Studies confirm potency of DryLab® Design Spaces in evaluating stationary phase chemistry

Berlin: – A series of recent studies have confirmed that liquid chromatography modelling using the Design Space in Molnár-Institute for Applied Chromatography’s DryLab® software platform has the potential to revolutionize pharmaceutical chemistry and research.

The studies repeatedly show how the DryLab® Design Space facility for quick and visual comparison of columns using retention modelling can enable faster and more robust separations and drastically reduce the time needed for analyses.

Incorporating QbD principles

One of the most-cited works on 3D visual comparison of various stationary phases, for proof-of-concept based on Quality by Design (QbD) principles was lead authored in 2013 by Dr. Robert Kormány of the Egis Plc in Hungary, along with Molnár-Institute Founder and President, Dr. Imre Molnár and Product Manager Dr. Hans-Jürgen Rieger.

Their paper ‘Exploring better column selectivity choices in ultra-high performance liquid chromatography using Quality by Design principles’ [1] was published in the Journal of Pharmaceutical and Biomedical Analysis (JPBA).

The study carried out separations of Amlodipine using QbD principles in DryLab® modelling to show how previous analysis time of 60 min could be reduced by 90% to under 6 min. Their method investigated 9 different UHPLC column chemistries, using 3D-Design Spaces of the Critical Resolution, based on 12 experiments, modelled in-silico using DryLab® v. 3.9 optimization software, consisting of the DryLab® Core-module, the PeakMatch® module and the 3D-Critical Resolution Space feature called the “Cube”. The DryLab® approach is based on the measurement of the retention behaviour of organic compounds in Reversed Phase HPLC (RP-HPLC).

The study paved new ground in establishing a scientifically reliable way of comparing HPLC-columns in a highly variable multifactorial Design Space, that reflects not only column chemistries, but also at the same time the influence of gradient time, pH, ternary eluent composition, flow rate, starting- and final organic eluent composition (%B). This new approach enabled researchers to gain insight of the fundamentals of multifactorial variabilities of UHPLC methods used in daily pharma routine.

The authors concluded that in method development for ultra-high performance liquid chromatography (UHPLC) separations, according to QbD principles, solutions for the best separations could easily be found for almost every column using retention modelling, with associated drastic reduction in analysis time.

“This is a great advantage in the rapid development of the best possible separation in industrial units, helping to develop new drugs faster for many diseases, which could not be treated before,” the paper concluded.

Besides of the scientific importance of precise predictions of chromatograms, the paper is also important for the application of reliable science at reduced costs in the pharmaceutical industry, helping to update older pharmacopoeia methods.

Improving method robustness by better control of mobile-phase properties

Dr. Kormány was a leading participator in a further study in the following year, led by Dr. Norbert Rácz of Budapest University of Technology and Economics, that also involved Prof. Dr. Jenö Fekete and Dr. Imre Molnár from the Molnár-Institute Berlin. This study ‘Establishing column batch repeatability according to Quality by Design (QbD) principles using modelling software’ was also published in the JPBA. [2].

This study sought to further improve column technology by applying intelligent modeling software to yield information on how to characterize batch-to-batch repeatability of columns. The team compared 12 columns from the same production process, but from different batches, studying retention parameters of these columns with real life samples, focusing the influence of gradient time, temperature and pH. Retention modeling in DryLab®4 allowed all substances to be separated independently from the batch and/or column packing material. Based on calculated results, the team was able to evaluate batch-to-batch repeatability of BEH columns with high robustness, defined as a success rate of >98% among the predicted 12×729 experiments for all 12 columns.

“Without using modeling software, it is very hard to find conditions, where the method is robust according QbD principles. Even in Reversed Phase Chromatography (RPC) small variations in surface chemistry result in differently deteriorated separations,” the study noted.

“Using resolution cubes help to evaluate column equivalency and show the best and most robust working conditions for a given set of solutes. In pharmaceutical analysis using HPLC the QbD approach is a basic need. The quality of stationary phase is one of the most important parameters and using an intelligent program gives a solid fundament to evaluate it,” the team concluded.

Identifying replacement columns

Dr. Kormány also led the next significant study to advance in-silico modelling, introducing the then Column Comparison DryLab® module, now called Design Space Comparison module, as a much easier method of finding alternative columns to assess better method life cycle management for increased robustness in daily Quality Control.

The 2017 study ‘A workflow for column interchangeability in liquid chromatography using modeling software and quality-by-design principles’, also involved researchers from the Universities of Geneva and Lausanne [3].

With thousands of liquid chromatographic columns from which to choose, researchers need automated assistance to speed method development in selecting the most suitable stationary phase for a given separation and find an appropriate replacement (alternative) column, as required for method validation in pharmaceutical regulatory guidelines.

The goal of the study was to develop a generic workflow to evaluate the chromatographic resolution in a large design space and easily find the correct replacement column for the method. To attain this objective from a limited number of initial experiments, DryLab®4 was employed to study the behaviour of the compounds and visually compare the parts of design spaces, obtained with different columns, fulfilling a given criterion of critical resolution. This allowed a zone of robust space to be identified, where design spaces overlapped each other.

The study succeeded in pioneering a workflow for comparing the resolution of an impurity profiling method, in a large Design Space of 3 measured and 3 calculated variables to find possible replacement columns for the method. This strategy based on the use of the DryLab®4 Design Space Comparison feature, allowed the team to visually compare different compartments of Design Spaces obtained with different columns, where the analytical target profile (ATP) for a selected critical resolution, mostly baseline resolution, is fulfilled. A section of robust areas could then easily be found by overlapping the corresponding Design Spaces.

The advantage of this approach is the mapping of the retention behaviour of the compounds of interest in an entire 3D Design Space, allowing replacement columns to be proposed at early stages in HPLC method development.

Pioneering a new method

The most recent study focusing on using Design Space in pharmaceutical method development was published in 2020. Led by Dr. Dániel Enesei of Egis Pharmaceuticals in Budapest, Hungary, the study  ‘Updating the European Pharmacopoeia impurity profiling method for Terazosin and suggesting alternative columns’ also involved Dr. Kormány as well as Dr. Szabolcs Fekete from the Universities of Geneva [4].

The study was motivated by the demand of European Directorate for the Quality of Medicines and Health- Care (EDQM) for a new liquid chromatographic (LC) method for terazosin impurity profiling to replace the old European Pharmacopoeia (Ph. Eur.) method, based on two different chromatographic separations.  The team’s goal was to reduce analysis time from the previous 90 minutes to below 20 min and to use the entire DryLab® suite of capabilities in modern method development and regulatory compliance.

Overcoming the problem of impurities insufficiently retained in reversed phase (RP) conditions, making an auxiliary ion-pair (IP) chromatographic separation necessary, the team found it possible to appropriately increase the retention of the two critical compounds using alternative stationary phases. Applying a pentafluoro-phenyl (PFP) stationary phase, it was feasible to separate and adequately retain all the impurities. The detection wavelength was also changed compared to the Ph. Eur. method and is now appropriate for the detection and quantification of all impurities using perchloric acid in the mobile phase at low pH.

Further goals of the 2020 study were to develop a generic workflow to evaluate the chromatographic resolution in a wide range of method variables and suggest some replacement columns for terazosin impurity profiling. The researchers used retention modelling to study the chromatographic behavior of the compounds of interest and visualize resolution for the different columns.

“Our strategy was based on the use of state-of-the-art chromatographic modelling software, allowing to visually compare the parts of response surfaces (resolution cubes) obtained with different columns, where the criterion for a selected critical resolution is fulfilled. A section of robust spaces can then easily be found by overlaying resolution cubes,” the study reported.

“Our real-life case study seemed to be a good and representative example to illustrate the implementation of an early stage robustness study. Based on 12 initial experiments – on a given column –, robustness could be accurately estimated (on the basis of retention modelling),” said the study.

“The fact, that retention modelling was involved makes the method adjustment more flexible, since only “adjustment” of the method variables is not considered to be a “change”. Therefore slight deviations of method variables can be done (allowed) in order to meet the criterion of the separation (e.g. when transferring the method). Thus, method revalidation is not necessary.

This study also demonstrates that common column tests are hardly applicable in practice to find alternative columns,” the team concluded.

References

1. Kormány R, Molnár I, Rieger HJ. Exploring better column selectivity choices in ultra-high performance liquid chromatography using quality by design principles. J Pharm Biomed Anal. 2013 Jun; 80:pp. 79-88. doi: 10.1016/j.jpba.2013.02.028. Epub 2013 Feb 28. PMID: 23528332.
2. Rácz N, Kormány R, Fekete J, Molnár I. Establishing column batch repeatability according to Quality by Design (QbD) principles using modelling software. J Pharm Biomed Anal. 2015 Apr 10;108:pp. 1-10. doi: 10.1016/j.jpba.2015.01.037. Epub 2015 Jan 25. PMID: 25703234.
3. Kormány R, Tamás K, Guillarme D, Fekete Sz. A workflow for column interchangeability in liquid chromatography using modeling software and quality-by-design principles. J Pharm Biomed Anal. 2017 Nov 30;146: pp.220-225. doi: 10.1016/j.jpba.2017.08.032. Epub 2017 Sep 1. PMID: 28886523.
4. Enesei D, Kapui I, Fekete Sz, Kormány R. Updating the European Pharmacopoeia impurity profiling method for terazosin and suggesting alternative columns. J Pharm Biomed Anal. 2020 Aug 5;187:113371. doi: 10.1016/j.jpba.2020.113371. Epub 2020 May 19. PMID: 32460215.

About MOLNÁR-INSTITUTE

Founded in 1981, Molnár-Institute develops DryLab®4, a software for UHPLC modelling for a world-wide market. Its powerful modules allow for the most sophisticated method development as required across pharma industries. Analytical scientists use DryLab®4 to understand chromatographic interactions, reduce analysis times, increase robustness, and conform to Analytical Quality by Design (AQbD) principles.

The Molnár-Institute is a registered partner to the US-FDA, CDC and many other regulatory bodies. DryLab®4 has pioneered AQbD long before regulatory agencies across the world encouraged such submissions. Widely implemented by thought leaders, the software contributes substantially to the paradigm shift towards a science and risk driven perspective on HPLC Quality Control and Assurance.

Further information at: http://www.molnar-institute.com/

Resources

Click on Exploring better column selectivity choices in ultra-high performance liquid chromatography using Quality by Design principles (2013) to access full study.
Click on Establishing column batch repeatability according to Quality by Design (QbD) principles using modelling software (2014) to access full study.
Click on A workflow for column interchangeability in liquid chromatography using modelling software and quality-by-design principles (2017) to access full study.
Click on Updating the European Pharmacopoeia impurity profiling method for terazosin and suggesting alternative columns (2020) to access full study.