Decades of chromatography research done with DryLab®.

Journal of Pharmaceutical and Biomedical Analysis · 2017

Separation of antibody drug conjugate species by RPLC: A generic method development approach

Szabolcs Fekete, Imre Molnár, Davy Guillarme

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Analytica Chimica Acta · 2015

Effect of temperature on acid–base equilibria in separation techniques. A review

Leonardo G. Gagliardi, Marcos Tascon, Cecilia B. Castells

Studies on the theoretical principles of acid–base equilibria are reviewed and the influence of temperature on secondary chemical equilibria within the context of separation techniques, in water and also in aqueous-organic solvent mixtures, is discussed. In order to define the relationships between the retention in liquid chromatography or the migration velocity in capillary electrophoresis and temperature, the main properties of acid–base equilibria have to be taken into account for both, the analytes and the conjugate pairs chosen to control the solution pH. The focus of this review is based on liquid–liquid extraction (LLE), liquid chromatography (LC) and capillary electrophoresis (CE), with emphasis on the use of temperature as a useful variable to modify selectivity on a predictable basis. Simplified models were evaluated to achieve practical optimizations involving pH and temperature (in LLE and CE) as well as solvent composition in reversed-phase LC.

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.

Chromatographia · 1987

Solvent-strength selectivity in reversed-phase HPLC

L. R. Snyder, M. A. Quarry, Joseph L. Glajch

Solvent-strength selectivity refers to the variation of band spacing by changing the %-organic in the mobile phase (ion-pair or reversed-phase HPLC). A review of the literature has been combined with new experimental data to illustrate the general potential of this approach for HPLC optimization. It appears that most samples exhibit significant changes in band spacing method development based on solvent-strength optimization plus computer simulation (DryLab software) are given for illustration. For relatively simple mixtures (10 or fewer components), it appears that solvent-strength optimization compares favorably with other methods such as mapping the organic-solvent selectivity of methanol, acetonitrile, tetrahydrofuran, and water.

Journal of Chromatography · 1989

Drylab® computer simulation for high-performance liquid chromatographic method development: II. Gradient Elution

J. W. Dolan, D. C. Lommen, L. R. Snyder

Computer simulation (DryLab software) as an aid for the development of gradient high-performance liquid chromatographic methods is reviewed. Several examples of its application are presented and the accuracy of such predictions is discussed.

HPLC 2023 · 2023

Software-supported HPLC Method Development for Separating Four Regioisomers of a Sugammadex-related Impurity

Dominika Herr, Arnold Zöldhegyi, Péter Soma Szakály, Erzsébet Varga

This research focuses on a simple, relatively inexpensive reversed-phase high-pressure liquid chromatography (RP-HPLC) application in combination with UV-detection aiming for the separation of four relevant regioisomers (marked as AB, AC, AD, AE) of Di-OH-SGM (Hexakis(6-deoxy-6-(2-carboxyethyl)thio)-gamma- cyclodextrin). The presence of these regioisomers in the synthesized Di-OH-SGM reference material has already been proven by MS and NMR spectroscopy. Thus, the main goal of our project was to baseline-resolve these regioisomers—as this is required for the qualification of the synthetized Di-OH-SGM material. Although our preliminary results on the selected C18 stationary phase provided a promising initial separation, only Rs,crit. = 0.9 of peaks could be realized. Hence, we decided to apply a systematic modeling approach (DryLab) to contextualize data and to try optimizing critical method parameters. We found that by lowering column temperature, better separation but still no baseline separation of peaks was possible—while the modeling software revealed some unique features of these molecules. The poster will present the current results of our software-supported development process, underlining the key benefits of modeling approaches in better understanding the complex nature and chromatographic behavior of regioisomers of Di-OH-SGM.

Journal of Pharmaceutical and Biomedical Analysis · 2021

Superheated reversed phase chromatography with ultrashort columns for the analysis of therapeutic proteins

Balázs Bobály, Alex Keresztfalvi, Thomas Gräber, Maria Anna Schwarz

Mild or elevated temperatures are routinely used for the analysis of therapeutic proteins by reversed phase liquid chromatography. Generic conditions can be used for the analysis of monoclonal antibodies, and may be adapted for species derived thereof, for instance their immuno-conjugates. Beyond platform monoclonal antibodies, many novel, non-covalent protein complexes are also frequently pursued as protein therapeutics. These complexes, in reverse phased chromatography, may require extremely harsh, superheated conditions to dissociate and elute as interpretable profiles. In order to minimize on-column degradation under superheated conditions, the analysis time has to be reduced as much as possible. Using ultrashort columns and fast gradients is a promising approach in achieving informative separations within a minute, or even faster. Here the applicability of this approach, which supports maintaining levels of degradation products close to the intrinsic sample composition without further on-column degradation is demonstrated. NISTmAb as conventional IgG, a bispecific homodimer and a bispecific homotetramer were used for demonstrating differences in the elution characteristics and the necessity of using the proposed approach. The analysis of the bispecific homodimer was discussed in detail as a case study.

Journal of Pharmaceutical and Biomedical Analysis · 2014

Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis

Szabolcs Fekete, Isabelle Kohler, Serge Rudaz, Davy Guillarme

In the last decade, an important technical evolution has occurred in pharmaceutical analysis with numerous innovative supports and advanced instruments that have been proposed to achieve fast or ultra-fast separations in LC with an excellent sensitivity and ease of operation. Among the proposed strategies to increase the throughput, the use of short narrow-bore columns packed with sub-3μm core–shell and porous sub-2μm particles have emerged as the gold standards. Nevertheless, to take the full benefits of these modern supports, a suitable chromatographic system has to be employed. This review summarizes the instrumental needs and challenges in terms of extra-column variance, dwell volume, maximum system pressure, detector data acquisition rate, and injection cycle time. In addition, because of their reasonable pressure drop, the use of columns packed with sub-3μm core–shell particles on a conventional LC instrument is discussed in detail. A methodology is proposed to check the compatibility between stationary phase and instrument, and some solutions are proposed to improve the performance of standard instruments. Finally, because the column technology is evolving faster than instrumentation, it is nowadays possible to purchase short, narrow-bore columns packed with 1.3μm core–shell particles. Micro columns (1mm I.D.) packed with 1.7–1.9μm porous particles are also available from several providers, which limit frictional heating effects and reduce solvent and sample consumption. However, it remains difficult to find instruments compatible with such column geometries and a severe loss of performance may be observed due to the system itself.

Journal of Chromatography Open · 2024

Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial

Szabolcs Fekete, Mateusz Imiołek, Matthew Lauber

With this work, we present a comprehensive tutorial for the analysis of oligonucleotides (ONs, 5 to 100 mer) using ion-pair reversed-phase liquid chromatography (IP-RPLC) on ultra-short columns (20 × 2.1 mm). We explore the impact of ion-pairing (IP) agents on ON retention and demonstrate that while IP agents significantly influence absolute retention, their effect on selectivity is often minimal. Our findings emphasize the utility of systematic method development, including software-assisted retention modeling, to optimize gradient steepness and temperature such that resolution can be optimized for both sequence and length variants. We recommend the use of low-adsorption column hardware to minimize nonspecific interactions, which is to the benefit of improving method robustness, peak shapes and recovery (especially of shortmer impurities). Our results confirm the utility of so-called ultra-short column formats as is demonstrated by way of the example, quick run time separations and high-throughput ON analyses. The study establishes practical guidelines for developing robust, reproducible, and high-efficiency IP-RPLC methods for ONs which will be of assistance to analysts working on new therapeutics and new sequencing and diagnostic reagents alike.

Journal of Chromatography A · 2024

Revisiting column selectivity choices in ultra-high performance liquid chromatography–Using multidimensional analytical Design Spaces to identify column equivalency

Arnold Zöldhegyi, Krisztián Horváth, Róbert Kormány

Current guides and column selection system (CSS) platforms can provide some helpful insights with regard to the selection of alternative phases. Their practical reliability however, can also turn out to be questionable, especially considering the lack of detailed specifics, such as a clear definition of points of equivalence—appropriate running conditions under which the given analytical mixture can be satisfactorily resolved on various stationary phases. In this context, the use of multivariate modeling tools can be highly beneficial. These tools, when applied systematically, are ideal for uniquely characterizing complex LC-separation systems, a fact supported by numerous peer-reviewed papers. Revisiting our earlier work [1] and the applied systematic workflow [2], we used a Design Space modeling software (DryLab), with the main focus on building and comparing 3-dimensional separation models of amlodipine and its related impurities to identify shared method conditions under which columns are conveniently interchangeable. Our study comprised 5, C18-modified ultra-high performance liquid chromatography (UHPLC) columns in total, in some cases with surprising results. We identified several equivalences between the Design Spaces (DSs) of markedly different columns. Conversely, there were cases where, despite the predicted similarities in column data, the modeled DSs demonstrated clear differences between the selected stationary phases.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Implementation of a generic liquid chromatographic method development workflow: Application to the analysis of phytocannabinoids and Cannabis sativa extracts

Szabolcs Fekete, Vida Sadat-Noorbakhsh, Cedric Schelling, Imre Molnár, Davy Guillarme, Serge Rudaz, Jean-Luc Veuthey

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps: i)The screening of primary parameters (i.e. stationary phase nature, organic modifier nature and approximate mobile phase pH) was carried out with a generic gradient on a short narrow bore column, using a system able to accommodate numerous solvents/buffers and columns. Instead of complete peak tracking, the number of peaks which can be separated was considered as a response at this level, to save time.ii)The optimization of secondary parameters (i.e. gradient conditions, mobile phase temperature and pH within a narrow range) requires only 12 initial experiments and the use of HPLC modeling software for data treatment. It allows to find out the best retention and selectivity for the selected compounds. Peak tracking was performed with a single quadruple mass detector in single ion recording mode, and UV detection (in a broad wavelength range).iii)The refinement step allows to further adjust column efficiency, by tuning column length and mobile phase flow rate. This can also be done virtually using HPLC modeling software.iv)The robustness testing step was also evaluated from a virtual experimental design. Success rate and regression coefficients were estimated in about 1 min, without the need to perform any real experiment. At the end, this method development workflow was performed in less than 4 days and minimizes the costs of the method development in liquid chromatography.

Journal of Chromatography · 1992

Application of the gradient elution technique: Demonstration with a special test mixture and the DryLab G/plus method development software

R. Däppen, I. Molnár

A mixture of ten compounds with an overlapping peak pair was analysed with a 90-min elution gradient. To improve the separation, two reversed-phase gradients differing by a factor of 3 in their run times, were applied. Contrary to expectation, two peak pairs were less well separated in the gradient run with the lower slope. The relative resolution map provided a rapid solution to the problem: a gradient with 16-min run time gave the best separation of the mixture. The simulated chromatogram was verified experimentally. The differences between the predicted and experimental retention times averaged 0.03 min. Further improvement was obtained using a segmented gradient, which adequately separately all peaks in only 9 min.

Journal of Chromatography · 1990

Use of high-performance liquid chromatography in the pharmaceutical industry

Fritz Erni

Requirements for new pharmaceutical products and their impact on applications of high-performance liquid chromatography (HPLC) are discussed. The strengths and weaknesses of HPLC in this context are evaluated and compared with current trends and expectation in separation science.

Chromatographia · 2012

Enhancing the Quality of Separation in One-Dimensional Peptide Mapping Using Mathematical Transformation

Róbert Berky, Szabolcs Fekete, Jenő Fekete

In this study, some practical examples are presented that show the quality of separations using very efficient columns packed with the latest generation of core shell sub-3 μm and fully porous sub-2 μm particles in one-dimensional peptide separations. This paper shows an approach for the analysis of proteins, such as high-resolution separations, and a data transformation process to improve peak recognition and analysis. Applying power functions on raw chromatographic data can be a neat tool in the field of biosimilar analysis, especially in comparability studies regarding the quality (primary structure) of proteins. Based on the results presented here, it can be stated that the use of power functions is beneficial for the comparison of chromatograms when peak areas are considered but has no effect when using peak heights. In this study, the new Acquity CSH columns (C18 and phenyl-hexyl) and the core–shell type wide pore Ascentis Express Peptide ES C18 material were applied with great success in peptide mapping. Finally, using phenyl-hexyl stationary phase in peptide separation seems to be a good alternative to the generally applied C18 or C4 phases.

Advanced Materials Technologies · 2020

A Novel Approach to Glycan Method Development for Biotherapeutics using Superficially Porous Particle Technology

Joseph L. Glajch, Andrew Harron, Benjamin P. Libert

In this white paper, Advanced Materials Technology presents a novel method for separating and identifying glycans. Glycans are instrumental in cellular signaling, therefore a specific protein’s glycosylation can influence the effectiveness of the biotherapeutic. Given the importance of glycosylation to the overall performance of a glycoprotein, there is a need to completely characterize and then monitor these glycans for both research and manufacturing purposes.

Journal of Pharmaceutical and Biomedical Analysis · 2018

Simultaneous optimization of mobile phase composition and pH using retention modeling and experimental design

Norbert Rácz, Imre Molnár, Arnold Zöldhegyi, Hans-Jürgen Rieger, Róbert Kormány

Chromatographic methods are progressing continuously. Increasing sample complexity and safety expectations lead to higher regulatory demands, hence challenges in liquid chromatography analysis are rising, even today, when faster and faster chromatographic systems are extensively employed and become widely accessible for successful method development. The goal of this study was to investigate the impact of mobile phase influences as important factors of selectivity tuning in method development. This would mitigate mobile phase-related robustness issues throughout the method’s lifecycle. To discover and understand these effects, a new module of chromatographic modeling software DryLab (ver. 4.3.4. beta) was introduced and a special experimental design (DoE) was tested, allowing the simultaneous optimization of solvent-dependent parameters, such as gradient time (tG), ternary eluent composition (tC) and pH, requiring 18 input experiments (2 × 3 × 3 = 18). Additionally, the model creation, using a UPLC system and a narrow bore column (50 × 2.1 mm), the entire experimental work could be finished in 2–3 hours. To demonstrate the applicability of this new design, amlodipine and its related pharmacopoeia impurities (A–H) were subjected to be used in a case study. Predicted vs. Experimental (or Verification) runs showed excellent agreement, average retention time deviations were typically less than 1 s. Modelled robustness testing was also performed, elucidating all important mobile phase and instrument parameters that could influence a method’s lifetime performance. Furthermore, as the in silico robustness testing is the least time consuming part of the method development process, it can be used extensively to evaluate robustness even at the very early part in stage 1 of the Method Life Cycle (MLC).

Journal of Chromatography A · 2026

Retention modeling of oligonucleotides on an amide-based HILIC column: A descriptor-driven approach

Jonathan Maurer, Balasubrahmanyam Addepalli, Fabrice Gritti, Matthew A. Lauber, Davy Guillarme, Szabolcs Fekete

Hydrophilic interaction chromatography (HILIC) has recently gained attention as a powerful tool for the analysis of nucleic acid–based therapeutics, offering high resolving power and excellent compatibility with mass spectrometry. However, the successful development of HILIC methods requires a clear understanding of the underlying retention mechanisms. Despite numerous studies, the respective contributions of hydrophilic partitioning, polar interactions, and ionic interactions to oligonucleotide retention remain poorly understood. In this work, the role of hydrogen bonding, dipole-dipole interactions, solvophobic effects, and ionic interactions in governing oligonucleotide retention have been systematically investigated. A series of targeted design-of-experiments studies, performed on a representative panel of samples to investigate the impact of oligonucleotide structure on retention, provided a refined framework for interpreting HILIC separations of oligonucleotides. All experiments were performed on an amide-bonded stationary phase, and the proposed descriptor-based retention model applies specifically to this surface chemistry. Our results demonstrate that solvophobic effects and hydrogen bonding are the dominant drivers of retention, whereas hydrophilic partitioning contributes negligibly under practical conditions. This mechanistic insight has important consequences for method development: introducing protic solvents into the mobile phase, conditions that would disrupt the water-rich layer, substantially increases selectivity and enables the resolution of closely related oligonucleotide species. Altogether, these findings shift the conceptual basis of HILIC for nucleic acids from a hydrophilic partitioning/interaction model toward a retention mechanism best described as a solvophobic–interaction‑dominated HILIC regime, in which protic solvents assist selectivity through H‑bond competition. This new perspective provides a more accurate foundation for designing, optimizing, and interpreting HILIC methods for oligonucleotide analysis.

Analytical Chemistry · 1986

Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution

Joseph L. Glajch, M. A. Quarry, J. F. Vasta, Lloyd R. Snyder

Journal of Chromatography · 1984

Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography: Verification of retention relationships

M. A. Stadalius, H. S. Gold, L. R. Snyder

The presently accepted theory for gradient separations of small molecules has been used to develop a predictive model for peptides and proteins as samples, using reversed-phase high-performance liquid chromatography. Given the experimental conditions (gradient time, flow-rate, temperature, etc.), the molecular weight of the sample, and certain column characteristics (Knox parameters, column dimensions, particle diameter, etc.), it is possible to calculate the overall results of a given separation by gradient elution: peak capacity or average resolution, peak volume or relative peak height, etc. This information can in turn facilitate the optimized separation of any sample. The present model assumes that isocratic and gradient retention are interrelated for peptide molecules, in the same fashion as for small molecules. This assumption has been verified for various peptides and proteins and further used to gain new insight into the control of retention and band-spacing in gradient elution.

Journal of Chromatography A · 2012

Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles

Stephanie A. Schuster, Barry E. Boyes, Brian M. Wagner, Joseph J. Kirkland

The separation range of superficially porous particles (Fused-Core®) has been extended by design of particles with 160Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7μm Fused-Core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100°C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10–20mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-Core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150min on coupled columns of HALO Peptide ES-C18 with a combined length of 250mm.