(c) 2002 Jeanne Moldenhauer1 Validation of Rapid Microbiology Systems Based upon PDA Technical Report No. 33

(c) 2002 Jeanne Moldenhauer2 Task Force Members Brian Bauer, Ph.D., Merck & Co. Mark Claerbout, Lilly Research Laboratories Warren Casey, Ph.D., GlaxoWellcome R&D Anthony M. Cundell, Ph.D., Wyeth-Ayerst Pharmaceuticals, Pearl River, New York (Chair) Martin Easter, Ph.D., Celsis Ltd., Edward Fitzgerald, Ph.D. Consultant, (USP Microbiology Subcommittee) Carol Gravens, BioMerieux, Inc. David Hussong, Ph.D., CDER, FDA Michael Korcynzski, Ph.D., PDA Training Institute (USP Microbiology Subcommittee) Robin Lerchen, American Pharmaceutical Partners Frederic J. Marsik, Ph.D. CDER, FDA Amy Meszaros, StatProbe Inc Jeanne Moldenhauer, Ph.D., Jordan Pharmaceuticals, Inc. Manju Sethi, Qualicon Scott Sutton, Ph.D., Alcon Laboratories (USP Microbiology Subcommitee) Martin Tricarico, Chemunex (USA) Amanda Turton, Millipore Corp Christine Vojt, Johnson & Johnson Diagnostics Inc. Kirsty Wills, Celsis Ltd. Jon Wuannlund, Becton Dickinson Microbiology Systems

(c) 2002 Jeanne Moldenhauer3 Disclaimer Presentation represents the views published in the PDA Technical Report May not in all ways be consistent with my personal views regarding the validation methods

(c) 2002 Jeanne Moldenhauer4 Document Contents Part I : Selection of New Microbiological Methods –1.0 Introduction –2.0 Technology Overview Generic Description of Types of Methods Technology Review Growth-based technologies Viability-based technologies Artifact based technologies Nucleic Acid-based technologies –3.0 Regulatory Review

(c) 2002 Jeanne Moldenhauer5 Document Contents Part II: How to Validate and Implement New Microbiological Methods –4.0 The Validation Process Equipment Model Validation Criteria Special Considerations for Microbiology –5.0 Glossary –6.0 References

(c) 2002 Jeanne Moldenhauer6 Implementation Requires Validation May require regulatory prior approval Many are not equivalent to traditional methods May be little or no guidance available for validation of microbial methods Many have associated computer-related systems and 21 CFR Part 11 issues New technology frequently requires new learning curves Reluctance to change

(c) 2002 Jeanne Moldenhauer7 What Validation is Not Method/technology selection Evaluation of feasibility of the method Proof of concept testing Vendor selection Doing it differently for economic considerations

(c) 2002 Jeanne Moldenhauer8 Typical Flow for Selection, Evaluation & Validation for a Rapid Method Is current compendial or industry standard method meeting all of your company’s needs? Determine what test requirements and specifications are (faster, less labor intensive, etc.) Look at alternate methods and see if the method can meet the specified requirements Perform sufficient feasibility proof of concept testing Does testing yield acceptable results? Plan and execute validation protocol. Were results acceptable? Submit regulatory supplement if required Implement test (after approval) Evaluate other alternate methods, rejecting the unacceptable method Continue using existing method No Yes

(c) 2002 Jeanne Moldenhauer9 Validation Definition Established documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting pre-determined specifications and quality attributes

(c) 2002 Jeanne Moldenhauer10 The Equipment Validation Model Method/Vendor Requirements (Defined) Validation Plan Design Installation Qualification (IQ) Operational Qualification (OQ) Performance Qualification (PQ) –Verify and document the system performs as specified using selected compendial organisms and environmental isolates for routine testing of batches of product.

(c) 2002 Jeanne Moldenhauer11 Intentions of Validation Demonstration data which shows: Accuracy of method (appropriateness) Reproducibility of method (continuity)

(c) 2002 Jeanne Moldenhauer12 Unique Concerns for Developing Validation Requirements for Rapid Microbiological Methods What type of validation support does the vendor have? (e.g., DMF, sample protocols) Is the new method “equivalent” to existing method If side-by-side studies are used, what is the impact of the rapid method being “out-of-specification”, when the traditional method is within limits? Is the new technology/method subject to different variabilities than traditional methods? Can the method handle the expected laboratory throughput?

(c) 2002 Jeanne Moldenhauer13 Unique Concerns for Developing Validation Requirements for Rapid Microbiological Methods (continued) USP definitions for validation terms (e.g., accuracy, precision, ruggedness... ) are written specifically for chemistry applications while automated microbiological methods probably should take these definitions into account {USP has drafted a chapter to resolve this} Are there other human interface concerns that are not present in the compendial methods? Difficulties in setting limits for the “unknown”

(c) 2002 Jeanne Moldenhauer14 Establishing Validation Criteria USP Chapter Validation of Compendial Methods gives criteria, but definitions are in terms of chemical methods Draft revision in Pharmacopeial Forum with criteria for Alternative Methods. PDA Technical Report 33 has issued microbiological definitions of these same criteria Since some tests are quantitative and others are qualitative (e.g., limits tests), not all criteria are applicable to each test type

(c) 2002 Jeanne Moldenhauer15 Validation Criteria

(c) 2002 Jeanne Moldenhauer16 Accuracy Definition: A measure of the exactness of the microbiological method that is true for all practical purposes Determination: Analyze at least five suspensions across the range of test. Calculate each suspension as a percentage dilution of the original. Acceptance criteria: Should be equivalent or better than existing methods (±30% are considered equivalent)

(c) 2002 Jeanne Moldenhauer17 Precision Definition: Precision may be a measure of either the degree of reproducibility of the microbiological method under normal operating conditions Repeatability refers to use of the method within the same laboratory over a short period of time using the same analysts and same equipment Reproducibility uses different analysts with the same equipment

(c) 2002 Jeanne Moldenhauer18 Precision (continued) Determination: at least five suspensions across the range of the test should be analyzed. Each suspension should be analyzed for at least ten replicates. Standard deviation or coefficient of variation should be calculated. Acceptance criteria: generally, a coefficient of variation in the 15-30% range is acceptable

(c) 2002 Jeanne Moldenhauer19 Precision (continued) For comparing precision of two methods, the F-test is recommended. If the critical value exceeds the statistical table, a significant difference exists between the methods.

(c) 2002 Jeanne Moldenhauer20 Specificity Definition: the ability of a method to detect a range of microorganisms which demonstrate that the method is fit for use Determination: screen against representative range of microorganisms and sample types Acceptance criteria: all selected organisms are isolated and enumerated from sample matrices

(c) 2002 Jeanne Moldenhauer21 Limit of Detection Definition: a parameter of a limit test which identifies the lowest number of microbes which can be detected, but not necessarily quantified in the test conditions Determination: multiple replicates, greater than or equal to five, should be tested, since one cannot reliably obtain samples with a count of one

(c) 2002 Jeanne Moldenhauer22 Limit of Detection (continued) Acceptance criteria: ideally, if one organism is tested, it will be detected in the time frame of the assay (use of CHI- square test)

(c) 2002 Jeanne Moldenhauer23 Limit of Quantification Definition: the lowest number of microbes which can be determined with acceptable precision and accuracy under the slated experimental conditions Determination: since there are no reliable methods to get a sample count of one, greater than or equal to five replicates should be used Acceptance criteria: if a single organism is present, it will be quantified in the time frame of the assay

(c) 2002 Jeanne Moldenhauer24 Linearity Definition: the ability to elicit results that are proportional to the concentration of microbes present in the sample within a given range Determination: at least five replicates across range of assay Acceptance criteria: correlation coefficient r 2 = 0.9, or better with a slope not diverging more than 20% from 1.0, i.e., r 2 =

(c) 2002 Jeanne Moldenhauer25 Range Definition: the interval between the upper and lower levels of microbes that have demonstrated to be determined with precision, accuracy and linearity, using the method as written Determination: the range of the method is validated by verifying that the analytical method provides acceptable precision, accuracy and linearity, when applied to samples at the extreme of the range and within the range Acceptance criteria: depends upon performance characteristics of the method

(c) 2002 Jeanne Moldenhauer26 Ruggedness Definition: degree of reproducibility of test results obtained by samples under variety of normal test conditions, e.g., different analysts, instruments, lots of reagents Determination: prepare a suspension of microbes and test at least ten replicates against each assay variable. Calculate standard deviation or coefficient of variation. Acceptance criteria: generally, a coefficient of variation in the 10-15% range is acceptable

(c) 2002 Jeanne Moldenhauer27 Robustness Definition: a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage Determination: the manufacturer investigates changes up to 20% on the critical reagent concentrations, instrument operation parameters and incubation temperatures Acceptance criteria: review results need to be reviewed against the manufacturer’s quality requirements and instructions for use

(c) 2002 Jeanne Moldenhauer28 Equivalence Definition: a measure of how similar the test results are to the method it is intended to replace Determination: should first be demonstrated in pure culture work. Requires the two methods be run in parallel for comparison. It’s important to have randomized experimental design. A large number of replicates should be used. Acceptance criteria: generally, a statistical test is used

(c) 2002 Jeanne Moldenhauer29 Microbiological Reference Standards Issues associated with identification and determination of material to be used as a reference standard for this type of testing. TR33 highlights the need to show equivalence to existing methods, but the technology for many of these systems is superior to the old method and may not show equivalence. Provides some guidance on preparing in-house standards.

(c) 2002 Jeanne Moldenhauer30 Method Validation Variability of Microbiological Methods Sample Distribution Error Cell Morphology Metabolic Activity

(c) 2002 Jeanne Moldenhauer31 Other Issues in Validation Use of multiple identical equipment within the laboratory and company Whether or not you are the first or fiftieth company to submit this test method Company’s regulatory compliance status Unique issues with microbiology equipment –Regulatory requirements, e.g., LAL –Impact on other system, e.g., sterility testing –Etc.