Key Criteria for the Selection of Rapid and Alternative Microbiological Methods

• Bio Products Laboratory Ltd

Introduction

Rapid microbiological method technologies aim to provide more sensitive, accurate, precise, and reproducible test results when compared with conventional, growth-based methods. Rapid methods normally involve some form of automation and the methods often capture data electronically. With several different technologies available on the marketplace, the microbiologist has a difficult, and sometimes expensive, choice to make in selecting the optimal method. This article outlines some of the considerations that need to be considered for their selection.

Changing World of Microbiology

Conventional microbiological methods, despite having served microbiologists well, have limitations, including time-to-result; culturability; and the issue of ‘viable but non-culturable (VNBC) microorganisms^1-3. These concerns with limitations of conventional methods, as well as the possibilities afforded by technological advances, led to a new generation of rapid and alternative microbiological methods emerging. Rapid microbiological methods (RMM) and alternative microbiological methods include any microbiological technique or process which increases the speed or efficiency of isolating, culturing or identifying microorganisms when compared with conventional methods⁴.

RMMs are essentially used as alternatives to four major types of conventional microbiological determinations^5,6:

1. Qualitative tests for the presence or absence of microorganisms (e.g. enrichment turbidity measurements of growth). For example, to determine if Escherichia coli is in a sample of water.
2. Quantitative tests for enumeration of microorganisms (e.g. plate count methods, to determine the bioburden of a sample).
3. Quantitative tests for Potency or Toxicity (e.g. what level of endotoxin is in the sample?)
4. Identification tests (e.g. biochemical and morphological characterization)

Types of Rapid Microbiological Methods

Rapid or alternative methods can be categorized in different ways. One way is based on technology or application.

Growth Based Methods

Growth based methods, where a detectable signal is usually achieved following a period of subculture (e.g. electrochemical methods). These methods generally involve the measurement of biochemical or physiological parameters that reflect the growth of microorganisms. Examples include impedance microbiology (measurable electrical threshold during microbial growth) and the utilization of biochemical and carbohydrate substrates.

Direct Measurement

Direct measurement, where individual cells are differentiated and visualized (e.g. flow cytometry). These methods generally use viability stains and laser excitation for the detection and quantification of microorganisms without the need for cellular growth.

Cell Component Analysis

Cell component analysis, where the expression of specific cell components offers an indirect measure of microbial presence (e.g. genotypic methods). These methods generally involve the detection and analysis of specific portions of the microbial cell, including ATP, endotoxin, proteins and surface macromolecules.

Optical Spectroscopy

Optical spectroscopy methods: utilize light scattering and other optical techniques to detect, enumerate and identify microorganisms (e.g. ‘real time’ airborne particle counters).

Nucleic Acid Amplification

Nucleic acid amplification technologies: such as PCR-DNA amplification, RNA-based reverse-transcriptase amplification, 16S rRNA typing, gene sequencing and other novel techniques. These methods are commonly used for microbial identification.

Micro-Electrical-Mechanical Systems

Micro-Electrical-Mechanical Systems (MEMS): utilize microarrays, biosensors, and nanotechnology, to provide miniaturized technology platforms.

Selection of Rapid Microbiological Methods

It is important that care is taken in choosing a rapid or alternative method for a particular application. The method must determine a product’s critical quality attribute and adhere to appropriate Good Manufacturing Practice principles and validation requirements⁷.

Guidance for the implementation of rapid methods is available from both the U.S. Pharmacopeia (USP) and the European Pharmacopeia (Ph. Eur.):

• USP<1223> “Validation of Alternative Microbiological Methods”⁸,
• Ph. Eur.. 5.1.6. “Alternative Methods for Control of Microbiological Quality⁹,
• Ph. Eur.. 2.6.27 “Microbiological Control of Cellular Products” ¹⁰.

There are several considerations to be made and steps to be taken for the implementation of rapid microbiological methods. An important consideration is to decide what is wanted from a rapid method. The first step is to consider the following questions:

• What do I want to achieve?
• How much budget do I have?
• Which technologies are available?
• Which technologies are ‘mature’? (who else is using them?)
• How ‘rapid’ is the rapid method?
• What papers have been published? (are these ‘independent’?)
• What have regulators said?

The above can form part of a risk-benefit consideration, focusing on¹¹:

• The defined purpose for the test method.
• The type and depth of information required.
• The limitations of the conventional method and what the rapid method might be able to offer

Next consider such factors as time, accuracy and automation.

• Time taken to prepare the test: is the rapid method faster, equivalent or slower?
• Time taken to conduct the test.
• Sample throughout.
• Time to result.
• Whether there is a reduction in the time taken to conduct complimentary tests.
• Whether more or less time is required for data analysis.
• Whether results reporting is simplified or more efficient?
• If the rapid method will lead to a reduction in human error.
• If there is a reduction in subjectivity.
• Whether the alternative method will detect more accurately in comparison to a conventional method?
• Whether there is a need for the rapid method to detect what a cultural method cannot?

Other considerations include:

• If there is a need for the electronic capture of data?
• Whether the method needs to be automation?
• If there is a need for connecting apparatus or linking the method to a Laboratory Information Management System?

With business issues, one of the key concerns is return-on-investment. This can be assessed by:

• Operating costs of the conventional method.
• Operating and investment costs of the rapid method.
• Cost savings and cost avoidances of the rapid method.

Other aspects that can support a business case include:

• Online/At Line systems can result in reduced microbiology testing and finished product release cycle times.
• RMM’s can assist in more immediate decisions on in-process material.
• Reduced repeat testing and investigations.
• Maximizing warehousing efficiencies by way of reduced inventory holding.
• Reduction in plant downtime/ return from shut downs.
• Increased production yield – shift to continuous manufacturing.
• Maximizing analyst output by eliminating waste activity.

Validation

When choosing an RMM consideration should be given to how it is going to be validated. Any methods that are being adopted need to yield results equivalent to or better than the method currently used. The following validation strategy is recommended¹²:

• Define the characteristic of the current test that the RMM is to replace.
• Statistically determine the relevant measures that establish equivalence of the RMM to the current method.
• Demonstrate the equivalence of the RMM to the current method in the absence of the product sample.
• Demonstrate the equivalence of the RMM to the established method in the presence of the test sample.

From this plan, equipment validation is normally achieved through appropriate Installation Qualification, Operational Qualification and Performance Qualification (IQ, OQ, PQ respectively). If a validated method is transferred to another laboratory (including third parties) appropriate change management should be in place.

Summary

This short article has outlined some of the key considerations to be made when selecting between the different types of rapid methods that are available. The article did not set out to differentiate between different technologies (this itself is a rapidly developing field), but more to offer general advice to those tasked with making the selection.

References

1. Sutton, S. (2011) Accuracy of Plate Counts, Journal of Validation Technology, 17 (3): 42-46
2. Gray, J.C., Staerk, A., Berchtold, M., Hecker, W., Neuhaus, G., Wirth, A. (2010) Growth promoting properties of different solid nutrient media evaluated with stressed and unstressed micro-organisms: Prestudy for the validation of a rapid sterility test. PDA J Pharm Sci Technol; 64:249-263
3. Sandle, T. (2011): A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA Journal of Pharmaceutical Science and Technology, 65 (4): 392-403
4. Duguid, J., Balkovic, E., du Moulin, G.C. (2011). Rapid Microbiological Methods: Where Are They Now?, American Pharmaceutical Review, November 2011: https://www.americanpharmaceuticalreview.com/Featured-Articles/37220-Rapid-Microbiological-Methods-Where-Are-They-Now/
5. Moldenhauer, J. (2008) Overview of Rapid Microbiological Methods, In: Zourob, M., Elwary, S. and Turner, A. (Eds.) Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems, pp49-79
6. Miller, M.J. (2005) Encyclopedia of Rapid Microbiological Methods. Parenteral Drug Association and Davis, Healthcare International Publishing, LLC, United States of America, p. 103-35
7. Denoya, C., Colgan, S., du Moulin, G.C. (2006). Alternative Microbiological Methods in the Pharmaceutical Industry: The Need for a New Microbiology Curriculum. Am. Pharm. Rev.; 9:10-18
8. USP <1223> Validation of Alternative Microbiological Methods. In: United States Pharmacopoeia. 34th Ed. Rockville, MD: The United States Pharmacopeial Convention, Inc.; 2011
9. Ph. Eur. Alternative Methods for Control of Microbiological Quality. In: European Pharmacopeia. 7th ed. Section 5. 1. 6. Strasbourg, FR: European Directorate for the Quality of Medicines; 2011
10. Ph. Eur. Microbiological Control of Cellular Products. In: European Pharmacopoeia. 7th ed. Section 2. 6. 27 Strasbourg, FR: European Directorate for the Quality of Medicines; 2011
11. Cundell, A.M. (2006). Opportunities for Rapid Microbial Methods. Eur. Pharm. Rev.; 1:64-70
12. Sandle, T. (2014) Approaching the Selection of Rapid Microbiological Methods, Journal of Validation Technology, 20 (2): 1-10

Author Biography

Dr. Sandle is the Head of Microbiology at Bio Products Laboratory Limited (a pharmaceutical organization). Dr. Sandle is a chartered biologist (Society for Biology) and holds a first class honors degree in Applied Biology; a Masters degree in education; and obtained his doctorate from Keele University.

Dr. Sandle has over twenty-five years experience of designing and operating a range of microbiological tests (including sterility testing, endotoxin LAL methodlogy, microbial enumeration, environmental monitoring, particle counting, bioburden, isolators and water testing). In addition, Dr. Sandle is experienced in microbiological and quality batch review, microbiological investigation and policy development.

Dr. Sandle is an honorary consultant with the School of Pharmacy and Pharmaceutical Sciences, University of Manchester and is a tutor for the university’s pharmaceutical microbiology MSc course. Dr. Sandle serves on several national and international committees relating to pharmaceutical microbiology and cleanroom contamination control (including the ISO cleanroom standards). He is chairman of the Pharmig LAL action group and serves on the Blood Service cleaning and disinfection committee. He has written over two hundred book chapters, peer reviewed papers and technical articles relating to microbiology; and delivered papers to over forty conferences.

Dr. Sandle is the editor of the Pharmaceutical Microbiology Interest Group Journal and runs an on-line microbiology website and forum (http://www.pharmamicroresources.com/). Dr. Sandle is an experienced auditor and frequently acts as a consultant to the pharmaceutical and healthcare sectors.