he highest possible quality of an end product, in compliance with requirements and regulations, can be attained only if quality assurance is not merely limited to final product testing. Rather, the entire manufacturing process, besides incoming quality control of the raw materials used, needs to be continuously monitored.
pharmaceutical industry, risk analysis of individual manufacturing steps
is performed and the results of this analysis are used to define
in-process quality control tests. Such QC tests permit timely detection
of inconsistencies or non-conforming items and, in particular, increases
in the bioburden as they occur in manufacture so that corrective action
can be promptly initiated. Even though the risk of contamination has
been considerably reduced by GMP-compliant production, decontamination,
and sterilization of the end products, as well as by strict hygiene
standards, quality control of the final product continues to be of prime
analysis of microorganisms involves counting the colony-forming units
(CFU), hence the term “microbial enumeration.” This number can be
expressed either as the total viable number of CFUs in general or of
certain product-relevant species of microorganisms. This is why
microbial limit tests are performed on various products from different
sectors, including the pharmaceutical, beverage, and waste water
industries, to ensure that defined limits are not exceeded. The accuracy
and reliability of microbial limit test results are essential as they
serve as the basis for the release of products, whether potable water or
pharmaceuticals, and the impact of undetected pathogens can be
potentially devastating on the health of consumers.
microbial enumeration, membrane filtration continues to be the method
of choice for reliable quantification of microorganisms in liquid
samples. The principle of this method is based on the concentration of
organisms—which are filtered out from relatively large sample volumes—on
the surface of a membrane filter and their subsequent cultivation by
incubating the filter with the retained microbes on a culture medium.
direct incubation of a sample, membrane filtration offers the advantage
that large sample volumes can be tested without individual
microorganisms going undetected. In addition, inhibitors, such as
antibiotics or preservatives, can be removed by rinsing the membrane
with buffer so that microbial growth is not suppressed.
Microbiological tests in the pharmaceutical industry
a microbiological viewpoint, pharmaceuticals can be subdivided into two
categories: non-sterile and sterile products. For both categories, the
potential risk resulting from microorganisms and their toxins on
patients’ health must be eliminated or at least mitigated. At the same
time, the quality and effectiveness of such pharmaceuticals must be
Products defined as sterile, such as eye drops,
physiological saline, antibiotics, etc., need to be tested for sterility
(USP Chapter 71 and EP, Chapter 2.6.1) in order to be verified as such.
Unlike sterile products, non-sterile end products are tested for their
number of viable microbes according to the microbial limit test (USP
Chapter 61 and EP Chapter 2.6.12). Furthermore, in the pharmaceutical
industry, in-process microbiological quality control tests are carried
out on raw materials, mostly water, as well as bioburden analysis during
Critical steps in microbial enumeration
classic equipment setup for performing membrane filtration consists of a
vacuum pump, a multi-branch vacuum manifold, membrane filters, reusable
funnel-type filter holders or single-use filtration units, culture
media, and tweezers.
In this method, the filter support of a
reusable filter holder is sterilized by flaming, and a membrane filter
is subsequently placed on this support. Then the funnel is attached to
the support and a sample is poured into this funnel. Filtration begins
when the tap on the vacuum source is opened. At the end of filtration,
tweezers are used to remove the membrane filter and transfer it to an
agar culture medium.
The culture medium is incubated for a
defined time at a predetermined temperature inside an incubator. At the
end of incubation, evaluation is done by enumerating the individual CFUs
and comparing their count with the permissible microbial limits for
each particular sample.
Flaming or disinfecting the filter
support poses an added risk of contamination due to the inherent
inaccuracy in performing these sterilization procedures. In particular,
maintaining the required time of contact with the flame or disinfectant,
the choice of disinfectant (not just a bactericide, but a sporicide)
and regular changing of the disinfectant are all critical factors in
determining whether sterilization is 100% effective. Besides
representing a health hazard for lab personnel, flaming also poses the
risk that not all areas contaminated by microbes are exposed to the
hottest point of the flame long enough in order to kill off these
Minimization of secondary contamination
single-use filter unit does not require any decontamination, provided
that a single-use filter base is used. As a result, the only especially
critical step that remains is transferring the membrane filter to an
agar medium, which increases the risk of secondary contamination and can
lead to false-positive results. The reason lies in the use of tweezers
to transfer the membrane. Although these tweezers are also flamed, i.e.,
sterilized, they can potentially carry over exogenous microbes when
used to grasp the membrane.
Single-use filter units increase the
safety and efficiency of microbiological quality control by eliminating
the need for disinfection or flaming of the filter support, as well as
for using tweezers to transfer a membrane to a culture medium. A system
comprised of single-use filter units and agar media dishes can increase
efficiency and reliable results.
The filter unit in this type
of system is a sterile, ready-to-use combination of a funnel, a filter
base, and a gridded membrane filter. This filter unit is connected to a
stainless steel multi-branch manifold in order to directly filter a
sample. Afterwards, the filter unit is easy to remove from the manifold
and eliminates the critical step of decontaminating the stainless steel
base of a reusable filter holder.
Agar media dishes are used for
microbial limit testing. They are pre-filled with different types of
agar medium, sterile-packaged and, when together with a single-use
filter, are ready to use immediately. In combination with a single-use
filter unit, these media dishes feature an active lid that permits
touch-free transfer of a membrane onto agar, without using any tweezers.
This active lid lifts the membrane filter from the base of the filter
unit so the filter can be safely transferred onto the pre-filled agar
dish. Once the medium dish is closed, the membrane is ready to incubate.
Solution for safe membrane transfer
combination of agar media dishes and filter units represents a new
membrane transfer and agar concept. As just a few steps are all it takes
to proceed from sampling to incubation, a single-use system of agar
media dishes and filter units accelerates workflows, making them
cost-efficient. At the same time, touch-free membrane transfer enables
even more reliable results to be obtained in analysis, while reducing
secondary contamination to an absolute minimum.