, the air binary gas test also maintains the convenience, safety, and environmentally friendly aspects of the air-water diffusion test. LAY ABSTRACT: To ensure that sterilizing and virus removal filters are free of defects, an integrity test is often conducted both before and after use of the filters. In the commonly used air-water diffusion integrity test, pressurized air is applied to the water wetted filter and the air flow rate across the filter is measured. A flow rate above a specified limit indicates a leak through the filter. The sensitivity of the test is limited by the level of background noise (integral flow rate) relative to the leak signal (excess flow rate). An enhancement to the air-water diffusion test is introduced here in which the sensitivity of the test can be improved by measuring the composition of the diffused gas. Oxygen permeates through water faster than does nitrogen, so the permeating gas will be enriched in oxygen. Compared to the flow rate, which can span a range of values for integral filters, the integral oxygen concentration is well defined, so even small deviations from the expected concentration signal a leak. Because air and water are used as the test materials, the developed approach achieved higher sensitivity without sacrificing the convenience, safety, and environmentally friendly aspects of the air-water diffusion test.
Subject(s)
Filtration , Reproducibility of Results , Signal-To-Noise Ratio , Sterilization , WaterABSTRACT
Membrane discs offer a convenient format for evaluating membrane performance in normal flow filtration. However, while pleated devices of different sizes tend to scale in close proportion to their contained areas, they do not necessarily scale in direct proportion from flat discs. The objectives of this study are to quantify differences in performance among sterilizing-grade membrane devices as a function of device type and size, to develop an understanding of the factors that affect device scalability, and to develop a mathematical model to predict a cartridge-to-disc scalability factor based on membrane properties and porous support properties and dimensions. Measured and predicted normalized water permeability scalability factors for seven types of pleated cartridges, including 0.1-micro and 0.2-micro rated PES, and 0.2-micro rated polyvinylidene fluoride (PVDF) sterilizing-grade filters in nominal 1-inch to 5-inch lengths, were determined. The results of this study indicate that pleated cartridge performance can be closely predicted based on 47-mm disc performance provided that a number of measured device parameters are properly accounted for, most importantly parasitic pressure losses in the filter device and plumbing connections, intrinsic membrane variability, true effective device filtration area, and the hydraulic properties of all porous support materials. Throughput scalability factors (discs to devices) tend to converge towards unity, especially for highly plugging streams. As the membrane fouls, the resistance through the membrane dominates other resistances, so the flux scales more linearly with membrane area and the overall scaling factor becomes close to one. The results of throughput tests on seven different cartridge types and five different challenge streams (with widely varying fouling characteristics) show that most of the throughput scaling factors were within +/-10% of 1.0. As part of this study, the effects of pressure and temperature were also evaluated. Neither of these factors was found to have a significant effect on scalability.