Cleaning validation sampling in the pharmaceutical industry has been a subject of increasing interest and scrutiny in recent Food and Drug Administration (FDA) inspections. The validation of the procedures used to clean the equipment used during the different steps of a manufacturing process is a clear requirement of current Good Manufacturing Practices (cGMP). For example, FDA inspectors now expect to see a cleanliness validation program in place with the proper documentation in place during their inspections.
The objectives of this article are to establish a broad basis for sampling in cleaning method validation policies and programs and to determine the requirements, procedures, acceptance limits, and working documents necessary to support this critically important activity.
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Cleaning Validation Protocol
Cleaning validation protocols must be developed, approved, and executed in accordance with the standard operating procedures covering these activities in effect at the time. A typical cleaning validation protocol should consist of the Objective, Sampling and Testing Methodologies, and Acceptance Criteria sections.
This section defines the intention and scope of the cleaning validation exercise. Additionally, it will include information such as equipment names, identification numbers, the name(s) and type(s) of the product being cleaned from the equipment, and the individual components of the product and equipment under investigation.
Sampling and Testing Methodologies
This section should generally include a step-by-step explanation of the sampling techniques and requirements, as well as the specific analytical procedures that will be used in the analysis of those samples. You must specify which laboratories will participate in the tests and the precautions to be taken during the validation exercise.
A visual check should be incorporated into the cleanliness evaluation. The sampling technique chosen to evaluate the effectiveness of the cleaning procedure should be the smear, the fluid rinsing of the samples, or a combination of both methods. The following sampling methods provide various levels of assurance regarding cleanliness:
- Active product contact parts of the equipment are individually examined (wherever possible) for cleanliness. This visual inspection allows the early localization and identification of any inadequacies in the cleaning procedure.
- Qualitative – dependent upon inspector and item sampled.
- Subjective – dependent upon inspector and item sampled.
Rinse water sampling and analysis:
- According to 2004 FDA “Guide to Inspections Validation of Cleaning Processes:” “Two advantages of using rinse samples are that a larger surface area may be sampled, and inaccessible systems or ones that cannot be routinely disassembled can be sampled and evaluated.”
- Analysis can be quantitative, using pH, conductivity, particle count, microbial count,
- Total Organic Carbon (TOC) determination, spectrophotometry, bioassays, or Limulus amebocyte lysate for pyrogens. ✓ The recovery factor is uncertain; it involves dilution.
Surface sampling and analysis:
- Eliminates adherent materials.
- The analysis can be quantitative.
- A precise definition of the sampled area is required.
Surface sampling from coupons:
- Depends on whether coupons are equivalent to the surface of interest.
- Requires removing coupons from the system.
Whenever possible, each piece of equipment should be disassembled into its individual components after cleaning and each part should be individually tested for cleanliness. In this way, any deficiencies in the cleaning process will be more easily identified and located. It may not be practical or desirable to dismantle large or clean-in-place (CIP) equipment. Regardless, sampling and validation testing should begin as soon as possible after the cleanup process is complete to reduce the possibility of contamination from external sources. Equipment that has just been cleaned must be covered immediately with suitable means to protect it from any contamination.
Aqueous or organic solvents used in the cleaning procedure should be sufficient to remove residue and, at the same time, should be minimized to reduce the risk of reaction or damage to equipment, or excessive dilution of the residue and the resultant. loss of analytical sensitivity. Samples must be collected in clean or sterilized containers. Sterile containers are suitable for this intended use. All validation samples must be properly labeled with complete information about the source of the sample, the name of the sampler, the date of sampling, the reference number, the name of the product, and the part of the equipment from which the sample was taken.
A sample of the rinse or swab solvent should always be included with the actual test samples to serve as a reagent blank for any chemical or microbiological determination when required. All types of samples, physical, chemical, or microbiological, must be collected in accordance with a written procedure and using techniques, reagents, equipment, and containers appropriate for the type of test to be performed. Only trained personnel should collect these samples.
The sampling method selection for cleaners involves choosing between rinse water sampling, swabbing surfaces, coupon sampling, or placebo sampling. Rinse water sampling involves taking a sample of an equilibrated post-final rinse that has been re-circulated over all surfaces. Rinse samples should be correlated to a direct measuring technique such as swabbing. Swabbing involves using a wipe or swab that is moistened with high purity water, such as Water-for-Injection (WFI) that is typically wiped over a defined area in a systematic multi-pass way always going from clean to dirty areas to avoid recontamination (e.g.: 10 cm side by side strokes vertically, 10 cm horizontally, and 10 cm each with the flip side of the swab in each diagonal direction).
For TOC analysis, very clean swabs or wipes and sample vials should be used. (All of these are commercially available). The amount of residue is known to be uniformly distributed on the smooth surfaces of equipment parts. Also, the most difficult to clean or “worst-case” areas of the equipment should be identified and specifically targeted for sampling whenever possible.
Selecting a method to detect cleaner residues can involve specific methods for specific cleaner ingredients such as High-Performance Liquid Chromatography (HPLC), ion-selective electrodes, flame photometry, derivative UltraViolet (UV) spectroscopy, Thin Layer Chromatography, enzymatic detection, and titration. It can also involve non-specific methods that detect the presence of a blend of ingredients such as: TOC, pH, and conductivity. The FDA prefers specific methods but will accept non-specific methods with adequate rationales for their use. For investigations of failures or action levels, a specific method is usually preferable.
Analytical validation of the cleaning procedure should be performed after the approval of visual inspection (absence of stains or any materiel residue). The specificity, sensitivity, and percentage of recovery of the test method should be adequate to meet acceptance criteria.
For the swab method it may be necessary to determine:
- The percentage recovery of the swab extraction procedure.
- The effectiveness of the swab at recovering residues from equipment parts surface.
- The interference of swab materials in the analysis. For the rinse solution method it may be necessary to determine:
- The percentage recovery of the rinse solution extraction procedure.
- The effectiveness of the rinse solution at recovering residues from equipment parts surfaces.
- The interference of the rinse solution in the cleaning procedure and analysis.
- A correction for recovery efficiency in calculations for acceptable residue level
Percentage Recovery = 100 x Sample Concentration / Standard Concentration
The percentage recovery is important because it will be applied when evaluating the final residual concentration according to the relation:
Percentage of the actual amount of residual = Calculated Amount x Percentage Recovery.
It is very difficult to establish acceptable fixed limits for recovery percentage due to the individual difference in solubility of residues, the solvent used, and the nature of the manufacturing surfaces. The following three factors contribute to the difficulty of establishing fixed limits for the recovery percentage:
- The residues behavior toward the solvent used.
- The solvent used.
- The nature of the manufacturing surfaces.
Some products such as proteins, for example, have very low solubility, so the percentage recovery may be as low as 10–20%. For soluble residue, a higher percentage recovery should be expected. In general, we can expect an ideal percentage recovery that falls between 60% and 90%. It is very important to continuously develop the sampling and swabbing methods and reproducibility to improve percentage recovery values.
A safety factor of not more than 1 / 1000 (0.1%) of the active under investigation (contaminant) found in a single unit of the lowest dosage form of the
next product should remain in the equipment after the cleaning procedure. A
list summarizing the batch size of products manufactured through the same
equipment should be prepared in order to determine the smallest batch size.
This is an important step to calculate the carryover limit.
The calculation of acceptance criteria should be based on the following
• Residual limit of active (contaminant) expressed in mg / cm2 : R
• 1/ 1000 of concentration of active (contaminant) per dose units: L
• Maximum allowable number of doses per day of next product (contaminated): D
• Smallest batch size in mg: B
• Concentration of active in unit dose of next product (contaminated) or the number of total dose units manufactured: C
• Total surface area of equipment parts in contact with the product
(contaminant) expressed in cm2 : T
• Surface swabbed in cm2 : S
Total surface area of equipment parts in contact with the product (contaminant) expressed in cm2 = 45000 cm2
Surface swabbed in ccm2 = 100 cm2
Residual limit of active (contaminant) in mg / cm2 =
30 x 1/1000 / 4 x 50 x 106 / 200 x100 / 45000
Residual limit of active (contaminant) in the equipment after cleaning =
4.17 mg / cm2
For the automated systems and where rinse is used and rinse volumes are known, the following equation could be used taking into consideration the total volume of final rinse in ml V:
R mg / ml = L / D x B / C x 1 / V
The active (contaminant) carryover from product A to the next product B (contaminated) per unit dose is calculated as follows:
For example, the concentration of active (contaminant) A per unit dose is 2 mg per day. Based on a safety factor of not more than 1 / 1000 (0.1%) of the active under investigation (contaminant) found in a single unit of the lowest dosage form of the next product, we can say:
The limit of concentration of active A found in total (standard) daily dose
units of next (contaminated) product B is:
1 / 1000 x 2 mg = 0.002 mg or 2 µg
Supposing that the total amount of contaminant product A found in the total product contacting parts of the equipment after cleaning is 9 mg (9000 µg). The next smallest batch size worst-case (contaminated) B is 100 Kg and B unit dose weight (tablet weight for example) is 90 mg. The total allowable daily dose of B is three doses. The concentration of active contaminant A carried over to unit dose of product B is calculated:
Contaminant µg / mg = 9000 µg / 100 x 106
Contaminant µg / mg = 9 x 10-5
Contaminant µg / Unit dose = 9 x 10-5 x 90
Contaminant µg / Unit dose B = 0.0081
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