	The International Organization for Standardization (ISO) published the long-awaited revisions to its standards for classification and monitoring of air cleanliness in cleanrooms on Dec. 15, 2015. ISO 14644-1:2015 “Cleanrooms and associated controlled environments Part 1:Classification of air cleanliness by particle concentration” (1) replaces ISO 14644-1:1999, and ISO 14644-2:2015 “Cleanrooms and associated controlled environments Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration” (2) replaces ISO 14644-2:2000.

	The 2015 editions are the result of a systematic review and include changes made in response to requests by users and experts in the cleanroom community. In particular, the requests for reviewing Part 1 were related to “the basis for the number of sampling locations and, most importantly, the whole statistical basis of classification of cleanliness using the Student T-test for one to nine sampling locations,” notes Gordon Farquharson, convenor of the ISO TC209 working group 1, which performed the review and revisions. Because Part 2 is closely aligned with Part 1, the committee reviewed both parts together.


	The addition of “by particle concentration” to the title of the standard is a long-overdue clarification, comments Karen Ginsbury, CEO at PCI Pharmaceutical Consulting. “For years, I have heard cleanroom contractors and practitioners alike wrongly describe ISO 14644-1 and 2 as cleanroom ‘validation’ or ‘qualification’ standards.” She notes that the standards only address airborne particles, not other factors crucial to cleanroom qualification, such as smoke tests to determine airflow patterns.

	The introduction of Part 1 explains, “This part of ISO 14644 specifies classes of air cleanliness in terms of the number of particles expressed as a concentration in air volume. It also specifies the standard method of testing to determine cleanliness class, including selection of sampling locations” (1).


	The primary changes to Part 1 involve the number of samples and the selection of sampling locations. “The number of samples will increase from what was required previously,” explains Marsha Stabler Hardiman, senior consultant at ValSource. “The minimum number of samples is now determined from a lookup table (instead of an equation), and that number is set to be statistically significant.”

	According to ISO, the new method for selecting the sites and number of sampling collections uses a more consistent statistical approach based “where samples are drawn randomly without replacement from a finite population. The new approach allows each location to be treated independently with at least a 95% level of confidence that at least 90% of the cleanroom or clean zone areas will comply with the maximum particle concentration limit for the target class of air cleanliness. No assumptions are made regarding the distribution of the actual particle counts over the area of the cleanroom or clean zone; while in ISO 14644-1:1999 an underlying assumption was that the particle counts follow the same normal distribution across the room” (1). The sampling locations are to be chosen representatively, meaning that “features such as cleanroom or clean zone layout, equipment disposition, and airflow systems should be considered when selecting sampling locations” (1).

	This representative selection of sample locations is a big change from the previous random selection, says Hardiman. “A company now has to have a rationale and justification for sample site location to ensure that the sample locations selected are representative of the characteristics of that section.  Companies will have to look at the new number of sample locations and then determine where the representative sample locations will be collected.  If contracting out the classification activities, you should make sure that your contractor is now using your new, representative sample locations.” 

Classification of macro and nano-particles


	The revision makes a change regarding large (≥ 5 µm) particles, which are required to be measured for some classifications in the EU Annex 1 GMP guidelines (3) and others. “The experts working on the revision of ISO 14644-1 were of the opinion that particles ≥ 5 µm diameter should not be used to classify ISO class 5 and cleaner environments because of the uncertainty associated with particle collection efficiency and accuracy of counting low concentrations,” says Farquharson. “In order that the European Union (EU), the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme, World Health Organization, and Chinese GMPs are not left without a classification tool for their Grades A  (at rest and operational) and B (at rest), ISO 14644-1:2015 provides a mechanism of extrapolating the macro-particle descriptor for class limits of 20 and 29 particles ≥ 5 µm.”

	The new document does not address nano-scale particles, which were formerly defined as ultrafine particles  in ISO 14644-1:1999, but will address these under a new Part 12, notes Farquharson, who explains, “These particles are measured using a different particle counter, and industries such as semi-conductor monitor for concentration of these very small airborne particles at critical control points. These particles are not generally of interest to the pharmaceutical and life sciences industries.”


	ISO 14644-2:2015 now requires monitoring to provide evidence of cleanroom performance, explains Farquharson. The standard addresses airborne particle concentration, airflow, and device pressure difference. New topics include monitoring of critical parameters and setting action and alert alarms.

	The revised standard now allows companies to use risk management to set their periodic classification testing schedules, notes Hardiman. “In the past, the retesting was prescribed in a table and the timing was based on the ISO class of the cleanroom or clean zone. Now, companies can put more emphasis on the day-to-day data that they generate in their own facilities to help determine the appropriate testing and frequencies needed for continued cleanroom compliance. If a company is generating great data and the risk to continued cleanroom compliance is low, then they can set a longer periodic classification frequency,” explains Hardiman. She notes that risk-based sample site selection is crucial for environmental monitoring. “The key is understanding your unique products and processes and selecting sites that best address the risks that your products and processes present. It is important to be able to identify all of the potential contamination sources in each cleanroom and to select environmental monitoring sample locations in close proximity to these sources. It is also very important to understand the people, material, and waste flows,” concludes Hardiman.

	Pharmaceutical cleanrooms typically already have monitoring plans, which are required by Annex 1 of the EU GMPs, says Ginsbury (3). She notes that users should, however, check with their cleanroom contractors to determine whether a contractor is qualified and familiar with the revisions, because the regulators reference the ISO standards and they must be followed, says Ginsbury. “I would recommend having the discussion now so that, by 2017, your contractors and in-house staff are fully up to speed and following the new standard,” she notes. “Even if you use contractors, the responsibility for review and approval of their results and compliance with regulatory standards lies with you. ISO may not require 5 µm particles and may let you use risk assessment to determine frequency of classification. However, EU Annex 1 still requires measuring 5 µm particles as part of classification, and current industry practice (EU [3] and FDA [4]) is to perform cleanroom classification twice a year for aseptic core. We haven’t heard the regulators on this one but I wouldn’t rush to reduce that frequency based on risk assessment,” cautions Ginsbury.

ISO 14644-1:2015, Cleanrooms and associated controlled environments – Part 1: Classification of air cleanliness by particle concentration

		ISO, ISO 14644-2:2015, Cleanrooms and associated controlled environments – Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration (Geneva, 2015).	

EudraLex Volume 4: Good manufacturing practice Guidelines

, “Annex 1, Manufacture of Sterile Medicinal Products,” (Brussels, 2008).	

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice 

Articles

Production Trends

Revised versions of ISO 14644 adopt changes to sampling procedures and monitoring plans for cleanrooms.

Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

Shipping temperature-sensitive pharmaceuticals such as biological products, has never been easy. Today, changing global requirements and a more complex supply chain are driving more conservative approaches to temperature control. For products such as cell therapies and tissue culture, cryogenic shipping is now preferred.  Other biopharmaceuticals are being shipped at lower tempertures, and even small-molecule-based pharmaceuticals that might have been shipped at ambient temperatures a few years ago, must now be kept at controlled temperatures.

	Transport has become much more than simply moving product from one place to another.  It’s now being seen as a “mobile form of storage,” says Volker Kirschner, director of temperature control solutions for World Courier Management Co, part of Amerisource Bergen. This is especially true in Europe, he says, where the European Union (EU) began strengthening its good distribution practice (GDP) guidelines three years ago.

	Nothing can be taken for granted.  “From political unrest to pandemic concerns, the possibility of changes in shipping patterns and logistics strategies is quite real,” he says, and developing a global transport strategy has become more complex.

	Specialists in cold-chain shipment are responding to regulatory challenges and uncertainty by investing in new technology and IT, and strengthening partnerships with specialty logistics providers. For their pharmaceutical and biopharm customers, they caution, careful risk-assessment is the only way to avoid costly product shipment problems. Many are adding services and products to help make this task easier.  


	The most pronounced recent change in the cold-chain market has been more complex global regulations, from the EU’s GDP guidelines for pharmaceuticals to the Drug Supply Chain Security Act in the United States. “It can be difficult for companies, especially smaller ones, to know where to begin, and how to revamp compliance processes to stay ahead,” says Wanis Kabbaj, director of global healthcare strategy for UPS.

	Additional requirements (e.g., for controlled room temperature) have increased compliance challenges. Many drugs that were once shipped at ambient emperatures must now be shipped at 15°F to 25°C, increasing costs at a time when most pharmaceutical companies are trying to reduce logistic and supply chain operations budgets, says Ariette Van Strien, chief commercial officer at Marken Global Life Science Supply Chain Solutions, which specializes in cold chain and logistics.  

	Compliance has become especially challenging for clinical trial supply shipments, she says, noting that suppliers must scrutinize their global network, enhance tracking capabilities, and improve quality standards.

	Of utmost importance, says Kirschner, are quality management systems (QMS) and documentation, personnel and training, and risk management, especially regarding standard operating procedures (SOPs); facilities and storage requirements; and transport. “Quality departments within manufacturers’ supply-chain operations are gaining more and more influence,” he says, noting that regulatory agencies expect pharmaceutical manufacturers to take a risk-based approach and enforce audits and quality agreements.


	The most critical aspect to maintaining stability and integrity is choosing the most appropriate temperature conditions for the product and, consequently, the best packaging for maintaining those conditions, says Mark Sawicki, chief commercial officer at the cold chain specialist, Cryoport.

	A perfect example, Sawicki says, is the distribution of critical biomarker samples. Under currently accepted practice, these samples are shipped on dry ice. However, he notes, this shipping method has been connected with a 15-30% product compromise or failure rate. As a result, more companies are opting to use cryogenic temperatures to ship biomarkers, to help eliminate potential risks associated with transit times and temperature excursions.

	PCI Pharma Services, a diversified contract services firm that offers cold-chain expertise, has seen a dramatic increase in demand for storing product at ultra-low temperatures, from -40°C down to -196°C, and has adapted its products and services to focus on supporting these temperatures for storage, packaging and shipments, says Samantha James, associate director of clinical services.

	Pharmaceutical manufacturers need to conduct more critical, comprehensive shipping studies on all materials transported in support of a clinical program, says Sawicki, not only looking at sample failure but the impact of the selected transport medium on assay or product performance.

	“We consult with every client to select the best system and temperature-control solution, whether dry ice, liquid nitrogen, or phase-change materials (PCMs) to ship pharmaceutical products, diagnostic specimens, biotherapeutics, and tissue samples, says Van Strien.  

	Generally, the most crucial factors to consider include therapeutic indication, route, mode of transportation, and product sensitivity, she says. “Because a pharmaceutical’s stability is not always known in early trial phases, additional caution may be necessary, and protection for any changes in environmental conditions.”  

	Van Strien advocates developing a thorough risk assessment to help ensure product stability from origin to destination, including studies of the following:

		Potential risks and mitigation strategy

	For clinical products that are only available in small samples, incorrectly handled shipping and transportation can put millions or even billions of revenue dollars at risk by compromising clinical studies, potentially delaying product commercialization, notes Kirschner. When minimizing risk, it is important for manufacturers to consider the package type and choice of temperature-control mechanisms, environmental concerns, value of the ingredients in the product, temperature requirements and variables, and overall shipping costs.

	Suppliers need to master nontechnical issues as well, including local politics and security issues, especially when conducting clinical trials in new geographic markets. Proactive planning is required, Kirschner says, to help protect the integrity of products and, ultimately, the safety of patients who use those pharmaceuticals.

	Generally, he notes, up-to-date methods should be used to assess risk.  For example, he says, failure mode and effects analysis (FMEA) can be conducted, and risk priority numbers (RPNs) assigned, based on the probabilities and severities of certain adverse events occurring during transport.  These RPNs would help determine whether mitigation is required, and what type would work best for the specific product, market and application.

	Temperature is not the only variable to consider. Many pharmaceuticals and biopharma manufacturers require a great deal of stability protection from temperature, shock, vibration, humidity, and light, explains Kabbaj.  “Healthcare logistics has little room for error, so specialized packaging, advanced temperature and location monitoring leveraging global control towers, and in-transit intervention are all critical.”

	In addition, Kabbaj says, extended time in transit when product is being transported inland may expose the packaging to temperature fluctuations that increase risks. Ocean transportation risk factors include container placement on the vessel, sunlight exposure, container insulation, and dwell time on the dock -- all introducing additional packaging stressors.  

	He suggests that pharmaceutical manufacturing professionals take the following steps to ensure that they are covering all the bases required for risk mitigation:

Test packaging, including seals, for endurance, and put prototypes through compression, impact, vibration, temperature, humidity, and shock tests.

 Package optimization should include validation through design qualification, operational qualification, and performance qualification protocols.

Optimize packaging and transportation together.

 Do not develop a cold-chain solution sequentially with assumptions based on transportation, and then the transportation solution that comes with pack-out expiry. Pairing a lighter and cheaper cold-chain packaging with expedited air transportation, for example, may prove to be more effective than opting for a slower mode of transportation (e.g., ground shipping) with complex, heavy, and bulky packaging.

Innovate continually to create better efficiencies.

 Optimize the shipping carton, minimizing unused space, and select more precise packaging configurations.

	“Pharmaceutical manufacturers that do not continually review, evaluate and update packaging best practices leave themselves open to product risk and cost inefficiencies,” Kabbaj says. “Leave room for new ideas, material innovations, and new packaging manufacturers by engaging cold chain partners in a collaborative mode.”


	Cold-chain service providers have been developing new technologies to help pharmaceutical companies better manage risk and respond to problems.  “New requirements are driving developments in packaging and monitoring technologies,” says Kirschner.  As an example, he points to newer semi-active packaging technologies that allow for longer durations of temperature control with lighter materials that have less environmental impact.

	In addition, he says, passive packaging  solutions have been developed that can provide proven temperature control over long distances. Independent testing has shown these solutions to perform five to seven times more efficiently than semi-active solutions, he says. Kirschner also points to new phase-change materials (PCMs), comprised of paraffin or salt-based solutions, that allow for more precise temperature control to maintain product stability over long distances or through extreme climates.

	Many packaging manufacturers are now developing their own vacuum-insulated panel (VIP) containers, combined with PCM solutions, for easier handling and storage. Additionally, he says, on the monitoring side, developments in global positioning software (GPS) and tracking equipment now include automatic start-up and shutdown mechanisms that can provide a real-time view into a shipment’s status. Many technology providers have invested in real-time, GPS-enabled data loggers. Combined with improved data access, through customer portals and better IT, the technology offers immediate insights into bottlenecks and delays.


	“These data loggers provide the ability to track both condition and chain-of-custody, in real time,” says Sawicki.  They can track, not just temperature, but also monitor the impact of external influences on container integrity including orientation and package damage, he adds, and can also tell whether the container has been opened.

	Examples of new cold-chain data loggers include Sensitech’s TempTale and Nexleaf’s ColdTrace systems. Contract service suppliers are also offering enhanced IT connections. PCI Pharma Services, for instance, has developed a portal to allow clients to access real-time data, 24/7. “They can monitor stock levels, and view and trace all live shipments associated with their project, with direct links to courier tracking,” says James.

	Last year, Marken introduced Sentry, a GPS-enabled sensor platform, designed specifically for pharma with real-time track and trace capability through custom-designed software that monitors, records, and reports on location, temperature, motion, shock, exposure to light (i.e., when the box opens), atmospheric pressure, and remaining battery life. The device transmits in real time and communicates through customized cloud-based software that connects with Marken’s Maestro IT system, says Van Strien.

	Today, major cold-chain trends include increased use of temperature- managed shipping, and an interest in evaluating transport  methods and equipment critically, to maximize sample integrity, says Sawicki. There is also an increased emphasis on protecting the products of cell, gene and immunotherapy, says Van Strien, and the complex supply chain logistics that these products require.


	A growing number of biopharm companies must now ship cell- and gene-based therapies, says Kabbaj.  Such products require cryogenic storage at temperatures -238°F and below. In the past, shippers relied on dry ice (which performs best at approximately  -108.4°C), he explains, but dry ice emits carbon gas, which can damage proteins in biologic shipments, and might be harmful to handlers and to the environment. “Not only is dry ice shipping subject to a wide range of international regulations, it simply does not provide a cold enough temperature for many new specialty therapies” he says.

	Yet another important trend, says Van Strien, is increased demand of remote shipping, for example, of clinical trial dosages to patients’ homes. “We will continue to develop further services to allow patients to participate in clinical trials from home, or remote sites of their choosing,” she says.  

	Marken has also introduced an enhanced online booking app for investigator sites that is pre-programmed by study. It has also integrated enhanced scanning technologies throughout its global network. “More mobile technology will help treat patients at locations of their choosing,” says Van Strien.  

:
	When referring to this article, please cite it as A. Shanley, “Cold Chain: Going the Extra Mile," 

Packaging

Cold-chain shipping for sensitive pharmaceuticals benefits from real-time GPS technology, better IT connections, and more conservative, controlled shipping temperatures.

Trends in Temperature-Controlled Shipping

	Pharmaceutical facilities follow a strict microbial contamination control program for cleanrooms. But, what if these solutions are a source of future contamination? It’s a problem in the pharmaceutical, biotech, and medical device industries, but one that’s easily remedied.

	Regulatory agencies, including FDA and the European Medicines Agency (EMA), expect GMP manufacturers to implement a robust decontamination program for their cleanrooms. The approach most routinely used by pharmaceutical manufacturers includes a rotation of one or more disinfectants with a sporicidal agent. These products are formulated with multiple ingredients that are essential for their performance and stability, but their regular repeated use can result in buildup on surfaces over time. This buildup poses visual, safety, and product integrity threats, including: sticky or slippery floors and doors, streaks and discoloration, and contamination. Something that doesn’t look clean can cause issues during an audit for general housekeeping. Residue could be a source of future contamination by giving microbes a place to hide and could potentially be a food source for bioburden. Residue can also lead to an increase in overall particle counts in the cleanroom, and particles can flake off curtains or other surfaces and get into drug products.


	In most cases, 70% isopropyl alcohol (IPA), purified water (PW), or water for injection (WFI) can be used for removing residue. IPA is most appropriate for glass windows, most cleanroom furniture and equipment, and stainless-steel equipment while purified water or WFI is better for residue removal on larger surfaces, such as walls, floors, and ceilings. These rinsing applications should be done only after the sporicide or disinfectant has been applied for the desired wet contact time and allowed to dry. If a surface is rinsed too soon, this can dilute the disinfectant and may actually reduce the effectiveness of the microbial control program.

	All cleaning and rinsing agents used in ISO 5 cleanroom operations should be sterile and applied with sterile equipment and supplies, such as wipes and mops.


	Residue removal is necessary for a complete cleaning and disinfection program and should be performed as needed, based on how the surface looks and feels. If it’s completed on a routine basis (i.e., weekly, monthly, or quarterly), water (alone) may be appropriate. If the removal step has never been implemented or there’s significant residue buildup or hard-to-clean soils, a formulated cleaner may be required. If a formulated cleaner is used, a water rinse step should follow.

	A well-defined residue removal program can be fairly simple to implement with the proper training. By incorporating a surface rinsing protocol into standard operating procedures, pharmaceutical manufacturers can prevent long-term buildup of disinfectant and sporicide residues in aseptic processing areas. Ultimately, this produces better aesthetics, improved safety, and a reduced risk of contamination in products.


	Jim Polarine is senior manager, Technical Services at STERIS Life Sciences, 

Troubleshooting

Regular removal of residues from disinfectants and sporicidals is important for improved aesthetics and safety in cleanrooms.

Equipment and Processing Report-03-16-2016

Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

Trends in Temperature-Controlled Shipping

Best Practice: The Importance of Removing Sporicidal and Disinfectant Residue