Assessment of Microbial Contamination And Spoilage

Assessment of Microbial Contamination And Spoilage

Early indications for microbial spoilage are gas production, pH changes, and organoleptic changes like smell, sour, fishy amines, bad eggs, earthy taste, bitter, etc. Microbial polymerization of sugars and surfactant molecules can produce slimy, viscous, masses in syrups, shampoos, and creams, and fungal growth in creams produces 'gritty’ textures. Acidic or basic microbial metabolites change the pH of the drug and also enhance microbial growth which is inhibited by the initial pH. This contamination and spoilage is assessed by quality control tests. As per the Pharmacopoeial and regulatory limits, the maximum permissible numbers of microorganisms in manufactured products or raw materials are not more than 100–1000 colony-forming units (cfu) per ml or gram. The risk of microbial infection and spoilage arises from microbial contamination during manufacture, storage, and use and that is eliminated by presenting all medicines in sterile, impervious, single-dosage units. Beyond these, some pathogens initiate infections resulting in detectable changes in chemical composition, physical appearance, or stability.


Assessment of microbial contamination and spoilage is also known as microbial quality control. This includes laboratory applications like sterility testing, bioburden determination, air monitoring, and product testing. These methods are conducted to:


• monitor microbial contamination of raw materials.

• monitor and confirm the efficacy of sterilization.

• control the risk from pathogenic microorganisms by confirming their absence.


Quality means meeting the predetermined requirements of users for a particular substance or service.


Quality includes: Total Quality management (TQM), Continuous Quality Improvement (CQI) which are under Good Pharmaceutical Manufacturing Practice (GPMP), and Quality Assurance (QA).


It is possible that the presence of the microorganisms can be detectable by:


• Their physical presence (cloudiness in liquid medicines, molds on, or in, creams and syrups, or discoloration of tablets stored in a damp environment).

• Changes in color (pigment production).

• Smell (due, for example, to amines, acetic or other organic acids, or sulfides from protein breakdown).

• Gas accumulation without any obvious odor (bubbles of carbon dioxide following sugar fermentation).


Quality Control:


The methods for counting and detecting some microbes in non-sterile products have poor accuracy and precision. For example, low-number microorganisms sometimes damage the product and cannot be isolated, products with active spoilage yield very low microbial count, or microorganisms in high numbers but it is not the primary spoilage agent nor pathogenic. The uneven distribution of microorganisms in viscous formulations provides a serious sampling problem. The type of culture medium and conditions of recovery and incubation may influence viable counts obtained. Pharmacopoeias (Ph Eur, BP, and USP) have included quantitative and qualitative standards for non-sterile products. The British Pharmacopoeia and the European Pharmacopoeia describe a single-challenge preservative test that routinely uses four test organisms (two bacteria, a yeast, and a mold) that are used for some specific situations. There are limits to maximum total microbial levels, for example: Oral product total viable count: 103 aerobic bacteria, 102 fungi/g or ml, and exclusion of specific microorganisms depending on the route of administration, e.g. E. coli and Salmonella. Higher levels are permissible if the product contains raw materials of natural origin. There is more interest in rapid methods: e.g. fluorescent dyes and epifluorescence, use of vital signs (e.g. detection of ATP using luciferase), and Endotoxin (pyrogen) levels in parenterals must be very low to prevent endotoxic shock. Formerly this was tested by injecting rabbits and noting any febrile response. Nowadays the test is performed using the Limulus Amoebocyte Lysate (LAL) test (gel clot method) where an amoebocyte lysate from the horseshoe crab Limulus polyphemus reacts specifically with microbial lipopolysaccharides to give a gel and opacity even at very high dilutions. Tissue culture tests are under development where the ability of endotoxins to induce cytokine release is measured directly.


Sterility Testing:


Sterility testing of pharmaceutical articles is required during the sterilization validation process as well as for routine release testing. It is a very tedious and artful process that must be performed by trained and qualified laboratory personnel. Sterility testing is an essential part of sterilization validation. It is designed and executed to eliminate false positive results. False positive results are generally due to laboratory contamination from the testing environment or technician error. These tests are suitable to reveal the presence of viable forms of bacteria, fungi, and yeast in pharmaceutical products or devices. It is a testing procedure to ensure the batch of products is sterile or has been sterilized, meaning free of viable microorganisms like bacteria, yeast, or molds.


Good Pharmaceutical Manufacturing Practice (GPMP):


Quality Control (QC) is the part of GPMP that deals with specification, documentation, and assessing conformance to specification. A high assurance of overall product quality is raised only from a detailed specification, control, and monitoring of all the stages that contribute to the manufacturing process. Parametric release is accepted as an operational alternative to routine sterility testing for batch release of some finished sterile products where the manufacturer can provide assurance that the product is of the stipulated quality, based on the evidence of successful validation of the manufacturing process and review of the documentation on process monitoring carried out during manufacturing.


Quality Assurance (QA):


It is a combined scheme of management that embraces all the procedures necessary to provide a high probability that medicine will conform consistently to a specified description of quality. It includes formulation design and development (R&D), good pharmaceutical manufacturing practice (GPMP), quality control (QC), and post-marketing surveillance. The risk of microbial infection and spoilage is raised from microbial contamination during manufacture and storage and hence preservatives are recommended for further protection against environmental microbial contaminants but it is relatively non-specific in their reactivity. Laboratory tests are devised to challenge the product by using ‘preservative challenge tests’ where relatively large inocula of various laboratory cultures are added to aliquots of the product and determine their rate of inactivation by viable counting methods (single challenge tests).


The Preservative Challenge Test (Antimicrobial Effectiveness Test):


It is required for the assessment of the microbial preservation of multiple used cosmetics and pharmaceutical products. They are added to products to prevent or limit microbial contamination, which occurs during normal conditions of storage and use. The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation, or by the formulation in which it is incorporated, or by the container and/or closure being used as the final packaging material. The test method is qualified for the product under evaluation to ensure that the correct diluents are used in assays for surviving microorganisms. The product is inoculated with a specified number of each challenge organism. The inoculated product is held at room temperature for 28 days and examined by the duplicate plate count method to determine the number of viable microorganisms that survive at each specified time interval.


Results are evaluated with the tabulated acceptance criteria of the relevant Pharmacopoeias or test protocols.


Post-market Surveillance:


It is the most important stage to follow up on a medicine that is smoothly floating in the market without any complaints by the customers. A proper quality assurance system is included for monitoring in-use performance and for responding to customer complaints. These are constantly followed up in great detail in order to decide carefully constructed and implemented schemes for product safety.

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