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Cleaning verification: A five parameter study of a Total Organic Carbon

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 Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Cleaning verification: A five parameter study of a Total Organic

Carbon method development and validation for the cleaning
assessment of residual detergents in manufacturing equipment
Xue Li, Imad A. Haidar Ahmad, James Tam, Yan Wang, Gina Dao, Andrei Blasko ∗
Novartis Pharmaceuticals Corporation, San Carlos, CA 94070, USA

a r t i c l e i n f o a b s t r a c t

Article history: A Total Organic Carbon (TOC) based analytical method to quantitate trace residues of clean-in-place
Received 9 August 2017 ® ®
(CIP) detergents CIP100 and CIP200 on the surfaces of pharmaceutical manufacturing equipment was
Received in revised form
developed and validated. Five factors affecting the development and validation of the method were
18 September 2017
Accepted 22 October 2017
identified: diluent composition, diluent volume, extraction method, location for TOC sample preparation,
Available online 26 October 2017 and oxidant flow rate. Key experimental parameters were optimized to minimize contamination and
to improve the sensitivity, recovery, and reliability of the method. The optimized concentration of the
Keywords: phosphoric acid in the swabbing solution was 0.05 M, and the optimal volume of the sample solution
Manufacturing equipment was 30 mL. The swab extraction method was 1 min sonication. The use of a clean room, as compared to
Cleaning optimization an isolated lab environment, was not required for method validation. The method was demonstrated to
Cleaning verification be linear with a correlation coefficient (R) of 0.9999. The average recoveries from stainless steel surfaces
Detergent residues at multiple spike levels were >90%. The repeatability and intermediate precision results were ≤5% across
Coupons the 2.2–6.6 ppm range (50–150% of the target maximum carry over, MACO, limit). The method was also
TOC shown to be sensitive with a detection limit (DL) of 38 ppb and a quantitation limit (QL) of 114 ppb.
The method validation demonstrated that the developed method is suitable for its intended use. The
methodology developed in this study is generally applicable to the cleaning verification of any organic
detergents used for the cleaning of pharmaceutical manufacturing equipment made of electropolished
stainless steel material.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction tently yield results that meet pre-determined specifications and

Detergents are commonly used to remove drug product residues
from the manufacturing equipment in the pharmaceutical indus-
try. These detergents can remain on the equipment surfaces if they
are not sufficiently rinsed during the cleaning process, contaminat-
ing the next manufactured product. Usually, a cleaning validation
master plan requires that detergent(s), used to clean the manufac-
turing equipment in the cleaning validation phase, is removed to
an acceptable level. A validation cleaning limit (CL) for detergent,
along with a suitable analytical method, is required to quantify the
carryover into the commercial product.
Cleaning verification establishes documented evidence with a
high degree of assurance that the cleaning process can consis-
∗ Corresponding author at: Novartis Pharmaceuticals Corporation 150 Industrial
Road San Carlos, CA 94070, United States.
E-mail address: andrei.blasko@novartis.com (A. Blasko).
quality characteristics. Cleaning verification for detergents is a
requirement by health authorities at the commercialization of drug
products and failing to comply with the requirements may result
in regulatory warning letters [1–7].
Assessment of residual detergents at the end of the equipment
cleaning process is challenging because the analytical method has
to address limit of quantitation at trace level (e.g., ppm) of ana-
lyte(s) that often lack ultraviolet (UV) chromophores. In addition,
suppliers may not disclose chemical compositions of the commer-
cial detergents, but may provide only total organic carbon (TOC)
content. Many studies have been reported on cleaning verification
of pharmaceutical residues; however, reports on cleaning valida-
tion of detergents used for the cleaning process are limited [8–20].
®
If the detergent contains a chromophore like the LpH se germicidal
detergent (with 4-tert-amylphenol and 2-phenylphenol), a HPLC-
UV method is suitable for the detection and quantitation [16]. If
a UV chromophore is absent, other detection techniques, such as
ion chromatography with conductivity detection [17] or charged

https://doi.org/10.1016/j.jpba.2017.10.021
0731-7085/© 2017 Elsevier B.V. All rights reserved.
34 X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39
aerosol detection [22] must be used. All these component specific
detection techniques are not applicable when chemical changes
occur during the cleaning process, as is always the case of biologics’
degradation during cleaning of manufacturing equipment.
Assessing detergent residues by TOC has been reported [18–21].
Compared to the conventional HPLC based methods, TOC meth-
ods are faster, simpler, and shorter; thus, the analytical method
development is simpler. The TOC technique is a nonspecific, total
sample combustion analysis that measures the total organic carbon
content from all sources, including that from residual API, excipi-
ents, detergents, and contaminants. The analyzer uses UV radiation
and a chemical oxidizing agent to oxidize the organic compounds
in the sample to form carbon dioxide (CO2 ). The generated CO2
is measured using a sensitive and selective membrane-based con-
ductivity detection technique [23], and the TOC result is reported
as concentration of organic carbon in the sample (ppm or ppb).
Despite the above advantages, a general, systematic, and prac-
tical approach to method development has not been reported.
Typically, there are several commercial products used for the
®
cleaning of manufacturing equipment [16,17]. CIP100 (base) and
®
CIP200 (acid) are the most commonly used for CIP systems or auto-
mated cleaning systems. In a typical cleaning process, the surface
of the manufacturing equipment is cleaned by the application of
hot water for injection (WFI), followed by the sequence of diluted
® ®
CIP100 , hot WFI, diluted CIP200 , and hot WFI. Stainless steel
coupons, with the same surface/finish as the manufacturing equip-
ment, are used to simulate equipment surfaces during laboratory
method validation studies of the cleaning verification methods [15].
The material of construction (MOC) of the coupons was not the
subject of this study. Considering the stainless steel material, MOC
might indirectly affect the recovery by not being able to be cleaned
properly. If MOC were a factor, after properly cleaning the coupons,
different types of molecules (small, large) with different electronic
surface density would interact differently with the metal surface,
which we demonstrated was not the case [15].
Previous reports addressed the problem associated with the
difficulty of recovering the sample from the equipment which
depended on the tools and the skills of the analyst [24–27]. Stud-
ies have reported challenges with using TOC such as low speed,
lack of specificity, limitation of use to water soluble compounds,
variability from one instrument to another, and inaccuracies that
may lead to false positives [24–26]. We believe that a majority of
the reported unreliability with TOC measurements is due to the
sample preparation and handling along with the precise control
of the instrumental parameter. These two issues, which may have
driven analysts to use techniques such as IR absorption and mass
spectrometry instead of TOC [27], are addressed in this paper.
Parameters such as temperature on oxidation efficiency and car-
rier gas flow rate are known to affect the accuracy, precision, limit
of detection, and calibration of the TOC analysis [28]. This paper
describes the influence of different parameters affecting the devel-
opment and validation of a TOC based analytical method to quantify
® ®
trace residues of CIP100 and CIP200 on the surface of manu-
facturing equipment. The methodology developed in this study is
generally applicable to the cleaning verification/quantitation of any
organic detergents used for the cleaning of pharmaceutical manu-
facturing equipment using TOC analysis based on the UV/persulfate
oxidation principle.
2. Experimental
2.1. Materials
The system suitability standard set was from GE Analytical
Instruments (Boulder, CO, USA), STD 31004-XX (containing: Work-
ing System Suitability 1,4-Benzoquinone Standard Solution (RSS),
Working Standard Sucrose (RS), and Reagent Water (RW)). Clean-
® ®
ing in place solutions (CIP100 and CIP200 ) were obtained from
Steris Corporation (Mentor, OH, USA). The sampling swabs with low
carbon, certified <50 ppb, part number TX714K, were from Texwipe
(Kernersville, NC, USA) and were used without pretreatment. The
sample diluent was 0.05 M phosphoric acid in water prepared by
dilution of concentrated (85%, w/w) phosphoric acid (EMD Milli-
pore, Darmstadt, Germany) in water. Each TOC vial (VWR (Radnor,
PA, USA), 89094-204, TOC <10 ppb) was filled with 30 mL of the
diluent. Pipettes were from Rainin (Oakland, CA, USA). Ultrapure
water was obtained using the in-house Millipore Milli-Q Advan-
tage Water Purification System (Darmstadt, Germany). HPLC grade
methanol and phosphoric acid were from EMD (Billerica, MA, USA).
Stainless steel coupons were 316L SS with a roughness average of
20, from GlobePharma, Inc. (New Brunswick, NJ, USA).
2.2. Instrumentation
The TOC analysis was conducted using a Sievers TOC ana-
lyzer, Model 900 with Autosampler (GE Analytical Instruments;
Boulder, CO, USA). Key instrument parameters were “Acid” flow
rate (0.8 ␮L/min) and “Oxid” flow rate (1.4 ␮L/min for blanks;
6.8 ␮L/min for the standard and sample solutions). Other parame-
ters were default values set by the instrument vendor [22]. System
suitability was tested at the beginning of each run according to the
vendor’s instructions using the system suitability standard set. The
background TOC values for the diluent blanks and reagent water
should not be more than 250 ppb.
2.3. Method development
2.3.1. Concentration of phosphoric acid in the diluent
To determine the appropriate concentration of the phosphoric
acid in the diluent, three concentrations of phosphoric acid were
prepared (0.05 M, 0.25 M, and 0.5 M) and their TOC values were
measured. Additionally, two clean swabs were added to 15 mL of
the diluents at each concentration. The swab blank extractions
were performed, and the TOC values of the resulting solutions were
determined.
2.3.2. Volume of the diluent
Cleaning sample solutions at the proposed maximum allow-
able carry over (MACO) limit were prepared with 15 mL or 30 mL
of the diluent, respectively. To determine the impact of vol-
ume/concentration of the sample solution on carryover, water
blanks were injected both before and after the injection of sample
solutions.
2.3.3. Extraction method
Each TOC vial containing two swab heads that were spiked at the
MACO limit was either sonicated or vortexed for 1 min, and then
shaken by hand to ensure thorough mixing. The extracted swab
heads were removed from the vials using a clean disposable pipette
tip, and the vial was re-capped.
2.3.4. Environment/lab location
Blank, clean coupons swabbing was performed at three differ-
ent locations: analytical lab (normal), analytical lab (isolated), and
clean room (ISO 8). The analytical lab was isolated by minimiz-
ing foot traffic into/out of the lab during the experiment and by
minimizing respirations/talking during sample preparation.
 X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39 35

Table 1
Calibration curve data.

Linearity Solution Target concentration of CIP200 (␮g/mL) Theoretical TOC value (ppm) Actual TOC reading (ppm) Correcteda TOC reading (ppm)

LS0 0.000 0.0000 0.0327 N/A

LS02 4.044 0.088 0.110 0.0773
LS05 10.11 0.22 0.247 0.2143
LS10 20.22 0.44 0.448 0.4153
LS15 30.33 0.66 0.677 0.6443
LS20 40.44 0.88 0.872 0.8393
LS50 101.1 2.2 2.08 2.0473
LS100 202.2 4.4 4.16 4.1273
LS150 303.3 6.6 6.15 6.1173
LS200 404.4 8.8 8.15 8.1173
a
Corrected TOC reading = Actual TOC reading − 0.0327 (TOC reading of LS0).

Table 2
®
Preparation of the CIP200 detergent simulated residue.

Levela Target concentration Volume of CIP200

(␮g/mL) stock solution (␮L)

0% (blank) 0 0
50% 101.1 202.2
100% 202.2 404.4
150% 303.3 606.6
a
The range covers from 50% to 150% of the target limit (333 ␮g dried detergent
®
content, equivalent to 6066 ␮g of the commercial CIP200 solution applied to a
50 cm2 surface area, corresponding to 4.4 ppm as TOC value).

2.3.5. Flow rate of the oxidant
Cleaning sample solutions at the MACO limit were analyzed at
various oxidant flow rates (1.4, 3.2, 6.8, 9.2, and 12.2 ␮L/min), and
the recovery was determined for each experiment.
Fig. 1. TOC value of diluent with different concentrations of phosphoric acid and
the sample solution (diluent + swab) after extraction from two clean swabs.
3. Results and discussion

2.4. Preparation of solutions 3.1. Method development

®
2.4.1. Stock CIP200 solution TOC is a non-specific analytical technique with high sensitivity
®
150.0 mg of the commercial CIP200 solution was transferred and high likelihood of environmental contamination with carbon
to a 10 mL volumetric flask, diluted to volume with Milli-Q water, containing compounds. Therefore, the quality of solvents, reagents
and mixed gently and thoroughly to obtain a 15.0 mg/mL solution. and proper sample handling techniques are important for robust
method development and validation. The focus of the method
development is to minimize contamination during sample prepa-
2.4.2. Linearity solutions (LS) ration and to maximize recovery for the trace levels of detergents.
The linearity solutions were prepared by serial dilution of the We have identified five factors that could negatively impact the
®
stock CIP200 solution with the sample diluent to obtain 30 mL of TOC analysis: the concentration and volume of the diluent, extrac-
the target concentrations listed in Table 1. tion method, location for TOC sample preparation, and oxidant flow
rate. These factors were evaluated to achieve a robust and reliable
2.5. Coupon spiking and swabbing analytical method suitable for its intended use.

Before spiking, the coupons need to be cleaned, following the
procedures as previously reported [15,29]. The spiked coupons
were prepared by applying the appropriate volumes of the stock
®
CIP200 solutions (Table 2) onto the coupons. Each concentra-
tion level was spiked in triplicate onto separate, freshly cleaned
coupons. The coupons were allowed to evaporate to dryness at
ambient conditions and swabbed as previously described [15]. The
swabbing procedure was performed as previously reported [15].
After swabbing, the swab heads are separated from the swab han-
dle at the pre-scored notch by forcefully bending the swab against
lip of the vial, avoiding extraneous contact and contamination. As
the swab breaks, the swab heads fall into the TOC vial containing
the sample diluent, and the vial is securely capped. Each TOC vial
containing two swabs was sonicated for 1 min, and then shaken by
hand to ensure thorough mixing. The extracted swab heads were
pulled from the vial using a clean disposable pipette tip, again tak-
ing care to avoid extraneous contact and contamination, and the
vial is re-capped.
3.1.1. Concentration of phosphoric acid in the diluent
Once the swabbing procedure is executed, the swab residues
need to be extracted in a proper diluent. Water has been used as
the extraction solution in previous reports [18,20]. However, in this
study, diluted phosphoric acid solution was chosen as the diluent
for sample preparation. Phosphoric acid (6 M) is also used as the
“Acid” reagent in the TOC analyzer to remove the inorganic car-
bon from the sample solution [23]. An acidic diluent solution helps
to effectively extract the analyte from the swab; however, if the
acid concentration is too high, there may be undesirable extraction
of organic carbon from the sampling swabs. To select the opti-
mal acid concentration, we evaluated the blank swab extractions
at three concentrations of phosphoric acid, 0.05, 0.25, and 0.5 M.
The results are summarized in Fig. 1. The TOC values were trending
higher as the phosphoric acid concentration increased. To minimize
the measured TOC background, 0.05 M of phosphoric acid in water
was chosen as the sample diluent for coupon recovery studies and
swab extraction. The value for 0.05 M corresponds to a TOC of the
36 X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39
Table 3
Influence of the volume of diluent.
Experiment number TOC blank reading (ppb)
15 mL diluent
Before sample injection
1 25
2 25
3 24
Fig. 2. The notch on the swab handle where it is broken to allow the swab head to
fall into the TOC vial.
Table 4
Influence of the extraction method.
Extraction method Average Recovery (%)
Vortexing 79
Sonication 76
swab extraction of 0.4 ppm, which is well below the cleaning limit
(4.4 ppm).
3.1.2. Volume of the diluent
In trace analysis, it is desirable to make the sample concentration
as high as possible to improve the limit of quantitation/detection.
The TOC vial capacity is 40 mL. Initially, 15 mL diluent was selected
for the swab extraction. This volume provides ample volume (four
injections) of the cleaning sample solution in each analysis. In addi-
tion, the notch in the swab handle stays above the surface of the
solution when the swab head is submerged into the diluent. This
avoids contamination of the sample solution from the swab handle
where the analyst hand makes contact during sample preparation.
By using the 15 mL sample volume, however, we observed that
the TOC value of the blank injections made after sample injection
were relatively high, suggesting carryover was taking place. The
carryover significantly decreased when 30 mL of diluent was used
(Table 3). Therefore, 30 mL was chosen as the diluent volume. With
this volume of diluent, the notch in the handle touches the solution
when the swab is submerged in the TOC vial. To avoid contamina-
tion from the handle, the swab head should be separated from the
handle (Fig. 2).
3.1.3. Extraction method
Vortexing and sonication are two commonly used extraction
methods. They were compared for extraction accuracy (% recovery)
and precision (repeatability) (Table 4). The recovery was calcu-
lated by comparing the measured TOC values with the theoretical
TOC values. The recoveries for the vortex and sonication extrac-
tions were 79% and 76%, and the relative standard deviations (Srel )
were 8% and 3%, respectively. Comparing the two methods, the
30 mL diluent
After sample injection Before sample injection After sample injection
187 25 54
165 25 64
200 25 48
Fig. 3. TOC reading of a clean stainless steel surface swab sampled under various
environmental conditions. The error bars represent the standard deviation of three
sample preparations (three coupons) in each case.
precision of the sonication method was much better than that of
the vortexing method, while the percent recoveries were within
Srel (%) the experimental variability. Therefore, sonication for 1 min was
chosen as the sample extraction method.
8
3
3.1.4. Location of sample preparation
The TOC analysis is extremely sensitive and cannot differentiate
the different sources of carbon (e.g., sample, contamination from
environment or handling, including respiratory exhalations). To
assess the influence of the lab environment we conducted spiking
and coupon swabbing experiments under different lab conditions.
We swabbed clean stainless steel coupons (50 cm2 ) in normal lab
environment with normal foot traffic and conversations of other
lab personnel nearby, in an isolated lab where entrance/exit was
restricted and the analysts’ talking (i.e., respiration) was minimal-
ized during the swabbing, and, finally, in a clean room (ISO 8) also
with minimal talking. As shown in Fig. 3, the TOC reading of the
coupon blank sample prepared under the normal lab condition was
the highest, suggesting appreciable contamination from the envi-
ronment. When the swabbing was conducted in an isolated lab,
the background TOC was significantly reduced. The contamination
from the environment was the least when the swabbing was con-
ducted in a clean room. However, because of the limited availability
of the clean room (for method validation), we chose to conduct
the experiments for method validation in an isolated lab environ-
ment. Compared to the proposed CL of 4.4 ppm, the TOC from the
environment, approximately 0.2 ppm, was deemed insignificant.
3.1.5. Flow rate of the oxidant
In a TOC experiment, the elemental carbon content of the ana-
lyte is oxidized to carbon dioxide using ammonium persulfate as
oxidizing agent [23]. The flow rate of the oxidant dictates the
amount of the oxidant in the reaction and should be optimized. Too
low of a flow rate will lead to an incomplete oxidation of the ana-
lyte; too high a flow rate could result in a lower measured TOC value
[30]. To optimize the oxidant flow rate, the recovery of the clean-
ing sample (prepared at the MACO limit) was determined at various
oxidant flow rates. As shown in Fig. 4, at 1.4 ␮L/min the oxidation
 X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39 37

Table 5
Accuracy at different spiking levels.

Level Expected TOC value (ppm) Actual TOC reading (ppm) Corrected TOC valuea (ppm) Recovery (%) Average recovery (%) Srel (%)

0% 0.0 0.328 N/A N/A N/A N/A

0.264
0.184
50% 2.2 2.14 1.881 94 94 3
2.06 1.801 90
2.17 1.911 96
100% 4.4 4.06 3.801 95 90 4
3.80 3.541 88
3.77 3.511 88
150% 6.6 6.17 5.911 96 97 1
6.26 6.001 97
6.30 6.041 98
a
The corrected TOC value was calculated by subtracting the average TOC reading of 0% level (0.259 ppm) from the actual TOC reading of each level.

® ® ®
CIP100 (3.35%, w/w) or of a mixture of CIP100 + CIP200 is higher
®
than that of CIP200 (2.173%, w/w). The lower carbon content in
®
CIP200 makes for the more stringent calculated CL in TOC value
corresponding to the MACO, as detailed below.
In this study, the method was validated for the CL correspond-
®
ing to 333 ␮g detergent/50 cm2 (MACO). The commercial CIP200
solution is a complex mixture. According to the formulation of
®
CIP200 provided by the vendor, there are three key components in
®
CIP200 that contribute to the total carbon content of a given sam-
®
ple. Thus, MACO expressed in ␮g of CIP200 commercial solution
2
per 50 cm is calculated using Eq. (1).
333
MACOCIP200 = = 6066g (1)
A1 × B1 + A2 × B2 + A3 × B3
where,
Fig. 4. % Recovery versus flow rate of the oxidizing reagent (ammonium persulfate)
of a sample prepared at the proposed MACO limit. The % Recovery is calculated by Ai % w/w of component i
comparing the measured TOC with the theoretical (expected) TOC at the CL. Bi % purity of component i
The values of A1 , A2 , A3 , B1 , B2 , and B3 were provided by the
vendor.
is incomplete and less than 60% of the carbon content in the sample Since the MACO of 6066 ␮g per 50 cm2 of commercial CIP200
®

was oxidized. There were no appreciable differences in the recov- solution is swabbed and sampled into a 30 mL volume, the TOC CL
eries at flow rates ≥ 3.2 ␮L/min, indicating that 3.2 ␮L/min oxidant is 4.4 ppm, calculated using Eq. (2).
flow rate is needed to ensure complete oxidation of the analyte. To
ensure consistent, quantitative analyte conversion to carbon diox- CCCIP200 × MACOCIP200 g
CL = = 4.4 = 4.4ppm (2)
ide, a higher value (6.8 ␮L/min) was chosen as the oxidant flow VT mL
rate. where,
®
CCCIP200 % carbon content in the CIP200 solution: 2.173% [16]
®
3.1.6. Calculation of the cleaning limit in TOC MACOCIP200 Maximum Allowable Carry Over of CIP200 solu-
2
tion per 50 cm : 6066 ␮g
The MACO dictates the threshold for the level of residual deter-
gent above which the cleaning process would be considered as a VT Total sample volume: 30 mL
failure [15]. According to EPA (40CFR156.62) toxicity standards,
® ®
CIP100 and CIP200 are considered slightly to moderately toxic 3.2. Method validation
based on LD50 (rat) data. This toxicity data justifies the amount
of residue detergents allowed to carryover of 100 ppm in the 3.2.1. Linearity
subsequent batch. The MACO is calculated based on this value, The linearity between the theoretical TOC value and the back-
®
subsequent batch size, and equipment surface area. In our case, ground corrected TOC value of CIP200 was examined over a range
®
the 100 ppm MACO limit corresponds to 333 ␮g detergent/50 cm2 from 4.0 to 404.4 ␮g/mL of CIP200 (88 ppb − 8.8 ppm of TOC val-
surface area. During the cleaning process, the surface of the manu- ues), corresponding to approximately 2% to 200% of the nominal CL
facturing equipment is cleaned by the application of hot water for concentration of 202.2 ␮g/mL, or 4.4 ppm TOC value. One prepara-
®
injection, followed by the rinse sequence of diluted CIP100 , hot tion of each linearity level solution was analyzed to determine the
®
water for injection, diluted CIP200 , and hot water for injection. TOC value. The TOC reading of the linearity samples were corrected
The volume of the hot water for injection used for the final rinse for the background reading of the blank. The correlation coefficient
®
is typically six times the volume of the diluted CIP100 solution. was 0.9999, and the y-intercept relative to response at 100% nomi-
®
With the multiple rinses with hot water and CIP200 solution, it nal CL concentration was 0.5%. The linearity data is summarized in
®
is reasonable to assume that CIP100 has been completely rinsed Table 1.
from the equipment surface at the end of the cleaning process and
®
that the detergent residue remaining only comes from CIP200 . 3.2.2. Accuracy
® ®
The TOC analysis of CIP200 presents the worst case scenario for Accuracy was demonstrated by the % recovery of CIP200 deter-
calculation of the TOC limit (ppm) because the carbon content of gent spiked in triplicate at 3 different levels onto the 50 cm2
38 X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39

Table 6 The method was evaluated for linearity, accuracy, precision, and
Pooled TOC data to assess intermediate precision.
sensitivity. The results demonstrated that the method is reliable
Level Pooled Average Pooled Standard Pooled Relative Standard and suitable for the cleaning assessment of residual detergents of
Recovery (%) Deviation (%) Deviation (Srel,pooled ) (%) the manufacturing equipment. The method was found to be lin-
50% 91 3 3 ear (R = 0.9999), accurate (recovery ≥ 90%), precise (Srel ≤ 4%), and
100% 92 3 3 highly sensitive (DL: 38 ppb, QL: 114 ppb). The methodology devel-
150% 96 5 5 oped in this study is generally applicable to the cleaning verification
of any organic detergents used for the cleaning of pharmaceutical
Table 7 manufacturing equipment made of electropolished stainless steel
Validation characteristics of the TOC analytical methods for cleaning assessment of material.
detergents.

Characteristics Present method ref [18] ref [19]

Linearity (R2 ) 0.9999 N/A
Range 4.0–404 ␮g/mLb N/A
Accuracy (%Recovery) 90–97% 92%a
Precision (Srel ) ≤4% 4.1%a
QL 114 ppbc N/A
DL 38 ppbc N/A
a
The values are for CIP-200 .
® [5] FDA warning letter (WL: 320-13-07), http://www.fda.gov/iceci/
b ®
The values are the concentration of CIP-200 .
c [6] FDA warning letter (WL: 320-08-02), http://wayback.archive-it.org/7993/
The values are the TOC reading.
®
stainless steel coupons. The spiked CIP200 was swab sampled
from the coupon surface and extracted with 30 mL of diluent, as
previously discussed. Recoveries were calculated against the deter-
mined concentrations of the solutions used to spike the coupons.
All sample TOC values were corrected for the diluent blank, back-
ground TOC value. The average recoveries were 90–97%, and the
individual recoveries were between 88 and 98%, with relative stan-
dard deviations (Srel ) of 1–4% (Table 5), comparable with previous
reports [18,20].
3.2.3. Precision: repeatability and intermediate precision
The repeatability of the method was demonstrated by the rel-
ative standard deviation (Srel ) of the % recovery of the surface
recovery samples (n = 3) at each of the three spiking levels (50%,
100% and 150%) and the Srel values were 1–4% (Table 5).
To demonstrate the intermediate precision, a second analyst
conducted the recovery experiments on a different day with a dif-
ferent TOC instrument. Results from both analysts were pooled
and summarized in Table 6. The results showed good day-to-day
accuracy and precision between two analysts.
3.2.4. Detection limit (DL) and quantitation limit (QL)
® [16] J. Zayas, H. Colı́on, O. Garced, L.M. Ramos, Cleaning validation 1: Development
The QL solution was established at 5.3 ␮g/mL of CIP200 solu-
tion (114 ppb TOC value) based on the linearity data. The DL, defined
®
as QL/3, was calculated as 1.8 ␮g/mL CIP200 solution (38 ppb TOC
value).
A summary of the validation characteristics is presented in
Table 7. By inspection of Table 7, excellent accuracy was obtained
considering that this is a trace analysis method. The linearity cov-
ers a two order of magnitude range with a detection limit of
38 ppb.
4. Conclusion
Five factors were identified affecting the development and vali-
dation of a TOC based analytical method to quantify trace residues
® ®
of CIP100 and CIP200 detergents on the surface of manufac-
turing equipment during the cleaning verification stage. These
factors were the concentration and volume of the diluent, extrac-
tion method, location for TOC sample preparation, and oxidant
flow rate. Key experimental parameters were optimized to min-
imize contamination and to improve the sensitivity, recovery, and
reliability of the method.
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