HARMONIZATION TEAM A6 STABILITY

SUMMARY OF RECOMMENDATIONS

 June  2012

- General recommendations - Reference values - Transferability of stability results - Treatment of failing stability results - Stock stability - Extract stability - Whole blood stability - Tissue stability - Urine stability - Incurred sample stability - Stability of endogenous analytes

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Overview

As analyses are usually not performed directly after sampling, but after samples have been processed and stored, it is essential that analyte stability be maintained over the various relevant storage conditions, in order to ensure that the concentration results obtained adequately reflect those directly after sampling. Stability is not only related to the chemical integrity of a molecule, but also to other issues that may affect its concentration during storage, such as solvent evaporation, adsorption to containers or collection materials, precipitation and non-homogeneous distribution over a sample. In addition, for biologicals quantified using ligand-binding assays, the three-dimensional biological integrity of the molecule also determines the response and should, therefore, be maintained. All conditions during sample collection and storage should be such that analyte stability is ensured. Appropriate stabilizing measures, if any (e.g. decreased temperature, protection from light and moisture, addition of stabilizers) should be defined during the stage of method development. Stability assessment during method validation should serve to confirm the suitability of the experimental conditions only.

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General recommendations

Ideally, stability results are available before the beginning of the (pre)clinical phase of a study, because running a study without knowing the appropriate storage and stabilizing conditions poses a risk to generate invalid concentration results. Whenever feasible, the biological matrix used to prepare the stability samples should be as fresh as possible to best mimic the situation of study samples directly after sampling. This is especially important for analytes that are known to be susceptible to enzymatic degradation in the matrix, e.g. by the action of esterases or proteases. Stability has to be assessed by subjecting spiked samples to a particular storage condition and subsequent analysis. The applicable storage conditions to be investigated have to be established for each individual application and should cover all relevant conditions to which bioanalytical samples will be subjected in practice. They typically include (but are not necessarily limited to): stability in the biological matrix e.g. bench-top storage, long-term frozen storage and repeated freeze/thaw cycles, stability in sample extracts (where applicable) and stability of stock and derived solutions that are used to prepare calibrators and quality control (QC) samples. There is no need to determine analyte stability at conditions that are not encountered in practice.

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General recommendations

The duration of a particular storage period has to be at least equal to the maximum period that will be used to store samples at the corresponding condition during the clinical or pre-clinical trial. Where necessary during the lifetime of a method, stability assessments can be added or storage periods prolonged to cover expanding needs. For biological samples and their extracts, the maximum allowed deviation of the analytical result from the reference value should be similar to the generally accepted maximum bias for (unstored) quality control samples, i.e. generally ±15% for chromatographic and ±20% for ligand-binding assays. Stability has to be assessed at a low and a high concentration within the applicable calibration range and it is recommended that these are equal to the low and high QC levels that are used to determine method precision and accuracy. For studies in which it is likely that higher concentrations will be frequently found and samples will need to be regularly analyzed after dilution (such as ascending dose studies), it is recommended that stability assessment be considered at a relevant over-curve concentration.

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General recommendations

A single stability assessment per storage condition per concentration level is considered sufficient, although it is realized that analysis after multiple time-points will provide more detailed information, which can be helpful to interpret the stability profile of an analyte. A sufficient number of replicates should be performed to obtain a reliable average result for any stability assessment. It is recommended that each stability assessment be performed in triplicate as a minimum for methods that use a single analysis to obtain a result, such as most chromatographic methods, or in duplicate as a minimum for assays using two analyses which are averaged to obtain a result, such as is typically the case for ligand-binding assays. For methods with a large intrinsic variability, increasing the number of replicates may be considered to increase the level of confidence in the result and avoid drawing incorrect conclusions. Stability of a particular compound is determined by temperature, exposure to light, the matrix (including anti-coagulant and the presence of stabilizing additives) and the material of the sample container. Stability results should not be extrapolated to other conditions. The effect of the counter-ion of the anti-coagulant is considered negligible and its change does not require additional stability assessment.

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General recommendations

Although it is recognized that accelerated stability testing (e.g. storage at 37°C) is a useful tool in method development to build up knowledge of the stability properties of a compound, it should not be used during validation to replace stability assessment at the actual storage temperature. For multi-analyte assays, all analytes should be added to the samples used for stability assessment. It is generally acceptable that all analytes are added at their respective low or high concentrations. For analytes that could be converted to one another during storage, it is recommended that stability assessment should also be performed with the single analytes, to be able to evaluate the degree of conversion to the other analyte and define the in vivo concentration ratio of the analytes for which conversion (if any) is considered acceptable. The recommendations given here are meant for full validations in regulated bioanalysis. Depending on the purpose of the analysis, a more limited validation and a less extensive stability assessment may be applicable.

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General recommendations

Expressing stability by comparison of the result after storage to the nominal value is scientifically correct, as long as due attention is paid to the effect that the bias originating from the spiking process may have on the final result. It is recommended that the concentrations of the stability samples be measured prior to subjecting these to storage to allow evaluation of the correctness of spiking. In order to avoid incorrect conclusions about stability, it is advisable to consider preparation of a new set of stability samples if the concentrations are outside a specific range. The values of this range will depend on the risk one accepts, but need to be more stringent than the usual 15% or 20% acceptable bias. Expressing stability by comparison to the corresponding t=0 value can be regarded as equally acceptable scientifically. Also in this case, it is advisable to check the t=0 concentrations and to consider preparation of a new set of stability samples if they are outside a specific range, to avoid that concentrations after storage are outside the normal acceptance criteria.

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Reference values

In some situations, comparison to the nominal value is impossible because the results cannot be directly derived from a calibration curve. Examples include the assessment of the stability of stock and working standard solutions, the stability in whole blood (if performed by preparation and subsequent analysis of plasma) and the stability of endogenous analytes. In these cases, comparison to the corresponding t=0 value is the only option. The use of freshly prepared calibrators for stability assessment is recommended for at least the determination of (long-term) frozen stability, where “fresh” is defined as prepared on the day of the stability assessment and stored using any necessary stabilizing precautions (such as protected from light or on ice).

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Reference values

For other stability assessments, the use of calibrators that are not freshly prepared but stored frozen until their use is deemed acceptable, if the stability of the calibrators over the period of frozen storage is confirmed. In particular, when calibrators and stability samples are prepared simultaneously and stored frozen under the same conditions until the actual performance of the stability assessment. Spiked quality control samples need to be included in the analytical runs used to analyze stability samples to evaluate the acceptability of the runs. Their concentration levels, the number of replicates and the acceptance criteria need to be similar to those used for regular bioanalysis and they should be within their demonstrated (frozen) stability period.

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Reference values

As analyte (in)stability is determined by physico-chemical parameters (temperature, time, matrix composition, exposure to light), stability results can be considered universally valid. Therefore, it is deemed scientifically justifiable to refer to stability results obtained elsewhere, if critical storage conditions are similar and the stability assessment has been performed in a scientifically sound and traceable way. For analytes with a clear stability profile, slight variations in storage conditions (e.g. because of seasonal fluctuations) will be an acceptable risk, both within a single laboratory and between different laboratories. For temperature- and/or light-sensitive compounds, that need special protective measures, it is advisable to take a cautious approach and repeat the stability assessment at each different bioanalytical site, if the exact reproduction of the original storage conditions cannot be guaranteed.

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Transferability

In case of larger variations in storage conditions, it is recommended to either repeat the stability assessment in a new lab or to cover the different storage conditions in the original lab. Whether or not a stability experiment will need to be repeated has to be considered case-by-case and will also depend on the effort it will take to cover the necessary storage conditions during the original validation, compared to simply repeating the experiment. Any properly validated method may be considered to yield reliable results and thus the acceptability of transfer of stability results does not need identical analytical methods at the different sites. An exception is any method that uses protein-protein interactions (such as LBAs) and that employ different reagents and/or incubation times.

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Transferability

If the calibration curve and/or analytical QCs included in the batch fail the acceptance criteria or if there is a documented error in preparation, then the results are invalid and the assessment should be performed again, regardless of whether the stability assessments would have passed. If the run is valid, but the stability assessment fails, the investigated storage conditions should be considered unsuitable. Where needed, shorter storage periods, lower storage temperatures and/or the addition of stabilizing agents may be investigated. If a failed assessment is suggestive of an analytical outlier, the recommended approach is to investigate this by repeating the experiment twice. Reporting all the valid data and making a scientific judgment of the total data set, based on pre-defined criteria, is appropriate.

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Failing results

In some cases, repeating the experiment may be unnecessary for the final decision. One example is a time course where the early and late time points pass the criteria while the middle time point fails. It may be scientifically justifiable to accept through the longer time without a repeated assessment depending on the magnitude of the discrepancy and overall data trend. The use of an outlier test to remove a deviating analytical result from a data set is only appropriate if the replicate results of a single sample (one concentration, one storage condition, one time-point) are statistically compared. Results of different storage conditions and/or time-points should not be combined and subjected to an outlier test.

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Failing results

Stock standard solution and working standard solution stability should be demonstrated to cover the actual conditions undergone – namely the storage solvent and temperature. It is recommended to perform stability assessments at the lowest and highest concentration encountered in practice, as solubility and adsorption effects may be concentration-dependent. To demonstrate stability for both analytes and internal standards, the maximum relative percent difference between the mean of the freshly-prepared solution results to the mean of the stability solution results should be no more than 10%. Similarly, the coefficient of variation for each set of replicates should be no more than 10%.

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Stock stability

Stable-isotope labeled forms of a molecule can be considered to be chemically identical to their unlabeled forms. Therefore, for stable-isotope labeled internal standards one can refer to the stability results obtained for the unlabeled analyte as long as it has been demonstrated that no isotope exchange occurs during storage. For reference material received as a solution, the stability information provided by the manufacturer is acceptable for use and does not need to be regenerated at the analytical lab, provided the manufacturer’s storage conditions are followed. When possible, working stock solutions should be prepared in the same solvent or buffer as the provided stock solution to build on the manufacturer’s stability documentation.

 

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Stock stability

Stability of sample extracts (post-preparative stability) needs to be determined for all conditions to which these will be subjected. On-instrument stability refers to the storage of sample extracts in the autosampler or any other instrument for serial analysis, prior to their actual analysis. It is tested by storing extracts on the instrument and analyzing them after storage, against freshly extracted calibrators and QCs. The stability of extracts during storage prior to placement in the autosampler, such as a few hours at room temperature, does not need to be assessed as a standard, but only if it actually occurs during analysis. Re-injection reproducibility refers to the possibility to re-inject a complete or partial batch of samples into the analytical instrument. This is typically done by analyzing a set of samples, re-injecting them together with the calibrators and calculating the result against the re-injected calibration curve and/or against a freshly extracted curve. It should be noted that this is in fact a reproducibility assessment rather than a stability assessment.

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Extract stability

Although analytes being measured in plasma or serum will be stored in whole blood for some time during the sampling process and information about their stability in this period of time would be needed, there is accumulating evidence that there is little difference between stability in plasma/serum and stability in whole blood except for some well-defined classes of molecules such as N-oxides and hydroxamic acids, which are subject to hemoglobin-mediated degradation. Whole blood stability testing may therefore not be strictly necessary in all situations as long as plasma/serum stability under the same storage conditions has been clearly demonstrated. It is recommended that stability in whole blood should be assessed at least for compounds belonging to these classes, for compounds with borderline stability in plasma/serum and for situations in which the storage conditions in whole blood are not covered by plasma/serum stability data. Two approaches to whole blood stability testing exist: (1) the analysis of plasma/serum after removal of cellular material and (2) the analysis of whole blood itself.

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Whole blood stability

The analysis of plasma/serum best mimics the actual sampling procedure and gives combined information about the stability and the in vitro (re)distribution of analytes over plasma and blood cells during the sample collection procedure. It allows the use of the validated plasma or serum method. The analysis of whole blood gives information about stability only. It allows the use of nominal concentrations as the reference. It may need a different analytical approach and additional method development. Both approaches are scientifically acceptable to assess stability in whole blood. If the analysis of plasma/serum gives acceptable results, stability in whole blood can be considered demonstrated. Where necessary, both methods can be combined to obtain more detailed information.

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Whole blood stability

As intact tissues cannot be homogeneously spiked, stability assessment has to be performed by preparing and spiking blank tissue homogenate and subsequently subjecting this to stability experiments. The composition and volume of the homogenization solvent are important for the resulting analyte stability profile. Stability will vary from tissue to tissue and species to species and needs to be established for each new matrix. It is advisable to homogenize tissues from dosed animals as soon as practical after collection and store the samples as homogenate, in order to cover as much of the storage period as possible by actual stability results. There might be logistical limitations that make it difficult in some cases to immediately homogenize tissue samples after collection. If this is the case, then it is advised to store the intact tissues at <-70 °C rather than at -20 °C until homogenization and to still try to limit the storage period until homogenization as much as possible.

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Tissue stability

Since the native pH of urine varies between ca 4 and 8.5, it is important that stability assessments take this into account. For any fully validated method, it is recommended that stability of the analyte in urine should be assessed at three pH values, e.g. pH 4, 6.5 and 8.5. Since adsorption problems in urine are likely to be more pronounced than in more protein-rich matrices, it is especially important that the same container material and additives, if any, will be used for the stability assessments and the actual urine sampling. The experiments can be conducted in smaller containers (e.g. 30 ml or even less), while sampling is done in larger containers (often 1 or 2 liters) as long as the container material is the same.

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Urine stability

Since usually a small urine aliquot is transferred to a secondary container which is subsequently sent to the bioanalytical lab, stability should also be tested in the secondary container, if this has a different material. In case of the (expected) presence of significant amounts of phase-2 metabolites in urine that can convert back to the analyte, additional stability experiments using samples spiked with relevant concentrations of these metabolites or using incurred samples should be considered, where it should be taken into account that stability of phase- 2 metabolites can be pH-dependent.

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Urine stability

The main difference between spiked and incurred samples with regard to stability properties is the potential presence of unstable metabolites that could convert back to the analyte during sampling and storage, sample preparation and analysis and cause the overestimation of analyte concentrations. Assessment of incurred sample stability (ISS) represents a way to obtain information about this effect. Alternatively, spiked stability experiments using reference standards from these metabolites could be done, provided that the blank matrix used is sufficiently fresh. The main stability dataset will be stability assessments done using spiked samples. ISS should serve to bridge a possible gap between spiked and incurred samples, when deemed necessary based on the physicochemical and/or metabolic properties of the analyte.

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Incurred sample stability

As the metabolite profile can be different over different species, it is recommended that the experiments should be conducted in all relevant species, whenever feasible. It should not be required to repeat experiments e.g. for different subject populations and disease states, unless there are indications that the abundance of potentially unstable metabolites is significantly increased. Should there be ISR failures in the conducted bioanalyses, then assessment of ISS could also be used as an investigation strategy for ISR failures, as these might be related to incurred sample instability. Timing of the experiments should be flexible. The ISS experiments can be conducted as soon as the study samples from the first study are available, but can also be conducted in a later stage of drug development, e.g. in phase 2 or 3. Should the experiments demonstrate that there are issues with the method when applied to incurred samples, then the impact of the issues with the method on the bioanalytical data generated using the method should be assessed and documented.

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Incurred sample stability

The stability assessment for endogenous analytes should follow the general approach whenever possible. It is essential that the samples used for stability determination should be authentic biological matrix spiked with authentic analyte, although it is recognized that calibration samples generally cannot be in that form because of the typical presence of analyte in the matrix. A typical approach would be to screen a number of lots of the authentic matrix for low analyte levels and select a sample to be used as the low concentration for stability assessment.

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Stability of endogenous analytes

The low-concentration validation sample could be spiked to prepare the high-concentration stability sample or, alternatively, an incurred sample with a suitable high concentration could be selected after screening a number of lots of the matrix. Stability will typically have to be expressed against a t=0 value because the nominal concentration generally is unknown If it is the intention not to prepare fresh calibration standards in each run for bioanalysis, stability should also be assessed in the calibration matrix.

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Stability of endogenous analytes