Current Methods and Approaches for Viral Clearance

• Bio Products Laboratory Ltd

Introduction

Biological and biotechnological products are at risk from chemical impurities, bacteria and fungi, and from viruses. With such products the potential for transmission of viral diseases is a real risk. With viral contamination, contamination can affect raw materials, cell culture processes, bioreactor contamination and downstream processing. It is for these reasons that pharmaceutical organizations need to practice viral safety and incorporate virus clearance into the manu-facturing process.

Viruses are composed of DNA (such as herpes viruses) or RNA (such as hepatitis viruses) encapsulated by a protein coat. 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.

The types of products for which virus safety include¹:

• 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 pathogenic viruses and residual pathogenicity. The process is also complicated by viral inactivation being rarely linear. It can also be that viruses are subsequently discovered several years after a batch has been manufactured. Therefore, whilst product testing can support control measures in terms of the showing that the probability of viral contamination is low, greater confidence is garnered through viral clearance strategies and associated risk assessment.

Both U.S. FDA and the European Medicines Agency have put in place requirements for viral safety in relation to biologics, and a degree of harmonization has been achieved through ICH². Furthermore, in recent years, there has been increased regulatory scrutiny of incidents relating to adventitious viruses in manufacturing processes.

This article assesses the key factors for viral clearance and considers the current methods used to remove viral particles from the product stream.

Viral Risks

Biologics are at risk from pathogenic viruses at various stages in the manufacturing process. Viral risks can occur though:

• 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.
• 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 viruses³.

The possibility of viral contamination or a drift towards 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 on⁴;
• 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. Risk assessment is best served through HACCP (Hazard Analysis and Critical Control Points) style approaches. Here it is necessary to determine critical process parameters (CPP) and the specific critical control points (CCPs) in the manufacturing process⁵.

Risk assessments should focus on:

• Selecting and testing source material for the absence of known detectable viruses;
• Testing the capacity of the manufacturing process to inactivate or remove viruses;
• Testing the product at appropriate time points and process stages for detectable viruses.

Various test method are available. Of these, qPCR is becoming the leading technology in determining clearance and removal, or to complement standard viral titration assays⁶.

Virus Clearance

Within the pharmaceutical process measures need to be put into place for virus clearance. These are commonly divided into^7,8:

• Viral inactivation methods, and
• Viral removal methods.

Examples of inactivation methods are:

• Solvent/detergent inactivation is a robust, well-established viral clearance method.
• Low pH inactivation is effective against enveloped viruses. This is a robust step for virus inactivation. With this the critical process parameters to observe are pH, time and temperature.
• Other methods, such as heat (pasteurization), microwave heating, irradiation and high-energy light are less commonly undertaken.

These methods are generally effective against enveloped viruses. However, solventdetergent, pH conditioning, and heating have limited effect on non-enveloped viruses. Instead, chromatography and virus filtration removal methods are effective with nonenveloped viruses.

With virus removal:

• Certain affinity steps (Protein A) and chromatographic separations can be optimized for virus clearance⁹. With this, several anionic chromatography mechanisms have proven effective in the removal of viruses in the downstream purification processes of biopharmaceuticals¹⁰. The effectivity is dependent upon the resin and binding mode. Flow rates, buffers, temperature and wash volumes can also affect viral removal efficiency.
• Another primary method is nanofiltration. Here viruses are removed from the product through size exclusion. Effective virus removal of large viruses (> 50 nm) is possible using both large- and small-pore-size filters. For the removal of small viruses (~ 25 nm), this remains process dependent even when small-pore-size virus filters are used. With filtration: time, temperature and pressure are key parameters. However, under optimal conditions filters can reliably achieve greater than 4 logs of reduction of small nonenveloped viruses.

Importantly no single approach is sufficient; it is only through a combination of controls and viral treatment steps that a suitable level of assurance is reached.

It is important that viral clearance is demonstrated through validation studies. The objective of such studies is to evaluate the ability of the manufacturing process to clear (inactivate or remove) identified viral contaminants. In addition, such studies assess the robustness of the manufacturing process to unknown viruses and equipment cycle life. With the latter, blockage of viral filters can occur due to denatured and aggregated proteins or chromatography column media may cease to function through successive cycles. Thus the assessment of technology as used in the manufacturing process is important.

The choice of virus(es) to use in virus validation studies will depend upon the type of product, process, and regulatory. The user will need to determine the level of virus clearance required (as per logarithmic reduction). It is good practice to use both enveloped and non-enveloped viruses and viruses of different shapes (parvovirus, for example, is icosahedral in morphology whereas HIV is spherical.)

New Technologies and Approaches

While the above methods are well-established, there is a growing interest in new technologies.

Viral removal

To remove viruses from the process, membrane chromatography is increasingly becoming a method of choice, especially in large-scale biopharmaceutical manufacturing processes. This type of pro-cess will remove impurities as well as viruses. The process works by removing viruses by adsorptive removal, such as ion-exchange membrane adsorbers with ligand–virusbinding properties (with a positive surface charge). The further advantage with this technology is that it has a relatively high flow-rate beyond that of conventional column chromatography.

In validating the process, factors that can influence the efficiency of virus removal include protein concentration, product characteristics (e.g., hydrophobicity), and the nature and amount of impurities present.

With solvent-detergent viral inactivation, procedures ordinarily involve incubating protein solutions in the presence of an organic solvent such as tri-(n-butyl) phosphate and a detergent. The removal of the solvent and detergent is necessary once the viral inactivation step has been completed; and the same time the treated proteins must be effectively collected through chromatographic mechanisms. The removal of these chemicals can be achieved through a sorbent, for example a polymer.

To test the robustness of the viral clearance process, it should be practiced with both new and aged chromatography media. This is important in order to establish the maximum number of cycles that can be run.

Virus filtration

With virus filtration progress has been made with filter design. Virus filtration is normally achieved through direct flow, and modern well-designed filters allow fast performance combined with good virus capture. A recent design is with an orthogonal removal mechanism. A further development is with higher area filters. These allow increased flow rates and throughputs.

To add to this, progress has been made with fully automated, single-use systems. These disposable technologies provide the advantages that single-use technology adds to the manufacturing process including savings on energy consumption and avoiding the complexities of cleaning validation¹¹. With larger scale, fixed systems turnaround between products can be made easier with clean-in-place systems.

The selected virus filter needs to be robust and deal with an appropriate run-time (larger processes require process times running into several hours.)

Other methods of inactivation

To safeguard in-coming materials like culture media, some pharmaceutical companies are using ultraviolet-C (UVC) light or hightemperature short-time (HTST) treatment for inactivation. UVC is low-dose radiation at a wavelength of 254 nm. It is an effective method for non-enveloped viruses.

With HTST liquid moves in a controlled, continuous flow while subjected to temperatures of 160°F to 165°F for approximately 30 seconds. As an alternative, virus reduction filtration processes are carried out.

Synergy

As indicated above, adopting one approach is rarely sufficient. Many manufacturers are moving towards a combination of membrane chromatography, filtration and solventdetergent; or they are putting into place a combination of UV inactivation and filtration.

Summary

Viruses present a risk to biologic products and this article has discussed the implications of these risks and where they might occur. In doing so the article has emphasized the importance of process controls and the targeting of controls through risk assessment. Control is achieved through the twin processes of viral removal and inactivation. In presenting these, the article has highlighted current approaches and techniques. Importantly, it is only through a combination of approaches that viral security can be achieved.

References

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2. 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
3. Willkommen, H., Schmidt, I., and Lower, J. “Safety Issues for Plasma Derivatives and Benefit from NAT Testing,” Biologicals 27, 325–331 (1999)
4. Onions, D. and J. Kolman, Massively parallel sequencing, a new method for detecting adventitious agents. Biologicals, 2010. 38(3): p. 377-80
5. 5. J. (Eds.) Contamination Control in Healthcare Product Manufacturing, Volume 1, DHI Publishing, River Grove: USA, pp423-474, 2013
6. Shi L, Chen Q, Norling LA, Lau AS, Krejci S, Xu Y. Real-time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification. Biotechnol Bioeng. 2004;87(7):884–896
7. ICH.Q8 (R2), Pharmaceutical Development. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; Current Step 4 version, August 2009
8. ICH.Q5A (R1),Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin. 1999.
9. Aoyama, S. Adsorption study of egg-derived influenza virus with cellufine sulphate affinity chromatography media, BioProcess Internaitonal, Industry Yearbook 2015-2016, p46, 2015
10. Norling, L. Impact of multiple re-use of anion exchange chromatography media on virus removal. J. Chromagr. A, 2005 1069 79–89
11. Sandle, T. and Saghee, M. R. (2011): Some considerations for the implementation of disposable technology and single-use systems in biopharmaceuticals, Journal of Commercial Biotechnology, 17 (4): 319–329

Author Biography

Dr. Tim Sandle has over twenty-five years experience of microbiological research and biopharmaceutical processing. This includes experience of designing, validating and operating a range of microbiological tests including sterility testing, bacterial endotoxin testing, bioburden and microbial enumeration, environmental monitoring, particle counting and water testing. In addition, Dr. Sandle is experienced in pharmaceutical microbiological risk assessment and investigation.Dr. Sandle is a tutor with the School of Pharmacy and Pharmaceutical Sciences, University of Manchester for the university’s pharmaceutical microbiology MSc course. In addition, Dr. Sandle serves on several national and international committees relating to pharmaceutical microbiology and cleanroom contamination control (including the ISO cleanroom standards). He is a committee member of the Pharmaceutical Microbiology Interest Group (Pharmig); serves on the National Blood Service advisory cleaning and disinfection committee; and is a member of several editorials boards for scientific journals.Dr. Sandle has acted as a consultant, expert witness and technical advisor to sterile and non-sterile manufacturing facilities, microbiology laboratories, the medical device industry and hospitals. Dr. Sandle has also undertaken several technical writing and review projects.Dr. Sandle has written over three hundred book chapters, peer reviewed papers and technical articles relating to microbiology. In addition, Dr. Sandle has written several books. Dr. Sandle has also delivered papers to over fifty international conferences and he is an active journalist.