1. In your opinion, what are the biggest challenges in viral detection, clearance, inactivation and removal?

I cannot speak for the whole industry for this question, but for our company we have encountered serious problems in virus filtration and virus inactivation at low pH for our IgG2 therapeutic mAbs. One problem is that these mAbs are unstable in terms of aggregation and loss of potency when kept at low pH (below 4). Another problem is that these mAbs have 10-times lower throughput in virus filtration than regular mAbs. Our preliminary investigation suggests that the extremely low filtration throughput is related to the high hydrophobicity of these mAb molecules, rendering the molecules self-associated to foul the filter. One interesting observation is that different virus filters from various vendors did not show much difference in the filtration throughput for these molecules. We have identified the critical factors to significantly enhance the virus filtration throughput of these molecules and integrated the low pH virus inactivation step with the virus filtration step as will be reported in my presentation.

Guihang Zhang, Ph.D., Purification Group Leader, Process Sciences, Agensys

Massive parallel sequencing is currently highly discussed as an effective tool for virus screening. In principle detection of even unknown viruses t low concentration is possible. However, the technology and particularly the data analysis is complex and obviously difficult to establish within a quality assured (GMP) environment.

New technologies for virus removal and inactivation without affecting sensitive biological become more and more attractive in the purification of recombinant products. UV treatment can inactivate small non-enveloped virus like PPV or MVM very effectively. Multimodal Chromatography and Membrane Adsorber do not only reduce the viral load significantly it also reduce the time and the number of process steps required for product purification

Horst Ruppach, Ph.D., Head of Viral/TSE Safety Studies, Biopharmaceutical Services, Charles River, Germany

One of the most significant challenges in viral detection is the general lack of available technologies to rapidly identify and characterize viruses. An emerging technology in this space is electrospray differential mobility analysis (ES-DMA). This technology is a rapid, quantitative, validated, label free platform for virus identification and characterization with sub-nanometer resolution of full multimodal size distributions. We have demonstrated that it can detect viruses by size, have worked out strategies for rapid virus identification, and can quantify the degree of viral inactivation. We look forward to discussing each of these advances in our presentation entitled "Electrospray Differential Mobility Analysis (ES-DMA): An emerging technology for viral identification, characterization and clearance" at the 2012 IBC Viral Safety for Conference.

Leonard Pease, Ph.D., Professor, Departments of Chemical Engineering, Pharmaceutics & Pharmaceutical Chemistry and Internal Medicine, University of Utah

2. In your opinion, what is an overlooked challenge in viral detection?

The assays used in viral testing should not be taken for granted. Many companies rely on CROs for performing their viral testing. These CROs typically have valuable expertise, but frequent and open communication between CROs and clients is essential to ensure that assays are updated to reflect new viral threats, and that the lab practices are in place to reduce the risk of false positives.

Mark D. Moody, Ph.D., Vice President, Analytical Services, Merrimack Pharmaceuticals

3. Which stage of the process has the most inherent risk of a viral contamination - upstream processing, downstream processing or manufacturing operations? Why?

Because a contaminating virus can replicate during cell culture, a single infectious virion could lead to massive contamination of a bioreactor. Therefore, attention should be focused on risks to the upstream process. And, because viruses such as MMV virus can persist in the environment, there are many potential opportunities for the virus to be introduced into the manufacturing process through contamination of materials or equipment.

Mark D. Moody, Ph.D., Vice President, Analytical Services, Merrimack Pharmaceuticals

The upstream process has the most inherent risk of virus contamination. The cell bank itself bears a risk depending on the history of the cell line. The raw material used in the culturing processes can introduce viruses. Finally, the culturing process itself can provide an ideal environment for very low virus contaminants to grow up to high titer. Low titer contaminations in the downstream process are less critical. They cannot grow up and might only be concentrated through the purification process. This level, however, will generally be much lower compared to fermentation process.

Horst Ruppach, Ph.D., Head of Viral/TSE Safety Studies, Biopharmaceutical Services, Charles River, Germany

I think it best if I leave this question to those who work directly in the manufacturing environments, as their insight would be more valuable to your participants than my second hand information.

Leonard Pease, Ph.D., Professor, Departments of Chemical Engineering, Pharmaceutics & Pharmaceutical Chemistry and Internal Medicine, University of Utah

4. How do virus filters help? What are the current technological advances happening with virus filters?

Virus filters are an important purification step because they add an orthogonal, size-based removal mechanism to compliment viral inactivation and chromatographic removal. In a relatively short amount of time, we have seen viral filtration move from tangential to normal flow, a shift from large to small viral retentive filters, and improvements in membrane design to prevent viral passage despite increased permeability decay. Looking forward, I think membrane design will continue to improve, leading to higher permeability / capacity filters while maintaining a high level of viral clearance. Other improvements, such as pre-filtration, chemical additives, or upstream process optimization, will continue to be investigated hopefully leading to more economic and robust filtrations.

Jerome Bill Jr., Engineer II, Purification Development, Genentech, Inc.

Virus filters are specifically introduced into the manufacturing process for virus removal. Some filter vendors have already launched second generation filters and other vendors are working on new filters. A higher product throughput and efficient virus reduction are targeted parameters. However, the currently discussed smallest virus - the porcine circovirus, which is smaller than the standard challenging viruses PPV and MVM - gives different results on different filters: Complete removal to no significant removal are reported.

Horst Ruppach, Ph.D., Head of Viral/TSE Safety Studies, Biopharmaceutical Services, Charles River, Germany

Virus filters are clearly helpful in removing viruses, but only if properly selected. The Parenteral Drug Association's Virus Filtration Taskforce has established standards to assist in filter selection, differentiating small virus filters from large virus filters. The bacteriophages pp7 and PR772 were selected as the test phages for the small and large virus filters, respectively. We have further shown that small differences in precisely measured virus sizes correlate with the breakthrough rate through some of these filters. We look forward to presenting this data at our presentation entitled "Electrospray Differential Mobility Analysis (ES-DMA): An emerging technology for viral identification, characterization and clearance" at the 2012 IBC Viral Safety for Conference.

Leonard Pease, Ph.D., Professor, Departments of Chemical Engineering, Pharmaceutics & Pharmaceutical Chemistry and Internal Medicine, University of Utah