Ongoing Analytical Procedure Performance Verification Using a Risk-Based Approach to Determine Performance Monitoring Requirements.

The analytical procedure life cycle (APLC) provides a holistic framework to ensure analytical procedure fitness for purpose. USP's general chapter <1220> considers the validation activities that take place across the entire analytical procedure lifecycle and provides a three-stage framework for its implementation. Performing ongoing analytical procedure performance verification (OPPV) (stage 3) ensures that the procedure remains in a state of control across its lifecycle of use post validation (qualification) and involves an ongoing program to collect and analyze data that relate to the performance of the procedure. Knowledge generated during stages 1 (procedure design) and 2 (procedure performance qualification) is used as the basis for the design of the routine monitoring plan to support performance verification (stage 3). The extent of the routine monitoring required should be defined based on risk assessment, considering the complexity of the procedure, its intended purpose, and knowledge about process/procedure variability. The analytical target profile (ATP) can be used to provide or guide the establishment of acceptance criteria used to verify the procedure performance during routine use (e.g., through a system/sample suitability test (SST) or verification criteria applicable to procedure changes or transfers). An ATP however is not essentially required to perform OPPV, and a procedure performance monitoring program can be implemented even if the full APLC framework has not been applied. In these situations, verification criteria can be derived from existing validation or system suitability criteria. Elements of the life cycle approach can also be applied retrospectively if deemed useful.

Selection of Analytical Technology and Development of Analytical Procedures Using the Analytical Target Profile.

A structured approach to method development can help to ensure an analytical procedure is robust across the lifecycle of its use. The analytical target profile (ATP), which describes the required quality of the reportable value to be produced by the analytical procedure, enables the analytical scientist to select the best analytical technology on which to develop their procedure(s). Once the technology has been identified, screening of potentially fit for purpose analytical procedures should take place. Analytical procedures that have been demonstrated to meet the ATP should be evaluated against business drivers (e.g., operational constraints) to determine the most suitable analytical procedure. Three case studies are covered from across small molecules, vaccines, and biotherapeutics. The case studies cover different aspects of the analytical procedure selection process, such as the use of platform method development processes and procedures, the development of multiattribute analytical procedures, and the use of analytical technologies to provide product characterization knowledge in order to define or redefine the ATP. Challenges associated with method selection are discussed such as where existing pharmacopoeial monographs link acceptance criteria to specific types of analytical technology.

Development and Validation of an In-Line API Quantification Method Using AQbD Principles Based on UV-Vis Spectroscopy to Monitor and Optimise Continuous Hot Melt Extrusion Process.

A key principle of developing a new medicine is that quality should be built in, with a thorough understanding of the product and the manufacturing process supported by appropriate process controls. Quality by design principles that have been established for the development of drug products/substances can equally be applied to the development of analytical procedures. This paper presents the development and validation of a quantitative method to predict the concentration of piroxicam in Kollidon® VA 64 during hot melt extrusion using analytical quality by design principles. An analytical target profile was established for the piroxicam content and a novel in-line analytical procedure was developed using predictive models based on UV-Vis absorbance spectra collected during hot melt extrusion. Risks that impact the ability of the analytical procedure to measure piroxicam consistently were assessed using failure mode and effect analysis. The critical analytical attributes measured were colour (L* lightness, b* yellow to blue colour parameters-in-process critical quality attributes) that are linked to the ability to measure the API content and transmittance. The method validation was based on the accuracy profile strategy and ICH Q2(R1) validation criteria. The accuracy profile obtained with two validation sets showed that the 95% ß-expectation tolerance limits for all piroxicam concentration levels analysed were within the combined trueness and precision acceptance limits set at ±5%. The method robustness was tested by evaluating the effects of screw speed (150-250 rpm) and feed rate (5-9 g/min) on piroxicam content around 15% w/w. In-line UV-Vis spectroscopy was shown to be a robust and practical PAT tool for monitoring the piroxicam content, a critical quality attribute in a pharmaceutical HME process.

Using the Analytical Target Profile to Drive the Analytical Method Lifecycle.

Quality by design (ICH-Topic Q8) requires a prospective summary of the desired quality characteristics of a drug product. This is known as the Quality Target Product Profile (QTPP), which forms the basis for the design and development of the product. An analogous term has been established for analytical procedures called the Analytical Target Profile (ATP). The ATP, in a similar fashion to the QTPP, prospectively summarizes the requirements associated with a measurement on a quality attribute which needs to be met by an analytical procedure. Criteria defined in the ATP relate to the maximum uncertainty associated with the reportable result that is required to maintain acceptable confidence in the quality decision made from the result. The ATP is used to define and assess the fitness of an analytical procedure in the development phase and during all changes across the analytical lifecycle. One or more analytical procedures can meet the requirements of an ATP. The ATP can be applied to any quality attribute across any pharmaceutical modality where an analytical procedure is used to generate a reportable result, and this paper provides examples from three of these modalities: small molecules, oligonucleotides, and vaccines. Some key performance characteristics will be discussed for each ATP, namely specificity, accuracy, and precision, taking into account the expected range of the analyte. The combination of accuracy and precision into a combined uncertainty characteristic is also discussed as a more holistic approach. The use of the ATP concept will help focus attention on the properties of a method which impact quality decisions rather than method descriptions and may enable greater regulatory flexibility across the lifecycle using established conditions based on method performance criteria as proposed in the Step 2 version of ICHQ12. The revision of ICHQ2(R1) and development of the new ICHQ14 guideline (Analytical Procedure Development) will provide a golden opportunity to harmonize the definition of new QbD concepts such as the ATP.

Avoid the perils of using rounded data.

From a purely technical point of view it is always better to work with recorded/unrounded data. This paper discusses reasons why despite this, rounded data used for reporting may have been used in the past in calculations and visualisation. If rounded data are used then it is recommended that a risk assessment of the impact of the rounding is performed. If it is not possible to use unrounded data (with many decimal places) then the authors recommend that the data used should have at least two decimal places more than the number of decimal places required in the reported value ("effectively unrounded data"). Examples are given to illustrate the importance of using effectively unrounded data in visualisation and numerical calculations. It is recommended that effectively unrounded data and, if required, reported data are listed in reports. The implications of using rounded data when making formal assessments against specification limits is also discussed and guidance is provided on the use of nominal and effective specification limits.

A risk-based statistical investigation of the quantification of polymorphic purity of a pharmaceutical candidate by solid-state 19F NMR.

The DMAIC (Define, Measure, Analyse, Improve and Control) framework and associated statistical tools have been applied to both identify and reduce variability observed in a quantitative (19)F solid-state NMR (SSNMR) analytical method. The method had been developed to quantify levels of an additional polymorph (Form 3) in batches of an active pharmaceutical ingredient (API), where Form 1 is the predominant polymorph. In order to validate analyses of the polymorphic form, a single batch of API was used as a standard each time the method was used. The level of Form 3 in this standard was observed to gradually increase over time, the effect not being immediately apparent due to method variability. In order to determine the cause of this unexpected increase and to reduce method variability, a risk-based statistical investigation was performed to identify potential factors which could be responsible for these effects. Factors identified by the risk assessment were investigated using a series of designed experiments to gain a greater understanding of the method. The increase of the level of Form 3 in the standard was primarily found to correlate with the number of repeat analyses, an effect not previously reported in SSNMR literature. Differences in data processing (phasing and linewidth) were found to be responsible for the variability in the method. After implementing corrective actions the variability was reduced such that the level of Form 3 was within an acceptable range of ±1% ww(-1) in fresh samples of API.

Method ruggedness studies incorporating a risk based approach: a tutorial.

This tutorial explains how well thought-out application of design and analysis methodology, combined with risk assessment, leads to improved assessment of method ruggedness. The authors define analytical method ruggedness as an experimental evaluation of noise factors such as analyst, instrument or stationary phase batch. Ruggedness testing is usually performed upon transfer of a method to another laboratory, however, it can also be employed during method development when an assessment of the method's inherent variability is required. The use of a ruggedness study provides a more rigorous method for assessing method precision than a simple comparative intermediate precision study which is typically performed as part of method validation. Prior to designing a ruggedness study, factors that are likely to have a significant effect on the performance of the method should be identified (via a risk assessment) and controlled where appropriate. Noise factors that are not controlled are considered for inclusion in the study. The purpose of the study should be to challenge the method and identify whether any noise factors significantly affect the method's precision. The results from the study are firstly used to identify any special cause variability due to specific attributable circumstances. Secondly, common cause variability is apportioned to determine which factors are responsible for most of the variability. The total common cause variability can then be used to assess whether the method's precision requirements are achievable. The approach used to design and analyse method ruggedness studies will be covered in this tutorial using a real example.

Acceptance criteria for method equivalency assessments.

Quality by design (ICH-Topic Q8) requires that process control strategy requirements are met and maintained. The challenging task of setting appropriate acceptance criteria for assessment of method equivalence is a critical component of satisfying these requirements. The use of these criteria will support changes made to methods across the product lifecycle. A method equivalence assessment is required when a change is made to a method which may pose a risk to its ability to monitor the quality of the process. Establishing appropriate acceptance criteria are a vital, but not clearly understood, prerequisite to deciding the appropriate design/sample size of the equivalency study. A number of approaches are proposed in the literature for setting acceptance criteria for equivalence which address different purposes. This perspective discusses those purposes and then provides more details on setting acceptance criteria based on patient and producer risk, e.g., tolerance interval approach and the consideration of method or process capability. Applying these to a drug substance assay method for batch release illustrates that, for the equivalence assessment to be meaningful, a clear understanding and appraisal of the control requirements of the method is needed. Rather than a single exact algorithm, the analyst's judgment on a number of aspects is required in deciding the appropriate acceptance criteria.

Design and analysis of method equivalence studies.

Method equivalence assessments should be considered when analytical methods are either modified or substituted. The TOST (two one sided tests) approach provides a sounder data driven method for testing equivalence than a simple comparative intermediate precision study which is typically performed as part of method validation. Prior to designing an equivalency study, an acceptance criterion (an acceptable bias between original and modified/changed method) must be chosen. The choice of acceptance criteria requires the identification of the smallest mean difference or bias between methods that is practically important. Equivalence testing in this manner is used to prove that the new method can generate data which continues to support previously established specifications. Once the acceptance criterion is decided, other aspects of the study can be designed following a set of design principles. When the design and acceptance criteria have been established, the collection of the data can commence. Demonstration of equivalence should not start until the validity of the observations has been confirmed such as assessment for outliers, normality, and comparison of variances. Once the suitability of the data is confirmed, the mean difference between the two data sets can be calculated along with a +/-90% confidence interval using the TOST approach. It can then be established whether equivalence of the two methods has been demonstrated.

Trace level impurity method development with high-field asymmetric waveform ion mobility spectrometry: systematic study of factors affecting the performance.

For the determination of trace level impurities, analytical chemists are confronted with complex mixtures and difficult separations. New technologies such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) have been developed to make their work easier; however, efficient method development and troubleshooting can be quite challenging if little prior knowledge of the factors or their settings is available. We present the results of an investigation performed in order to obtain a better understanding of the FAIMS technology. The influence of eight factors (polarity of dispersion voltage, outer bias voltage, total gas flow rate, composition of the carrier gas (e.g. %He), outer electrode temperature, ratio between the temperatures of the inner and outer electrodes, flow rate and composition of the make-up mobile phase) was assessed. Five types of responses were monitored: value of the compensation voltage (CV), intensity, width and asymmetry of the compensation voltage peak, and resolution between two peaks. Three types of studies were performed using different test mixtures and various ionisation modes to assess whether the same conclusions could be drawn across these conditions for a number of different types of compounds. To extract the maximum information from as few experiments as possible, a Design of Experiment (DoE) approach was used. The results presented in this work provide detailed information on the factors affecting FAIMS separations and therefore should enable the user to troubleshoot more effectively and to develop efficient methods.

Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API).

This work describes the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a highly toxic impurity, FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine), in paroxetine active pharmaceutical ingredient (API), followed by the subsequent validation of the methodology and transfer into a global production/quality control environment. The method was developed to achieve a detection limit of 10ppb mass fraction of FMTP in paroxetine API. An LC-MS/MS method was chosen because it provided the required sensitivity and selectivity with minimal sample preparation. This paper discusses the issues with transferring such complex methodology to a production environment. Linearity, repeatability and reproducibility of the method were demonstrated. This work shows that it is possible using the same approach that would be used for the transfer of any analytical method from R&D to a manufacturing environment.

Investigation into the factors affecting accuracy of mass measurements on a time-of-flight mass spectrometer using Design of Experiment.

The results of an investigation of the parameters which have the most significant effect on the accuracy of mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (q-oaToF) are reported. The influence of eight factors is investigated: ion abundances of reference and analyte compounds, mass difference between analyte and reference compounds, quality of calibration, number of reference acquisitions averaged and TDC (time-to-digital converter) settings (resolution, Np multiplier (number of pushes correction factor), minimum number of points, i.e. minimum acquisition width which defines a peak). To extract the maximum information from as few experiments as possible, a Design of Experiment approach was used. The data will be used as a basis for developing guidance on accurate mass measurement on q-oaToF instruments.

Determination of selectivity differences for basic compounds in gradient reverse phase high performance liquid chromatography under high pH conditions by partial least squares modelling.

The retention behaviour of compounds in a chromatographic system is believed to be multivariate by nature, i.e. many physico-chemical properties of an analyte can influence its retention. Principal component analysis (PCA) and partial least squares (PLS) can therefore be particularly useful tools for visualising, exploring and modelling the complex interactions between solutes and the mobile and stationary phase. PCA allows the relationships between compounds (the observations) and their retention parameters (the variables) to be visualised in usually just two or three dimensions. PLS can be used to model quantitative structure-retention relationships (QSRRs) and may lead to better understanding of retention and selectivity changes in chromatographic systems. The objective of the study was to investigate the chromatographic behaviour of basic compounds under optimised gradient conditions using octadecyl high performance liquid chromatography (HPLC) columns designed for high pH separations. Three pharmaceutical mixtures were analysed by linear gradient reverse phase HPLC (RP-HPLC) at high pH using ammonia as a pH modifier, and methanol and/or acetonitrile as the organic modifier. The separations were carried out on three octadecyl columns: Waters XTerra MS C18, Agilent Zorbax Extend C18 and Thermo Hypersil-Keystone BetaBasic-18. Multivariate PCA and PLS modelling were employed to explore and explain the differences in selectivity between the chromatographic systems studied when the basic compounds were analysed under the high pH conditions. The interactions between the analytes and the mobile-stationary phases were described by relating the compound molecular descriptors with the selectivity of each chromatographic system. The selectivity differences between the chromatographic systems were identified.

Toward single-calibrant quantification in HPLC. A comparison of three detection strategies: evaporative light scattering, chemiluminescent nitrogen, and proton NMR.

There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.

Comparative performances of selected chiral HPLC, SFC, and CE systems with a chemically diverse sample set.

Pharmaceutical companies have a continuous need to resolve new racemates. Analysis may be required in aqueous and nonaqueous media, or in the presence of several different sets of potentially interfering compounds. There is often a preparative requirement. For these reasons analysts may require a number of different separation systems capable of resolving a given pair of enantiomers. We wished to improve upon existing approaches that address this situation and undertook a program of work to screen over 100 racemates, selected for their chemical diversity, on over 100 different chiral HPLC, SFC, and CE systems. Here we report results of this comparison and illustrate the use of rapid gradient screening as a valuable tool for chiral method development.