Sterility testing is carried out to detect the absence of microbes in pharmaceutical preparations. There are two main methods for sterility testing - membrane filtration and direct inoculation. The membrane filtration method involves filtering the product sample and incubating the filter membrane in culture media to check for microbial growth. The direct inoculation method involves directly transferring the product sample to culture media and incubating. Both methods require observing the culture media for any signs of microbial growth over a period of incubation. A positive result indicates the product failed the sterility test.
Sterility testing is carried out to detect the absence of microbes in pharmaceutical preparations. There are two main methods for sterility testing - membrane filtration and direct inoculation. The membrane filtration method involves filtering the product sample and incubating the filter membrane in culture media to check for microbial growth. The direct inoculation method involves directly transferring the product sample to culture media and incubating. Both methods require observing the culture media for any signs of microbial growth over a period of incubation. A positive result indicates the product failed the sterility test.
Sterility testing is carried out to detect the absence of microbes in pharmaceutical preparations. There are two main methods for sterility testing - membrane filtration and direct inoculation. The membrane filtration method involves filtering the product sample and incubating the filter membrane in culture media to check for microbial growth. The direct inoculation method involves directly transferring the product sample to culture media and incubating. Both methods require observing the culture media for any signs of microbial growth over a period of incubation. A positive result indicates the product failed the sterility test.
DEFINITION: Sterility Testing: It is a procedure carried out to detect and
conform absence of any viable form of microbes in or on pharmacopeia preparation or product. PRINCIPLE : Sterility testing only shows that organisms capable of growing in selected conditions are absent from the fraction of batch that has been tested. If the microorganism are present in the product can be indicated by a turbidity in the clear medium. OBJECTIVE OF STERILITY TESTING: For validation of sterilization process. To check presence of microorganisms in preparation which are sterile. To prevent issue of contaminated product in market. K.I.P.M. GIDA, GORAKHPUR, U.P. STEPS INVOLVED IN STERILITY TE TESTING 1) Sampling 2) Selection of the quantity of the product to be used 3) Method of sterility testing i ) METHOD 1 Membrane filtration method ii) METHOD 2 Direct inoculation method 4) Observation and interpretation Must be carried out under aseptic condition. K.I.P.M. GIDA, GORAKHPUR, U.P. Sampling The sample must be representative of the whole of the bulk material & a lot of final containers. MAINLY FOLLOWED BY TWO RULES: A fixed percentage of the final container are selected. A fixed number of container are taken independent of the lot or batch size. K.I.P.M. GIDA, GORAKHPUR, U.P. According to Indian Pharmacopoeia following guidelines for determining the minimum number of items are: Selection of the quantity of the product to be used Selection of the quantity of the product to be used for sterility testing depends mainly on the volume or weight in the container. K.I.P.M. GIDA, GORAKHPUR, U.P. Method of sterility testing Membrane filtration method (METHOD 1): Membrane filtration Appropriate for : (advantage) Filterable aqueous preparations Alcoholic preparations Oily preparations Preparations miscible with or soluble in aqueous or oily (solvents with no antimicrobial effect) All steps of this procedure are performed aseptically in a Class 100 Laminar Flow Hood K.I.P.M. GIDA, GORAKHPUR, U.P. Membrane filter 0.45 porosity Filter the test solution After filtration remove the filter Cut the filter in to two halves First halves (For Bacteria) Second halves (For Fungi) Transfer in 100 ml culture media (Fluid Thioglycollate medium) Incubate at 30-35 0 C for not less then 7 days
Transfer in 100 ml culture media (Soyabeans-Casein Digest medium) Incubate at 20-25 0 C for not less then 7 days
Observe the growth in the media
Observe the growth in the media
K.I.P.M. GIDA, GORAKHPUR, U.P. Suitable for samples with small volumes volume of the product is not more than 10% of the volume of the medium suitable method for aqueous solutions, oily liquids, ointments an creams Direct inoculation of the culture medium suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium & incubate for not less than 14 days. K.I.P.M. GIDA, GORAKHPUR, U.P. Direct inoculation method (METHOD 2): Observation and results Culture media is examined during and after at the end of incubation. The following observations are possible: 1) No evidence of growth Pass the test for sterility. 2) There is evidence of growth Re-testing is performed same no. of sample, volume & media as in original test No evidence of growth Pass the test for sterility. 3) There is evidence of growth isolate & identify the organism. Re-testing is performed with twice no. of sample if: No evidence of growth Pass the test for sterility. There is evidence of growth Fail the test for sterility
K.I.P.M. GIDA, GORAKHPUR, U.P. Particulate matter monitoring Particulate matter is defined as unwanted mobile insoluble matter other than gas bubble present in the product. If the particle size of foreign matter is larger than the size of R.B.C.. It can block the blood vessel. The permit limits of particulate matter as per I.P. are follows:
Source of particulate matter: 1. Intrinsic contamination: The material which are originally present in the parenteral solution e.g. Barium ions leach in parenteral & react with sulphur ions in the product to form barium sulphate crystals. 2. Extrinsic contamination: The material which comes from the environment e.g. Shedding of material from cloth, body, & cotton, paper, rubber, tissue etc. K.I.P.M. GIDA, GORAKHPUR, U.P. Methods for monitoring particulate matter contamination: 1) Visual method 2) Coulter counter method 3) Filtration method 4) Light blockage method Identification of particulate matter: 1) Microscopy 2) X-ray powder diffraction 3) Mass spectroscopy 4) Polarized light spectroscopy 5) Scanning electron microscopy (SEM) K.I.P.M. GIDA, GORAKHPUR, U.P. Faculty seal packaging / leaking testing The sealed ampoules are subjected to small cracks which occur due to rapid temperature changes or due to mechanical shocks.
Vials & bottles are not suitable for this test because the sealing material used is not rigid. K.I.P.M. GIDA, GORAKHPUR, U.P. Filled & sealed ampoules Dipped in 1% Methylene blue solution Under negative pressure in vacuum chamber Vacuum released colored solution enter into the ampoule Defective sealing Pyrogen Testing Pyrogen = Pyro (Greek = Fire) + gen (Greek = beginning). Fever producing, metabolic by-products of microbial growth and death. Bacterial pyrogens are called Endotoxins. Gram negative bacteria produce more potent endotoxins than gram + bacteria and fungi. Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not present in cell-free bacterial filtrates Stable to at least 175 o C; steam sterilization ineffective Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm) so endotoxins can easily pass through 0.22m filters Sources: Water (main), raw materials, equipment, process environment, people, and protein expression systems if using gram negative bacteria. K.I.P.M. GIDA, GORAKHPUR, U.P. Biological properties of endotoxin Pyrogen elevate the circulatory levels of inflammatory cytokines which may be followed by fever, blood coagulation, hypotension Low doses of Pyrogen: asymptomatic inflammation reaction Moderate doses: fever & changes in plasma composition High doses: cardiovascular dysfunction, vasodilation, vasoconstriction, endothelium dysfunction, multiple organ failure & finally death. K.I.P.M. GIDA, GORAKHPUR, U.P. Sources of pyrogen 1) Equipment 2) Containers (Glass , plastic , metal) 3) Solvent 4) Solute Elimination of pyrogens Dry heat sterilization : For glass wares, metal equipments, powders, waxes, oils, heat stable drugs 650 o C temp - 1 min 250 o C temp - 30 min 180 o C temp - 240 min Ultra filtration Reverse osmosis : RO membrane is composed of cellulose acetate phthalate/ polyamide Distillation Adsorption method
K.I.P.M. GIDA, GORAKHPUR, U.P. Principal: Rabbits are used to perform this test because their body temp increases when pyrogen are introduced into their bodies by parenteral route 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are selected Do not use any rabbit having a temp higher than 39.8 o C Showing temp variation >0.2 o C between two successive reading in the determination of initial temp Sham test is performed within 7 days of actual test Animal showing temp increase over 0.6 o C should be removed from pyrogen testing K.I.P.M. GIDA, GORAKHPUR, U.P. Method : Dissolve the subs being examined in, or dilute it with a pyrogen free saline solution Warm the liquid being examined to approx. 38.5 o C temp before injection The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body weight Withhold water during test Clinical thermometer is inserted into the rectum of rabbit to record body temp 2 normal reading of rectal temp are should be taken prior to the test injection at an interval of half an hr & its mean is calculated- initial temp The solution under test is injected through an ear vein Record the temp of each rabbit in an interval of 30 min for 3 hrs The difference between initial temp & maximum temp is recorded- taken as response K.I.P.M. GIDA, GORAKHPUR, U.P. Interpretation of results K.I.P.M. GIDA, GORAKHPUR, U.P. Bacterial endotoxin (LAL) test ) To detect or quantify endotoxins of gram-ve bacterial origin Reagent: amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). The name of the test is also Limulus amebocyte lysate (LAL) test
K.I.P.M. GIDA, GORAKHPUR, U.P. Mechanism of LAL Test:
The test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located with the crab's amebocyte blood cells endotoxin
Initiation of an enzymatic coagulation cascade
proteinaceous gel Test performance (short) Avoid endotoxin contamination Before the test: interfering factors should not be present equipment should be depyrogenated the sensitivity of the lysate should be known Test: equal Volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube Incubation at 37C, 1 hour remove the tube - invert at (180) observe the result pass-fail test K.I.P.M. GIDA, GORAKHPUR, U.P. LAL test Three different techniques: 1. The gel-clot technique - gel formation 2. The turbidimetric technique - the development of Turbidity after cleavage of an endogenous substrate 3. The chromogenic technique - the development of color after cleavage of a synthetic peptide- chromogen complex K.I.P.M. GIDA, GORAKHPUR, U.P. Advantages of LAL test Fast - 60 minutes vs. 180 minutes Greater Sensitivity ,Less Variability Much Less False, Positives ,Much Less Expensive Alternative to Animal Model, cheaper, particularly useful for: Radiopharmaceuticals and cytotoxic agents Blood products Water for injection Production facilities of parenterals The production area where the parenteral preparation are manufactured can be divided into five sections: Clean-up area Preparation area Aseptic area Quarantine area Finishing & packaging area K.I.P.M. GIDA, GORAKHPUR, U.P. Clean-up area: It is not aseptic area. All the parenteral products must be free from foreign particles & microorganism. Clean-up area should be withstand moisture, dust & detergent. This area should be kept clean so that contaminants may not be carried out into aseptic area. Preparation area: In this area the ingredients of the parenteral preparation are mixed & preparation is made for filling operation. It is not essentially aseptic area but strict precautions are required to prevent any contamination from outside.
K.I.P.M. GIDA, GORAKHPUR, U.P. Aseptic area: The parenteral preparations are filtered, filled into final container & sealed should be in aseptic area. The entry of personnel into aseptic area should be limited & through an air lock. Ceiling, wall & floor of that area should be sealed & painted. The air in the aseptic area should be free from fibers, dust and microorganism. The High efficiency particulate air filters (HEPA) is used for air. UV lamps are fitted in order to maintain sterility.
K.I.P.M. GIDA, GORAKHPUR, U.P. Quarantine area: After filling, sealing & sterilization the parenteral product are held up in quarantine area. Randomly samples were kept foe evaluation. The batch or product pass the evaluation tests are transfer in to finishing or packaging area. Finishing & packaging area: Parenteral products are properly labelled and packed. Properly packing is essential to provide protection against physical damage. The labelled container should be packed in cardboard or plastic container. Ampoules should be packed in partitioned boxes
K.I.P.M. GIDA, GORAKHPUR, U.P. Lyophilization or freeze drying Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; Freezing, Primary drying (sublimation), and Secondary drying (desorption).
K.I.P.M. GIDA, GORAKHPUR, U.P. The advantages of Lyophilization include: Ease of processing a liquid, which simplifies aseptic handling Enhanced stability of a dry powder Removal of water without excessive heating of the product Enhanced product stability in a dry state Rapid and easy dissolution of reconstituted product Disadvantages of Lyophilization include: Increased handling and processing time Need for sterile diluent upon reconstitution Cost and complexity of equipment
K.I.P.M. GIDA, GORAKHPUR, U.P. The Lyophilization process generally includes the following steps: Dissolving the drug and excipients in a suitable solvent, generally water for injection (WFI). Sterilizing the bulk solution by passing it through a 0.22 micron bacteria-retentive filter. Filling into individual sterile containers and partially stoppering the containers under aseptic conditions. Transporting the partially stoppered containers to the lyophilizer and loading into the chamber under aseptic conditions. Freezing the solution by placing the partially stoppered containers on cooled shelves in a freeze-drying chamber or pre- freezing in another chamber. Applying a vacuum to the chamber and heating the shelves in order to evaporate the water from the frozen state. Complete stoppering of the vials usually by hydraulic or screw rod stoppering mechanisms installed in the lyophilizers.
K.I.P.M. GIDA, GORAKHPUR, U.P. There are many new parenteral products, including anti-infectives, biotechnology derived products, and in-vitro diagnostics which are manufactured as lyophilized products. Additionally, inspections have disclosed potency, sterility and stability problems associated with the manufacture and control of lyophilized products.