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Rinse Sampling for
Cleaning Validation Studies
Destin A. Le Blanc
CALGON VESTAL
Rinse-sampling procedures that draw from the final
rinse solution can provide upper-limit estimates of
potential contamination in subsequently manufactured
products. The process can be improved by separating
the process rinse from the sampling rinse. This may
result in more accurate actual estimates, more
flexibility in selecting analytical procedures, and more
flexibility in choosing a sampling rinse solution that is
different from the process rinse solution. Procedures
for such determinations depend on adequate rinse
recovery studies. This article describes these studies.
Destin A. Le Blanc is vice-president of scientific technical support,
Calgon Vestal Division, Steris Corp., PO Box 147, St. Louis,
MO 63166, tel. (314) 535-1187, fax (314) 535-1083, e-mail
(destin_leblanc@steris.com).
V
alidation of cleaning processes used for product contact
surfaces in pharmaceutical manufacturing is required by
FDA. The 1993 decision of US v. Barr Laboratories gave
FDA the right to mandate that cleaning is a critical process
that must be validated (1). Although this decision focused mainly
on issues related to retesting after out-of-specification results, it also
held that FDA had the right in this case to mandate validation of
cleaning processes. In July 1992, while this case was being litigated,
the agency’s Mid-Atlantic Region staff developed an internal in-
spection guideline for cleaning validation (2). In July 1993, the more
official and still current “Guide to Inspections of Validation of Clean-
ing Processes” was issued (3).
The 1992 Mid-Atlantic guide discusses three types of sampling:
direct surface, rinse, and placebo. Direct surface is rated most de-
sirable, rinse is considered less desirable, and placebo is considered
generally unacceptable. In the 1993 guide, although not explicitly
stated, the relative ranking of these methods is assumed to hold —
with direct surface being most desirable and placebo being accept-
able only if direct surface or rinse sampling is also done. Two rea-
sons support the less-desirable designation for rinse sampling. The
first issue is whether the residue of concern is soluble in the rinse
solution. One analogy is that of a dirty pot. If the concern is
whether or not a washed pot is clean, does one examine the rinse
water or the pot itself? The second concern is the analytical strategy
used. Some pharmaceutical companies previously analyzed the
rinse water and tested it merely for USP compendial specifications
for purified water or for water for injection. FDA argued (quite
rightly) that manufacturers should have a target residue and mea-
sure that target residue. It is possible that the rinse water meets USP
specifications and yet may not reasonably indicate contaminants
in the rinse water because the target residue is present either at a
level or as a chemical species that cannot be adequately detected by
USP water specifications.
The 1993 document does offer some guidance on acceptable
residue levels by citing a paper from scientists at Eli Lilly about
their procedures for setting residue limits (4). These residue limits are
based on the possible contamination of product subsequently manu-
factured in the same equipment. The Eli Lilly scientists proposed
limits in the subsequent product that would produce 0.001 of a min-
imum daily dose of the drug residue in a maximum daily dose of
the next drug product. They also proposed that if the calculated value
based on the 0.001 daily dose is more than 10 ppm, then a default
value of 10 ppm residue in the subsequent product should be used.
Based on possible contamination of the subsequent product, it
is then possible to calculate — using parameters such as batch size,
surface area, and sampling technique — items such as allowable
contamination per surface area or per swab sample. Although
swab-sampling residue limits have been discussed in numerous
papers (5,6,7), rinse sampling has not been covered as rigorously.
............................................................................
Based on the author’s experience, manufacturers have often
made assumptions about acceptable rinse water limits without
adequate consideration of the potential for contamination of
the subsequently manufactured product. In many cases, a rinse
water limit of 10 ppm has been automatically selected and as-
sumed to represent an acceptable residue limit in the rinse so-
lution. The rest of this article focuses on the critical relation-
ship between rinse-sampling results and potential contamination
of the subsequent product. Issues covered include rinse sam-
pling as it is currently done (i.e., grab sample at the end of the
rinsing process), rinse-sampling processes that may provide
more scientifically justified residue limits, and methods that
may adequately determine rinse-sampling recovery.
SINGLE-POINT FINAL-RINSE SAMPLING
In this method, manufacturers obtain a sample at the end of the
final rinsing process. If the rinsing medium is water, then this is
obviously a water sample; if a solvent is used for the final rinse,
then this is a solvent sample. The volume of rinse sample taken
will depend on the analyses done but typically is in the 500–1000-
mL range. The sampling point is usually identified as that point
at the end of the cleaning circuit at which the water exits the
cleaning circuit. Assuming that a specific residue is targeted, as
FDA recommends, what does it mean if a chemist measures a
target residue in the grab sample and measures a level of, for ex-
ample, 5 ppm? Can it be reasonably assumed that a level of 5 ppm
in this rinse sample corresponds to a potential contamination of
5 ppm in the subsequent product? The existing literature for
cleaning validation does not provide a series of calculations to
go through, such as with swab sampling, that can lead to a scien-
tifically based correlation between rinse sample analysis and po-
tential contamination of the subsequently manufactured product.
How can this relationship be established? For swab samples,
factors such as batch size of subsequent product, shared sur-
face area, area sampled, and amount of solvent used for de-
sorbing the swab must be considered to obtain an accurate rela-
tionship between the analytical result in the desorbed swab
sample and the corresponding contaminant levels in the subse-
quent product. What are the corresponding factors for rinse
samples? How do these factors relate to what is really of inter-
est — that is, potential contamination of the next product?
One way to establish a relationship is to make assumptions
about what rinsing represents. The first and critical assumption
is that the target residue as it exists on the equipment surfaces is
solubilized in the rinse solution by the rinsing process. If this is
true, then the levels found in the rinse are indicative of the levels
on the equipment surfaces. The second assumption is that the
rinse process adequately cleans all surfaces and any sample mea-
sured is therefore representative of the entire system. It should
be noted that the notion of worst-case locations does not apply to
cleaning validation rinse sampling because the entire product con-
tact surface is sampled. Several more assumptions that depend
on the types of rinsing process will be discussed later.
Two scenarios of interest are possible. The first case involves
a continuous rinse process, such as would be found in a CIP
operation. The other process involves discrete rinses or batch
rinsing. Batch rinsing is considered first because it is the simpler
of the two. In a batch-rinsing process, the manufacturing equip-
ment is filled with rinse solution, the solution is circulated or
otherwise agitated until it is uniform, and then it is dumped.
For rinse sampling, a portion of that final rinse is sampled, and
then that sample is analyzed for the target residue. If X liters
are used in the final rinse, the subsequently manufactured prod-
uct is made with a batch size of Y liters, and Z ppm (adjusted
by a recovery factor that will be discussed later) residue is mea-
sured in the last rinse, then the level of residue C that can po-
tentially contaminate the subsequent product can be reasonably
assumed to satisfy the following limit:
C  (Z)(X)  (Y) [1]
For example, for a 1000-L vessel, it may take 1100 L of rinse
water to adequately rinse the vessel in a batch-rinsing process
(the extra water is associated with the pumping lines as the rins-
ing solution is recirculated). If the measured residue is 5.0 ppm
in that final rinse solution, and if the batch size of the subse-
quently manufactured product is 900 L, then the maximum po-
tential contamination in the next product will be
(5.0 ppm)(1100 L)  (900 L)  6.1 ppm [2]
The measured level in the rinse water is less than the potential
maximum contamination level in the next product because the
volume of the rinse water is greater than the volume of the sub-
sequently manufactured product.
This is a maximum and not a specific amount. If the sam-
pling is done on the final rinse, then any rinse done subsequently
should represent a significantly lower residue level. In other
words, if the nth rinse resulted in a 5-ppm residue level, then the
(n  1)th rinse should produce a value that is considerably less
than 5 ppm. This is because the sampling of the nth rinse does
not represent the contamination of the surfaces after the nth
rinse, but rather the contamination after the (n  1)th rinse. This
is adequate justification for saying that the residue in the subse-
quent product in this example will be 6.1 ppm. If the limit
for that target residue is 10 ppm, then one can be assured of be-
ing under that limit. On the other hand, if the limit in the subse-
quent product is 3.2 ppm, then one may or may not be under that
limit after the nth rinse, and more work must be done to either
confirm the accuracy of the 6.1 ppm calculation or to find
whether the actual value is less than 3.2 ppm. If the acceptance
limit in the subsequent product were actually 3.2 ppm, then the
data are insufficient to give adequate assurance. However, if
the residue is soluble in the rinse solution, then it is reasonable
to speculate that the results after one additional rinse might be
acceptable because the (n  1)th rinse will give a true measure
of what is present after the nth rinse.
A continuous rinsing procedure is more commonly used in
the pharmaceutical industry. In this method, the rinsing is on a
continuous, once-through-to-drain basis. If the rinsing procedure
is for m minutes, then at the end of m minutes, a grab sample is
obtained and analyzed for the target residue. An analysis similar
to the one done in the batch-rinsing process can be done in a con-
tinuous rinsing procedure, although the assumptions are some-
what more tenuous. The first assumption is that the sample col-
lected at the end of m minutes is the worst case, and any
subsequent sample provides a lower level (also assuming, as we
have throughout, that the residue is solubilized in the rinse solu-
............................................................................
tion by the rinsing process). If the total amount of rinse solution
collected is assumed to equal the volume of the next batch, then
the analysis of that total volume would give a residue level less
than or equal to the residue measured in the grab sample at the
end of m minutes. In this case, the potential contamination in
the subsequent batch is no greater than that in the grab sample
at the end of the rinsing process. If the grab sample has a level
that is below the acceptance limit for the target residue in the
subsequent product, then the residue level in the subsequent
product can be assumed to be at an acceptable level. On the other
hand, if the level in the grab sample is above the acceptance limit
in the subsequent product, the residue levels in the equipment
may or may not be acceptable, and further work must be done.
REFINEMENTS IN RINSE SAMPLING
The rinse-sampling methods described above can only provide
an upper limit for residue levels either because the equipment
is not tested after the final rinse (as in the case of the batch-
rinsing procedure) or because a representative sampling volume
is not adequately defined (as in the case of the continuous rinse
procedure). This is not to say that these procedures are unac-
ceptable. The purpose of residue testing is to determine whether
the residue is below the acceptance limit and not necessarily to
define the exact level of contamination. If the above methods
show acceptable results for residue levels, then these procedures
are sufficient. On the other hand, if the upper limit of the mea-
sured residue is above the acceptance limit or if the analytical
tools do not adequately measure at those residue levels (e.g., the
analytical method analyzes a solution 25 ppm, but the limit
in the subsequent product is 5 ppm), then a refined sampling
process can provide more useful results. These sampling re-
finements also offer the option of using a sampling rinse that is
different from the final rinse solution.
To more accurately measure residue with a rinse solution, one
must first define the sampling process so that rinse sampling is
conducted only after the rinsing process is completed and then
to carefully define the rinse-sampling volume. By measuring the
residue in the final rinsing solution using the assumptions pre-
viously discussed, pharmaceutical companies are actually es-
tablishing a worst-case scenario. However, rinse sampling may
be able to estimate residue levels more accurately.
A batch-rinsing process is considered first. The key here is
that if the rinsing process in the SOP calls for n discrete process
rinses, then an additional rinse should be done (the sampling
rinse) only for the validation studies, and that sampling rinse so-
lution should be analyzed. Such an analysis of the (n  1)th
rinse should provide an accurate measurement of the residue
on the equipment after the final (nth) process rinse if the sam-
pling rinse solution provides adequate recovery. This approach
may offer measured residue levels in the analyzed solution that
are lower than those in the final rinse sample. Of course, the
same equation (equation 1) for calculating potential contami-
nants in the subsequent product must be used. Because the sam-
pling volume and the volume of the subsequent product are the
same, the only factor that can provide lower residues is lower
amounts measured in the (n  1)th rinse. Therefore, this method
cannot be used to resolve problems in which the limit of detec-
tion of the analytical method is the limiting factor.
On the other hand, if the problem is solubility of the target
residue in the process rinsing solution, then it may be neces-
sary to complete the (n  1)th rinse with a solvent that more
readily solubilizes the target residue instead of with the process
rinsing solvent. For example, in aqueous cleaning with a batch-
rinsing process that involves six rinses with USP purified wa-
ter, as soon as the rinsing process is complete, one can begin
sampling with a rinse that uses a different solvent. This solvent
could merely be water at a different pH if the target residue is a
drug active that dissolves more readily at a higher or lower pH.
It could be a 70% isopropanol solution or some organic solvent
if the target residue is more readily soluble in an isopropanol so-
lution or that specified organic solvent.
Regardless of the solution used for this sampling rinse, it should
be noted that this sampling rinse is to be used only as part of the
validation studies. The (n  1)th rinse is not part of the cleaning
SOP but rather is truly part of a sampling procedure. Another fea-
ture of this approach is that it targets all surfaces in the equip-
ment. The positive side is that this not only ensures that all surfaces
are sampled, but it also effectively averages the contaminant levels
from different locations. Although it does not release a company
from the obligation to clean effectively and to achieve visibly clean
surfaces, this averaging does reduce the possibility that one aber-
rant result in a swab-sampling program could invalidate a cleaning
validation study. This averaging process also assumes that the
transfer of contaminants to the subsequently manufactured product
is a uniform process. In the case of nonuniform contamination, this
method gives a measurement of the total system contamination,
but other techniques must be considered to evaluate how that con-
taminant is transferred to the subsequent product.
The second situation involves a continuous rinsing procedure.
In this case, an approach that begins the sampling rinse only af-
ter the rinsing process is completed may provide considerable
advantages in analytical method selection. Once process rinsing
is finished, the rinse sampling is started. Note that in actual prac-
tice the process rinse and sampling rinse may, in fact, be con-
tiguous. However, there is nothing wrong with stopping the rins-
ing process and at a later time beginning the sampling rinse. This
is no different from swab sampling in which after the rinsing
process is completed, the sampling process is begun at a later
time. What this means practically is that, if the SOP is written
with a process rinse time of 10 min, then a sampling rinse is be-
gun at the end of 10 min. The length of time needed to collect a
sample (i.e., the volume of sampling rinse collected) depends
on the amount of sampling rinse necessary to adequately con-
tact all surfaces within the equipment. For a CIP system, this
amount will typically be the total volume of cleaning solution
in the CIP circuit. For example, if the vessel being cleaned is a
1000-L vessel, it may take 100–200 L of cleaning solution in
the CIP circuit. This includes the heel in the manufacturing ves-
sel, the solution in the CIP cleaning solution tank, the solution
in associated piping, and the solution on the dome and sidewalls
of the vessel. If the amount of cleaning solution in the circuit dur-
ing the cleaning (recirculating) step is 150 L, then it can be rea-
sonably assumed that the circulation of 150 L of sampling rinse
would contact and adequately sample all surfaces. The next step
is to collect that sampling rinse (all 150 L), agitate it until uni-
form, take a subsample of it, and analyze that subsample.
............................................................................
Such a sampling procedure can use equation 1 to measure po-
tential contamination in the subsequent product. To continue with
the above example, suppose a sampling rinse of 150 L is adequate
to sample a 1000-L vessel and the target residue in that sampling
rinse was measured at 5.0 ppm. What would this correspond to
in terms of contamination of a subsequently manufactured prod-
uct of batch size 900 L? Using the equation, the calculation gives
(5.0 ppm)(150 L)  (900 L)  0.8 ppm [3]
In other words, 5 ppm in the sampling rinse solution corresponds
to only a maximum of 0.8 ppm residue in the subsequent prod-
uct. If the acceptable residue limit in the next product is 10 ppm,
then an analytical method that measures at around 60 ppm could
acceptably measure that residue. If analytical limits of detection
are a problem, one solution is to move to this type of rinse-
sampling procedure to leverage the analytical method. Compared
with the batch-rinsing procedure, the surface is sampled with a
smaller volume of liquid, thus effectively concentrating the
residue in the sampling solution. Certainly the same primary
concern applies in this procedure as with a batch-sampling rinse
process, namely, the residue must be solubilized in the sampling
rinse solution during the rinse-sampling process. The possibility
also exists for using a sampling rinse solution that is different
from the process rinse solution. This may result in more rele-
vant and defendable cleaning validation processes.
DETERMINATION OF SOLUBILITY IN SAMPLING RINSE
A key in any sampling procedure is that it quantitatively removes
the residue from the surface for subsequent analysis. In swab
sampling, this is done by a swab-recovery study. Such a study
determines not only the removal of residue by the swab but also
the subsequent desorption of the residue from the swab. Al-
though swab-recovery studies are common, it is also necessary to
perform sampling rinse–recovery studies to verify that a sam-
pling rinse procedure can adequately recover residues from sur-
faces. The best way to approximate a sampling rinse–recovery
is to conduct lab studies that involve coupons. As with swab
sampling, a coupon of the substrate of interest (e.g., stainless
steel) is spiked with the target residue of interest. Depending on
the application, the spiked residue may be dried or baked onto
the surface to simulate how it may exist in actual processing con-
ditions. There are two methods that can simulate rinsing. Both
depend on a ratio of rinse-sampling solution to surface area that
is no larger than the expected ratio under actual rinse-sampling
conditions. The temperature for the recovery study should be
the same temperature as that of the rinse-sampling solution (or
less, if the solubility increases with increasing temperature).
One method involves positioning the spiked coupon over a
beaker to collect the rinse-sampling solution, then pipetting the
rinse-sampling solution over the coupon, and finally letting the
solution collect in the beaker below. After this simulated sampling
rinse, the collected solution is analyzed for the target residue and
© Reprinted from PHARMACEUTICAL TECHNOLOGY, May 1998
STERIS® Corporation 5960 Heisley Road Mentor, OH 44060-1834 USA 440-354-2600
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compared with the amount spiked onto the coupon. As with spik-
ing studies in swab sampling, issues such as potential interference
sources must be addressed. If the analytical method is total or-
ganic carbon, then a control for the rinse sample solution should
be run as a background subtraction. This blank should not be the
rinse-sampling solution itself but rather the solution that has been
used to sample a clean, unspiked coupon.
The second procedure requires placing the coupon in an ap-
propriate amount of sampling solution into the bottom of a
beaker and gently swirling for 30 s. The coupon and rinse so-
lution should be separated immediately to quench the dissolution
process, and then the solution should be analyzed. This method
has the handling disadvantage of dealing with the underside of
the coupon. Also, it is more difficult to remove the coupon from
the sampling solution to quench the rinsing process. But these are
not insurmountable problems. As with swab-sampling recovery,
the level of residue spiked onto the coupons represent those lev-
els expected to produce test solutions around the acceptance cri-
teria for the analytical test solution.
DISCUSSION
In a cleaning validation study, current single-point, grab-sampling
procedures for a rinse solution analysis at the end of the rinsing
process can provide adequate estimates of potential contamination
levels in a subsequently manufactured product. If a rinse recovery
study demonstrates residue recovery, and if proper assumptions
are made regarding the relationship between the residue level in the
rinse solution and the potential contamination in the subsequent
product, then the rinse-sampling procedure is adequate. Sampling
the process rinse solution gives an upper limit estimate of the av-
erage overall contaminant level in the process equipment.
If a more rigorous treatment of rinse sampling is desired, if
the use of a sampling solution other than the process rinse so-
lution is necessary, or if the analytical limits of detection are
inadequate to provide results that are below the acceptance cri-
teria, then the use of a sampling rinse separate and distinct from
the process rinse should be considered. Such an approach that
uses a distinct sampling rinse does not obviate the value of rinse
water monitoring as a routine QC technique.
REFERENCES
1. United States v. Barr Laboratories, 812 F. Supp. 458 (DNJ 1993).
2. FDA, “Mid-Atlantic Region Inspection Guide: Cleaning Valida-
tion,” 28 July 1992.
3. FDA, “Guide to Inspections of Validation of Cleaning Processes,” July 1993.
4. G.L. Fourman and M.V. Mullen, “Determining Cleaning Validation
Acceptance Limits for Pharmaceutical Manufacturing Operations,”
Pharm. Technol. 17 (4), 54–60 (1993).
5. K.M. Jenkins and A.J. Vanderweilen, “Cleaning Validation: An
Overall Perspective,” Pharm. Technol. 18 (4), 60–73 (1994).
6. J.M. Hyde, “Cleaning Validation Strategies,” paper presented at
ISPE CIP/SIP Seminar, Atlanta, Georgia, June 1994.
7. Cleaning and Cleaning Validation: A Biotechnology Perspective,
J.R. Voss, Ed. (PDA, Bethesda, MD, 1996).
Printed in U.S.A.
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