In the pharmaceutical industry, defining
the control strategy for a new
active pharmaceutical ingredient
(API) is a main goal during development
of the process for its commercial
manufacture. This task includes
defining measures to limit microbial
content and specifying microbial
quality attributes as needed to ensure
patient safety. International Conference
on Harmonization (ICH) Guideline
Q6A1 provides a framework for
determining on a case-by-case basis
when microbial limits should be
established. The guidance document
identifies information to be assembled
during development, and provides a
decision tree for determining the need
for microbial testing of the drug substance.
This paper illustrates how such
data may be collected and evaluated
to ensure a robust microbial control
strategy that is consistent with ICH
Guideline Q6A.
Microorganisms may be introduced
into APIs via the materials used in
their manufacture and through various
environmental sources during
processing. Once microbes are present,
proliferation may occur when
conditions are favorable. Likewise,
there are key control points that
can effectively limit bioburden (the
number of microorganisms present
in a material) and control microbial
growth. A comprehensive microbial
control strategy involves limiting the
potential for contamination during
API manufacture, relying on the bactericidal
conditions of the chemical
synthesis to limit viability of microorganisms,
and appropriately controlling
water in the API that is available
to sustain microbial growth during
storage. These opportunities for
control are discussed in the context
of establishing a microbial control strategy for nonsterile APIs produced
through chemical synthesis.
Engineering controls provide the
means to minimize the potential
for microbial contamination during
processing. In general, materials are
manufactured in a high containment
facility. The room air is filtered (bag
filter with a high-efficiency particulate
air [HEPA] prefilter), heated as
required, and distributed throughout
the building. Personnel wear protective
equipment that protects the API
from human-sourced microbial contamination.
Personnel gowning areas
and equipment air locks are part of
the facility design to control the flow
of airborne particles. Equipment is
designed to minimize dead legs and
is sloped to allow drainage to reduce
opportunities for water to be trapped
in the equipment, which could permit
microbial growth. Manufacturing
equipment is typically cleaned using
antimicrobial agents and dried by
purging with nitrogen.
The manufacture of an API via chemical
synthesis involves a sequence
of operations that transform starting
material(s) into the desired drug
substance. Raw materials including
organic solvents may be utilized at various
points in the chemistry. Processing
conditions such as temperature or
pH are often manipulated to control
reactions and isolate products. Steps
in the synthesis are typically performed
in an oxygen-free environment to
ensure safety and minimize oxidative
by-products.
While essential for control
of the chemical synthesis, each of
these variables also offers the potential
to affect bioburden of the API.
Table 1 - Solvent–microbial viability relationship*
Microorganisms cannot thrive in nonaqueous
environments. In fact, many
of the organic solvents commonly used
in the manufacture of nonsterile APIs
are bactericidal. The solvents listed
in Table 1 were evaluated for their
antimicrobial properties. The microorganisms
tested represent various
classes of organisms commonly found
as contaminants in the manufacturing
environment. In general, the solvents
inoculated with Gram-positive bacterial
endospores did not show a significant
antimicrobial effect toward these
microorganisms. Bacterial endospores
contain dipicolinic acid in the spore
coat, which enables the spores to resist
some heating, chemicals, and desiccation
effects when other microorganisms
are killed by these conditions.
Vegetative bacteria and the fungal
spores experienced nearly instantaneous
lethality due to the organic solvents’
antimicrobial activity.
Processing conditions such as temperature
and pH can have a significant
effect on microbial viability.2–4 In general,
microorganisms commonly found
in the API pharmaceutical manufacturing
environment thrive at pH 6–8.
Conditions outside this pH range are
usually deleterious to organisms. While
the temperature required to destroy a
microorganism depends upon the individual
organism and the other environmental
conditions, temperatures above
60 °C are effective in killing most vegetative
bacterial cells, and temperatures
above 100 °C kill most microorganisms.
In 100% aqueous media, microorganisms
grow very little or not at all at
temperatures of 0–5 °C.
Many of the common microorganisms
require oxygen for growth. Executing
the chemical process in an
oxygen-free environment obtained
by purging with nitrogen and processing
under a nitrogen headspace effectively reduces oxygen content
and limits microbial viability.
Packaging of the active pharmaceutical
ingredient is a seemingly simple
but important parameter to consider
in protecting the material from microbial
proliferation during storage. All
microorganisms require water to grow
and survive.4–6 Water is the one key
ingredient needed for metabolic and
nutritive purposes and to maintain
the structural integrity of the cell wall.
Packaging the drug substance with
a foil-based outer liner provides an
effective moisture barrier.
Given the means to limit bioburden
and control microbial growth afforded
through engineering controls, synthetic
process conditions, and packaging
components, there remains potential
for microbial proliferation in the API
during storage when water is present.
Water may be part of the crystalline
lattice (“bound” water) or adsorbed to
the surface (“free” water). Of the two
types of water described, it is the free
water, not the bound water, that is
required for microbial growth. Since
total water in a compound is not necessarily
directly related to the potential
for microbial proliferation, a technique
to measure the free water is needed to
assess the quality of material.
Water activity describes the amount of
free water in a material that is available
for microbial or chemical reactions. It
is defined as the ratio of partial pressure of water above the material (p) to the
partial pressure of pure water (pw) at
the same temperature.4,5,7
Water activity instruments measure
the equilibrium relative humidity
of a material in a sealed or
enclosed container (as compared to
hygrometers, which measure relative
humidity under dynamic conditions).
The test material is placed
in a closed cell fitted with humidity
and temperature probes. Following
an equilibration period, the instrument
measures the temperature and
vapor pressure generated by moisture
present in the test material to
determine the water activity.