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Cleaning Verification of the Manufacturing Equipment is an FDA Requirement

Updated: Apr 9, 2022

Cleaning verification for detergents

In this blog, I will talk about the cleaning assessment of residual detergents in manufacturing equipment. Detergents are commonly used to remove drug product residue from the manufacturing equipment; however, detergents can remain on the equipment surfaces if they are not sufficiently rinsed during the cleaning process, contaminating the next manufactured product. Usually, a cleaning validation master plan requires that detergent(s), used to clean the manufacturing 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 for detergents is a requirement by health authorities at the commercialization of drug products (21 CFR 211.67, and failing to comply with the requirements may result in regulatory warning letters. Assessment of residual detergents at the end of the equipment cleaning process is challenging because the analytical method must address limit of quantitation at trace level (e.g., ppm) of analyte(s) that often lack UV chromophores.

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Clean-in-place detergents are the most used for automated cleaning

Clean-in-place (CIP) detergents CIP100® (base) and CIP200® (acid) are the most used for CIP systems or automated 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 equipment, are used to simulate equipment surfaces during laboratory method development and validation of the cleaning verification methods.

Total Organic Carbon (TOC) based analytical methods are the best choice for cleaning assessment

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, excipients, 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 conductivity detection technique, and the TOC result is reported as concentration of organic carbon in the sample (ppm or ppb).

High likelihood of environmental contamination with carbon containing compounds requires attention to the quality of solvents, reagents, and proper sample handling techniques. The focus of the method development is to minimize contamination during sample preparation and to maximize recovery for the trace levels of detergents.

Several factors can affect the development and validation of the TOC methods

Most of the reported unreliability with TOC measurements is due to the sample preparation and handling along with the precise control of the instrumental parameter. Parameters such as temperature on oxidation efficiency and carrier gas flow rate are known to affect the accuracy, precision, limit of detection, and calibration of the TOC analysis.

Factors that could negatively impact the TOC analysis are the concentration of the phosphoric acid and volume of the diluent, extraction method, location for TOC sample preparation, and oxidant flow rate (J. Pharm. Biomed. Anal. (2018), 149, 33–39).

Concentration of phosphoric acid in the diluent

Once the swabbing procedure from the stainless-steel coupons is executed, the swab residues need to be extracted in a proper diluent. Diluted phosphoric acid solution is the best chois 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 carbon from the sample solution. 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 minimize the measured TOC background, 0.05 M of phosphoric acid in water is recommended as the sample diluent for coupon recovery and swab extraction. This level of phosphoric acid concentration corresponds to a TOC of the swab extraction of 0.4 ppm, which is well below the cleaning limit (e.g., 4.4 ppm).

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. A low sample volume (e.g., 15 mL) leads to a relatively high carryover after the blank injection. The carryover significantly decreases with the diluent volume (30 mL) increase. With this volume of diluent, the notch in the swabbing handle touches the solution when the swab is submerged in the TOC vial. To avoid contamination from the handle, the swab head should be separated from the handle (Figure 1).

Figure 1. The notch on the swab handle to be broken, for the swab head to fall into the TOC vial.

Extraction method

Vortexing and sonication are two commonly used extraction methods. They were assessed by comparing the extraction accuracy (% recovery) and precision (repeatability). The % recoveries for the vortex and sonication extractions were 79% and 76% and the relative standard deviations (Srel) were 8% and 3%, respectively. Comparing the two methods, the precision of the sonication method was much better than that of the vortexing method, while the percent recoveries were within the experimental variability. Therefore, we recommend a 1 min sonication for the sample extraction method.

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). Under normal lab condition the TOC reading of the coupon-blank sample are higher, with appreciable contamination from the environment (Figure 2). In an isolated lab, where entrance/exit was restricted and the analysts’ talking (i.e., respiration) was minimalized during the swabbing, the background TOC is reduced. The contamination from the environment was the least when the swabbing occurs in a clean room (ISO 8). However, there is a limited availability of the clean rooms for method validation. Compared to the proposed CL of 4.4 ppm, the TOC contribution from the environment of approximately 0.2 ppm, is insignificant and we recommend the use of an isolated lab.

Figure 2. 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.

Flow rate of the oxidant

In a TOC experiment, the elemental carbon content of the analyte is oxidized to carbon dioxide using ammonium persulfate as oxidizing agent. 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 analyte; too high a flow rate could result in a lower measured TOC value. The oxidant flow rate needs to be optimized for the recovery of the cleaning sample prepared at the maximum allowable carry over (MACO) limit. We found that a 3.2 µL/min oxidant flow rate is needed to ensure complete oxidation of the analyte (Figure 3). To ensure consistent, quantitative analyte conversion to carbon dioxide, a higher value (6.8 µL/min) is recommended for the oxidant flow rate.

Figure 3. % 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.

MACO limit in TOC

The MACO dictates the threshold for the level of residual detergent above which the cleaning process would be considered as a failure. 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, the subsequent batch size, and the equipment surface area. During the cleaning process, the surface of the manufacturing 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.

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