By Eric Lingenfelter, Wes Atkins, and Henry Evans
It’s Clean, but Can You Prove It?
Validation and revalidation are key when establishing cleaning methods
Editor’s Note: This article is the second in a two-part series on cleaning validation methodology. Part one, “How to Improve Cleaning Processes,” appeared in our June issue.
Method limits, selection of cleaning techniques, and selection of method detection were addressed in the first part of this article. Part two will address method validation, the importance of stability for cleaning validation samples, when revalidation of a cleaning method is necessary, the use of correction factors, and how to handle failing results.
Once the basic cleaning procedure elements have been established (establishment of limits, cleaning procedures, master plan, cleaning protocols, development of analytical method), the method is ready to be validated.1 This section outlines typical components utilized to validate the analytical method. The validation components presented below are based upon International Conference on Harmonisation (ICH) and United States Pharmacopeia (USP) guidelines.
Accuracy/Precision: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or an accepted reference value and the value found. The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.2
Accuracy/precision should be assessed using a minimum of three concentration levels, each prepared in triplicate. Accuracy/precision is typically performed with concentrations ranging from 80 to 120% of the final theoretical sample concentration (based upon the maximum contamination limit or MCL), although a wider range may be more appropriate in certain instances.
There are various categories of accuracy/precision that need to be established as part of the method validation.
- Solution accuracy is the measurement of the compound of interest added directly to the extraction solution. Reference standard solution is spiked directly into the diluent to prepare the three levels of concentration. Solution accuracy is performed as a control—usually in triplicate—to prove recovery of the analyte from the extraction solution. This recovery can then be compared to both swab and surface accuracy recoveries. If rinseates are being analyzed, this test is the only one needed to prove accuracy/ precision.
- Swab accuracy determines the method’s ability to recover the compound of interest directly from the swab head. These studies are performed by directly adding standard material to the swab head and then extracting as per the analytical method. Typically, three replicate-spiked swabs are prepared at the high and low concentrations, while six replicates are prepared at the 100% level.
- Surface accuracy determines the method’s ability to recover the compound of interest directly from a defined surface. Coupons of the defined surface material are spiked with reference standard at the three concentration levels mentioned above. The area of the coupon spiked with standard is dependent on the actual cleaning procedure and the surface area typically sampled after manufacturing. Typical surface areas sampled are 25 to 100 cm2.
Acceptance criteria should be evaluated and determined during the development of the analytical methods. Many factors influence the establishment of appropriate criteria, including the surface being swabbed, MCL, type of swab, and instrumentation. Intermediate accuracy/precision should be performed by a second analyst repeating the accuracy/ precision tests listed above. If multiple surfaces are involved in the validation, the second analyst can perform accuracy/precision on select surfaces (worst case) if appropriate.
Linearity: The linearity of an analytical procedure is its ability, within a given range, to obtain test results that are directly proportional to the concentration (amount) of analyte in the sample.2
A minimum of five concentration levels are typically evaluated, with duplicate injections at each level. Concentrations ranging from the limit of quantitation to 200% of the MCL are typically evaluated during validation. Acceptance criteria are generally based upon either the correlation coefficient or the coefficient of determination of the linear plot. In addition, criteria can be established around both the slope and Y-intercept of the plot.
Specificity: Specificity is the ability to assess the analyte unequivocally in the presence of components that may be expected to be present.2
Swab type and surface type are typically evaluated to determine if interferences are present in the method. Although each of these components is typically examined during method development, they should be included in the validation process and shown, under protocol, to have little or no interference. Swabs and surfaces are prepared as per the method without the introduction of the analyte of interest. Interference from either the swab or surface should be less than 10% of the MCL. Lower limits for specificity may be appropriate depending on the method conditions.
Limits of Detection and Quantitation: The limit of detection (LOD) of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The limit of quantitation (LOQ) of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy.2
Both the LOD and LOQ should be verified by a suitable number of preparations known to be prepared near the respective limit being evaluated.4 LOD and LOQ can be estimated using a signal-to-noise approach with typical values of three-to-one for LOD and 10-to-one for LOQ.
During method validation, standard solutions are prepared at the estimated LOD and LOQ (three preparations for LOD and three preparations with duplicate analyses for LOQ). Typical acceptance criteria for LOD require that the analyte be detected in each analysis. For LOQ, the percent recovery is determined for each of the six measurements and should fall between 75% and 125% recovery. The relative standard deviation is also determined for the six measurements and should be less than 25%.
Robustness–Chromatographic Conditions: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.2
Instrument and reagent variations, for example, may be examined as part of robustness to ensure that the method provides reliable data under varying conditions. Robustness is not a critical validation component according to ICH guidelines, but should be considered on a case-by-case basis. Robustness may be determined during development of the analytical procedure, and if measurements are susceptible to variations in analytical conditions, these should be suitably controlled, or a precautionary statement should be included in the procedure.3
Stability–Stock Standard, Working Standard, Working Swab: Stability of stock standards, working standards, and working swab or rinseate samples are evaluated as part of the validation. Stability can be evaluated under various conditions such as refrigeration or protection from light, but ambient conditions are preferred. This stability period is necessary to ensure that cleaning validation samples can be collected, shipped to the testing facility, and analyzed. The length of stability is particularly important for cleaning validation sample solutions and should be at least one week old, preferably two weeks.
Table 1 lists a summary of the validation components involved in a typical cleaning validation, along with examples of typical acceptance criteria that can be set for those tests. Please note that the acceptance criteria are listed for informational examples only and that the actual acceptance criteria must be determined on a case-by-case basis, depending on validation specifics.
Cleaning validation procedures should be revalidated when the equipment train of the manufacturing process is changed. Possible changes in the equipment train include the surface type utilized and/or surface area, which can lead to the establishment of a new MCL. A full validation can usually be avoided, and only certain elements of the cleaning validation need to be revalidated. If the new limit is within the previously established linear range, only surface recoveries (bracketing the new limit) and surface residue specificity need to be revalidated. These same two elements must be revalidated if a surface type is changed.
If the new limit is outside the previously established linear range, linearity must be extended above or below the new limit, and swab recovery, surface recovery, and surface residue specificity need to be revalidated. For a new limit below the established linear range, a new standard concentration at this level may be recommended. If the method is not linear through the new level, however, a new standard concentration is necessary. A new standard concentration requires a full revalidation.
Other possible but less likely reasons to revalidate swab recovery, surface recovery, and surface specificity include a change in the type of swab or swabbing pattern. For a change in swab type, swab specificity also needs to be revalidated. For any of the previously listed changes, elements that do not require revalidation are LOD and LOQ.
The prior revalidation discussion assumes that the validated method was for swab samples and not for rinse samples. For rinse samples, validation elements involving swabs and surfaces do not need to be conducted. Additionally, any changes in the synthesis of the drug substance, changes in the composition of the finished product, or changes in the analytical procedure require revalidation according to ICH guidance.2
Sometimes in cleaning validation studies, it is determined that not all the residue on a surface can be fully recovered, thus producing lower recoveries. In these instances, it may be necessary to apply a recovery factor. If a recovery factor is deemed appropriate, several issues must be considered before it is set:
- Recovery factors are usually not applied if recovery results are above 70%; however, there is no standard limit.
- Recovery factors must be set under sound scientific justification.
- Recovery factors should not be used if recoveries are too low. (For example, if recoveries are consistently around 10%, a 10X factor would not be appropriate.)
- Recovery factors need to be set prior to or during validation, not during routine monitoring.
- All results used to determine the recovery factor need to be consistent and reproducible.
Recovery factors are often seen as a last resort to salvage a mediocre method. Recovery method optimization should always be explored as an alternative prior to using recovery factors.
No matter which scientific field you are in, the question of how to handle failing data during routine sample testing will arise; the world of cleaning validation is no different. The best way to approach this issue is to address it before it becomes a problem. When developing the cleaning validation master plan or protocol, dedicate a section to appropriate handling of failing results. Here, a step-by-step investigation of the results can be laid out in advance, so that decisions won’t be made based on instance-by-instance circumstances. When reviewing data, regulatory agencies like to see that failing results were handled in a consistent and systematic manner.
All data that do not meet protocol or master plan acceptance criteria need to be treated as a deviation. They must be handled by first being verified, resolved, and approved. This may require that samples be retested. Sometimes more samples may need to be collected to verify outlying results. If results indicate that a criterion or limit is not attainable under set conditions, modifications to the method, protocol, standard operating procedure, or master plan may be entertained. Again, all of these scenarios should be investigated during the feasibility/method development/validation stage of the cleaning validation study. n
Lingenfelter, Atkins, and Evans are senior chemists in the method development and validation group at Lancaster Laboratories Inc. For more information, reach Lingenfelter at (717)656-2300 begin_of_the_skype_highlighting (717)656-2300 end_of_the_skype_highlighting, ext. 1449, or at email@example.com.
- Active Pharmaceutical Ingredients Committee (APIC). Cleaning validation in active pharmaceutical ingredient manufacturing plants. Washington, DC: APIC; 1999. Available at: http://apic.cefic.org/pub/4CleaningVal9909.pdf. Accessed July 10, 2009.
- International Conference on Harmonisation (ICH). Harmonised tripartite guideline: validation of analytical procedures: text and methodology Q2(R1). Geneva, Switzerland: ICH; 2005. Available at: http://www.ich.org/LOB/media/MEDIA417.pdf. Accessed July 10, 2009.
- United States Pharmacopeia/National Formulary. USP 32/NF 27, General Chapters: <1225>Validation of Compendial Procedures. Rockville, Md.: United States Pharmacopeial Convention; 2009.