Validation of Microbiological Methods Expectations

Validation of Microbiological Methods Expectations for Regulatory Compliance Rajesh K. Gupta, Ph.D. Biologics Quality & Regulatory Consultants, LLC Analyze, Strategize & Operate – Different & Smart

[email protected] 1 240 246 0126

European Microbiology Conference Prague, Czech Republic, 7-8 May 2014

Outline of Presentation  Microbiological Methods used during Manufacture of Pharmaceutical Drugs

 Verification, Qualification or Validation  What is Required and How much?  Understanding Regulations

 Relevance of ICH Q2(R1)  Expectations and Common Practices  Examples

 Equivalency or Superiority Alternate Rapid Methods to Compendial Methods  Challenges with Validation of Microbiological Methods 2

Biologics Quality & Regulatory Consultants, LLC

Microbiological Methods  Clinically Important (Safety & Efficacy of Drugs)  Direct Effect on Health  Immediate (Disease & Death in a Short Period)

 Long Term (Cancer, Autoimmune & Other Diseases)

 Stability of the Drug (Chemistry & Pharmacology)  Impact on Safety and Efficacy

 Wide Variety of Methods  Testing and Monitoring

 Larger Variability (up to 2-fold Common or more)  Usually Do Not Provide Absolute Result  Provide Assurance for Absence from Specific Microbes (Bacteria, Fungi, Mycoplasma, Adventitious Viruses) 3


Role of Microbiological Methods Assure Safety & Purity of the Product

 Raw Materials, Starting Materials & Utilities  Bioburden/Sterility  Adventitious Agents

 Intermediate Components (Sterile or Non-Sterile)  Adventitious Agents (Viruses, Mycoplasma)  Residual Live Organisms

 Environmental Monitoring  Specifications based on Product – Sterile/non-Sterile

 Final Product Testing  Sterility/Bio-burden  Endotoxin 4


Role of Microbiological Methods Assure Potency, Yield & Stability

 Raw Materials & Starting Materials  Viable Counts (Titers, CFU, PFU, TCID50)

 Intermediate Components  Microbial Yields  Microbial Products (Bioassays)

 Final Product Testing  Potency Tests  Anti-Microbial & Preservative Effectiveness

 Titers

5


Microbiological Methods – Safety Assurance  Not Provide Complete Absence of Viable Organisms  Absolute Absence Cannot be Demonstrated without Completely Destroying the Product  Assurance of Absence based on Volume Tested & LOD

How to Assure Microbial Safety?  Testing, Validation, Monitoring & Verification  Terminal Sterilization  Biological Products Generally Not Terminally Sterilized

 Microbial Removal/Reduction/Inactivation  Validation of Manufacturing Process

Build Microbial Quality into a Product Don’t Build a Product and Test for Microbial Safety 6


Building Microbial Quality Into Product  Validation, Monitoring & Verification  Sterilization  All Equipment Used in Aseptic Processing Sterile (e.g. Formulation, Filling, Lyophilization Operations)  All Materials Sterile or Aseptically Processed

 Manufacturing  Removal/Reduction/Inactivation of Microbes  Aseptic Processes – Media Fill Studies  Container Closure Integrity

 Environment, Utilities, Cleaning

 Analytical Methods  Appropriate Microbial Tests at Different Stages 7


Method Verification  Most Microbiological Methods are Compendial  Need Verification (Do NOT Need Validation)  To Demonstrate that Method Can be Performed Reliably (Accurate & Precise), as described in Compendium o Documentation of Appropriate Environment, Qualified Equipment, Trained Personnel, Qualified Media, Laboratory Quality Systems o Results with Authentic Cultures, Standards, References, Controls o Traceability of Standards and Reagents

 Compendial Methods Need Qualification with Matrix  Qualification of Matrix with Specified Cultures (Compendia), Environmental Isolates, Reference or Positive Controls o Bacteristasis and Fungistasis for Sterility (Challenges with Recovery) o Similar for Mycoplasma & Adventitious Agents o Maximum Valid Dilution for Endotoxin Test 8


Method Qualification & Validation  Required for New or Alternate Methods  Purpose of Method Qualification & Validation is to Demonstrate that Method is “Suitable for Intended Purpose”  Method Qualification Provides Flexibility in Conduct of Studies & is Useful during Clinical Development  Scientifically Sound with data on Accuracy, Precision, Linearity, LOQ (if required), etc.  A Research Study without a Protocol

 Method Validation as per ICH (Q2R1) Parameters with a pre-approved Validation Protocol and Defined Acceptance Criteria for Validation Parameters 9


Status of Methods During Product Life Cycle  Validated Methods  Release Testing of Licensed Product  Raw Materials, Intermediates, Final Bulk (DS), Final Container (DP)

 Environmental Monitoring (Air, Surfaces, Personnel)  Utilities Monitoring (Water, Gases, etc.)  Stability Program  Safety Evaluation – All Stages of Clinical Development (Sterility, Adventitious Agents)  Viral Clearance Studies

 Cleaning Validation 10


Status of Methods During Product Life Cycle  Preferred to be Validated  Process Validation Studies  Equipment Qualification

 Qualified (Scientifically Sound Providing Reliable Results)  Phase 1 and 2 Clinical Studies (Except Safety Assays need Validation)  Characterization and Other Tests (For Information)  Varying Degree of Qualification  Scientifically Sound 11


Rapid Microbiological Methods (RMM)  RMM Used in Clinical & Pharmaceutical Microbiology  In Pharmaceutical Microbiology – For In-Process Testing, Use in Release Testing Not Common yet

 Role of RMM in Pharmaceutical Microbiology  Manufacturing Aspects  Faster Testing of Raw Materials and In-Process Samples,

 Faster Investigations, Corrective Actions  Faster Resolution of Process Issues, PAT

 Changing Nature of Products (Biologicals)  Shorter Shelf Lives  Smaller Quantities Available to Test

 Faster Availability of Product  Emergency Use (Influenza Pandemic, Bioterrorism) 12


Validation of Microbiological Methods Regulations and Guidance ICH Q2(R1) Validation of Analytical Procedures  “to provide some guidance and recommendations on how to consider the various validation characteristics”  “Approaches other than those set forth in this guideline may be applicable and acceptable”  “Due to their complex nature, analytical procedures for biological and biotechnological products in some cases may be approached differently than in this document”

 Useful Document on Validation Characteristics – Definitions and Data Required  Broader Guidance on Methods Validations  Information of Validation Parameters Required Depending upon Type of Method 13


Validation of Microbiological Methods Regulations and Guidance USP Chapter Validation of Alternative Microbiological Methods (Under Revision)  “guidance for validating methods for use as alternatives to the official compendial microbiological methods”

 Discusses “Qualitative Tests for the Presence or Absence of Microorganisms”, “Quantitative Tests for Microorganisms” & “General Concerns”

 Provides Valuable Guidance on Validation Characteristics, as defined in ICH Q2 (R1), as applicable to Microbiological Methods  Discusses Challenges in Variability in Microbiological Methods & Comparability with Compendial Methods 14


Validation of Microbiological Methods Regulations and Guidance EP Chapter 5.1.6 Alternative Methods for Control of Microbiological Quality  Provides guidance on alternative microbiological methods

PDA TR 33, Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods  Discusses “challenges with microbiological methods – long

times to get results, the potential inability to recover microorganisms, particularly stressed, dormant, linking microbiology to QbD & quality risk management principles”

 Discusses guidance documents, changes in policy & regulations, validation expectations etc.  Discusses value of RMM, Continuous Improvements & Risks 15


Validation of Microbiological Methods Regulations and Guidance New Sterility Rule 21 CFR 610.12  Provides Flexibility to Demonstrate New Method “Suitable for Intended Purpose”  Comparative Evaluation with Compendial Test Not Necessary

 Demonstration of Validation of Method for Limit of Detection, Specificity, Precision, Robustness, as appropriate  Suitable for Intended Use Based on Risk-Benefit Ratio of Product Comparative Evaluation of Limit of Detection & Specificity with Compendial Method (Same Level of Assurance)? 16


Comparative Evaluation with Compendial Methods  Compendial Microbiological Methods – Growth Based & Currently Reference Method (Gold Standard)  Challenges in Establishing Comparability between Growth Based and Non-Growth Based Methods

 Comparative Evaluation of Alternate Microbiological Methods with Compendial Methods Essential  Equivalent or Superior in Terms of Providing Assurance for Microbial Safety of the Drug  Specifications may be Different  Comparative Risk-Benefits Analysis

 Comparability Protocols and Discussions with Regulatory Agencies 17


Considerations for Qualification & Validation of RMMs for Release Testing  Use of Compendial Organisms & Environmental Isolates  Appropriate Representative and Stressed Organisms  Limit of Detection Studies to Evaluate below 1 CFU Levels  Spiking in Product Matrix, also with Preservatives, Excipients

 Capability to Identify Contaminant  Non-Destructive Technology for Detection of Contaminant  Same or Complimentary Technology for Identification

 Practical Aspects  Adaptable to Current Technology  For example, for Sterility Membrane Filtration System

 Not Complex, Easy to Perform, Rapid

 Automation Preferable, Equipment Qualified (IQ/OQ) 18


Examples of Modern Methods for Microbiological Testing (Sterility & Bioburden)  Growth Based (Propagation of Contaminant) o ATP Bioluminescence (Rapid Milliflex Detection System, PALL Chek) o CO2 Detection (BACTEC, BacT/Alert)

o Solid Media (Schaedler Blood Agar, TSA + 5% Blood) o Growth Direct Method

 Non Growth Based (Non-Propagation of Contaminant) o Genomics Based (MilliPrep + PCR or RTPCR) o Solid Phase Cytometry (Scan RDI)

What is “Recommended”?

Spiking Studies - LOD & Specificity (at least up to 0.1 CFU) including Environmental Isolates & Stressed Org. 19


Examples of Modern Methods for Test for Mycoplasma  Growth Based (Propagation of Contaminant)  Hybrid – Broth Culture with qPCR/PCR  MDCK Cells with qPCR

 Non Growth Based (Non-Propagation of Contaminant)  Genomics Based (MilliPrep + PCR, RTPCR or TMA)

What is Required?  Demonstration of Degree of Assurance Similar to the Current Method  Appropriate Studies for Method Validation  Representative Strains Characterized by CBER and available from ATCC (Biologicals 2013, 41 (6), 377-383) 20


Examples of Modern Methods for Adventitious Viruses  Degenerate PCR (Mixed PCR or Family Specific Primers to detect wide variety of Potential Adventitious Viruses)  Virus Microarray 

Virochip DNA Microarray – Designed to Detect All Known & New Viruses

 Broad Microbial Identification System using Broad Range PCR Combined with Mass Spectrometry 

PLEX-ID Molecular System (Abbott)

 Massively Parallel Sequencing, Next Generation Sequencing or Deep Sequencing 

Roche 454, GS FLX Titanium, Illumina MiSeq & HiSeq, Life tech Solid 4, Helicos Bio Heliscope, Pacific Biosciences SMRT)

 Molecular Bar Coding  21

NanoString Technolgies Biologics Quality & Regulatory Consultants, LLC

Modern Methods for Adventitious Agents and Challenges  Tests for Adventitious Agents – Not Absolute  Provide a Level of Assurance, Based on Sampling

 TEM (Low Sensitivity, 106 Particles/ml) and PCR based Methods (Small Volume Limitations)  Not Same Level of Assurance as Current Methods

 Selection of Appropriate Representative Organisms  Cannot Use Pathogen or Select Agents  May not be Known (Potential Risk)  Selection should Cover Wide Characteristics – Size, Sensitivity of Inactivation, Growth Requirements, etc

 Challenges with Methods Validations (Discussed Later) 22


Challenges in Validation of Microbiological Methods  Growth Based (Viable) & Non-Viable  Establishing Comparability (Superiority or Equivalence) between Growth-based and Non-Viable Methods

 Generation of Stressed Organisms and Maintaining these in Stressed Conditions

 Determination of Absolute Limit of Detection & Specificity – Challenging  Variability in Microbiology Methods  Large Number of Experiments Required to get Meaningful Results

    23

Data Not Normally Distributed Use of Appropriate Statistics Most Probable Number (USP ) CFU is Relative – Based on Media, State of Organisms Biologics Quality & Regulatory Consultants, LLC

Challenges in Validation of Microbiological Methods  Level of Assurance (Volume)  Restrictions with Amount of Volume with Certain Methods (PCR)

 Isolating and Identification of Contaminant  Certain Methods are Destructive for the Isolate

 Complexity in Technology and Methodology  False Positive (Concern for Manufacturers)  Background  Detection of Non-viable Organism (e.g. DNA)

 False Negative (Concern for Patients, Regulatory Agencies)  Low Metabolic State (P. acnes)

 Case by Case Evaluation for “Suitable for Intended Use” Based on Product Risk/Benefit Ratio 24


Addressing False Positives – 2 Stage Test First Stage Test – Release Product, if no False Negative 2nd Stage Test – Re-test, Compendial Method with Appropriate Number of samples to Provide Same Level of Assurance

Number of Samples – Probability of Accepting Lots Samples Tested

25

Per cent Lots Pass with Contamination at 0.1% 1% 10%

20 (CFR) 20 – Re-test

98 99

82 99

11 58

100

74

37

0.5

500

61

1

0


Example – Statistics for LOD (Sensitivity) of RMMS vs Compendial Method Methods

Positive/Total Experiments (% Positive Experiments) 10 CFU

1 CFU

0.1 CFU

134/138 (97)

101/138 (73)

20/89 (22)

Compendial Direct Inoculation

53/56 (94)

36/56 (64)

10/50 (20)

Rapid Milliflex Detection Sys (TSA/SDA)

75/77 (97)

66/77 (86)a

21/70 (30)

Rapid Milliflex Detection System (SBA)

40/40 (100)

39/40 (97)a

29/33 (87)a

BACTEC

74/77 (96)

38/77 (49)b

12/57 (21)

BacT/Alert

89/95 (94)

47/97 (49)b

10/56 (18)

Compendial Membrane Filtration

Data was evaluated statistically by Chi square analysis. a Significantly more sensitive from Compendial membrane filtration method p < 0.05 b Significantly less sensitive from Compendial membrane filtration method p < 0.05 26


Example – Precision & Basic Statistics  Data Must be Normally Distributed for Relying on  Mean  Standard Deviation (SD)  Coefficient of Variation (CV) or Relative SD (%RSD)

 ± 1 SD = 67% Confidence Intervals (CI)  ± 2 SD = 95 % CI  ± 3 SD = 99 % CI

+ 3 SD (99% CI) + 12 SD (67% (95% CI) x 27


Understanding Inherent Variability  SD Depends upon Numbers  CV or % RSD Normalizes Data  Understanding CV  10% CV or RSD = ± 20% Variability at 95% CI ± 30% Variability at 99% CI

 For Log-transformed Data, Calculation of CV Misleading  Calculate Geometric Coefficient of Variation (GCV)

 If Data Not Normally Distributed, SD, CI, Not Useful  Use Non-Parametric Statistics 28


Example – Management of Variability by Assay Design Example Virus Titration (TCID50) in Cell Cultures (Absolute Quantitation)

 ±0.5 Log Variability Acceptable (95% CI)  1 Log (10-fold) Variability – Too High

 1 in 20 Tests Have Larger Variability  95% Confidence Intervals – Tests  1 Test – ±0.5 log

 2 Tests – ±0.3 log  3 Tests – ±0.2 log  9 Tests – ±0.05 log

 20 – 30 Tests Close to True Value 29


Application in Design of Test for Manufacture (Formulation) & Testing  Requirement 106 TCID50/ml  Bulk Conc. – 9 Tests for Formulation (±0.05)  Example, 108 (107.95 to 108.05)

 Formulate at 106.5 TCID50/ml

 Passes by 1 Test – 97.5% (106 to 107)  Release Test – 3 Tests (106.2 to 106.8 99% CI)

 Stability – 1 Test (~2.5% OOS Risk) 30


Summary and Conclusions  Microbiological Methods Important to Assure Safety, Purity, Potency and Stability of Drugs and Biologics  Appropriate Microbiological Methods during Manufacture are Necessary in Building Quality  Multiple Overlapping Strategies (Validation, Monitoring, Testing & Verification) Required to Assure Microbial Safety  Understanding Science, Regulations and Challenges Key to 

Developing a Robust Testing & Environmental Monitoring Profile for Microbial Safety



Assuring Methods are “Suitable for Intended Use”

 Regular Communications with Regulatory Agencies Essential to Implement New Alternate Methods  Use of Powerful Modern Technologies may Increase Assurance about Microbial Safety of Drugs & Biologics 31


Validation of Microbiological Methods Expectations

Validation of Microbiological Methods Expectations

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