Effectivity of HEPA Filters to Remove Viruses from Air Entering Cleanrooms

Introduction

In the manufacture of certain pharmaceuticals, viral removal or destruction is a key part of manufacturing (such as products produced from cell lines or from blood or plasma, as per ICH Q5A).¹ This requires a combination of viral secure areas and viral removal steps. In a previous American Pharmaceutical Review article, this author examined the ways by which viruses can be removed or inactivated from pharmaceutical products (including solvent-detergent; low pH inactivation; heat; chromatographic separation; and nanofiltration).² In terms of detection, nucleic acid-based assays such as Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS) provide rapid and sensitive detection of adventitious and endogenous viruses in constituent materials and finished products.³

This follow-up article considers how viruses can be excluded by HEPA filtration, thereby minimizing the entry of viruses and viral particles into cleanrooms. Aspects of this review may also be of interest in the wider context of the 2019-2020 novel coronavirus pandemic (caused by the virus SARS-CoV-2).⁴

Viral Secure Areas

Products where viral exclusion and inactivation is important for maintaining patient safety require processing in a viral secure area, especially within multi-product facilities for downstream purification processes. Viral secure areas achieve control through the use of dedicated spaces; control of personnel (such as wearing gowns dedicated to such areas only); through the control of materials (focusing on disinfectant transfer protocols); and with the use of airlocks, to control any interaction with non-viral secure areas. Viral secure areas typically process intermediate products that have been through a virus removal or inactivation step and here the objective is to prevent re-contamination of such products from other products and materials that have not been subject to such virus elimination methods. Typically, bacteriophages like PR 772 and PP7 are deployed as surrogates in place of mammalian viruses to assess viral removal, exclusion, and inactivation steps in relation to viral clearance and for defining the process design space.⁵

A further aspect in relation to maintaining viral safety is with minimizing the viral challenge through cleanroom air supply. For this, effective air filtration through appropriately graded high efficiency particulate air (HEPA) filters is required.

Air Transmission Risks

The extent of any viral airborne transmission of risk will depend on whether viruses enter the area intended to be excluded and then in part on the concentration of viral genetic material and viral particles (virions) contained within the cleanroom air.

To a degree, a cleanroom’s heating, ventilation, and air conditioning (HVAC) system will function to prevent the spread of airborne viruses by diluting (dilution ventilation, as assessed through air change rates per hour) and removing (exhaust ventilation) air from the cleanroom.6 Further air protective measures include controlling the direction of airflow and the air flow patterns in a building.

Direct risks from personnel, as with viruses contained within droplet nuclei (aerosols of about 1 to 5μm in diameter) from nasal or oral excretions can be managed through effective face mask control (ensuring masks are of an appropriate size exclusion standard and that masks are changed when they become damaged or wet, and changed at the end of every shift).

However, these control and removal measures are best served by engineering solutions that prevent an excessive viral load from entering the cleanroom in the first place. HEPA filters can aid in this intent.

HEPA Filtration

HEPA filters present an important aspect of contamination control across all pharmaceutical cleanrooms. HEPA filters are composed of a mat of randomly arranged fibers (manufactured using microfiber borosilicate glass on wet laid web-forming machines similar to those used for manufacturing paper). The fibers are typically composed of fiberglass and the activity of filtration removes microorganisms (bacteria and fungi) through the processes of impaction, interception and diffusion. In higher-classified cleanrooms HEPA filters are designed to remove around 99.97% of particles (normally particles of a 0.3 μm size) from air (the actual level of particulate control depends upon the grade of the filter). This is through the following mechanisms:⁷

• Inertial Impaction – against large, heavy particles suspended in the flow stream. As fluid changes direction to enter the fiber space, the particle continues in a straight line and collides with the HEPA filter media fibers where it becomes trapped.
• Diffusion - against smaller particles, which collide with the HEPA filter fibers and are collected.
• Interception - against particles in a mid-range size. Such particles become intercepted when they touch a fiber.
• Sieving – this mechanism captures particles that are too large to fit between the fiber spaces.

HEPA filters are protected from blockage by pre-filters which remove up to about 90% of particles from air; furthermore, in many facilities air handling systems re-circulate up to 80% of the air supplied to cleanrooms. Therefore, the initial challenge to the HEPA filters, as might be presented from outside air, is reduced.⁸

HEPA filters of the appropriate grade can also remove a high-proportion of airborne viruses (one of the earliest references to HEPA filter and viral removal dates back to 1976).⁹ The effectiveness of this depends upon the number of viral particles and the relative size of the virus. Although most cleanroom HEPA filters are certified against the ability to remove particles from the air of a size of 0.3 μm and greater, filters are capable of removing particles of a smaller size. Many HEPA filter manufacturers have undertaken testing in relation to virus removal (although different types of viruses and different viral challenges will have been used, in the absence of any standardization). There are different ways to assess viruses, including special bioaerosol samplers and electrostatic precipitators, with assaying using plaque assays (for bacteriophages) and for viral nucleic acids using quantitative PCR assays.

Viruses are of different morphologies (shapes and sizes). Some examples of virus sizes are:¹⁰

• SARS coronavirus: 0.08 to 0.16 μm
• SARS CoV-2 (the ‘novel’ coronavirus): 0.6 to 0.14 μm
• MERS-CoV coronavirus: 0.08 to 0.16 μm
• Swine Flu A(H1N1) virus: 0.08 to 0.12 μm
• Avian Flu A(H5N1) and A(H7N9) virus: 0.1 μm

As indicated above, viruses generally range from about 0.1 to 0.2 μm in size, although they often cluster or attach to larger particles in the airstream (such as from coughing or sneezing, where particles range from 0.5 to 3.0 μm). The reason why HEPA filters can capture particles smaller than 0.3 μm is due to one of the mechanisms of particle capture discussed above – diffusion. This was demonstrated in one study where silver particles – at 5 nanometers – were shown to be captured with a 99.99% efficiency using a cleanroom grade HEPA filter,¹¹ assessed using a Ultrafine Condensation Particle Counter.¹² At 5 nanometers, the challenge particles were smaller than most viruses (including the 2019 novel coronavirus). A second study, conducted at NASA, showed HEPA filters to be capable of capturing ultrafine particles of a dimeter of less than 0.01 micrometers.¹³ Equivalent efficacy to HEPA filters has also been shown with nanofiber filters.¹⁴ A third study showed 99% retention through a HEPA filter from a challenge of a T7 virus (size 0.04 microns) at concentrations of 5x10⁸.¹⁵

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Given that no HEPA filter can guarantee complete viral removal, technologies are available for rendering viral DNA and RNA inactive using ionization.

Ionization

The ability of ionization to inactivate viruses has been known since the 1950s;16 however, the application to enhance air filtration systems represents a more recent development. To enhance the capabilities of HEPA filters, ionization technologies can be used, such as Multifunction Ion Air Cleaning.¹⁷ Ionizers can be placed either prior to the HEPA filter or located within a room. These methods use a combination of the ionizing processes and electrostatic attraction of particles >0.003 μm which include cells of bacteria, fungi and many types of viruses.¹⁸ Inactivation of viruses is achieved through the either generation of negative ions, where the functional unit is the hydroxyl (a molecule comprised of one oxygen molecule and one hydrogen atom), or by both positive and negative ions (unipolar).¹⁹

At the heart of such technology is an ion generator, which uses an alternating plasma discharge to split water molecules into positively and negatively charged ions. As these ions are emitted into the air, they become surrounded by water molecules and this leads to the formation of cluster ions, which become attracted to airborne particles. As the cluster ion surround airborne particles, and the positive and negative ions undergo a reaction to form hydroxyls. The hydroxyls function to remove an airborne particle’s hydrogen atom, and this inactivates the particle by producing a hole in the viral particle’s outer protein membrane.²⁰

To assess airborne reduction a study utilized a carbon-fiber ionizer upstream of a HEPA filter to generate air ions. The ions which were used to charge the virus aerosols and hence increase the filtration efficiency. With the antiviral efficiency, this was shown to be a product of increased with ion exposure time and ion concentration. When the ionizer was operated in a bipolar mode and the number concentrations of positive and negative ions were 6.6×10⁶ and 3.4×10⁶ ions/cm3, respectively, and the antiviral efficiencies (against a bacteriophage MS2 challenge) was calculated as 97.4% after an exposure time of 45 minutes.²¹ A different study looked into placing negative ionization devices within rooms and assessing viral reduction using real-time PCR to enable sensitive detection of airborne viruses. The device deployed used an ionizing device operating at 12 V and this was used to generate negative ionizations in an electric field. The assessment was using Influenza A virus in a room of 19 m³.²² While there are variables relating to flow rate, residence time, and power consumption, ionization technologies have a role to play in aiding HEPA filters in reducing viral challenges into cleanrooms. The complexity of applying this on an industrial scale will require design optimization, performance evaluation, and the prediction of the power consumption of the selected ionizer system.

Alternative Methods

An alternative method is to use germicidal light. Ultraviolet (UV) light may be effective against viruses, although this has been less widely researched. One of the complexities with UV light to inactivate pathogens is the variables relating to the intensity of the light, the duration of exposure, and the relative humidity of the environment, offset against the fact that different pathogens require different light intensities and durations of exposure.²³ For most viruses the experimental applications include UV-C irradiation produced from a monochromatic light at 254 nm. This form of light targets viral nucleic acids. In one experiment, UV-C light resulted in complete inactivation of a viral challenge after 15 minutes from a distance of 3 cm (in contrast, a different wavelength using UV-A exposure did not demonstrate any significant effects on virus inactivation over the same duration of time).²⁴

A further alternative is with ozone gas, which can be used in empty rooms to decontaminate surfaces, however in rooms with people present ozone cannot been used due to its toxicity.²⁵ Other mechanisms, although not suitable for air decontamination, as tested out on the original SARS coronavirus, include heat, and extremes of pH. However, gamma irradiation has not been shown to be sufficient to inactivate the virus.²⁴

Summary

Viral contamination is a potential safety threat common to all animal and human-derived biologics and it follows that ensuring virological safety is challenging. Contamination of the production system can occur, and the processes of viral removal are complex and require regular assessment (to avoid the incomplete inactivation or removal of viruses). A further challenge arises with creating viral secure areas. As well as the control of materials and personnel, and important factor in maintaining such an area is through effective air filtration.

As this article has highlighted, higher-grade HEPA filters are effective at removing most viruses from the air through diffusion and retaining them within the media matrix. However, while some tests have been conducted, viral capture does not form part of conventional HEPA filter certification. Additional viral inactivation methods include the ionization of air, or alternative ultraviolet light and ozone methods. To achieve viral control for certain types of pharmaceutical processing, this requires a combination approach. This article makes reference to these approaches. Aside from specific products (like cell-culture and plasma products), the current rise in cases of SARS-CoV-2 (and the associated disease COVID-19) may make aspects of this article of wider interest to the cleanroom user.

References

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