GDD002_008 Aventis 27/8/04 15:38 Page 129
A u t h o r
Dr Joachim Ermer is responsible for coordinating,
supporting, and advising the quality control departments
of Aventis in analytical aspects. This also includes the
establishment of company guidelines and training modules.
Ermer is deputy head of the working group in quality
control/pharmaceutical analytics of the German
Pharmaceutical Society and a member of the working
group quality assurance of the International Association
of Pharmaceutical Technology.
VALIDATION
OF CLEANING
METHODS
Dr Joachim Ermer, director of analytical
processes and technology for Aventis quality
operations, discusses the performance
parameters for cleaning methods and suitable
calculations, tests and acceptance criteria.
he prevention of cross-contamination of drugs in
T pharmaceutical production must be avoided, therefore
the cleaning of the manufacturing equipment is an
important aspect of good manufacturing practice. The process
of demonstrating the efficiency of the cleaning procedure
is known as cleaning validation. Of course, the analytical
method applied must be validated itself.
Validation of cleaning methods should follow the same rules as
any other procedure in pharmaceutical analysis, but some aspects
need special consideration, as regulated by the intended application.
Therefore, sensitivity and recovery are of importance.
It is crucial to realise that the sampling procedure is an integral,
if not the dominating, part of the cleaning method.
Cleaning validation and verification
Often, the efficiency of the cleaning procedure is investigated by
swabbing a defined area of the cleaned equipment surfaces with
an appropriate material. The residual substance(s) sampled from
the cleaned surface are then extracted and their amount analysed.
Another approach is to analyse the final rinse solvent, applicable
if the equipment surface is not accessible and the equipment is
difficult to dissemble. However, the risk of tightly absorbed residues
that are not dissolved by means of rinsing should be considered.
With respect to chemical substances, residues include primarily
active ingredients and cleaning agents, while degradants, raw
materials, intermediates, or excipients may also be of concern.
The maximum acceptable amount of residue is dependent on
the pharmacological or toxicological activity of the respective
substance – often considered as a safety factor – as well as the
batch sizes and dosages of the previous and next product, and
the equipment surface. Often, a maximum limit of 10ppm in
the next batch is established. This ‘residual cleaning limit’ is
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then normalised with respect to the sampled equipment area,
as ‘specific residual cleaning limit’ (SRCL). Reported SRCL are
between 4ng/cm2 and 3µg/cm2. Usually, before any quantitation,
the surface must show no visually detectable traces of residues.
Analytical technique
The selection of the analytical technique is dependent on the
properties of the residue and sensitivity, as required by the cleaning
limit. Selective methods such as high performance liquid
chromatography, gas chromatography, high performance thin layer
chromatography, and immuno assays are preferred for active (and
related) residues. Non-selective methods, such as pH-measurement,
conductivity and total organic carbon, are mainly used for detergents.
As a rapid, sensitive and selective method, ion mobility spectrometry
was introduced, particularly for cleaning verification. This technique
(used in airports to detect explosives) is based on the ionisation of the
sample directly from the swab and measures the characteristic speed of
the ions moving through an electric field under atmospheric pressure.
Interferences from the
sampling procedure must
be taken into account
Validation characteristics
Development and optimisation of the analytical procedure and its
validation is an iterative process in which the influence of the cleaning
solvent, swab material, swabbing solvent, sampling technique, and
extraction of the analyte on specificity and recovery is investigated.
In this explorative stage, the recovery at one single concentration,
preferably at the defined limit, is sufficient. During this stage, the
robustness of the analytical technique should be investigated.
With respect to specificity, interferences from the sampling procedure
must be taken into account. This should include blank extractions of
the swab material, as well as blank swabbings of the respective surface.
The target substance might be altered during the cleaning process and
the original substance replaced by a degradant. This can be
investigated by using aged samples. Specificity is usually demonstrated
by sufficient chromatographic resolution, or lack of interference.
In linearity, the intended calibration model is the target. The
corresponding requirements are: for a single-point calibration
(external standardisation), a linear response function and a zero
intercept have to be demonstrated; for a linear multiple-point
calibration, only a linear function.
A widespread misinterpretation is to report the correlation co-
efficient as demonstration of a linear response function. However,
this parameter is no proof of linearity, but requires a linear response
function to have any meaning. Basically, it cannot distinguish
between random and systematic deviations from the linear function,
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but the latter is the primary objective. Therefore, the first step
should be to inspect the experimental data for systematic deviations

Paul GmbH from the applied regression function. Numerical parameters are
only suitable afterwards. The relative standard error of slope is
recommended because such a normalised (percentage) parameter
allows a straightforward evaluation by comparison with an

Film only acceptable precision.

After the conditions of sampling and sample preparation are

optimised, accuracy, precision, and quantitation limits can be validated
simultaneously in a range of at least 50 to 120 per cent of the defined
cleaning limit, using at least nine spikings. In cleaning validation, no
authentic, homogeneous samples are available. Therefore, precision of
the analytical procedure must be obtained from spiked samples.

Recovery is performed from the spiked surface(s) of identical

equipment material, often from spiked swabs. The latter may be
omitted if the former recovery is acceptable. If excipients can be
suspected to interfere, the spiking of the active should be performed in
their presence. Due to the potentially large influence of the sampling,
the robustness of the recovery, which will also include the swabbing,
should be investigated by repeating the recovery with another operator.

The contribution of other factors, such as analytical instrument and

reagents, may be investigated, but can be expected to be small
compared to the sampling. Dependent on surface properties and
material, the analyte can often be only partly recovered. Values of
larger than 80 per cent and 50 per cent are regarded as good and
reasonable, respectively, while less than 50 per cent is questionable.

Recoveries
If relevant (with respect to the precision), the recovery factor should
be used to correct the results of the cleaning method. To allow a
straightforward evaluation, the recoveries are preferably presented
graphically as percentages with respect to the spiked concentration.
This plot should be inspected for a concentration-dependent
behaviour. In case of no or only slight dependency, the average and
the relative standard deviation of all recoveries can be calculated. If
the spikings are analysed in an independent series, the calculation
can be performed as an analysis of variances. Here, the precision
contributions within and between the series are obtained separately,
and conclusions can be drawn on the robustness of the procedure.

The overall average from the intermediate recovery study is then

used as the recovery factor. In case of a concentration dependency,
either a concentration-dependent recovery factor is used, or it is
calculated at the cleaning limit as the relevant concentration. If
a sufficient number of determinations is available (at least five),
average and relative standard deviation can be calculated for each
concentration level. However, it is bad practice to calculate, say, a
standard deviation from three values only, because the upper limit of
its 95 per cent confidence interval is 4.4 times the calculated value.

Acceptable precisions may be linked to the quantitation limit (QL),

for example, repeatability <2xQL – 25 per cent, <10xQL – 15 per cent,

For further information circle 72

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M a n u f a c t u r i n g & P r o c e s s i n g

<20xQL – 10 per cent and >20xQL – 5 per cent. For intermediate

Figure 1. Recovery and precision of meclizine from
precision, an extra 5 per cent is granted for each concentration level.
swabs and stainless steel plates
In Figure 1, an example is given for the recoveries of a drug substance
from swabs and stainless steel plates. Meclizine, for example,

recovery (%)
1-(p-chloro-a-phenyl-benzyl)-4-m-methylbenzyl)piperazine 96
Recovery from swabs: 88.5% (RSD 4.2%)
dihydrochloride, is practically insoluble in water and slightly 94
Recovery from plates: 89.2% (RSD 2.7%) 1.3%
soluble in diluted acids. The SRCL was established to 0.50µg/cm2. 92
The system precision of the LC assay of 0.21 per cent and 0.41 per 0.5%
cent was obtained from five repeated injections at a concentration 90
0.9%
corresponding to 200 per cent SRCL. The recoveries from five swabs 88
and five plates at each of the three concentration levels were obtained. 4.3%
0.9%
86
1.8% Spiked swabs
No relevant difference is observed between the recoveries from swabs 84
and plates. Therefore it can be concluded that meclizine can be Spiked plates
82
recovered almost completely from the stainless steel surface, and
the main loss is due to absorption on the swab material. However, 80
the recoveries are acceptable, with only a slight concentration 0 50 100 150 200 250
dependency. Therefore, overall parameters can be calculated. spiked concentration (% SRCL)

The determined limit is strongly

Figure 2. Stability of meclizine under
dependent on the equipment, various (sample) conditions
conditions and calculation
procedure applied
110
Recovery (%) for 1µg/cm2 )

For the QL, various approaches can be applied, based either on the plates
blank (signal-to-noise ratio, standard deviation of the blank) or 100 swabs
extraction solution
analyses of the analyte in the low concentration range (standard
deviation of the intercept, residual standard deviation of the linear 90 Av.: 89.9%
regression). It is important to be aware that the determined limit RSD: 1.15%
(DL) is strongly dependent on the equipment and conditions as 80
well as on the calculation procedure applied. The minimum
requirement for QL is approximately 50 per cent of the SRCL.
70
When calculating DL/QL from a calibration line, a frequent
mistake is to use a too-large concentration range or even 60
extrapolation. Due to non-constant variances, higher 0 1 2 3 4
concentrations will dominate the dispersion parameters used to Storage time (d)
calculate QL. Therefore, the maximum concentration should not
exceed the 10- to 20-fold of DL. The range and mean of three determinations are indicated,
as well as the overall average and the relative standard
Stability considerations deviation of the swab samples.
An important aspect in cleaning validation is the ageing of the
sample at the various stages of the process to define the
conditions appropriately. In the given example, the ageing effect
was investigated with respect to the analyte on dry stainless steel such a case, the time between cleaning and sampling from the steel
plates, on moistened swabs, and in the extraction solution from surface and the shelf life of the extraction solution needs to be
the swabs (Figure 2). limited in the cleaning validation process. No changes were observed
for the storage of the moistened swabs. Due to the intermediate
Large effects can be observed for the ageing on plates and in the recovery conditions in the stability study, this average of 89.9 per
extraction solution, both with respect to recovery and variability. In cent is suitable to define the recovery factor. END

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