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.