Quantitative Open-Access HPLC Analysis

The use of open-access high-performance liquid chromatography (HPLC) systems in drug discovery and pharmaceutical development has steadily increased during the past decade. This growth has enabled chemists and engineers to perform a large proportion of their own analysis without dedicated support from analytical scientists. Within chemical development at GlaxoSmithKline (GSK), for example, about half of chromatographic analyses (250,000 samples per year) are performed by process chemists or chemical engineers using open-access systems. The uptake of these systems has been high and the application of traditional techniques for monitoring reactions such as thin-layer chromatography (TLC) has been superseded in chemical development at GSK. Implementation of a novel, global open-access approach using generic fast-LC and custom analytical methods on more than 100 HPLC-ultra-violet (UV) instruments has been described by Roberts et al. (1).

BORMAN ET AL.

Most HPLC open-access systems within chemical development laboratories are used to generate impurity-profile information using area–percentage responses on non-quality critical samples of active pharmaceutical ingredients (APIs), intermediates, and starting materials. In addition to obtaining this data, synthetic chemists must generate purity data on samples to support route- and process-development. A new tool developed by GSK, the Yieldaliser, enables the rapid and reliable estimation of yields in solution and assays of isolated solids (in addition to generating impurity profiles) without any requirement for the user to prepare standards. The authors discuss the calibration approach, which is performed using an unrelated standard material analyzed at an optimal wavelength which may be different to the compound of interest.

External standardization across different wavelengths

Several standardization methods were investigated during the development of the tool with a focus on automation. External and internal standardization were explored using single and multiple standard sets, which were analyzed at a single wavelength or multiple wavelengths. To analyze compounds that contain chromophores in the typical UV range used in HPLC (210–300 nm), the standard and sample compound had to be analyzed at their respective λMax or λOpt (i.e., the most optimal/robust region within the UV spectrum). Additional functionality was integrated into the open-access HPLC platform (this term is used by Roberts et al. to describe a collection of actively managed and supported open-access instruments [1]), which enables chemists and engineers to perform their own assay analysis.

To generate assay data on a particular compound using the platform, an analytical chemist (i.e., the lead user) must first determine an accurate response factor for the compound of interest (analyzed using the compound's λOpt) against the benzophenone external standard (analyzed using its λmax). The Yieldaliser software uses the response factor in its calculations.

Benzophenone was chosen as the external standard because it is stable, can be weighed out accurately, and is commercially available at high purity. Benzophenone is also very soluble in compatible solvents, has good chromatographic properties on open-access gradient HPLC methods, and has a high extinction coefficient at its λmax of 254 nm.

Integrating a standardized approach. To ensure that the HPLC system is functioning satisfactorily, a test mixture and blank solution are automatically injected early in the day. Immediately afterward, six standard vials of a solution of benzophenone of known concentration (typically 0.35 mg/mL made up in methanol [MeOH]) are injected and the percentage relative standard deviation (i.e., measure of injector precision) and average response of the benzophenone (i.e., measure of accuracy) are checked against predetermined limits. If the performance does not fall within these limits, then the system will automatically repeat the standardization. If the calibration fails again, then the lead user is notified by email and the system is checked for problems such as poor column performance, solvent leaks, or autosampler issues. The assay functionality is disabled if the standardization criteria are not met. Figure 1 shows a screenshot of the customized interface on the laboratory computer that is connected to the instrument. The user selects either the 3- or 8-minute gradient method and then chooses to perform an analysis of impurities or assay (the assay analysis option is highlighted in red in Figure 1 and only runs on the 8-minute gradient).

Figure 1: Sample log-on interface of the Yieldaliser software (GlaxoSmithKline, GSK). (ALL FIGURES ARE COURTESY OF BORMAN ET AL.)

Developing sample preparation protocols

Two protocols were developed to enable the analysis of liquid samples (for generating yield in solution data) and solid samples (assay data). These protocols were created to meet users' expectations. For example, sample preparation had to be quick, easy to follow, and use vessels that could be disposed of at minimal cost. Chemists needed to be able to to generate data that were sufficiently precise (within ± 3% w/w of the actual purity) for the majority of their applications. Trained analytical chemists can perform assays with greater precision (e.g., typically ± 0.5% w/w), however this is far more resource intensive.

Liquid protocol. The liquid protocol was aimed at analyzing solution concentrations ranging between 20 mg/mL (50 volumes) and 200 mg/mL (5 volumes); these concentrations are typical of those used in reactions. Because the 8-minute HPLC gradient method requires sample concentrations ranging between 0.003 and 0.35 mg/mL, accurate dilutions of approximately 1000-fold were needed. Previous experience had shown that electronic displacement pipettes (EDPs) were accurate and easy to use. To achieve a 1000-fold dilution and retain a suitable sample volume, the developed protocol involved adding 20 microliters of solution to 20 mL of solvent in a scintillation vial. Analytical solution concentrations are quite high, thereby requiring a low injection volume (1 µl). The low injection volume facilitates protocols that do not rely on significant serial dilution, have minimal peak splitting due to solvent mismatch, and do not significantly compromise the repeatability. Although acceptable accuracy and reproducibility were demonstrated when developing a protocol for most of the commonly used solvents (e.g., toluene), certain solvents such as dichloromethane could not be dispensed with sufficient accuracy. Because dichloromethane is a commonly used solvent, a modification to the same protocol was made by replacing the EDP pipette with a 25-microliter syringe. A Pressmatic (Bibby Scientific, Staffordshire, UK) dispenser was used to deliver the 20 mL of solvent (typically methanol or acetonitrile) to the scintillation vial (this device can be attached directly on top of a bottle of solvent and is sufficiently accurate and precise when delivering the solvent). Feedback from users also indicated that there was a need to analyze compounds across a wider range of concentrations to support the analysis of more concentrated reaction mixtures and diluted solutions typical of mother liquors or wash solutions to optimize yields. These protocols used the same principles to ensure that samples could be easily prepared (see Figure 2).

Figure 2: Screenshot showing the Yieldaliser (GSK) software's assay interface.

Solid protocol. The requirements for establishing a solid protocol were similar to those used to create the liquid protocol. The developers initially used four-decimal-place balances in the laboratories, Pressmatic dispensers, and EDP pipettes. The final protocol calls for ~15 mg of solid that is weighed accurately on a four- or five-decimal balance into a disposable polypropylene container. Fifteen miligrams was found to be the minimum weight required for achieving an accurate measurement. One mililiter of dimethyl sulfoxide (DMSO) was added to the solid with an EDP pipette, and then 40 mL of solvent was added from the Pressmatic dispenser. DMSO was chosen as the predissolution solvent based on its aproticity, which maximizes solubility while remaining compatible with the system.

Adding and selecting compounds. A similar protocol to the solid protocol can be used to assess the suitability of a compound for the Yieldaliser and to ensure accuracy in determining the response factor. A full assessment of safety, stability, repeatability, and applicability of the compound (e.g., presence of appropriate λOpt ) should be performed. Using five-decimal-place balances and volumetric glassware to determine response factors provides improved accuracy over the liquid and solid protocols described above with a percentage relative standard deviation of < 1.5% (n = 3). Accurate determination of the response factors is crucial for meeting customer expectations of the system's performance.

The left side of Figure 2 shows the compounds (stored under each project) for which the Yieldaliser is set to analyze. The right side displays the custom choices a user can make when performing yield in solution- or solid-assay analysis. Figure 2 shows the screen that appears after selecting the assay option.

Figure 3: A proof-of-concept study demonstrated chemists can generate data with acceptable precision and accuracy.

Proof of concept. A proof-of-concept study was performed to demonstrate that chemists can produce data within ± 3% of the actual purity (chemists' expectations) for the previously defined protocols. Four trained chemists were able to generate yield in solution and assay results for samples of known concentration within these limits for several compounds. Figure 3 provides some of the data generated from the proof-of-concept study. More than 90% of the data were within the desired ± 3% limits. The clusters of results that fell outside the limits represent results from chemists who were not familiar with the sample preparation devices. After receiving additional training, the data from these chemists improved in precision and accuracy.

Application

Following the implementation of the Yieldaliser across chemical development at GSK—including sites within the US, UK, and Italy—a wide range of tasks were performed by chemists and engineers using the approach. Three types of analysis—mass-balance determiniation, crystallization endpoints, and solubility curves—exemplify how data provided by the Yieldaliser can be useful in chemical development.

Mass-balance determinations. These determinations involve identifying where major material losses occur during solubilization. For example, during a recrystallization of an API, it is useful to gain an understanding of the efficiency of the initial crystallization along with the subsequent solvent wash steps.

Crystallization endpoints. The Yiedaliser can be used to determine whether visually determined API crystallization endpoints are sound by analyzing the supernatant. Results can be used to determine whether the input batch and crystallization parameters affect the rate of crystallization.

Solubility curves. To aid in the development of the final API-stage crystallization, accurate solubility curves (concentration versus temperature) are required. Several curves can be generated using Yieldaliser data. These curves represent the solubility at several crucial solvent-mixture ratios. Key points in the process can be identified (e.g., seeding composition, point of supersaturation, and completion of crystallization).

Figure 4 shows solubility curves of an API that is crystallized from acetone after adding water. GSK chemists used several curves, which represented the solubility point at several acetone:water ratios. A saturated slurry was prepared at 0 °C and equilibrated for 1 h before sampling and assaying the supernatant. This process was repeated at 10 °C intervals, going up to 50 °C, and the results were plotted. Four solubility curves were determined to model key points in the process (e.g., starting composition 10:0.5 acetone:water, seeding composition 10:3 acetone:water, isolation composition 10:6 acetone: water). To further extend the temperature range, the exponential trend lines were extrapolated to provide a temperature range of –10 to 60°C.

Figure 4: Active pharmaceutical ingredient solubility at various acetone:water ratios, including exponential trendline (–10 to +60 °C).

Key points in the crystallization process are shown in Figure 4. For example, the line labeled "post-distillation" represents the concentration and composition of the solution at the end of the atmospheric distillation. The line shows that there is an approximate 7 °C window between the solution cooling to the point of supersaturation (dark blue line) and the boiling point. Also marked in Figure 4 is the seeding point at 53 °C, which is approximately 5 °C inside the saturated solubility curve (pink line) for this composition. The red line represents the isolation composition and shows that very little material (1.2 mg/mL) is left in solution once the slurry has been cooled to –10 °C. This same line shows that most of the API has crystallized during the water addition and before the cool-down from 47 ° to –10 °C. Therefore, it is more important to examine changes when water is added. Users were able to define the final process parameters with the help of these solubility curves.

Continuous improvement

The integration of the Yieldaliser into the open-access HPLC platform has enabled chemists to generate impurity-profile and assay data simultaneously from a single analysis. The additional assay data obtained from the same sample preparation and chromatographic run is therefore generated with little additional cost. Users can perform the sample preparation in ~1 minute, thereby enabling results to be generated and emailed to their desks within 10 minutes of the sample preparation (i.e., when the sample queue is empty). A common open-access HPLC platform also allows chemists to generate Yieldaliser data that can support processes that have been transferred from the laboratory scale into larger production facilities regardless of where in the world the analysis is required.

To continually improve the Yiedaliser, the software was written to enable ongoing collection of metrics, thereby monitoring system usage. At the GSK facility in Stevenage, there has been sufficient demand during the past eight years to ensure that four open-access systems are equipped to generate Yieldaliser data. Figure 5 displays the number and percentage of the different types of assay analysis performed by ~50 users across the four systems in 2009.

Figure 5: Annual metrics collected using the Yieldaliser from GSK's Stevenage, Hertfordshire, United Kingdom, facility in 2009.

Figure 5 highlights the most frequent types of assay analysis performed by users at GSK's Stevenage site. More than 97% of the analyses focused on generating yield in solution using the typical protocol or dilute protocol (see Figure 2). The data also show that chemists that used the calibration tool throughout 2009 often did not need to determine the purity of isolated solids (more precise data is often required on solids and therefore generated by trained analytical chemists on dedicated HPLC systems).

Future plans

The examples documented by the authors demonstrate the use of the Yieldaliser within GSK's chemical development division. Implementation of an equivalent validated system, applicable to current good manufacturing/laboratory practice analysis, may help standardize operating methods for determining HPLC assays. Evolving quality by design approaches for analytical methods will potentially provide greater flexibility for changing methods that are already registered (2, 3). Improvement to the sample-preparation protocols is also under investigation (e.g., use of liquid handling devices that can better handle 20 µl over a wide range of solvents) to automate further sample analysis and improve accuracy and precision. Lastly, automated methodology is being explored for automating fault detection and troubleshooting across the open-access HPLC platform (4–6).

Phil Borman* and John Roberts are managers in analytical sciences/chemical development at GlaxoSmithKline (GSK), Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom, tel. +44 1438 763713, fax +44 1438 764414, phil.j.borman@gsk.com. Barbara O'Reilly is a statistical programming analyst at Roche (Welwyn Garden City, UK). Robin Attrill is a manager in synthetic chemistry/chemical development at GSK in Stevenage. Ian Barylski is a manager and Keith Freebairn is a director, both in particle/process sciences and engineering/chemical development, at GSK in Stevenage.

References

1. J. Roberts et al., Amer. Pharm. Rev.13 (2), 38–44 (2010).

2. P. Borman et al., Pharm. Technol.31 (10), 142–152 (2007).

3. M. Schweitzer et al., Pharm. Technol.34 (2), 52–59 (2010).

4. H. Eriksson and P. Larses, J. Chem. Inf. Comput. Sci.32, 139–144 (1992).

5. T. Wessa et al., Organic Process R&D4, 102–106 (2000).

6. L. Kaminski et al., J. of Pharm. and Biomedic. Anal.51 (3), 557–564 (2010).

Editor's Note: This article is being simultaneously published in Pharmaceutical Technology Europe's October 2010 issue.