Decades of chromatography research done with DryLab®.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatoraphy, part 2: Optimization of the phase system

Alessandra Cusumano, Davy Guillarme, Alain Beck, Szabolcs Fekete

The goal of this second part was (i) to evaluate the performance of commercially available HIC columns and (ii) to develop a fast and automated “phase system” (i.e. stationary phase and salt type) optimization procedure for the analytical characterization of protein biopharmaceuticals. For this purpose, various therapeutic mAbs (denosumab, palivizumab, pertuzumab, rituximab and bevacizumab) and a cysteine linked ADC (brentuximab-vedotin) were selected as model substances. Several HIC column chemistries (butyl, ether and alkylamide) from different providers were evaluated in four different buffer systems (sodium acetate, sodium chloride, ammonium acetate and ammonium sulfate). As stationary phases, the historical TSK gel Butyl NPR phase and the brand new Thermo MAbPac HIC-10 were found to be the most versatile ones in terms of hydrophobicity, peak capacity and achievable selectivity. As salt types, ammonium sulfate and sodium acetate were found to be particularly well adapted for the analytical characterization of mAbs and ADCs, but it is important to keep in mind that a concentration 2 to 3-times higher of sodium acetate versus ammonium sulfate is required to achieve a similar retention in HIC. After selection of the most appropriate phase systems, the optimization of the separation can be carried out by computer assisted retention modeling in a high throughput manner.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products

Szabolcs Fekete, Jean-Luc Veuthey, Alain Beck, Davy Guillarme

Hydrophobic interaction chromatography (HIC) is a historical strategy used for the analytical purification and characterization of proteins. Similarly to what can be done in reversed-phase liquid chromatography (RPLC), HIC is able to separate protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform separations under non denaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature and no need for organic solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theoretical and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technology, applications and future perspectives are described and critically discussed.

Journal of Pharmaceutical and Biomedical Analysis · 2016

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase

Marta Rodriguez-Aller, Davy Guillarme, Alain Beck, Szabolcs Fekete

The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼1%.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Automated UHPLC separation of 10 pharmaceutical compounds using software-modeling

Arnold Zöldhegyi, Hans-Jürgen Rieger, Imre Molnár, Liliya Fekhretdinova

Human mistakes are still one of the main reasons of underlying regulatory affairs that in a compliance with FDA’s Data Integrity and Analytical Quality by Design (AQbD) must be eliminated. To develop smooth, fast and robust methods that are free of human failures, a state-of-the-art automation was presented. For the scope of this study, a commercial software (DryLab) and a model mixture of 10 drugs were subjected to testing. Following AQbD-principles, the best available working point was selected and conformational experimental runs, i.e. the six worst cases of the conducted robustness calculation, were performed. Simulated results were found to be in excellent agreement with the experimental ones, proving the usefulness and effectiveness of an automated, software-assisted analytical method development.

LC GC Europe · 2002

Optimizing Multilinear Gradients in HPLC

Tom H. Jupille, Lloyd R. Snyder, Imre Molnár

Multilinear gradients have not been widely used in general purpose high performance liquid chromatography, in part because of experimental inconvenience in method development. Even with the use of computer modelling, identifying an optimum set of breakpoints has primarily been done by trial and error. Combining a spreadsheet with controllable chromatography modelling software has allowed us to implement a systematic approach to bi- and tri-linear gradient optimization. Application of this approach to 10 randomly generated sample sets suggests that the improvement in critical resolution that can be obtained is considerably smaller than that obtained through the use of a second selectivity variable. Because no additional experimental time (and only limited computational time) is required once an optimum linear gradient has been modelled, however, any improvement thus gained is essentially "free".

Wiley · 2010

Equipment

Lloyd R. Snyder, J. J. Kirkland, John W. Dolan

Journal of Pharmaceutical and Biomedical Analysis · 2014

In silico robustness testing of a compendial HPLC purity method by using of a multidimensional design space build by chromatography modeling—Case study pramipexole

Alexander H. Schmidt, Mijo Stanic, Imre Molnár

Purity testing of the active pharmaceutical ingredient (API) pramipexole is performed using an official (compendial) and harmonized method published in the European Pharmacopeia (E.P.) and United States Pharmacopeia (USP). According to this monograph the successful chromatographic separation of the API from impurities is achieved on a C18 column with gradient elution of an ion pairing buffer of pH 3.0 (mobile phase A) and acetonitrile (mobile phase B). Although not recommended in general, compendial methods are often adapted for purity testing of generic formulations. In this paper a novel approach to evaluate method robustness of an adapted method – prior of full method validation – is described. Based on Quality-by-Design (QbD) principles, a small number of experiments are performed, which after entering them into a chromatography modeling software allow to visualize a multidimensional “Design Space”, a region, in which changes in method parameters will not significantly affect the results as defined in the ICH guideline Q8(R2) leading to a more flexible method handling in routine analysis. For two different recommended C18 columns a multidimensional Design Space (Method Operating Design Region, MODR) was constructed to study the robustness of the adapted method with a newly developed Robustness Module. In a full factorial design the following six parameters were varied at three levels (low, nominal, high): gradient time, temperature, pH of the aqueous eluent (A), flow rate, start- and end concentration of the organic mobile phase component (eluent B). The resulting 36=729 experiments were performed in silico from the previously constructed models for Design Space in less than 1min and showed that the required resolution of 2.0 could not be reached in all experiments for the two columns which were recommended by the E.P. (failure rate 25% and 16%, respectively). However, by adjusting the gradient time, we were able to fulfill the requirements with a failure rate of zero. For the aqueous eluent a separate “Eluent Design Space” study was performed, which allows the construction of ionic strength vs. ion pairing concentration models to identify the optimum combination of the concentrations for the buffer and the ion-pairing reagent.

Journal of Pharmaceutical and Biomedical Analysis · 2017

Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass spectrometric peak tracking

Róbert Kormány, Imre Molnár, Jenő Fekete

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography–Mass Spectrometry (LC–MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured Rs,crit −values.

Journal of Pharmaceutical and Biomedical Analysis · 2013

Using an innovative Quality-by-Design approach for development of a stability indicating UHPLC method for ebastine in the API and pharmaceutical formulations

Alexander H. Schmidt, Imre Molnár

A stability-indicating ultra high performance liquid chromatographic (UHPLC) method has been developed for purity testing of ebastine and its pharmaceutical formulations. Successful chromatographic separation of the API from impurities was achieved on a Waters Acquity UPLC BEH C18, 50mm×2.1mm, 1.7μm particle size column with gradient elution of 10mM acetate buffer pH 6.2 and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Incorporating Quality by Design (QbD) principles to the method development approach by using the chromatography modeling software DryLab®4 allows the visualization of a “Design Space”, a region in which changes to method parameters will not significantly affect the results as defined in the ICH guideline Q8 (R2). A verification study demonstrated that the established model for Design Space is accurate with a relative error of prediction of only 0.6%. The method was fully validated for specificity, linearity, accuracy and precision, and robustness in compliance to the ICH guideline Q2 (R1). The method was found to be linear in the concentration range from the quantification limit (LOQ) to 125% of the specification limit for ebastine and each of the impurities with correlation coefficients of not less than 0.999. The recovery rate was between 98.15 and 100.30% for each impurity. The repeatability and intermediate precision (RSD) were less than 3.2% for ebastine and each of the impurities. The robustness of the developed method was studied by varying the six parameters: gradient time, temperature, ternary composition of the eluent, flow rate and start and end concentration of the gradient at 3 levels (+1, 0, −1). The resulting 729 experiments were performed in silico from the previously constructed model for Design Space and showed that the required resolution of 2.0 can be reached in all experiments. To prove the stability-indicating performance of the method, forced degradation (acid and base hydrolysis, oxidation, photolytic and thermal stress conditions) of ebastine was carried out. Baseline separation could be achieved for all peaks of the impurities, the degradation products and the API. Total run time was only 4min, which is an impressive 40-fold increase in productivity in comparison to the method published in the Ph. Eur. monograph and allowed purity testing of more than 360 samples per day.

The Column · 2011

Experimental Combination of Method Development Strategies in a Working Environment of Different Instrumental Set-ups

Imre Molnár, Kate E. Monks, Björn-Thoralf Erxleben

Experimental Combination of Method Development Strategies in a Working Environment of Different Instrumental Set-ups

World Scientific Publishing Europe Ltd · 2019

Software-assisted method development in high performance liquid chromatography

Imre Molnár, Szabolcs Fekete

"This handbook gives a general overview of the possibilities in recent developments in chromatographic retention modeling. As a result of the latest developments in modeling software, several new features are now accessible, opening a new level in HPLC method development. Many of these current possibilities in software assisted liquid chromatographic method modeling for analytical purposes are presented. Several modes of chromatography, including Reversed-Phase Liquid Chromatography (RPLC), Ion Exchange Chromatography (IEX), Hydrophobic Interaction Chromatography (HIC), and Hydrophilic Interaction Liquid Chromatography (HILIC) are explained in detail. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Beside the industrial and practical benefits of retention modeling, the possibilities in teaching and education are also illustrated. Finally, numerous representative industrial examples are shown, to highlight the benefits, time and cost savings offered by state-of-the-art software assisted HPLC method development."--Publisher's website

The Column · 2012

Practical Application of Quality by Design Principles to The Development of an HPLC Assay Method for an API and Impurities

Kate E. Monks, Imre Molnár, Hans-Jürgen Rieger

A method for the assay of an active pharmaceutical ingredient (API) and five known and unknown impurities was developed in accordance with Quality by Design (QbD) principles. Highly influential separation parameters were simultaneously and systematically studied so that the quality of the HPLC method could be understood, controlled and ensured. Consequently, during routine analysis out of specification (OOS) results can be corrected more effectively.

Journal of Pharmaceutical and Biomedical Analysis · 2015

Establishing column batch repeatability according to Quality by Design (QbD) principles using modeling software

Norbert Rácz, Róbert Kormány, Jenő Fekete, Imre Molnár

Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (Rs,crit) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 36=729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments.

Journal of Chromatography A · 2025

Extended multidimensional design space studies: Comparing volatile and non-volatile buffer systems in UPLC

Arnold Zöldhegyi, Barnabás Soós, Krisztián Horváth, Imre Molnár, Róbert Kormány

Journal of Chromatography A · 2026

Addressing method transfer and robustness challenges using design space modeling

Arnold Zöldhegyi, Krisztián Horváth, Imre Molnár, Róbert Kormány

In high-performance liquid chromatography (HPLC), robust and easily transferable analytical methods are essential to ensure consistent performance throughout the entire lifecycle of an analytical procedure, including unforeseen future applications. Nevertheless, robustness-, and ruggedness-related issues are frequently reported in industrial practice, particularly during method transfer between laboratories. These challenges often arise from incomplete method understanding and insufficient consideration of HPLC system-related effects, such as dwell volume, extra-column contributions and other, at first less evident, instrument-to-instrument variations in solvent delivery, mixing behavior and column thermal control. In this context, modeling approaches aligned with Analytical Quality by Design (AQbD) principles provide valuable support for both proactive method development and reactive troubleshooting. In the current work, we used DryLab modeling Design Spaces (DSs) to create digitized modeling fingerprints of various instruments, enabling extensive in silico investigations, including in-depth assessment of method transferability and evaluation of subtle, instrument-dependent robustness characteristics.