Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing.

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.

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 phi6 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 laidlawii and 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 cm(2)) 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.

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.

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.