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2013 Viral Clearance Symposia - Session 5. Conference Summary: Key Discussion and Outcomes, Pending Questions, and Proposed Experiments Johannes Blümel and David Roush

PDA J Pharm Sci and Tech 2015, 69 206-210 Access the most recent version at doi:10.5731/pdajpst.2015.01044

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2013 Viral Clearance Symposia - Session 5. Conference Summary: Key Discussion and Outcomes, Pending Questions, and Proposed Experiments JOHANNES BLU¨MEL1,* and DAVID ROUSH2,* 1

Paul-Ehrlich-Institut; and 2Merck, Sharp and Dohme, Inc. ©PDA, Inc. 2015

The 2013 conference structure extended the framework from the preceding conferences (2009 and 2011), focusing on the key gaps and associated developments, but also including additional cases studies on integrated viral clearance strategies, linkage to upstream, and ways to improve the efficiency of the overall adventitious agent strategy. This report provides a high-level summary of the key gaps identified from the 2011 meeting, key outcomes and gaps based on the research presented at the 2013 Symposia, and new areas identified for future improvements. The first four sessions (1.1 to 1.4) provided an overview of Established Unit Operations including Viral Retentive Filtration (VRF), Anion Exchange (AEX) Chromatography, Protein A Chromatography, and Viral Inactivation. Subsequent discussions covered Alternative Viral Clearance and Inactivation Strategies (Session 1.5), Integrated Strategies including a Linkage to Upstream (Sessions 2.0 and 3.0, respectively) and an Overall Integrated Viral Clearance and Adventitious Agent Control Strategy (Session 4.0). More detailed discussion of the specific research presented is provided in the reports from the respective sessions. The new areas identified during the 2013 Symposia provide a framework for future discussions at the 2015 Viral Clearance Symposium.

Impact of virus purity of performance.

Impact of depressurization— how to study, intermediate tools to predict filterability.

Key Outcomes ●

A bracketed approach for lab-scale validation was proposed to potentially support multiple cycles of “stop-go” viral filtration within a harvest batch in the production. In analogy to validation of chromatographic columns, virus is added only to the initial and final cycle of filtration. However, it remains unclear whether health authorities accept such an approach.

Filter performance and/or virus retention effectiveness can be potentially affected by multiple parameters such as throughput (g/m2 or L/m2), product concentration, pH, level of impurity (process-related or product-related), and quality of virus preparations. Diffusion of virus particles under low pressure/flow might cause virus breakthrough in certain virus filter brands.

Virus clearance capacity determined by laboratoryscale virus reduction studies can be influenced by the amount of spiked viruses. Higher log10 reduction values (LRVs) were obtained when using lower amount of parvovirus spike (⬍8 log10).

Correlation between parvovirus breakthrough and low pressure or depressurization was reported by multiple companies, further indicating the importance of control/monitoring of the pressure in the production.

Session 1.1 Viral Retentive Filtration (VRF) Key Items Discussed ●

Sampling handling including freeze and thaw can have significant impact on performance.

*Corresponding Authors: David Roush, Merck, Sharp and Dohme, Inc., e-mail: [email protected]; Johannes Blu¨ mel, Paul-Ehrlich-Institut, e-mail: [email protected] doi: 10.5731/pdajpst.2015.01044


Pending Questions and Proposed Mechanistic Experiments ●

How to quantify the impact of depressurization on breakthrough (worst-case conditions)?

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New analytical/biophysical tools to predict filterability (capacity, flux).

Is the “bracketed” approach acceptable for regulatory filings?

How to justify the amount of spiking viruses in the lab-scale studies to ensure the validity and representativeness of the LRV achieved?

Follow-up experiments are needed in order to confirm that virus break-through correlates with breakthrough of impurities in multiple monoclonal antibody (MAb) systems. ●

Potential impact of interactions between MAb and virus spike on virus clearance.

Session 1.3 Protein A Chromatography

Post-Meeting Note

Key Items Discussed

Mechanism of Xenotropic murine leukemia virus (XMuLV) partitioning in the protein A step.

Alternate approaches combining virus inactivation with partitioning of viruses at protein A chromatography (part of Integrated Strategy, Session 4).

Initial work on the impact of virus quality for porcine parvovirus (PPV) for a specific virus filter recently published: Slocum, A.; Burnham, M.; Genest, P.; Venkiteshwaran, A.; Chen, D.; Hughes, J. Impact of virus preparation quality on parvovirus filter performance. Biotechnol. Bioeng. 2013, 110 (1), 229 –239.

Session 1.2 Anion Exchange (AEX) Chromatography

Key Outcomes ●

Most virus spike does not bind to the protein A column. However, a portion of virus can interact with the MAb bound to the resin, resulting in poor or moderate removal of XMuLV by protein A chromatography.

Key Items Discussed ●

Impact of AEX step in process on viral clearance.

Potential use of phage as a surrogate for virus partitioning in AEX.

Additives can be used to inactivate virus while bound to protein A column.

Impact of impurities on AEX viral clearance.

Partitioning of virus in protein A chromatography appears to vary depending on the MAb employed.

Key Outcomes Pending Questions/Experiments ●

Breakthrough of Impurities (host cell proteins or HCP, DNA, and potentially aggregates) correlates with poor LRV. Breakthrough of impurities could be used as a surrogate for breakthrough of virus. However, additional work is required for understanding this correlation and exploring the limits of tolerable breakthrough of impurities (and virus). When AEX was placed as a second purification step, impurities occasionally affected virus reduction. When AEX was placed as the third step in the purification scheme, there was in many cases consistent performance of virus reduction. This might be explained by the low levels of impurities at this stage.

Pending Questions/Experiments ●

Some variability in LRV was observed when AEX flowthrough (F/T) was the second operating step.

Vol. 69, No. 1, January–February 2015

Additional mechanistic experiments to map LRV variability in protein A chromatography.

Explore the benefit (increase in LRV) of additional regents (salt washes, washes with detergents) at more MAb/virus systems.

Session 1.4 Viral Inactivation (Low pH, Detergent, UV-C) Low pH Key Outcomes ●

Differences in inactivation kinetics have been observed across various buffer systems. An acetate buffer system (compared to a glycine or citrate buffer system) was the most robust, enabling complete in207

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activation across a pH range (pH 3.3–3.9) independent of protein concentration and temperature. ●

Inactivation of MuLV in the other two buffer systems is robust at pH 3.3; however, inactivation at higher pH values is more difficult to predict.

to a proposed standard by ASTM (ASTM WK39883). ●

Evaluate inactivation kinetics for Triton side-byside with alternative detergents.

Characterize robustness of inactivation of a wider range of viruses with varying properties using Triton X-100 at low concentration and alternative detergents.

Collaboration with detergent manufacturers to establish safety of ingredients for new generation detergents (e.g., tolerable daily exposure values).

Discrepancies in performance with citrate have been observed among various companies.

Pending Questions/Experiments ●

Mechanistic experiments to provide a better understanding of the role of buffer types in virus inactivation (e.g., more robust virus inactivation in acetate beneficial versus less robust inactivation in citrate buffer).

Session 1.5 Alternative Unit Operations ●

The American Society for Testing and Materials (ASTM) has proposed a standard procedure for inactivation of retroviruses by low pH (WK36552) and specific buffer systems have not been defined. Since specific buffer/product systems have not been defined in the proposed standard, the impact of different buffer systems on the procedure should be investigated.

Harvest Key Items Discussed and Outcomes ●

The retrovirus-like particle (RVLP) content entering the downstream process (i.e., RVLP content of the clarified harvest) was found to be approximately 2.1 logs lower than would be predicted by measuring RVLPs in unprocessed bulk, with approximately 1.7 logs of clearance being observed across the harvest step.

Samples were taken were from clinical trials support batches (at scale production lot) and therefore are representative and relevant.

Detergents Key Items Discussed and Outcomes ●

Since Triton X-100 is toxic to aquatic organisms and is subject to disposal restrictions at some sites, there is a need for alternative detergents for virus inactivation. Several non-toxic alternative detergents were identified with good inactivation of XMuLV and PRV. These alternatives can also be effectively cleared during subsequent Protein A purification and have no negative impact on product quality. There is potential to reduce levels of Triton X-100 to below the widely-used concentration of 1% (e.g., 0.2% to 0.8%). The time to XMuLV inactivation in harvested cell culture fluid and lysed cell supernatant is quick, regardless of concentration (0.1% or 0.3%). However, the amount of host cell impurities can affect the minimum concentration of Triton X-100 needed to achieve complete inactivation.

Pending Questions/Experiments ●

Review databases to determine if a critical level of Triton (e.g., 0.5%) can be determined and linked


Pending Questions/Experiments ●

Continue work comparing RVLP concentration, by transmission electron microscopy (TEM) assay, from unprocessed bulk versus clarified cell culture supernatant.

It was suggested that measuring the TEM value post– harvest clarification is more representative of the true RVLP content entering the downstream process and is more sensitive for the detection of RVLPs. There was no consensus on the appropriateness for the use of this for clinical trial applications. One could argue that clarified bulk is relevant for ICH Q5A risk assessment. Data reviewed were collected from production-scale processes. Hence, evaluation of the impact of the primary recovery step on TEM values would be beneficial.

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Membrane Adsorbers

HTST (High-Temperature/Short-Time)

Key Items Discussed and Outcomes

Key Items Discussed and Outcomes

Membrane chromatography continues to gain adoption for use in the manufacture of recombinant protein therapeutics. A comparison of AEX membrane chromatography to traditional packed bed AEX resins operated in a flowthrough mode was presented. The results indicated that AEX membranes can provide viral clearance properties similar to traditional AEX resins.

HTST treatment of cell culture media is widely used to reduce the risk of adventitious virus contamination. The results indicate that HTST treatment at 102 °C for 10 seconds provides robust parvovirus inactivation [data presented for minute virus of mouse (MVM)] in a variety of cell culture media and provides an effective viral barrier.

However, the impact of impurities on performance of virus reduction (LRV) was observed.

The potential to establish a standard procedure was discussed.

Pending Questions/Experiments

Pending Questions/Experiments

The impact on impurities on LRV for several viruses should be further investigated. Specifically, the evaluation of the impact of DNA and HCP separately (data presented included both HCP and DNA).

FDA data base summary comparison of AEX and adsorbers— consistent performance (2/18 outliers with low LRV). The data base should be updated accordingly.

Key Items Discussed and Outcomes

The impact of ramp-up and ramp-down of temperature on performance needs to be assessed.

Sessions 2, 3, and 4

Adventitious Agent (AA) Control Gap ●

The sensitivity gained by extending in-vitro testing for Master Cell Bank (MCB) and Un-Processed Bulk (UPB) was discussed.

In vitro viral assays for adventitious viruses screening typically employ three or more cell lines and can be carried out with either a 14 day or 28 day duration. The additional passage in the 28 day assay improves the assay sensitivity but may increase the risk of introducing viral contaminants during culture passage. For UPB testing, the 14day assay may be a compromise as it provides adequate virus detection and minimizes the chance of potential false positives.

Sponsor should use Fetal Bovine Serum (FBS) that has been treated with effective virus elimination methods (e.g., gamma irradiation).

Mixed Mode Key Items Discussed and Outcomes ●

Ceramic hydroxyapatite (CHT) chromatography can provide additional virus clearance capacity to the downstream process. The partitioning pattern for CHT indicated that viral clearance is generally achieved through virus binding to the resin and being retained during product elution. The weakest retention was found with PPV, while both XMuLV and PRV showed stronger retention to the resin. Reduction of Simian virus 40 (SV40) was more variable.

Pending Questions/Experiments ●

The mechanism of virus reduction on CHT has not been fully clarified (balance of hydrophobic and electrostatic properties). Impact of impurities needs to be further characterized (robust with low impurity levels—third chromatography step).

Vol. 69, No. 1, January–February 2015

Pending Questions/Experiments ●

Quantitative comparison of detection sensitivity for multiple model viruses between 14 day and 28 day in vitro viral testing. 209

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Impact of Cell Line on Adventitious Agent (AA) Strategy

Other Virus Safety Aspects

Key Items Discussed and Outcomes

Key Items Discussed, Outcomes and Pending Questions



Per.C6 cells can be considered as Case A category of the ICH Q5A guideline, if tested negative for viral transcriptase (reverse transcriptase, RT) activity. Testing RT by product-enhanced RT assay sometimes shows background activity, while conventional RT assay and TEM assay are less sensitive.

Risk assessment is challenging for unknown AA risk.

RVLP preparations from side fraction obtained at purification of monoclonal antibodies have been used as spikes in virus clearance. Comparable reduction of Chinese hamster ovary (CHO) cellderived RVLPs and XMuLV at chromatographic purification steps and virus filtration was observed. Using RVLPs preparations for spiking studies has the advantage that no laboratory with specific biosafety requirements would be needed. However, a suitable CHO cell line producing high numbers of RVLPs is essential. At the discussion, the question was raised whether similar results could be obtained with RVLPs from other rodent cell lines such as mouse myeloma (NSO).

The potential to utilize RVLP preps in other unit operations (e.g., low-pH inactivation) and means to reduce the limit of quantification (LOQ) for the assay were also discussed.

Baculovirus ●

Baculoviruses and parvoviruses can be reduced from the product stream by AEX chromatography.

Differences between PPV and MMV clearance were observed, but it was not finally clarified whether this result is caused by a different binding strength of both parvoviruses, virus preparation (including impurities in the spike), or is related to the detection methods [infectivity assay versus quantitative polymerase chain reaction (qPCR)].

Virus Retentive Filtration ●

Parvoviruses are much smaller than retroviruses and LRVs obtained from studies with a parvovirus at virus filtration can be used as a claim for retrovirus removal.

Post-meeting note: Evaluation of use of a parvovirus (MVM) for worst case in virus retentive filtration was recently published: Gefroh, E.; Dehghani, H.; Mcclure, M.; Connell-Crowley, L.; Vedantham, G. Use of MMV as a single worst-case model virus in viral filter validation studies. PDA J. Pharm. Sci. Technol. 2014, 68 (3), 297–311.

Pending Questions/Experiments ●

Determination of isoelectric points (pI) for virus panel (including PPV). The pIs for MVM and XMuLV (pI is 5.8) have been determined. Expand virus panel to include determination of pI for other viruses (e.g., circoviruses?).

Initial estimate for PPV pI is 5.5 (communication by G. Miesegaes).

Post-Meeting Note: Recent literature publication on Rhabdovirus contamination in insect cells: Ma, H.; Galvin, TA.; Glasner, DR.; Shaheduzzaman, S.; Khan, AS. Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines. J. Virol. 2014, 88 (12), 6576 – 6586; early/2014/03/20/JVI.00780-14.


Column Lifetime and Carryover Studies ●

The potential to simplify the approach for validation of re-used columns (virus removal at end of column lifetime and sanitization demonstrated by virus carry-over studies) was discussed. Further exploration is needed.

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2013 viral clearance symposia - session 5. Conference summary: key discussion and outcomes, pending questions, and proposed experiments.

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