Optimizing a Viral Testing Strategy

Dr. Tim Sandle, PhD - Head of GxP Compliance and Quality Risk Management, Bio Products Laboratory Limited

Introduction

Viruses are found in almost every ecosystem on Earth and are the most numerous types of biological entity. As such, viruses pose a challenge to many aspects of biopharmaceutical manufacturing. These challenges not only relate to viruses per se or viruses that pose a particular problem to different cell lines or production processes, but also from the varied nature of viruses themselves – the genetic material (molecules of DNA or RNA that encode the structure of the proteins by which the virus acts); the nature of the protein coat (the capsid, which surrounds and protects the genetic material) and whether or not the virus possess an outside lipid envelope.¹ The shapes of these virus particles range from simple helical and icosahedral forms to more complex structures. These geometrically sophisticated architectures are influencing factors where nanofiltration is used as a viral inactivation step. Given the risk presented from both pathogenic viruses in starting materials and from adventitious agents arising during bioprocessing, the testing strategy becomes an essential part of the viral contamination control strategy.

In a previous issue of American Pharmaceutical Review, a strategy was outlined for viral reduction and virus clearance within a facility.² This companion article considers the testing component of the overall viral strategy. 

Virus Contamination

With the exception of Adeno-Associated Viruses (AAV) vectors as platforms for gene delivery for the treatment of many human diseases, specific viruses are unwanted in bioprocessing. Viral contamination is a potential safety threat common to all animal and human-derived biologics and it follows that ensuring virological safety is challenging. Despite advances in processing technology, the use of cell culture to produce recombinant proteins continues to be susceptible to contamination with viruses. In particular, viral infection of mammalian cell culture presents a continued risk.³ At risk products and manufacturing stages include:

• Products produced from in vitro culture of cells lines of animal or human origin;
• Products produced from in vivo culture of cell lines;
• Products produced from organs or tissues of human origin;
• Products produced from blood or other human fluids.

Complete viral safety, as defined by absolute freedom from extraneous viral agents, is not easy to achieve and some would argue that it is an impossible task due to the difficulties in testing for the full range of viruses and residual virion activity. What is important is excluding or clearing viral pathogens of concern (by viral inactivation methods and viral removal methods).⁴ When contamination with pathogenic viruses occurs, such contaminations can potentially cost millions of dollars, especially when a facility needs to be decontaminated and a recovery process developed. Most importantly, enforced shutdown can lead to patients not receiving therapies.

In terms of viruses of concern, the contaminated cell type, contaminating virus and suspected source of contamination varies. Perhaps the most common are: herpesvirus, human adenovirus type 1, parainfluenza virus type 3 and reovirus type 3. In addition:

• Minute virus of mice (MVM), of which there are different infective species. An example is Rodent protoparvovirus. These viruses are common infectious agents of laboratory mice, easily spread between populations. In processing, the primary risk is to mammalian cell culture, especially Chinese hamster ovary cell-based production of biologic drugs. Also, at risk are NS0 cells. These are a model cell line derived from the non-secreting murine myeloma used in biomedical research and commercially in the production of therapeutic proteins.
• Vaccinia virus is a large, complex, enveloped virus belonging to the poxvirus family. This virus has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes. The most common control mechanisms are effective HVAC systems.
• Rhabdovirus, which is one of the emergent novel viruses of note. This virus is from a family of viruses responsible for rabies and vesicular stomatitis of cattle and horses. The virus has been associated with contamination of some cell culture: Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), used in insect cell culture for recombinant protein production.

Viruses can be enclosed in an envelope made of proteins (capsid), carbohydrates, and lipids (enveloped) or alternatively they are nonenveloped. An example of an enveloped virus is herpes; an example of a non-enveloped virus is parvovirus. Generally non-enveloped viruses are the most difficult to remove in viral clearance studies.

Testing

With the detection of viruses, while there are some indicators such as a change in cell culture parameters as a leading indicator of a contamination, only a robust testing strategy can determine presence and provide an estimation as to the titer and all major viral contamination events can be virus-specific Polymerase Chain Reaction (PCR) assay, provided a suitable method is selected and recovery can be proven through validation. Viruses are composed of DNA (such as herpes viruses) or RNA (such as hepatitis viruses) encapsulated by a protein coat, and there are PCR approaches for both. PCR approaches are superior to traditional infectivity assays and electron microscopy.

PCR is a laboratory method used for making a very large number of copies of short sections of DNA from a very small sample of genetic material.⁵ This process is called “amplifying” the DNA and it enables specific genes of interest to be detected or measured. With viral RNA, the single stranded nucleic acid molecule needs to be made into DNA before it can be amplified. This is achieved by the enzyme called Reverse Transcriptase (RT) and an antisense (reverse) primer. PCR assays can be developed relatively quickly, which is advantageous should a novel pathogen emerge. Furthermore, primers can be combined to allow for the identification of multiple viruses in a single test. This is normally up to a maximum of four, in terms of reliability for beyond this test sensitivity decreases. PCR methods are also affected by mutations, which also affect sensitivity, and which can lead to false negative results.

An alternative to PCR is Next Generation Sequencing (NGS). In terms of the advantages and disadvantages of the test approaches, real-time PCR is arguably easier to use and more tolerant of variable DNA quality. However, it has a more limited multiplex capability (that is, the ability to screen for multiple targets). NGS, in contrast, allows simultaneous analysis of various genomic loci and it can reveal the exact sequence changes.⁶ An example is shotgun metagenomics and such technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA.⁷ These methods have opened up our understanding of virus discovery, identification, sequencing, and manipulation. However, NGS methods are generally more expensive to run than PCR methods and not always as practicable. All assays should include appropriate controls to ensure adequate sensitivity and specificity.

Testing Strategy

The approach to viral testing needs to be planned out and this should connect with process understanding, preventative measures, and virus clearance steps.⁸ These should be brought together within a contamination control strategy. The testing strategy should be considered from the outset of new product development. Failure to account for later stage regulatory requirements⁹ in the early development phases is a common source of project delays and failures.

Once established, testing should involve starting materials and cell cultures, including master and working cell banks. In addition, a risk-based framework should be used to establish the use of analytical tools used for materials, upstream and throughout the process.¹⁰ This is to:

• Ensure the manufacturing processes can handle the product and stay free from viral contamination. This is especially when used upstream and throughout the process.
• Consider how real-time testing (as with qPCR methods) can shorten the time required for batch release.

Risk assessment is best served through the application of HACCP (Hazard Analysis and Critical Control Points). Here it is necessary to determine Critical Process Parameters (CPP) and the specific Critical Control Points (CCPs) in the manufacturing process. An effective HACCP will begin with prevention, then consider where contamination could occur as it tracks the process downstream. Examples of prevention include selecting cell lines and other raw materials, including media components, based on the likely absence of undesirable viruses which may be pathogenic (and confirming this through testing).

The risk approach can be enhanced by knowing the viral titer upstream and how the downstream process can deal with that viral load before lot disposition. Here it is good practice to demonstrate the capability of the manufacturing process to remove or inactivate potential contaminants. This will involve a small-scale simulation and with using four to six viruses with a sufficient starting titer to demonstrate a four-log reduction or greater. Alternatively, other models utilizing bacteriophages or nanoparticles can be used. These carry the advantage that higher reduction values can be measured.

The risk-based approach should aid in the identification of points of viral contamination including raw materials, cell culture processes, bioreactor contamination and downstream processing. Key risk areas include:

• Incoming materials and contaminated excipients, including animal-derived additives such as bovine serum albumin. Many incidents of viral contamination stem from using poorly characterized materials.
• Contamination of cell lines.  
• Purification and formulation reagents.
• Presence of impurities leading to viral stability in the process.
• Failure of controls within a viral secure area.
• Accidental contamination of a production system. Adventitious contamination can arise from a variety of sources such as reagents, the use of contaminated biological reagents such as animal serum components, or from human operators
• Incomplete inactivation of live viruses used in biopharmaceutical production.
• With blood and plasma products an additional complication arises with the source material: human plasma. Thus, an important additional factor is the profile of the donor and the tests carried out to confirm the donated plasma is free from pathogenic viruses.

Minimum test points would comprise of unprocessed bulk, purified bulk, and final products. With more sophisticated bioprocessing, additional stages should be considered through the use of suitable risk framework. Inputs into the assessment will include:

• The nature of the cell lines used to produce the desired products.
• The results and extent of virus tests performed during the qualification of the cell lines.
• The cultivation method.
• Raw material sources.
• Results of viral clearance studies.
• Trend data from batch manufacturing.

To enhance the accuracy of the data, the sample taken should be representative of the manufacturing stage. Thought must also be given to the sample size given that a sample of a few milliliters may or may not contain infectious particles.

Increased viral risk can occur when:

• Changes in critical process parameters that alter the safety profile take place;
• Failures of virus detection systems to detect low levels of viruses. Weaknesses with current molecular methods include limited assay sensitivity (enhanced by the low volume of sample volumes assayed); limitations with detection methods; unavailability of permissive cell lines to detect viral variants, and so on;
• Data errors, for example, with the extrapolation of viral inactivation data;
• New and emerging viral risks.

With manufacturing, it is not sufficient merely to note the risks. These risks need to be identified through a formal risk management strategy. This involves a combination of risk reduction steps and selecting process stage points for testing. The term ‘combination’ is relevant not only for fusing together viral clearance and testing, but also because no single approach of viral clearance sufficient; it is only through a combination of controls and viral treatment steps that a suitable level of assurance is reached. Additional contamination control practices like the adoption of single-use technologies can also be factored into the control strategy. Hence, adopting one approach is rarely sufficient. Many manufacturers are moving towards a combination of membrane chromatography, filtration, and solvent detergent; or they are putting into place a combination of UV inactivation and filtration. While testing is an important element it must be considered alongside multiple controls (including prevention, detection, and viral clearance) throughout the process. The biggest risks are the viral load moving downstream since clearance becomes more challenging. Here a well-defined testing strategy can support this, and it is good practice to obtain as much information that can be gathered from in-process testing.

Test Validation

Assessing assay reliability is important for understanding how well controls are actually working, for release testing, and to demonstrate robustness to regulatory authorities. While there is still work to be done to harmonize divergent global practices for method development, qualification, and sample analysis.

One of the variables is with different test methods and test kits that form part of the same method, from different manufacturers. There are variations with scientific interpretation and in terms of reaction volumes or DNA quantities required. In addition, some variants of PCR, like digital PCR, require considerably longer method development time due to the need for assessment of various conditions not necessarily required for traditional qPCR (which is something that needs to be balanced with the additional advantages of digital PCR).

Much of the method development is aimed toward optimizing the detection of a specified sequence. To start method validation it is important to gather information regarding the type of cell or gene therapy product (target DNA) to be tested, the host species and strain to be treated with the test articles, and the standard DNA to be used for determining the amount of target DNA within each sample.

Common problems with method validation include:¹¹

• Issues of dilution.
• The accuracy of the standard DNA concentration is essential for successful qPCR analysis – good practice to develop methods in quadruplicate.
• Challenge with amplification efficiency.
• The importance of ensuring the lower limit of quantification (such as with most qPCR assays, which require ≤50 copies of vector test article per 1 μg of gDNA).

As well as specific methodological issues, test error needs to be avoided such as contamination from adventitious viruses. The separation of workstations and control of contamination through thorough disinfection can aid the avoidance of contaminants.

Viral Testing: In-House or Outsourced?

The location of viral testing capabilities is very much an independent decision for a particular manufacturer. In house gives direct control and enables the demands of process variability to be met. Moreover, having employees working according to specific requirements saves time and often ensures compliance with local and regulatory compliance. Outsourcing, on the other hand, can help to achieve better quality of testing where the contract facility employs analysts who are experts in different areas of software testing methodologies. Selecting between these two options requires an understanding of the range of viruses that pose a risk to the process, which are likely to be present, and hence need to be detected and from this the capabilities to conduct the testing.

Summary

This article has considered viral risk to biopharmaceutical manufacturing and the use of risk assessment to determine where control and testing are required. In particular, risk assessments should focus on selecting and testing source material for the absence of known detectable viruses and assessing the capacity of the manufacturing process to inactivate or remove viruses. The strategy must also extend to testing the product at appropriate time points and process stages for detectable viruses. While various test method are available, qPCR is probably the optimal technology for determining viral clearance and virus removal in a close approximation to real-time, enabling faster responses and actions to reduce the risk to a specific batch or to the entire manufacturing facility.

References

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