Validation of Microbiological Assays

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• Validation of Microbiological Assays
• - Microbial Recovery
• - Alternate Methods
• Scott Sutton

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Validation of Recovery

• Types of Preservatives Available
• Classes available
• Sites of action
• Microbial Resistance to Preservatives

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Classes of Preservatives Available

• Alcohols
• Benzyl alcohol, Chlorbutol, Phenylethanol,
  Bronopol
• Aldehydes
• Formaldehyde, Glutaraldehyde
• Biguanides
• Chlorhexidine, PHMB
• Halogens
• Chlorine, Hypochlorite, Chloroform, Iodine

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Classes of Preservatives Available

• Heavy Metals
• Mercurials
• H2O2 and Peracid Compounds
• Phenols
• Surface-active agents (surfactants)
• Anionic
• Cationic
• Ampholytic

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Sites of Action for Preservatives
From Hugo, W.B. 1992. Mode of Action of
Non-Antibiotic Antibacterial Agents. IN
Pharmaceutical Microbiology, W.B. Hugo, and A.D.
Russel (eds) pp 288-294.
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Microbial Resistance to Preservatives

• Permeability
• EDTA
• Porins
• Growth Rate a major factor - fast growing cells
  are less sensitive
• Biofilm formation
• Spores

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lt1227gt Validation of Microbial Recovery

• Importance
• Scope
• Neutralization of preservatives
• Accuracy of plate counts
• Lower Limit of Resolution
• Upper Limit
• Pharmacopeial Preview in Nov/Dec 1996 Pharm Forum
• In-process Revision in in Jan/Feb 1998 Pharm
  Forum
• In-process Revision in in Jan/Feb 1999 Pharm
  Forum
• Final in 28th Interim Announcement - Mar/Apr
  1999 Pharm Forum (5/15/99)

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Validation of Microbial Recovery

• Neutralization of Preservatives
• Inactivation of Preservatives by Chemical Means
  Bacteriostasis/Fungistasis
• Inactivation of Preservatives by Dilution
• Filtration
• Concentration Exponent

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Validation of Microbial Recovery

• Properties of an Inactivating Agent
• It should not have an inhibitory effect on
  microorganisms
• It should overcome completely the activity of the
  inhibitor (disinfectant).
• If it combines with, and neutralizes, the
  inhibitor, the resultant product should be
  non-toxic to microorganisms.

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Population Comparisons
Test Group
Neutralizer Efficacy
Peptone Control Group
Neutralizer Toxicity
Viability Group
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The Concentration Exponent

• Cnt k
• Where
• C is the concentration
• t is the time required to kill a standard
  inoculum
• k is a constant
• n is gradient of the plot of log t against
  log C

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Validation of Recovery

• Types of Recovery Strategies
• Agar
• Membrane Filtration
• In Liquid
• Recovery of Injured Organisms

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Validation of Microbial Recovery

• Accuracy of Plate Counts
• Upper Limit of Resolution What is TNTC?
• Lower Limit of Resolution Is 1 CFU/plate
  really different from 2?

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Validation - Upper Limit of Plate Count

• Where
• HC is the average higher count, and
• LC is the average lower count.

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Validation - Lower Limit of Plate Count
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ltXXXXgt Validation of Alternate Microbiological
Methods
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Validation of Alternative Microbiology Methods

• Three Types of Microbiology Assay
• ID - What is there?
• Qualitative - Is something there?
• Quantitative - How many are there?
• How to Demonstrate Equivalence to Compendial
  Method?
• lt1225gt Validation of Compendial Methods

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lt1225gt Validation of Compendial Methods

• Accuracy
• Precision
• Specificity
• Limit of Detection
• Limit of Quantification

• Linearity
• Range
• Ruggedness
• Robustness

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Alternative Technology Component Matrix
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Definitions

• Accuracy
• The accuracy of a microbiological method can be
  defined as the closeness of the test results
  obtained to the value obtained by the compendial
  method.
• Precision
• The precision of a method in comparison to a
  compendial method is the degree of agreement
  between the two when the procedures are applied
  repeatedly to different lots of the same product.

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Definitions - Specificity

• Qualitative Tests
• The specificity of an alternative quantitative
  microbiological method is its ability to detect a
  range of microorganisms.
• Quantitative Tests
• The specificity of a microbiological method is
  its ability to detect a range of microorganisms
  that demonstrate the method is fit for its
  intended purpose. This is demonstrated using the
  compendial organisms.

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Definitions - Limit of Detection

• Qualitative Test
• The lowest number of microorganisms in a sample
  that can be detected under the stated
  experimental conditions.
• Quantitative and ID Tests
• Not Applicable

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Definitions

• Limit of Quantification
• The lowest number of microorganisms, which can be
  determined with acceptable precision and accuracy
  under the stated experimental conditions.
• Linearity
• The linearity of a microbiological test is its
  ability to provide results that are proportional
  to the concentration of microorganisms present in
  the sample within a given range.

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Definitions

• Range
• The range of a microbiological method is the
  interval between the upper and lower levels of
  microorganisms that have been demonstrated to be
  determined with precision, accuracy and linearity
  using the method as written.
• Ruggedness
• The ruggedness of a microbiological method is the
  degree of precision of test results obtained by
  analysis of the same samples under a variety of
  normal test conditions.

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Definitions

• Robustness
• 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.

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Major Alternative Technologies

• Qualitative
• Celsis - ATP Bioluminescence
• Millipore - ATP Bioluminescence
• Organon Teknika - CO2 Production
• Chemunex- Vital Dye

• Quantitative
• Celsis
• Chemunex
• ID
• Vitek
• MIDI
• Qualicon

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Validation of Alternative Methods- Identification
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Tests for Presence of Microorganisms
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ATP Bioluminescence

• Celsis
• Millipore MicroStar

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BacT/Alert - Organon Teknika

• Direct Transfer System - 10-20 mL per vial
• Minimal Training
• Colorimetric sensor on cap to read production of
  CO2
• BacT/Alert automatically reads color change,
  generates growth curve for culture
• Standard format includes bar coding wand and
  labels

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Chemunex - Scan RDI

• Filtration system
• Viability substrate taken up by cell,
  enzymatically cleaved to release fluorochrome
• Counterstaining procedure uniformly labels
  background material
• Membrane filter is scanned, specifically labeled
  microorganisms are counted