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European Biopharmaceutical Review

Ensuring Success

One of the most challenging tasks facing an analyst in pharmaceutical drug development is the growth of stability when indicating high-performance liquid chromatography (HPLC) methods for drug substances and products. The main requisite for this type of method is selectivity between the main peak and often a large number of impurities (process impurities as well as degradants). In order to find the best separation, different combinations of HPLC stationary and mobile phases need to be tested. Performing this in a serial, trial-and-error mode can often lead to long development timelines, which do not fit with the speed needed to bring compounds through drug development – especially in the early/mid-phase.

At the same time, one of the biggest problems for early-phase projects is the lack of robustness. Because the approach is often to invest little time and effort in early/mid-phase method development, the performance of these methods during initial stability studies – where it needs to be used for the first time in consecutive periods – often proves to be a real challenge, leading to either inconsistent result trends with time, or repeated sample analysis for investigation due to poor method performance and low-quality data. This will then continue to be an issue for the project until the compound progresses to the point where redevelopment of the methods is considered to achieve suitable robustness.

The validation of developed methods is mandatory to ensure the required quality of generated data to be submitted to regulatory agencies as part of the dossiers for clinical trial applications. International Conference on Harmonisation guidelines for validation represent a worldwide accepted standard for tests to be performed to demonstrate the method suitability for its intended use, modulated by development phases.

Validation is usually a time- and effort-consuming activity, leading to a large amount of data that needs to be generated, elaborated, verified, statistically evaluated, stored and, if electronically generated, compliant with 21 CFR Part 11 of the Code of Federal Regulations requirements.

In order to ensure maximum quality of our analytical methods while reducing development and validation timelines during all drug development phases, Aptuit follows a structured analytical strategy based on Quality by Design (QbD) principles, aided by the latest generation software tools – such as DryLab, Chromsword and Empower Method Validation Manager (Empower MVM) – to support both development and validation tasks.

This analytical development strategy consists of six steps:

1. Definition of the method target attributes and business requirements. Phase of development and drug pharmaceutical form are key factors to consider here
2. Evaluation of the physico-chemical characteristics of the target analytes, including the understanding of chemical structure, polarity and acid/base equilibria, solubility screening, and the assessment of degradation pathways and kinetics to identify the compounds to be detected, separated and quantified, ensuring the stability-indicating power of the method (this is usually followed by a forced degradation study)
3. Selection of the separation and detection techniques that suit the outcomes of step 1 and step 2
4. Identification and scouting of the key method parameters – for example, buffer pH, organic modifier, stationary phase gradient and temperature – affecting the method performance and, in predefined ranges, the method design space
5. Selection of a set of optimal conditions within the method design space
6. Verification/validation to demonstrate suitability of the method conditions for its intended purpose and its robustness

To support the analytical development strategy and maximise the output of each experiment, scientists are equipped with method development workstations combined with a number of chromatography modelling software packages and computer assisted tools. During the last 15 to 20 years, these software packages have become commercially available and their use has proven effective in overcoming the limitations of the trial-and-error approach.

The major computer systems available at Aptuit for method development are DryLab and Chromsword. To support method validation to a high standard of data quality, ensuring controlled data management and storage, scientists can combine either of these with Empower MVM.

DryLab

In a QbD approach, the use of DryLab as a modelling tool to predict separation upon the change of one, two or three variables in the chromatography of a mixture of components has found extended use in the industry, generating a map of results in the design space. Robust methods have been obtained quickly on many different compound families (1-3).

DryLab’s underlying theory and software development dates back to the late 1980s (4). A collection of applications and a summary of key fundamentals of Drylab can be found in a review by Molnar Institute (5).

In the case presented in Figure 1, DryLab was used to determine a set of improved chromatographic conditions for a mixture of 13 compounds, including synthetic and degradation impurities of an active pharmaceutical ingredient of low polarity and medium molecular weight. A pH – gradient time combination of experiments (six chromatographic runs) – was used to generate the resolution map, helping to identify the expected range of robustness for the method.

The optimal chromatographic conditions determined by DryLab modelling are then experimentally verified and tested for robustness. As a result, methods can be optimised within a few experiments, combined with simulations, thus minimising development lead time and cost for solvents and running instruments.

Chromsword

Chromsword is a software used for automated chromatographic method development, enabling rapid progress of separation methods in liquid chromatography with a minimum set of experiments (6, 7). It has a free-running capability of acquiring data, analysing results and automatically generating new sets of conditions to optimise the results in the following run (8-10). It can also be used for optimising separations in reversed-phase and normal-phase.

Figure 2 demonstrates a case where method redevelopment for a 14-compound mixture suffered from poor resolution and early elution in the original method. Chromsword software was used for the first screening of conditions, generating a series of sequential runs in order to identify the best HPLC test mix trace within a few hours of experiments. Based on these initial outcomes, a pre-selection of columns was created in order to go ahead with the ‘optimisation’ mode. The second step aimed to test different HPLC gradients in order to further optimise the best HPLC parameters. This was achieved by using DryLab software for collecting gradient performance information at different pH levels and gradient steepness on the columns previously identified.

The final method conditions were then identified by intelligently combining the two software tools at the aim of column, buffer and gradient screening of conditions (see Figure 3).

Empower MVM

Empower MVM allows the performance of chromatographic method validation – from protocol planning through to final reporting. It helps to generate analytical sequences for individual or combined validation tests, set the test acceptance criteria and evaluate results against them, displaying the status of ongoing validation studies and highlighting failures. Statistical evaluation of data is performed for each test at a defined significance level. Empower MVM is validated for Good Manufacturing Practice use and compliance with electronic records and signature requirements.

Figure 4 shows the screenshot of a typical validation study and some validation results. The data elaboration performed by Empower MVM and presented in the reports includes data plots and summary tables, statistical elaboration of data and evaluation of results against set acceptance criteria for each validation test.

Conclusion

A structured, straightforward and reliable approach for method development and validation, based on QbD principles, can deliver effective analytical solutions for each pharmaceutical development phase. Highly experienced analytical scientists and a software-assisted strategy for method development and validation enables delivery of high performance, robust and reliable stability-indicating HPLC methods.

Acknowledgement

The authors would like to thank Ameriga Fanigliulo, Nicola Vaccari and Francesca Capelli for their support and assistance in preparing this article

References

1. Bonfichi R, J of Chromatogr A 678: pp213-221, 1994
2. Krisko RM et al, J of Chromatogr A 1122: pp186-193, 2006
3. Jayaraman K et al, Analytica Chim Acta 696: pp116-124, 2011
4. Snyder LR, Dolan JW and Lommen DC, J of Chromatogr 485: pp65-112, 1989
5. Molnar, J of Chromatogr A 965: pp175-194, 2002
6. Galushko SV et al, J of Chromatogr A 660: pp47-59, 1994
7. Beinert WD et al, Am Laboratory 33: pp14-15, 2001
8. Hewitt EF, Lukolay P and Galushko S, J of Chromatogr A 1107: pp79-87
9. Xiao KP et al, J of Chromatogr A 1163: pp145-156, 2007
10. Zheng J and Rustum AM, J Pharm Biomed Analysis 51: pp146-152, 2010


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About the authors

After her PhD in Pharmaceutical Science, Riet Dams started her career in the industry in 2003, working for MDS Pharma Services before joining GlaxoSmithKline. At Aptuit, she initially took on the role of manager of the Analytical QC group. Riet currently heads the company’s Product Development Team in the Pharmaceutical Sciences department, which encompasses formulation, analytical and stability teams. She is accountable for the development of finished dosage forms and is head of the quality control laboratories.

Chris Bland trained as a Pharmacist at the University of Bath and completed a PhD at the University of Nottingham. He has over 20 years of experience in a career spanning formulation development, clinical trial manufacturing and packaging. Chris held senior roles in investigational drug product manufacturing and supply chain management before joining Aptuit in 2011. He is a former Chairman of the ISPE Investigational Products Council and the Parenteral Society Freeze Drying Committee.

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