Quality control of parenteral preparations cont.

Bacterial endotoxins ● To detect or quantify endotoxins of gram-negative bacterial origin. ● A test sample is incubated with amoebocyte lysate from the blood of the horseshoe crab (Limulus polyphemus). ● The name of the test is known as Limulus Amoebocyte Lysate (LAL) test. ● This test is more sensitive, more rapid, and easier to perform than the rabbit test.

Mechanism of LAL The test is based on the primitive blood-clotting mechanism of the horseshoe crab proteins located with the crab's amebocyte blood cells initiation of an enzymatic coagulation cascade proteinaceous gel endotoxins

4- Sterility testing for parenteral products Definition : Sterility testing attempts to reveal the presence or absence of viable micro- organisms in a sample number of containers taken from batch of product. Based on results obtained from testing the sample a decision is made as to the sterility of the batch. ► The purpose of a sterility test is to determine the probable sterility of a specific batch.

● Is made after the product exposition to the one of the possible sterilization procedures. ● Can only provide partial answers to the state of sterility of the product batch under test. ● Is inadequate as an assurance of sterility for a terminally sterilized product. ► The USP official tests are the direct inoculation of the culture media with the product to be examined, and technique of membrane filtration method. ► Appropriate negative controls are included. Sterility test

● The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test. ● Membrane filtration, use membrane filters having a nominal pore size not greater than 0.45µm whose effectiveness to retain micro- organisms has been established. ● Cellulose nitrate filters, are used for aqueous, oily and weakly alcoholic solutions. Cellulose acetate filters, for example used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products, e.g. for antibiotics.

● The filtration apparatus and membrane are sterilized by appropriate means. ● The apparatus is designed so that the solution to be examined can be introduced and filtered under aseptic conditions. ● The direct inoculation of the culture medium ◘ Transfer the quantity of the preparation to be examined directly into the culture medium so that the volume of the product is not more than 10% of the volume of the medium. ◘ If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of the culture medium. ◘ When it is necessary to use a large volume of the product it may be preferable to use a concentrated culture medium prepared in such a way that it takes account of the subsequent dilution. Where appropriate, the concentrated medium may be added directly to the product in its container.

● two sterile culture media: a-Thioglycollate which incubated at 32 ºC for 2 weeks b-Soybean casein digest incubated at 22 ºC for 2 weeks Observation and interpretation of the results: ● At intervals during the incubation period and at its conclusion, examine the media for macroscopic evidence of microbial growth. ● If the material being tested renders the medium turbid so that the presence of microbial growth cannot be readily determined by visual examination, 14 days after the beginning of incubation transfer portions (each not less than 1ml) of the medium to fresh vessels of the same medium and then incubate the original and transfer vessels for not less than 4 days. ● If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. ● If evidence of microbial growth is found the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined.

● If the test is declared to be invalid it is repeated with the same number of units as in the original test. ● If no evidence of microbial growth is found in the repeat test the product examined complies with the test for sterility. ● If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.

► The interpretation of sterility results is divided into two stages by the USP relative to the type of sterility failure if one occurs. ● If sterility failure of the test samples occurred because of improper aseptic technique, or as a fault of the test itself. Stage 1 may be repeated with the same sample size. ● Sample size is doubled in a stage 2 testing, which is performed if microbial growth is observed in stage 1, and there is no reason to believe that the test was invalid. ● The only absolute method to guarantee the sterility of a batch would be to test every vial or ampoule.

● Confidence in the sterility test is dependent on the fact that the batch has been subjected to a sterilization procedure of proved effectiveness. ● Records of all sterility tests as well as temperature recording and records from autoclaves, ovens, or other equipment used during the manufacturing process must be maintained. ● All sterilizing equipment must be validated to ensure that the proper temperatures are obtained for the necessary time period. ● These validations are obtained by the use of thermocouples, chemical and biological indicators, sealed ampoules containing culture medium with a suspension of heat-resistant spores, and detailed sterility testing.

Major factors of importance in sterility testing ► The environment in which the test is conducted ►The quality of the culture conditions provided ►The test method ►The sample size ►The sampling procedure

5- Leaker testing and sealing verification ● Ampoules that have been sealed by fusion must be tested to ensure that a hermetic seal was obtained. ● The leaker test is performed by immersing the ampoules in a dye solution, such as 1% methylene blue, and applying at least 25 in. (64 cm) of vacuum for a minimum of 15 minutes. ● The vacuum on the tank is then released as rapidly as possible to put maximum stress on weak seals. Next, the ampoules are washed. ● Defective ampoules will contain blue solution.

● Another means of testing for leaker is a high-frequency spark test system developed by Nikka Densok Company of Japan, which detects pinholes in ampoules. ● Some advantages of this system include higher inspection accuracy, higher processing speed, and eliminating the possibility of product contamination.

● Bottles and vials are not subjected to such a vacuum test because of the flexibility of the rubber closure. However, bottles that are sealed under vacuum may be tested for vacuum by striking the base of the bottle sharply with the heel of the hand to produce a "water hammer " sound. ● Another test is the spark test, in which a probe is applied outside the bottle. When it reaches the air space of the bottle, a spark discharge occurs if the headspace is evacuated. ● The microbiological integrity of various packages, such as vials and stoppers, disposable syringes, and plastic containers, should be determined.

Labeling The label states the name of the preparation (in case of a liquid preparation) The percentage content of drug in a specified volume (in case of dry preparation) The route of administration Storage condition Expiration date Name of the manufacturer The lot number ● The labeling for parenteral dosage forms are integral and critical parts of the product. ● The labeling must be legible, and clearly identify the drug, its concentration, handling or storage conditions, and any special precautions.

Containers for injection that are intended for use as dialysis, or irrigation solution are labeled to indicate that the contents are not intended for use by iv infusion Injection intended for veterinary use are labeled to that effect The containers are so labeled that a sufficient area of the container remains uncovered for its full length to permit inspection of the contents