A validated biological manufacturing process includes the demonstration that the process adequately removes or inactivates potentially known or unknown viral containments. The International Conference on Harmonization (ICH) Q5A guideline, Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, describes the viral safety testing and evaluation of biotechnology products derived from human or animal origin that should be submitted within the biological registration package. ICH Q5A discusses complementary approaches to control potential viral contamination; however, the ICH guideline lacks specificity on how to conduct and perform appropriate viral clearance studies and has not been revised since 1999.
The United States Pharmacopeia-National Formulary (USP-NF) contains general information chapters (numbered from 1000 to 1999) that are for informational purposes only and are intended to provide best practices for manufacturers, regulators, and laboratories who are developing, manufacturing, testing, and releasing drug substances and products. A new General Chapter, <1050.1> Design, Evaluation and Characterization of Viral Clearance Procedures will soon publish in the 1st Supplement of USP39-NF34 and will become official August 1, 2016. This chapter provides practical insight and strategies for conducting viral clearance studies. General Chapter <1050.1> is a companion document to General Chapter <1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, which was adapted, essentially unchanged, from the ICH Q5A guidance. Chapter <1050.1> provides users with current, practical points to consider regarding the design, evaluation, and characterization of viral clearance procedures for products derived from the same class of manufacturing processes. The viral clearance study principles in 1050.1> will help generate meaningful data to demonstrate the suitability of the overall production and purification processes to remove or inactivate a broad spectrum of viral types that may affect the safety of recombinant products.
Chapter <1050.1> includes best practices beyond that of Q5A and discusses the viral clearance design plan, selection of virus, process clearance capability, downstream processing steps (including the need for different or orthogonal mechanisms of clearance), scale down, sampling time points, assay selection, matrix effects and storage. For process clearance capability, the chapter states that specific clearance targets must be established and justified. Generally, a log reduction factor (LRF) of 4 or more of clearance must be demonstrated for a single virus clearance step to be considered effective. In contrast, if a clearance step only demonstrates 1–3 LRF it is considered a supportive step. At least two independent experiments should be performed to demonstrate the reproducibility of an effective step or reproducibility should be supported by the process development history (or experiences).
When discussing the viral clearance design plan, Chapter <1051.1> emphasizes the importance of detailed documentation prior to starting the study which includes the following:
- the steps that will be tested
- the sampling plan for each step
- sample identification
- sample handling and storage
- sample shipment (if required)
- critical operating parameters
- process scale
- rationale for worst-case conditions (if they have been established), and
- appropriate controls for each step.
Regarding samples for testing, the chapter recommends (at a minimum) including samples for determination of 1) the spiking virus titer, 2) the spiking virus titer after freeze–thaw (if applicable), 3) the virus titer in the production solution before processing, and 4) the virus titer after processing. Additional samples may also be necessary, depending on the step under test and the phase of product development. Various types of viral quantitation assays can be used for testing, including infectivity assays such as tissue-culture infectious-dose assays and plaque quantitation assays. In addition, quantitative polymerase chain reaction (qPCR) assays are often helpful to detect and quantify the nucleic acids of both infectious and noninfectious viruses.
The choice and number of viruses that may be used in a viral clearance study are determined by an understanding of the history and details of the production cell line and any raw materials (e.g., animal-derived materials) that may be a source of viruses. At least two viruses, one enveloped (typically a retrovirus, e.g., MuLV) and one nonenveloped (preferably a parvovirus, e.g., MVM), are recommended in the early clinical phases of product development, but three or more viruses are more common for registration-enabling studies. The chapter includes a table of examples of viruses that can be used in viral clearance studies for biotechnology products derived from cell cultures. This table is harmonized with a comparable table in Q5A but has been updated with more recent viruses of concern.
Manufacturers and contract laboratories often struggle when developing suitable qualification and scale-down of purification steps so a portion of the chapter is devoted to this topic. The chapter states that any scaled-down purification process step must be qualified and found to be comparable to the full-scale production process by all relevant, measurable criteria. Comparability should be demonstrated using representative raw materials and intermediates from production, and equipment with process parameters through appropriate scaledown principles.
In a typical biomanufacturing process, viral clearance can be accomplished by virus inactivation (e.g., pH treatment, heat treatment, or solvent and detergent treatment) or by virus removal (e.g., filtration or column chromatography). Chapter <1050.1> also provides practical examples and general approaches for testing and sampling suggestions for viral clearance studies using inactivating agents, filtration and column chromatography. Although general in nature, the examples in Chapter <1051.1> provide practical insight and application of ICH Q5A to viral clearance sampling and testing in the biomanufacturing processes of biologics.
The chapter explains that virus inactivation is complex and involves a fast initial phase followed by a slower phase, making it necessary to take samples at different times in order to construct an inactivation time curve. Samples collected for inactivation studies should include the planned process time, and at a minimum, time zero; a time point or a suitable number of greater than zero but less than the minimum inactivating-agent exposure time; and a time point equal to the minimum inactivating-agent exposure time. In addition, a time point beyond the minimum exposure time might be helpful in cases where there is a slow inactivation curve. Additional time points may be particularly important when there is no prior experience with virus inactivation kinetics. Sampling points for virus-removal steps, such as chromatography and filtration, should include the feed-stream process solution that will be applied to the step, as well as the resulting process pool that is then processed in the subsequent steps.
Sample matrix effects testing should be performed before the spiking studies to confirm that the results of the viral clearance spiking studies are actually due to viral clearance and not other intrinsic or extrinsic factors yielding a false-negative result. If process solutions are used within their established hold times, stability studies should be performed to better understand the effect of holding or storing the solutions on the clearance step, or on the virus (e.g., aggregation).
If the samples from the spiking study are stored frozen before virus quantitation, analysts should perform stability studies using the viral infectivity result as an endpoint. Ideally, this should be done before the viral spiking study by spiking dilutions of each process solution with a known amount of virus and then freezing each sample.
The chapter includes a series of figures regarding the design and sampling of viral clearance studies that are often encountered in biomanufacturing situations. The viral clearance techniques described in the figures include virus inactivation, filtration, and chromatography. When viral clearance by inactivation occurs, two scenarios generally are possible: when load material does not contain an inactivating agent (e.g., the inactivating agent is added after viral spiking), and when load material contains an inactivating agent (e.g., Protein A column eluate at low pH). Suggested sampling of the process intermediates with and without inactivants are illustrated in the figures. Additional figures cover general approaches for design and sampling for viral clearance performed via filtration and chromatography. In these situations, virus spiking can be associated with difficult-to-predict aggregation effects and practical user approaches are offered as well as chromatographic considerations.
USP has also developed two other general chapters related to this topic: General Chapter <1237> Virology Test Methods and General Chapter <1240>Virus Testing of Human Plasma for Further Manufacture. Chapter <1237> contains a variety of test methods (immunochemical, nucleic acid testing, in vivo, and in vitro cell-based methods, etc.) that are commonly used to identify and quantify viral contaminants. These tests are independent of sample source and much like in chapter <1050.1>, there is a significant section on demonstrating that the methods are suitable for the particular sample types. Since ICH Q5A (chapter <1050>) states that blood derived products are out of scope, USP also developed chapter <1240> for plasma-derived materials intended for further manufacture since no ICH guidance specifically covers this topic. The chapter covers U.S. and European regulations and guidances for testing of these donated materials using immunoassays and nucleic acid tests. In the future, USP plans to develop a new chapter for demonstration of viral clearance in plasmaderived products, analogous in focus to that of <1050.1>, but covering the unique challenges presented by these types of studies.
References
International Conference on Harmonization (ICH) Q5A guideline, Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5A_R1/Step4/Q5A_R1__Guideline.pdf.
Author Biographies
Dr. Maura C. Kibbey is the Director of Science & Standards in USP’s Global Biologics department. Dr. Robert G. Bell is the Chair of the USP Viral Clearance Expert Panel, a member of the USP General Chapters- Biological Analysis Expert Committee, and the President/Owner of Drug & Biotechnology Development, LLC.