Objective
To understand
To Implement
Ultimate Goal
Bioburden
Bioburden is normally defined as the number of bacteria living on a surface that has not been sterilized The term is most often used in the context of bioburden testing, also known as microbial limit testing, which is performed on pharmaceutical products and medical products for quality control purposes. Products or components used in the pharmaceutical or medical field require control of microbial levels during processing and handling. Bioburden or microbial limit testing on these products proves that these requirements have been met. Bioburden testing for medical devices made or used in the USA is governed by Title 21 of the Code of Federal Regulations and worldwide by ISO 11737.
According to United States Pharmacopeia (USP) <1227>; ISO 11737 PARTS 1&2 Natural bioburden is defined as: The amount of living microorganisms on an item (e.g. tissue or medical device) prior to and after manufacturing Bioburden can be described both in terms of QUANTITY and TYPE of microorganisms Why is knowing your bioburden important? Sterilization Dose Aseptic processing Process Capabilities (manufacturing of pharma and non-pharma products like food,beverages etc.) CONTROL!!! Prevention of infection in medical aid, hospitals, surgery etc.)
Bioburden
Bioburden: Population of viable microorganisms on a raw material, component, a finished product, and/or package. Measured in CFU (colony forming units) per unit of product
Sources of Bioburden
Raw Material Processing Equipment Environment Personnel Packing Material
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Material Degradation & Life Cycle Reduction Affect experimental data & Results
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Material Sourcing
Material inherently high in bioburden Aqueous liquids- with / without salts & sugars Natural Products Yeast Extract, Peptone Water- Fresh Every Day
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Process Equipment
Use bioburden reduction agents where ever possible NaOH, Acids, Hyprochloride
Use Autoclaved glassware for critical operations
Periodically clean equipment thoroughly for removing soil/ grime from hard to clean areas
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Keep Lab area clean Use Laminar Flow Hood for all critical sample Aliquot when possible to reduce risk Following good handing procedures Sanitize hands before critical material handling Hold containers away from mouth/ brim Keep your body and other things away from the product
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Filter Buffers and solutions Filter Intermediates- for storage Filter intermediates before loading on to the columns Filter all critical DS/ DP Avoid multiple opening and handling Tubing are good source of bioburden Wet, difficult to clean
Packing Material
Use Autoclaved Glassware and handling tools Always use sterile tips and pipettes
Falcon tubes are sterile as long as you handle them that way
Bioburden Applications
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Quality assurance and manufacturing controls shouldbe such that organisms capable of proliferation and contamination of the product are within acceptable limits.
Quality control test for non sterile pharmaceuticalproduct includes the microbiological testing of raw material , finalproducts .
Plant animal , mineral based formula ..absence of salmonella Orally administered product .. absence of E coli Topical pharmaceutical preparations absence ofS.aureus and Ps. aeruginosa Vaginal,rectal and urethral ..absence of molds and yeasts
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Plant animal , mineral based formula ..absence of salmonella Orally administered product .. absence of E coli Topical pharmaceutical preparations absence ofS.aureus and Ps. aeruginosa Vaginal,rectal and urethral ..absence of molds and yeasts
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Antimicrobial preservatives are often added to compendial products for the following reasons: 1. To prevent proliferation or limit microbial contamination that may occur subsequent to the manufacturing process. 2. To inhibit growth of microorganisms during normal conditions of storage and use when microorganisms might be introduced inadvertently from repeated withdrawing of individual doses from containment. 3. To prolong the life span of the product. The Antimicrobial Effectiveness Test is performed to prove that the added preservative provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use. This test is not to be used for routine control purposes but it designed to put a challenge on a compendial preparation in its final container with a prescribed inoculums of suitable microorganisms.
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notice that Compliance with the tests for sterility individually cannot certify absolute assurance of freedom from microbial contamination. Tests for sterility are adequately designed to reveal the presence of microorganisms in the 'samples' used in the tests.
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Sterility testing
All products labeled sterile must pass the sterility test as they have ben subjected to an effective process of sterilization as per International Pharmacopoeia and USP These tests are suitable to reveal the presence of viable forms of bacteria, fungai and yeasts in a pharmaceutical products or devices
Extraneous microorganisms should be excluded throughout the test procedure and period
The sterility testing of human and veterinary products is conducted by specific procedures Test is based on assumptions that Microorganisms grow on the provided culture medium Limitations (different organism have different nutritional requirements, temp. for growth, spores take more time to grow)
Antimicrobial precautions
To avoid any accidental contamination use Laminar Flow Hood Already present Microorganisms on the product must be killed Working area should be monitored periodically both
Air Surface
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(b) Staphylococcus aureus (c) Klebsiella aerogenes (d) Enterobacteria. the membrane filtration is to be preferred exclusively in the case of the following (i) an oil or oil-based product, (ii) an ointment that may be put into solution, (iii) a non-bacteriostatic solid that does not become soluble in the culture medium rapidly, and (iv) a soluble powder or a liquid that essentially possesses either inherent bacteriostatic or inherent fungistatic characteristic features
reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.
incubated at 60 C for 1 hr
Formation Formerly
rabbit test was used where dilution was injected into a rabbit and rabbit watched for pyrexia
Environmental monitoring
Environmental Monitoring (E/M) is a program designed to demonstrate the control of viable (living microorganisms) and non-viable particles in critical areas. These areas include clean-rooms for drug fill/ finish, formulation tank rooms, laminar flow hoods, molding machines, kit assembly lines, Intravenous (IV) compounding areas and sterile packaging.
Viable monitoring is :
Testing
for the detection and enumeration of bacteria, yeast and mold. It includes the monitoring of personnel, air and area surfaces for microbial contamination. Non-viable environmental monitoring is particle counts measured by a laser counter.
Environmental monitoring
The items that are sampled in a manufacturers clean room include 1. Personel
1.Personnel
2. Air
3. Surface
- Personnel are the biggest source of contamination in clean areas. Personnel harbor bacteria, carrying them with them everywhere they go. Gowning is the most effective way to protect the cleanroom environment from ourselves.. Personnel monitoring employs contact plates to assess microbial contamination of clean room personnel.
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Environmental monitoring
Settling plates (passive air sampling) Petri dishes containing sterile growth media are exposed to the environment for a specific period of time, usually between 30-60 minutes but can be exposed up to four hours before compromising the integrity of the media itself. Viable microorganisms which settle onto the media surface will grow after the plates are incubated. However, passive air sampling is tending to be phased out because it does not reflect microbial contamination with an accurately measured volume of air.
Environmental monitoring
3. Surfaces (including floors, walls, equipment, etc.) are cleaned and monitored
on a regular basis for viable counts by using specially designed contact plate
Two methods for surface monitoring in a Clean Room 1. Contact Plates -are special Petri dishes which contain sterile growth medium prepared in a manner so the surface of the media protrudes above the rim of the plate. These plates that contain a growth medium called Trypticase Soy Agar (TSA) and Sabouraud Dextros Agar (SDA, TSA at 30-35C which is mainly the optimal growing temperature for most environmental bacteria, and 20-25 C which is the optimal growing temperature for most mold and yeast species. The contact plate is pressed against any flat surface that needs to be sampled. Any viable microorganisms on the surface will stick to the agar surface and will grow upon proper incubation. This technique reveals the number of viable microorganisms on a surface. 2. Swabs - are sterile and stored in a suitable sterile liquid. The swabs are rubbed over the test surface. The microbiologist can determine the type of microorganisms on the swab by subculturing it to media. Swabs are used for surfaces that are not flat, and can be used to sample hard to reach areas of machinery that could not be sampled with a contact plate. Swabbing is more qualitative than quantitative.