Microbial analysis of water system and endotoxin estimation
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The document presents an analysis of microbial quality in various grades of water used in veterinary vaccine manufacturing, emphasizing the importance of microbiological control. It describes methods for testing water samples including microbial limit tests and bacterial endotoxin tests, highlighting potential pathogens and results from different water sources. Overall, the findings confirm that potable water, purified water, and water for injection meet microbiological safety acceptance limits essential for vaccine production.
Microbial analysis of water system and endotoxin estimation
- 1. A presentation on MICROBIAL ANALYSIS OF WATER SYSTEM AND ENDOTOXIN ESTIMATION FOR VETERINARY VACCINE MANUFACTURING FACILITY Internal Guided By: Ms. ROSHANI K CHAUDHARY Assistant Professor Department of Biotechnology MUIS External Guided By: Mr. HARDIK MISTRTY Assistant Manager Quality Assurance Hester Biosciences Ltd. Presented By Asha P Patel M.Sc Microbiology 16064511028
- 2. To study importance of different grades of water in veterinary vaccine manufacturing industry. To study uses and preparation techniques of different grades of water used in industry. To enumerate the number of bacteria present in water samples by different methods.
- 3. Water is very widely used in the pharmaceutical, biopharmaceutical and biological industry for various purpose like as Raw material, Ingredient as a solvent in processing, formulation and manufacturing of the wide range of the finish products, as API and intermediates and analytical reagents. There are many different types of water described in USP monographs that specify uses, acceptable methods of preparation, and quality attributes. Types of water used in Vaccine Manufacturing industry are Non-Potable, Potable, Purified and Water for Injection prepared through different techniques such as distillation, Reverse osmosis, Deionization, Filtration, Softening etc. Due to its criticality in industry, microbiological control of water is of great importance. Veterinary vaccines are considered as the sterile parenteral dosage form and water used for the various purpose in the veterinary vaccine manufacturing facility must have to comply with the regulatory requirements.
- 4. Importance and Uses of Water in Pharmaceutical Industry Different Grades of Water used in Vaccine Manufacturing Industry Uses and Preparation Techniques of various grades of Water Microbiological Control of Water
- 6. Grades of water Uses Preparation techniques Non potable Water Cleaning of floor Obtained from natural sources Potable Water Equipment cleaning and rinsing Filtration Softening Iron removal Purified Water(PW) •For the production of non- parental preparation •As an excipient •Deionization •Reverse osmosis Water for injection (WFI) •For the production of parenteral preparation •For cleaning of parenteral product contact component • In preparation of vaccine diluents •Distillation •Reverse osmosis Uses and preparation techniques of various grades of water
- 7. Microbial Limit Test (MLT) Microbial Enumeration Tests (MET) Membrane Filtration Plate Count Method Most Probable Number (MPN) Tests for specified Microorganisms Determination for the presence or absence of Particular Pathogens METHODOLOGY
- 8. Flow chart of presumptive MPN 1) MOST PROBABLE NUMBER (MPN) Water Sample
- 9. Flow chart of Confirmed and Completed MPN
- 10. 2) Plate Count Method
- 11. 3) Bacterial Endotoxin Test BET tests detect and quantify bacterial Endotoxin which is extracted from the outer membrane of Gram negative bacteria. The critical component of the LAL (Limulus Amebocyte Lysate) reagents used in Endotoxin tests is derived from blood cells (amebocytes ) of the horseshoe crab, Limulus Polyphemus. It contains the proteins of the blood clotting mechanism, which is triggered by Endotoxin. LAL reagents are primarily used to test for Endotoxin in injectable pharmaceuticals, biological products, and medical devices.
- 12. 4) Membrane Filtration Method The MF Technique was accepted by the U.S. EPA for microbiological testing of water in the 11th edition of Standard Methods for the Examination of Water and Wastewater. TTypical colonies
- 13. 5) Pathogen Detection Test In qualitative estimation the four more commonly found pathogenic bacteria are: 1. Escherichia coli 2. Staphylococcus aureus 3. Pseudomonas aeruginosa 4. Salmonella abony Initial process: 1. Firstly samples are collected 2. Now aseptically transfer 10 ml of prepare sample into 100ml of Soyabean Casein Digest Media(SCDM). 3. Incubate at 35-37° C for 18-48 hours. 4. Observe after incubation, if growth is present carry out the pathogen testing on selective media.
- 14. Water Samples Combination of positive tubes MPN Index per 100 ml Non-Potable Water 5 3 3 180 Potable Water(Generation Point) 4 4 3 54 Potable Water(User Point) 4 2 3 38 Purified Water(Generation Point) 0 0 0 0 Purified Water(User Point) 0 0 0 0 Water for Injection(Generation Point) 0 0 0 0 Water for Injection(User Point) 0 0 0 0 From MPN Index 1) Most Probable Number RESULT
- 15. Confirmed test Isolation of Microorganisms on EMB agar plate Figure 5.1 Non-Potable Water Figure 5.2 Potable Water (Generation point) Figure 5.3 Potable Water (User point) Completed Test Negative for Lactose gas production Positive for Lactose gas Production
- 16. 0 50 100 150 200 250 300 350 10¹ 10² 10³ NumberofColonies Dilution Factor Non-Potable Water Potable Water Purified Water Water for injection Graph ( dilution factor v/s number of colonies) For Generation Point 0 50 100 150 200 250 300 350 10¹ 10² 10³ Numberofcolonies Dilution Factor Non-Potable Water Potable Water Purified Water Water For Injection For User Point 2) plate count method
- 17. 1) Non-Potable Water Dilution Colony CFU/ml 10¹ 298 2.98 × 104 CFU/ml 10² 270 2.70 × 105 CFU/ml 10³ 189 1.89 × 106 CFU/ml 2)Potable Water (Generation Point) 10¹ 127 1.27 × 104 CFU/ml 10² 87 8.7 × 104 CFU/ml 10³ 62 6.2 × 105 CFU/ml 3) Potable Water (User Point) 10¹ 102 1.02 × 104 CFU/ml 10² 85 8.5 × 104 CFU/ml 10³ 43 4.3 × 105 CFU/ml 4)Purified Water (Generation Point) 10¹ 78 7.8 × 103 CFU/ml 10² 58 5.8 × 104 CFU/ml 10³ 33 3.3 × 105 CFU/ml 5) Purified Water (User Point) 10¹ 53 5.3 × 103 CFU/ml 10² 42 4.2 × 104 CFU/ml 10³ 31 3.1 × 103 CFU/ml CFU Counting
- 18. 3) Bacterial Endotoxin Test SAMPLES RESULT Non-potable water Positive Potable water (GP) Positive Potable water (UP) Positive Purified water (GP) Positive Purified water (UP) Negative Water for injection (GP) Negative Water for injection (UP) Negative Gel Formation
- 19. 4) Membrane Filtration Method Non-Potable Water Potable Water Potable Water (Generation Point) (User point) Purified Water WFI WFI (User Point) (Generation Point) (User point) Purified Water (Generation Point)
- 20. 5) Pathogen Detection Test MEDIUM APPEARENCE/ GROWTH EOSIN METHYLENE BLUE (EMB) AGAR Greenish Metallic sheen under reflected light and blue black appearance under transmitted light. 1) Escherichia coli Non-Potable Water Potable Water Potable Water (Generation Point) (User Point)
- 21. MEDIUM APPEARENCE/ GROWTH Mannitol salt agar(MSA) medium Yellow colour colonies are produced with yellow zone 2) Staphylococcus aureus Non-Potable Water Potable Water (Generation Point)
- 22. MEDIUM APPEARANCE/ GROWTH Citramide Agar(CA) medium Greenish-Yellow pigment 3) Pseudomonas aeruginosa Non-Potable Water Potable Water (Generation Point) Potable Water (User Point) Purified Water (Generation Point)
- 23. MEDIUM APPEARANCE/ GROWTH TYPES OF WATER SAMPLES RESULT xylose-lysine - deoxycholate agar(XLDA) Red colonies with or without black centre Non-potable water Growth observed Potable water(GP) No growth Potable water(UP) No growth Purified water(GP) No growth Purified water(UP) No growth Water for injection(GP) No growth Water for injection(UP) No growth 4) Salmonella abony Non-Potable Water
- 24. Conclusion In vaccine manufacturing industry, different grades of water are used for different purposes. Testing of the Potable water, Purified water and Water for injection used for the manfuacturing of the veterinary vaccines at the different stage of the process done for the generation points as well as the user points to be done using the MPN method, Plate count method and Filtration method. So as far as the microorganisms are concerned based on the testing and its respective results reveals that Potable water, Purified water and Water for injection used for the manfuacturing of the veterinary vaccines comply with the acceptance limit of the microbiology and states that the control for the microorganism in water system is there .
- 25. REFERENCES Mehta G, Prabhu SM, Kantawala D. Industrial wastewater treatment-The Indian experience. J. Indian. Assoc. Environ. Management. 22, 1995, 6-287. Gilbride, K. Molecular Methods for the Detection of Waterborne Pathogens. In Waterborne Pathogens, Detection Methods and Applications; Bridle, H., Ed.; Elsevier B.V.: London, U K 2014, p. 387 APHA Standard Methods For the Examination of Water and Wastewater, 21st ed., Washington DC: American Public Health Association, American Water Works Association, and Water Environmental Federation, 2005. Jorgensen, J.H., Lee, J.C., Pahren, H.R.,. Rapid detection of bacterial endotoxins in drinking-water and renovated wastewater. Applied and Environmental Microbiology 32,1976, 347-351. Traeger, H, Pharmaceuticals, The Presence of Bacteria, Endotoxins, and Biofilms in Pharmaceutical Water, Ultrapure Water, March 2003, 98-102. Howe RHL, Nicoles RA. Waste treatment for veterinary and plant science research and production at Eli Lilly Laboratories. Proceedings of the 14th Industrial waste conference, 1959, 25-30. Geldreich EE, Taylor RH, Blannon JC, Reasoner DJ. Bacterial colonization of point-of-use water treatment devices. J Am Water Works Assoc 1985,77(2):72-80.