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Over the past several decades, biologics such as monoclonal antibodies (MAbs) and recombinant proteins have provided therapeutic benefits and efficacy for the treatment of human disease. Completion of the human genome project (launched in 1990) produced a draft of the genome in 2001. A full sequence was published on the 50th anniversary (2003) of the initial publication of Watson and Crick’s papers on the double-helical structure of DNA (1). That large volume of genetic information has been translated into usable scientific knowledge, helping researchers in their hunt for better therapeutic approaches and establishing the functional relationships between genes and clinical disorders. Ongoing research has led to new understandings, which have paved the way for many new designs of recombinant biologics.

Today, most biopharmaceutical products are proteins expressed in recombinant hosts. Microbial systems are attractive platforms because of their low cost, high productivity, and rapid implementation. And bacterial expression systems present no concerns about adventitious mammalian viruses.

However, many biopharmaceutical molecules are simply too big or too complex to be made by bacteria. For example, properly folded proteins (including MAbs with appropriate glycosylation) require posttranscriptional metabolic machinery that is available only in mammalian cells. And because such cells can add relevant human-like posttranslational modifications, they have become the preferred host for production of complex protein therapeutics.

It has been more than a decade after the completion of the human genome sequence and more than 30 years after regulatory approval of the first genetically engineered drug. On 28 October 1982, the FDA approved the first recombinant medicine produced using genetic engineering: recombinant human insulin, Humulin, developed by Eli Lilly and Company and Genentech. The drug was expressed in bacteria. Today, such biologics represent a major portion of the pharmaceutical market, with most approved in the United States and Europe as glycosylated proteins produced by mammalian cell culture.

Patents are expiring soon for more than 20 first-generation blockbuster biologics. As a result, an increasing number of biosimilars (which can be similar but not identical to innovator biologics) are expected to be submitted for approval. In addition, biobetters, (considered improved versions of innovator biologics) are gaining popularity.

Pharmaceutical companies developing biosimilars and biobetters must confirm that the efficacy, safety, and purity of their products are similar to those of original innovator- approved drugs (2). In this process, viral safety is of upmost importance.

Because production of all biopharmaceutical products carries an inherent risk of potentially introducing adventitious viruses, viral safety is an essential consideration in the development and manufacture of these products. Viruses possessing diverse characteristics can potentially contaminate therapeutic agents, so different methods of virus inactivation and removal are required to guarantee a safe drug supply.

Although viral safety is a focal point for regulatory authorities, the potential for transmission of diseases — specifically viral — through any biologic that uses a mammalian- derived component in its manufacturing process is a real risk.

Current state of the art precludes claiming absolute absence of viral presence in biologics. The practical approach applied by most regulatory bodies is evaluating safety case by case. Such a strategy considers current, real, and perceived virus contamination concerns on the basis of source materials used for manufacturing each product as well as availability of adequate technologies for detection and clearance of viral agents.

Through international collaborations, regulatory guidelines stress a holistic approach that includes appropriate sourcing of materials; demonstrating the capability of a manufacturing process for viral clearance; and testing in process, among others. Critical use of an effective viral safety program involves careful selection and testing of cell lines and original raw materials for adventitious viruses. In addition, in-process testing can identify contaminants such as endogenous retroviral particles, which are typically associated with mouse and hamster cells. Viral testing is complemented by viral clearance studies, which demonstrate the capacity of manufacturing steps to inactivate or remove potential viral contaminants.

A Market in Transition
Over the past decades, the development and production of medicinal products for human use has undergone a major shift. Among factors affecting the global market for pharmaceutical manufacturing is a paradigm shift in the need for medicinal products. Demands and patient needs are shifting from blockbuster drugs to specialized, even personalized medicine (3). Because many innovator drugs are going off patent in the next few years, the industry is moving toward developing biosimilars and biobetters.

A September 2013 report published by IMS Health suggests that the global market for biologics is expected to grow from US$169 billion in 2012 to $221 billion in 2017 (4). The authors of that report expect that biologics will continue to outpace the growth of overall pharmaceutical spending and expect it to be driven by increased demand for MAb innovator drugs and recombinant human insulin. The authors also predict that by 2016, the market for biosimilars will be worth about $5 billion, with expected exponential growth afterward.

Complexity
The manufacturing process of biologics is challenging and complex because such products are derived from plants, animals, or mammalian cells or supplemented with reagents derived from living systems or cell lines. The process requires a high level of reproducibility in commercial development and manufacture to guarantee high-quality, consistent biological products. The level of complexity is generally influenced by a host of factors, including individual cell characteristics, growing environment, and nutrients provided during manufacturing (5–8).

Whereas synthetic small-molecule pharmaceuticals rely on a chemical analysis of components to assess purity, the quality, safety, and efficacy of complex biologics depend on careful control of process inputs and operating conditions. Processing involves many manufacturing stages and parameters, as well as stringent control mechanisms and process control (9).

Although biopharmaceutical production may be challenging, guaranteeing virological safety is even more complex. Aided by advanced detection, donor selection, raw materials screening, virus removal, and inactivation, biomanufacturers have greatly increased the safety profile of their products. Such therapies include tissue-, blood- and plasma-derived products, MAbs, recombinant proteins, vaccines, and animal products carrying an inherent risk of transmitting viruses based on the source material, manufacturing processes, and routes of administration.

But despite stringent efforts on the part of manufacturers, companies continue to face threats of new and emerging viruses. Unfortunately, endogenous and adventitious viruses can escape detection. That might be the result of limited assay sensitivity or limitations in detection methods, which require increased efforts of viral clearance. As part of such work, viral clearance studies are designed to help demonstrate virus reduction, achieved by a number of steps in the manufacturing process in case process intermediates are contaminated.

Vulnerability
Sourcing of raw materials is one of the greatest concerns for regulators and biopharmaceutical manufacturers. Most incidents of viral contamination result from using poorly characterized materials. This risk is intensified by the sourcing of raw materials from multiple vendors. And because large volumes of process media and gases are used to produce biologics, viral contamination is not necessarily a rare occurrence.

Evaluation of raw materials has become a critical component of any risk mitigation strategy. Such a strategy can require evaluating source materials beyond what can generally be expected or is reported about the source. For example, many processes now limit or entirely move away from animal-derived materials for producing recombinant cell lines or media formulations. But there is still a risk that drug products have been exposed to animal-derived materials at one or multiple points in the supply chain. Furthermore, it is not always possible to move away from animal- derived materials, which presents a risk of introducing virus contamination into a manufacturing system.

Rigorous evaluation and mitigation are required to eliminate potential risks of contamination. Some manufacturers use virus inactivation methods such as UV-C inactivation, high-temperature short-time (HTST) treatment, or removal (virus reduction filtration) procedures for culture media or media components. The viral reduction potential of such risk-mitigation procedures can be evaluated in a viral clearance study. Similarly, the viral reduction potential of downstream manufacturing process steps (designed to complement rigorous upstream screening and mitigation steps) also can be evaluated in a viral clearance study.

Lessons learned from past incidents show the importance of thoroughly understanding the complexity of (and possible risks in) the supply chain. Regulatory authorities can use such understanding — as well as the knowledge of the extent to (and the nature in which) biologics include animal-derived components — to assess how much viral clearance is required.

Mammalian Cells
With its intrinsic complexity, the production of recombinant biopharmaceuticals poses a series of unique manufacturing challenges. Viral safety is particularly challenging when mammalian cells are used. Although iatrogenic incidents have occurred in the past decade as a result of biologics contamination, incomplete inactivation of viruses, or contamination of excipients in a nonrecombinant product, only a limited number of known and reported events have taken place. (To date there has been no transmission of an infectious virus to a patient from a recombinant biotech product. However, such transmissions have occurred with plasma-derived products, and some have resulted in disease. Furthermore, although contamination events for products derived from mammalian cells have been reported, they were detected before the drugs were administered.)

In 2010, Kerr and Nims reported that during a specific 10-year period, biologics derived from a number of mammalian cell culture processes were evaluated using in vitro virus screening assays. Reported results showed that only reovirus type 2 and Cache Valley virus (a bunyavirus contaminant) were detected in biologics manufactured in Chinese hamster ovary (CHO) cells. Other reports showed that the murine parvovirus mouse minute virus and contamination with vesivirus 2117 had also been detected in biologics manufactured using CHO cells (10).

Myth and Solution
As a result of stringent regulatory oversight, biologics manufacturers are required to show that their products are safe for human use. And although biopharmaceutical scientists and regulatory authorities understand that absolute freedom of viral contamination is impossible, much can be done to ensure viral safety. This is reflected in the regulatory landscape.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) is a unique collaboration of regulatory authorities and the pharmaceutical industries of Europe, Japan, and the United States. The organization has developed guidelines requiring manufacturers of biologics to demonstrate their ability to remove or inactivate viral contaminants (11– 14).

The European Medicines Agency (EMA), US Food and Drug Administration (FDA), and other authorities have adopted guidelines that focus on testing and evaluating viral safety of biotechnology products derived from characterized cell lines of human or animal origin. Those guidelines provide a general framework for testing production cell banks and bulk harvests for viral contaminants and designing viral clearance evaluation studies. The EMA also has adopted guidelines concerning clinical trials and submissions based on an investigational medicinal product dossier that includes viral safety evaluation (15).

Investigational Products
The EMA has provided a regulatory guideline governing virus safety for investigational medicinal biotechnology products (15). This document, which became effective in 2009, applies to clinical trial material derived from well-characterized cell lines and harmonizes the regulatory expectations throughout the European Union. This guideline acknowledges that, for clinical trial materials, the manufacturing process and full product characterization is still in development. It offers reduced viral safety testing and viral clearance study scope compared with requirements for marketing authorization provided in ICH Q5A.

The viral safety of products intended for clinical trials is a priority. However, the EMA guideline acknowledges that working cell banks may not yet be established, and it lifts the requirement for testing cells at the limit of in vitro age. For viral clearance studies, a reduced virus panel can be used (a retrovirus and a parvovirus). Robustness studies are not required for products at this stage of development, again acknowledging that process parameter ranges may not be established yet. If a product used for a clinical trial has been manufactured using relatively new chromatography resins, then clearance studies on aged resins and resin sanitization studies are not expected. These guidelines were developed with input from industry and other regulatory agencies that worked together to assure viral safety.

Risk Management
A risk-based approach to viral safety is consistent with the approach that is now used to ensure quality in biopharmaceuticals and is outlined in several guidance documents. Such documents include ICH Q8, which includes process analytical technology and quality by design; ICH Q9, which includes quality risk management; and ICH Q10, which presents guidelines on pharmaceutical quality system (12–14).

Use of a risk-based approach to viral safety is consistent with the viral safety “tripod.” Selection and characterization of source materials will define potential contaminants that may be present. New technologies such as massively parallel sequencing are powerful tools for detecting and identifying contaminants that may be present, especially for novel or complex source materials. The resulting information can provide a framework for more directed routine testing.

Testing provides key information about the design of a viral clearance study. Understanding the potential contaminants will guide selection of a virus panel for study so that models of potential contaminants are included. Testing data also will direct the level of clearance that is expected from manufacturing processes. For example, retroviral-like particles are quantitated in bulk harvests from CHO cell lines. Viral clearance for a process should be high enough that the risk of that contaminant in a final dose of product is less than one in a million.

For many years, the focus of viral clearance has been on downstream manufacturing. Although downstream viral clearance steps are still essential, many manufacturers are looking at upstream viral clearance steps. Including a viral inactivation or removal step for cell culture media or media components is another way to mitigate the risk of viral contamination. Steps such as UV-C inactivation, HTST, and even virus reduction filtration can provide yet another layer of viral safety for a manufacturing process.

Facility Design
One often-underestimated approach to viral safety is the design of a manufacturing facility. To comply with regulatory directions, it is important to consider where viral clearance takes place in the production and manufacturing process and which steps provide effective viral inactivation or removal. Well-designed facilities ensure adequate segregation of process intermediates that have been through an effective viral reduction step from those intermediates that have not. Also, dedicated viral clearance must be planned in the manufacturing process to alleviate safety concerns.

As demonstrated by viral incidences with vesivirus 2117, the possible introduction of undetected viruses from unknown sources is a great risk. Once a viral contaminant enters the manufacturing process, the only effective control involves robust viral inactivation or removal steps.

Lessons Learned
The most important lessons to be learned from regulatory guidelines — and a general principle that can be applied to the manufacture of all biopharmaceuticals — is that viral clearance studies must be reviewed case by case. Each study involves product-specific risk factors. If a modular approach to viral clearance is used for similar products and processes, you must demonstrate the absence of product-specific influences on viral clearance. That may involve verification of processing parameters, analytical testing of process intermediates, and/or some evaluation of viral clearance. Testing source materials will help you identify potential contaminants and the level of virus reduction to be achieved in manufacturing.

Past incidents have shown that the correct approach ultimately helps prevent costly mistakes. Such mistakes can delay regulatory approval of new biologics and halt current production and manufacturing of already approved drugs.

Importance of Viral Clearance
Effective viral clearance is required to guarantee the availability of a safe supply of drug products for human use. Because adventitious viruses present in original materials can contaminate biologics during processing, manufacturers must follow regulatory requirements to ensure adequate virus removal. During the past decade, viral clearance studies have received increased regulatory scrutiny and become important factors to consider in the design of manufacturing processes.

Viral clearance in downstream purification can involve a number of methods to inactivate or remove viruses from a final therapeutic product. Because contamination incidents also can occur in bioreactors (adding associated regulatory implications and costs associated with decontamination), some manufacturers have chosen to also include upstream barriers in their processes.

Biomanufacturing processes are varied and can include a number of upstream and downstream steps. Virus- removing (nano-) filters (designed for the removal of almost all virus types through a size-exclusion mechanism and chromatographic resins in column or membrane configurations) are typically included downstream. Upstream steps may include UV-C inactivation and HTST. Exposure to low pH or solvent/detergent treatment steps provides inactivation of enveloped viruses downstream.

In addition to viral clearance, a typical manufacturing process also contains steps to purify a biopharmaceutical product. Athough such steps are not necessarily designed to provide virus reduction, they may have the potential to do so. Chromatography is a good example of a purification operation that can help achieve viral clearance. However, because a number of operating parameters can influence viral reduction, chromatography is generally less robust than viral reduction by other methods such as inactivation.

In the conclusion of this two-part series, I discuss effective planning of viral clearance studies as well as the implementation of viral clearance strategies.

References
1 Collins FS, et al. A Vision for the Future of Genomics Research. Nature 422, 2003: 835–847.

2 Islam R. Bioanalytical Challenges of Biosimilars. Bioanalysis 6(3) 2014: 349–356.

3 Weiler A. A New Dawn for Western CMOs. Pharm. Technol. 1 February 2014: S14– S17.

4 The Global Use of Medicines: Outlook Through 2017. IMS Institute for Healthcare Informatics: Danbury, CT, September 2013.

5 Vyas VV, et al. Clinical Manufacturing of Recombinant Human Interleukin 15: Production Cell Line Development and Protein Expression in E. coli with Stop Codon Optimization. Biotechnol. Prog. 28(2) 2013: 497–507.

6 Hofland P. An Introduction to Recombinant Biologics and Immunotherapy. TVCC, Almere, The Netherlands, January 2003.

7 Schellekens H. Biosimilar Therapeutics — What Do We Need to Consider? NDT Plus 2(sup 1) 2009: i27–i36.

8 Zhang J. Mammalian Cell Culture for Biopharmaceutical Production. Manual of Industrial Microbiology and Biotechnology, Third Edition. Baltz RH, Davies JE, Demain AL, Eds. American Society of Microbiology: Washington, DC, 2010; 157–178.

9 Lee JF, Litten JB, Grampp G. Comparability and Biosimilarity: Considerations for the Healthcare Provider. Curr. Med. Res. Opin. 28(6) 2012: 1053–1058.

10 Kerr A, Nims R. Adventitious Viruses Detected in Biopharmaceutical Bulk Harvest Samples Over a 10-Year Period. PDA J. Pharm. Sci. Technol. 64(5) 2012: 481–485.

11 ICH 5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Geneva, Switzerland, October 1997.

12 ICH Q8: Pharmaceutical Development. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Geneva, Switzerland, August 2009.

13 ICH Q9: Quality Risk Management. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Geneva, Switzerland, November 2005.

14 ICH Q10: Pharmaceutical Quality System. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Geneva, Switzerland, June 2008.

15 Guideline on Virus Safety Evaluation of Biotechnological Investigational Medicinal Products. Evaluation of Medicines for Human Use: London, UK, July 2008.

Kathryn Martin Remington, PhD, is a principal scientist, development services, clearance, at BioReliance; kathy. remington@bioreliance.com.