Containment Levels and Facility Design

By Ian Pearson

Containment, by definition, is the action of preventing a hostile force from expanding into other areas. In the biotech and pharmaceutical arenas, this takes on significant proportions, particularly in clean room or laboratory environments, extending to the very fundamentals of building design, validation and process management, and determining the success of pivotal research and development (R&D) projects.
Containment encompasses the procedural steps required to manage biological or chemical agents within a known and fixed parameter; this includes the measures employed to prevent both the release of these agents, which often present a hazard to the surrounding environment, and the ingress of contaminants into a sensitive and controlled process.

Figure 1: Containment level one (CL1).

These umbrella statements cover many different disciplines and doctrines, but essentially convey a simple message of 'safety first.' The main areas of application for containment facilities are:

• high containment laboratories for minor organisms
• contagious disease units
• potent drug manufacturing
• emergency outbreaks and bioterrorism attacks
• biotechnology R&D.

The practicalities of fulfilling a containment policy mean that biopharm and pharmaceutical management teams must complete meticulous and careful planning before developing or building holding envelopes around primary containment isolators or enclosures. It also requires the establishment of specific validation and performance criteria, and the assurance of intrinsically safe and flexible operations.
Depending on the level of containment required, this could materialize in the form of designated airline supply points within movement zones, ergonomically designed containment suites, dedicated fumigation ports and/or HVAC (heating, ventilating, air conditioning) HEPA (high efficiency particulate air) filtration. The determining factor is the level to which the facility must be adequately contained.
Levels of containment There are four primary facility containment levels for the control of biological agents. Each contains explicit stipulations that have a fundamental impact on the design and planning of new and existing clean room or laboratory environments.

Figure 2: Containment level two (CL2).

Containment level one (CL1). As the first level of containment is intended for organisms or processes that present a low risk to researchers and the surrounding environment, CL1 areas typically require few specially designed features beyond those that would normally be specified for a microbiology laboratory (Figure 1). For example, within CL1 containment zones, there is no requirement for biologically safe cabinets, meaning that work can be done on open worktops. Containment is essentially achieved through good practice and standard operating procedures (SOPs). Other CL1 specifications include washable walls covered with epoxy or emulsion paint, laminate-faced doors, epoxy, polyurethane and vinyl floors, recessed or teardrop light fittings and trespa, laminate, corian or epoxy lab worktops.

Key points for successful containment

Containment level two (CL2). Similar to CL1, this designation relates to agents and organisms that present a moderate risk but are usually not transmitted in air. In such areas, care should be taken to avoid the generation of splashes or aerosols, which can settle on work surfaces and become an ingestion hazard by contamination of the skin; hand washing should be actively encouraged. Similar to CL1 areas, there is no need for an airlock or mechanical ventilation, although inward airflow and safety cabinet ventilation are often recommended (Figure 2). Primary containment is achieved through biological safety cabinets, personal protective equipment and centrifuges with sealed rotors or safety cups. Once again, washable walls, laminated doors, epoxy, polyurethane and vinyl floors, and recessed lights are among the standard fitting requirements.
Unlike CL1, however, CL2 areas require hand washing sinks and decontamination facilities (autoclaves), SOPs and provisions for containing leakage from spillage and fumigants. Secondary containment is controlled and contained by the physical properties of the facility.

Figure 3: Containment level three (CL3).

Containment level three (CL3). CL3 containment generally applies to diagnostic, research and clinical laboratories, production facilities, or teaching laboratories handling agents such as Bacillus anthracis (anthrax), Brucella abortus (bovine brucellosis) and Brucella canis (canine brucellosis), that can be transmitted in air and often need only a low infectious dose to produce serious or life-threatening diseases. Consequently, the demands placed on CL3 facilities are far more stringent. For example, additional primary and secondary barriers are necessary to minimize the possibility of infectious organisms reaching the immediate laboratory and the outside environment (Figure 3). Specific measures include respiratory protection, HEPA filtration of exhausted laboratory air and strictly controlled access to laboratory areas. In addition, all cultures and regulated wastes should be decontaminated before disposal by an approved method, such as autoclaving, and strict fumigation procedures to decontaminate the laboratory should be initiated after each session.
The severity of the organisms contained in CL3 areas means laboratory suites must be partially isolated and include a separate airlock, shower room and changing facilities. Other requirements include airtight perimeter HEPA filtration and steriglazed washable walls.
Containment level four (CL4). The most rigorous containment classification is CL4, which relates to dangerous and exotic agents that pose a high individual risk. These agents include Lassa fever and Ebola, which have the potential for aerosol transmission and can produce serious or fatal diseases at low infectious doses. Often, with such agents, there is no treatment or vaccine available.

Figure 4: Containment level four (CL4).

CL4 encapsulates the maximum containment safeguards and follows the doctrine embracing unit isolation, negatively pressurized environments and Class III biological safety cabinets. CL4 stipulations are so extreme that there are fewer than ten facilities in the world that have so far been validated to this level. This level of containment is normally represented by an isolated unit within an existing facility that is functionally and, when necessary, structurally independent of other areas (Figure 4). CL4 emphasizes maximum containment of the infectious agent through complete sealing of the facility, with a negative pressure environment (270 Pa). Research scientists must be isolated from the pathogen either by containing individuals in positive pressure suits or containing the pathogen within a Class III biological safety cabinet; Class II biological safety cabinets can also be used with one-piece positive pressure personnel suites that are ventilated via HEPA-filtered circulatory unit packs.
All liquid wastes must be contained and treated before release into a dedicated decontamination holding/treatment vessel. Solid wastes have to be bagged and autoclaved at source before leaving the laboratory and then be incinerated.
Planning and design The planning and design strategy will be driven by the processes or products involved; the type, performance and specification of the equipment available; and the production throughputs and flexibility required. In principle, the process is generally straightforward and comprises concept design, client definition, performance design and detailed design - with each stage becoming increasingly rigorous; for example, working from concept layouts to full definition of the engineering solution.
Although the principle may be straightforward, the implementation can be complex, as people, product variables, regulatory and pharmaceutical legislation and operational and budget requirements are introduced.
Ultimately, there may be many systems, structural configurations, technologies and products involved, so it is important that all relevant personnel, from drug development, production, quality, logistics and maintenance departments, as well as process and construction engineering, are involved from the outset in the facility design. Achieving buy-in from all parties at an early stage will minimize overall costs and the time required before the facility is fully operational; for example, eliminating the need for reworking design layouts or the risk of incorrectly specified equipment.
The design contractors should also be involved as part of the overall team, helping to develop the initial user requirement specification (URS). This needs to define the processes, equipment, operations, capacities and environmental criteria required for the facility, reflecting appropriate standards and legislation.
Material and people flows In the URS, the design and construction engineers and contractors, plus facility managers and users need to review the flows around the containment areas and boundaries to assess the optimum layout for regulatory compliance, efficient operation and to minimize cross-contamination. It is generally easier to simplify the flows in new facilities, but can be more difficult in retrofit projects, where compromises may need to be made because of space or cost constraints. Here, special procedures or controls may have to be put in place to avoid cross-contamination where waste, people, raw materials and finished goods have to share common areas.
Process equipment Typically, this will include both the specific equipment required for handling and processing products in the laboratory, and the systems and structural equipment, such as decontamination showers, autoclaves and freezers. The URS, therefore, needs to define clearly which equipment is to be used, what options it will have and, if a new process, what downstream or associated areas still need to be developed. This will enable the design team to anticipate and accommodate equipment changes. The decision whether to make a piece of equipment a fixed part of the structure or skid mounted (common with biopharmaceutical equipment) also needs to be taken and reviewed early in a project, as it will have a significant impact on the layout, programme and costs.
It is also important to consider the impact that different containment levels will have on the complexity and possible cost of the project. For example, process systems may have to be adapted to accommodate the collection and treatment of effluent and vent gasses, whereas the need for high specification equipment seals, which are resistant or impermeable to aggressive liquids and gasses, may add considerably to start-up costs.
Buildings The URS should also include the materials and finish of the structure; for example, walls and ceilings may need to be impervious, with coved abutments, whereas floors are likely to require special coverings, doors to be constructed from steel, GRP (glass-reinforced plastic) or be gas-tight and, in CL3 and CL4 suites, windows are not a preferred option, particularly within CL4 environments.
Similarly, the facility may need to be designed to encompass positive and negative pressure systems, with changing and decontamination areas, whereas the required sampling and materials handling methodologies will also need to considered at an early stage - as they may have a considerable impact on the layout of the facility.
One final issue that is often overlooked at the primary design stage of high containment facilities is the impact of fumigation processes on the operability and downtime of each laboratory. It should, for example, be recognized that a typical fumigation procedure will take at least 12 h, with area preconditioning to a determined and stable temperature, fumigation, soak, degassing and purging.
Standards and validation Regardless of the level of containment, the laboratory should, in addition to the mandatory requirements that have to be addressed, be designed to meet standards or guidelines produced by bodies such as the Advisory Committee on Dangerous Pathogens (ACDP), Advisory Committee on Genetic Modification (ACGM), the Health and Safety Executive (HSE), the British Standards Institution (BSI) and relevant professional organizations. Additionally, process equipment must comply with the international and, possibly, national standards of the country in which the facility operates; for example, BS EN 1822 or Eurovent 4/4 for HEPA filters and BS EN 12469:2000 for biosafety cabinets will be required for CL1 and above, and COSHH (control of substances hazardous/harmful to health) 1999, ACDP Guidance (CL1–CL4) and HAZOP (hazard and operability) will be applicable at all levels.
Perhaps as importantly, consideration must be given both to performance criteria and validation processes for the laboratory, as it is essential that these are set up and operated effectively if the facility is to be truly accountable. Similarly, the issues of production flexibility and facility security and, in the event of a breach of security, remedial measures, are of utmost importance and should be given a high priority from the outset.
To achieve comprehensive validation and compliance, a facility must satisfy criteria relating to training, documentation, resource management and equipment validation - to name but a few. Managing the smooth progression of a validation study, and thereby avoiding costly setbacks, is an often-difficult feat to achieve and is frequently left to a single project manager to complete. However, as we have seen with containment classification, the influence of rudimentary considerations extends throughout the entire process, and it is, therefore, more effective, in terms of both results and cost control, to recruit a management team that can oversee the entire project and formulate an end-to-end solution.
Lessons learnt During the past few years, our consultants have been involved at all levels in many different containment projects. As a result, there are a few lessons that have been learnt and that should be considered for new projects.
From the outset, make use of the most appropriate tools to deliver optimum results quickly and within budget; if manual analysis is the most effective route then do not allow the project to be sidetracked by unnecessarily complex computer-based processes; conversely, processes such as CAD (computer-aided design) and CFD (computational fluid dynamics) can provide valuable early stage solutions to evaluate process and ventilation flows.
The impact of noise and vibration, both on working conditions and, potentially, on the long-term integrity of processes and the facility itself, are often overlooked and should be considered from the outset. Similarly, it is essential to be realistic about the ongoing operation of the containment facility, so that future expansion, modification or possible decontamination are all factored into the design phase - a small increase in investment during the initial construction stage may save a far larger sum in the future.
In terms of process engineering, the cost of treating liquid and solid effluents will invariably account for a significant proportion of the budget, particularly in CL3 and CL4 facilities. In addition, the challenge of interfacing information technology (IT) systems, particularly when existing legacy systems are involved and the connections between process equipment are from different manufacturers, can often take a disproportionate amount of time to resolve.
One final word of advice: no matter how simple or complex the project appears, success or failure depends on the degree of consideration given to the detail. It is often easier to focus on wider strategic or technical issues than the day-to-day planning, construction, operation and maintenance of a containment facility. Yet, it is the details that must be dealt with effectively and in a planned, structured manner. Achieve this successfully and you will meet your objectives - be they commercial, technical or marketing.

Figure 1: Containment level one (CL1).
Figure 2: Containment level two (CL2).
Key points for successful containment
Figure 3: Containment level three (CL3).
Figure 4: Containment level four (CL4).

Economy by design

By Gail Sofer,Guenter Jagschies

For biopharmaceutical chromatographic processes

One of the latest trends in biotechnology is the application of the concept of quality by design (QbD). QbD entails designing therapeutic products to meet patients' needs and then consistently producing them. Consistency is achieved by understanding the impact of starting materials, critical process parameters and product quality attributes, and controlling variability. In today's marketplace, concomitant with designing in quality, companies producing biotherapeutics need to consider economy. "Quality" does not have to mean "expensive" because economy and quality can be built into a process at the same time. It isn't logical to design a high-quality biotherapeutic that no one can afford.
Fortunately, today's technology enables reasonable production costs. For example, in the manufacture of monoclonal antibodies (mAbs), the cost for producing one gram is estimated to be $100–300 (€68.32–204.95), depending on scale of operation. Lower costs have been published based on a production estimate of 10 tons per annum. More specifically, the cost for Protein A resins used in a capture step for mAbs is normally 3% of the total costs when used for more than 30 batches. The cost for ion exchangers in antibody purification is in the order of $0.5–1.0 (€0.34–0.68) per gram.¹

Table 1 Six heuristics for economy by design.

More than 15 years of successful production in the biotechnology industry have resulted in downstream processing heuristics (i.e., rules-of-thumb) that point towards economical purification strategies (Table1). We will look at these individually. Address current and future costs in development
For companies with a history of producing biopharmaceuticals, the cost-effectiveness of various production tools is usually known. However, this is not the case for many start-ups, especially those in which the downstream process developer has little or no industrial experience. (For information on protein purification heuristics, see Protein Purification Handbook 18-1132-29 [GE Healthcare, Sweden.]) Cost-effectiveness is designed by understanding processing needs.
In some downstream processes, utilization of reusable chromatography media and filters may be necessary to obtain good process economy. This is particularly true for large-scale production as multiple batches per year are required to meet market needs. In one economic evaluation of reuse, media and validation costs were calculated. The greatest cost for establishment of reuse was related to its validation. Incremental annual savings were approximately $113 million (€77.18)/year for 10 lots, $15 million (€10.25)/year for 30 lots, but by 90 lots the savings were down to $0.7 million (€0.48) and the validation costs had risen significantly.²
For large volume processes, throughput requirements are often critical, especially to reduce initial volume and minimize contact with proteases. In this case, chromatography media with higher capacity and higher flow properties will enable a more economical process design. By reducing initial volume, buffer, including costly water-for-injection (WFI), consumption needs will be lowered. A reduction of buffer consumption by one-third has been estimated to reduce cost by 6%.¹
For other processes, disposables afford better economy as they can provide sanitary, ready-to-use processing equipment that reduces time to first-in-human (FIH) studies. Replacing chromatography and filtration media after each use may be less costly than trying to validate cleaning routines, particularly early in process development. Disposables are often the most economical solution for multiproduct facilities.
Storage of dilute buffers can also increase costs, but these can be minimized by using an automated in-line dilution of concentrated solutions. Clearly, there are many cost-saving measures that can be taken. During development, evaluate the process transfer capabilities of the technologies intended to decrease processing costs. If they cannot be transferred and new tools are needed, there could be a significantly negative impact on costs. Evaluate the costs for buffers and other processing agents; column re-use, repacking production columns; storage; cleaning; extractable studies for disposables; automation and personnel costs. For example, packed column storage costs include facility space, storage solutions, removal of storage solutions and their disposal, and testing for bioburden and column integrity after storage.
Design a process that is suitable for intended use and anticipated dose of product
An economical process should be capable of producing a quality product. Defining the level of acceptable quality is certainly a challenge in initial development. However, industry experience and regulatory expectations dictate some commonalities for purification of therapeutic proteins and other biological molecules; for example, DNA plasmid vaccines. Target values for removal of host cell proteins and DNA; cell culture media and processing agents should be dictated by product indication and its maximum predicted dose.³ Over-specifying product attributes can be very costly. Keep in mind that purer is not always better, and a more highly purified product may alter safety, efficacy and potency.
For products derived from mammalian cell substrates, human or animal sources such as plasma or transgenic animals, demonstration of viral clearance is required. If the cell substrate is well-characterized and has no infectious viral particles, all raw materials controlled, and manufacturing protocols performed in a suitable environment with proper adherence to GMPs, then striving to achieve an excessively high overall log reduction value may not be warranted.⁴ Extra steps inserted into a process for virus inactivation and removal are usually very costly. Consider an inactivation step. There may be product loss as a result of aggregation; a subsequent unit operation may be required to remove an inactivating agent; increased analytical methods may be needed; more WFI and other raw materials might be necessary; processing time extended, and so forth. Regulatory authorities are not likely to request removal of a step so this step becomes "grandfathered" into the process, often at an unrealistic cost.
Of course, it is essential to consider patient safety during process design. Regulatory holds are very expensive, perhaps delaying shareholders' first-to-market expectations. Part of an economical design strategy is to perform a risk assessment that addresses impurities, potential adventitious agents, patient population and dose.
Evaluate regulatory compliance levels for early clinical trials through license application
During the last few years, there has been an increased emphasis on safety for patients enrolled in clinical trials. This is especially true in Europe where legislation was passed requiring inspections and a greater level of GMP compliance for manufacturing investigational medicinal products.^5,6 However, in the US there has been a push to get more investigational products to patients in need of alternatives.⁷ One outcome has been a draft document on reduced compliance with cGMP for Phase 1 clinical studies.⁸ (Note, although the ruling for implementation of this approach was rescinded, the draft guidance still provides relevant information for phasing-in compliance.)
In a global economy, it is important to consider regulatory compliance expectations for all regions in which you want to market a biotherapeutic. Designing a process and product with quality attributes that only satisfy one region of the world could limit profitability by requiring expensive redevelopment, new clearance studies and even repeated clinical trials.
Minimize the number of processing steps
Every unit operation adds to production costs. By minimizing the number of intermediate steps (i.e., dilution, concentration and buffer exchange), these expenses can be reduced. The number of unit operations should be determined by a risk assessment and process capabilities. Consider current expectations for removal of impurities and control over adventitious agent contamination, and utilize analytical methods to design a process with as few steps as possible. Evaluate new analytical tools as they become available for process development; quantitative polymerase chain reaction (Q-PCR), for example, has enabled evaluation of DNA and virus removal at a reasonable cost during process development.

Table 2 Comparison of a 2-step process using MabSelect SuRe and Capto adhere with a 3-step process using MabSelect SuRe, Capto S and Capto Q.

The optimal time to evaluate how many purification steps will be needed for a new process is during its development, as decreasing the number of steps for a licensed product may present a regulatory challenge. For example, recent advances in mAb purification have enabled a reduction in the number of purification columns after a Protein A capture step. Instead of two columns following Protein A, one multimodal chromatography media can be used to achieve a two-step purification process for many monoclonal antibodies.⁹ This approach can provide savings in both operating costs and time, yet still remove impurities (Table 2). While removal of dimers (and other aggregates) and Protein A is the same for both processes shown in Table 2, the overall removal of host cell proteins is greater in the three-step process. However, the level of 7.5 ppm achieved with the two-step process might be sufficient. That judgment would be based on preclinical and clinical data and a risk assessment. Design a process that is robust and transferable
Designing robust purification operations is an iterative process requiring both empirical work and design of experiment (DoE).¹⁰ A robust process requires an understanding of what each step accomplishes; for example, level of reduction of each impurity and preservation of product integrity. A robust process is one in which reasonable control parameters are utilized. As more sophisticated analytical tools become available, especially those that enable feedback with process control, the application of PAT will become more common in downstream processing, which is likely to enhance process robustness.
A robust process offers an economic advantage by preventing product batch failures that might occur in a process that is defined too narrowly to account for unavoidable variability. Take, for example, buffer preparation and column qualification measurements. In the case of buffer preparation, a reasonable range for pH might be ±0.1 pH unit. This is an achievable value for large-scale manufacturing. For column qualification, an acceptable height equivalent to a theoretical plate (HETP) value might be much larger for a capture step than for a final polishing step in which high resolution is needed.
During the design of a robust process, cleaning, sanitization and storage should be considered. Contamination with microorganisms during purification poses a significant economic risk to the product and a safety risk for its ultimate customer; a human patient. The selection of chromatographic media and raw materials is paramount to providing a high-quality product and an economical process. Ensuring minimal carryover from column runs is essential, and selecting materials that can be cleaned and sanitized in place allows the use of sufficiently stringent conditions.
High-quality raw materials should always be used. An inferior, potentially contaminated, or impure raw material can only lead to costly batch failures and possible product adulteration, which can incur a loss in product sales, and even a loss of reputation if that product gets into the marketplace.
Equipment requirements should also be designed into a downstream process. Leachables from resins and filters, as well as potential extractables from polymers in contact with product must be evaluated. It is more economically effective to use materials for which some data and analytical methods exist to address these issues. While the cost of raw materials with supporting documentation may be higher than others, the cost for designing and performing the analysis is usually even greater. Also consider the sourcing of all raw materials, as the concerns related to potential BSE contamination must be addressed.^11,12 Variability in the strictness with which this issue is enforced varies worldwide and a risk assessment can be a cost-effective approach in demonstrating an insignificant risk.
As a process is being designed, think about how it will be transferred to pilot plant, full-scale production or contract manufacturer. If the process used in very early development requires that only one specific operator be present to make decisions, it is likely that scale changes and technology transfer will be problematic. A transferable process is one that has defined operating procedures and is understood as much as possible. Understanding a downstream process requires the use of orthogonal analytical methods that are qualified and reasonably robust. The application of in-process analytical methods that are transferable to manufacturing is clearly an advantage for smooth technology transfer.
Transferring a process to another company can be quite challenging; this includes transfer to a contract manufacturer. The product owner may not want to divulge too much confidential information, which often leaves the contract manufacturer with insufficient information to successfully run the process. The process development scientist should provide available protocols, descriptions of reagent quality and other necessary know-how. For example, packing large columns requires a certain level of skill. Transferring that skill often takes more time than anticipated and may lead to costly production delays. If the process developer has defined reasonable acceptance criteria for column packing, the technology transfer can be expedited. Other considerations include system wetted materials and configuration. When a different chromatography skid design is used, variation in sensitivity of in-line controls, such as those for UV and conductivity, may alter chromatographic performance.
Another technology transfer occurs when small-scale clearance studies are conducted at a contract testing laboratory; for example, for viral clearance. It is clear that scale changes will impact the technology transfer. The best approach is for the process developer to validate the scale-down at the site where the appropriate analytical methods are available. Otherwise, shipping of samples must be validated — further adding to the cost. For hazardous materials, such as virus or prion proteins, a mock spike (i.e., one without the actual agent being tested) should always be evaluated prior to the actual clearance study to determine the effect of volume, and any of the other components, on the spike material that may alter performance.
Communicate, plan for change and plan for validation
As process understanding increases during development, changes are made. This is particularly true for Phase 1 and 2 clinical studies. Once in Phase 3, making changes can be excessively costly — mostly because of the need for bridging studies and the ability to interpret clinical data when more than one product is being used for the same study. One of the greatest hurdles seems to be convincing upper management that process development costs and sufficiently allocated time are good business strategies. The costs for repeating a clinical study or having to implement a bridging study are huge compared with those for good process development that applies know-how based on industry experience with biopharmaceutical production.
Whenever changes are made during development, it is necessary to assess comparability, which requires extensive analysis, in-process and API retention samples, and working standards. For licenced products, change is inevitable as technology improves and expectations from regulatory bodies evolve. Changes in regulatory opinions often arise from the finding of a new risk factor or introduction of a technology that can enhance patient safety. New technologies may enhance quality, but they can also significantly add to production costs.
It is important to always evaluate new technologies, especially those that enhance patient safety, increase productivity and reduce overall cost while still maintaining production of a high-quality product. At the same time, consider the economics of making a change in conjunction with potential regulatory issues that might lead to delays in implementation. Clearly there is a balance between making a change in productivity and preventing release of a batch. In the US, comparability protocols provide a mechanism for implementing changes without excessive delays.¹³ ICH has also produced a guideline on comparability for biotech products,¹⁴ while in the EU there is a mechanism for making changes to a marketing authorization application (MAA).^15,16 The importance of the application of risk management when production changes are made is illustrated by the EMEA guideline that came into effect in 2005, which requires that when an application for a biotech product involves a new manufacturing process, a risk management plan must be submitted along with the application.¹⁷
Wasted time as a result of poor communications is costly and communications within an entire organization are essential. Manufacturing capabilities need to be understood by process development, just as manufacturing must understand what was done in clearance studies at small-scale to avoid moving the process away from the acceptance criteria of those studies.
Take advantage of the regulatory agencies' willingness to communicate for developing projects. Both the US and EU have mechanisms for asking questions. FDA encourages pre-IND meetings, and even provides a list of frequently asked questions that can be used to prepare for the meeting.¹⁸ In Europe, EMEA now offers support for small- and medium-sized enterprises.¹⁹ Although EMEA charges, the advice could provide a significantly greater cost-savings derived from taking the right strategies to produce your biotech product.
A validation master plan can prevent inadvertent omissions. The terminology used for qualification and validation is often inconsistent, even within one firm, and this can lead to costly delays. A PDA technical report on process validation of protein manufacturing and the ICH Q7A guideline on APIs provide a platform for consistent terminology that can be used worldwide to enable more rapid implementation of successful validation.^20,21
Conclusions
The heuristics provided here offer a strategy for economical development and licensure of high-quality products. Designing in quality can also mean designing in economy. Economic processes can be "smart" processes that remove impurities, build in safety margins for adventitious agent clearance and maintain product integrity in a minimum number of steps. Such processes should also be robust and capable of being transferred. Both robustness and transferability require that the process is understood. That understanding, in turn, requires the use of multiple, reliable analytical methods — some of which should be capable of being placed in, or at, line in manufacturing.
A patient safety risk translates into a business risk. Product may be lost and, significantly, an adverse event because of poor product quality is likely to harm the value of a company. In this highly regulated industry, an economical process has to be one acceptable to regulatory bodies charged with making decisions about whether the product should be introduced into humans and whether is it manufactured consistently. Understanding and accounting for worldwide regulatory expectations in process design can enhance marketability of a biotherapeutic.
Acknowledgments
The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F. Hoffman-LaRoche. A licence to use the PCR process for certain R&D activities accompanies the purchase of certain reagents from licensed suppliers.
Gail Sofer is director of regulatory compliance, Fast Trak life sciences, at GE Healthcare Bio-Sciences (NJ, USA). She is a cochair of the PDA advisory board and also serves on several other advisory boards. She is co-author of a recently published book on process chromatography.
Guenter Jagschies is in his 22nd year with GE Healthcare Life Sciences (former Amersham) and has held senior management positions in sales, marketing, training and consulting within the bioprocess segment of the company business supplying the industry with downstream processing solutions for development and manufacturing. His current role is senior director biopharma technology working globally with industrial collaborations and as business advisor for the life sciences R&D team. He is based in Uppsala (Sweden).
References
1. L. Hagel, G. Sofer and G. Jagschies, Handbook of Process Chromatography: Development, Manufacturing, Validation and Economics (Elsevier, The Netherlands, 2007).
2. A.S. Rathore and G. Sofer, "Life span studies for chromatography and filtration media", Process Validation in Manufacturing of Biopharmaceuticals (CRC Press/Taylor & Francis, Boca Raton, FL, USA, 2005) pp 169–204.
3. H. Simmerman and R.P. Donnelly, BioProcess Int., 3(6), 32–40 (2005).
4. ICH Q5A — Viral Safety Evaluation (1997). www.ich.org
5. European Commission, Volume 4, Annex 13, Manufacture of Investigational Medicinal Products (2003). ec.europa.eu
6. EMEA/CHMP/BWP/398498/2005-corr, Guideline on Virus Safety Evaluation of Biotechnological Medicinal Products, Draft (2006). www.emea.europa.eu
7. FDA Guidance for Industry, Investigators, and Reviewers, Exploratory IND Studies (January 2006). www.fda.gov
8. FDA Guidance for Industry, INDs: Approaches to Complying with CGMP During Phase 1, Draft (January 2006). www.fda.gov
9. GE Healthcare, Capto adhere data file 28–9078–88AA www6.gelifesciences.com
10. G. Sofer and M. Ahnfelt, BioPharm Int., February 2, Supplement (2007).
11. EMEA, Note for Guidance on TSEs EMEA/410/10-Rev. 2 (2002). www.emea.europa.eu
12. FDA, BSE/TSE Action Plan, Federal Register, 66(163), 44146–44149 (2001).
13. FDA, Guidance for Industry, Comparability protocols: chemistry, manufacturing, and controls information (2003). www.fda.gov
14. ICH Q5E — Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process (2004). www.fda.gov
15. EMEA Post-Authorisation Guidance for Users of the Centralised Procedure (2007). www.emea.europa.eu
16. The Rules Governing Medicinal Products in the European Union (2007). ec.europa.eu
17. EMEA/CHMP96268/2005 — EU risk management plan (EU-RMP) (2005). www.emea.europa.eu
18. Frequently Asked Questions on the Pre-Investigational New Drug (IND) Meeting (2005). www.fda.gov
19. Addressing the needs of small- and medium-sized enterprises (SMEs). www.emea.eu.int
20. PDA Technical Report 42 — Process Validation of Protein Manufacturing (2005). www.pda.org
21. ICH Q7A — Good Manufacturing Practices for Active Pharmaceutical Ingredients (2000). www.fda.gov

Table 1 Six heuristics for economy by design.
Table 2 Comparison of a 2-step process using MabSelect SuRe and Capto adhere with a 3-step process using MabSelect SuRe, Capto S and Capto Q.

In-Process Control Methods for the Manufacture of APIs

By Jose Zayas,Victor Sanchez,Michelle Talley

The authors propose a strategy for classifying and validating in-process testing methods.

In-process methods are key components of quality control in a chemical manufacturing plant. These methods ensure that a production reaction step conducted by trained operators within the entire validated process will produce a quality chemical entity in the expected yields. The presence of impurities and related compounds (derived from the reaction or secondary reactions) is a critical parameter that determines a synthetic material's quality.
Chemical processing differs from product manufacturing. For example, the manufacture of a finished product typically involves a molecular entity that is stable under normal conditions and can be stored for prolonged periods without losing its physical and chemical characteristics. Most chemical reactions, however, require very tight controls and close monitoring of their progress because any of several potential result paths may be followed if conditions are not monitored closely. Other factors such as temperature and pressure are critical parameters for the successful completion of the chemical conversion process.
Each chemical reaction is unique. Consider the combination of reactants and the resulting end products, for example. One must examine conditions such as temperature, light, heat, environment and the reaction vessel's surface. In addition, whether the reaction is chemical or biological is an important factor. Therefore, each process must be analysed separately and classified according to the International Conference on Harmonization (ICH) Q7A guidance. This task is important because a reaction step may generate an impurity that may be carried over to the active pharmaceutical ingredient (API), regardless of how far apart that process may be from the API.
ICH's Q7A guidance briefly mentions analytical methods validation and does not discuss the in-process control methods for each reaction. The guidance does indicate, however, that as the process gets closer to the manufacture of the key intermediate and the API, the current good manufacturing practices (cGMP) requirements become more demanding.
This article outlines a plan for classifying and validating in-process testing methods and is intended as a foundation for assessing the parameters and acceptance criteria needed for validation.
Matrix and Interferences
To characterize a reaction by means of an analytical method, it may be necessary to prepare a matrix, which functions similarly to a placebo for a finished product. A matrix is the combination of the reactants without the main component or precursor being converted. Because of the nature of some reactions, the combination of reagents may not be possible. However, adducts or complexes can be formed, which would not otherwise be formed in the presence of a component being converted into a product. The preparation of the matrix must be judged by the scientists working with the reaction process.
Classification of Methods
For monitoring purposes, analytical methods can be classified according to the manufacturing step in which they are applied. The document indicates that the GMP requirements become more stringent as the synthesis steps approach the API. Beginning with the introduction of the starting material into the process, manufacturing processes can be divided into three classes, which reflect the practices established in the ICH Q7A guidance:

• intermediates production (e.g., alkylation, hydrogenation)
• isolation and purification (e.g., washing, crystallization)
• physical processing and packaging (e.g., micronization).

Figure 1. The classes to which an API manufacturing process is divided, according to the ICH Q7A guidance document.

For the purposes of this article, intermediates production is subdivided into intermediates and key intermediates production. The classification of the methods (e.g., in-process controls and intermediate-release methods) is determined by how far the stage or step is removed from the API. Figure 1 represents the application of the ICH guidance to these classes. The ICH Q2B guidance enables chromatographic resolution to be used as an indicator of specificity for critical separations, which means that peak purity is not necessary. Furthermore, peak purity should not be a consideration because samples are submitted only to confirm the disappearance of the starting material and the formation of the desired adduct. No peak will be as pure as required when the analysis is a crude reaction mixture.
Solution and standard stability should be included as part of some of the studies. The length of the stability study is defined by the process requirements. Class 1. The Class 1 classification is exclusive to methods used for in-process control (and monitoring) of intermediate steps during an API manufacturing process. The classification pertains to reactions that are at least two steps from the processing of the key intermediate. Because the formation or source of impurities should be known and each impurity identified, it is possible that in some instances, the classification becomes Class 2 several steps before the key intermediate production.

Definitions

Class 2. Class 2 is exclusive to methods used for in-process control (and monitoring) of intermediate steps during an API manufacturing process. The classification pertains to those reactions that precede the formation of the key intermediate. Class 3. This classification includes methods used as intermediate-release methods when the product formed is an intermediate that will be used further after isolation or supplied as a starting material for another synthesis. This classification pertains to the key intermediate or isolated entity that will eventually be converted to an API.

Figure 2. An example of how processes can be classified.

An example of how processes would be classified is shown in Figure 2. Substance D is the key intermediate, one step before the API formation. Substance E represents the final API molecule before purification. Substance B, for example, could be subject to intermediate-release method testing if the material is isolated and the starting material is used in a parallel synthesis. In this case, the purification step does not involve any chemical conversion and the API is structurally identical to Substance E. The purification step can be recrystallization, micronization or any other physical manipulation of the active that does not involve a chemical conversion or change in chemical structure. In addition, a reaction sequence may involve the isolation of an intermediate that is several steps away from the formation of the key intermediate. The intermediate would be classified as an intermediate-release method or Class 3. Validation of Methods. The suitability of all methods used as in-process control methods and as intermediate-release methods should be verified and documented under actual conditions of use. Each category has a recommended suitability procedure defined. The degree of analytical validation performed must reflect the purpose and stage of the API production process. All analytical equipment must be qualified before it is used for method validation. Complete records must be maintained for any and all equipment modifications made to validate analytical methods.
The validation process may require that intermediates be characterized, isolated and used as reference markers for establishing the relative retention times. The preparation of a matrix or reaction mixture without the active may help establish unknown peaks and potential interferences. The matrix must be treated according to the procedures established for in-process control monitoring methods.

Table 1 Summary of the requirements, per classification, for chromatographic methods.

If an intermediate is not isolated, but reacted in situ to a later step, isolation may not be necessary for its characterization if it is an unstable entity. Should it be a stable molecule, however, its isolation and characterization may be necessary. Chromatographic Methods
Chromatographic methods are validated according to their classification, as previously discussed. The method validation protocol should include a discussion of the method's classification and the justification for the classification. The validation described for each classification is for quantitative chromatographic methods. Chromatographic identification and semiquantitative techniques such as thin-layer methods must be validated (described later in this article). These methods require the determination of accuracy in their semiquantitative level. Table 1 summarizes the requirements of each classification.
Class 1. Description. Class 1 methods are limit tests and must be validated accordingly. This validation should include a demonstration of the method's detection limit specificity and determination because these steps are far removed from the formation of the key intermediate and API. Therefore, one must be able to identify the peak of interest, properly resolved from the starting materials (reactants). The method should confirm the disappearance of the reactants or the formation of the adduct.
Requirements. The testing needed to monitor the intermediate steps during API manufacture falls into Class 1. According to ICH Q7A, this classification suits reaction steps far removed from the formation of the API and for which cGMP requirements are not as demanding.
As is typical for every method, system suitability is a requirement and usually includes injecting a marker for methods using response normalization for quantitation. Precision is not necessarily a requirement at this stage. Knowledge of the relative retention times of the reactants being monitored is essential, however.
The method's sensitivity should be established once the target entity monitored is quantified and the limit is established. The limits can be established by following ICH Q2B recommendations, which suggest a linearity experiment. At this stage, running a three-point linearity experiment around the target value can be used to estimate the detection limit. For example, consider a reaction in which the transformed or consumed reactant will be monitored until the area of the peak of its signal is 0.5% of the product being formed. The linearity experiment can be executed to include 0.25, 0.50 and 0.75% of the reactant. Linear regression results would provide the intercept's slope and standard error, and thus, the estimation of the detection limit for this classification. Alternatively, estimation solutions can be prepared. When a signal-to-noise ratio (3.3:1) is obtained for the solution, the figure becomes the detection limit for the method.
Class 2. Description. Class 2 method validation includes the limit-test requirements previously described (specificity, detection limit), the determination of the method quantitation limit and the demonstration of the method linearity covering the entire range of the method (i.e., reactants and adduct being formed to determine the specified limit). Thus, the detection limit and quantitation limit values should be calculated from the linearity data generated during the execution of the validation protocol. The testing required for monitoring the formation of the key intermediate falls under Class 2.
As with Class 1, a marker solution can be injected to establish the system-suitability criteria such as resolution and tailing factors. Again, precision may not be an issue because the criticality of this monitoring is the appearance or disappearance of a reactant or an adduct relative to each other.
The determination of the quantitation limit would be part of the limit-test requirement. In this case, because we are further into the ICH 7A-suggested criteria for cGMPs, it is recommended that the linearity experiment be conducted over its full range. This range would be from the quantitation limit to at least 125% of the concentration range monitored. If the target value lies far from the quantitation limit (e.g., the quantitation limit is 0.01% and the target concentration is 5%), then a three-point calibration should be run close to the quantitation limit. This step would be followed by a normal five-point linear regression from 50 to 125% of the target. It is useful to compare the precision between the curves and to compare the response factors obtained from both curves.
Solution and standard stability are essential components at this stage of the synthesis. The cGMP requirements mandate that these solutions be stable for the time of use. Any degradation could generate false data and miscalculations/misidentifications of unknowns. The length of stability will be defined by the process requirements.
Class 3. Description. In essence, Class 3 methods are finished-product (release) methods that control the final product of the manufacturing process. As such, the extent of validation for this type of method approximates the extent of validation for the finished product methods described in the US Pharmacopeia (USP). Class 3 methods validation includes linearity, accuracy, precision, specificity and intermediate precision. The ranges applied to each parameter are subject to internal procedure requirements. The rationale for the ranges used must be clearly explained in the methods validation protocol. The impurity method parameters will be applied when the reactant is the quantified chromatographic component.
Requirements. The testing required for the monitoring reactions for the formation of the final API molecule falls into Class 3. The following are the specific requirements for validating such methods:

• The standard must be fully characterized by scientifically recognized methods.
• USP standards should be used if they are available.

Semiquantitative thin-layer chromatography methods are in this category. This technique is a two-dimensional chromatographic procedure. As such, the system must be tested and the procedure must be clear.
The accuracy and linearity of the technique being qualified as a semiquantitative method must be verified. The reference standards must be prepared at concentrations ranging from a quantitation limit to at least 125% of the concentration targeted in the analysis. This information is a visual calibration for the method.
Conversely, specifications must be set for the response factor (Rf) of the spots of reference material and any other reference substances that are critical for the test. This process will establish the conditions for system suitability. Furthermore, the sensitivity of the method must be determined to enable visual calibration to be established and a reliable limit to be set for the test.
Along these lines, the composition of the mobile phase and the length of the plate run are critical to this test. The concentration established for the test must be such that the separation yields good resolution (R) between the closest eluting components of the mixture. The process also involves changes in chromatographic plates and analyst-to-analyst comparison. Therefore, robustness is also required.
A comparison of a unique Rf for the main substances monitored or quantified with the Rf of other major components of the reaction would yield the test's specificity. Once again, standard and solution stability are critical as controls for the methodology and for ensuring reliable results.
Nonchromatographic Instrument Methods
Among nonchromatographic methods, titration can be used for monitoring reaction sequences. Titration methods will require validation beyond equipment qualification.
Linearity and accuracy for the technique are important. Linearity is established by preparing three-point calibration curves. Regarding accuracy, the titration would require the determination of matrix contributions, if any, to the end point of the procedure. The preparation of spiked solutions, as well as the titration of blanks containing matrix elements, is required for validating the reliability of the technique. Thus, precision can be assessed from the experiments conducted under linearity and accuracy.
The reaction and its stoichiometric relationship can be presented to assess the specificity of the method. If a method is not specific, an explanation or the rationale for using a nonspecific method must be provided in the method validation protocol.
In-process Spectrophotometric Methods
With the exception of chromatographic methods, in-process spectrophotometric methods are those for which the end detection technique is spectrophotometric. When these methods are used for identification purposes, only sensitivity, standard and solution stability data are required. The calibration of each technique is required before analytical work can begin.
The validation of atomic absorption techniques should include linearity, accuracy, precision and specificity. Although specificity is inherent to the technique, the protocol and its report should indicate the analysis specificity of the particular method.

Table 2. Summary of the requirements for nonchromatographic methods.

Furthermore, the sensitivity (detection limit) and standard and solution stability should also be included. For system suitability, the %relative standard deviation (RSD) of a standard's replicate sampling measurements must usually be reported. At minimum, most systems include a calibration curve and a standard confirmation reading. Systems that do not report %RSD values and/or include a calibration curve must be addressed in the methods validation protocol and report. Table 2 summarizes the validation requirements for nonchromatographic methods. UV-vis spectrophotometry should include linearity, accuracy, precision, sensitivity (detection limit) and standard and solution stability. This technique is nonspecific. A calibration curve is an essential component for analyses such as the American Pharmaceutical Association's standard for absorption of light (colour) and requires the preparation of primary standards. If a calibration curve must cover a range of time (e.g., 7 days), the statement's validity must be established and documented.
Conclusion
Monitoring chemical processes for the formation of an API is the first step to ensuring quality in pharmaceutical manufacturing. Having reliable and reproducible methods will enable the production plant to guarantee the consistency of drugs batch after batch. Furthermore, it may simplify the characterization of such processes and their chemical profile.
Through the years, vast publications and general information have been presented to pharmaceutical industry specialists about the validation of analytical methods. Federal and international regulatory groups have published various guidelines to shed light on analytical method validation. No such emphasis has been given, or guidances described, however, the validation of in-process control methods. This article intends to establish a starting point for discussions about the validation of in-process methods.
Jose Zayas is a technical director at Zaycor Industries Corp., Puerto Rico.
Victor Sanchez is an analytical support supervisor at Schering-Plough, Spain.
Michelle Talley is a consultant at the Mintanciyan Consulting Group, USA.
References
1. ICH, ICH Quality Guidelines: Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients Q7A (ICH, Geneva, Switzerland, 2001).
2. US Food and Drug Administration, Guidance Document for Industry, "Analytical Procedures and Methods Validation," (FDA, Rockville, MD, 2000).
3. ICH, ICH Quality Guidelines Validation on Analytical Procedures: Methodology Q2B (ICH, Geneva, Switzerland, 1996).
4. General Chapter (1225) "Validation of Compendial Methods," USP 27–NF 22 (US Pharmacopeial Convention, Rockville, MD, 2003).
This article was first published in Pharm. Technol. 29(4), 154–162 (2005) and is reproduced with kind permission.

Figure 1. The classes to which an API manufacturing process is divided, according to the ICH Q7A guidance document.
Definitions
Figure 2. An example of how processes can be classified.
Table 1 Summary of the requirements, per classification, for chromatographic methods.
Table 2. Summary of the requirements for nonchromatographic methods.

Keep it simple

Validating a Facility Monitoring System

Figure 1. An FMS solution configured with alarm notification.

To fulfil regulatory requirements, a clean room must be within certain specifications and meet the required standards. Clean rooms must be regularly monitored to ensure constant awareness of environmental conditions, such as temperature, humidity, pressure and particle levels. Digital outputs for alarms must also be monitored to ensure that an alarm will be raised if there is a change in any of the parameters (Figure 1). Validating a facility monitoring system (FMS) can be challenging. In this column we will concentrate on a systematic, yet simple, approach to validation. The principles described can be used in most validation projects, but we will not mention all documents or aspects regarding a validation project. However, we will highlight areas that will simplify FMS validation. Let's start with what may be thought of as non-traditional thinking.
Non-traditional/practical approach
A company has a heating, ventilation and air conditioning (HVAC) system controlling and monitoring the environment in its facility, including non-good manufacturing practice (GMP) controlled areas. This HVAC solution consists of five different tailor-made systems, the oldest dating back to the late 1970s. The quality assurance (QA) department then decides that the system needs revalidating because the validation documentation is outdated or parts of the system are not validated. The company now faces a huge validation task.
If you recognize yourself in this scenario, you're not alone. This is a common situation often resulting in redesigning the entire system, which is time-consuming and costly. Is it possible to avoid an expensive and complicated validation of the entire HVAC system? Well let's review what the International Society for Pharmaceutical Engineering (ISPE) guidelines suggest on the topic:¹

• "While it is good practice to monitor the performance of equipment such as fans and coils and control components, it is not a regulatory requirement."
• "It is possible to monitor, record, and/or alarm with portable or other instrumentation which is not a part of the HVAC control system."
• "Critical parameters should be monitored, either through the HVAC control system, a process control automation, or by manual methods..."

This means the environmental monitoring can be separated from the HVAC system, and it is, therefore, possible to monitor and validate only the GMP-critical parameters. Furthermore, through the FMS solution you can monitor the performance of the HVAC system. Figure 1 shows how such a system could be configured
Even if you separate the environmental monitoring from the HVAC system you will have to validate the FMS solution, but the validation effort will be significantly less. How can you avoid over-validating or ending up in a bottleneck of requirements and tests?
Systematic validation approach
As discussed in a previous issue of Pharmaceutical Technology Europe, the practical approach is to reuse and update existing system validation documentation, thereby practising the principles of the O-model.² This is very convenient and practical — particularly regarding future system changes. Details regarding specific documents are explained here.
Validation plan. All validation activities must be well planned. The key elements of any validation projects should be documented in a validation plan (VPL). The VPL should be brief and clear including, but not be limited to, the following:

• Validation policy, including company and regulatory requirements.
• The organization structure of validation activities.
• Summary of FMS equipment subjected to the validation (overview of the system).
• Planning and scheduling, including prerequisites for approval of protocols.
• References to existing and future documents.

When more then one facility or FMS solution is being validated, it is appropriate to write a validation master plan (VMP) covering and controlling several VPLs addressing each specific system. Specifications. The most important document of any proposed system is the user requirement specification (URS). The URS is part of the required documentation to support the validation process, which is defined by FDA as: "Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes."³
The URS ensures that both the user and supplier understand what is required. The URS specifies what the system is supposed to do and the functional specification (FS) specifies how the system will do it. For example, one requirement in the URS could be that the system must generate three different alarm levels. The FS will then specify how this function is implemented. When the URS and FS are written you move on to the design of the system/design specification (DS). This specification should be a top-level document that clearly states what items are required, and how mechanical and software items are to be connected to fulfil the function specification requirements.
Implementation
FAT. The factory acceptance test (FAT) enables you to see how the system functions before delivering it to the site. The system is fully built using all system components and then tested to an approved set of protocols, which verifies that the system meets the acceptance criteria and system design.
After testing, the system is installed at site. If there are any test failures at this stage the supplier is obligated to fulfil the requirements specified in the URS. It is far simpler (and less expensive) to solve problems on the supplier's site than on the user's. When the FAT and the issues raised during the tests are resolved, a site acceptance test (SAT) can be performed.
IQ. The installation qualification (IQ) should include, but not be limited to, the following:⁴

• All items listed in the FS have been delivered and are of the correct type. IQ should secure that all support systems (user manuals, technical manuals, system diagrams etc.) are in place.
• All instruments have been calibrated and the calibration is still valid.
• All inputs/outputs of a data collection unit are operational and all switches, lights, transducers are functional.

Once it has been documented that the system has been supplied as detailed within the FS, operational qualification (OQ) can start.
OQ. This verifies that the system operates as stated within the URS and FS. There should be traceability from specifications to the OQ functionality test. OQ should include, but not be limited to, the following:⁴

• testing functions described in specifications
• testing the alarm limits of the calibrated sensors
• a specific part of the OQ should address 21 CFR Part 11⁵
• backup and recovery procedures should be tested
• procedures regarding operator training and preventative maintenance requirements should be in place.

The OQ should permit a formal release of the system. It is important to ensure that once the system has been validated it is maintained as such.
Baseline. Baseline could be defined as a snapshot of the hardware and software configuration. Baseline would normally be taken at key milestones, such as prior to:

• formal testing
• IQ
• OQ
• release
• after change.

Why is baseline essential? Imagine a company having 250 sensors connected to an FMS solution. Baseline will control and monitor all components connected to the system. Furthermore the baseline document will specify all system parameters, sensor tag names and alarm levels. Changes to the system are usually processed with a change control procedure. Together with the change control, a baseline snapshot of the hardware and software configuration will provide an overview of the system configuration and components.
Maintaining the validated state
When a system is validated and in operation, measures should be taken to ensure that it remains in a validated state. This maintenance not only involves the integrity of the hardware and software, but also the documentation. The maintenance of a validated system includes many activities, which the user is responsible for. These could include:

• Standard operation procedures: should address alarm limits for each area and required corrective actions if any of the areas show a deviation from expected results. They should also detail how the system is backed up and the frequency of this.
• Training: all operators and QA staff using the system should attend training in system functionality. All training activities must be documented.
• Service agreements: all service agreements should include parts of the system that you are unable to address yourself (program updates or patches). In some cases it should include calibration of equipment (such as particle counters etc).
• Operational change control: all changes proposed during the operational phase should be subject to formal change control processes and be followed by baseline management.
• Instrument calibration: calibration routines must be implemented. Calibration and maintenance of instrumentation should be performed to approved procedures and standards.

Conclusion
Any pharmaceutical company facing a validation of its HVAC system should consider the possibility of separating the GMP-critical environmental monitoring sensors from the system and focusing the main validation effort on those sensors. Key words in any validation project are 'thorough specifications', 'test traceable to specification' and importantly 'baseline'. By following these principles you are well on your way to having a validated FMS.
References
1. Pharmaceutical Engineering Guide for New and Renovated Facilities, Volume 2 Oral Solid Dosage Forms Baseline Guide, ISPE (3109 W. Dr. Martin Luther King, Jr. Blvd., Suite 250, Tampa, FL 33607, USA, 1998).
2. C. Stage, Pharm. Technol. Eur. 17(8), 13–15 (2005).
3. General Principles of Software Validation; Final Guidance for Industry and FDA Staff (1987) www.fda.gov/cdrh/comp/guidance/938.html
4. Final Version of Annex 15 to the EU Guide to Good Manufacturing Practice pharmacos.eudra.org/F2/eudralex/vol-4/pdfs-en/v4an15.pdf
5. Title 21 Code of Federal Regulations (21 CFR Part 11) Electronic Records; Electronic Signatures www.fda.gov/ora/compliance_ref/part11

Figure 1. An FMS solution configured with alarm notification.

A Compliance Perspective on Dissolution Method Validation for Immediate-Release Solid Oral Dosage Forms on Automated Instrumenta

 By David Fortunato
An automated dissolution method can be a powerful tool to test drug products at all phases of their development. With minimal automated method validation, this tool can be used early in the drug-evaluation process. And with additional validation efforts, an automated method can be extended to the testing of Phase IV stability batches. Validating an automated dissolution-test method requires an understanding of the potential effects from filtration parameters, system interference, carry-over, cleaning..

As the pace of product development accelerates, the approach to dissolution-method development must advance beyond a manual method and an assay. A natural progression of the method-development process must include the transfer of the manual method onto automated instrumentation.

Validating the automated method is the primary challenge when transferring from the manual method. A dissolution scientist must understand the potential effects from filtration, system interference, carry-over, cleaning parameters, and media replacement. Automated dissolution instrumentation can help generate good manufacturing practice (GMP) data only when validated testing parameters can negate these influences, so the dissolution scientist can be confident that results do not differ between the manual and the automated methods. In addition, all laboratory equipment used to support or generate GMP data about automated instrumentation must follow an instrument "chain of compliance." Proper documentation must exist that proves each piece of equipment has been properly qualified and calibrated for its intended use.

Product development life cycle


Tips for validating automated dissolution parameters
The level of automated dissolution-method validation depends upon a product's phase of development. For early-phase products, minimal validation is required to screen the initial batches. Typically, filtration parameters must be established first to ensure that no amount of active pharmaceutical ingredient (API) is lost with filtration. The initial filtration parameters could be established manually and then transferred to the automated instrument. Next, automated dissolution-profile testing is completed to screen several different dissolution media. Profile sampling could be performed at 10, 20, 30, 45, and 60 min and the results compared. Selecting the various dissolution media that are used in the evaluation depends upon the solubility and stability of the API in each media. This process allows a dissolution chemist to determine quickly the media having the potential to provide the most discriminating dissolution performance for the product. Once these initial parameters have been established, a dissolution chemist can use the automated instrumentation to quickly screen early formulations and help formulators direct their future formulation efforts.

As the life cycle of the product progresses, automated dissolution-method parameters must be validated if the generated data have the potential to be included in any type of GMP submission. At this phase of development, a dissolution chemist should have substantial experience performing both manual and automated dissolutions on a product. This experience can be useful to select automated dissolution method parameters, which should be able to generate results equivalent to those of manual dissolution tests.


Ensuring instrument qualification
Because the manual test is considered the "official" dissolution test, side-by-side dissolution-profile testing should be conducted manually and with automation. Results could be compared at 10, 20, 30, 45, 60, and infinity minutes. At the infinity time point, a final sample is taken after the dissolution has progressed with a stirring apparatus speed of 250 rpm for an additional 30 min after the Q time point. The chosen acceptance criteria for the comparison between the two methods should reflect and compensate for both nonvariable and highly variable drug products. The results generated at the earlier time points are the most significant because they have the highest potential for variation between the manual and automated methods. If comparable results are obtained between the two methods at the earlier time points, a dissolution chemist is more likely to be assured that accurate data are obtained at all time points of the automated dissolution-profile testing. At later time points, the percent drug released typically approaches 100%; therefore, not as much variation between the two tests would be expected at these later time points. Acceptance criteria must be established for results generated at the earlier time points (<85% dissolved) and the later time points (>85% dissolved) with tighter acceptance criteria established at the later time points.

Validation of automated method parameters

It is advantageous for a dissolution chemist to validate individual automated parameters even if comparable results are obtained between the automated and manual dissolution tests. This process provides additional information about the product and the automated procedure, which may be useful as the formulation evolves. In addition, by validating individual automated parameters, a dissolution chemist can demonstrate to the US Food and Drug Administration a high level of control and understanding of the automated procedures used to evaluate the performance of the drug product. The individual automated parameters to be validated should include filtration, system interference, carry-over, cleaning parameters, and media replacement for off-line sample collection. A dissolution chemist always must be aware that the dissolution of the product itself is validated, not a particular formula. With this fact in mind, it is beneficial to conduct the validation experiments after final formulas have been determined. If future formulations change drastically, experience and scientific judgment must be used to determine the necessity of revalidation of individual automation parameters.

Filtration. Although automated filtration parameters should be established first, the filtration procedures for manual dissolutions may not always transfer exactly to the automated instrument. Incompatible or inadequate automated filtration procedures are the most likely cause for different results between the manual and automated dissolution tests. A side-by-side comparison between a manual test and an automated test is the quickest way to evaluate compatibility between the two methods. Because results typically approach 100% drug released at later time points, samples taken at the earlier time points provide the best information. At minimum, it is advantageous to test two lots of drug product at the highest and lowest dosage strengths.

Experience in performing manual dissolutions should help determine the type of experiment to be conducted. The comparison can be performed two different ways. For highly variable batches, where the result relative standard deviation is >20% at the 10-min time point or >10% at later time points, USP recommends performing an automated dissolution and collecting manual samples simultaneously during the automated sampling. To ensure that the automated sampling probe is not affecting the hydrodynamics of the dissolution, however, a dissolution scientist should first perform the automated dissolution using only manual sampling. These results could be compared with the manual dissolution. For less variable batches, USP recommends comparing the results between separate manual and automated dissolutions tests. The acceptance criteria proposed under USP "<1092> Intermediate Precision guidelines state the difference should not exceed 10% with less than 85% dissolved and the difference should not exceed 5% for remaining time points above 85% dissolved (1, 2).

System interference. After acceptable filtration parameters have been established, a dissolution chemist should determine whether system interference is affecting the results generated with the automated instrumentation. This parameter is an important variable to investigate and hopefully eliminate as a potential source of difference between the manual and automated tests.

An example of system interference is any binding of the API to tubing or sampling needles when using sampling parameters described in the automated method. Once automated sampling occurs, the API often must travel through very long lines of tubing, thus generating the potential for system interference. The test may be performed by preparing a 20% API and a 100% placebo solution. The amount of API is measured with and without the automated system. A portion of the prepared API solution is placed into each of the six dissolution vessels. Sample aliquots should be withdrawn manually and filtered simultaneously as the automated system is sampling. The difference between the two responses should not exceed 2.0%. Because system interference is more likely to be observed with a 20% API solution, the information generated from this experiment is useful when analyzing results from dissolution-profile testing. Low levels of system interference from higher API concentrations may be less likely to be noticed. If system interference is not observed with a 20% API solution, a dissolution chemist is more likely to be assured that accurate data are obtained at earlier time points of automated dissolution profile testing. Conversely, if the results between the manual and the automated dissolution tests are not equivalent, especially at the earlier time points of profile testing, it may be determined that system interference is the cause of the nonequivalent automated results. Although only the results at Q time will pass or fail a batch, system interference is an important parameter to evaluate because it demonstrates to FDA a high level of control and understanding of the automated procedures used to evaluate the performance of the drug product.

Carry-over. If it has been determined that system interference is not a factor for the product, the elimination of carry-over must be validated for an automated dissolution testing system. Carry-over of an API may occur between sampling time points during dissolution-profile testing and between batches during multiple batch runs. To determine whether carry-over exists between sampling time points, perform a sampling sequence using solutions equivalent to 100% of the highest dosage form and a blank solution. The solutions must be sampled according to the already established automated filtration and sampling procedures. The sequence of the sampling should be as follows: 100% solution, blank, 100% solution.

The API in the blank solution should not exceed 1.0%, and the result for the second 100% sampled solution must be equivalent to 99.0–101.0% of the result of the first 100% sampled solution. If results exceed these acceptance criteria, increased sampling flush volumes, filter changes between time points, different filters, or any combination of these three parameters may need to be altered to obtain acceptable results.

Potential drug product carry-over between batches must be validated and eliminated if possible. This process allows the automated testing system to be used to its fullest potential so that multiple batches can be tested in a single run. The validation can be conducted by performing dissolution tests with the highest dosage strength batch followed immediately with a blank batch (no dosage forms). The API in the blank batch must not exceed an average of 1.0% for six vessels. The samples should be taken and compared at the infinity time point when results are expected to approach 100% API released. Cleaning parameters may need to be increased if results exceed this acceptance criterion.

Cleaning parameters. It is important to clean the automated dissolution-testing system between batches of a single run and between product changes. Clean the dissolution vessels, stirring shafts, sampling needles, and the entire length of all sampling lines. Potential problems may occur, especially between product changes, if a surfactant was used previously. Results from future batches may be inaccurately high if surfactant remains in the system from a previous run.

Adequate cleaning procedures must be validated to ensure no carry-over occurs between batches of a single run or after product changes. Validation of the cleaning parameters may be determined at the same time as the carry-over between batches experiments. If the carry-over results between batches exceed the acceptance criteria, increased cleaning parameters may solve the problem. The amount of dissolution media or hot water flushed through the lines at the end of a dissolution test may be set at the maximum allowable volume for that particular automated dissolution-testing system. In addition, the highest number of vessel washes and the volume of dissolution media or hot water used for the vessel washes may be set at the maximum allowable volume for that particular automated system. If even the most extensive cleaning parameters do not prevent an acceptable level of carry-over from an API, a dissolution chemist may decide that the dissolution procedure for this particular product is not "automatable."

Media replacement. A media-replacement process between time points for off-line sample collection should be validated with an automated dissolution-testing system. The media-replacement option corrects for sampling loss. This option allows a dissolution chemist to replace fresh media into each dissolution vessel after each sampled time point. The replacement media may be the primary dissolution media or a secondary media, typically used to affect the pH of the media already present in the dissolution vessel. The secondary media may be used for enteric-coated products that require a media pH change.

Automated off-line sampling collection differs greatly from manual sampling. Typically, larger sample volumes are removed for automated sampling, which has the potential to affect results for dissolutions with multiple time points. For each time point, a cumulative sample volume is removed. The total volume removed includes the flush volume, the tubing dead volume, the filter-deaeration volume, and the sample-collection volume. The flush volume is the volume of sample used to saturate the filter to prevent loss of the API on the filter. The tubing dead volume is the amount of sample that must fill the lines between the dissolution vessels and the sample-collection vials. The filter-deaeration volume is the amount of sample that is used to prepare the filter for filtration (used in certain automated dissolution testing systems). The sample-collection volume is the amount of sample that is collected for the off-line assay. A dissolution chemist must take into consideration the entire sample volume removed at each time point and decide whether an equivalent amount of fresh media is to be replaced into each vessel after each sampling time point. The large amounts of sample volumes removed and replaced may affect dissolution results. Potentially, large amounts of undissolved drug substance are removed for each sample, which may inaccurately lower the results of subsequent samples. Alternately, large amounts of replacement media may inaccurately dissolve the dosage form, which may affect the results of subsequent samples.

Validation is required to determine the necessity of media replacement. A dissolution chemist should perform dissolutions with and without media replacement and compare the results to manual dissolutions. The technique that produces results that more closely resemble manual results should be used in the automated dissolution test.

Instrument qualification and calibration

Equipment qualification. In addition to all of the validation work that must be completed for each product tested on the automated dissolution system, an instrument "chain of compliance" must be established and well documented for all primary, secondary, and tertiary instruments used to support GMP data generated by the automated system. Instrument qualifications and calibrations must be completed for all components on the entire automated system. These components may include the dissolution apparatus, any on-line ultraviolet or high-performance liquid chromatography instrumentation, and any ancillary equipment. The supporting equipment used to calibrate each component periodically on the automated system includes balances, weights, stopwatches, timers, thermometers, eccentricity meters, and vibration meters. Each piece of supporting equipment must maintain a documented and current calibration status. Although the company ultimately is responsible for GMP compliance when using automated instrumentation, the company may choose to follow qualification acceptance criteria established by the US Pharmacopeia, the instrument vendor, or their own company standard operating procedures (SOPs).

Initially, an installation qualification (IQ) and operation qualification (OQ) must be completed successfully and documented for each component of the automated dissolution-testing system. The customer should request from the vendor the test-script documentation that will be followed to complete initial qualifications. It is advantageous to have the compliance department review the documentation to be certain it fulfills the requirements for the instrument to be used in a GMP environment. If one chooses to have the vendor complete the IQ and OQ activities, one must be certain the company provides training documentation for their service technicians indicating that they are qualified to complete the qualification activities.

Preventive maintenance. The qualification practices do not end after the instrument is initially installed, qualified, and calibrated. Periodic preventive maintenance and calibration schedules must be established according to company SOPs. A qualified vendor is the best choice to perform the preventive-
maintenance activities for the automated instrument. These activities may include a periodic performance qualification of the instrument, which evaluates the overall performance and operation of the system. The preventive maintenance may also include the replacement of general components necessary for the continued smooth operation of the system. These components may include tubing, belts, sampling lines, lamps, and so forth. It is important to understand that the level of maintenance or repair performed on the instrument may necessitate a requalification, recalibration, or a change control. As stricter requirements are placed on the GMP environment, company SOPs should be reviewed to ensure that preventive maintenance activities do not push the instrument out of compliance.

Calibration checks. A periodic calibration schedule must be established for each component on the automated system. The schedule may include semiannual, quarterly, weekly, and daily activities designed to confirm the proper operation of the system. For the automated dissolution-testing system, the quarterly activities may include balance and temperature-probe calibrations. In addition, weekly or daily calibrations may include a quick balance check. For the dissolution apparatus, a semiannual performance calibration must be completed using USP calibrators. Trial dissolutions must be performed on disintegrating (e.g., prednisone) and nondis-integrating (e.g., salicylic acid) USP calibrators. Each dissolution test must pass the USP acceptance criteria established for the lot of drug tested. Semiannual physical testing must also pass USP acceptance criteria. The physical specifications include shaft and basket eccentricities, bath level, shaft verticality, and vessel and shaft centering. In addition, even though USP acceptance criteria have not yet been established for vibration, bath vibration is an important variable that should be measured periodically, especially if mechanical components have been changed on any of the components of the automated system. New mechanical components may increase bath vibration, which may increase dissolution results inaccurately. Daily physical specifications that must pass USP acceptance criteria include proper paddle–basket height, initial and final temperatures in all vessels, and shaft rotational speed (rpm).

Conclusion

Automated instrumentation for dissolution testing offers several advantages such as the ability to perform unattended testing and the ability to screen several batches with varying parameters. But, automated instrumentation also poses challenges for a dissolution chemist, including the need to have an overall understanding of the the automated system. Parameters such as filtration, system interference, carry-over, cleaning parameters, and media replacement are factors that must be addressed and validated to ensure equivalent results are obtained with manual and automated methods. The automated system can be used to generate GMP data only if all components on or supporting the system maintain a documented and current qualification and calibration status.

Acknowledgments

The author thanks Ron Mamajek, John Ballard, Ronnie McDowell, Dr. Michael Breslav, Dr. Daniel Kroon, Dr. Weiyong Li, and Dr. Brigitte Segmuller for their valuable suggestions.

References

1. "<1092> The Dissolution Procedure: Development and Validation," Pharmacopeial Forum, 30 (1), 351–363 (Jan.–Feb. 2004).

2. "<1092> The Dissolution Procedure: Development and Validation," Pharmacopeial Forum, 31 (5), 1463–1475 (Sep.–Oct. 2005).

David Fortunato is a scientist in the Chem Pharm division of Analytical Development, US, Johnson and Johnson Pharmaceutical Research and Development, LLC, Welsh and McKean Rds., Spring House, PA 19477, tel. 215.628.5098, fax 215.540.4684, dfortuna@PRDUS.JNJ.com [dfortuna@prdus.jnj.com]

Submitted: Feb. 22, 2006. Accepted: Apr. 7, 2006.

Keywords: Analytical testing, process automation, regulation validation and compliance, solid dosage forms

Tips for validating automated dissolution parameters
Ensuring instrument qualification