Shivani Rao

Stability studies are performed on all pharmaceutical products before they
are marketed. These include the exposure of the products to stress conditions
for short time period.

Preservatives or antimicrobials are added in some of
the products in view of protecting it against contaminants during the usage
or storage by the consumer.
This renders the testing of the efficacy of the preservative as a necessity
during stability studies. Preservative efficacy testing can be done by analytical
or microbiological methods.  The article highlights importance of preservative
efficacy testing during stability studies, the regulatory status and need
for harmonization.

The rapid
methods for testing preservative testing are also described.

Antimicrobial Preservation

Antimicrobial
preservatives are substances added to non-sterile
dosage forms to protect them from microbiological growth or from microorganisms
that are introduced during or subsequent to the manufacturing process^{1, 2}.
In the case of sterile articles packaged in multiple dose containers,
antimicrobial preservatives are added to inhibit the growth of microorganisms
that may be introduced from repeated withdrawal of individual doses.

The preservative
has no influence on the ingress of organisms into the product, which is
generally due to container design or inability to follow good manufacturing
practices (GMP) during production. The function of preservative is to shield the product from spoilage
during use.

The safety of
consumer is the vital purpose of the preservative. A product that is
susceptible to microbial spoilage is deficient in several respects.

• It may pose an infection hazard to the consumer if it supports the
growth of pathogens.
• Degradation of the active ingredient may be ensued due to microbial
metabolism leading to loss of the product.
• The metabolites of microorganisms may modify the excipients chemically
resulting in physical instability.
• The organoleptic properties may be altered due to microbial growth
rendering the product unacceptable

A pathogen contaminated product can have severe consequences both for the
user and the manufacturer. To avoid these consequences it is necessary to
protect the product against these effects with the aid of adequate preservative.
It should be noted that the preservative should not be used for compensation
of the manufacturing deficiencies arising due to non compliance with the GMP’s.

Selection of preservative

The preservative must be largely undissociated at the pH of the formulation.
Product storage temperature must not harm, as well as other active substances
should not interact with the preservatives, this may alter the effective concentration
required for its function³.

Importance of antimicrobial efficacy testing during dosage form stability
studies

Pharmaceutical
products are subjected to long term as well as short term (stress testing)
stability studies to forecast the time for which product remains within
specifications. The short term stability studies are used for rapid assessment
of stability and as support to long term studies ⁴. International
Conference for Harmonization advocates short term stability testing of new drug
products at 40 ºC and 75 % relative humidity. Since the preservative, an
antimicrobial substance, is aiding in the keeping quality of the product it is
necessary to ensure the activity of the same is maintained and that effective
concentration is maintained till the expiry of the product. This necessitates
analyzing the concentration of the preservative during stability studies.
Relying on chemical assays to provide the necessary assurance of preservative
protection would not be sufficient. Since the chemical assay value may be
unchanged but the biological activity is altered. This may arise due to,

• Modification in the antimicrobial activity in presence of the excipients
in the formulation.
• The preservative may lose its activity due to adsorption onto suspended
solids or partition into the non aqueous phase of an emulsion.
• Leaching of the ingredients from the container may interact with the
preservative and modify its activity.
• The level of bioburden associated with the excipients is one common
source of contamination in nonsterile pharmaceuticals if it is derived from
animal, botanical or mineral sources.
Apart from the concentration of the antimicrobial preservative the activity
may be influenced due to pH and redox potential which may change during storage.

• The preservative may degrade resulting in an enhanced activity due
to its degradation.
• Presence of two or more type of preservative system increases the
complexity of the analytical assays.

 These factors clearly points to the requirement of the assessment of the
preservative efficacy biologically as well as analytically during stability
studies.^{5, 6}

Microbiological test design for efficacy testing of antimicrobials:

The articles
to be tested are classified based upon categories assigned by the regulatory
authorities² and accordingly are tested as per
flow chart on page no 18

The test design includes the challenge of the preservative with the
organism and determination of the growth or the inhibition of the microorganism
by counting the cfu/ml in comparison with the reference and the positive control.
This is done either by direct inoculation of the challenge organism in the
original containers of product or if the use of the final container does not
permit inoculation and mixing of the inoculum (e.g. cream and ointment tubes),
then the product should be expelled from the market pack into a sterile borosilicate
glass container capped biological containers of suitable size.7   The inoculum
to be added is as per the table no 1

Adequacy of neutralization of Preservative at the time of sampling

Since the
counting will be done from the samples containing the microorganism, it is
necessary to neutralize the preservative present to ascertain the exact count
of the microorganism.

To achieve
this, the counting may be done by the following ways

• Dilution of the sample to make the concentration of the preservative
negligible.( May lead to dilution of the contaminants also)
• Addition of a non-toxic neutralizing agent of the preservative.
• Usage of membrane filters (provided the sample passes through it)
to adsorb the contaminants on it and on washing remove the preservative.

Factors influencing reproducibility

The preservative efficacy tests may be influenced by many parameters as

• Test organisms used are intended to be examples of those that might
be realistically represent contaminants of the product from the user.
• An infection hazard posed by the products intended use.
• The raw materials used in the manufacturing and its contaminants.
• Presence of contaminants which survive under unfavorable conditions

Major difference in approach of EP and USP is that EP recommends efficacy
testing as requirement in product development documentation while USP recommends
the same as mandatory product performance testing.

Preservative efficacy assessment by rapid methods:

Since the tests for preservative efficacy is time consuming, they require
a 28 day sampling period (in addition to incubation time) and lot of operator
input. They are not quantitative, cannot be extrapolated to statistical evaluation,
and utilize large volumes of materials; it has become the need of the hour
to develop a testing procedure that would help to diminish above shortcomings.

Various attempts are made to address these issues. The methods are so designed
so as to be useful in new product development process⁸. These tests
are not replacements but prelude to Pharmacopeial testing procedures. The
term rapid assumes two different meanings:

1)  One uses traditional microbiological technique but short sampling
period which are extrapolated to predict efficacy over 28 days. The fact that
microbial inactivation follows first order kinetics makes it possible to use
linear plot of log of viable cell count vs. exposure time and use D (decimal
reduction values) to predict surviving fraction after longer exposure. Such
procedures are reported.^9. Such procedures have limitations such
as small error in calculation of D value or slope is magnified in large error
in calculated percentage of survivors projected over 28 days. Test organisms
may grow back after initial decline to higher than initial value. Many organisms
may not follow first order kinetics of inactivation or pseudo first order
in case of gross excess of preservative. Most probable number technique (MPN)
using microtitre plates have also been used for this. ¹⁰

2)
Procedures where sampling period is not shortened but measure of viability is
completed within shorter time than that required for plate count. These employ
alternative to traditional counting procedures based on biomedical indexes.
These are the substances which are present in a living cell which can be used
as marker for detection of these organisms. The methods used to detect these
markers include bioluminescence, viability staining using dye reduction and
conductance impedance methods that detect electrical changes in culture media
resulting from microbial growth.^{11, 12, 13, 14, 15, 16.}

Biochemical Indexes

The measurement of microbial biochemical residues or enzymes to index the
level of contamination has been gaining popularity with industrial applications
over the last decade.¹⁷

The biochemical indexing methods can provide results that are truly
useful. For example, a 10³ CFU contamination level will be detected
by biochemical indexing methods within a time frame to add additional biocide
or take other measures to prevent the establishment of gross contamination of
critical system components, while time duration required for obtaining results
using culturing methods the count of 10³ CFU can grow 500-fold.

Adenosine Tri-Phosphate (ATP)
is the most popular biochemical index as it is ubiquitous in cellular life
forms and can be detected rapidly using
bioluminescence reactions.
Bioluminescence is the emission of light by biological methods using Luciferase
enzyme¹⁸. Luciferase, together with its co-factors D-Luciferin and
Oxygen, produces light in the presence of ATP according to the following
reaction:

Luciferase + D-Luciferin + O₂ +
ATP --> Luciferase + oxy-luciferin + CO2+ AMP + PPi+ Light

The amount of light is proportional to the concentration
of ATP in the original sample. The ATP concentration in a sample is, in turn,
related to the number and types of organisms within the sample. Thus a relative
index of the amount of contamination can be generated using firefly bioluminescence
within a few minutes of sampling. Other chemicals can be used to index the
levels of microbial contamination in addition to ATP.

The enzyme Adenylate Kinase
(AK) is another compound which can be used as a biochemical index. AK is an
enzyme that is constitutively expressed by cellular life forms regardless of
growth phase (with the possible exception of spores). AK catalyses the
following reaction:

ADP + ADP --> ATP + AMP

By supplying a buffer containing an excess of ADP, the
presence of AK in the sample produces significant quantities of ATP which, in
turn, is consumed by fire-fly luciferase to produce light and ADP. The
luciferase will also detect the ATP already present in the sample in addition
to that produced by AK. Thus, the signal generated by AK/luciferase system
includes both the ATP from the sample and the AT P produced as a result of the
AK catalytic activity.

Advantage of using AK over ATP as biomedical index:

• AK is constitutively produced by organisms in any growth phase (except
spores) and is generally produced at a fairly consistent level. ATP, in contrast,
may vary considerably in intra-cellular concentration depending on growth
phase and nutrient availability.
• A single molecule of AK enzyme can produce an amplified signal because
of its catalytic activity. Specifically, a single molecule of AK can produce
copious amounts of ATP during a short incubation period. Because of this amplification,
as few as 10 cells will produce a signal that is detectable over background.

• Both ATP and AK indexes are rapid (1 min. for ATP; 1 hr for AK) and
provide “real-time” results.

Impedance is the term defined as the resistance to flow of an
alternating current through a conducting material. In an electrolyte solution
exposed to electric field the
amount of current is proportional to the extent of the concentration and
mobility of ions¹⁹.

In simple terms, an impedance system measures the net changes in impedance in the culture medium at
regular intervals. The
test values are pre-determined for a particular system in use. If the values
measured exceed these limits, the system detects growth.

An increase in conductance, which is the reciprocal of
the resistance, corresponds to decrease of impedance and an increase in current which is measured in Siemens (S) units. There are several systems
commercially available that use this technology for enumeration of
microorganisms, e.g., Bactometer® (bioMerieux), BacTrac® (Sy-Lab), Malthus®
System (Malthus Instruments), RABIT® (Rapid Automated Bacterial Impedance Technique, Don Whitley
Scientific.

The composition of microbial growth media is relatively large
uncharged or weakly charged molecules, e.g., carbohydrates, fats and proteins.
During the process of microbial metabolism, these large molecules are broken
down into smaller and more highly charged components, e.g., fatty acids, amino
acids and other organic acids. The highly charged molecules cause a change in
the media’s electrical
conductivity / resistance. These changes indicate the presence of
microorganisms in the original sample.

Time to Detection
(TTD) is the point at which the changes in biomedical index measurements are recognized as a result of
microbial activity. This is measured at a point where the cellular growth has
reached a critical mass, usually about 10⁶cells/mL. There is a known
relationship between the numbers of cells present in the original sample to the
TTD which corresponds to the growth curve.

Furthermore, the results of TTD are reproducible, i.e., using traditional methods for
enumeration of microorganisms like serial dilution will yield a standard curve
against the TTD, if all the samples are exactly the same material. This allows
for one to give a direct estimate of the number of microorganisms present in
the sample and the growth rate of the microorganisms.

For pharmaceutical applications, there are a wide array
of dosage forms and concentrations making it difficult to select one standard
curve for all product types. This necessitates validation by product type or by
use of a wide range of products in the validation to establish the standard
curve. The reason that this is necessary is because the microorganism’s growth
rate and metabolic conditions vary from sample to sample and result in a
corresponding change in the TTD.

There are several factors that influence the TTD
including:

•Initial microbial population

•Growth kinetics of the organism

•Properties of the test medium

•TTD only correlates when the generation time of the test
population is more or less constant under the experimental conditions, which
implies that the incubation temperatures have to be kept constant as this
affects generation time.

This method can
be used in the various microbiological testing of the pharmaceutical products
and the components used for its preparation e.g., raw material
bioburden, in-process bioburden, microbial limits testing, quantitative and
qualitative analysis of antimicrobial activity, susceptibility to antibiotics,
sterility testing, generating predictive models of cellular growth, detection
of pathogens etc.

Conclusion

The stability studies for pharmaceutical products must
also include testing of antimicrobial preservative effectiveness. To achieve
this, rapid methods for determination of preservative efficacy must be
employed. Use of these methods will ensure that time and labor required for the
testing will drastically reduce. Since these rapid methods are based upon the
presence of living organisms they are the most reliable, especially the
electrical method as well as the luciferase system seems to be the most
reliable and convenient. In due course, it is necessary to implement these
methods in the pharmaceutical industry with harmonization in the approach of
the regulatory authorities. Disadvantage of the use of ubiquitous biochemical
agents as indexes is that they do not differentiate between classes of
microbes. There have been some recent advances in the ability to index enzymes
specific to certain species that may allow the rapid enumeration of specific microbes without culturing, but these
tests are currently not user-friendly. Because of the number of specific advantages
of usage of biochemical indexing
methods, number of people using
these methods may well influence even the most reluctant entities in their
opinion of these tools.

Acknowledgements:

The authors sincerely
acknowledge the support and guidance of Dr.K.G. Bothara Principal of AISSMS
College of Pharmacy, Pune.

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Microbiological test design for efficacy testing of antimicrobials: (Flow
Chart)

The
requirements for antimicrobial effectiveness are met if the criteria specified
under table no 2 are attained.

Table no 1:     Pharmaceutically
useful preservatives