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Analysis journal homepage: www.elsevier.com/locate/jpba Cleaning verification: A five parameter study of a Total

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

Pharmaceuticals Corporation, San Carlos, CA 94070, USA a r t i c l e i n

a r t i c

l e

i n f o

Article history:

Received 9 August 2017 Received in revised form 18 September 2017 Accepted 22 October 2017 Available online 26 October 2017

Keywords:

Manufacturing equipment Cleaning optimization Cleaning verification Detergent residues Coupons TOC

a b s t r a c t

A Total Organic Carbon (TOC) based analytical method to quantitate trace residues of clean-in-place

(CIP) detergents CIP100 ® and CIP200 ® on the surfaces of pharmaceutical manufacturing equipment was

developed and validated. Five factors affecting the development and validation of the method were identified: diluent composition, diluent volume, extraction method, location for TOC sample preparation, 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

phosphoric acid in the swabbing solution was 0.05 M, and the optimal volume of the sample solution was 30 mL. The swab extraction method was 1 min sonication. The use of a clean room, as compared to an isolated lab environment, was not required for method validation. The method was demonstrated to

be linear with a correlation coefficient (R) of 0.9999. The average recoveries from stainless steel surfaces

at multiple spike levels were >90%. The repeatability and intermediate precision results were 5% across

the 2.2–6.6 ppm range (50–150% of the target maximum carry over, MACO, limit). The method was also 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

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).

https://doi.org/10.1016/j.jpba.2017.10.021

0731-7085/© 2017 Elsevier B.V. All rights reserved.

tently yield results that meet pre-determined specifications and 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

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 (CO 2 ). The generated CO 2 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.

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. Experimental

2.1. Materials

The system suitability standard set was from GE Analytical Instruments (Boulder, CO, USA), STD 31004-XX (containing: Work-

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

Table 1 Calibration curve data.

35

Linearity Solution

Target concentration of CIP200 ( g/mL)

Theoretical TOC value (ppm)

Actual TOC reading (ppm)

Corrected a 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.

Level a

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 cm 2 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.

2.4. Preparation of solutions

2.4.1. Stock CIP200 ® solution

150.0 mg of the commercial CIP200 ® solution was transferred to a 10 mL volumetric flask, diluted to volume with Milli-Q water, and mixed gently and thoroughly to obtain a 15.0 mg/mL solution.

2.4.2. Linearity solutions (LS)

The linearity solutions were prepared by serial dilution of the stock CIP200 ® solution with the sample diluent to obtain 30 mL of the target concentrations listed in Table 1.

2.5. Coupon spiking and swabbing

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.

contact and contamination, and the vial is re-capped. Fig. 1. TOC value of diluent with different

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

3.1. Method development

TOC is a non-specific analytical technique with high sensitivity and high likelihood of environmental contamination with carbon containing compounds. Therefore, the quality of solvents, reagents 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- ration and to maximize recovery for the trace levels of detergents. We have identified five factors that could negatively impact the TOC analysis: the concentration and volume of the diluent, extrac- tion method, location for TOC sample preparation, and oxidant flow rate. These factors were evaluated to achieve a robust and reliable analytical method suitable for its intended use.

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

Table 3 Influence of the volume of diluent.

X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39

Experiment number

TOC blank reading (ppb)

15 mL diluent

30 mL diluent

Before sample injection

After sample injection

Before sample injection

After sample injection

1

25

187

25

54

2

25

165

25

64

3

24

200

25

48

2 25 165 25 64 3 24 200 25 48 Fig. 2. The notch on the

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 (%)

S rel (%)

Vortexing

79

8

Sonication

76

3

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 (S rel ) were 8% and 3%, respectively. Comparing the two methods, the

8% and 3%, respectively. Comparing the two methods, the Fig. 3. TOC reading of a clean

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 the experimental variability. Therefore, sonication for 1 min was chosen as the sample extraction method.

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 cm 2 ) 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

Table 5 Accuracy at different spiking levels.

37

Level

Expected TOC value (ppm)

Actual TOC reading (ppm)

Corrected TOC value a (ppm)

Recovery (%)

Average recovery (%)

S rel (%)

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.

level (0.259 ppm) from the actual TOC reading of each level. Fig. 4. % Recovery versus

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 comparing the measured TOC with the theoretical (expected) TOC at the CL.

is incomplete and less than 60% of the carbon content in the sample was oxidized. There were no appreciable differences in the recov- eries at flow rates 3.2 L/min, indicating that 3.2 L/min oxidant flow rate is needed to ensure complete oxidation of the analyte. To ensure consistent, quantitative analyte conversion to carbon diox- ide, a higher value (6.8 L/min) was chosen as the oxidant flow rate.

3.1.6. Calculation of the cleaning limit in TOC

The MACO dictates the threshold for the level of residual deter- gent above which the cleaning process would be considered as a failure [15]. According to EPA (40CFR156.62) toxicity standards, CIP100 ® and CIP200 ® are considered slightly to moderately toxic based on LD50 (rat) data. This toxicity data justifies the amount of residue detergents allowed to carryover of 100 ppm in the subsequent batch. The MACO is calculated based on this value, subsequent batch size, and equipment surface area. In our case, the 100 ppm MACO limit corresponds to 333 g detergent/50 cm 2 surface area. During the cleaning process, the surface of the manu- facturing equipment is cleaned by the application of hot water for injection, followed by the rinse sequence of diluted CIP100 ® , hot water for injection, diluted CIP200 ® , and hot water for injection. The volume of the hot water for injection used for the final rinse is typically six times the volume of the diluted CIP100 ® solution. With the multiple rinses with hot water and CIP200 ® solution, it is reasonable to assume that CIP100 ® has been completely rinsed from the equipment surface at the end of the cleaning process and that the detergent residue remaining only comes from CIP200 ® . The TOC analysis of CIP200 ® presents the worst case scenario for calculation of the TOC limit (ppm) because the carbon content of

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 cm 2 (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 per 50 cm 2 is calculated using Eq. (1).

MACO CIP 200 =

333

A 1 × B 1 + A 2 ×

B 2 + A 3 × B 3 = 6066 g

(1)

where, A i % w/w of component i B i % purity of component i The values of A 1 , A 2 , A 3 , B 1 , B 2 , and B 3 were provided by the vendor. Since the MACO of 6066 g per 50 cm 2 of commercial CIP200 ® solution is swabbed and sampled into a 30 mL volume, the TOC CL is 4.4 ppm, calculated using Eq. (2).

CL = CC CIP 200 × MACO CIP 200

V T

where,

= 4 . 4 g

mL

= 4 . 4ppm

(2)

CC CIP200 % carbon content in the CIP200 ® solution: 2.173% [16]

MACO CIP200 Maximum Allowable Carry Over of CIP200 ® solu-

tion per 50 cm 2 : 6066 g V T Total sample volume: 30 mL

3.2. Method validation

3.2.1. Linearity

The linearity between the theoretical TOC value and the back- ground corrected TOC value of CIP200 ® was examined over a range from 4.0 to 404.4 g/mL of CIP200 ® (88 ppb 8.8 ppm of TOC val- ues), corresponding to approximately 2% to 200% of the nominal CL concentration of 202.2 g/mL, or 4.4 ppm TOC value. One prepara- tion of each linearity level solution was analyzed to determine the TOC value. The TOC reading of the linearity samples were corrected for the background reading of the blank. The correlation coefficient was 0.9999, and the y -intercept relative to response at 100% nomi- nal CL concentration was 0.5%. The linearity data is summarized in Table 1.

3.2.2. Accuracy

Accuracy was demonstrated by the % recovery of CIP200 ® deter- gent spiked in triplicate at 3 different levels onto the 50 cm 2

38

X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 33–39

Table 6 Pooled TOC data to assess intermediate precision.

Level

Pooled Average

Pooled Standard

Pooled Relative Standard

Recovery (%)

Deviation (%)

Deviation (S rel,pooled ) (%)

50%

91

3

3

100%

92

3

3

150%

96

5

5

Table 7 Validation characteristics of the TOC analytical methods for cleaning assessment of detergents.

Characteristics

Present method

ref [18]

ref [19]

Linearity (R 2 )

0.9999

N/A

0.9998

Range

4.0–404 g/mL b

N/A

50–500 g/mL b

Accuracy (%Recovery)

90–97%

92% a

95%

Precision (S rel )

4%

4.1% a

1.8%

QL

114 ppb c

N/A

151 ppb c 50 ppb c

DL

38 ppb c

N/A

a The values are for CIP-200 ® . b The values are the concentration of CIP-200 ® . c 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 (S rel ) 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 (S rel ) of the % recovery of the surface recovery samples (n = 3) at each of the three spiking levels (50%, 100% and 150%) and the S rel 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)

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.

The method was evaluated for linearity, accuracy, precision, and sensitivity. The results demonstrated that the method is reliable and suitable for the cleaning assessment of residual detergents of the manufacturing equipment. The method was found to be lin- ear (R = 0.9999), accurate (recovery 90%), precise (S rel 4%), and highly sensitive (DL: 38 ppb, QL: 114 ppb). The methodology devel- oped 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.

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