Pharmaceutical Microbiology Lab Layout & Functions.pptx
- 1. Presenter Muhammad Imran Hafiz Umar Pharmaceutical Microbiology Lab
- 2. Agenda Pharmaceutical Microbiology and GMP Guidelines Core Design Features Overview of Pharmaceutical Microbiology Lab Microbiological tests for pharmaceuticals
- 3. is a specialized branch of microbiology that plays a pivotal role in ensuring the safety and efficacy of pharmaceutical products. Pharmaceutical Microbiology
- 4. Pharmaceutical Microbiology The primary goal of pharmaceutical microbiology is to prevent and control microbial contamination during the manufacturing process, storage, and distribution of pharmaceuticals. Rigorous testing protocols are employed to assess the sterility, stability, and overall microbiological quality of pharmaceutical formulations.
- 5. GMP is the part of quality assurance that ensures quality products are consistently produced. Good manufacturing practice (GMP) :
- 6. Create a controlled environment. Minimize contamination risks. Optimize workflow and efficiency. Ensure high quality of product. Prioritize personnel safety. Why is GMP required?
- 7. GMP Guidelines for Microbiology Laboratory: 1. Personnel : All personal must be wear apron, gloves. 2. Environment: Control air, temperature & humidity. 3. Equipment: Must be cleaned up & stored. 4. Validation: To ensure stability & conformity.
- 8. Did You Know that…
- 9. Sources of Contamination • People/Personnel • Air/Environment • Equipment • Surfaces
- 10. Particle Size Range 0.2mm 0.5mm 1mm 5mm 50mm Virus smog mold dust bacteria pollen Particle size Color Guide = biological = inert = virus
- 11. Clean Rooms & Microbiology – Activities vs. Particles Generated Sitting Quietly Moving Walking Particles shed per minute 100,000 1 Million 5 Million
- 12. Clean room Classifications Furniture & Surface Materials Core Design Features
- 13. Clean room Classifications: GMP Eu Std At Rest >0.5 um/M³ At Rest >5um/M³ Operational >0.5um/M³ Operational >5um/M³ Equivalent ISO Equivalent Fed209E at rest /operational A 3,520 20 3,520 20 ISO-5 100 B 3,520 29 352k 2,900 ISO-6 1000 C 352k 2,900 3,520k 29k ISO-7 10k/100k D 3,520k 29k not defined not defined ISO-8 100k/na
- 14. CFUs are determined 48 hours after the incubation at 30 – 35 o C
- 15. Pharmaceuticals processes and clean room Grade A Filling – Aseptic & Terminal Sterilization B Background for Grade A – Aseptic C Background for Grade A – Terminal Sterilization. Preparation of solutions to be filtered. D Supporting Clean Areas (Cleaning of equipment, Handling and assembling of components, equipment, and accessories after washing and before sterilization)
- 16. Aseptic Process
- 18. Surface finishes (floors, walls, and ceilings) should be smooth, non -shedding, non- porous, and resistant to sustaining microbial growth Chemical resistant, easy cleaning and disinfection surfaces (epoxy-coated surfaces) Building, Furniture & Surface Materials
- 19. Crack and crevice -free construction Building, Furniture & Surface Materials
- 20. Sinks and drains prohibited in Grade A and B Areas All furniture should be stainless steel or non-absorbing and non-shedding Building, Furniture & Surface Materials
- 22. Bench surface sealed to wall Microbiology Laboratory
- 23. Floors covered to walls for easy decontamination Microbiology Laboratory >>
- 24. Overview of Pharmaceutical Microbiology Lab Desing lab
- 29. Preparation Room Glassware ( Class A )
- 32. Culture Room Laminar Air Flows (Culture Room )
- 34. Environmental Monitoring and Microbiological Testing Air Sampler (Active air ) Settling plates (Passive air) Contact plates (Rodac Plate) Swabs Finger plates (staff) Gown plates
- 38. Outlines Introduction to non-sterile pharmaceutical microbiology Environment monitoring Microbiology of non-sterile products Validations in non-sterile pharmaceutical products Microbiology of sterile products
- 39. 39 Introduction to non-sterile pharmaceutical microbiology
- 40. Introduction to non-sterile pharmaceutical microbiology To maintain the safety of pharmaceutical product, To monitor processes, products and personnel to prevent microbial contamination in non sterile products The acceptance criteria of pharmaceuticals products such as tablets, capsules, syrups, inhalers, gel, creams and ointments should be strictly maintained according to the recommended specifications given by USP, BP, EP, JP. Good Manufacturing Practice should be implemented during the manufacture, storage, and distribution of pharmaceutical preparation to maintain low bioburdens in finished dosage forms USP-NF <1111>
- 41. 41 Environment for non sterile manufacturing
- 42. Environment for non sterile manufacturing Manufacturing Environment classification Classification is performed in terms of concentration of air born particles in non- sterile manufacturing area. Particle sizes being tested are ≥0.5µ and ≥5µ. Particle count is performed by particle counter. For clean room classification, following prerequisites are required:
- 43. ENVIRONMENT FOR NON STERILE MANUFACTURING CLASSIFICATION CONTINUES… Selection of sampling location (Particulate Matter) Select the number of locations based on size of clean room. Then divide the whole clean room into defined no. of locations (equal sections) . Select within each section a sampling location considered to be representative of the section (Proposed to center of section). Example
- 44. ENVIRONMENT Monitoring Routine Environment Monitoring. Following parameters should be considered for routine environment monitoring Microbiological Parameters Viable Environment monitoring Surface Monitoring (Personnels, Equipment's, Wall and floors) Non Viable Particulate Monitoring • Physical parameters Temperature/Relative humidity Pressure Differentials
- 45. Viable Environment Monitoring Techniques Active air sampling by Air samplers Passive air sampling by settle plate technique Parameters TAMC (at 30°C to 35°C for 72 hour) TYMC (at 20°C to 25°C for 5 days) Media Tryptone Soya agar ENVIRONMENT Monitoring Routine Environment Monitoring…..
- 46. ENVIRONMENT Monitoring Viable Environmental monitoring….. Sampling Locations for viable environment monitoring. Sampling locations should be selected based on risk evaluation followed by a general hygienic survey of the environment. High-traffic areas where materials are transferred into and out of the area may be particularly prone to transient microbial contamination. Microbiological monitoring is not required in areas beyond the point where product has been placed into primary packaging containers.
- 47. ENVIRONMENT Monitoring Surface Monitoring (Personnels, Equipment's, Wall and floors) Technique Surface monitoring should be performed by contact plate method or swab test methods
- 48. ENVIRONMENT Monitoring Non-Viable particulate matter monitoring. Non-viable particulate matter monitoring should be performed during periodic qualification or whenever required.
- 49. References USP-NF <1115> Bioburden control of nonsterile drug substances and products USP-NF <1116> Microbiological Control and Monitoring of Aseptic Processing Environments USP-NF <795> Pharmaceutical compounding—nonsterile preparations WHO Technical report series , 1010, 2018 Annex 8: Guidelines on heating, ventilation and air-conditioning systems for non-sterile pharmaceutical products WHO Technical report series , 937, 2006 Annex 4: Supplementary guidelines on good manufacturing practices: validation. (Appendix 1)
- 50. Microbiology of non-sterile products
- 51. Microbiology of non-sterile products Application of Microbiological Testing. This involves selecting, sampling, and testing of: Purified Water Certain Raw materials where specifies in pharmacopieas Packaging materials with specified frequency Testing of the final product with some defined frequencies
- 52. Microbiological Enumeration Test This test is further comprised of two test: Total aerobic microbial count (TAMC) and Total yeast and molds count (TYMC). Techniques for Microbiological Enumeration Test Membrane Filtration method Pour plate method Microbiology of non-sterile products
- 53. Microbiology of non-sterile products Microbiological Enumeration Test continue……. Sample Quantity: Unless otherwise directed, use 10 g or 10 mL of the product to be examined taken with the precautions. For fluids or solids in aerosol form, sample 10 containers. For transdermal patches, sample 10 patches. Microbiological Media and incubation conditions For TAMC, Soybean–Casein Digest Agar is used and incubated at 30°C – 35 °C for 72 hours. For TYMC, Sabouraud Dextrose Agar Is used and incubated at 20°C to 25°C Interpretation of results. The results are interpretated as CFU/g both for TAMC and TYMC.
- 54. Microbiology of non-sterile products Test for specified Microorganism The test for specific organisms is to determine the absence, or limited occurrence of specified microorganisms in a given pharmaceutical sample, that may be detected under the test conditions. Pharmacopeas focus on screening for one or more of the following organisms: Escherichia coli: natural inhabitant of gut flora. Some species are pathogenic and causediarrhoea. If recovered, the organism indicates fecal contamination; Salmonellae: common inhabitant of gut flora. If recovered, the organism indicates fecal contamination and of high pathogenicity
- 55. Microbiology of non-sterile products Test for specified Microorganism continues….. S. aureus: common inhabitant of human skin and nose, detectable in feces. If recovered it indicates high pathogenicity potential. There may also be a risk to product quality due to resistance to preservatives. The bacterium has a low nutrient demand can grow to high numbers in certain materials P. aeruginosa: a common water inhabitant, especially of stored water. If recovered it indicates high pathogenicity potential;
- 56. Microbiology of non-sterile products Quality control of Microbiological media Whenever Microbiological media is prepared in lab, This media should qualified for following test for any microbiological test Physical appearance Change in color pH of media Should be tested before and after sterilization of media Sterility of media Check for sterility of media after sterilization Growth Promotion Test
- 57. Microbiology of non-sterile products Growth Promotion test continues….. For General Purpose Microbiology Media Media Name Test Organism ATCC No Incubation Conditions Soybean–Casein Digest Broth (Tryptic Soya Broth) S aureus P aeruginosa B subtilis 6538 9027 6633 30°C–35°C ≤3 days for Soybean–Casein Digest Agar (Tryptic Soya Agar) S. aureus P. aeruginosa B. subtilis 6538 9027 6633 30°C–35°C ≤3 days for C. albicans A. brasiliensis 10231 16404 20°C–25°C ≤5 days for Sabauraud Dextrose Agar C. albicans A. brasiliensis 10231 16404 20°C–25°C ≤5 days for R2A agar P. aeruginosa B. subtilis 9027 6633 30°C–35°C ≤3 days for
- 58. Microbiology of non-sterile products Growth Promotion test continues….. Media Name Test Organism ATCC No Expected physical Growth Characteristics Incubation Conditions RVS Broth S. typhimurium S. aureus 14028 6538 Growth Promotive: Turbid Growth Growth Inhibitory: No Growth 30°C to 35°C for 18 to 24 hours XLD agar S. typhimurium 14028 Growth Promotive: Red color, black center colonies 30°C to 35°C for 18 to 48 hours MacConkey Broth E. coli S. aureus 8739 6538 Growth Promotive: Turbid Growth, Color change to yellow Growth Inhibitory: No Growth 42°C to 44°C for 24 to 48 hours MacConkey agar E. coli S. aureus 8739 6538 Growth Promotive: Pink to red color colonies Growth Inhibitory: No Growth 30°C to 35°C for 18 to 72 hours Cetrimide agar P. aeruginosa E. coli 9027 8739 Growth Promotive: green pigmented color colonies Growth Inhibitory: No Growth 30°C to 35°C for 18 to 72 hours Mannitol salt agar S. aureus E. coli 6538 8739 Growth Promotive: Yellow color colonies Growth Inhibitory: No Growth 30°C to 35°C for 18 to 72 hours
- 59. Microbiology of non-sterile products References USP-NF <51> / BP Appendix VI C / (Ph.. Eur. General text 5.1.3): Antimicrobial effectiveness testing USP-NF <60> Microbiological Examination of Nonsterile Products: Tests for Burkholderia Cepacia Complex USP-NF <61> / BP Appendix VI B / (Ph.. Eur. method 2.6.12): Microbiological examination of nonsterile products: Microbial enumeration tests USP-NF <62> / BP Appendix VI B / (Ph.. Eur. method 2.6.13): Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms USP-NF <64> Probiotics test USP-NF <81> Antibiotics—microbial assays USP-NF <1111> BP Appendix VI D / (Ph.. Eur. General text 5.1.4): Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use USP-NF <1113> Microbial Characterization, Identification, and Strain Typing
- 60. Microbiology of sterile products
- 61. Microbiological Test Methods 1. Microbial limit test. 2. Sterility testing. 3. Bacterial Endotoxin Test. 4. Growth promotion test 5. Water Microbiological analysis. 6. Microbial identification Microbiological tests for pharmaceuticals fall into several categories :
- 62. Bioburden Testing (Microbial Limits Test) : This test is used to estimate the total number of viable microorganisms. The microbial content of the product includes the total bacterial count (TBC), total yeast and mold count (TYMC).
- 63. Sterility Testing. Sterility testing is done on wide range of pharmaceutical products as eye drop, antibiotic, and medical devices.
- 64. Bacterial Endotoxin (LAL Testing) Endotoxins are natural compounds released by the cell wall of gram negative bacteria. that are potentially toxic to humans.
- 65. Bacterial Endotoxin (LAL Testing) The test for bacterial Endotoxin is used to detect or quantify endotoxins using Limulus Amoebocyte Lysate (LAL) LAL is an extract of blood cells from the horseshoe crab (Limulus polyphemus)
- 66. The Growth Promotion test It is a quality control test of media. It is measure the ability of any media used to support growth when the inoculum contains a small number of microorganisms.
- 67. Water Testing Water is one of the major supplies consumed by the pharmaceutical industry. Total viable count is studied to rule out microbial contamination.
- 68. Microbial identification E.COLI TEST SALMONELLA TEST PSEUDOMONAS AERUGONSA TEST STAPHYLOCOCCUS AURUES TEST Detection, isolation and identification of microorganism :
- 70. Microbiologist Responsibilities To prepare standard operating Procedures according to latest guidelines To perform Microbiological testing of Products To perform environmental monitoring To perform suitability studies of microbiological testing procedures To prepare and test Microbiological culture media To maintain Microbiology reference ATCC cultures To perform identification of microorganisms
- 71. Microbiology Lab equipment Sr # Equipment Sr # Equipment 1 Weighing Balance 10 Digital Colony Counter 2 Universal Oven for sterilization 11 Air Sampler for viable monitoring 3 Hot Incubator 12 Microscope Binocular 4 Cool Incubator 13 Micropipette 5 Incubator 14 Autoclave for sterilization of fresh media and glassware 6 Water Bath 15 Autoclave for sterilization of waste media disposal 7 Air Borne Particle Counter 16 Pharmaceutical Refrigerator for storage Fresh media and reagents 8 Biological Safety cabinet 17 Pharmaceutical Refrigerator for storage of reference ATCC Cultures 9 pH Meter 18 Manifold Filtration assemblies
- 72. Microbiology Lab equipment High pressure diffusers for compressed gases monitoring
Editor's Notes
- #64: This material is pyrogenic (causing high fevers in humans).