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...

Keyword Year

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.

Computer assisted liquid chromatographic method development for the separation of therapeutic proteins

Eva Tyteca, Jean-Luc Veuthey, Gert Desmet, Davy Guillarme, Szabolcs Fekete
Analyst (2016)

Keywords: Separation of Therapeutic Proteins and mAbs, Retention modeling, Biopharmaceuticals, RPLC, IEX, HIC


This review summarizes the use of computer assisted liquid chromatographic method development for the analytical characterization of protein biopharmaceuticals. Several modes of chromatography including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX), hydrophobic inter- action chromatography (HIC) and some perspectives are discussed. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Then, the retention models that were applied in the literature in RPLC, IEX and HIC are described and critically discussed. Finally, some representative examples of separation of therapeutic proteins and mAbs are shown, to illustrate the possibilities offered by the retention modeling approach. At the end, the reliability of the models was excellent, whatever the chromatographic mode, and the retention time prediction errors were systematically below 2%. In addition, a significant amount of time can be saved during method development and robustness testing.

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

Szabolcs Fekete, Jean-Luc Veuthey, Alain Beck, Davy Guillarme
J. Pharm. Biomed. Anal., 130, 25 October, 3-18 (2016)

Keywords: Hydrophobic interaction chromatography, Antibody-drug-conjugate, Therapeutic antibody, Method development, Columns, DryLab, Method modeling


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.


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