JVT

Approaching the Selection of Rapid Microbiological Methods | IVT

Peer Reviewed: Microbiology


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 paper, whilst addressing some of the emerging technologies, is not so much about the different rapid microbiological methods that are available; it is more concerned with the considerations that need to be considered for their selection. As such, the paper provides some advice for the microbiologist to consider when drawing up a rationale for the selection of a rapid or alternative microbiological method.

Changing World of Microbiology

Conventional microbiological methods; including those long-established and described in the European, Japanese, and United States Pharmacopoeia; have served microbiologists well over the past century and have helped to ensure the production of microbiologically safe products. For example, a wide range of microbiological methods have been successfully verified using plate count methods to enumerate and identify microorganisms (within an accepted margin of error [1]). However, conventional methods have limitations. These limitations include the time taken to produce a result and the inability of many methods to recover all of the microorganisms that might be present in a sample.

Considering these issues further, the time taken to produce a result relates to the incubation period required for conventional methods, which rely on agar as a growth medium, or for microorganisms to grow in broth culture. Such methods are relatively slow, and results are only available after an incubation period (somewhere between two to 10 days, depending on the application) (2).

A further limitation is culturability and the issue of “viable but non-culturable (VNBC) microorganisms.” Many bacteria, although maintaining metabolic activity, are non-culturable due to their physiology, fastidiousness, or mechanisms for adaptation to the environment. Some research suggests, for example, that less than 10% of bacteria found in cleanrooms are culturable (3). Thus, it stands that some rapid microbiological methods (RMMs), especially those that do not rely on growth, may provide a higher recovery count as compared with traditional methods. Some rapid methods produce results where the number or types of microorganisms can be measured. With rapid methods that do not directly “grow” microorganisms, such as those that detect metabolic activity, it is possible to correlate the new measurements, such as a fluorescing unit, with the old measurement (that is, the 'colony forming unit') and establish new acceptance levels.

These concerns with limitations of conventional methods, as well as the possibilities afforded by technological advances, led to an emerging new generation of rapid and alternative microbiological methods. RMMs and alternative microbiological methods include any microbiological technique or process that increases the speed or efficiency of isolating, culturing, or identifying microorganisms when compared with conventional methods (4).

As to what rapid methods are, according to US Food and Drug Administration (5):

“RMMs are based on technologies which can be growth-based, viability-based, or surrogate-based cellular markers for a microorganism (i.e., nucleic acid-based, fatty acid-based). RMMs are frequently automated, and many have been utilized in clinical laboratories to detect viable microorganisms in patient specimens. These methods reportedly possess increased sensitivity in detecting changes in the sample matrix (e.g., by-products of microbial metabolism), under conditions that favor the growth of microorganisms.”

Although the use of the word “rapid” is often used to describe the range of techniques employed, some of the methods included within this collective do not give a more rapid result; they instead provide a more accurate, precise, or detailed result (and thus the term “alternative” is employed).

RMMs can be applied to a range of microbiological tests, including raw materials, water, intermediate products, final products, and environmental monitoring. There is a sufficient range of RMMS to provide an assessment of the microbiological quality throughout an entire production operation. RMMs may also be used by research and development. For example, in understanding formulations better in terms of microbial robustness, RMMS can be used in the support of marketing claims and communicating product benefits to consumers.

RMMs are essentially used as alternatives to four major types of conventional microbiological determinations (6-8):

  • 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.
  • Quantitative tests for enumeration of microorganisms (e.g., plate count methods to determine the bioburden of a sample).
  • Quantitative tests for potency or toxicity (e.g., what level of endotoxin is in the sample?)
  • Identification tests (e.g., biochemical and morphological characterization).

Advantages of Rapid Methods

Another advantage afforded by rapid methods, aside from the time-to-result, is throughput. Most rapid systems allow for higher volumes than the traditional method. In environments with considerable volumes of raw ingredients, in-process batches, and final products to test, a high throughput can confer an important advantage for maintaining manufacturing uptime and moving inventory as quickly as possible.

Furthermore, RMMs can assist with:

  • Designing more robust processes that could reduce the opportunities for contamination (fitting in with some quality-by-design objectives)
  • Developing a more efficient corrective and preventative action process
  • Confirming that the process is in a continuous state of microbiological control through “real-time” monitoring (that meets some process analytical test objectives)
  • Assisting with continuous process and product improvement.

Other advantages include labor efficiency and error reduction. Reducing errors is one of the greatest potential benefits of rapid enumeration. While some methods require extra human intervention and thus create greater potential for mistakes, others automate the most error-prone processes. Microbial counting, incubation changeovers, and data entry can all become far more reliable given the right equipment.

Arguably, RMMs enable a proactive approach to be taken to instances of microbial contamination, especially in relation to out-of-specification results. Here, RMMs enable quicker responses to out-of-trend situations through providing real-time or near real-time results. This allows for corrective actions to be taken earlier.

Furthermore, when considering a RMM, the new method must offer a higher level of quality assurance. There needs to be a clear and demonstrable benefit in adopting the alternative method. Examples of this include: 

  • The ability to make critical business decisions more quickly  
  • The prevention of recalls through greater method sensitivity to microorganisms
  • The detection of “objectionable” microorganisms
  • Recovery of higher or more accurate microbial numbers
  • Potential reduced stock holding through faster release times
  • Improvement in manufacturing efficiency
  • A more proactive, rather than reactive, decision making.

It is because of these advantages that RMMs attract interest from microbiologists and are areas of considerable investment by vendors.

Regulatory Acceptance

RMMs are accepted by the major global regulatory agencies. For example, in 2011, FDA published their new Strategic Plan entitled Advancing Regulatory Science at FDA (9). In Section 3, FDA seeks to “Support New Approaches to Improve Product Manufacturing and Quality.” With regard to control and reduction of microbial contamination in products, FDA supports those who:

  • Develop sensitive, high-throughput methods for the detection, identification, and enumeration of microbial contaminants and validate their utility in assessing product sterility 
  • Develop and evaluate methods for microbial inactivation/removal from pharmaceutical products that are not amenable to conventional methods of sterilization
  • Evaluate the impact of specific manufacturing processes on microbial contamination 
  • Develop reference materials for use by industry and academia to evaluate and validate novel methods for detecting microbial contamination.

Types of Rapid Microbiological Methods

Rapid or alternative methods can be categorized in multiple means. One way is based on technology or application. Here, based on the European Pharmacopeia, the RMMS can be grouped into six categories.

Growth-based Methods

Growth-based methods are those 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. These methods aim to decrease the time at which once can detect actively growing microorganisms. The methods continue to use conventional liquid or agar media. In summary, they include: 

  • Impedance microbiology (measurable electrical threshold during microbial growth)
  • The detection of carbon dioxide (CO2
  • The utilization of biochemical and carbohydrate substrates
  • The use of digital imaging and auto-fluorescence for the rapid detection and counting of micro-colonies 
  • Fluorescent staining and enumeration of micro-colonies by laser excitation 
  • Selective media for the rapid detection of specific microorganisms.

Direct Measurement

Direct measurement is 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. These methods include:

  • Demonstration of direct labeling of individual cells with viability stains or fluorescent markers with no requirement for cellular growth
  • Flow cytometry (individual particles are counted as they pass through a laser beam)
  • Solid-phase cytometry (staining and laser excitation method).

Cell Component Analysis

Cell component analysis is 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. The methods include:

  • ATP bioluminescence (the generation of light by a biological process)
  • Endotoxin testing (LAL)
  • Fatty acid analysis (methods that utilize fatty acid profiles to provide a fingerprint for microorganism identification)
  • Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry (microbial identification).

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). These methods include:

  • Real-time and continuous detection, sizing, and enumeration of airborne microorganisms and total particles. These methods are applied to the monitoring of cleanrooms.

Nucleic Acid Amplification

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

  • Riboprinting: 16S sequence of rRNA is highly conserved at the genus and species level. 
  • PCR methods for targeting specific microorganisms (millions of copies of the target DNA in a short period of time).
  • Gene sequencing (specific dye labeling).

Micro-Electrical-Mechanical Systems

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

  • Microarrays (DNA chips), evolved from Southern Blot technology, to measure gene expression (e.g., mycoplasma detection).

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 requirements (10).

In some ways, the process of applying introducing a rapid or alternative method does not differ significantly when compared with implementing a conventional method. The key points of ensuring the method is validated and shows acceptable recovery rates or accurate identification does not differ whether rapid or conventional methods are used (11).

When choosing to implement a RMM, it is important to ensure the new method is appropriate for the company’s formulations, facilities, and personnel. For example, the introduction of a method with a higher level of sensitivity needs to be aligned with the existing bioburden in raw materials, environment, and finished products.

Guidance for the implementation of rapid methods is available from both United States Pharmacopeia (USP) and Ph. Eur. (c):

  • USP <1223>,Validation of Alternative Microbiological Methods (12) 
  • Ph. Eur. 5.1.6., Alternative Methods for Control of Microbiological Quality (13)
  • Ph. Eur. 2.6.27, Microbiological Control of Cellular Products (14).

In addition, FDA has published Guidance for Industry Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products, defining validation criteria for growth based rapid or alternative microbiological methods. From an industry perspective, the Parenteral Drug Association (PDA) published a useful guide for implementation (15).

There are several considerations to be made and steps to be taken for the implementation of rapid microbiological methods. These are discussed below.

Key Considerations

An important consideration is to decide what is wanted from a rapid method and to consider this alongside a cost-benefit analysis. The first step is to consider the following questions:

  • What do I want to achieve?
  • What budget do I have?
  • What technologies are available?
  • What technologies are ‘mature’? Who else is using them?
  • How "rapid" is the rapid method?
  • What papers have been published on the subject? Aare these ‘independent’?
  • What have regulators said?

The above can form part of a risk-benefit consideration. Risk-benefit analysis should focus on (16):

  • 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, a more detailed assessment should be undertaken. This includes considering such factors as time, accuracy, and automation.

With time, factors to consider are: 

  • 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?

With accuracy, issues to consider include:

  • 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 automated?
  • If there is a need for connecting apparatus or linking the method to a Laboratory Information Management System (LIMS)?

Internal Company Obstacles

The conventional microbiological methods currently used are, generally, already approved and provide meaningful data. Consequently, there may be reluctance within companies to change procedures and adopt RMMs. Thus, arguments relating to the benefits of implementing RMMs may need to be explored.

Furthermore, there may be reluctance to adopt RMMs because of the capital investment in equipment, training, and possible adaption of current manufacturing processes as well as the time and cost of the important validation required before use. The financial implications are naturally important considerations, and it is recommended that discussions on whether employing new RMMs should involve multidisciplinary personnel (e.g.. senior management, quality unit, microbiology, production, business development, finance, and members of supply chain). With business issues, one of the key concerns is return-on-investment. This can be assessed by considering the following:

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

The following questions can help with this step:

  • How much will the validation cost?
  • How long will the validation take?
  • How many personnel will the validation require?
  • How many tests will be needed to run for the validation?
  • Does the validation require a comparison with another (existing) method?
  • How will the data be analyzed and reported?

The cost of implementation should not be considered in isolation; the cost/benefit to the business in terms of higher quality assurance, reduced stock inventory, and quicker release of product may generate cost reduction to the business in excess of the cost of implementation. Capital outlay and running costs will depend upon the RMM chosen and the equipment purchased.

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.
  • RMMs can assist in more immediate decisions on in-process material.
  • Reduced repeat testing and investigations.
  • Maximized warehousing efficiencies by way of reduced inventory holding.
  • Reduction in plant downtime/ return from shut downs.
  • Increased production yield—shift to continuous manufacturing.
  • Maximized 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 that already gives an acceptable level of assurance. In addition, the RMM and the method currently used should be run in parallel for a designated time period as a condition of approval.

Validation will be centered on two key aspects: the assessment of the equipment and an assessment of the materials that the rapid method will assess to demonstrate that microorganisms can be recovered from the material under test (17).

The validation strategy should reflect the RMM selected. Some methods that are based on analytical chemistry will suit validation criteria that include accuracy and precision, specificity, limit of detection, limit of quantification, linearity and range, and ruggedness and robustness. However, microbiology methods do not necessarily lend themselves to this approach to validation (in that not all of these criteria will be applicable), as FDA indicates (18):

"While it is important for each validation parameter to be addressed, it may not be necessary for the user to do all of the work themselves. For some validation parameters, it is much easier for the RMM vendor to perform the validation experiments."

Therefore, the following validation strategy is recommended: 

  • Define the characteristic of the current test that the RMM is to replace.
  • Determine the relevant measures that establish equivalence of the RMM to the current method. This may require statistical analysis. 
  • 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.

More specifically, with certain groups of methods, these various validation considerations can be interpreted as:

  1. Qualitative Methods
    • Accuracy and Precision, a presence absence test: Low number of positives of a low microbial count (<10 cfu).
    • Specificity: Growth promotion test.
    • Limit of Detection: Inoculate at less than 5 cfu in both the pharmacopeia method and the rapid method to be tested over several replicates.
    • Robustness: Different variations of the normal test conditions (e.g., different analysts, different instruments, and different reagent lots).
  2. Quantitative Methods
    • Accuracy: Suspensions at the upper end of the expected range and then serially diluted down and testing alongside the compendial method. The level of agreement should not be less than 70% compared with the compendia test.
    • Precision: A statistically significant number of replicates should be used. The level of variance should generally be within the 10–15% and should not be larger than that found within the pharmacopeia method.
    • Specificity: Carried out using a range of microorganisms.
    • Limit of Quantification: The lowest number of microorganisms that can be reliably counted. 
    • Linearity: A directly proportional relationship between the concentration of microorganisms used and those expressed in the rapid method.
    • Range: The results found in precision, accuracy, and linearity can be used here in order to determine the upper and lower limits of the rapid method’s detection.
    • Robustness: Different variations of the normal test conditions (e.g., different analysts, different instruments, and different reagent lots).

During the course of validation, deviations from the established criteria may occur. The implications of these will depend upon the seriousness of the issue and the degree of drift from established parameters. The deviation may or may not lead to a recommencing of the validation after an appropriate change has been made. In the most serious cases, the deviation can lead to the abandonment of the qualification and the rejection of the equipment or system. All deviations require a deviation report to be generated. Deviation reports must be reviewed by a competent expert and be accepted by quality assurance.

With the equipment qualification aspect, validation normally begins with the validation plan (VP). The VP is a document that describes how and when the validation program will be executed in a facility. The VP document will cover some or all of the following subjects:

  • Introduction
  • Plan origin and approval 
  • Derivation 
  • Scope of validation activities 
  • Validation objectives 
  • Validation plan review
  • Roles and responsibilities 
  • An overview of activities 
  • Division of responsibilities 
  • System description 
  • Overview of system 
  • Overview of process
  • System description 
  • Validation approach 
  • Site activities 
  • Documentation and procedures 
  • Scope of documentation
  • Validation schedule of activities 
  • Project master schedule
  • References.

From this plan, equipment validation is normally achieved through appropriate installation qualification, operational qualification, and performance qualification (IQ, OQ, and PQ, respectively) (19). Here:

  • IQ provides documented evidence that the equipment has been provided and installed in accordance with its specification. The IQ demonstrates that the process or equipment meets all specifications, is installed correctly, and all required components and documentation needed for continued operation are installed and in place.
  • OQ provides documented evidence that the installed equipment operates within pre-determined limits when used in accordance with its operational procedures.
  • PQ provides documented evidence that the equipment, as installed and operated in accordance with operational procedures, consistently performs in accordance with predetermined criteria and thereby yields correct results for the method.

Method Transfer

If a validated method is transferred to another laboratory (including third parties), appropriate change management should be in place. Full validation of the equipment (IQ/OQ/PQ) will need to be carried out. Full validation of the method may not be required, but, as a minimum, it needs to be demonstrated that the method gives equivalent or comparable results to the original laboratory. Any changes to formulations need to be assessed to determine if full or partial revalidation of the method is required.

Training

It is important that when RMMs are introduced, sufficient training is provided to ensure a successful and complete implementation of the new methods. This should include the microbiologists and other personnel involved in the running of the tests and should also take account of the laboratory or manufacturing facilities. Different rapid methods may also require different steps for sample preparation. Rapid methods that require different preparation steps than traditional methods will require additional training and standard operating procedure updates.

Qualified microbiologists will still be required to interpret and manage the data, continue to develop the method, and ensure that correct decisions are made. This should form part of the overall microbial quality management system.

Expectations from the Vendor

Outside of the suitability of the technology, there are a number of points that need to be satisfied in considering a specific technology, most notably the experience of the vendor itself. The following points can be useful:

  • What is the vendor’s expertise to date?
  • Is the vendor in a position to support your validation process?
  • Does the vendor have the relevant QMS procedures in place?
  • What stage is the vendor at in terms of development? For example, is the company financially sound?
  • Is the technology known to regulators?
  • Has the vendor made any product filings to regulators?
  • Does the vendor supply relevant documentation with the technology? For example, design of documents, providing material standards, and so forth.
  • Does the vendor provide training to analysts?
  • Is the vendor in a position to react with a reasonable response time to technical issues? 
  • How often does the vendor envisage system/software updates, and how will these be handled?

Summary

This paper has outlined some of the key considerations to be made when deciding whether to adopt a rapid method and the subsequent selection between the different types of rapid methods that are available. The paper 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 and undertaking the work required to qualify the method so that it is available for the laboratory or process area to use.

References

  1. S. Sutton, “Accuracy of Plate Counts,” Journal of Validation Technology 17 (3), 42–46, 2011.
  2. J.C. Gray, A. Staerk, M. Berchtold, W. Hecker, G. Neuhaus, and A. Wirth, “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 Journal of Pharmaceutical Science Technology 64, 249–263, 2010.
  3. T. Sandle, “A Review of Cleanroom Microflora: Types, Trends, and Patterns,” PDA Journal of Pharmaceutical Science and Technology 65 (4), 392–403, 2011.
  4. J. Duguid, E. Balkovic, G.C. du Moulin, “ Rapid Microbiological Methods:  Where Are They Now?,” American Pharmaceutical Review, 2011, available here.
  5. FDA, Guidance for Industry Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products (Rockville, MD, 2008).
  6. J. Moldenhauer, “Overview of Rapid Microbiological Methods,” in M. Zourob, S. Elwary, A. and Turner, eds. Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems, pp 49–79, 2008.
  7. M.J. Miller, Encyclopedia of Rapid Microbiological Methods. Parenteral Drug Association and Davis, Healthcare International Publishing, LLC, p. 103–135, 2005.
  8. R.T. Noble, S.B. and Weisberg, “A Review of Technologies for Rapid Detection of Bacteria,” Journal of Water and Health 3, 381–391, 2005.
  9. FDA, Strategic Plan for Regulatory Science (Rockville, MD 2011).
  10. C. Denoya, S. Colgan, G.C. du Moulin, “Alternative Microbiological Methods in the Pharmaceutical Industry: The Need for a New Microbiology Curriculum,” American Pharmaceutical Review 9, 10–18, 2006.
  11. M.W. Griffiths, “Rapid Microbiological Methods with Hazard Analysis Critical Control Point,” Journal of AOAC International 80 (6), 1143–1150, 1997.
  12. USP <1223> Validation of Alternative Microbiological Methods, 2011
  13. Ph. Eur. Alternative Methods for Control of Microbiological Quality, 2011
  14. Ph. Eur. Microbiological Control of Cellular Products, 2011.
  15. PDA Technical Report No. 33, Revised 2013 (TR 33) Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods 
  16. A.M. Cundell, “Opportunities for Rapid Microbial Methods,” European Pharmaceutical Review 1, 64–70, 2006.
  17. S. Sutton, “ Validation of Alternative Microbiology Methods for Product Testing Quantitative and Qualitative Assays,” Pharmaceutical Technology, 118–122, 2005.
  18. B. Riley, “A Regulators View of Rapid Microbiology Methods,” European Pharmaceutical Review 16 (5), 3–5, 2011.
  19. M. Miller, “The Implementation of Rapid Microbiological Methods,” European Pharmaceutical Review, 24–26, 2010.



Product Added Successfully

This product has been added to your account and you can access it from your dashboard. As a member, you are entitled to a total of 0 products.

Do you want access to more of our products? Upgrade your membership now!

Your Product count is over the limit

Do you want access to more of our products? Upgrade your membership now!

Product added to cart successfully.

You can continue shopping or proceed to checkout.

Comments (0)

Post new comment

The content of this field is kept private and will not be shown publicly.
  • Web page addresses and e-mail addresses turn into links automatically.
  • Lines and paragraphs break automatically.
  • Use to create page breaks.
Image CAPTCHA
Enter the characters shown in the image.
Validation Master Plan Download banner