Literature

So far, more than 230 peer reviewed papers have been published on the application of DryLab – a complete list of which you can find here.

DryLab draws on the philosophy described in the three most famous Solvophobic Theory papers IIIIII of Csaba Horváth, which were developed in the years 1975-1977 at Yale University (see also literature by Dr. Imre Molnár).

Read more about the Fundamentals of DryLab and its History.

Keyword Year

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
J. Chromatogr. A, 1599, 136-143 (2019)

Keywords: Polysorbate 80, Design of experiments, Quantification, Oleic acid, UHPLC-MS Peak separation, Cis/trans separation

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http://https://doi.org/10.1016/j.chroma.2019.04...

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.


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

Evelin Farsang et. al
J Pharm Biomed Anal., 168, 10 May, 138-147 (2019)

Keywords: Cation-exchange chromatography, pH gradient, Salt gradient, Salt-mediated pH gradient, Monoclonal antibody, Method development

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http://doi.org/10.1016/j.jpba.2019.02.024

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.


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
J. Chromatogr. A, 1586, 8 February, 149-153 (2019)

Keywords: Site-specific antibody drug conjugates, Hydrophobic interaction chromatography, Reversed phase liquid chromatography, Mass spectrometry, Biopharmaceuticals

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http://doi.org/10.1016/j.chroma.2018.12.020

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.


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
J Pharm Biomed Anal., 164, 5 February, 395-401 (2019)

Keywords: Retention modeling, Antineoplastic agent, Drylab, Method development, Reversed phase liquid chromatography

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http://doi.org/10.1016/j.jpba.2018.11.014

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.


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

Sándor Görög
Trends Anal. Chem., 101, April 2018, 2-16 (2018)

Keywords: Impurity, Degradant, Chromatography, Spectroscopy, Hyphenated methods, Enantiomer

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http://doi.org/10.1016/j.trac.2017.09.012

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 toxicimpurities 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.


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

Szabolcs Fekete et. al
J Pharm Biomed Anal., 155, 5 June 2018, 116-124 (2018)

Keywords: Cannabinoids, Cannabis, Method development, Robustness test, DryLab, UHPLC

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http://doi.org/10.1016/j.jpba.2018.03.059

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativaextracts. 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.


Automated UHPLC separation of 10 pharmaceutical compounds using software-modeling

A.Zöldhegyi, H.-J.Rieger, I.Molnár, L.Fekhretdinova
J Pharm Biomed Anal., 156, 15 July, 379-388 (2018)

Keywords: Automated resolution modeling, In-silico method development, Reduction of human error in (U)HPLC, Data integrity, Time efficiency, DryLab

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http://doi.org/10.1016/j.jpba.2018.03.039

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.

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