Jerold Martin was the senior vice president of global scientific affairs at Pall Life Sciences and the chairman of the Board and Technology Committee at Bio-Process Systems Alliance.

Jerold M. Martin

A broad issue for single-use filter users and suppliers across Europe and North America is the pre-use integrity testing of sterilising filters in single-use systems. The subject has received considerable discussion lately, with differences in regulatory requirements and risk assessments being considered. The properties and suitability of gamma-radiation sterilised single-use filtration systems, and the flexibility of these designs, were not considered when regulatory guidelines were written, but now provide additional options for manufacturers. At the moment, there is no harmonised guidance on the pre-use integrity testing of sterilising filters. FDA does not formally require it, stating in its aseptic processing guidance that "Integrity testing of the filter(s) can be performed prior to processing..." (1).  

In contrast, the EMA states in EU GMP, Annex 1 that, "The integrity of the sterilised filter should be verified before use..." (2). So, while integrity testing after use is a common requirement, pre-use testing, whether before or after sterilisation, is not a universal requirement. This situation is further complicated in Europe where different national authorities apply EU GMP differently. Some European inspectors demand that pre-use, poststerilisation integrity testing be conducted because it is specifically required in EU GMP, while others are more flexible and do not require it.  

The topic was discussed during a workshop at the May 2012 PDA–PIC/S conference in Geneva. Arguments were expressed both for and against testing, but insufficient time was allotted to fully explore the technical issues and no resolution could be drawn. This situation leaves manufacturers with inconsistent international regulatory expectations, and more work is needed to ensure that inspectors have a common understanding. 

The issue is relevant to single-use disposable technology because EMA's published rationale for requiring pre-use, poststerilisation in integrity testing is based on the risks of filter damage or other loss of integrity during high-temperature sterilisation processes, such as steam-in-place or autoclaving.  

Hot topics at the IBC Single-Use Applications Conference			

EMA states, "The filter sterilisation process may be physically stressful for the filter. For example, high temperatures during the process may cause the filter to distort, potentially leading to fluid pathways that allow the passage of particles greater than 0.2 μm in size. The performance of a filter can improve with use, as particles begin to block individual pathways and remove larger pathways that smaller particles could successfully navigate" (3). 

Although such filter distortions have been reported for some prototype membranes, commercialised sterilising-grade filters are validated to maintain integrity and not undergo fluid path distortions (enlargement) during steam sterilisation that might enable initial bacterial penetration. Furthermore, sterilisation by gamma irradiation, which is typically applied to single-use filtration systems, can be considered a less stressful process (low heat, low pressure), and has not been implicated in the distortion of fluid pathways or loss of filter integrity so long as the membrane material is compatible with gamma radiation and that gamma dosage does not exceed specified maxima. These parameters are typically certified with every filter or single-use system lot. Many users question whether the possible risk of introducing contamination by performing a pre-use, poststerilisation integrity test on a sterile filter in a gamma-sterilised single-use filtration system is higher than the risk of not performing the test. 

The decision on whether or not to perform pre-use integrity testing should be made on a risk-assessment basis. Many users consider the filter manufacturer's integrity test prior to release to be sufficient confirmation that the filter is integral as made and is ready to use upon installation and sterilisation. It is rare for users to experience filter-integrity failures post use, so any user pre-use testing, before or after sterilisation, seems unnecessary. Experience with steam-sterilised filters, however, suggests that most instances of post-use filter-integrity failures, when they do occur, are due to gross filter damage incurred during poorly controlled steam-in-place or occasionally autoclave cycles. Where steaming processes are not well characterised and controlled to avoid filter damage, damage (if it occurs) can be recognised prior to product filtration if a pre-use/poststerilisation integrity test is performed.  

For gamma presterilised single-use systems, where the filter manufacturer has already validated that gamma irradiation does not compromise filter integrity, the risk of filter damage during sterilisation appears even more remote. The primary risk of product loss due to integrity failure of gamma-sterilised filters in a single-use filtration system would most probably be from filter damage incurred after sterilisation, for instance, during shipping, handling, and system installation. The lower risk of filter damage and product loss in this case may be further ameliorated if the process is a bulk filtration and, in the event of post-use filter integrity failure, refiltration is qualified and documented in the SOP.  

Alternatively, a redundant filtration scheme may be used to minimise risk of product loss in the event of a filter integrity failure discovered only postuse. Finally, the cost of the drug product or process fluid may be sufficiently low that an infrequent disposal is considered inconsequential (e.g., buffers and inexpensive small-molecule drug products).  

Although the potential for product loss due to filter nonintegrity may be very low, the effect can be great if the product is expensive and cannot be refiltered, such as with biopharmaceuticals and vaccines. Avoiding catastrophic batch loss, as well as possible product shortages, is a justifiable reason to perform pre-use, poststerilisation filter-integrity testing on single-use filtration systems. 

Another advantage of single-use systems compared with stainless steel is that versatile system designs can readily accommodate the necessary flushing to prewet filters before the pre-use integrity test. Stainless-steel tanks for the supply and collection of wetting liquid, which must be cleaned and steam sterilised in place, or autoclaved and aseptically connected, can be easily replaced with preassembled flush supply and collection bags that are gamma sterilised in place. Sterilisation validation is provided by the supplier. Where product is used to prewet the filter for integrity testing, even the side-flush collection bag can be eliminated in some cases. Additional schemes are also available to enable flushing the filter to reduce leachables and any particles that may be present on the downstream side of the filter, along with wetting them for pre-use integrity testing. Filters can be flushed with product to wet the filter, reducing filter leachables and potential particles while recovering the product to maximise yield.   

is senior vice-president of Global Scientific Affairs at Pall Life Sciences, Port Washington, NY (US), and chairman of the Board and Technology Committee at Bio-Process Systems Alliance. Tel. +1 516.801.9086 

Sterile Drug Products Produced by Aseptic Processing —Current Good Manufacturing Practice

Guide to GMP, Annex I — Manufacture of Sterile Medicinal Products

 (Brussels, Belgium, revised Feb. 2008, effective Mar. 2009). 

3. J. Wood, presentation at the IBC Single-Use Conference (San Francisco, CA, Jun. 4–6, 2012). 

4. J. Martin, presentation at the IBC Single-Use Conference (San Francisco, CA, Jun. 4–6, 2012). 

6. K. Lear, presentation at the IBC Single-Use Conference (San Francisco, CA, Jun. 4–6, 2012). 

Articles

There is no harmonized guidance on pre-use integrity testing of sterilizing filters, prompting discussion among users as to whether such testing is necessary.

Pre-use Filter-Integrity Testing: To Test or Not To Test?

Gamma rays are a form of electromagnetic radiation—like x-rays, but with higher energy. The primary industrial sources of gamma rays are radionuclide elements such as Cobalt 60, which emit gamma rays during radioactive decay. Gamma rays pass readily through plastics and kill bacteria by breaking the covalent bonds of bacterial DNA. They are measured in units called kiloGreys (kGy) 

Gamma irradiation provides a number of benefits in cost and sterility assurance. It can be applied under safe, well-defined and controlled operating parameters, and is not a heat- or moisturegenerating process. Consequently, there is no heat stress and condensate drainage or outgassing are not required. Most importantly, there is no residual radioactivity after irradiation. 

Beyond having a different lethality mode, characterising the radiation sensitivity of the product bioburden is another key difference from moist heat (steam) sterilisation. Radiationresistant biological indicators are not used. After the mean bioburden is quantified and sensitivity to a low radiation dose (~8-10 kGy) is established, a statistically determined higher dose (typically >25 kGy) can be applied to provide the appropriate sterility assurance safety margin for every unit in the batch. This safety margin is similar to that of moist heat sterilisation, where a target of <10–6 probability of a non-sterile unit (Sterility Assurance Level, SAL) is established. 

A third difference is that the gamma dosage can be measured in each batch using detectors called dosimeters, which enable parametric release. Product batches subjected to gamma radiation do not need to be lotsample sterility tested for release. 

Standards for validation of gamma sterilisation

Validation procedures for the sterilisation of single-use systems via gamma irradiation are well established and based on widely used industry standards. These standards are recognised by regulatory agencies globally in lieu of any specific regulatory guidance.  

The international standards are harmonised among three official standards bodies: the American National Standards Institute (ANSI), the American Association of Medical Instrumentation (AAMI) and the International Standards Organization (ISO). Their common document is ANSI/AAMI/ISO 11137, Sterilization of Health Care Products — Radiation (1).

ANSI/AAMI/ISO 11137 comprises three parts: Part 1 covers requirements for development, validation and routine control of a sterilization process, Part 2 covers establishing the sterilization dose, and Part 3 provides guidance on dosimetric aspects, the measurement of the radiation dose. Part 2 describes 3 methods for establishing a sterilizing dose (with SAL <10-6). Methods 1 and 2 were designed with small medical devices in mind and involve determination of bioburden and multiple dose analyses that require over 100 or 200 units respectively, both for initial validation and for quarterly dose lethality audits. When we consider large single-use systems, which are made in relatively small batches, both of these methods can be very costly and time consuming. However, the standard provides a third method called VDmax (VD stands for verification dose). Rather than determining the minimum dose to achieve a SAL of <10–6, the VDmax Method substantiates the suitability of a predetermined dosage level, specifically 25 kGy or, for plastic devices with lower gamma tolerance, 15 kGy. 

In conjunction with the publication of the VDmax method for doses of 25 or 15 kGy, additional doses were qualified and published by AAMI in their Technical Information Report 33:2005 (2). This is considered a supplement to ANSI/AAMI/ISO 11137 and they will likely be merged at the next scheduled revision. It expands the VDmax method to seven additional dosages; 17.5, 20, 22.5, 27.5, 30, 32.5 or 35 kGy, enabling flexibility of minimum sterilising dosage based on mean bioburden levels for the product. 

The VDmax method still requires at least 40 systems; 30 for bioburden testing (10 from each of three lots) and 10 units for sterility confirmation after low dose exposure. That's a lot of systems and a primary reason to consider simply irradiating at >25 kGy and claiming microbial control wherever a validated sterile claim is not required. 

Once the mean bioburden and minimum validated sterilising dose is established, and the product goes into production with a sterile claim, quarterly dose audits are conducted to confirm that the levels of bioburden and/or their sensitivity to gamma irradiation have not changed over time. These quarterly dose audits require an additional 20 units from a current lot each time; 10 for mean bioburden analysis and 10 for irradiation at the low verification dose and sterility testing. If any of the irradiated units are found to be nonsterile, the test must be repeated at a higher verification dose, which will then qualify a new, higher production sterilisation dose for subsequent batches to return the process to a <10–6 SAL. 

Addressing technical difficulties with testing of single-use systems

Because of their size and complexity, single-use systems can present some technical difficulties when assessing bioburden and sterility after irradiation. Fortunately, the standard provides some practical strategies by allowing for the grouping of similar products into families so that validation only needs to be performed with a "worst case" or representative unit. Product families are defined by common nature and source of raw materials, components and product design and size, along with assembly process, equipment and environment, etc. A single Master Product can then be identified or constructed to be the representative or "worstcase" version of all the products in the family. Sterilisation of comparable products in the family can then be rationalized as equivalent to the validated Master Product. 

While this reduces the number of different systems that need to be qualified, bioburden recovery and sterility testing of large complex systems can still present formidable technical challenges. Therefore, it is common to only validate sterility of the internal product fluid contact pathway (with closures at any ports or openings) The system exterior and inner packaged space, which receives the same radiation dose, can be considered microbially controlled, but not validated as sterile.  

To further ease the technical challenge of aseptic handling for bioburden and sterility testing, the Master Product can also be broken into smaller subunits, termed Sample Item Portions (SIP). These can be tested separately and have their mean bioburden and sterility results combined to establish the validated sterilising dose for the fully configured system.

Radiation steriliser — dose mapping and quarterly audit

Cobalt 60 can be stored safely in a pool of water, while the chamber above the pool is surrounded by a thick concrete barrier that prevents gamma rays from escaping when the gamma source is elevated into the irradiation chamber. Product intended for sterilisation is packaged, palletised and transported into the irradiation chamber using a conveyor. 

Once a minimum sterilising dose is established for the Master Product, a pallet load configuration and density is established. Dose measuring devices called dosimeters are distributed throughout the packaged load to confirm that the minimum sterilising dose is reached throughout the batch. Because the received dose can vary based on density, materials are typically qualified to withstand up to 50 kGy. On a quarterly basis, the process is audited by, again, determining bioburden in 10 current product, Master Product or SIP samples. Verification dose sterility tests are conducted on 10 additional samples.  

The sterilisation validation and irradiator batch data support both the supplier's claim for sterility for the singleuse system(s) and the user's claim for cellculture process control and finished product sterility. Several documents serve to support both system and finished product sterile claims. First, the supplier should provide a letter explaining the rationale for the sterile claim of each specified system by part number, based on actual or Master Product validation. Suppliers can insert a Certificate of Quality within the unit packaging stating that the product is sterile after irradiation. External irradiation indicators that change colour upon exposure are not sufficiently quantitative to confirm sterility. A summary Sterilisation Validation Report supporting the minimum sterilising dose, as well as the Dose Mapping Study Report and most current Quarterly Dose Audit Report generated by the irradiator, should be provided by the system supplier. Original data and irradiator certificates, however, may only be available during supplier audit. Lastly, the supplier should provide a Certificate of Irradiation for the system lots in the batch, certifying the minimum dose recorded by the batch dosimeters. 

This combination of sterilisation validation rationale, minimum dose sterilisation validation, batch load dose map, batch irradiation dose certificate and quarterly dose audit serves to support the ongoing sterility of the single use system and the sterile products produced with them. For more information on this subject, a good resource is available free of charge in the form of the BPSA's Guide to Gamma Irradiation and Sterilization (3). 

1. AAMI/ANSI/ISO 11137:2006, "Sterilization of health care products — Radiation — Part 1: Requirements for the development, validation and routine control of a sterilization process for medical products; Part 2: Establishing the sterilization dose; Part 3: Guidance on dosimetric aspects"(2006).  

2. American Association of Medical Instrumentation, "Sterilization of health care products—Radiation—Substantiation of a sterilization dose—Method VDmax", Technical Information Report TIR33:2005 (2005). 

3. BPSA, "Guide to Irradiation and Sterilization of Single-use Systems" (2008). 

This column will address some of the questions on how single use systems are sterilized by gamma irradiation and what documentation may be requested by regulators.

Understanding Gamma Sterilisation

How have single-use systems advanced in recent years and what impact has this had on cleanroom environments?

Single-use systems have only recently been introduced into final-filling cleanroom environments. A key strength is their recognition as “closed systems” by drug manufacturers and regulatory authorities. Many drug and vaccine manufacturers see this as a great opportunity to work in non-classified or lower-classified areas (e.g., Grade C or D/Class 100,000/ISO 8), and some have already applied this approach in new facilities and expansions. A key enabler of single-use systems in final-filling areas has been the innovative development of sterile connectors, which enable sterile connections in nonclassified environments. These sterile connectors are valued as providing higher assurance of containment than traditional aseptic connections that must be performed under Grade A/Class 100/ISO 5 conditions. Although perhaps having minimal impact on existing cleanroom facilities, these advanced capabilities are stimulating designers of new aseptic filling facilities to consider reduction in cleanroom spaces and classifications, greatly reducing the potential cost to put up new aseptic filling suites. 

When designing and building a new cleanroom facility, what consideration should be given to single-use systems? 

Implementing single-use systems in a new cleanroom facility can enable reduction in the size of the cleanroom by allowing for downgrades at every stage, working in grey areas for most of the process and limiting Class A (Grade 100, ISO 8) areas to around the filling needles. This is further facilitated through the use of complementary technologies within cleanrooms, for example, single-use port approaches on isolators that enable single-use system integration into Grade 100 areas. The primary benefit of these technologies is a reduction in controlled environment class and areas, which can greatly decrease facility build cost and time, as well as labour resources, time and the costs of environmental monitoring, maintenance and other ongoing quality control activities. 

How do single-use, closed systems help reduce or, in some cases, eliminate the need for classified cleanroom environments? 

Closed systems eliminate the risk of external contamination—including chemical, microbial or particulate. Sterile connectors have been shown to maintain sterile pathways even when externally contaminated with up to one million bacteria per unit and in bacterial aerosols far higher than would occur even in an uncontrolled environment. Because clean sterile fluid pathways are maintained independent of the environment, classified cleanroom conditions are only needed at points of filling. Conservative practices and tradition may preclude complete elimination of controlled spaces, but single-use systems will enable manufacturing in smaller and lower classification areas, greatly reducing facility construction and operating costs. Regulatory acceptance of sterile connection technologies is increasing at the pace of single-use technology implementation, such that in the coming years, both the user base and data for sterile connector technology will only further increase. 

The pharma industry is still heavily reliant on stainless steel. How are manufacturers integrating stainless steel and single-use components and what impact does this have on sterile environments? 

In existing facilities, it is common for biopharmaceutical manufacturers to develop a “hybrid” system of both existing stainless steel and new single-use components. In build-outs or new facilities, particularly at small to moderate scale, more single-use is being introduced. At the larger scale, hybrid systems again dominate with large fermenters, chromatography columns and tangential flow filtration (TFF) systems continuing in stainless steel, while buffer and intermediate/final product hold tanks and filters are introduced as single-use systems. These hybrid systems will be used in large volume production facilities for quite some time, particularly in the downstream area despite progress made in operation technologies such as single-use TFF. Most biosimilar production today is focused on large volume drugs where stainless steel will still predominate, especially for large bioreactors and chromatography columns. In filling areas, however, we are seeing a lot of interest in single-use formulation and filling systems, but we have to take into account the large installation base of stainless steel filling equipment. 

Functionally, manufacturers integrate stainless steel and single-use components or systems using linking technologies that are typically steam sterilisable (“steam-to” or “steam-through” connectors). The linking technology is steamed with the stainless portion of the process. Then the single-use flow path (which is typically pre-sterilised by autoclave or gamma irradiation) is opened to the stainless portion. 

What are the current hurdles to the increased use of single-use systems in aseptic environments? What is the regulatory perspective? 

Regulatory requirements are spelled out in existing GMP documents: process equipment and bulk drug product containers must not adversely affect final drug product quality or safety. Drug manufacturers must demonstrate that single-use systems used in aseptic environments do not introduce leachables, particles or microbes to the drug product that could compromise the product or patient safety. Hurdles include understanding how supplier extractables data, when suitable, can be applied to assess potential toxicity of leachables, while minimizing or even eliminating the need for additional testing, and how and when additional leachables testing should be applied. Drug manufacturers also need to better understand how to apply extractables and leachables data in the context of final dosage levels and understand potential toxicological impact (or, more commonly, substantiate the absence of toxicity above established safety threshold levels). Microbial safety (i.e., sterility) is ensured by validated sterilisation using gamma irradiation, but drug manufacturers need to know what supplier documentation must be submitted to regulatory agencies and understand how the valid sterilized state is maintained for each batch of single-use equipment. Particles from single use systems are generally controlled by in-line filters, but in final filling manifolds and aseptic systems without sterilizing filters, control of particles by the component and assembled system supplier, as well as implementation of flushing protocols where appropriate, must be considered. Another potential hurdle is understanding how to flush and perform integrity tests on in-line sterile filters prior to use when required. Lastly, as there is currently no test to ensure the leak or microbial barrier integrity of assembled single-use systems, components and assembly must be designed and controlled to minimize risk of leaks, and users must learn to trust these assurances in the absence of an on-site pre-use system integrity test correlated to microbial barrier performance. These are key areas where drug manufacturers must work with their supplier partners to design single-use systems and operating procedures that facilitate appropriate use and meet quality and safety requirements. 

Jerold Martin, senior vice president global technical affairs, at Pall Life Sciences, explains the importance and impact of single-use systems in sterile environments.

The Impact Of Single-Use Systems

At the IBC Single Use Applications meeting, held in Boston (6–9 June), I gave a talk entitled "Standardisation of Single-Use...Pros, Cons and Possibilities". The topic of standardisation has appeared in many surveys as one of the top 10 needs for expanding single use implementation. However, there is little definition of what is meant by standardisation, which could be applied to many areas. 

One particular area of discussion is the interchangeability of sterile connectors to enable single-use unit systems with sterile connectors from different suppliers to be linked. In preparing my talk, I also discovered that there is already an interchangeable "standard sterile connector" recognised by industry. If you search for this phrase in Google Images, you'll find a photo of a traditional two-way hosebarb fitting. 

In my talk, I discussed how standardisation of advanced sterile connectors must begin with the end-user's company. Different systems' assemblers and integrators can generally source whatever advanced sterile connector is specified, so the first effort must be for the end-user's company to decide what its preferred sterile connector will be. Since the introduction of the first advanced sterile connector (Pall's Kleenpak), there are now five additional designs on the market from other suppliers, but there is still no industry consensus on which design might become the "standard" of the future. Unlike the easily copied tri-clamp style hygienic flange connector, which is commonly used in stainless steel and some single-use systems, or the aforementioned hosebarb fitting, many advanced single-use sterile connectors incorporate patented technology and designs. Users calling for the standardisation of sterile connectors have yet to address which design should dominate or why the patent-holder should license their design to competitive suppliers. Once bioprocessers agree on a preferred design within their own facilities or companies, once there is consensus on an industry-preferred design (such as happened with tri-clamp style connectors) and once patents expire, options will appear to either standardise to one of the original sterile connector designs, or perhaps users will prefer an innovative proprietary design that is superior to current designs, but has yet to be seen. Until then, care has to be taken to avoid inhibiting innovation and it may not be realistic to expect meaningful voluntary standardisation from the bioprocessing supply industry. 

One area where there is an industry initiative underway to standardise connectors, however, is for polymeric 'tri-clamp' style hygienic flange seal connectors. In response to stimulus letter from the BPSA, the American Society of Mechanical Engineers' Bioprocess Equipment group (ASME-BPE) has formed a task group to review the requirements for single-use polymeric tri-clamp style connectors and how the ASME–BPE Standard for stainless steel hygienic flange seal connectors can be modified in response to accommodate their use. Unlike their stainless steel counterparts, single use polymeric tri-clamp connectors do not require cleanable finishes, crevice-free cleanable seals, or thermal resistance and mechanical strength suitable for steaming in place. They also do not require tolerances that are readily achieved with stainless steel on a lathe, but are difficult to achieve with plastic moulding, thus incurring unnecessary mould costs. Expansion and relaxation of the ASME–BPE standard to cover the specific requirements for single-use polymeric tri-clamp style connectors will be a benefit to both suppliers and users. 

The PDA held its first Single-Use Workshop in Bethesda on 22–23 June. The main focus was to preview the draft PDA Technical Report on Single-use Manufacturing, which is currently in development, and to solicit attendee feedback. As a member of the PDA Single-use Task Force, I served on the Planning Committee and as a moderator and presenter on the report for this workshop.  

The purpose of the new report is to provide the reader with critical concepts and topics to consider when implementing a single-use manufacturing strategy for drug or vaccine production. The draft report discusses single-use systems that may be in direct or indirect contact with raw materials, intermediates, intermediate products, pharmaceutical drug substances or the drug product. The primary goals for developing a single-use manufacturing strategy are customer-based and focus on patient safety and product availability, as well as product and process understanding and control.  

The workshop programme focused on QbD principles and other high-level topics to guide users on their initial decision, selection, validation and implementation of single-use processes. The workshop included several opportunities for Q&A and open discussion, which gave the task force valuable feedback from attendees on the draft report. Key take-away points included:  

The report should aim to help end-users move away from gut-feeling to fact-based decisions. 

Control of suppliers will come under more scrutiny and supplier audits can be expected to increase. 

More detail on materials and manufacturing methods (e.g., films) may be required. 

Partnership between the supplier and end user was stressed; those companies that truly embrace partnership will be the ones most likely to achieve success. 

The industry will expect suppliers to have pharmaceutical standard quality systems in place, particularly with regards to having an appropriate materials change control and change notification programme. 

Training in single-use will also be a key requirement/capability from suppliers. 

Clear communication strategies encompassing quality, trust, track record, openness and security will be in focus. 

The target date for the report's publication is by the end of this calendar year. Current activities entail completion of the manuscript incorporating the feedback from the June workshop and final technical review of completed sections. Follow-on workshops are planned for Uppsala (Sweden) on 28 November 2011 and Phoenix (AZ, USA) on 18–19 April 2012 (see 

Regulatory GMP focus explained at BPSA conference 

The third important single-use conference of the summer was the BPSA International Single-Use Summit, which was held in Washington, DC on 27–29 July. This inaugural conference for BPSA served as a forum for suppliers and users to highlight the business model for single-use. In addition to business leader speakers, the summit featured J. David Doleski, the consumer safety officer at FDA Center for Drug Evaluation and Research. Doleski opened with a review of relevant FDA regulations that impact single-use manufacturing, including 21 

 211.65 on Equipment, 211.94 on Drug Containers, 600.3 on Biological Purity and 600.11 on Biological Control. Each of these has similar statements about assuring that process equipment and containers do not adversely affect the drug or biological product. Additional compendial standards noted were USP General Chapters <87> and <88> on Biological Reactivity, <661> on Plastics and <381> on Elastomers, as well as relevant FDA guidances, including cGMP for Phase I Investigational Drugs (July 2008) and Container Closure Systems for Packaging Human Drugs and Biologics (May 1999). 

Advantages of single-use manufacturing were recognised as:  

Reduced need for cleaning and sterilisation systems and validation

Potentially greater control over aseptic operations (as facilitated with sterile connectors/disconnectors and tube welders/sealers). 

Doleski's talk included an excellent overview of topics that should be incorporated in process documentation and FDA filings. Initially highlighted were considerations for vendor partnerships and materials control, included establishment of manufacturing quality agreements, vendor audits, notifications of changes in product (i.e., materials or design), certificates of analyses, and flow path testing for endotoxin, particulates and bioburden (where necessary). With regards to sanitisation (e.g., irradiation for microbial control) or sterilisation, bioprocessers should note where sterilisation is performed (i.e., contract irradiator) and provide documentation on the sterilisation validation method, sterilisation records, impact on materials (supplier validation data) and repeated sterilisation where applied (note that single-use systems are generally not suitable for multiple irradiations at doses >25 kGy). 

Extractables and leachables studies are performed to assess the potential impact of leachables on product quality, efficacy and safety. This can begin with compatibility and extractables data from the supplier, testing with additional model solvents under manufacturing process parameters (e.g., temperature, pH, pressure and time) where needed, considering the cumulative effect of all manufacturing equipment and conducting further risk assessments to determine if a leachable study is necessary (e.g., for final product formulation).  

Process validation considerations should take into account the full range of the manufacturing process, and incorporate multiple unit operations and actual manufacturing parameters, such as mixing speed and duration of perfusion culture. Sterile media simulations should be conducted for filling of sterile product (i.e., bulk or unit dosage). When fluids are stored in single-use containers, validation should include the length of time and temperature range, with assessments of the impact of fluid on materials, and the impact of materials on product, buffers or media and container integrity (leakage) after storage. Where bulk fluids are shipped, considerations should include the effect of pressure changes, such as altitude, effect of motion (i.e., acceleration or vibration) and the protection offered by external containers. Other environmental considerations can include light, chemicals and other mechanical forces that may impact the contained fluid. 

Despite commonly cited concerns, leak integrity issues with today's improved biocontainer designs are rare. However, possible issues should be qualified and noted, such as movement or shipping of storage containers, operator error (i.e., handling training), improper operation parameters (i.e., tube welding) and exposure to extreme temperature. 

Doleski summarised his talk by saying that the FDA recognises the importance of the user's relationship with their single-use equipment suppliers and expects users to work with suppliers to develop knowledge of their single use equipment, understand their product and processes, consider potential issues, conduct a corresponding risk assessment, perform appropriate validation and establish proper quality systems to maintain a state of control. 

June and July saw three major US conferences on implementing single-use technologies: the IBC Single-use Applications meeting, the PDA Single-use Workshop and the Bio-Process Systems Alliance (BPSA) International Single-use Summit (ISUS). Jerold Martin highlights some of the key topics discussed at these meetings.

Focus on Standardisation, Quality by Design and Regulatory GMP

Biopharmaceutical and vaccine manufacturers are increasingly considering the use of single-use systems for final formulation and filling. These applications have stimulated many questions about the particulate quality of single-use systems and their suitability for use under GMP. Particles in singleuse systems have not historically been an issue because most applications were for media or buffer preparation, intermediate hold, or other applications upstream of final filters. Recently, however, drug developers and manufacturers have begun to apply single-use technology to final formulation and filling, which are downstream of final filters. In this column, I will summarise the regulatory requirements for particles in finished drug products, discuss how they can be related to quality requirements for particles from final filters and single-use system fluid paths, and suggest what suppliers and users can do to ensure that finished drug products meet the regulatory requirements for particulate quality. 

Existing drug and biologics GMP regulations and guidelines that address particulate quality apply either to the external cleanroom environment or to the finished drug product itself, after filling in its final dosage container. In addition to the inspection of final dosage units for visible particles (e.g., per USP <1> Injections), injectable drug products are also subject to lot sample testing for microscopic particles (e.g., per USP <788> Particulate Matter in Injections) (1,2). Similar tests exist under the European Pharmacopoeia 2.9.19 and Japan Pharmacopoeia 6.06 (3,4). For global harmonisation, these tests have also been incorporated into International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q4B (5). 

In contrast to finished injectable dosage units, process equipment, including single-use systems, is not directly regulated under drug or biologics GMP. Consequently, there are no regulations or guidelines for the particulate quality of final filter effluents, single-use filling equipment fluid paths, final bulk drug containers, or even finished drug product dosage containers prior to filling. By themselves, these components and systems do not fall within the scope of GMP regulations, which focus on finished drug products.  

However, regulatory authorities recognise the potential influence of the process equipment fluid path when in contact with final bulk drug formulation. For example, US 21 CFR Part 211.65 states, "Equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements"(6). 

While particulate quality of process equipment is not specified, drug manufacturers must ensure that process equipment does not adversely impact the quality of the final drug product. This requires the single-use system user to put reasonable controls are in place to ensure that process equipment does not cause the drug product to fail particulate quality specifications. 

According to most GMPs worldwide, injectable drug products are required to be essentially free of visible particles. USP General Chapter <1> Injections states, "Each final container of all parenteral preparations shall be inspected to the extent possible for the presence of observable foreign and particulate matter (otherwise termed 'visible particulates') in its contents. The inspection process shall be designed and qualified to ensure that every lot of all parenteral preparations is essentially free from visible particulates" (1). Similarly, the European Pharmacopoeia (2.9.20) states, "Injectable solutions, including solutions constituted from sterile solids intended for parenteral use, should be essentially free from particles that can be observed on visual inspection" (7). 

The observation of a "visible particle" during inspection of filled drug product will depend on the drug and container clarity, lighting, particle size, optical density, refractive index, colour and contrast, and operator or automated inspection sensitivity and reliability. "Essentially free" is also not defined. Because only those individual drug product containers observed to contain visible particles are subject to rejection, limits can vary for the number of unacceptable drug product containers with one or more visible particles per batch, which can be rejected to meet an interpretation of "essentially free of visible particles" for released dosage units. Whether the regulatory expectation of "essentially free of visible particles" applies to particles recovered from single-use fluid paths that may or may not be ultimately visible in drug products, and to what level, is open to interpretation. 

Suppliers' procedures, processes and qualifications of single-use systems to minimise particulate contamination vary with components and systems. 

Sterilising-grade filters are typically preflushed during manufacturing to wet prior to 100% integrity testing. This flushing also serves to minimise downstream particles. Preflushing of particulate filters used as final filters should be confirmed with the supplier. Final filters and re-usable downstream fluid paths or containers are also flushed by users before filling of drug product containers to further minimise particulate and leachable contamination before final drug filling. This flushing may be done for various other reasons, including pre-use integrity testing of the sterilised filter, priming of the filling lines, and checking for final container fill weights and other quality control tests. Filter manufacturers typically recommend that final filters and their associated downstream fluid paths be flushed with a suggested liquid volume before filling of final dosage containers to minimise leachables and downstream particles that would otherwise be introduced into injectable drug products. Initial filter or system flushing is optional per FDA GMP, but indirectly required by EMA GMP Annex 1, which specifies that the sterilised filter should be integrity tested before drug product filtration. Such integrity testing requires inline flushing to wet the filter, which would also serve to minimise downstream particulates if the flush effluent is discarded before finished drug product filling. 

FDA GMP requires that the final filters used to manufacture injectable drugs are "non-fiber-releasing" in accordance with US CFR Title 21, Parts 210.3 (b) and 211.72 (6–8). While this requirement originates from the time when filters contained asbestos fibers, it helps ensure that sterilising grade filters contribute a minimum amount of fibers of any kind (typically from nonwoven polyester or polypropylene support layers). Filter manufacturers may certify that either the finished element or just the membrane meets this requirement. Downstream cleanliness of supplied cartridges, especially those only claiming the membrane is "non-fiber-releasing" 

 the finished cartridge, should be assessed as part of product qualification and selection. Filter samples from each manufacturing lot may also undergo effluent particulate analysis by the supplier.  

Despite these effluent cleanliness qualifications, users should recognise that filters are subsequently dried, packaged, shipped, unpacked, handled and installed prior to use. All these manipulations carry the potential for visible particles to be generated after the manufacturing flush and quality control tests. Visual inspection of the downstream core of a filter capsule for particles is not possible; thus, most filter manufacturers recommend filters to be flushed prior to use to ensure minimum downstream particles and leachables prior to drug product filling.  

Unlike the manufacture of filters, film extrusion is not an intrinsically particle-generating process. Films used to manufacture biocontainers are typically handled in a Class 10000 cleanroom where fluid contact surfaces are minimally exposed to a controlled environment. During biocontainer assembly (seam and port welding), sealed biocontainers are typically subject to inspection for visible particles. Samples of assembled biocontainers may also be subjected to rinse and analysis per USP <788> (2). Biocontainers integrated into single-use systems may be further screened for visible particles during system assembly. Visibility of particles may vary with film clarity. 

Sterile connectors and disconnectors are often employed downstream of final sterilising filters and could potentially contribute particles from the fluid path into final dosage containers. To control this risk, suppliers should sample and inspect moulded parts for visible particulates upon receipt, and may also subject samples of assembled connectors and disconnectors to rinse analysis for microscopic particulates. Sterile connectors and disconnectors should be validated to not be significant sources of particles during actuation. When installed on single-use systems, sterile connectors and disconnectors can again be screened for visible particulates. Optically-clear connectors and disconnectors can facilitate the observation of visible particles that may be in the fluid path. 

Tubing and fittings are typically not manufactured with specific claims for particulate cleanliness. However, as with biocontainer films, their manufacturing processes (i.e., extrusion and moulding) are not intrinsically particle generating, and particulate contamination can be minimised if manufactured and packaged under controlled, clean conditions. Incoming tubing and fittings from qualified suppliers can be screened for visible particles on a sampling basis and then rescreened more thoroughly during assembly into single-use systems. Visual inspection for particles within tubing and fittings can be limited, however, by translucency or opacity of the tubing material. Additional testing for particulates in lot samples of tubing and fittings may be performed as part of the quality control procedures for assembled single-use systems.  

Even where particulate matter in single-use components is minimised, there remains a risk for particles entering the fluid path from the cutting of tubing and insertion of hosebarbs into tubing. Systems can be assembled in a Class 10000 cleanroom, but tube cutting and assembly methods should also be qualified by the supplier to demonstrate that visible particles are not generated and introduced into the fluid path during system assembly.  

To assess the particulate quality of assembled tubing systems, some system suppliers have established quality-control programmes where single-use system lot samples, or master systems with "worst case" tubing connections and component combinations, are subjected to rinse fluid analysis for particles. Results can demonstrate that the fluid path rinse fluids meet USP <788> limits for microscopic particles and quantify any visible particles detected.  

Many drug developers have embraced the plug-and-play concept of single-use systems to mean that pre-use flushing of the fluid path can also be eliminated. Whether or not flushing of single-use final filtration and/or filling systems is required to minimise particles in the fluid path must be determined by the end user, as defined by process requirements for drug product quality. Where flushing with water for injection or buffer is desired, systems can be designed with flush collection bags and protocols to minimise residual fluid path hold-up volumes that could dilute initial drug product effluent. Where sterilising filters are installed, this same procedure can be applied for wetting the filter before pre-use integrity testing, an increasing requirement for the marketing of drug products to some European countries. Alternatively, where appropriate, filters and the downstream fluid path can be flushed with product to minimise losses. Sterilised sterilising grade filters can be pre-use integrity tested "product wet" with the flush going to initial filled containers that may be used for quality control weight/volume checks or formulation analysis. In that way, initial flush can be accommodated to minimise particles without separate loss of product.  

Particulate quality of post-filtration single-use system fluid paths for final filtration and filling should be considered as part of design requirements. Assessments of recoverable particle sizes and levels should be made in advance of clinical and GMP production. 

If you are considering single-use disposable systems for final filling or any other applications, please email me your questions or suggestions for future Disposable Advisor topics. 

1. USP 34–NF 29 General Chapter <1>, "Injections."  

2. USP 34–NF 29 General Chapter <788>, "Particulate Matter in Injections." 

3. EurPh, 5.0, 2.9.19 "Particulate Contamination: Sub-Visible Particles" (EDQM, Strasbourg, France, 2005), 253–255.  

4. Japan Pharmacopoeia XV, 6.06, "Foreign Insoluble Matter Test for Injectables," in General Tests, Processes, and Apparatuses Section, p. 110–113.  

5. ICH, Q4B, Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions — Annex 3 Test for Particulate Contamination: Subvisible Particles, General Chapter (Jan. 2009). 

6. Code of Federal Regulations, Title 21, Food and Drugs (Government Printing Office, Washington DC), Part 211.65, "Equipment."  

7. EurPh, 5.0, 2.9.20, "Particulate Contamination: Visible Particles" (EDQM, Strasbourg, France, 2005) 255–256. 

8. Code of Federal Regulations, Title 21, Food and Drugs (Government Printing Office, Washington, DC), Part 211.72 "Filters." 

Strategies to minimise particle levels in the finished product when using single-use technologies downstream of final filters.

Particulate Quality Of Single-Use Filling Systems

The use of single-use systems has become one of the biggest trends in biopharmaceutical manufacturing since the introduction of complex and sensitive biomolecules, such as monoclonal antibodies and recombinant protein biotherapeutics. In particular, single-use technologies have been widely adopted by global vaccine manufacturers as a means to eliminate cleaning and cleaning validation issues following the influenza vaccine shortage of 2005, which was attributed to contamination issues in a production facility located in the UK.  

There has already been much discussion on single-use systems at US conferences and in US publications during the past 10–15 years, but interest is now expanding further across the globe to Europe and Asia. Despite this growth, there have only been a few dedicated conferences in these regions that focus on single-use systems, even though there has been significant implementation in European vaccine and biopharmaceutical manufacturing facilities. However, this trend may be changing. This year, I've been privileged to speak at and chair several international conferences focusing on single use. Such conferences provide an important opportunity for industry participants to share experience, concerns, and best practices. 

On February 1–2, 2011, I visited Brussels (Belgium) to chair the International Quality and Productivity Center's (IQPC) Pharma–IQ Disposables Solutions for Biomanufacturing Conference, which was attended by nearly 100 European manufacturers and suppliers. The conference was Pharma–IQ's third Annual European Disposables meeting. The previous two annual conferences were held in London (UK), and the event is shaping up to be a key European conference for single-use activities. This year's meeting featured speakers from several European-based vaccine, biopharmaceutical and contract manufacturers, including Novartis, Vivalis, Genzyme, CMC Biologics, Cobra Biomanufacturing, Innogenics, Crucell, Eli Lilly, GlaxoSmithKline, Pfizer and Merck Serono, along with supplier presentations from Pall Life Sciences, Sartorius-Stedim Biotech, Novasep, JM Bioconnect, GE Healthcare, and ATMI Life Sciences. 

National European regulatory authorities and the EMA have not made public statements about the acceptability or requirements for single-use technologies, but this has not deterred European drug sponsor and contract manufacturing facilities from implementing such systems for both clinical and production batches. Conference topics, for example, focused on everything from comparability of product quality and reduction of carbon footprints to implementation of single-use systems for buffer filling processes and preparation of regulatory submissions. The conference also featured several roundtable discussion sessions that facilitated the sharing of questions and experiences among participants. 

Shortly after Pharma–IQ, I visited Tokyo in Japan (just before the earthquake and tsunami occurred) to give a presentation on single-use technologies and quality requirements at the International Society for Pharmaceutical Engineering (ISPE) Japan Sterile Products Processing Community of Practice meeting. Compared with Europe and the US, implementation of single-use systems in Japan has not been as extensive. However, Japanese pharmaceutical companies are very interested in new applications for single-use technologies. I also met with representatives from Japan's Ministry of Health, Labor and Welfare, who were eager to learn about best practices for implementing single-use systems in biopharmaceutical manufacturing. 

At the end of February, I attended the ISPE Tampa Conference in Florida (USA), where I spoke at an aseptic processing seminar about sterilising filtration. One of the hot topics discussed related to European GMP Annex 1 recommendations, which call for integrity testing of sterilised filters prior to use and double filtration schemes to enhance sterilisation assurance. These recommendations can lead to complex systems and procedures, but are possible when using properly designed single-use systems (as opposed to stainless steel systems). One concern that the EMA cites, however, is a potential subtle change in membrane retention properties that might occur with exposure to moist heat (steam sterilisation), but this issue can potentially be resolved by filter plugging during the process.   

With single-use filtration systems, sterilisation is most commonly performed with gamma irradiation, which does not introduce heat and has not been associated with changes to membrane filter porosity or bacterial retention property (so long as the filter materials are gamma compatible and validated for gamma irradiation stability). Single-use filtration systems are typically supplied presterilised, so that a poststerilisation integrity test is the only pre-use test a user can apply. Even without the hypothesised potential changes in membrane flow path and retention property caused by moist-heat sterilisation conditions, it can still be economically prudent to conduct a pre-use (poststerilisation) integrity test in many single-use filtration processes. This step ensures that the filter has not been damaged during shipment and handling. Such damage is not repairable by plugging and is a more valid reason to conduct pre-use integrity testing of the sterilised filter, especially where a drug product is sterile filtered through single (nonredundant) filtration schemes and filled into multiple final containers, or that cannot otherwise be refiltered in the event of post-use filter integrity failure. When designing single-use single or double filtration systems, it is important to consider how the filters will be initially wetted, how they will be tested and whether or how to displace the wetting liquid before product introduction. Approved system design should be discussed with the filter supplier, system-design engineer, and system integrator.  

In March, I finished my single-use tour by chairing the second annual IBC Biomanufacturing and Single-Use Systems Asia Conference, which took place in Shanghai. Last year, the event was held in Seoul (South Korea). I was immediately struck by the progress in appreciation for the benefits of single-use manufacturing by many of the Chinese participants. Single-use manufacturing has been a difficult concept for many Chinese biopharmaceutical manufacturers to accept because they have historically focused on minimising the cost per batch, rather than the degree of cleaning and cleaning validation required in the West, or other beneficial aspects of single-use systems, such as reduced carbon footprint. At this meeting, participants heard case studies and considerations for single-use technologies from companies across Asia, including Simcere Pharmaceuticals, Shanghai Kanda Biotech, Wison and Autekbio, (all in China); Novartis and Green Cross (South Korea); Innobioventures (Malaysia); as well as Piramal Healthcare and Intas Biopharmaceuticals (India). In Asia, much of the biopharmceutical focus is on the development of biosimilars. Although the cost of single-use technologies versus cleanable and reusable stainless steel is still an issue for many process developers in Asia, disposable bioreactors and single-use tangential flow filtration are now being seen as potential ways to accelerate biosimilar development. 

For those involved in or considering single-use processes, two upcoming US conferences may be of interest. The PDA Single-Use Workshop, taking place 22–23 June in Bethesda (MD), will include details about PDA's technical report on single-use technologies using quality-by-design principles. Workshop attendees will have the opportunity to preview and discuss the draft report during the workshop so that industry input can be incorporated into the final version, which will be published by PDA in Autumn 2011. More details are available at 

The Bio-Process Systems Alliance International Single-use Summit, which will be held on 27–30 July 27–30 in Washington, DC, is meant for biopharmaceutical business leaders and will focus on the impact of single-use manufacturing on the future of healthcare and biopharmaceuticals. The meeting will feature speakers from PricewaterhouseCoopers, Venable LLP, and FDA, as well as international single-use end-users and leading suppliers. More details are available at 

Regions around the world are starting to follow the US trend in disposables and are working to solve common challenges.