Evaluation of a viral transcriptome Next Generation Sequencing assay as an alternative to animal assays for viral safety testing of cell substrates.

The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample inoculation in animals and embryonic eggs. Following the 3Rs principles of replacement, reduction, and refinement of animal-use methods, such techniques are intended to be replaced not only for ethical reasons but also because of their inherent technical limitations, their long turnaround times, and their limits in virus detection. Therefore, we have compared the limit and range of sensitivity of in vivo tests used for viral testing of cells with a transcriptomic assay based on Next Generation Sequencing (NGS). Cell cultures were infected with a panel of nine (9) viruses, among them only five (5) were detected, with variable sensitivity, by in vivo tests. The transcriptomic assay was able to detect one (1) infected cell among 103 to 107 non-infected cells for all viruses assessed, including those not detected by the conventional in vivo tests. Here we show that NGS extends the breath of detection of viral contaminants compared to traditional testing. Collectively, these results support the replacement of the conventional in vivo tests by an NGS-based transcriptomic assay for virus safety testing of cell substrates.

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.

Twenty plus Years of Data Demonstrating Virus Filtration as an Effective and Robust Step for Large Virus Removal.

Virus filtration has been demonstrated to be an effective and robust dedicated viral clearance step that is used in biopharmaceutical manufacturing processes. Here we present virus filtration data from a multicompany collaboration with data compiled from WuXi Advanced Therapies' and Charles River Laboratories' internal viral clearance databases spanning more than 25 years. The data were sorted by virus removal and type and then further subdivided into murine leukemia virus only, pseudorabies virus only, and reovirus type 3 only categories to allow for analyses of viral clearance results. A total of 2311 virus filtrations were analyzed, composed of 1516 murine leukemia virus, 385 pseudorabies virus, and 410 reovirus type 3 virus filtrations. These data provide clear evidence that will help supplement both internal and industry-wide initiatives focused on using prior knowledge for the creation of modular claims for small virus retentive filters and allow better allocations of resources typically spent on potentially unnecessary studies.

Viral safety for biotherapeutics and biosimilar.

This review summarizes the viral safety concepts applied for cell line derived recombinants including biosimilars. The major aspects - material sourcing, testing, and viral clearance - are outlined and essentials per aspect to be considered described in more detail. The principles of viral clearance are explained in more detail like the background of viral removal or inactivation, model virus selection and definition of virus reduction capacity.

Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells.

The utilization of the current combination of in vitro, in vivo and PCR assays for the identification of adventitious viruses in production cells has a limited range of detection. While Next Generation Sequencing (NGS) has a broader breadth of detection, it is unable to differentiate sequences from replicating viruses versus background inert sequences. In order to improve NGS specificity, we have designed a new NGS approach which targets subsets of viral RNAs only synthesized during cell infection. In order to evaluate the performance of this approach for detecting low levels of adventitious viruses, we selected two difficult virus/cell systems. This included B95-8 cells persistently infected by Human herpesvirus 4 (HHV-4) and serially diluted into HHV-4 negative Ramos cells and Madin-Darby bovine kidney cells with an early infection produced via a low dose of Bovine viral diarrhea virus. We demonstrated that the sensitivity of our RNA NGS approach was equivalent to targeted PCR with an increased specificity for the detection of viral infection. We were also able to identify a previously undetected Murine Leukemia Virus contaminant in Ramos cells. Based on these results, we conclude that this new RNA NGS approach is suitable for conducting viral safety evaluations of cells.

Virus study for continuous low pH viral inactivation inside a coiled flow inverter.

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.

Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers.

Pore-size distribution (PSD) is the most critical parameter for size-exclusion virus removal filters. Yet, different dry- and wet-state porometry methods yield different pore-size values. The goal of this work is to conduct comparative analysis of nitrogen gas sorption (NGSP), liquid-liquid and cryoporometry with differential scanning calorimetry (CP-DSC) methods with respect to characterization of regular and cross-linked virus removal filter paper based on cellulose nanofibers, i.e. the mille-feuille filter. The filters were further characterized with atomic force and scanning electron microscopy. Finally, the removal of the worst-case model virus, i.e. minute virus of mice (MVM; 20 nm, nonenveloped parvovirus) was evaluated. The results revealed that there is no difference of the obtained PSDs between the wet methods, i.e. DSC and liquid-liquid porometry (LLP), as well as no difference between the regular and cross-linked filters regardless of method. MVM filtration at different trans membrane pressure (TMP) revealed strong dependence of the virus removal capability on applied pressure. It was further observed that cross-linking filters showed enhanced virus removal, especially at lower TMP. In all, the results of this study highlight the complex nature of virus capture in size-exclusion filters.

Achieving a Successful Scale-Down Model and Optimized Economics through Parvovirus Filter Validation using Purified TrueSpikeTM Viruses.

UNLABELLED: This article describes a four virus panel validation of EMD Millipore's (Bedford, MA) small virus-retentive filter, Viresolve® Pro, using TrueSpike(TM) viruses for a Biogen Idec process intermediate. The study was performed at Charles River Labs in King of Prussia, PA. Greater than 900 L/m(2) filter throughput was achieved with the approximately 8 g/L monoclonal antibody feed. No viruses were detected in any filtrate samples. All virus log reduction values were between ≥3.66 and ≥5.60. The use of TrueSpike(TM) at Charles River Labs allowed Biogen Idec to achieve a more representative scaled-down model and potentially reduce the cost of its virus filtration step and the overall cost of goods. The body of data presented here is an example of the benefits of following the guidance from the PDA Technical Report 47, The Preparation of Virus Spikes Used for Viral Clearance Studies. LAY ABSTRACT: The safety of biopharmaceuticals is assured through the use of multiple steps in the purification process that are capable of virus clearance, including filtration with virus-retentive filters. The amount of virus present at the downstream stages in the process is expected to be and is typically low. The viral clearance capability of the filtration step is assessed in a validation study. The study utilizes a small version of the larger manufacturing size filter, and a large, known amount of virus is added to the feed prior to filtration. Viral assay before and after filtration allows the virus log reduction value to be quantified. The representativeness of the small-scale model is supported by comparing large-scale filter performance to small-scale filter performance. The large-scale and small-scale filtration runs are performed using the same operating conditions. If the filter performance at both scales is comparable, it supports the applicability of the virus log reduction value obtained with the small-scale filter to the large-scale manufacturing process. However, the virus preparation used to spike the feed material often contains impurities that contribute adversely to virus filter performance in the small-scale model. The added impurities from the virus spike, which are not present at manufacturing scale, compromise the scale-down model and put into question the direct applicability of the virus clearance results. Another consequence of decreased filter performance due to virus spike impurities is the unnecessary over-sizing of the manufacturing system to match the low filter capacity observed in the scale-down model. This article describes how improvements in mammalian virus spike purity ensure the validity of the log reduction value obtained with the scale-down model and support economically optimized filter usage.