Virus filter scalability: Demonstration of consistent viral clearance across laboratory and manufacturing scales.

Virus removal filtration processes in biopharmaceutical manufacturing are developed, optimized and validated for viral clearance using laboratory scale filters. Thus, the scalability of these filters is critical for accurately extrapolating filtration performance and reliably extending viral clearance to manufacturing scale. Virus removal filter manufacturers generally validate scalability of filtration performance based on various filtration parameters, and virus removal capability is extended to manufacturing scale filters using inert, size-appropriate particles such as gold nanoparticles to avoid the risks associated with using mammalian viruses in large feed volumes. In this study, we use bacteriophage PP7 as a parvovirus model to directly demonstrate viral clearance on Planova™ BioEX virus removal filters across all scales, including manufacturing scale. Filters with hollow fibers from three spinning series with filter sizes ranging from 0.0003 to 4.0 m2 were tested for virus removal, flux, and protein recovery performance using BSA spiked with PP7. Complete viral clearance was observed across all filter sizes with PP7 LRV of ≥4.7 or higher. Flux and protein recovery were also consistent. These results demonstrate the scalability of filtration performance and consistent virus removal at all sizes, supporting the use of laboratory scale filters to validate viral clearance at manufacturing scales.

Integrity testing of Planova™ BioEX virus removal filters used in the manufacture of biological products.

Confirmation of virus filter integrity is crucial for ensuring the safety of biological products. Two main types of virus filter defects may produce inconsistent and undesirable performance in virus removal: improper pore-size distribution across the membrane; and specific damage, such as tears, broken fibers, or pinholes. Two integrity tests are performed on each individual filter manufactured by Asahi Kasei Medical to ensure the absence of these defects prior to shipment. In this study, we verified that typical usage of Planova™ BioEX filters would not improperly shift the pore-size distribution. Damage occurring during shipment and use (e.g., broken fibers or pinholes) can be detected by end-users with sufficient sensitivity using air-water diffusion based leakage tests. We prepared and tested filters with model pinhole defects of various sizes to develop standard acceptance criteria for the leakage test relative to porcine parvovirus infectivity logarithmic reduction values (LRVs). Our results demonstrate that pinhole defects at or below a certain size for each effective filter surface area have no significant impact on the virus LRV. In conclusion the leakage test is sufficiently sensitive to serve as the sole end-user integrity test for Planova™ BioEX filters, facilitating their use in biopharmaceuticals manufacturing.

Diagnostic usefulness of ribosomal protein l7/l12 for pneumococcal pneumonia in a mouse model.

The capsular antigen detection (CAD) kit is widely used in clinics to detect Streptococcus pneumoniae infection from urine, because it is rapid, convenient, and effective. However, there are several disadvantages, including false-positive results in children colonized with S. pneumoniae and prolonged positive readings even after the bacteria have been cleared. RP-L7/L12 is a component of the 50S ribosome that is abundant in all bacteria and is specific for each bacterial species. We investigated whether RP-L7/L12 could be used to accurately diagnose pneumococcal pneumonia infection in mouse models of pneumonia and colonization generated by infecting CBA/JN or CBA/N mice, respectively, with S. pneumoniae strain 741. RP-L7/L12 detection by enzyme-linked immunosorbent assay accurately assessed active lung infection, as RP-L7/L12 levels decreased simultaneously with the bacterial lung burden after imipenem administration in the pneumonia mouse model. Based on the data, antibodies detecting RP-L7/L12 were applied to rapid immunochromatographic strips (ICS) for urine sample testing. When we compared the ICS test with the CAD kit in the pneumonia model, the results correlated well. Interestingly, however, when the lung bacterial burden became undetectable after antibiotic treatment, the ICS test was correspondingly negative, even though the same samples tested by the CAD kit remained positive. Similarly, while the ICS test exhibited negative results in the nasal colonization model, the CAD kit demonstrated positive results. Bacterial RP-L7/L12 may be a promising target for the development of new methods to diagnose infectious disease. Further studies are warranted to determine whether such a test could be useful in children.

In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells.

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 ß-lactamase (BLA) can result in fusion proteins in which ß-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to ß-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.

Morphogen-defined patterning of Escherichia coli enabled by an externally tunable band-pass filter.

BACKGROUND: Gradients of morphogens pattern cell fate - a phenomenon that is especially important during development. A simple model system for studying how morphogens pattern cell behavior would overcome difficulties inherent in the study of natural morphogens in vivo. A synthetic biology approach to building such a system is attractive. RESULTS: Using an externally-tunable band-pass filter paradigm, we engineered Escherichia coli cells to function as a model system for the study of how multiple morphogens can pattern cell behavior. We demonstrate how our system exhibits behavior such as morphogen crosstalk and how the cells' growth and fluorescence can be patterned in a number of complex patterns. We extend our cell patterning from 2D cultures on the surface of plates to 3D cultures in soft agarose medium. CONCLUSION: Our system offers a convenient, well-defined model system for fundamental studies on how multiple morphogen gradients can affect cell fate and lead to pattern formation. Our design principles could be applied to eukaryotic cells to develop other models systems for studying development or for enabling the patterning of cells for applications such as tissue engineering and biomaterials.

An externally tunable bacterial band-pass filter.

The current paradigm for tuning synthetic biological systems is through re-engineering system components. Biological systems designed with the inherent ability to be tuned by external stimuli will be more versatile. We engineered Escherichia coli cells to behave as an externally tunable band-pass filter for enzyme activity and small molecules. The band's location can be positioned within a range of 4 orders of magnitude simply by the addition of compounds to the growth medium. Inclusion in the genetic network of an enzyme-substrate pair that functions as an attenuator is a generalizable strategy that enables this tunability. The genetic circuit enabled bacteria growth to be patterned in response to chemical gradients in nonintuitive ways and facilitated the isolation of engineered allosteric enzymes. The application of this strategy to other biological systems will increase their utility for biotechnological applications and their usefulness as a tool for gaining insight into nature's underlying design principles.

uPAR (CD87) as a biocompatibility marker of dialysis membrane.

BACKGROUND/AIMS: Hemodialysis (HD) therapy may lead to functional changes in patient leukocytes. For example, the upregulation of inflammatory cytokines, such as IL-1beta and TNFalpha, has been well characterized. However, these findings do not explain the entire response of leukocytes in HD. In this study, we carried out a comprehensive gene expression analysis in leukocytes treated with various dialysis membranes using DNA microarrays. The identified gene has the potential to be a new marker for testing dialysis membrane biocompatibility. METHODS: Gene expression profiles were compared between a group of leukocytes treated with various dialysis membranes and an untreated group by using DNA microarray analysis. Expression was confirmed by quantitative RT-PCR. The expression of the gene product (leukocyte surface protein) was examined in 20 chronic HD patients by flow cytometry. RESULTS: In addition to the inflammatory cytokines, the urokinase plasminogen activator receptor (uPAR or CD87) gene was induced in leukocytes treated with each dialysis membrane. The extent of induction depended on the membrane's material composition. The expression of the uPAR (CD87) protein on leukocytes was markedly increased in patients undergoing dialysis therapy. The magnitude of uPAR (CD87) protein expression was correlated with clinical findings, i.e., the degree of leukopenia and the expression of adhesion molecules. CONCLUSIONS: The gene and protein expression of uPAR (CD87) depended on the dialysis membrane material and correlated closely with clinical findings. These results suggest that uPAR has the potential to serve as a marker not only for clinical use but also for the development of new dialysis membranes.