QUALITY CONTROL MICROBIOLOGY Maggie Bryans Sheila Byrne John Hasyn BIOMAN 2011 MiraCosta College Oceanview, California Quality The degree to which a set of inherent properties of a product, system or process fulfills requirements. FDA Guidance for Industry Q9 Quality Risk Management Quality Control (QC): • Testing performed during biopharmaceutical manufacturing to verify that appropriate standards of product quality are attained • The actual organizational group (QC unit) who execute this work Biopharm QC Testing Scheme • Figure 7-2 P-257 “ Quality should be built into the product and testing alone cannot be relied on to ensure product quality.” Testing Performed by QC Microbiology • • • • • • • • • Environmental monitoring Utility testing Sterility testing Microbial content testing – Bioburden – Microbial Limit Bacterial Endotoxin (LAL) Microbial identification Antimicrobial Effectiveness Testing Cleaning validation Media fills Environmental monitoring includes….. 1) Water and clean steam monitoring, 2) Air monitoring- non-viable 3) Air monitoring – viable 4) Microbial surface testing using RODAC plates, 5) Gown and fingertip RODAC testing. Air Monitoring Particulate (Non Viable) Air Monitoring How large is a human hair? How large is a particulate? Cross section of a hair Guidelines for Clean Rooms Federal Standard 209 FS 209 E ISO 14644-1 ISO 14644-2 1963 First comprehensive guideline to clean room classification. English units. 1992 Fifth revision added metric or SI units FS 209 Class 1 to 6 2001 International Society for Standardization ISO Class 1 to 9 Classification of Clean Rooms - Federal Standard 209 ≥ 0.1µm ≥ 0.2µm ≥ 0.3µm 3 3 Particles/ft3 Particles/ft Particles/ft ≥ 0.5µm Particles/ft3 Class 1 35 7.5 3 1 Class 10 350 75 30 10 750 300 100 Class 100 ≥ 5.0µm Particles/ft3 Class 1000 1,000 7 Class 10,000 10,000 70 Class 100,000 100,000 700 Selected Equivalent Classes FS 209 Classes Class 1 Class Class Class 10 100 1,000 Class Class 10,000 100,000 ISO 14644-1 Classes Class 3 Class Class Class 4 5 6 Class 7 Class 8 ISO 14644-1 Class is equivalent to FS 209 Class above it. Particle Detection • The validation of a clean room is ongoing • The air quality of a clean room must be monitored • An optical particle counter is used to monitor air quality – “Real-time” test results Types of Particle Counters Facilities Maintenance System Portable Particle Counter Microbial Air Monitoring • Passive - Settle plates are exposed for specified time period. • Active - Electric pump draws preset sample volume of air onto nutrient media plate. Pharmaceutical Applications • Trend analysis of aseptic filling areas • Determine microbiological quality of laminar flow hood air • Assess decontamination procedures Inspection of Agar Plate and Count • Total microbial count – – • Bacteria Mold The colonies are counted and reported as colony forming units (CFU) per cubic meter of air FDA Guidance For Aseptic Processing FS 209 CLASS ISO >0.5 CLASS PARTICLES/m3 ACTION LEVELS cfu/m3 100 5 3520 1 1000 6 35200 7 10,000 7 352000 10 100,000 8 3520000 100 RODAC Plate Acceptable / Action / Alert - Levels Environmental Monitoring Testing Results Action Level: a test result that is ___?__ . 140 120 Action Level Alert Level: indicates ___?__. 100 80 Alert Level 60 Passing Level: are __ ? __ results. 40 20 0 Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Test #7 Knowledge Management ICH Q10 Microbial Identification What Do We Identify? - Bacteria - Yeast - Mold What Is An Identification? Determination of the genus and species, e.g. Escherichia coli When Do We Identify? • When the # of microorganisms exceeds an acceptable level – Class 100 – Class 10,000 • When a microorganism is recovered from a presence/absence test Identification Methods / Systems (Phenotypic Methods) • Bacteria • Conventional Method • Standardized Identification Systems • Automated Identification Systems Conventional Method • • • • Colony morphology and Gram stain Series of biochemical tests Read reactions Refer to Bergey’s Manual Colony Morphology Size, shape, texture, and color Biochemical Tests • • • • Fermentation of carbohydrates Production of catalase Production of indole Production of hydrogen sulfide gas Limitations of Conventional Method • Time consuming / labor intensive • Dependent on the bacteria’s ability to use the biochemicals • Requires a high level of technical knowledge Standardized Identification Systems • API Strips® • Enterotube® API Strips® Miniaturized biochemical tests API Strips® - Method • • • • • Gram stain Prepare a suspension of the bacteria Inoculate with the suspension Incubate strip Read the pattern of reactions (color changes) • Refer to index API® Strips Benefits • Convenient • Easy to use • Low cost per ID ($6) Limitations • Small database • Subjective • Dependent on the bacteria’s ability to use the biochemicals Automated Identification Systems • • Vitek® Biolog® Genotypic Methods • Molecular Microbiology (genotypic methods) is the wave of the future. • No single method or system is ideal for all identifications Endotoxin Testing Endotoxin What is it? A lipopolysaccharide Where does it come from? The outer bacteria. membrane of Gram negat Endotoxin Testing Which products are tested? • injectable drugs and medical devices which will contact blood or spinal fluid • includes raw materials, water and in process monitoring The USP now recognizes two tests – • The Pyrogen Test conducted with rabbits • Bacterial Endotoxins Test, also termed the Limulus Amebocyte Lysate (LAL) Test. Pyrogen Assay • USP XIX considers a solution to be pyrogenic when 10 ml/kg is injected into a rabbit and there is a rise of temperature of 0.6 C or more for any rabbit, or a total rise of more than 1.4 C for three rabbits in a three rabbit test group. LAL Test • Limulus amebocyte lysate test - based on clotting reaction of horseshoe crab (Limulus polyphemus) blood cell (amebocyte) lysate to endotoxin • Developed in 1960’s by Drs. Bang and Levin • Faster, more economical, more sensitive than rabbit pyrogen test Types of LAL Tests • Gel Clot • Turbidimetric • Colorimetric Gel Clot Method • Original method • The official “referee test” • The specimen is incubated with LAL of a known sensitivity. Formation of a gel clot is positive for endotoxin. Chromogenic Method • Endotoxin concentration is measured as a function of color intensity • LAL contains enzymes that are activated in a series of reactions in the presence of endotoxin. The last enzyme activated in the cascade splits the chromophore, paranitro aniline (pNA), from the chromogenic substrate, producing a yellow color. Turbidimetric Method • In the presence of endotoxin LAL becomes turbid and, under appropriate conditions, forms a solid gel-clot. • In the kinetic turbidimetric LAL method, endotoxin concentration is measured as a function of either the rate of increase in turbidity or the time taken to reach a particular level of turbidity. Comparison of Methods Gel Clot Chromogenic Endpoint Chromogenic Kinetic Turbidimetric Semiquantitative Quantitative Quantitative Quantitative Simple, Least expensive, Requires 37°C bath Requires spectrophotometer or plate reader Requires incubating plate or tube reader Requires incubating plate or tube reader Manually read and recorded Manual or can be automated, allows electronic data storage Is automated, allows electronic data storage Is automated, allows electronic data storage Sensitive down to 0.03 EU/ml Sensitive down to 0.1 EU/ml Sensitive down to .005 EU/ml Sensitive down to .001 EU/ml * * (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)