Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Viral contamination in biologic manufacture and implications for emerging therapies.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

Meeting report-workshop on spike characterizations and virus removal by filtration: trends and new developments.

This workshop was held on June 3, 2013, in Berlin, Germany, in conjunction with the PDA Virus & TSE Safety Forum 2013. A total of nine speakers presented on key considerations of virus filtration, including a historical overview and emerging trends in evaluating parvovirus filters. Several talks addressed understanding the mechanism of virus capture and breakthrough by filters, as well as addressing this risk by carefully controlling transmembrane pressure. Improvements to validation studies were proposed via the use of highly purified virus preparations, more relevant models such as Chinese Hamster Ovary retrovirus-like particles, as well as new assays for virus quantification. The workshop ended with a panel discussion covering a range of topics including virus breakthrough, up-stream media treatment, virus spike preparation quality control, and regulatory expectations.

Meeting report--workshop on virus removal by filtration: trends and new developments.

The workshop was held on 27 June 2011 in Barcelona, in conjunction with the PDA Virus & TSE (transmissible spongiform encephalopathy) Safety Forum 2011. Virus-retentive filters are important tools to assure a high virus safety level of biological medicinal products. Important parameters such as properties of virus spike preparations, mechanism of virus retention by different filter brands, use of prefilters to improve the filtration performance, and, finally, strategies to select the most appropriate filter for a specific product were discussed on the workshop. The panel discussion at the end of the workshop that involved speakers and regulators from different global areas came to following conclusions: The major mechanism of virus retention is size exclusion; filtration, however, is complex and protein and virus can interact with the membrane in multiple ways. Pressure interruption during filtration resulted in enhanced virus passage. It has never been reported that murine leukemia virus (MuLV) passes a parvovirus filter. It makes sense that a small virus can be used to provide a claim for a large virus like MuLV. This relies on the assumption that there is no aggregation or interaction of the model parvovirus with proteins leading to aggregates larger than retroviruses. Several prefilters are under investigation to improve flow rate and throughput of filtration in large-scale manufacture. It was discussed whether the prefilter and the virus-retentive filter can be viewed as one unit operation so that virus retention by both can be claimed as the viral clearance capacity of this manufacturing step. This question engendered some controversy: whereas some saw the combination as a correct reflection of manufacturing conditions, others discussed the different mechanisms of virus retention, which need to be studied separately. All together, the workshop was seen as a valuable forum for the discussion between regulators and industry; it was proposed that such forum should be provided again if possible in connection with one of the next PDA Virus & TSE Safety Conferences. LAY ABSTRACT: The workshop was held on 27 June 2011 in Barcelona, in conjunction with the PDA Virus & TSE (transmissible spongiform encephalopathy) Safety Forum 2011. Virus-retentive filters are important tools to assure a high virus safety level of biological medicinal products. Important parameters such as properties of virus spike preparations, mechanism of virus retention by different filter brands, use of prefilters to improve the filtration performance and, finally, strategies to select the most appropriate filter for a specific product were discussed on the workshop. At the end of the workshop, aspects of the discussion were summarized by the following: The major mechanism of virus retention is size exclusion, but interactions are complex. Pressure interruption during filtration resulted in enhanced virus passage. It has never been reported that murine leukemia virus (MuLV) passes a parvovirus filter, and thus the parvovirus may provide a claim for a large virus like MuLV. Combination of prefilter and the virus-retentive filter are seen by some panelists as a correct reflection of manufacturing conditions; others discussed the different mechanisms of virus retention, which need to be studied separately. All together, the workshop was seen as a valuable forum for the discussion between regulators and industry.

The Pix pilus adhesin of the uropathogenic Escherichia coli strain X2194 (O2 : K(-): H6) is related to Pap pili but exhibits a truncated regulatory region.

Adhesins provide a major advantage for uropathogenic Escherichia coli in establishing urinary tract infections (UTIs). A novel gene cluster responsible for the expression of a filamentous adhesin of the pyelonephritogenic E. coli strain X2194 has been identified, molecularly cloned, and characterized. The 'pix operon' contains eight open reading frames which exhibit significant sequence homology to corresponding genes in the pap operon encoding P pili, the prevalent E. coli adhesins in non-obstructive acute pyelonephritis in humans. Although a pixB gene corresponding to the PapB regulator was identified, a papI homologue could not be found in the pix operon. Instead, a fragment of the R6 gene of the highly uropathogenic E. coli strain CFT073 was identified upstream of pixB. The R6 gene is located in a pathogenicity island containing several pilus-encoding sequences and shows homology to a transposase of Chelatobacter heintzii. In a pixA-lacZ fusion system it was demonstrated that the expression of Pix pili is regulated at the transcriptional level by the R6 gene sequence. A significantly reduced transcription was observed by deleting this fragment and by lowering the growth temperature from 37 to 26 degrees C. In contrast to other filamentous adhesin systems, Pix pili are mainly expressed in the steady state growth phase and were not repressed by the addition of glucose.