Literature | MOLNÁR-INSTITUTE

Journal of Pharmaceutical and Biomedical Analysis · 2020

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Julian Kopp, Florian B. Zauner, Andreas Pell, Johanna Hausjell, Diana Humer, Julian Ebner, Christoph Herwig, Oliver Spadiut, Christoph Slouka, Reinhard Pell

Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E. coli. The overall goal was to develop a fast, robust and mass spectrometry compatible method which can baseline resolve and quantify each protein of interest. Method development consisted of three steps, oriented on an Analytical Quality by Design (AQbD) workflow: (i) the stationary phase as primary parameter was chosen based on state-of-the art technology thus minimizing protein on-column adsorption and providing high efficiency, (ii) secondary parameters (i.e. gradient conditions and column temperature) were optimized applying chromatographic modeling, and (iii) the established Method Operable Design Region (MODR) was challenged and confirmed during robustness testing, performed in-silico and experimentally by a Design of experiment (DoE) based approach. Finally, we validated the RPLC method for pivotal validation parameters (i.e. linearity, limit of quantification, and repeatability) and compared it for protein quantification against a well-established analytical methodology. The outcome of this study shows (i) a protocol for RPLC development using an AQbD principle for new method generation and (ii) a highly versatile RPLC method, suited for quick and straightforward recombinant protein titer measurement being applicable for the detection of a broad range of proteins.

Journal of Chromatography A · 2020

Rapid analysis of polycyclic aromatic hydrocarbons

Justin M. Godinho, Jason Lawhorn, Barry E. Boyes

Polycyclic aromatic hydrocarbons are a continuing environmental and health concern. The analytical methods developed to analyze this class of compounds have relied on reversed phase liquid chromatography and are often on the order of tens of minutes. Reduction in analysis times through the application of sub-2 μm fully porous and superficially porous support materials can increase the throughput of these LC separations. Herein, we demonstrate similar selectivity between a fully porous 1.8 μm and a 2.7 μm superficially porous material. Separations were individually developed with in silico modeling for a given flow rate determined by the fully porous column's backpressure requirements. Since the 2.7 μm superficially porous materials inherently require less backpressure to achieve similar levels of efficiency as the 1.8 μm fully porous materials, a marked increase in throughput is possible with elevated flow rates. Good resolution for a standard 16-component sample mixture is demonstrated in a sub-minute separation.

Journal of Pharmaceutical and Biomedical Analysis · 2019

Computer-assisted UHPLC–MS method development and optimization for the determination of 24 antineoplastic drugs used in hospital pharmacy

Nicolas Guichard, Szabolcs Fekete, Davy Guillarme, Pascal Bonnabry, Sandrine Fleury-Souverain

This study reports the use of retention modeling software for the successful method development of 24 injectable antineoplastic agents. Firstly, a generic screening of several stationary and mobile phases (using various organic modifiers and pH) was achieved. Then, an optimization procedure of mobile phase temperature, gradient profile and mobile phase binary composition was conducted through only 28 real experiments using retention modeling software for data treatment. Finally, the optimized separation was achieved with a mobile phase consisting in 10 mM acetic acid at pH 5.1 (A) and acetonitrile (B). A Waters CORTECS® T3 column (100 × 2.1 mm, 1.6 μm) operated at 25 °C with a gradient time of 17.5 min (0–51%B) at a flow rate of 0.4 mL/min was used. The prediction offered by the retention model was found to be highly reliable, with an average error lower than 1%. A robustness testing step was also assessed from a virtual experimental design. Success rate and regression coefficient were evaluated without the need to perform any real experiment. The developed LC–MS method was successfully applied to the analysis of pharmaceutical formulations and wiping samples from working environment.

Journal of Chromatography A · 2019

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Valentina D’Atri, Reinhard Pell, Adrian Clarke, Davy Guillarme, Szabolcs Fekete

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species.

Journal of Pharmaceutical and Biomedical Analysis · 2019

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation

Evelin Farsang, Amarande Murisier, Krisztián Horváth, Alain Beck, Róbert Kormány, Davy Guillarme, Szabolcs Fekete

Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl. This two buffer system provides an equivalent or slightly better separation than the standard, mobile phases for therapeutic mAbs. It was also demonstrated that working with salt-mediated pH gradients, allows to extend the possibilities in method development, since the concentration of salt in the mobile phase has a significant impact on selectivity. Using HPLC modeling software (Drylab), it was possible to successfully develop CEX methods for authentic mAb samples within only 6 h, by optimizing the gradient steepness and salt concentration in the B eluent.

Journal of Chromatography A · 2019

Development of a design of experiments optimized method for quantification of polysorbate 80 based on oleic acid using UHPLC-MS

Julia Puschmann, Dirk-H. Evers, Christel C. Müller-Goymann, Michael E. Herbig

The aim was to develop a straightforward UHPLC-MS quantification method for polysorbate 80 using oleic acid as surrogate marker, which was the commonest substance within the emulsifier. However, hydrolysis of polysorbate 80 and subsequent analysis of fatty acids revealed a co-elution of oleic acid and an isomer while all the other fatty acids were successfully separated by varying retention times and mass-to-charge ratios. For identification and separation of the isomer a derivatization method was evaluated. Oxidation to the corresponding dihydroxystearic acids with potassium permanganate resulted in peak separation of cis/trans and structural isomers of the 18:1 fatty acids. Hydrolyzed and derivatized polysorbate 80 was quantified indirectly in the range of 0.046–5.83 μg/mL (R2 > 0.997) with a limit of detection of 11.4 ng/mL. Quantification of polysorbate 80 using oleic acid as a surrogate marker showed good reproducibility and linearity. As all isomers of the 18:1 fatty acids were successfully separated, the previously co-eluting peak was identified as elaidic acid and was found as a component in the mixture of the emulsifier polysorbate 80. Additionally, cis-vaccenic acid was separated as a second co-eluting isomer. Therefore, derivatization led to successful chromatographical separation of cis/trans and structural 18:1 fatty acid isomers.

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

Software-assisted method development in high performance liquid chromatography · 2019

Modeling of HPLC Methods Using QbD Principles in HPLC

Szabolcs Fekete, Imre Molnár, Imre Molnár, Hans-Jürgen Rieger, Róbert Kormány

HPLC method modeling is becoming a powerful tool to be used in the communication about method quality in HPLC between different labs, different companies, and between companies and regulatory agencies. The understanding of simple rules of peak movements will facilitate the development of new drugs, which are badly needed for smaller patient populations. The new features of HPLC modeling software, such as 3D resolution map, the modeled robustness testing, a practicable method transfer, or a method knowledge management offer a closed loop of all information about the birth and practical use of a method, and it further suggests the use of such software solutions in regulated laboratories to make analyst's life easier-especially in the pharmaceutical industry.

Journal of Chromatography A · 2018

Structure assisted impurity profiling for rapid method development in liquid chromatography

Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria Kristina Parr

The use of trial-and-error principles is a frequently used technique in method development. This may lead to the fact that analytical methods are used routinely without developers and users having gained extensive and well-founded knowledge about the robustness of their analytical methods and the influence of critical key parameters. This very often leads to unnecessary problems for analysts. A simple way in reverse phase chromatography to simulate the effects of pH value changes on the separation and retention of substances is the pH-dependent calculation of the logD value. With this tool, model substances were used to show how the time requirement for method screening can be considerably reduced in silico and, in addition, extended knowledge about the separation mechanics can be generated. Based on this knowledge, a new method for the purity analysis of carbamazepine was developed within a very short period of time, which improves the performance of the official Ph.Eur. monograph by far. Furthermore, the extremely high robustness of the new method was demonstrated. Using the logD based approach, Quality-by-Design is applied in method development and kept pace with the increasing requirements of regulatory authorities in the pharmaceutical industry.

Food Chemistry · 2018

Identification of cis/trans isomers of menaquinone-7 in food as exemplified by dietary supplements

Arkadiusz Szterk, Adam Zmysłowski, Katarzyna Bus

For the first time, the cis/trans isomer content of menaquinone-7 in food products has been identified and marked. A novel method of marking isomers of vitamin K2MK-7 in dietary supplements was developed and validated. Five different isomers of cis/trans vitamin K2MK-7 were identified. Identification of cis/trans isomers was performed by HRMS-QTOF, whereas their quantities were determined by using CAD and DAD detectors. In the majority of cases, the content of biologically active all trans vitamin K2MK-7 was below its declared content. The content of all trans K2MK-7 was in the range between 5.5 and 49 µg in pills. In one of the studied supplements, this vitamin was not found, regardless of the claim on the product label. The content of cis/trans isomers in certain dietary supplements exceeded the content of all trans K2MK-7 by up to 3.7 times.

NPC Natural Product Communications · 2018

Optimization of Thin-layer Chromatography and High-Performance Liquid Chromatographic Method for Piper guineense Extracts

Eunice Ego Mgbeahuruike, Heikki Vuorela, Teijo Yrjönen, Yvonne Holm

In this study, a thin-layer chromatography (TLC) and a high performance liquid chromatographic (HPLC) methods were developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts. To obtain a chromatogram with satisfactory resolution and favorable retention time, DryLab software was used to simulate and optimize a HPLC method for the analysis of P. guineense extracts. The aim was to achieve the best possible overall resolution while keeping the analysis time and solvent consumption to a minimum. With the optimized method, a total of 16 main components in the extract were separated with favorable resolution. Optimal TLC conditions were also developed using solvents of various solvent strength (ST) and solvent selectivity (PS) values. The mobile phase composition was systematically tested using various proportions of solvents differing in ST and PS values under the same experimental conditions. During the optimization, emphasis was set on achieving the best possible overall separation of the main components of the extracts (for example piperine). In addition, the effects of the developing chamber was tested using three types of unsaturated chamber conditions: horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. During the study, a TLC method was developed, and the best mobile-phase composition giving favorable resolution of the bands was toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The developing chamber conditions did not affect the TLC separation efficacy in the analysis of P. guineense extracts. The HPLC method was applied to determine the percentage content of piperine in P. guineense. The piperine content was 0.43 % w/w, linearity (0.997), interday precision (% relative standard deviation (RSD), 1.6), intraday precision (% RSD, 2.7 – 5.9), recovery (98.4%), limit of detection (0.001 μg /mL) and limit of quantification (0.003 μg /mL).

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 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 Pharmaceutical and Biomedical Analysis · 2018

An overview of experimental designs in HPLC method development and validation

Prafulla Kumar Sahu, Nageswara Rao Ramisetti, Teresa Cecchi, Suryakanta Swain, Chandra Sekhar Patro, Jagadeesh Panda

Chemometric approaches have been increasingly viewed as precious complements to high performance liquid chromatographic practices, since a large number of variables can be simultaneously controlled to achieve the desired separations. Moreover, their applications may efficiently identify and optimize the significant factors to accomplish competent results through limited experimental trials. The present manuscript discusses usefulness of various chemometric approaches in high and ultra performance liquid chromatography for (i) methods development from dissolution studies and sample preparation to detection, considering the progressive substitution of traditional detectors with tandem mass spectrometry instruments and the importance of stability indicating assays (ii) method validation through screening and optimization designs. Choice of appropriate types of experimental designs so as to either screen the most influential factors or optimize the selected factors’ combination and the mathematical models in chemometry have been briefly recalled and the advantages of chemometric approaches have been emphasized. The evolution of the design of experiments to the Quality by Design paradigm for method development has been reviewed and the Six Sigma practice as a quality indicator in chromatography has been explained. Chemometric applications and various strategies in chromatographic separations have been described.

Journal of Chromatography A · 2018

Enantioselective multiple heartcut two-dimensional ultra-high-performance liquid chromatography method with a Coreshell chiral stationary phase in the second dimension for analysis of all proteinogenic amino acids in a single run

Ulrich Woiwode, Stefan Neubauer, Wolfgang Lindner, Stephan Buckenmaier, Michael Lämmerhofer

A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 μm C18 phase (100 × 2.1 mm ID column) in the first dimension (1D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 μm Coreshell particle column (50 × 3 mm ID) in the second dimension (2D). Pre-column derivatization has been performed with Sanger’s reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 μL fractions of the 1D peaks were sampled into the 2D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 μL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, β-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (1D and 2D). All 2D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in 2D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.

Journal of Chromatography A · 2018

Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and related products

Balázs Bobály, Matthew Lauber, Alain Beck, Davy Guillarme, Szabolcs Fekete

A wide-pore silica-based superficially porous material with a high coverage phenyl bonding was evaluated for the analysis of monoclonal antibodies and antibody-drug conjugates. This new material is based on 2.7 μm particles having a shell thickness of 0.40 μm and average pore size of approximately 450 Å. Various important features of this reversed phase column technology were explored, including kinetic performance for large biomolecules (i.e. speed of analysis, efficiency and peak capacity), recovery of proteins, selectivity for resolving modifications, and the possibility to reduce the amount of trifluoroacetic acid in the mobile phase. A systematic comparison was also performed with other existing modern wide-pore phases possessing differences in structure/morphology and chemistry. If all these figures of merit are considered, it is clear that this phenyl bonded wide-pore superficially porous stationary phase is one of the most promising materials to have been developed in recent years. Indeed, it offers kinetic performance comparable to the most efficient wide-pore SPP column on the market. In terms of protein recovery, this new phase was found to be superior to silica-based and silica-hybrid C4 bonded materials, particularly with separations performed at sub-80 °C temperature. Under such conditions, it in fact shows recoveries that are quite similar to a divinyl benzene (DVB) polymer-based material. More importantly, due to its unique, high coverage phenyl bonding, it offers additional steric effects and potentially even π-π interactions that yield advantageous selectivity for mAb sub-unit peaks and ADC species as compared to commonly used C4 or C18 bonded phases. Last but not least, mobile phases consisting of only 0.02–0.05% trifluoroacetic acid can be successfully used with this column, without significant loss in recovery and peak capacity.

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.

TrAC Trends in Analytical Chemistry · 2018

Critical review of reports on impurity and degradation product profiling in the last decade

Sándor Görög

Drug impurity and degradation profiling mean the detection, structure elucidation and quantitative determination of impurities and degradation products in bulk drug materials and pharmaceutical formulations. This is today one of the most important fields of activities in pharmaceutical analysis. The reason for this is that unidentified, potentially toxic impurities are health hazards, and in order to increase the safety of drug therapy, impurities should be identified and determined by selective methods. The aim of this review is to characterise the state-of-art in the field of impurity and degradation profiling of drugs based on papers published in the last decade. The separation and determination of impurities and degradants with a known structure are discussed, but emphasis is placed on the structure elucidation and determination of new (unknown) impurities and degradation products by off-line and on-line chromatographic–spectroscopic methods. The analytical aspects of enantiomeric purity of chiral drugs are also discussed.

Journal of Pharmaceutical and Biomedical Analysis · 2017

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Balázs Bobály, Valentina D’Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

Journal of Pharmaceutical and Biomedical Analysis · 2017

A workflow for column interchangeability in liquid chromatography using modeling software and quality-by-design principles

Róbert Kormány, Katalin Tamás, Davy Guillarme, Szabolcs Fekete

The goal of the present study was to develop a generic workflow to evaluate the chromatographic resolution in a large design space and easily find some replacement column for the method. To attain this objective from a limited number of initial experiments, modern LC modeling software (Drylab) was employed to study the behaviour of the compounds and visually compare the parts of design spaces obtained with different columns, where a given criterion of critical resolution is fullfilled. A zone of robust space can then easily be found by overlapping design spaces. By using 50×2.1mm columns packed with sub–2μm fully porous particles (UHPLC), the resolution in the entire design space can be modeled on the basis of only 2–3h experimental work per column. To demonstrate the applicability of the developed procedure, amlodipine and its related pharmacopeia impurities were selected as a case study. It was demonstrated that two columns from different providers (Waters Acquity HSS C18, Thermo Hypersil Gold C18) can be interchanged, providing a sufficient resolution at the same working point and a high degree of robustness around this condition.

Decades of chromatography research done with DryLab®.

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Rapid analysis of polycyclic aromatic hydrocarbons

Computer-assisted UHPLC–MS method development and optimization for the determination of 24 antineoplastic drugs used in hospital pharmacy

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation

Development of a design of experiments optimized method for quantification of polysorbate 80 based on oleic acid using UHPLC-MS

Software-assisted method development in high performance liquid chromatography

Modeling of HPLC Methods Using QbD Principles in HPLC

Structure assisted impurity profiling for rapid method development in liquid chromatography

Identification of cis/trans isomers of menaquinone-7 in food as exemplified by dietary supplements

Optimization of Thin-layer Chromatography and High-Performance Liquid Chromatographic Method for Piper guineense Extracts

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

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

An overview of experimental designs in HPLC method development and validation

Enantioselective multiple heartcut two-dimensional ultra-high-performance liquid chromatography method with a Coreshell chiral stationary phase in the second dimension for analysis of all proteinogenic amino acids in a single run

Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and related products

Automated UHPLC separation of 10 pharmaceutical compounds using software-modeling

Critical review of reports on impurity and degradation product profiling in the last decade

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

A workflow for column interchangeability in liquid chromatography using modeling software and quality-by-design principles