8.G.1 Requirements for method validation

"The analytical methods should be validated before the Cleaning Validation Study is carried out. The analytical methods used to detect residuals or contaminants should be specific for the substance to be assayed and provide a sensitivity that reflects the level of cleanliness determined to be acceptable by the company. The analytical methods should be challenged in combination with the sampling methods used, to show that the contaminants can be recovered from the equipment surface and to show the level of recovery as well as the consistency of recovery. This is necessary before any conclusions can be made based on the sample results. A negative result may also be the result of poor sampling techniques. "
(PIC/S PI006, section 7.10).

The PIC/S guideline thus attaches great importance to 3 aspects of the method validation:

• Specificity
• Sensitivity
• Recovery

The validation of analytical methods usually also includes the following aspects (see chapter 14.F Validation of analytical methods):

• Precision
• Accuracy
• Limit of detection and limit of quantitation
• Selectivity
• Linearity and linear range
• Robustness

The following section only discusses the aspects which have particular significance for regulatory purposes in the validation of analytical methods as part of the cleaning validation.

Specificity

A distinction can generally be made between specific and non-specific analytical methods. Whereas specific methods relate to a specific substance, non-specific analytical methods record chemically different substances with the same properties. Some specific and non-specific analytical methods are listed in figure 8.G-1.

The requirement for specificity of the analytical procedure for determining active pharmaceutical ingredient residues can generally be fulfilled without problems, as there already exist appropriate procedures for the starting and final release analysis, which can be adapted to the residue determination requirements for the cleaning validation.

However, it is more difficult to implement specific analytical procedures when determining cleansing agent residues. Due to the chemical structure of many ingredients of cleaning agents, a direct specific determination of assay is often not possible. A specific determination of content can be customised for some ingredients using derivisation and/or special detectors, but the effort and cost of this are not inconsiderable.

Sensitivity

The sensitivity of an analytical method indicates how strongly the measurement will react to changes in concentration. The larger the measurement change in the event of a change in concentration, the more sensitive is the procedure. If there is direct proportionality, the sensitivity corresponds to the gradient of the calibration line.

It is to be assumed that the requirement for sufficient sensitivity with regard to the defined level of purity in PIC/S guideline PIC/S PI006 goes beyond this classic definition of the concept: to ensure that the smallest amount of a substance permissible in a sample can be reliably detected and determined, the limits of detection and quantitation must be determined.

The limit of detection is the smallest quantity of a substance in a sample (matrix) that can be detected but not quantified exactly (see chapter 12.3 LOD = Limit of Detection).

The Limit of quantitation is the smallest quantity of a substance in a sample that can be quantified as an exact value (see chapter 12.4 LOQ = Limit of Quantitation).

The limit of detection and quantitation can be derived from the calibration line. The sensitivity, linearity and limits of detection and quantitation are closely linked to each other with regard to analytical method validation.

Recovery

The requirement in PIC/S guideline PIC/S PI006 for determining the recovery rate comprises two issues:

• How much of the residue adhering to the surface is measured by sampling and analysis?
• How well can the procedure be reproduced?

This addresses two aspects of classic method validation:

Accuracy is the measurement of conformance of the measured value to the true value and therefore a measurement of systematic errors (see chapter 12.2 Accuracy).

Precision is a measurement of reproducibility of the result of an analysis under exactly the same conditions. The variations of the measured values from the average which occur on repetition are also designated as random errors (see chapter 12.1 Precision).

The recovery rate determined by taking into account the sampling procedure can be affected by a range of systematic and random errors. If the recovery rate is constantly too low, there are mainly systematic errors. If the results of the recovery trials vary significantly, increasingly random errors occur in addition to any systematic errors.

The reasons for a (too) low recovery rate may lie in either the sampling or in the sample preparation. To differentiate between these, the determination of the recovery rate must be divided into two sections:

• Determination of the recovery rate from the sample
• Determination of the recovery rate from the surface

Determination of the recovery rate from the sample

First it has to be assured that recovery from the sample is guaranteed. Proceedings can be shown in figure 8.G-2.

It is important that the reference solution is prepared with the same solvent that is used for sampling (wetting the swab in the swab test, rinsing the surface in the rinse test).

The systematic errors that can occur, especially during sample preparation of swab samples and the options available for remedying them are shown in figure 8.G-3 with some examples.

Determination of the recovery rate from the surface

The recovery from the surface must be incorporated in the next step of the trial. You can proceed as described in figure 8.G-4 and figure 8.G-5:

As the sample surface for the swab test is usually rather small (25-100 cm²), the trial can be carried out on a test plate, which material and surface rawness correspond to the equipment surface to be sampled.

Conversely, larger equipment surfaces are usually sampled in a rinse test. It is advisable - if possible - to carry out the recovery trial on the production equipment to ensure that the experimental conditions are comparable.

The substance quantity per sample for determining the recovery rate must be selected so that the quantity approximately corresponds to the acceptable limit for the relevant critical substance in the sample. The recovery rate may also be determined for a second, smaller value.

To be able to draw a conclusion about the reproducibility of the recovery, the trials must be repeated 3-5 times for each substance quantity investigated. If the sampling procedure recovery rate is still too low, even though the sample preparation has been improved, the error must be looked for in the sampling itself. Examples of causes of errors and how they can be eliminated are summarised in figure 8.G-6.

If the sampling procedure is developed in tight correlation with the analytical methods, time-consuming trial-and-error tests can be avoided. It is advisable to create an experimental plan, which can be used to systematically investigate several variables (e.g. different swabs, solvents, extraction conditions).

The recovery rate results in a correction factor by which the sample results must be multiplied to determine the actual residue quantity.

A universally valid specification of the extent of the individual recovery rates cannot be given here, as these can vary considerably depending on the substance properties and the analytical methods used. It is useful to establish a minimum requirement for the recovery rate beforehand, based on knowledge of the substance and methods, and develop and validate the method accordingly.

8.G.2 Selection of the appropriate analytical method

Active pharmaceutical ingredient residues

Even if there are already testing methods for quantification of the critical substance, these cannot generally be used without adaptation for the determination of residues as part of the cleaning validation. The reasons for this lie in the different operating ranges and sample matrices, as well as in the different sample preparation procedures.

However, the same analytical procedure can be used in most cases. For many active pharmaceutical ingredients, the combination of swab test and HPLC will be the procedure to choose.

In view of the large quantity of samples when swab tests are carried out, the analytical procedure should meet the following requirements:

• Simultaneous processing of several samples during sample preparation
• High degree of automation for sample preparation and analysis

Cleansing agent residue

The requirement for specific determination methods (e.g. HPLC, GC, DC) is more difficult to fulfil for cleansing agents than for active pharmaceutical ingredients and, in most cases, involves considerable effort and cost. The reason for this is that most of the organic compounds used in cleansing agents have long hydrocarbon chains without functional groups and can therefore not be detected using the UV/VIS detectors usually used in conjunction with chromatographic procedures. In principle, it would indeed be possible to determine the cleansing agent specifically with derivisation and/or the use of special detectors, but the analysis is expensive and time-consuming.

Non-specific analytical methods for detection of residues are suitable for many cleansing agents due to their characteristical properties, as long as the experimental conditions are correctly selected.

This allows residues of alkaline or acid cleansing agents which contain ionic components to be detected by measuring the conductivity. This method is quick, economical and can be implemented online. The sensitivity of the method is generally sufficient to determine residue where the measurement must be compared to that of pure demineralised water.

Residues of cleansing agents containing tensides can be detected by measuring the surface tension. This method also has the advantage that it is quick and economical to implement. In addition, it is very sensitive and has low associated equipment costs. Pure demineralised water should also be measured for comparison.

Another possibility for determining cleansing agent residues which is being increasingly applied is the determination of total organic carbon (TOC). Sufficient experience is required to use this method due to the high margin of error. However, if it is carried out correctly it has the advantage that it applies to a wide spectrum of substances. The measurements are characterised by their rapidity and their high sensitivity and can also be carried out online.

When a non-specific procedure is being used, bear in mind that results below the acceptance limit always prove that the equipment is sufficiently free from residue, whereas, if the results are above the acceptance limit, it is necessary to clarify whether the poor result is due to the cleansing agent which is to be detected or to other contamination with similar substance properties.

The following aspects must be investigated when the method is developed and/or validated:

• Limit of detection and limit of quantitation
• Calibration function
• Reproducibility of the individual measurements
• Recovery rate

If water-soluble cleansing agents are to be detected by measuring the conductivity or surface tension, the rinse test is the most suitable sampling procedure.

When taking the sample, it must be assured that the volume of solvent used is sufficient to sample the whole surface.

For residue in the sample solution to be reliably detected and determined, the assumption has to be made that there is as much residue on the sampled surface as is permissible for that surface and which has been quantitatively transferred to the sample solution. Taking into consideration the limits of detection and quantitation, the volume of solvent must be selected in such a way that a concentration above the limit of detection results, presumed that the acceptable amount of residue is present (see figure 8.G-7).

If this means that the volumes and/or concentrations resulting from these considerations differ from each other considerably, they must be adapted during the sample preparation.

The sample solution prepared in this way, which concentration corresponds theoretically to the surface-related residue limit is measured against a reference solution of the cleansing agent to be detected, the concentration of which is actually the same as the surface-related residue limit.

If the conductivity of the sample solution is lower (or the surface tension higher) than the reference solution, the residue concentration from the sampled surface is below the acceptable limit. A sample of the water used for sampling is measured as a comparison or blank value.

The trial procedure described and the calculations on which it is based are illustrated below by way of an example (see figure 8.G-7):

This procedure is based on the following reasoning: if the residue concentration measured using a non-specific procedure is less than the permissible residue concentration, compliance with the limit for the substance to be detected can be guaranteed in every instance.