Herbert Lutz

Performance of a Novel Viresolve NFR Virus Filter

Mammalian cell‐expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus‐like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus‐free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter Φ 6 bacteriophage and the 80–110 nm diameter Xenotropic Murine Leukemia Virus (X‐MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70–85 nm diameter Reovirus 3 (Reo3), 70–90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawiiand the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95–100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm2) disks, which are shown to scale linearly to the manufacturing scale pleated‐filters. Viresolve NFR filters provide consistent water permeability performance (34–37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.

Development and Qualification of a Novel Virus Removal Filter for Cell Culture Applications

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum‐free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage ΦX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by ≥8.8 LRV, and the bacteria Brevundimonas diminuta by ≥9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.

Qualification of a novel inline spiking method for virus filter validation.

Virus filters are widely used in bioprocessing to reduce the risk of virus contamination in therapeutics. The small pores required to retain viruses are sensitive to plugging by trace contaminants and frequently require inline adsorptive prefiltration. Virus spiking studies are required to demonstrate virus removal capabilities of the virus filter using scale down filters. If prefiltration removes viruses and interferes with the measurement of virus filter LRV, the standard approach is to batch prefilter the protein solution, spike with virus, and then virus filter. For a number of proteins, batch prefiltration leads to increased plugging and significantly lower throughputs than inline prefiltration. A novel inline spiking method was developed to overcome this problem. This method allows the use of inline prefiltration with direct measurement of virus filter removal capabilities. The equipment and its operation are described. The method was tested with three different protein feeds, two different parvovirus filters, two virus injection rates; a salt spike, a bacteriophage spike, and two mammalian virus spikes: MMV and xMuLV. The novel inline method can reliably measure LRV at throughputs representative of the manufacturing process. It is recommended for applications where prefiltration is needed to improve throughput, prefiltration significantly reduces virus titer, and virus filter throughput is significantly reduced using batch vs. inline prefiltration. It can even help for the case where the virus preparation causes premature plugging.

An alternate diafiltration strategy to mitigate protein precipitation for low solubility proteins

Application of the minimum diafiltration (DF) time solution for a monoclonal antibody resulted in a 20‐h process time rather than the expected 12 h. Further investigation indicated high turbidity associated with a product solubility issue that caused a flux decline. As a result, the gel flux model and the associated minimum DF time were not predictive. Multiwell plate solubility screening confirmed that the protein passed through a region of low solubility during the ultrafiltration step. Multiple approaches to address this issue were considered and a new strategy involving variable volume diafiltration (VVDF) was developed. Process modeling and simulation were used to predict performance and to select a value of the DF ratio control parameter (buffer flow/permeate flow = 0.65). Feasibility testing at the bench and pilot scales confirmed that the new strategy reduced solubility issues, fit within existing manufacturing tank volume and system area constraints, matched model predictions, and did not present significant implementation issues. Recommendations are made regarding the general value of this strategy, when it should be used, and how to implement it.