Rapid Microbiological Methods and the PAT Initiative

The methods used in most microbiological test laboratories originated in the laboratories of Koch, Lister, and Pasteur. While numerous changes have occurred in the chemistry laboratory, there have been limited improvements in methods used for microbiological testing.

In the past decade, many researchers have focused on the study and implementation of improved methods for isolation, early detection, characterization, and enumeration of microorganisms and their products. This translates into better methods, automated and miniaturized methods, methods that require less time or those that are less costly. All of these changes are collectively grouped into the category known as rapid microbiological methods (RMM). In some compendia, these are also called alternative microbiological methods. Although these methods are called rapid microbiological test methods, many of them have their roots in other sciences, e.g., chemistry, molecular biology, biochemistry, immunology, immunochemistry, molecular electronics, and computer-aided imaging.

RMMs provide significant advantages for pharmaceutical companies to obtain data that may be significantly better than traditional methods, may be more cost effective, may provide marketing advantages, and may allow for coordinated process analytical technologies to be fully integrated within a facility.

Slow to Adopt New Methods

While science moved forward in the development of new microbiological methods, industry was slow to accept and implement them. One fear originates from a concern that regulators would not recognize these methods as superior to traditional methods. Another concern was that companies would not be allowed to change test limits based upon the test method, i.e., they would use a superior method that was likely to detect more organisms and not be allowed to adjust the limits to accommodate the sensitivity of a new method.

A concern among other companies was that the first company to submit a new technology for regulatory approval would face a much more difficult time obtaining approval than companies that submitted later.

Regulatory Framework

In recent years, a variety of documents have been issued or drafted to aid the microbiologist in selection, purchase, implementation, and regulatory submission of RMMs.

• Industry Guidance PDA TR No.33 The Parenteral Drug Association (PDA) was one of the first organizations to develop guidance for the evaluation, implementation, and validation of RMMs. Guidance information was published as Technical Report Number 33.¹ This document was developed by a committee of individuals from industry, regulatory agencies, compendial groups, and instrument vendors. This guidance provided definitions in microbiological terms for validation criteria similar to the information in USP <1225> for chemistry methods.
• USP Proposed Chapter <1223> on Validation of Alternative Microbiological Methods The USP proposed a draft monograph <1223> that defines various validation criteria to be used for RMMs, along with definitions of these criteria in terms of microbiology. The proposal also identifies how to determine which criteria are applicable to different technologies, based upon the type of testing being performed.²
• GMPs for the 21st Century The Food and Drug Administration (FDA) initiated a program to modernize requirements for pharmaceutical manufacturing and quality. This modernization included encouraging early adoption of new technologies, facilitation of industry application of modern quality management technologies, encouraging implementation of risk-based approaches in critical areas, ensuring that policies for review of a submission, compliance, and facility inspection are based upon state-of-the-art technologies, and enhancing the consistency and, coordination of FDA regulatory programs.³ This resulted in an initiative entitled Pharmaceutical cGMPs for the 21st Century – A Risk-Based Approach in 2004.³
• FDA Guidance on Aseptic Processing 2004 In 2004, FDA published a guidance document on aseptic processing of pharmaceutical products.⁴ It includes a provision for the use of alternative microbiological test methods:

"Other suitable microbiological test methods (e.g., rapid test methods) can be considered for environmental monitoring, in-process control testing, and finished product release testing after it is demonstrated that the methods are equivalent or better than traditional methods (e.g., USP)."

This is one of the first regulatory documents that specifically recognizes the potential use of alternative RMMs.

• Process Analytical Technologies (PAT) Guidance The concept of PAT is described in FDA's Guidance for Industry – PAT A Framework for Innovative Pharmaceutical Development, Manufacture and Quality Assurance.⁵

PAT is defined here as: "Systems for analysis and control of manufacturing processes based on timely measurements, during processing, of critical quality parameters and performance attributes of raw and in-process materials and processes to assure acceptable end product quality at the completion of the process."⁵ PAT expects faster, more accurate test methods capable of producing real-time or near real-time data for process control, rather than reliance on finished product testing. Traditional microbiological test methods usually cannot deliver these results, making them unsuitable for PAT applications. RMMs were included by the FDA PAT subcommittee on PAT in October 2002 following input from industry practitioners.

• EP Proposed Chapter on RMM PHARMEUROPA published a draft chapter 5.1.6. Alternative Methods for Control of Microbiological Quality in 2004.⁶ This chapter provided an overview of some RMMs available and potentially applicable to pharmaceutical processes, and how they may be used for microbiological control of products and processes. It also provides guidance on how to choose and validate an appropriate method.

Does RMM = PAT Application?

In most cases, the definition of PAT includes collection of real-time data, typically in-line, to make decisions about the quality of a product earlier in the production process. Although there have been great advances in the RMMs in recent years, most methods developed to date are still conducted on the laboratory bench, off-line. Samples are collected and taken to a lab for testing. While this may not be as advantageous as many of the chemistry applications developed, it is a significant improvement over the traditional microbiological methods, where instead of days or weeks to obtain microbiological test results, they may be available in a period of a few hours to a few days. As such, implementation of these methods makes it possible to achieve many of the savings available from other systems.

Traditional Methods

Classical microbiological test methods used are frequently divided into three general categories, based upon the test function performed, e.g., presence or absence of microorganisms (e.g., pathogen detection, absence of objectionable organisms, sterility testing), enumeration of microorganisms (e.g., bioburden testing), and identification of microorganisms.

This classification of methods answers three specific questions: "Is something there?" (Presence/Absence); "How much is there?" (Enumeration); and "What is there?" (Identification)

RMMs

The classification systems for rapid methods are based on how the technology works, e.g., growth of microorganisms, viability of microorganisms, presence/absence of cellular components or artifacts, nucleic acid methods, traditional methods combined with computer-aided imaging, and combination methods.

• Growth-based Technologies These methods are based on measurement of biochemical or physiological parameters that reflect the growth of the microorganisms. Examples include: ATP bioluminescence, colorimetric detection of carbon dioxide production, measurement of change in head- space pressure, impedance, and biochemical assays.
• Viability-based Technologies These types of technologies do not require growth of microorganisms for detection. Differing methods are used to determine if the cell is viable, and if viable cells are detected, they can be enumerated. Examples of this technology include solid phase cytometry and flow fluorescence cytometry.
• Cellular Component or Artifact-based Technologies These technologies look for a specific cellular component or artifact within the cell for detection and/or identification. Examples include: fatty acid profiles, mass spectrometry (Matrix Assisted Desorption Ionized – Time of Flight, MALDI-TOF), enzyme linked immunosorbent assay (ELISA), fluorescent probe detection, and bacterial endotoxin-limulus amebocyte lysate test.
• Nucleic acid-based technologies These technologies use nucleic acid methods as the basis for operation. Examples include: DNA probes, ribotyping/molecular typing, and polymerase chain reaction (PCR).
• Traditional Methods with Computer-aided Imaging This involves using a classical method for most of the processing of a sample, and using imaging software to detect the growth earlier than methods requiring visual detection of growth. In most cases, detection of growth using human vision typically requires growth to 10⁵ or 10⁶ cells. Computer-aided imaging can detect growth at much lower levels of cellular growth.
• Combination Methods This term describes systems that use more than one type of methodology or test to achieve a final result.

Technology Types

GROWTH-BASED

• Adenosine Tri-Phosphate (ATP) Bioluminescence Premise of Technology: Adenosine triphosphate (ATP) is present in all living cells. In the presence of the substrate D-luciferin, oxygen and magnesium ions, the enzyme Luciferase will utilize the energy from ATP to oxidize D-luciferin and produce light.

• Adenylate Kinase Premise of Technology: Adenylate kinase is a cellular component that allows for microbial detection. The adenylate kinase released from cells is reacted with ADP to form ATP. The ATP is detected using an ATP bioluminescence method.⁷

• Changes in Head-space Pressure Premise of Technology: Electronic transducers are used to measure positive or negative pressure changes in the head-space of each culture bottle. Changes are caused by microbial growth. If the growth produces significant production and/or consumption of gas, the samples are flagged as positive.

• Colorimetric Detection of Carbon Dioxide Production Premise of Technology: As microorganisms grow, they produce carbon dioxide. Test samples are placed in culture bottles and are incubated, agitated, and monitored for the presence of microorganisms. These systems use colorimetric detection of CO₂ production from the growth of organisms.

• Conductivity Premise of Technology: Works like impedance methods, the measurement taken in conductance.

• Conventional Methods with Computer-Assisted Imaging Premise of Technology: Images are collected using a charge coupled device camera; the collected images are digitized on a computer utilizing image processing software that has programming capabilities (alternatively, some systems collect the data directly with a digital camera). The digitized picture is processed to detect colonies present, and separated colonies are counted.

• Fluorescent Detection of Carbon Dioxide Premise of Technology: This technology allows for continuous monitoring for contamination using a fluorescent carbon dioxide system.

• Impedance (also known as an Electrochemical Method) Premise of Technology: Microbial detection systems based on impedance technology are classified as either direct or indirect impedance systems. Direct impedance systems work by detecting changes in electrical conductivity of growth media when an AC current is passed across two electrodes. Indirect impedance systems detect carbon dioxide produced by metabolizing organisms. As the carbon dioxide is ionized, changes in impedance occur.

• Turbidimetry Premise of Technology: As microorganisms grow, one can detect changes in the capacity of the growth medium. Optical density measurements detect the differences in opacity at specified wavelengths using a spectrophotometer. Another version of this methodology uses microtitre plate readers with continuous detectors, to detect organism growth earlier.⁶ A common usage for this type of test us to determine microbiological suspension or inoculum sizes.

CELL-COMPONENT BASED

• Biochemical Assays and Physiological Reactions Premise of Technology: Pure culture suspensions are tested with biochemical substrates or subjected to analysis to generate a spectrum. The results are compared to a database of expected results. Comparisons allow the user to identify the microorganism.

• Endospore Detection Premise of Technology: A major component of the spore case is Ca (dpa). Dipicolinate anions (dpa²-) are only present in bacterial endospores. Ca (dpa) and (dpa²-), when dissolved, are not photoluminescent. It has been shown that Terbium (Tb³⁺) is able to complex with dpa²- forming a photoluminesecent complex.⁸

• Enzyme Linked Immunosorbent Assay (ELISA) Premise of Technology: One can use an antigen-antibody reaction to detect unique microorganisms or cellular components.

• Fatty Acid Profiles (Fatty Acid Methyl Esters [FAMEs]) Premise of Technology: Fatty acids are present in microorganisms. The fatty acid composition is typically homogeneous within different taxonomic groups. Isolates are grown on a standard media and selected for testing.The testing procedure includes saponification of fatty acids, methylation, and extraction, resulting in FAMEs. FAMEs are measured using gas chromatography. Measurements are then compared to a library of known organisms.

• Fourier Transformed Infrared Spectroscopy (FTIR) Premise of Technology: An FTIR can generate an infrared spectrum of microorganisms. Patterns are generated and compared to a database of spectra of known microorganisms.

• Gram Stains Premise of Technology: This technology uses a single solution without fixatives and washes. The method can be used with mixed cultures and results are obtained in a few minutes.

• Immunological Methods Premise of Technology: Antigen-antibody reaction can be used to detect unique microorganisms or cellular components. These systems are useful for identification and pathogen detection. In some cases, the systems may not be able to distinguish whether the cells detected are viable.⁶

• Limulus Amebocyte Lysate Endotoxin Testing (LAL) Premise of Technology: Amebocyte Lysate recovered from horseshoe crabs (Limulus) have similarities in blood coagulation to humans. This similarity has allowed the use of this reagent to detect the presence of bacterial endotoxins. This technology has been available for many years as a replacement for the rabbit pyrogen test. Many systems are available, which have widespread acceptance by regulators.

• Mass Spectrometry (Matrix-Assisted Laser Desorption-Time Of Flight MALDI-TOF) Premise of Technology: When microbial isolates are heated in a vacuum, the gaseous breakdown products can be analyzed using mass spectrometry. A spectrum is generated. The spectrum is compared to a database of known organisms for identification. The size of the database is important in evaluating the effectiveness of system use. This technology has been used for microbial identifications.

• RAMAN Spectroscopy Premise of Technology: A RAMAN Spectrophotometer can be used to generate a spectrum unique to the microorganism. Studies performed in clinical settings indicated that identifications could be made with about five hours incubation.

NUCLEIC ACID-BASED

• Nucleic Acid Probes Premise of Technology: Data available from nucleic acid sequencing are used to select a desired nucleic acid. The desired nucleic acids are extracted, immobilized to a solid phase, and hybridized to a labeled probe.⁹

• Polymerase Chain Reaction (PCR) Premise of Technology: PCR works like a copier machine, making "Xerox" copies of nucleic acid fragments. Nucleic acid fragments are amplified using polymerization techniques. This technology is widely used in other sciences such as anthropology and forensics.

• Ribotyping/Molecular Typing Premise of Technology: This technology utilizes restriction fragment length polymorphisms (RFLPs) of nucleic acids from bacterial genomes. The size-separated RFLPs are hybridized to a ribosomal RNA probe. Digital information is captured, data are extracted and compared to a database of known patterns for identification. Molecular typing is considered the "gold standard" in identification of microorganisms.

VIABILITY-BASED

• Direct Epi-fluorescent Filter Technique (DEFT) Premise of Technology: Samples are filtered and stained using a fluorescent viability indicator. Sensitivity of technique depends on the volume filtered and the number of fields viewed under the microscope. The robustness of the test can be affected by the distribution of the microorganisms on the membrane. This methodology is best suited for low viscosity fluids, although it may be possible to use pre-filtration to allow testing of other solutions.¹⁰

• Flow Cytometry (fluorescence) Premise of Technology: Using flow cytometry, microorganisms are labeled in solution with a non-fluorescent marker. The marker is taken up into the cell and cleaved by intracellular enzymatic activity to produce a fluorescing substrate. The labeled sample is automatically injected into a quartz flow cell, which passes each microorganism past a laser excitation beam for detection.

• Microcalorimetry Premise of Technology: The process of microbial catabolism results in heat that can be measured by microcalorimetry. A calorimeter can be used to establish growth curves. When high levels of contamination are present, one may need to use flow calorimetry.⁶ This technology cannot be used to determine if a single contaminant is present or used on samples with mixed contaminants.

• Solid Phase Cytometry Premise of Technology: Solid phase cytometry uses membrane filtration to separate potential microbial contaminants from filterable samples prior to labeling of the captured cells with a universal viability substrate. Solid phase cytometry eliminates the need for cell multiplication. Solid phase cytometry has been accepted for pharmaceutical water testing by FDA in February 2004 and also has been accepted by the United Kingdom in 2000.

COMBINED TECHNOLOGIES

• Concentric Arcs of Photovoltaic Detectors with Laser Scanning Premise of Technology: The system is comprised of five concentric arcs of photovoltaic detectors, almost in an orb-like platform. The sample being evaluated is suspended in a liquid or gas inside a collection device, placed near the center of the orb. A laser beam of red, solid state composition is passed through the sample. Identification occurs within a few milliseconds after the particle passes through the beam.¹⁰

• Fluorescent Probe Detection Premise of Technology: Nucleic acid probes are designed to bind to specific target sites on or in cells. Probes contain a molecule that is capable of fluorescing when stimulated by an energy source. Some of these systems have restrictions on the sample size allowed.

• Lab-on-a-Chip (LOC), Arrays, Microarrays, and Microchips Premise of Technology: The use of an array of data to perform tests has been used in many microbiology applications. Each microchip is like a miniature laboratory and often referred to as a "lab on a chip" device. Typical microbiological reagents include oligonucleotides, proteins, DNA, etc.⁷ One application of this technology is the antibody dot or microspot assay. A small amount of antibody is placed on the bottom surface of a plastic well. This antibody dot is used as the capture antibody in a microimmunoassay.

OTHER TECHNOLOGIES

Arrays, Microarrays, Microchips

• Biosensors and Immunosensors Premise of Technology: Immunological reagents are combined with sensor detection systems to produce an immunosensor. These types of systems are used for pathogens (including bioterrorism organisms).⁷

RMM Applications

When evaluating a system for use, consider a variety of factors:

• Type of technology considered versus the type of microbiological test being performed
• Initial system cost
• Cost per test on an on-going basis
• Can the system handle the type of products manufactured, filterability, sample size, detection limits appropriate for the test
• System through-put
• Level of automation required and available

Disclaimer

There are reports of thousands of systems in development for use in place of traditional microbiological methods. This article introduces some of the technologies available. Inclusion or exclusion of methods is not meant to confer credibility, endorsement, or acceptance of one method over another. This article was condensed due to space limitations. It will be published in its entirety in an upcoming issue of BioPharm International.

Other Resources

There are a variety of other resources available for use by microbiologists in gaining information on RMM. A limited number of references are listed here for use:

• www.fda.gov has guidance documents available on PAT and information on speaker presentations.
• www.rapidmicrobiology.com includes information on vendors, technologies, and press releases.
• Rapid Microbiology User's Group™ (RMUG™), information available at www.vectech.com, on seminars, newsletters, and support information.
• Pharmaceutical Microbiology Forum (PMF), www.microbiol.org is for pharmaceutical microbiologists and includes an e-mail discussion group and virtual library.

Acknowledgements

Special thanks to Casey Costello and Vicky Strong for their aid in compiling this information.

Jeanne Moldenhauer, Vechtech Pharmaceutical Consultants, Inc.,24543 Indoplex Circle, Farmington Hills, MI 48335 248.478.5820, Fax: 248.442.0060, jeannemoldenhauer@yahoo.com

References

1. Parenteral Drug Association. 2000. Technical Report 33, Evaluation, validation and implementation of new microbiological testing methods. J.Pharm. Sci. Technol. 54(3), Suppl. TR33 (May–June 2000).

2. Proposed Chapter <1223> Validation of Alternative Microbiological Methods, Pharmacopeial Forum, Vol. 29 (1) Jan-Feb 2003, 256 – 264.

3. FDA, September 2004, "Pharmaceutical cGMPs for the 21st Century – A Risk-Based Approach", Final Report – Fall 2004, Department of Health and Human Services, U.S. Food and Drug Administration.

4. FDA, September 2004, "Pharmaceutical cGMPs for the 21st Century – A Risk-Based Approach", Final Report – Fall 2004, Department of Health and Human Services, U.S. Food and Drug Administration.

5. FDA, Guidance for Industry – PAT A Framework for Innovative Pharmaceutical Development, Manufacture ad Quality Assurance, Government Printing Office, Rockville, MD, 2004.

6. PHARMEUROPA, October 2004. "5.1.6. Alternative Methods for Control of Microbiological Quality," 16(4): 555-565.

7. Kricka, L.J. "New Technologies for Microbiological Assays," in Rapid Microbiological Methods in the Pharmaceutical Industry, ed.M.C. Easter, Interpharm/CRC, Washington. D.C., 2003, p.233-248.

8. Rosen, D.L., Fell Jr., N.F. and Pellegrino, P.M., 2003, "Spectroscopic Detection of Bacterial Endospores Using Terbium Cation Reagent," In. Rapid Analytical Microbiology: The Chemistry and Physics of Microbial Identification, Edited by W. P. Olson, Bethesda, MD and Godalming Surrey, UK: Parenteral Drug Association and Davis Horwood International Publishing, Ltd., 230-235.

9. Rudi, K., "Application of Nucleic Acid Probes for Analyses of Microbial Communities," in Rapid Analytical Microbiology The Chemistry and Physics of Microbial Identification, Edited by W. P. Olson, Bethesda, MD and Godalming Surrey, UK: Parenteral Drug Association and Davis Horwood International Publishing, Ltd., 13-40.

10. DeSorbo, M.A.,"Rapid Contamination Detection Technology Patent Granted", www.cleanrooms.com.