4X-image/E+/Getty ImagesUnderstanding and evaluating the level of containment achieved by a containment system or equipment is crucial for the processing of APIs, particularly potent pharmaceutical compounds. Based on toxicological evaluations, APIs can be placed into hazard or control bands depending on their potency and toxicological effects. This is the first step in the identification of the hazard potential of the new chemical entity (NCE) or API and the associated exposure controls to ensure personal protection and minimize product cross-contamination. Each hazard/control band should be associated with safe handling guidelines that outline the appropriate facility, equipment, and administrative controls to ensure exposure is maintained below the occupational exposure limit (OEL) for the NCE/API. In general, a potent API is defined as one with an occupational exposure limit (OEL) at or below 10 µg/m

 and a highly potent API as having an OEL below 1 µg/m

Good industrial-hygiene practice dictates that engineering controls are the primary means to control personal exposure. Containment equipment, such as isolators, contained transfer systems, and other contained chemical and pharmaceutical process equipment, are examples of engineering controls in use today. Containment systems and equipment with integrated containment systems are being designed and used by biopharmaceutical manufacturing facilities for all pharmaceutical operations (i.e., chemical production of bulk API to product formulation to packaging) and for all dosage forms, including solid, parenteral, and others, including inhalation and dermal, to control personal exposure and minimize cross contamination in a multi product facility. 

The process of selecting containment equipment and systems should include:

Performing a process review of the process steps, unit operations, and tasks

Identifying the APIs, intermediates, and finished products to be handled and processed, including their associated OELs and/or hazard/control bands

Setting a containment performance target (CPT) for the process

Specifying and selecting the containment equipment based on the task list and the CPT

Verifying containment performance by performing a factory acceptance test (FAT) and site-acceptance test (SAT) 

Evaluating containment performance and occupational exposure to workers during actual operations processing the NCE or API.

To provide consistent and repeatable data to evaluate the equipment and/or containment system prior to installation, upon installation, and during operation, it is crucial that testing be performed following a standard methodology. Because the potential exists for both airborne emissions and surface contamination, industrial-hygiene sampling methodologies for both air and surface sampling are used to evaluate the potential emissions that may be generated during normal processing and cleaning of containment systems and equipment (2,3). 

The Good Practice Guide: Assessing the Particulate Containment Performance of Pharmaceutical Equipment

, from the International Society of Pharmaceutical Engineering (ISPE) describes methodologies for evaluating the containment capability of containment systems and processing equipment in the pharmaceutical and biotechnology industries under defined conditions. (4) This updated guide, first issued in 2005, addresses a broad selection of containment technologies and processing equipment and provides technical guidance and consistent methodologies for evaluating the particulate containment performance (i.e., particulate emissions). The guide defines current good practices and provides information to allow organizations to benchmark their practices and improve on them. Specifically, it provides a methodology to derive data associated with the handling of pharmaceutical ingredients that is useful in the assessment of potential risks. The revised guide was the culmination of a two-year process by the ISPE Containment Community of Practice to revise the original document. 

Since first published, the guide has been used by containment-equipment manufacturers, pharmaceutical companies, and industrial-hygiene consultants to perform containment verification assessments for FAT, and/or SAT of containment systems and equipment. It also has been used to evaluate and compare equipment from different suppliers. 

Containment-performance-assessment studies

The revised guide identifies crucial issues that need to be addressed prior to, during, and/or at the completion of containment-performance-assessment studies. It guides the user to perform repeatable containment verification testing to assess accurately the containment capabilities of various containment systems and process equipment. Sections provide information and guidance on test protocols, test environment, test material, air and surface sampling, sample analysis, sample interpretation, and reporting. 

 Table I: Containment equipment test protocols.The guide specifies a methodology using a surrogate material to verify containment performance and specifies several surrogate materials that may be used, including lactose, naproxen sodium, manitol, acetaminophen (paracetamol), insulin, riboflavin, and sucrose. Of these, lactose and naproxen sodium are the most commonly used. The guide also provides containment equipment test protocols to follow when performing the containment verification assessment. These seven protocols are listed in 

.As noted previously, prior to selection of a containment system or device, a CPT should be identified for the process, providing the containment system supplier with a containment target for the design and construction of the unit. A CPT should also be identified for off-the-shelf units purchased for use in standard applications such as sample weighing or dispensing. If a CPT has not been established, containment verification testing is still applicable to ensure that unit is providing adequate containment for the activities or processes to be performed within the unit. 

 Figure 1: Area sampler positions for Protocol 5 Ventilated Enclosure, from ISPE Good Practice Guide. Table II shows the corresponding personal, real-time and surface samples.The guide outlines containment verification at the supplier’s site (i.e., FAT) and when installed at the user’s site (i.e., SAT). The purpose for containment verification being performed at the supplier’s site is to identify and correct any deficiencies that may impact the meeting of the CPT prior to delivery. Containment performance verification testing should only be performed by qualified industrial hygienists, preferably under the direction of a certified industrial hygienist. 

Table II: Sampling details.The seven protocols in the guide state the recommended positions for air, real-time air monitoring, and surface samples. 

 from the ISPE Good Practice Guide shows the sampler positions for a ventilated enclosure. Protocol 5, Ventilated Enclosure, specifies the sampling locations for performing the containment verification testing. 

Purchasing a containment system or equipment from a reputable supplier should ensure the achievement of containment objectives. Proper planning and testing, however, is needed to obtain accurate reliable data during a containment-verification test. The following steps can help ensure a successful containment-verification test: 

1. Set a CPT as an airborne concentration (µg/m

) based on the OEL of the API to be handled. 

2. Select a surrogate material to use during containment performance verification. Some of the chemical/physical properties to be considered in the selection of the surrogate include:

3. Develop a sampling strategy that includes:

Type of sampling (air and/or surface sampling) 

The types of samples (area and/or personal) to be taken

Utilization of different operators (if possible) 

4. Use an accredited laboratory for the analysis. Verify there is a validated method for the airborne and surface surrogate samples with sufficient sensitivity to evaluate the containment performance. >

5. Review the containment equipment and/or system with the users prior to testing. >

6. Perform a dry run without the surrogate material prior to testing. >

7. Keep the surrogate material in an area or room outside the test room or process room. >

8. The containment-verification report should describe the testing methodology and provide an objective interpretation of the results. The data collected should be analyzed consistently and according to accepted statistical methodologies. An appropriate number of samples as noted previously (minimum of three runs) should be taken to reduce the variability and maintain consistency when evaluating the containment equipment. Finally, the use of traditional statistical measures such as the 95th percentile (if a sufficient number of samples are taken) or using European Standard EN 689:1986 or Bayesian analysis to confirm that airborne release concentrations will not normally exceed the CPT. >

Containment-verification testing of equipment and containment systems is an accepted means for evaluating the containment capability of containment systems and processing equipment in the pharmaceutical and biotechnology industries under defined conditions. ISPE’s 

Good Practice Guide: Assessing the Particulate Containment Performance of Pharmaceutical Equipment

 describes the methodologies for performing containment verification testing for both FAT and SAT scenarios. The guide specifies a methodology using a surrogate material to verify containment performance by setting a CPT and then performing air and surface sampling as outlined in standard protocols for containment systems. By following the guidelines, users can generate accurate and repeatable data to perform statistical analysis and confirm that airborne-release concentrations will not normally exceed the CPT. >

1. B.D. Naumann and E.V. Sargent, “Setting Occupational Exposure Limits for Pharmaceuticals,” 

2.  OSHA,  Sec. II: Ch. 1, “Personal Sampling for Air Contaminants,” in 

3. OSHA, Sec. II: Ch. 2, “Occupational Skin Exposure V. Wipe Sampling Methodology,” 

All figures and tables courtesy of the International Society of Pharmaceutical Engineering.

Articles

A Good Practice Guide from the International Society of Pharmaceutical Engineering provides a framework to assess how well pharmaceutical equipment contains hazardous APIs.

Containment-Verification Testing For Pharmaceutical Equipment Performance

ANDRZEJ WOJCICKI/Getty ImagesNanotechnology is an important area of research, particularly in certain therapeutic areas, such as anticancer therapies to control dosing and target drug delivery to the tumor site. A review of the literature shows several interesting developments in this field.

Superparamagnetic iron oxide nanoparticles

Swiss researchers have discovered a method that allows for the controlled release of an active agent on the basis of a magnetic nanovehicle. The research was conducted as part of the National Research Program “Smart Materials,” a cooperation between the Swiss National Science Foundation and the Commission for Technology and Innovation. The specific work was conducted by researchers at ETH Lausanne, the Adolphe Merkle Institute, and the University Hospital of Geneva in Switzerland.

The researchers demonstrated the feasibility of using a nanovehicle to transport drugs and release them in a controlled manner as explained in a Jan. 24, 2013 press release from the Swiss National Science Foundation. The nanocontainer used was a liposome with a diameter of 100 to 200 nm. The membrane of the vesicle was composed of phospholipids and the inside of the vesicle offered room for the drug. Superparamagnetic iron oxide nanoparticles (SPIONs) were integrated into the liposome membrane; the SPIONs become magnetic in the presence of an external magnetic field. Once they are in the field, the SPIONs heat up. The heat makes the membrane permeable, and the drug is released. The SPIONs also are a contrast agent in magnetic resonance imaging (MRI). A simple MRI shows the location of the SPION and allows for the release of the drug once it has reached the targeted spot (1, 2).

In their study, the researchers noted that liposomes have been characterized by cryogenic electron microscopy (CryoTEM) as well as in combination with nanoparticles in SPIONs incorporated inside the liposomal membrane. CryoTEM maintains the native state of the liposomes. The quick freezing of the sample immobilizes particles and liposomes exactly at their position in the suspension, which allows localization information to be extracted from the images. The researchers reported on the analysis of cryoTEM images of liposome-particle hybrids, including the estimation of the contrast transfer function (CTF) and electron dose as well as the correct positioning of the sample holder and tomography for accurate localization (1, 2).

Another challenge was to reach a temperature sufficiently high to open up the liposomes, according to the release. This problem was addressed by increasing the size of the SPION from 6 to 15 nm. The membrane of the vesicles had a thickness of only 4-5 nm. The researchers regrouped the SPION in one part of the membrane, which made the MRI detection easier. Before starting 

 tests, the researchers plan to study the integration of SPION into the liposome membrane in greater detail, according to the release.

Researchers at Yale University developed nanolipogels as a new drug transport technology to deliver anticancer therapies. The nanolipogels are nanoscale, hollow, biodegradable spheres, which are capable of holding chemically diverse molecules, according to a July 15, 2012 press release by the National Science Foundation (NSF), which is providing funding for the research. The nanolipogels contained transforming growth factor (TGF-β) and interleukin-2 (IL-2). Specifically, the researchers developed nanoscale liposomal polymeric gels (i.e., nanolipogels) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers to deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines. The nanolipogel releasing TGF-β inhibitor and IL-2 significantly delayed tumor growth, increased survival of tumor-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8+ T-cell infiltration (3, 4).

“The cytokine can be thought of as a way to get reinforcements to cross the dry moat into the castle and signal for more forces to come in,” said Fahmy in the NSF release. “In this case, the reinforcements are T-cells, the body’s anti-invader ‘army.’ By accomplishing both treatment goals at once, the body has a greater chance to defeat the cancer,” he said.

An important aspect of the nanolipogels is their ability to “package” two completely different kinds of molecules--large, water-soluble proteins, such as IL-2, and smaller water-phobic molecules, such as the TGF-β inhibitor--into a single delivery vehicle, according to the NSF release. The outer shell of each nanolipogel is made from a biodegradable, synthetic lipid that degrades in a controlled manner, can encapsulate a drug-scaffolding complex, and is easy to form into a spherical shell. Each shell surrounds a matrix made from biocompatible, biodegradable polymers that are impregnated with the TGF-β inhibitor molecules. Those near-complete spheres are put in a solution containing IL-2, which gets entrapped within the scaffolding, a process called remote loading, according to the NSF release. The result is a nanoscale drug-delivery vehicle that is small enough to travel through the bloodstream but large enough to be entrapped for delivery to the tumor sites (3, 4). 

Researchers at the Massachusetts Institute of Technology (MIT) developed nanoparticles that can be controllably triggered to synthesize proteins. The hope is that particles could be used to deliver small proteins that kill cancer cells and eventually larger proteins such as antibodies that trigger the immune system to destroy tumors. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photo-labile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. 

 protein synthesis was spatially and temporally controllable and could be initiated by irradiating micron-scale regions on the timescale of milliseconds (5).

The researchers designed the new nanoparticles to self-assemble from a mixture that includes lipids, which form the particles’ outer shells, plus a mixture of ribosomes, amino acids, and the enzymes needed for protein synthesis. Also included in the mixture are DNA sequences for the desired proteins. The DNA is trapped by DMNPE, which reversibly binds to it. This compound releases the DNA when exposed to ultraviolet light. In this study, particles were programmed to produce either GFP or luciferase. Tests in mice showed that the particles were successfully prompted to produce protein when UV light shone on them, according to an Apr. 9, 2012 MIT press release. Although more testing must be done to show that the nanoparticles can reach their intended destination in humans and that they can be used to produce therapeutic proteins, the research is an interesting start. The researchers are working on particles that can synthesize potential cancer drugs by targeting protein production that could be turned on only in the tumor, thereby avoiding side effects in healthy cells. The team also is working on new ways to activate the nanoparticles. Possible approaches include production triggered by acidity level or other biological conditions specific to certain body regions or cells, according to the MIT release.

Researchers at the University of Sydney in Australia, led by Nial Wheate, senior lecturer in the Faculty of Pharmacy, and researchers from the University of Strathclyde and the University of Glasgow in Scotland recently reported on delivering the anticancer drug cisplatin using gold-coated iron oxide nanoparticles for enhanced tumor targeting (6, 7). The researchers used this approach to overcome some challenges of cisplatin, namely poor bioavailability, severe dose-limiting side effects, and rapid development of drug resistance. The iron oxide core was coated in a protective layer of gold before the anticancer drug cisplatin was attached to the gold coating using spaghetti-like strings of polymer, according to a May 31, 2012 University of Sydney press release.

The iron oxide nanoparticles (FeNPs) were synthesised via a coprecipitation method before gold was reduced onto its surface (Au@FeNPs). Aquated cisplatin was used to attach {Pt(NH3)2} to the nanoparticles by a thiolated polyethylene glycol linker forming the desired product (Pt@Au@FeNP). The nanoparticles were characterized by dynamic light scattering, scanning transmission electron microscopy, UV visible spectrophotometry, inductively coupled plasma mass spectrometry, and electron probe microanalysis. Nanoparticle drug loading was found to be 7.9 × 10-4 moles of platinum per gram of gold. External magnets were used to show that the nanoparticles could be accumulated in specific regions and that cell-growth inhibition was localized to those areas (6, 7).

, online, DOI:10.1038/nmat3355, July 15, 2012.

., online, DOI: 10.1021/nl2036047, Mar. 20, 2012.

, online, DOI10.1016/j.ica.2012.05.012, May 30, 2012. 

Parenteral drug delivery offers a variety of challenges but also opportunities. The author examines recent developments in nanotechnology-based drug delivery and other advances in injection-based drug delivery.

Delivering Complex Drugs With Nanotechnology-Based Solutions

High-temperature, short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization are continuous-flow thermal processes that have been established and highly refined in other industries for many years. Their precision and minimal impact enable the manufacture of products that cannot be made using batch technologies. HTST and UHT are traditionally used for heat-sensitive products. As continuous-flow processes, they are effective against vegetative cells, viruses, and heat-stable endospores.  These characteristics and their continuous-flow nature make them potentially useful as part of the trend toward methods of continuous manufacturing of bio/pharmaceuticals. 

 Microthermics, Inc.Technologies frequently evolve separately, often independently, in unrelated industries and transfer between them. This transfer is often how industries and technologies advance and take significant steps forward. Identifying a technology that is proven, highly refined, and fully supported industrially and regulatorily, however, is less common. This is the case for high-temperature, short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization. HTST and UHT are continuous flow, thermal processes that have been used to pasteurize and sterilize liquids (e.g., foods) for more than 60 years. The processes have been developed as tightly controlled systems and refined to reliably produce high quality products at low cost. HTST and UHT have been optimized to reach high assurance levels for inactivation of vegetative cells, viruses, and heat-stable endospores, all while retaining quality that could not be maintained using batch processes, such as autoclaving. 

It seems somehow concerning to use a process that has evolved for food products, such as milk and juices, and use it for highly refined pharmaceuticals, but let’s consider the fundamentals. The chemical-reaction kinetics that describe how and why these processes inactivate bacteria but retain the quality in a biological fluid (e.g., milk) are the same as in any other biological fluid. Commercially, these processes are well established and used for products ranging from juices to baby food and even products as sensitive as liquid whole egg. These processes have annual capacities measured in hundreds of billions of packages per year. Commercial equipment for HTST and UHT processes commonly operate at flow rates ranging from roughly 5 gallons to more than 100 gallons per minute. Commercial capacities, however, do not lend themselves to the batch sizes and rapid cycles of research and development. The need to conduct thorough research and to optimize treatments (e.g., hold time, temperature, and heat transfer) for different products has triggered the development of miniaturized research equipment and experimental methods for this purpose. These tools have enabled R&D professionals to address potential manufacturing issues early and avoid losses and costly problems while also helping these processes to become better understood. Optimization of these processes has led to development of a wide assortment of time and temperature treatments as well as highly refined tools to test products and deliver these treatments. As a consequence, small-capacity systems have been developed for lower flow rates, bringing the benefits of HTST pasteurization and UHT sterilization to the high-value, low-volume materials of pharmaceuticals. 

Sterilization in continuous manufacturing

Continuous manufacturing has been described as a manufacturing breakthrough and as the method of the future by Konstatine Konstantinov, vice-president of commercial process development at Genzyme (now part of Sanofi), and Robert Bradway, chairman and CEO of Amgen (1, 2). The trend toward using this method is increasing as manufacturers of bio/pharmaceuticals strive to meet growing demand, reduce floor space, improve manufacturing flexibility and capacity, and reduce costs.The adoption of continuous manufacturing for biopharmaceuticals emphasizes the need to inactivate microorganisms continuously at rates consistent with these new processes. The adoption of HTST and UHT continuous processing and surrounding technologies is a natural fit. Early adopters in the biotechnology and biopharmaceutical industries have begun to deploy these processes. The question remains, however, what are the reasons to adopt HTST and UHT in these industries? Are their benefits simply a function of the continuous process or are there additional benefits that make HTST and UHT even more desirable?  

Figure 1: Flow diagram for continuous-flow thermal processes.The benefits of HTST and UHT processes result from their continuous flow nature and their use of different and more highly refined time and temperature conditions. To understand their benefits, it is useful to consider an example process like that shown in 

. The product is pumped continuously through the process at constant flow and is heated to the process temperature under steady-state conditions. It flows through the hold tube, which is of sufficient length to ensure that the product is hot for the time needed for the required lethality, before it is cooled as it exits the system. The result is that the product experiences a controlled, well-defined time–temperature exposure. This time–temperature history (TTH), conceptually shown in 

, is usually less than two minutes from start to finish. Although there are relatively few rules linking the terms “pasteurization” or “sterilization” to specific temperatures, for the sake of this discussion, pasteurization is usually conducted at hold-tube temperatures between 70 °C and 121 °C.  Sterilization hold temperatures range from 128 °C to 150 °C.Hold times most commonly range from 2 to 30 seconds.

 Figure 2: Conceptual plot of product timeâtemperature history.Because HTST and UHT continuous-flow processes are closed systems that operate at steady state, the impact of the thermal process on product quality and lethality is uniform and independent of batch and container size. In contrast, the thermal exposures delivered to liquids being pasteurized or sterilized in large-scale fermenters or vessels in autoclaves/retort are not as uniform because the heat exposure varies with the container size and location. In comparison, it becomes apparent that, in the continuous process, several major sources of variation and potential points of failure have been eliminated and overprocessing has been greatly reduced.

As thermal processes, HTST and UHT processes are effective against viral contamination. These processes are especially useful for emulsions and suspensions that are not compatible with filtration. They provide real-time monitoring and record-keeping of processing conditions. They are precise, and the actual process time and temperature conditions can be adjusted to optimize the retention of key components and the delivered lethality.  This precision can be important to maximize retention of key media components being fed into a fermentation process or of a desired active agent resulting from a different step of manufacture.

Unlike scale-up of batch operations, scale-up of HTST and UHT processes is often unnecessary because processing more material is linked only to the processing time, not the vessel size. Larger volumes of product are processed by simply running the equipment longer. Thus, multiple systems may not be needed for different batch sizes. When scale-up is necessary within the same general style of HTST or UHT equipment, it is a matter of duplicating the TTH. If a different style system is used, the detailed matching of the TTH may require more powerful mathematical and modeling tools for thermal process evaluation.

In the food industry, these processes are used to make many high quality products that would not be viable using longer-time and lower-temperature methods, such as autoclaving, because of poor quality. These examples demonstrate the potential to pasteurize or sterilize many bio/pharmaceutical materials that are also not well-suited to autoclaving. In simpler terms, these are enabling technologies. 

The effectiveness of HTST and UHT processing can be explained by thermal-process reaction kinetics. The exposure time and temperature in these processes exploit the differences between the reaction kinetics of the inactivation of microorganisms and of the reactions that define product quality. Quality can be broadly defined by components ranging from nutrient content to enzyme activity, but the overall relationship remains. 

Table I:  Equations for determining reaction kinetics, t = time, T = Temperature.The Bigelow and Ball method or Arrhenius kinetics can be used to describe the reaction kinetics supporting these processes, as shown in 

. Microbial inactivation and the loss of quality both follow first-order kinetics. The decimal reduction time (D value) is the time needed to reduce a microbial population by one log cycle at one temperature. Both the D value and the reaction rate (k) change exponentially with temperature. This temperature sensitivity is described by the z-value and the activation energy (E

The important point is that the reactions for inactivating microorganisms accelerate exponentially more than those defining quality, regardless of the mathematical model. The z values are smaller for microbial inactivation than for the destruction of most qualitative components. Correspondingly, the E

s are larger for microbial inactivation than for the destruction of most qualitative components.  

Figure 3: Plot of the log of process time (t) vs. 1/T or (Tr-T) showing optimization region of quality and process lethality, T = temperature, Tr- = reference temperature.This gap provides the opportunity to select conditions that optimize product safety and quality. Consider pasteurization or sterilization where the target organism and the microbial reduction (calcuated by log (N

 is the initial microbial count and N is the microbial count), reference temperature (T

), and z are all known. Using either Bigelow and Ball or Arrhenius kinetics, the resulting equation yields a constant or iso-process line representing all the time and temperature combinations that provide the microbial reduction for pasteurization.  Each pair of time and temperature conditions on the line delivers the same microbial kill.  More than that, they define a boundary, which is shown in 

. Treatments higher in temperature or longer in time (i.e., those left or above the microbial log reduction line in 

) have higher microbial kill and higher assurance levels. All other treatments do not have sufficient lethality. 

Similarly, the iso-process line for a qualitative factor (e.g., nutrient retention) can be calculated. As shown in 

, this line has a different slope because the reactions inactivating nutrients do not have the same sensitivity to temperature change as those inactivating microorganisms.All the points below and right of this line (i.e., lower temperature and time) retain more nutrients. 

 demonstrates why HTST and UHT processes retain quality and deliver proper lethality. Tracing the pasteurization/sterilization line toward higher nutrient retention leads to higher-temperature and shorter-time processing conditions. The higher- temperature and shorter-time processes retain more nutrients than the longer-time and lower-temperature processes, such as autoclaving. In addition, a treatment combination between the lines that increases both microbial inactivation and nutrient retention can be selected. 

The significance of these relationships and this analysis are considerable. Having the reaction-kinetic parameters for the target organism and the qualitative factor (in this case, nutrient retention) enables the selection of optimal processing time and temperature conditions. Taken another way, determining the thermal-destruction kinetics of qualitative materials allows them to be screened for suitability to preselected conditions. Either way, determining the thermal reaction kinetics for the target organism and the materials that are to be retained in the actual product is valuable because it allows prediction of their performance under a wide range of conditions using simple math and avoids problems at the production level.  

The learning curve in bio/pharmaceuticals

The technologies of HTST pasteurization and UHT sterilization have been refined for many years in other industries. Details of validation, maintenance of proper safety assurance, and documentation have also been well defined. The tools to generate thermal-destruction kinetics, test products, and optimize operating conditions simplify the adoption of these technologies for bio/pharmaceutical manufacture.

These technologies have been developing for many years, and their adoption involves the use of the existing tools to optimize them for each application. It also requires specific efforts to implement them properly. As continuous-flow processes that are new to a manufacturing environment, they require training and a different outlook to support this transition. It is a cultural change, which extends from manufacturing through maintenance, quality assurance, engineering, and management.Experience in other industries has shown that thorough initial testing of products is important. Screening to determine the suitability of materials and selecting processing conditions are significant initial steps. Test-processing products thoroughly is essential to obtain data supporting optimization of these conditions. It is equally essential that testing demonstrates the performance of the product with down-line unit operations, especially in continuous manufacturing.

Industry participants are finding that HTST pasteurization and UHT sterilization provide another tool for the manufacture of bio/pharmaceuticals. They have found products for which theses processes are strikingly successful and those for which they are not suitable. More importantly, perhaps the most interesting benefit is the ability to facilitate the development of entirely new products.

1. K. Weintraub, “Biotech Firms Race For Manufacturing Breakthrough,” 

, Jan. 30, 2013, www.technologyreview.com/news/509336/biotech-firms-in-race-for-manufacturing-breakthrough.

High-temperature, short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization are potentially useful as part of the trend toward methods of continuous manufacturing of bio/pharmaceuticals.

Applying Continuous-Flow Pasteurization and Sterilization Processes

Alfred Pasieka/Getty Images; Dan WardRestructuring and manufacturing rationalization have been the order of the day for the large bio/pharmaceutical companies during the past several years. Small-molecule and solid-dosage operations have been the largest target for such cost cutting, but there are bright spots on the manufacturing investments. Several Big Pharma companies are investing in biologic drug-substance, vaccine, and parenteral drug finished product manufacturing. 

. Bristol-Myers Squibb is investing approximately $250 million to expand its large-scale biologics manufacturing facility in Devens, Massachusetts. The expansion will introduce biologics development and clinical-trial manufacturing capabilities to the site while adding approximately 350 employees to the Devens workforce over time. The Devens site is home to the company’s large-scale bulk biologic manufacturing facility. Construction of the Devens site was completed in 2009. It represented the company’s largest single capital investment ($750 million) and provided Bristol-Myers Squibb with large-scale bulk biologics production capacity. In May 2012, the company received FDA approval to manufacture its arthritis drug Orencia (abatacept) at the Devens facility. 

The new $250-million investment will be used to construct two new buildings on the 89-acre Devens campus: one building for process development and one for clinical manufacturing. Together, the two buildings will add approximately 200,000 ft

 of laboratory and office space to the Devens site, which is now comprised of six major buildings in a 400,000-ft2 complex. Work on the expansion is expected to begin in late 2013 and be completed in 2015. In the interim, Bristol-Myers Squibb has leased 30,000 ft

  of laboratory space in nearby Hopkinton, Massachusetts, to begin moving some biologics process-development functions closer to Devens during construction. The company intends to maintain this space until construction is completed. 

. In the fourth quarter of 2012, Novartis announced the planned construction of a new biotechnology production site in Singapore with an investment valued at more than $500 million. The new facility will focus on drug- substance manufacturing based on cell-culture technology. Construction will begin in 2013, and the site is expected to be fully operational in 2016. It will be colocated with the company’s pharmaceutical production site in Tuas, Singapore. In the future, Novartis expects its Singapore site to be a technological competence center for both biotechnology and pharmaceutical manufacturing. 

In December 2012, Novartis acquired a 16,000-m

  FDA-approved manufacturing facility in Morris Plains, New Jersey from the biopharmaceutical company Dendreon for $43 million. Novartis purchased all fixed assets at the site, including all equipment, machinery, utilities, and cell- therapy-related plant infrastructure. The land and building will continue to be leased from a third party. The facility, and certain former Dendreon personnel that were retained will support clinical and commercial production of potential new products and therapies that emerge from the Novartis–University of Pennsylvania (Penn) collaboration announced in August 2012. These drugs include CTL019, a drug in Phase I/11 development for treating certain blood cancers. Under the Novartis–Penn pact, the parties will research, develop, and commercialize targeted chimeric antigen receptor immunotherapies for the treatment of cancers as well as build on the Penn campus in Philadelphia the Center for Advanced Cellular Therapies, which will be dedicated to developing and manufacturing adoptive T-cell immunotherapies. 

Novartis also is proceeding with a multiyear vaccine-production project. In 2008, its Vaccines and Diagnostics Division broke ground on a new rabies and tick-borne encephalitis $330-million manufacturing facility in Marburg, Germany. Construction is complete and the facility is in the process of executing the necessary validation activities with regulatory  approvals for products planned for 2013. 

In 2009, Novartis opened a new cell culture-based influenza vaccine-manufacturing site in Holly Springs, North Carolina. As of Dec. 31, 2012, the total amount spent on the project was $426 million, net of grants reimbursed by the US government. The total investment in this new facility is expected to be at least $900 million, partly supported by grants from the US government and prior investments in influenza cell-culture technologies at the Novartis vaccines site in Marburg, Germany.

The company is also proceeding with the building of a new $475-million vaccine-manufacturing facility in Recife, Brazil. The manufacturing plant is part of Novartis vaccines’ strategy to enter the Brazilian market and is aligned with the government’s goal to become self-sufficient in vaccines. The technical start-up of the facility is planned for approximately 2015. 

. As part of its growth strategy in diabetes, Sanofi’s Frankfurt, Germany site, its principal manufacturing center for Sanofi diabetes products, is being equipped with a new aseptic processing area that uses isolator technology to significantly improve the aseptic-filling process. This investment will be operational in 2016. The Sanofi diabetes industrial network also is expanding its manufacturing footprint in emerging markets, both in Russia and in China (Beijing), where a new facility that was inaugurated in 2012 has begun assembly and packaging of SoloSTAR, the prefilled injection system for Lantus (insulin glargine). Also, during 2012, Sanofi’s Ankleshwar Pharma site in Gujarat State, India, handled packaging and quality control through to release the first commercial batches of AllSTAR, the company’s insulin pen specifically intended for the India market.

Its Frankfurt site is one of three dedicated biotechnology hubs that Sanofi is developing in Europe. In 2012, its facility in Vitry-sur-Seine, its biggest integrated cell-culture facility, produced the first technical batches of aflibercept, the API in the company’s anticancer drug Zaltrap. Its facility in Lyon Gerland, France, is a new world center dedicated to the production of thymoglobulin, a drug to prevent and treat transplant rejection. During 2012, teams at Lyon prepared a dossier for the healthcare authorities as part of the process of transferring production to this site. 

In the United States, Sanofi, through its subsidiary Genzyme, has major investments underway, including at its Framingham, Massachusetts biologics site, which was approved by FDA and EMA in 2012 for the manufacture of Fabrazyme (agalsidase beta) to treat Fabry disease. Its site at Allston, Massachusetts has initiated a major investment program in connection with the implementation of its compliance-remediation workplan, approved by FDA in January 2012. In 2012, Sanofi’s Genzyme acquired the Bayer Healthcare facility in Lynnwood, Washington, which specializes in the manufacture of Leukine (sargramostim).

On the vaccine side, Sanofi Pasteur, the company’s vaccine arm, is undergoing a major investment phase, which includes a new dedicated dengue fever vaccine facility in Neuville, France, which is scheduled to produce its first batches in 2014. Two new, dedicated influenza vaccine-manufacturing facilities are in the start-up phase. Sanofi’s facility in Shenzhen, China is currently testing its production processes, and its facility in Ocoyoacac, Mexico was approved by Mexican regulatory authorities at the start of 2012 and began production in time for the Mexican influenza vaccination program in September 2012. In response to observations made by FDA during routine inspections conducted in 2012 at its facility in Toronto and Marcy l’Etoile, France, Sanofi Pasteur initiated a compliance program to addres quality issues that had been identified. 

. Roche is investing CHF 240 million ($257 million) at its facility in Penzberg, Germany to expand raw-material manufacturing for its Elecsy immunoassays, to be completed by the end of 2014, as well as to increase compounding, filling, and lyophilization capacity, which is planned for 2016. At its sites in Basel and Kaiseraugst, Switzerland Roche is investing approximately CHF 230 million ($246 million). Three projects were completed in 2012: a new pharmaceutical quality control and assurance building, expansion of cold-chain storage capacity, and a filling line upgrade for Herceptin (trastuzumab) subcutaneous formulation. The company is also expanding capacity to increase capacity for high-potency drugs. 

. GlaxoSmithKline (GSK) announced plans to invest more than £500 million ($798 million) in the UK across its manufacturing sites, which included selecting Ulverston in Cumbria as the location for the first new GSK manufacturing facility to be built in the UK in almost 40 years. The company also will invest in sites in Montrose and Irvine, Scotland. GSK’s announcement followed plans by the UK government to implement a “patent box” to encourage investment in R&D and related manufacturing in the UK by introducing a lower rate of corporation tax on profits generated from UK-owned intellectual property. Following a feasibility study conducted through 2011, GSK said it will locate a new £350 million ($559 million) biopharmaceutical manufacturing facility in Ulverston, Cumbria. Detailed planning and design of the new facility is underway with an anticipated start date for construction of 2014–2015, dependent on portfolio timing and obtaining necessary planning and related consents. Once construction starts, it is likely to take at least six years before the plant is fully operational.

. In 2012, Eli Lilly reported that it is investing EUR 330 million ($430 million) in a new biopharmaceuticals facility at its Kinsale campus in Cork, Ireland. 

Despite overall manufacturing rationalization in the pharmaceutical/biopharmaceutical industry, the large pharmaceutical companies continue to invest in biologic-based manufacturing. The author provides an update in recent investment activity among the pharmaceutical majors in biologic-drug substance, vaccine, and parenteral drug manufacturing.

Big Pharma's Manufacturing Investments in Biologics

Pharmaceutical product development activities are highly interconnected in nature. For parenteral drug products, the prefilled syringe is generally considered a challenging container closure system, particularly in the case of vaccines with complex product design requirements. The authors describe a holistic and integrated approach to focus on the linkage of the prefilled syringe with the four phases of product design, development, operation, and control. 

 Maciej Frolow/Getty ImagesThe product design phase involves integration of prefilled syringe as a crucial part of the target product profile (TPP) when considering the complex health policy and user requirements imposed by different global geographic regions and regulatory authorities. The product development phase focuses on the prefilled syringe as an integral part of the vaccine drug product. Particular attention needs to focus on multiple syringe component attributes such as silicone oil and tungsten that may create potential interactions with the diverse molecular properties of antigens. Emphasis of the product operation phase is to link prefilled syringes to the product performance per design requirements and supplier process capability and quality control. Lastly, processing and supplier quality control must be implemented within regulatory compliance framework to ensure delivery of quality vaccine product to patients. 

Vaccine product development in prefilled syringes—drivers and complexity

The goal of pharmaceutical product development is to design and establish the formulation composition and its manufacturing process to consistently deliver a drug product with all the appropriate quality attributes required for its intended efficacy and safety profile. A systematic approach according to the quality-by-design (QbD) concept is applied to achieve both the information and material deliverables during the product development lifecycle (1). Before work begins on the development of a new product, it is crucial to clearly define the quality attributes of the product with consideration of the end user’s needs in mind, commonly referred to as the target product profile (TPP) in the pharmaceutical industry.A TPP can be used to facilitate discussions with regulators regarding the anticipated label claims for a product (2). It can also be used for the private sector market, to help determine competition and associated pricing strategies (3, 4). Alternatively in the case of vaccines, a TPP can be used as a tool to engage and align with public health policy organization by working together to achieve the intended health impact (4, 5). 

The definition of TPP according to the target disease/health policy and user requirement drives the design of the dosage form with selection of the primary container-closure system as an integral aspect to meet the delivery requirement. Drug products packaged in prefilled syringes essentially remove the withdrawal step from another container, such as a vial or ampoule, prior to administration. As a result, prefilled syringes offer a more convenient alternative to vials for the preparation and administration of injectable drugs. This results in improved provider/patient convenience and compliance, as well as product differentiation when combined with auto-injector delivery devices.There are many drivers and good reasons to have vaccines in the prefilled syringe presentation. First and foremost vaccines are complex biological entities; therefore, they must be administered by injection due to the lack of significant advance in alternative delivery technologies. From the technical point of view, one can imagine it is absolutely crucial to reduce the risk of wrong dose and also better dose precision; therefore, one can see the advantages of prefilled syringe as compared to vials. From a practical point of view, vaccines are sometimes driven by public health outbreaks because many doses could be given in a crowded setting, allowing simple administration with limited manipulation. This provides for less probability of handling errors in a prefilled syringe presentation, which is preferred. Lastly, there is a different economic factor than for example biologics and is driven by key stakeholders in the public health sector. The minimal overfill in a prefilled syringe as opposed to vial can also provide an economic advantage, because costs per unit must be low. In summary, there are clear scientific, practical, and economical drivers for developing vaccines products in prefilled syringes. 

Vaccine product development is complex in many aspects. First, vaccine products are prophylactic as opposed to therapeutic, and more often than not, the target population is children and pregnant women, so the regulatory standard is high. Second, the product types are diverse such as recombinant proteins, glycoconjugates, virus-like particles, attenuated viruses, and live viruses. The dosing requirement is also challenging, in that doses are typically very small. Multiple antigens are often combined to reduce the number of injections, and adjuvants are added in some cases to boost the immune response. Lastly, the complex properties and interaction with prefilled syringe results in an acute challenge to vaccine manufacturers. 

Challenges of vaccine product development in prefilled syringes

The prefilled syringe is a complex system where the component characteristics often depend on the material of construction and processing conditions, which results in variability of the component attributes such as silicone oil level and distribution, as well as residual tungsten. Specifically, silicone oil and tungsten has been implicated in the induction of protein aggregation (6). It is also important to understand whether aggregation is a prerequisite for a cascade phenomenon leading to the visible particulate formation, which is not acceptable for a parenteral product. Not only does this create stability issues, proteinaceous aggregates presented in a highly arrayed structure of sub-visible size range, such as might be found in large non-denatured aggregate species, have been known to potentially interact with the immune system to enhance immune responses (7, 8). This interaction causes great concern and has been critically scrutinized by the regulatory bodies for therapeutic proteins. However, this should not be falsely perceived as a “hand waving favorable” immunogenicity risk assessment for vaccine products. It is categorically essential to ensure that product quality attributes including aggregate species, must be under strict control to demonstrate the intended immune response of the vaccine for the target disease and patient population. As stated earlier, the prophylactic nature of vaccine products impose a higher and more rigorous regulatory standard. 

 Figure 1: Silicone interaction and formation of visible particulates.In particular, the glass syringe barrel contains silicone oil for functionality purposes, which could migrate into the drug product solution during shelf-life storage. When protein interacts with silicone oil, there are potentially two phenomena that could lead to visible particulates formation. First, hydrophobic interaction could enhance protein aggregation as a prerequisite for visible particulates formation (6). On the other hand, the dispersed silicone oil droplets, although at low level, may be charged dependent on the formulation condition. The charge effect could lead to colloidal instability and flocculation of the silicone oil emulsion together with the protein, which has been reported in literature (9). The protein and silicone oil droplets are quite different in size microscopically, which can be characterized by a recently developed new technique of suspended microchannel resonator (SMR) (10). Silicone oil droplets are charged particles where surface properties have a large effect on emulsion stability. Both the concentration (i.e., quantity) and total surface area (i.e., number and size of oil droplets) are critical attributes to understand the protein silicone interaction phenomenon. Depending on the isoelectric point of the protein and pH of formulation, protein adsorption can change the surface charge properties, and therefore, kinetic instability of the silicone oil emulsion. As a result, the flocculation and coalescence of protein adsorbed silicone oil droplets can lead to the formation of visible particulates. This potential mechanism of action is illustrated in 

Figure 2: Charge heterogeneity of three glycoconjugates A, B, C and carrier protein as control measured by isoelectric focusing gel. These concepts are also highly relevant in the case of a complex vaccine product, for example, glycoconjugates. Effective immunization against encapsulated bacteria can be achieved by a type of vaccine based on oligosaccharide conjugated to a carrier protein, for example CRM197, a non-toxic mutant of diphtheria toxin (11). For a particular target disease, different serotypes with different polysaccharides would be needed to achieve majority coverage of the population. For example, serogroups A, B, C, W135, Y, and recently X account for the majority of meningococcal disease in humans (12). Bacterial polysaccharides are high molecular weight and mostly negative-charged carbohydrate molecules. Semi-synthetic glycoconjugates are complex molecules composed of protein and carbohydrate moieties linked together by chemical reactions. The conjugation reactions involve different chemistry and, therefore, different linking sites on the surface of the protein and along the saccharide chain. Moreover, the polysaccharides are high molecular weight carbohydrate moieties with some degree of polydispersion, which increases the heterogeneity of the resulting glycoconjugates. 

The polysaccharides are mostly negatively charged, and there is also significant charge heterogeneity of these glycoconjugate vaccines. 

 shows an example of the charge heterogeneity of three glycoconjugate vaccines as compared to the carrier protein measured by pI. The pI is a well-defined single band for the carrier protein but there is a significant dispersion of isoelectic point (pI) representing charge heterogeneity for the three different glycoconjugates. Each of these highly charge heterogeneous molecules are expected to interact with silicone in a different manner both individually in a monovalent and collectively in a trivalent formulation, which could lead to decrease colloidal stability of the low concentration silicone oil emulsion in a prefilled syringe. It is likely that visible particulates form when the silicone oil droplets associated with these glycoconjugates coalescence. 

At present, there is little reported in the literature on systematic and comprehensive characterization of silicone glycoconjugate interactions under a variety of formulation conditions to support the successful development of these types of important vaccine products. On the other hand, it is also important to consider the silicone variability associated with the prefilled syringe components, such as the intra- and inter-batch variability plus the migration during storage before product is filled. After the product is filled, product storage, handling, shipment, agitation, and excursion all contribute to potential significant intra- and inter-batch variability. 

In summary, a vaccine product such as glycoconjugate has additional complexity and challenges for prefilled syringe development when compared to therapeutic proteins. It is logical and reasonable to recognize that a large part of development effort should focus on understanding the components, and a standardized toolbox can help build such understanding and a better strategy to control the variability.  

Integrated prefilled syringe toolbox for vaccine development

Successful development of a vaccine product in prefilled syringes requires careful evaluation of the interaction between the formulation and primary packaging components. In support of each vaccine, a QbD approach will be used to design systematic product and process development studies and ensure a comprehensive data package. It is, therefore, crucial that a platform approach for these development efforts be implemented, in which there is detailed characterization and fundamental understanding of the various component attributes and associated variability within the platform prefilled syringe systems. In addition, the prefilled syringe system selected for the platform approach must also fit well with the existing manufacturing and production capability. To achieve a robust and flexible prefilled syringe platform, it is also beneficial to form a strategic alliance with a reliable supplier that has a good reputation of quality control. Processing and quality control compliance are vital for a successful vaccine product development and subsequent licensure. In summary, we propose a holistic and integrated approach to focus on the linkage of prefilled syringe in the following areas:

Target disease/health policy/user requirement

Manufacturing process (site and equipment) 

Regulatory and quality compliance requirement. 

 Figure 3: Integrated platform toolbox approach for vaccine product development in prefilled syringe.It is important to recognize there is significant interdependence between these areas, where an integrated platform toolbox is warranted and these six aspects are connected into the four phases of product design, development, operation, and control (

). The product design phase involves integration of the prefilled syringe as a crucial part when considering the complex health policy and user requirements imposed by different global geographic regions as well as private market competition. The product development phase focuses on the prefilled syringe as an integral part of the vaccine product due to the multiple contacts and potential interactions with the diverse molecular properties of antigens. In particular, particles characterization pertinent to silicone interactions is crucial because vaccines are prophylactic treatment to elicit an immune response before sick. 

The emphasis of product operation phase is to link the prefilled syringe to the product performance per design requirements and supplier process capability and quality control. As a result, a key focus will be on supplier collaboration to understand their process capability and improvements. Lastly, quality control of supplier with respect to incoming components, as well as manufacturing process with respect to both site and equipment, must be implemented with periodic review mechanism within the regulatory compliance framework to assure quality vaccine products delivered to the patients. 

Figure 4: Fishbone diagram of detail attributes as basis for risk assessment for selection and implementation of prefilled syringe toolbox.In addition, the six areas are divided into nine categories with detail attributes under each category based on technical considerations, prior project experience, and learning. These attributes can be summarized in a fishbone diagram, as shown in 

, to form the basis of risk assessment to select and implement different feasible options for a standard toolbox approach. In summary, the standard work stream, when combined with a toolbox approach, will drive down the development risk with information that allows plug and play, as well as supply of standardized component material that result in lower cost of goods. Furthermore, the platform approach can be improved and is expandable with accumulated product experience in the future after joint efforts with a formulation group and suppliers. 

The benefit of using a standard set of prefilled syringe components consistently for a variety of products is that a solid and robust data package can be established to support licensure application. These categories of information include:

Description, general information, material of construction, method of manufacture 

Suitability according to the FDA container closure guidance document which encompasses protection, compatibility, safety and performance 

The quality control aspects such as incoming specifications, industry standards, etc. 

Stability results, for example, will reveal some of the common stability issues and challenges depending on the product types, etc. 

Manufacturability when new products in the same set of prefilled syringe are being transferred to production facility, where deep process knowledge of the platform prefilled syringe systems have been accumulated with definitive enhanced production efficiency and quality control. 

This is important because it’s expected to provide all of this information in a submission as summarized in 

Table I: General submission information requirement.A holistic and integrated prefilled syringe platform toolbox approach will streamline development with higher probability of success even in the complex and challenging case of vaccine products. More importantly, such an approach is not only beneficial to technical challenges, but also provides economic benefits. We sincerely hope that the cost of innovation for a new prefilled syringe system is not so prohibitive that vaccine products cannot afford to achieve the intended health impact to society. 

We like to thank Anna Coslovi and Francesca Beccai for their preliminary results to characterize the charge heterogeneity of glycoconjugate vaccines. 

1. A-VAX Case Study, PDA, www.pda.org/Home-Page-Content/CMC-VWG-A-VAX.aspx, accessed Apr. 9, 2013. 

6. L.S. Jones, L. S., A. Kaufmann and C.R. Middaugh, 

12. L. Jodar, I.M. Feavers, D. Salisbury, D.M. Granoff, 

The authors describe a holistic and integrated approach to focus on the linkage of the prefilled syringe with the four phases of product design, development, operation, and control.

An Integrated Prefilled Syringe Platform Approach for Vaccine Development

 Ingram Publishing/Getty ImagesThis article presents a control strategy for maintaining a low level of particulates, thereby a low bioburden in cleanrooms, and for conducting environmental monitoring to demonstrate the ongoing state of control. Consideration will be given to current and proposed regulatory guidance and other cleanroom standards and monographs. In particular, the revised United States Pharmacopeia (USP) general chapter <1116> on microbiological control of cleanrooms and other controlled environments will be discussed. Changes to the ISO standard 14644 for cleanroom commissioning and qualification are also underway and will be addressed. Finally, some pointers for good practices in contamination control will be presented.

In 2012, mold contamination was found in supposedly sterile vials recalled from the marketplace after hundreds of people were infected with fungal meningitis; the compounding pharmacy responsible for manufacture of the product had clear warning signals. According to one newspaper report, their environmental monitoring program showed extensive contamination by mold and bacteria throughout the production area but they failed to implement effective corrective actions (1). 

In environmental control, it is essential to keep microbes out of a cleanroom environment. 

) has a doubling time of 20 minutes and one E. coli in a facility at 08:00 in the morning, means 8 by 09:00, and 16 million by 16:00 under ideal conditions. Therefore, to keep the environment clean, microbes must be excluded from the environment. 

To exclude microbes from the cleanroom, a control strategy is needed.  A control strategy is defined as “a planned set of controls, derived from … process understanding, that assures process performance and product quality” (2), or in other words, risk mitigation. Having identified the risk as one of contamination, the source being microbes that are carried on particulates, a whole host of measures is implemented to avoid contamination, starting with facility and equipment and primarily designed, wherever feasible, to separate the operator from exposed product.  

Where complete separation is not possible, another set of measures is implemented with the intent of containing as much as possible of the particulate contamination that is shed by humans. Low levels of particulates (i.e., viable and total counts—the latter includes viable and non-viable but we cannot distinguish) are designed into the facility by having a sealed building shell with flush fittings and no gaps or cracks.  High efficiency particulate air (HEPA) filters remove not less than 99.97% of particles greater than or equal to 0.3-micron diameter, which in the absence of humans in the area, gives a sterile environment with respect to microbes. Very high volumes of air are pushed through the clean area every hour ensuring many (usually more than 100) air changes per hour in the aseptic core. Cleaning and disinfection procedures are carefully designed and applied to remove any contamination that does find its way into the area, before it can multiply or settle. Operators undergo scrupulous gowning procedures to contain their bodies such that any particles shed are caught inside the cleanroom apparel and only removed outside the aseptic core. Operators are trained and qualified to perform operations in a cleanroom in a manner that does not disturb air currents and that minimizes shedding. Each of the individual measures that are part of the microbial control strategy is potentially crucial to the sterility of finished product especially where such product is aseptically filled (i.e., undergoes no terminal sterilization process in its final container). 

Breach of any measure can potentially result in contaminated product, although this is not by any means a one-to-one relationship. Nevertheless, it is a risk that needs to be monitored, which is where environmental monitoring enters the picture. Environmental monitoring is like an intelligence service. It is a risk-monitoring tool that allows us to collect useful data, analyze the data, and feedback the information into our environmental monitoring program. The Central Intelligence Agency (CIA) defines their mission as:

Collecting intelligence through … appropriate means

Correlating and evaluating intelligence related to (national security) and providing appropriate dissemination of such intelligence

Providing overall direction for and coordination of the collection of national intelligence

Creating special, multidisciplinary centers to address high priority issues. 

Shouldn’t every pharmaceutical company have a director of environmental monitoring responsible for assuring the existence of an appropriate cleanroom environment by collecting and evaluating data related to the state of control of the environment and providing overall direction (based on analysis of the data collected) regarding the continued collection (e.g., when special sampling and/or corrective actions might be needed)? Most importantly, such a director could create special, multidisciplinary task forces that can get into action fast when high priority issues are identified. 

 chapter <1116> (3) states that when operators are present in the aseptic processing operation, an expectation of zero contamination at all locations during every operation is not technically possible and is, therefore, unrealistic. This statement is problematic because, while scientifically correct, regulatory guidance (such as FDA’s 2004 Aseptic Processing Guide and Annex 1 of the EU GMPs) give “less than one colony forming unit (CFU)/m3 or per plate as the limit for an ISO 5 (Grade A/class 100) area, and FDA goes further, stating that ISO 5 should routinely yield zero counts. Indeed, it is true that in today’s aseptic processing environment it should be possible to routinely yield zero counts, which does not contradict the USP statement that zero at all locations during every operation is impossible.  However, in order not to mislead newcomers to a highly regulated environment, it is important to emphasize the regulatory limits as stated. This means that any count at all in an ISO 5 area would require a product impact assessment of some kind if there were production activity when the count was retrieved. 

The breakthrough in the USP chapter is the statement that “assessment of risk associated with aseptic operation must be assessed over a significant period of time and the contamination recovery metric based on actual data collected in the facility. The contamination rate can then be used to track the state of control of the facility/the ongoing performance and to allow early identification of trends and corrective actions and refinements to the overall control strategy” (3). 

The USP chapter states that once optimal conditions are established (not necessarily immediately after performance qualification [PQ] is finished—optimal conditions may come later when operators are more experienced and therefore more competent), the contamination recovery rate should become stable within a known range of variability. This concept is a basic tenet of quality assurance, and at that point, even small excursions from the range should be treated as alarm signals requiring close scrutiny and possible aggressive corrective actions to return to the previous state of control. The point being made is that when there is a change in the recovery rate, this is generally indicative of breach of one or more of the risk mitigation measures described above and, therefore, signals a potential breakdown of the control strategy.  It is a serious matter and must be addressed. 

The revised USP chapter requires a carefully documented investigation when recovery rates increase, description of corrective actions, and monitoring of effectiveness. A change in the contamination recovery rate might be a reason to convene a multidisciplinary task force that would be disbanded only after confirmation that the previous levels of recovery have been achieved once again. The focus should be on containment measures to ensure uniformly low recovery rates and a company should be vigilant in trying to reduce the recovery rates over time. 

Cautions when reading the revised chapter

The number 15 cfu is mentioned in a manner which, in the absence of an understanding of the issue, might be misinterpreted as suggesting product could be released if a single recovery of 15 cfu were found in an ISO 5 area, provided there was a uniformly low recovery rate for all preceding days in the same month. This author does not believe that is the USP’s intent—certainly not without exceedingly robust safeguards—and it will be interesting to hear regulators respond to the revised chapter.  In any case, the ultimate and sole responsibility for the safety of product released to the marketplace is that of the company producing it; therefore, there must be a documented impact assessment for any excursion outside the predetermined acceptance criteria. 

ISO 14644-1 and 2 guidelines address the design and operation of cleanrooms and are currently under revision. These guides only address particulate contamination of clean environments and do not discuss microbial contamination. They are often mistakenly quoted as addressing air velocities, changes, airflows, and pressures, which is not the case. The current revisions are intended to simplify testing to remove the need for evaluating the 95% upper confidence limit (UCL) at 2–9 locations, which is common practice. The target date for publication of the ISO documents is December 2013–January 2014. There is a proposal to delete the requirement for testing greater than or equal to 5 micron particles, which will take the EU Grades A and B out of the scope of the guide for classification purposes if the proposal passes into the final standard. Current issues still under discussion include:

The number of locations is still not class sensitive

How to deal with risk-based critical locations

Maintaining the quality of the environment in a cleanroom requires continual and relentless investment of energy in the facility and equipment maintenance and cleaning; in personnel competence (not just check the box “training and qualification”); in a garmenting program; in cleaning and disinfection; and in environmental control. Maintaining quality is about continual improvement. The environmental monitoring program provides the data to assess how well a risk management program is working. Response to risk must be rapid, and aggressive, and one must monitor the effectiveness when contamination recovery rates change. 

1. Sun L., “Compounding Pharmacy Linked to Meningitis Outbreak Knew of Mold, Bacteria Contamination,” 

3. USP, General Chapter <1116> Microbiological Control of Cleanrooms and Other Controlled Environments, 

A control strategy can maintain a low level of particulates, and thereby a low bioburden, in cleanrooms

Risk Mitigation and Microbial Control and Monitoring of Cleanrooms

Drug delivery is a multidisciplinary field. Researchers recently developed a drug-delivery system to mitigate some problems associated with jet-injection drug delivery, and also improved on the design and operation of microscale actuators as a possible drug-delivery method. 

Researchers at the Massachusetts Institute of Technology (MIT) recently developed a jet-injection drug-delivery system based on a custom high-stroke Lorentz-force motor to mitigate some of the problems typically associated with controlling drug delivery through jet injection. The researchers noted that needle-free drug delivery by jet injection typically is achieved by ejecting a liquid drug through a narrow orifice at high pressure, thereby creating a fine high-speed fluid jet that can readily penetrate skin and tissue. The researchers assert that such injection systems use force- and pressure-generating principles that cause injection in an uncontrolled manner with limited ability to regulate delivery volume and injection depth. To address these shortcomings, the researchers developed a controllable jet injection device, based on a custom high-stroke linear Lorentz-force motor that is feed-back controlled during the time-course of an injection (1).

The key part of the design is the Lorentz-force actuator, a magnet surrounded by a coil of wire that is attached to a piston inside a drug ampul, according to a May 24, 2012 MIT press release. When a current is applied, it interacts with the magnetic field to produce a force that pushes the piston forward, ejecting the drug at very high pressure and velocity out through the ampul’s nozzle. The speed of the coil and the velocity can be controlled by the amount of current applied. The resulting waveforms generally consist of two distinct phases: an initial high-pressure phase in which the device ejects drug at a high-enough velocity to breach the skin and reach the desired depth, then a lower-pressure phase where drug is delivered in a slower stream that can easily be absorbed by the surrounding tissue, according to the MIT press release. Through testing, the group found that various skin types may require different waveforms to deliver adequate volumes of drugs to the desired depth (1).

The team is also developing a version of the device for transdermal delivery of drugs ordinarily found in powdered form by programming the device to vibrate, turning powder into a fluidized form that can be delivered through the skin much like a liquid, according to MIT release. Such a powder-delivery vehicle may help mitigate the problems associated with needing a cold-chain system for delivering vaccines and other biopharmaceuticals.

In another development, researchers at the US Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California, Berkeley, have developed an new microscale actuator based on an vanadium dioxide that expands and contracts in response to a small temperature variation and which show promise for drug delivery among other applications. 

Piezoelectric actuators are the industry-standard for mechanical actuation on micro scales, but they are complicated to grow, need large voltages for small displacements, and typically involve toxic materials such as lead, according to a Dec. 2012 Berkeley Lab press release. The researchers’ device is simpler, uses nontoxic materials, and creates a displacement that is larger at a lower driving voltage. It works equally well in water, making it suitable for biological and microfluidic applications. The researchers envision using the microactuators as tiny pumps for drug delivery (2) .

1. A. Taberner, N.C. Hogan, and I.W. Hunter, 

, online, DOI/10.1016/j.medengphy.2011.12.010, Jan. 13, 2012.

Researchers recently developed a drug-delivery system to mitigate problems associated with jet-injection drug delivery, and also improved on the design and operation of microscale actuators as a possible drug-delivery method.