Development of pharmaceutical formulations incorporates a complexity of collaborative knowledge from ongoing partnering of scientists, integrated to ensure that quality attributes of a formulation lead to efficacy, purity and patient safety. A partnership of a formulator and a microbiologist is one example of the way knowledge can be shared and expressed to build robust formulation design. The old paradigm of final product testing (and the myth of ‘testing into quality’) is no longer appropriate. Recent industry and regulatory practice of ‘risk-based’ concepts and principles is intended to produce a ‘desired state’ [1, 2] of development and production of pharmaceuticals that have higher levels of consistency in meeting the objectives of efficacy, purity and safety. Integral to pharmaceutical development, Microbiology crosses multiple critical areas of concern in the development of robust formulations, such as material control, manufacturing design, formulation assessment, and packaging design.
A Strategy That Works
The terms of engagement of a pharmaceutical scientist cover a broad spectrum of specific scientific acumen, depending on the experience and education of the scientist. Many are dedicated to the field of pharmacy, chemistry or biology, and spend most of their time developing pharmaceutical substances and compounds, manufacturing materials, or clinically testing these materials. Their objective is to discover, develop and produce clinically effective, chemically pure and patient-safe drugs. The microbiologist in the pharmaceutical industry has a less visible engaging role, yet must be critically integrated to ensure biological purity and safety are met.
The mission of a microbiologist is to develop in the pharmaceutical organization a foundation for understanding of microbial origin, and parameters for proliferation and survival; to continuously improve/ embed the concepts for protection, exclusion, reduction, removal or destruction of contaminating microbiological entities. The strategy to accomplish this mission is based on two main roles in Microbiology: teaching and testing. Industry relates to teaching in terms of ‘training’, so I will use this term for now, even though the intent (of teaching) is to both educate and train by the most effective means available, with the beneficial result of learning of the basic concepts for effective microbiological control. Testing in microbiology is mainly verification, with the understanding that variability is inherent, distribution is often random, and high precision is limited because the living entities (cells) do not always follow the ‘rules’ in any given sample! Testing can also provide support for verifying success of approaches to microbiological control.
Relationships across multiple functional areas in the organization are a priority and are necessary to accomplish the strategy. The multiple areas include, for example, such functions as analytical chemistry, process chemistry, manufacturing, engineering, materials management, quality assurance, biotechnology and sanitation. Microbiologists have to have a very broad skill set, including education in biology, chemistry and physics, along with applied understanding of microbiology in humans, animals, plants and the environment. These provide competency in understanding microbiological control and in conveying the concepts (by training) to other scientists.
Testing or Training
Microbiological testing is necessary, mainly to provide a point-in-time understanding of the presence/absence of microbiological entities in a sample. Yet, it is mainly a complementary approach to adequate microbiology understanding and practices, which is more critical to actual control of microbiological quality of materials, people and the environment. Testing for microbiological quality is, at best, a verification process. As mentioned earlier, the inherent variability of results is due, in part, to the lack of sufficient and precise means to identify microbial cells and their life cycle phase. Even with current new technologies, indications of viability and identification of all possible cells in a sample is not simple. Also, heterogeneity of microbial populations in pharmaceutical environments/materials is a routine problem that has not been solved effectively by any statistical sampling regime.
Training in microbiological control can be accomplished in many forms, all of which can be both cost-effective and efficient. There are less formal, yet most commonly used, approaches in pharmaceutical organizations such as giving advice, consulting on projects, sharing knowledge in ad hoc conversations or meetings, and writing training recommendations for inclusion in other functional training curricula. There are also formal approaches that utilize the internal expertise from the Microbiology function, such as writing reports and proposals, and providing workshops or specific function training which can be accomplished face to face, or using virtual meetings or on-line (computer software) tools, both instructor-led or self-directed.
Key Areas of Learning
Microbiological Risk
Sharing knowledge is the key to organization learning. Understanding the factors which support microbiological survival and replication will enhance the ability of scientists to consider ways of manipulating those same factors to reduce microbiological risk.
There are some significant issues that re-direct scientists from dependence on final product testing.
Contamination by microbial entities is often heterogeneously
distributed, unlike counterparts of chemical contaminants. The size of a sample required for compendial testing (such as United States Pharmacopeia, European Pharmacopoeia, or Japanese Pharmacopeia) is quite small compared to the relative batch size – for example, 1 or 10 grams sample of an excipient taken from a subpopulation batch size of 75 kilograms which is part of a larger batch of 4 tons. This is such an insignificant sample [3] that it adds risk if there is inadequate understanding and a lack of knowledge of control by the supplier.
The most commonly performed microbiological tests are intended to recover microorganisms that are growing, or are culturable in the selected media of each test. Thus, many species of microorganisms are not, and cannot be recovered for counting or detection using the common methods we perform on most product samples.
To add to the concern of risk, there are different methods of final processing chosen to produce clean products that can be detrimental to the drug, as well as possibly hiding poor microbiological quality.
The following are some examples of processing and testing options that could minimize or reduce the ability to detect microbial problems.
- The use of terminal sterilization or aseptic filtration alone does not remove bacterial endotoxins (which can have pyrogenic impact on patients).
- If bioburden of pre-sterilized product is not kept under strict control and low levels, it can overwhelm some filtration processes.
- The sterility test is not a statistically valid test, and thus does not and cannot assure that every unit in a batch is ‘sterile’.
- When the sole expectation and release criteria for a nonsterile product is to meet‘compendial’ target microbiological levels (such as stated in a pharmacopeia) these levels could contain objectionable microorganism species.
Thus, we have a real practical reason for quality-by-design in Microbiology. Microbiological quality must be built into products. The patients depend on this.
Design Space and Critical Factors
The following is a microbiologist’s thinking about design space [4].
When manufacturing a parenteral product, the design space must be intended to result in ‘sterility’ and a ‘non-pyrogenic’ product. These are the criteria for release as well as for developing the criteria and methods of process control.
Sterile products which are not parenterals, such as ophthalmics, otics, wound topicals, and some inhalation products have design space variables intended to result in just ‘sterility’, unless proven circumstances of use or weakened human barriers indicate the need for an added ‘non-pyrogenic’ parameter.
Non-sterile products commonly are produced under stringent controls in the pharmaceutical industry, yet there is a wide range of acceptable microbial levels which are product and patient population dependent. The fact that they are non-sterile is problematic, and thus the design space must be intended to result in nothing less than ‘commercially sterile’ which really means “no objectionable microorganisms (by level or type) “. Note: The concept called ‘commercially sterile’ originates from the canned food industry, for thermally processed foods, and is defined as product which contains viable microorganisms that do not increase in numbers, do not cause physical product change, and are not pathogenic.” [5].
Taking a simple approach to formula design and developing a better understanding of microbiological control integrates learning in the key areas of formulation, process development, environmental control and personnel control, which in combination promise the development of a highly microbiologically pure product.
Formulation
Education leading to understanding the biological and physical factors that support microbial growth has significant impact. Knowledge about how microorganisms utilize water and nutrients provides a basis for understanding the impact of excipients and the physical nature of a formulation. For example, water is the diluent or menstruum in which microorganisms live, grow, multiply, and die, providing conditions which allow movement of nutrients into the cell and waste out of the cell. Nutrients, for any cells, are the building blocks for the cellular growth and survival; the most commonly known nutrients are proteins, carbohydrates, and lipids.
Developing a robust, microbiologically pure formulation, requires us to also understand much more about the excipients, the active ingredient, the diluent used, and the intended dosage form.
Excipients
Excipients are one of the two critical parts at the beginning of the supply chain.
As mentioned earlier, sampling and testing excipients can provide some or little useful information. So, complementary knowledge generation is critical for better understanding of risk.
A full quality assessment should be performed on each and every excipient in the formulation. Assessments can be performed by questionnaire, survey, audit, review, testing and any combination of these. Information that helps simplify microbiological decision-making about excipients includes:
- The origin of an excipient can indicate potential or lack of source of microbial contamination. Natural or biological origin is a common reason for inherent microbial levels. Soil or water origins will almost ensure that material carries viable or residual microbial contamination.
- The starting materials used to manufacture the excipients should be known since they also can be indicators of sources of microbial contaminants.
Excipients originate from all around the world, in many different forms, and with many different attributes. Learning about an excipient as if it were your own product is critical to design. The manufacturer should be subjected to a quality audit. The manufacturing process should be assessed by audit and review of process flow.
Starting with an understanding of factors that allow microbial growth, one can deduce factors that are likely to be inhibitory to microbial growth. Thus, the process should be assessed to identify what factors exist that can function as microbiological reduction steps. Some of those factors include heating, drying, adjusting pH and use of organic solvents.
An assessment of the supplier’s quality testing is critical, since Certificates of Analysis are only as good as the integrity of the laboratory testing that is performed. The supplier’s laboratory testing should be assessed to determine if appropriate sampling plans, type of samples and frequency will meet the intended purpose – to monitor control of microbiological purity. Testing methodology must be relevant, appropriate for the intended control measure, and for the type of product intended by the user (the formulator). The formulator and microbiologist must understand and relate the issues of the intended formulation to the excipient supplier to ensure they are on board with the same information.
Active Ingredients
The second critical part at the beginning of the supply chain is the Active Pharmaceutical Ingredient (API), or drug substance. Following similar thinking to that mentioned earlier to assess excipients, the same characteristics and process assessment should be performed for APIs.
Origin of the API and starting materials can help develop understanding of microbiological contamination potential. The process steps and controls for chemically-derived drug substances may be highly controlled for chemical purity, but may not be understood much with regards to microbiological purity – so this is where a microbiologist and a synthetic chemist can partner to teach each other the process and controls that are relevant and significant. Developing a biopharmaceutical drug substance has intended microbial reduction/removal steps, so process understanding is usually much higher. Since API manufacture is sometimes a unique set of steps that are not commonly used in the excipient industries, there can be some steps that are highly inhibitory to microbial survival such as: high temperatures for long time periods, use of solvents for extractions and separations, some chromatography which can be just as reductive as filtration, and drying is common when producing drug substance.
Assessment of sampling and quality testing are again important to ensure relevance and appropriateness to measuring purity.
An important consideration when assessing excipients and API is that, due to the global nature of our business, third party sourcing has become routine and accepted. Therefore, it is crucial to understand the supply chain in each and every case. Complexities in the supply chain can cause a minimization or loss of information and thus loss of control if the intention is to become more educated to design better. We must understand and fully know the supply chain for excipients and API to ensure we understand where weaknesses exist so that we can plan to implement better controls.
Diluent
The diluent is a key factor to supporting microbial growth, when the diluent is water. This is why the diluent is a critical part of designing a microbiologically robust product. Water is, of course, the most commonly used diluent. A formulation may or may not contain water. Water could be used in the manufacturing process and then removed at a later stage prior to finished drug preparation. For product manufacture, appropriately high purity water must be used. Its presence enhances opportunity for microbial growth, but a well-controlled high purity water system [6] can keep levels of microorganisms low. Water systems must be validated and monitored routinely for microbiological control. They can be simple systems or they can be highly complex systems.
There is no excuse for not producing the highest purity water necessary for intended dosage form. Engineers, chemists and microbiologists work together to ensure validation is thorough and production of pharmaceutical grade water is of the highest quality. If water is not the diluent used in a formulation, other substances that are used should be assessed for microbial purity and potential inhibition to microbial growth.
Dosage Form
Dosage form is another important factor in formulation design. The route of administration is key to patient use and efficacy. Knowledge about the route of administration is a way to assess the risk level that any product could have on a patient, relative to microbial contamination.
The numbers of different dosage forms, and their intended route of administration for the patient, are growing. From simple tablets and capsules, topical treatments, inhaled and intranasal administration, to subcutaneous, intramuscular and injection products, the dosage form carries inherent risks for protection of the patient from microbiological impact.
Once the intended route of administration is determined, we can identify the potential level of risk it carries.
Microbiological risk is low with doses containing low water activity doses (such as tablets, capsules, and caplets). The risk increases as the water activity increases and/or the route of administration passes the human body’s natural protective barriers. So, understandably, when an injection passes the barriers of the skin, the subcutaneous layers, and then the blood vessels, this brings with it the highest risk for contamination, and thus sterility is critical.
Better understanding of formulation and how to reduce origin of microbial contamination is key to designing a robust product. Manipulating factors that support microbial growth, use of clean excipients, and high purity diluents will lead to quality dosage forms.
This discussion has so far provided the key aspects of microbiology on formulation development. Other areas of control where microbiological design can also be highly productive in reducing risk are process development, environmental control and personnel.
Process Development and Control
Process control is often familiar territory for engineering and validation scientists, but it has issues that must be accounted for in microbiology. Processes can be very simple or complex, depending on the facility capabilities, the formulation, and the final dosage form. The use of Hazard Analysis Critical Control Points (HACCP), a quality tool [7], can be very valuable for assessment of excipient and API suppliers. For instance, a biopharmaceutical process with multiple bulk drug substance processing steps (e.g., cell banking, fermentation, separations, filtrations, etc.) has a very well planned process flow where HACCP can provide value. The critical control points (CCPs) can be easily identified, and the monitoring or quality tests are used to measure or verify microbiological purity at each critical control point. Once the CCPs are identified, a HACCP plan can be documented and followed. Measurement frequency, gathering monitoring data, trending, and follow-up then occur to show that the measurement and control system works, and if not adequate, the HACCP plan is reviewed for possible improvement. This is how process measurement and understanding can be continuously determined.
Another quality tool commonly used in process development is Failure Modes and Effects Analysis (FMEA). This is a prevention-based tool that helps identify potential failures, potential causes of failures, and actions to take to mitigate failures. The tool uses the concept of quantifying the severity, the frequency of occurrence and the ability to detect failure. As with the HACCP tool, this should be a collaborative team exercise with one of the results being a living document with high impact and ongoing value [8]. Used either in early stages of process design or later stages of process assessment, FMEA is another organized approach to developing a better understanding of microbiological risk.
Environment
Earlier, I discussed the impact of water as an ingredient to product microbiological risk. Other uses of water, such as for cleaning equipment and facilities, contribute to an environmental impact in a facility manufacturing pharmaceuticals. In addition, the use of compressed gases and filtration (to varying degrees) of air can contribute microbiological risk. Environmental microbiology is the study of microorganisms in natural or artificial environments [9]. Control in a manufacturing environment is both engineered and logistically developed based on an understanding of microbiology, because both natural and artificial origins of microbial contaminants contribute to the flora diversity in this type of environment. The conditions and ways that the environment is controlled must be measured to ensure microbiological contamination does not enter the manufacturing processes. Engineered design of processing equipment, rooms and flow of personnel can build robust control of microbial entry paths. Restrictions on types of process activities, approaches to cleaning of equipment, and levels of air pressure differentials between designated process areas contribute to better control practices. Measuring some of these controls for resulting microbiological levels can be just as variable and unpredictable as sampling excipients. So, the most logical and scientific approach is to design areas and activities to give appropriate microbial control and measure the consistency of the design(s) using physical parameters and measurement devices, while generating a broad snapshot view of the microbial levels using microbiological sampling methods and devices. A combination of the physical methods and microbiological methods can produce better assurance of the success of manufacturing control design.
Personnel
Another area of risk that definitely contributes to the manufacturing environment is the human factor. Personnel are going to have an effect on microbiological quality control under most conditions – from sampling to testing, and from formulation to packaging. Except where fully automated systems are present, human factors will impact quality. Logistics, as mentioned in the process control section, and gowning are keys to controlling cross-contamination by humans. Manufacture of sterile product depends on aseptic practices of operators. In non-sterile product manufacture, aseptic practices can be a significant way to reduce potential entry of microbial contaminants into a process or product. Appropriate gowning is paramount to control practices.
Training is one of the first steps to improvement for reduction of human error. Education supports the ability or capabilities of human operators in a laboratory or manufacturing environment. Ongoing training, by microbiologists, to keep operators and scientists involved in the continuous need for aseptic practices is necessary to imbed this critical learning. Consistent oversight, supervision and recurring qualification are crucial to an evaluation that should also be ongoing to ensure competency and skill sets of scientists, operators and technicians is up-to-date.
Conclusion
Recent industry and regulatory support of ‘risk-based’ concepts and principles are leading to a ‘desired state’ of industry development and production of pharmaceuticals that have a higher level of consistency in meeting purity, safety and efficacy than in prior history. Microbiologists’ role as trainers and expert partners in the pharmaceutical environment will continue to imbed a better understanding of how and where to implement microbiological control of processes, and microbiological purity can be built into products for all patient populations.
Acknowledgement
I would like to thank a colleague, Michael McBride, for his timely and valuable review.
References
1. ICH. Q8 Pharmaceutical Development. Harmonised Tripartite Guideline, Current Step 4 Version, 2005. www.ich.org
2. Pharmaceutical CGMPs for the 21st Century-A Risk-Based Approach Final Report, US Food and Drug Administration, September 2004
3. Singer, D.C., “Risky Business”, 2011. Quality Progress 44(7)
4. S. Langille, L. Ensor, and D. Hussong, ”Quality by Design for Pharmaceutical Microbiology,” Am. Pharm. Rev. 12 (6), 80-85 (2009)
5. Code of Federal Regulations. 1987. Part 113, Title 21. U.S. Govt. Printing Office, Washington, D.C.
6. Water for Pharmaceutical Purposes <1231>. 2012. U.S. Pharmacopeia 35, The United States Pharmacopeial Convention, Rockville, MD
7. The Quality Auditor’s HACCP Handbook. 2007. ASQ Food Drug and Cosmetic Division. Quality Press, Milwaukee, WI
8. Kubiak, T.M., and D.W. Benbow. The Certified Six Sigma Black Belt Handbook, 2nd ed. 2009. Quality Press, Milwaukee, WI
9. Manual of Environmental Microbiology, 2nd ed. 2002. ASM Press, Washington, D.C.
Author Biography
Donald C. Singer is a Global Lead Manager, R&D Microbiological Quality for GlaxoSmithKline, a member of the USP Microbiology Committee of Experts, and a Senior member of the American Society for Quality (ASQ). He is a Certified Specialist Microbiologist (NRCM), Certified Pharmaceutical GMP Professional (ASQ), an adjunct instructor in Biopharmaceutical Quality at University of Maryland Baltimore Campus, and a former Malcolm Baldrige Award Examiner.