Conventional pharmaceutical manufacturing is generally accomplished using batch processing with laboratory testing conducted on collected samples to ensure product and process quality. This approach has been successful in providing quality pharmaceuticals to the public. However, significant opportunities exist for improving the efficiency of manufacturing and quality assurance through the application of novel product and process development, process controls, and modern process analytical tools, including rapid microbiological methods.
Rapid microbiological methods may offer faster, easier, or better technologies and results when compared with conventional methods. They may be automated or miniaturized, offer increased throughput, and be more sensitive, accurate, precise, and reproducible. These methods have also been shown to detect slow-growers and/or viable but non-cultural microorganisms as compared with standard methods used today. Furthermore, rapid microbiological methods can provide significantly reduced time-to-result (e.g., hours instead of days) and for some technologies, results in real-time. For many in the industry, obtaining data within a single shift is considered as true, in-process microbiological testing, which can aid the laboratory in making immediate business decisions on in-process and finished product batches. The end result may allow for a reduction in product release time and rapid results during critical laboratory and/or manufacturing investigations.
Unfortunately, the pharmaceutical industry has been hesitant to introduce new technologies and innovative systems into the manufacturing sector. Our laboratories utilize centuries-old techniques based on the recovery and growth of microorganisms, using solid or liquid growth media. These methods are limited by slow microbial growth rates, unintended selectivity of cultures, and inherent variability of microorganisms in their response to culture methods. The reasons for our hesitancy include scientific and technical concerns (e.g., lack of clear guidance regarding the demonstration of equivalence to existing methods and validation of equipment, compatibility of new methods with product and/or processes, required sensitivity and specificity, degree of operator qualification and vendor support) and economic issues (e.g., cost per test compared with current method, initial capital/validation costs, and the return on investment). But most importantly, there has been an ever-growing uncertainty with respect to regulatory acceptance and understanding. We, as an industry, are apprehensive to implement rapid methods because of the potential for increased sensitivity in recovered microbial counts, concerns with changes in regulatory filing requirements, the perception that our existing regulatory system is rigid and unfavorable to the introduction of new technologies, and the impression that some inspectors may be unfamiliar with recent advances in microbiology.
Our hesitancy to broadly implement new pharmaceutical manufacturing technologies is undesirable from a public health perspective because the health of patients depends on the availability of safe, effective, and affordable medicines, and efficient pharmaceutical manufacturing is a critical part of an effective health care system. We will need to employ innovation, cutting-edge scientific and engineering knowledge, along with the best principles of quality management to respond to the challenges of new discoveries (e.g., novel drugs and nanotechnology) and ways of doing business (e.g., individualized therapy, genetically tailored treatment). At the same time, regulatory policies must also rise to this challenge. Fortunately, the U.S. Food and Drug Administration (FDA) has recognized the need to free our industry from its hesitant perspective and has recently introduced regulatory initiatives that promote the use of innovative process analytical tools, including rapid microbiological methods.
The first initiative is Pharmaceutical cGMP’s for the 21st Century: A Risk-Based Approach. This initiative is designed to use an integrated systems approach to regulating pharmaceutical product quality, and is based on science and engineering principles for assessing and mitigating risks related to poor product and process quality. We are encouraged to use the latest scientific advances in pharmaceutical manufacturing and technology to achieve the FDA’s vision of the future desired state of pharmaceutical manufacturing:
a) Product quality and performance are ensured through the design of effective and efficient manufacturing processes
b) Product and process specifications are based on a mechanistic understanding of how formulation and process factors affect product performance
c) Continuous real-time quality assurance
d) Relevant regulatory policies and procedures are tailored to accommodate the most current level of scientific knowledge
e) Risk-based regulatory approaches recognize the level of scientific understanding of how formulation and manufacturing process factors affect product quality and performance and the capability of process control strategies to prevent or mitigate the risk of producing a poor quality product
The second initiative, Process Analytical Technology (PAT), is intended to facilitate progress to the desired future state of pharmaceutical manufacturing. PAT is a system for designing, analyzing, and controlling manufacturing through timely measurements of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality and improving manufacturing efficiencies. This initiative operates under the principle that quality cannot be tested into products; rather, it should be built-in or should be by design. Companies that introduce PAT into their routine operations may realize reduced production cycle times by using on-, in-, and/or at-line measurements and controls, an elimination of rejects, scrap, and re-processing, the use of real-time release, and an increase in automation that improves operator safety and the reduction of human errors.
The FDA understands that to enable successful implementation of PAT, flexibility, coordination, and communication with manufacturers is critical. The recommendations provided in this initiative are intended to alleviate the fear of delay in approval as a result of introducing new manufacturing technologies. PAT may be implemented through reduced reporting strategies, the use of comparability protocols, and through inspections by the PAT team or PAT-certified inspector.
The third initiative, Sterile Drug Products Produced by Aseptic Processing – cGMP, updates the 1987 guidance primarily with respect to personnel qualification, cleanroom design, process design, quality control, environmental monitoring, and review of production records. This guidance document recommends the use of rapid genotypic methods for microbial identification, as these methods have been shown to be more accurate and precise than biochemical and phenotypic techniques. Furthermore, the document states that other suitable microbiological tests (e.g., rapid methods) can be considered for environmental monitoring, in-process control testing, and finished product release testing after it has been demonstrated that these new methods are equivalent or better than conventional methods (e.g., USP).
The opportunities for implementing rapid microbiological methods in the pharmaceutical industry are comprehensive. Applications include, but are not limited to, raw material and component testing,
in-process and pre-sterilization/filtration bioburden, purified/process water testing, environmental monitoring (e.g., surface, air, compressed gases, personnel), bacterial endotoxin testing, microbial limits, antimicrobial effectiveness testing, biological indicator survival assessments, sterility testing, media fill failures, and contamination investigations.
Current rapid method technologies can detect the presence of diverse types of microorganisms or a specific microbial species, enumerate the number of microorganisms present in a sample, and can identify microbial cultures to the genus, species, and sub-species levels. The manner in which microorganisms are detected, quantified, or identified will be dependent on the specific technology and instrumentation employed. For example, growth-based technologies rely on the measurement of biochemical or physiological parameters that reflect the growth of microorganisms. Viability-based systems use viability stains and/or cellular markers for the detection and quantification of microorganisms with no cell growth required. Artifactbased technologies analyze cellular components or use probes for specific target sites. Finally, nucleic acid-based technologies may utilize PCR-DNA amplification, 16S rRNA typing, or gene sequencing techniques. The next few paragraphs will describe some of the currently available rapid method systems and the science behind their technologies.
Impedance microbiology is based on the premise that microbial growth results in the breakdown of larger, relatively uncharged molecules into smaller, highly charged molecules (e.g., proteins into amino acids, fats into fatty acids, and polysaccharides/sugars into lactic acid). Growth may be detected by monitoring the movement of ions between electrodes (conductance), or the storage of charge at an electrode surface (capacitance). Impedance systems can detect changes in measurable electrical threshold in liquid media (during microbial growth) when microorganisms proliferate in containers that include electrodes. Growth may, therefore, be detected prior to the liquid media showing any signs of turbidity.
Microorganisms, when grown in liquid culture, also produce carbon dioxide. In a closed container, microbial growth may be detected by monitoring changes in the amount of carbon dioxide present. One system that is commercially available allows carbon dioxide to diffuse into a liquid emulsion sensor and the resulting color change alerts the user that microbial growth has been detected.
Bioluminescence is the generation of light by a biological process and is most commonly observed in the tails of the American firefly Photinus pyralis. In the presence of D-luciferin and luciferase, bioluminescence occurs when Adenosine Triphosphate (ATP) is catalyzed into Adenosine Monophosphate (AMP). One of the reaction byproducts is light (in the form of photons), which can be detected and measured using a luminometer. Because all living cells store energy in the form of ATP, cellular ATP can be used as a measure of microorganism viability. Current bioluminescent technologies use ATP-releasing agents to extract cellular ATP from microorganisms that may be present in a sample, and following the addition of bioluminescent reagents, can detect the amount of light emitted from the sample. Depending on the technology used, the amount of light emitted can indicate the presence of microorganisms, or may be correlated with actual cell counts.
A number of identification technologies measure the ability of microorganisms to utilize biochemical and carbohydrate substrates dehydrated in a microtiter plate format. A number of systems monitor changes in kinetic reactions or turbidity, the latter indicating microbial growth. One technology includes tetrazolium violet dye in the same wells that contain a dehydrated carbon source and if a microorganism utilizes that carbon source, the well will turn purple. The resulting data (normally in the form of positive and negative responses) are compared with an internal database or reference library for that specific system and a microbial identification is provided.
Viability-based technologies can differentiate living cells from dead cells and can target specific microorganisms using nucleic acid, enzymatic, or monoclonal antibody probes. In many cases, direct labeling of single cells is possible with no cell growth required. Furthermore,
spores, stressed cells, fastidious organisms, and viable, but non-culturable organisms can be detected and/or enumerated. Because viability labeling of microorganisms can occur within hours, near realtime results may be attained.
One type of viability-based technology utilizes flow cytometry. Cells are labeled with a viability marker and passed through a flow chamber, one cell at a time. Fluorescence and light scatter signals are detected, and individual cells are counted as they pass through a laser beam. The sample size required for flow cytometry systems are usually low (e.g., less than 1 mL). Because this technology can use fluorescent dyes and microbe-specific probes (e.g., antibodies or rRNA/PNA), simultaneous enumeration and detection of target organisms is possible.
Another viability-based technology, called solid-phase cytometry, uses a different type of labeling and detection technique. Samples are passed through a filter, and the filter is stained with a non-fluorescent viability substrate. Within the cytoplasm of metabolically active cells, the substrate is enzymatically cleaved to liberate a free fluorochrome. It should be noted that only viable cells with intact membranes have the ability to perform this cleavage and retain the fluorescent label. The entire membrane surface is subsequently laserscanned in a few minutes and labeled microorganisms are quantified. The system can also use antibodies and nucleic-acid probes to detect and quantify specific microorganisms.
Viability-based technologies measure cell viability, and do not rely on an organism’s ability to grow on media. For this reason, microbial counts using these technologies may be higher than those obtained using conventional, growth-based enumeration methods. This may be further enhanced for environmentally-stressed organisms or for cells exposed to sanitizing agents or preservatives.
Artifact-based technologies detect specific cellular components, such as antibodies or antigens when using an enzyme-linked immunosorbent assay (ELISA) or endotoxin by means of a Limulus Amebocyte Lysate (LAL) assay. Another cellular componentdependent technology relies on the analysis of fatty acid content to identify microorganisms. The cellular membrane contains lipid biopolymers, and the fatty acid profiles are different for distinct microorganisms. Fatty acids that are extracted from a microbial culture may be separated using gas chromatography, and the resulting peaks can be compared with an internal library.
Matrix-assisted laser desorption ionization, time-of-flight (MALDITOF) mass spectrometry is another artifact-based technology that has recently been introduced. Different bacteria, when exposed to an energy source, generate a variety of charged molecular weight patterns or spectra. These patterns are based on the macromolecules normally expressed on the surface of a particular bacterial species. Intact cells from a primary culture are smeared across a stainless steel target plate and allowed to co-crystallize with a UV-absorbing matrix. After drying, the target is placed into a mass spectrometer and exposed to a nitrogen laser. The matrix absorbs energy from the laser, and macromolecules from the surface of the microorganisms are desorbed, ionized, and mass analyzed. The resulting mass fingerprints
are compared with an internal database and an identification provided.
Nucleic acid-based technologies utilize DNA and RNA probes, as well as polymerase chain reaction (PCR) to aid in the rapid detection and identification of microorganisms. One technology extracts bacterial DNA and uses a restriction enzyme to cut the DNA into fragments. The fragments are then separated according to size by gel electrophoresis and immobilized on a nylon membrane (this is commonly referred to as a Southern Blot technique). The double-stranded DNA is denatured to single-stranded DNA, and the membrane is subsequently hybridized with a DNA probe (derived from the E. coli rRNA operon). Finally, an antibody-enzyme conjugate is bound to the probe and a chemiluminescent agent is added. Light emitted by the fragments is captured, and the image pattern is compared with patterns stored in the system database. If the pattern is recognized, a bacterial identification is provided.
Another nucleic acid-based technology identified microorganisms by sequencing the first 500 base-pairs of 16S rDNA. Extracted DNA is amplified using PCR, reverse and forward cycle sequencing is performed, and the extension products are analyzed by electrophoresis.
The resulting sequence is then compared with 16S rDNA sequences in the system database.
When a company is ready to explore the use of rapid microbiological methods, it is necessary that a desired technology be matched with its intended use and application. It is not uncommon to find a company that has purchased a system and spent considerable time, resources, and expense in validating the equipment and software only to find, at a later date, that the technology is incompatible with the process and/or product being evaluated, or that the sensitivity and/or specificity of the system is not what was originally anticipated. Once a company has completed its due diligence in identifying an appropriate technology, the task of preparing a meaningful validation plan is the next important step. A successful validation plan will include a GxP and 21 CFR Part 11 assessment, a complete User Requirements document, Functional and Design Specifications, and a Requirements Traceability Matrix. Appropriate training in the technology and instrumentation is required prior to initiating validation activities, and formal procedures should have been written, approved, and in place. Accuracy, precision, specificity, linearity, range, and limits of detection and quantification are evaluated during the operational and performance qualification stages of the validation plan, as well as equivalency studies (e.g., compared with the current or conventional method) and computer system validations. Guidance on how to conduct validation studies for rapid microbiological methods may be found in PDA Technical Report No. 33, Evaluation, Validation and Implementation of New Microbiological Testing Methods, in the proposed USP chapter <1223>, Validation of Alternative Microbiological Methods, and in the proposed EP chapter 5.1.6, Alternative Methods for Control of Microbiological Quality.
In closing, rapid microbiology methods offer in-process, real-time microbiology testing with a broad range of pharmaceutical manufacturing applications. The use of rapid methods is encouraged in current FDA initiatives, and numerous technologies exist for the detection, quantification, and identification of microorganisms. The use of rapid microbiological methods can assist our industry in facilitating progress to the desired future state of pharmaceutical manufacturing, with the ultimate goal of ensuring final product quality and improving manufacturing efficiencies.
Dr. Michael J. Miller is a Senior Research Fellow for Eli Lilly and Company, and is responsible for technical leadership in microbiology and sterility assurance within Manufacturing, Quality, Engineering, and Product Development. He has authored over 60 technical publications, poster sessions, and presentations in the areas of rapid microbiological methods, ophthalmics and sterilization, and has served as Chairperson for numerous rapid microbiological methods technical conferences in the U.S. and Europe. Dr. Miller was the Program Chairperson for the 2001 PDA Spring Meeting on Modern Pharmaceutical Microbiology, is the Program Chairperson for the 2005 PDA Annual Meeting, and is currently serving on PDA’s Strategic Planning and Program Committees. He also serves as the Chairperson for USP Technical Committee 18, Working Group 6, which is responsible for developing a general chapter on rapid microbiological methods. Dr. Miller has a Ph.D. in Microbiology and Biochemistry from Georgia State University, and has served as an adjunct professor at GSU and the University of Waterloo School of Optometry.
Author correspondence should be addressed to: [email protected]