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

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

Szabolcs Fekete, Imre Molnár, Davy Guillarme
J Pharm Biomed Anal., 137, 15 April, 60-69 (2017)

Keywords: Retention modeling, Antibody drug conjugate, Brentuximab vedotin, Method development, DryLab, Reversed phase liquid chromatography


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.

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

Róbert Kormány, Imre Molnár, Jenő Fekete
J Pharm Biomed Anal., 135, 20 Februar, 8–15 (2017)

Keywords: Terazosin, Design of experiments, Quality by design, DryLab, HPLC, UHPLC, QDa detector, Method development, Method modeling


An older method for terazosin was reworked in order to reduce the analysis time from 90 min (2 × 45 min) to below 5 min. 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. 0.5 s and there was no difference between the predicted and measured Rs,crit −values.

Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates

Balázs Bobály, Giuseppe Marco Randazzo, Serge Rudaz, Davy Guillarme, Szabolcs Fekete
J. Chromatogr. A, 1481, 20 January, 82-91 (2017)

Keywords: Hydrophobic interaction chromatography, Antibody-drug-conjugate, Method development, Retention modeling, Non-linear gradient, DryLab, Method modeling


The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC. Another important advantage of the logarithmic gradient, is its peak focusing effect for the DAR0 species, which is particularly useful to improve the quantitation limit of DAR0.

Finally, the logarithmic behavior of DAR species of ADC in HIC was modelled using two different approaches, based on i) the linear solvent strength theory (LSS) and two scouting linear gradients and ii) a new derived equation and two logarithmic scouting gradients. In both cases, the retention predictions were excellent and systematically below 3% compared to the experimental values.


In-silico optimisation of two-dimensional high performance liquid chromatography for the determination of Australian methamphetamine seizure samples

Luke M. Andrighetto et. al
Forensic Sci. Int., 266, September, 511-516 (2016)

Keywords: DryLab, 2D-HPLC, Ephedrine, Methamphetamine, HPLC method modeling


In-silico optimisation of a two-dimensional high performance liquid chromatography (2D-HPLC) separation protocol has been developed for the interrogation of methamphetamine samples including model, real world seizure, and laboratory synthesised samples. The protocol used DryLab® software to rapidly identify the optimum separation conditions from a library of chromatography columns. The optimum separation space was provided by the Phenomonex Kinetex PFP column (first dimension) and an Agilent Poroshell 120 EC-C18 column (second dimension). To facilitate a rapid 2D-HPLC analysis the particle packed C18 column was replaced with a Phenomenex Onyx Monolithic C18 withought sacrificing separation performance. The DryLab® optimized and experimental separations matched very closely, highlighting the robust nature of HPLC simulations. The chemical information gained from an intermediate methamphetamine sample was significant and complimented that generated from a pure seizure sample. The influence of the two-dimensional separation on the analytical figures of merit was also investigated. The limits of detection for key analytes in the second dimension determined for methamphetamine (4.59 x 10-4 M), pseudoephedrine (4.03 x 10-4 M), caffeine (5.16 x 10-4 M), aspirin (9.32 x 10-4 M), paracetamol (5.93 x 10-4 M) and procaine (2.02 x 10-4 M).


A platform analytical quality by design (AQbD) approach for multiple UHPLC-UV and UHPLC–MS methods development for protein analysis

Jianmei Kochling, Wei Wu, Yimin Hua, Qian Guan, Juan Castaneda-Merced
J Pharm Biomed Anal., 125, 130-139 (2016)

Keywords: Analytical quality by design; Method development; Robustness; Design of experiments; Statistical analysis; Platform approach for multiple methods


A platform analytical quality by design approach for methods development is presented in this paper. This approach is not limited just to method development following the same logical Analytical quality by design (AQbD) process, it is also exploited across a range of applications in method development with commonality in equipment and procedures. As demonstrated by the development process of 3 methods, the systematic approach strategy offers a thorough understanding of the method scientific strength. The knowledge gained from the UHPLC-UV peptide mapping method can be easily transferred to the UHPLC–MS oxidation method and the UHPLC-UV C-terminal heterogeneity methods of the same protein.

Using the DryLab simulation, the cost saving was tremendous. It shortened the method development time from the typical 1-3 months to about 1 week. Importantly, the unknown factors in chromatography became more predictable. The direct cost saving involved labor, consumable, and instrument time. Even more significant were the lateral benefits, as the productivity of scientists could be increased by multiple factors.

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
J Pharm Biomed Anal., 118, 25 January, 393-403 (2016),

Keywords: Hydrophobic interaction chromatography, Monoclonal antibody, Antibody-drug-conjugate, Method development, Brentuximab-vedotin


The goal of this work is to provide some recommendations for method development in HIC using mon- oclonal 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 soft- ware, 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%.

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
J Pharm Biomed Anal., 121, 20 March, 161–173 (2016), doi:10.1016/j.jpba.2016.01.037

Keywords: Hydrophobic interaction chromatography, Monoclonal antibody, Antibody-drug-conjugate, Method development, Brentuximab-vedotin


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.

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