Decades of chromatography research done with DryLab®.

Journal of Pharmaceutical and Biomedical Analysis · 2012

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: theory and applications

Szabolcs Fekete, Jean-Luc Veuthey, Davy Guillarme

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

Journal of Chromatography · 1987

Predicting bandwidth in the high-performance liquid chromatographic separation of large biomolecules: II. A general model for the four common high-performance liquid chromatography methods

M. A. Stadalius, B. F. D. Ghrist, L. R. Snyder

A general model for describing gradient elution separations of peptides and proteins by reversed-phase high-performance liquid chromatography (HPLC) has been presented previously. This model has now been modified so that it can be applied to any of the four HPLC methods used for separating biological macromolecules: reversed-phase, ion-exchange, hydrophobic-interaction and size-exclusion chromatography, carried out in either an isocratic or gradient elution mode. The role of sample molecule structure and the particular column used has been further studied, so that previous empirical parameters for different column/sample choices can now be estimated from three physical properties of the sample and the column: (a) sample molecular weight, (b) native vs. denatured sample, (c) column packing pore diameter. This eliminates much of the empiricism of our preceding model, and minimizes the number of experimental runs now required in order to apply the model in practice. The final model has been tested for (a) several hundred runs involving peptides and proteins in the molecular weight range 600–162000, (b) all four of these HPLC methods, (c) in both isocratic and gradient elution modes, and (d) using data from several different laboratories (including our own). The model is able to predict bandwidth in HPLC separations of proteins and peptides with an accuracy of ±17% (1 standard deviation), for the case of “well-behaved” separations. Separations that are not “well-behaved” will give wider bands than predicted by the model.

Journal of Chromatography A · 2022

Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals

Simon Jaag, Chunmei Wen, Benjamin Peters, Michael Lämmerhofer

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

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 Chromatography Open · 2025

Weak to strong ion-pair gradients to expand the selectivity of oligonucleotide separations in reversed phase liquid chromatography - A proof of concept

Szabolcs Fekete, Mateusz Imiolek, Matthew Lauber

This study introduces a novel ion-pair reversed-phase liquid chromatography (IP-RPLC) method using a dual gradient approach to enhance the separation of oligonucleotides. By combining a weak ion-pairing (IP) agent in the starting (weaker) mobile phase with a strong IP agent in the final (stronger) mobile phase, this method widens the elution window, improving the resolution and selectivity for complex ON mixtures. The approach outperforms traditional single IP systems, particularly in separating sequence and size variants of ONs. Experimental results demonstrate that the weak-to-strong gradient yields better peak separation and higher selectivity compared to conventional methods. This innovative gradient strategy, combined with concave gradients, offers significant potential for optimizing ON separations in the development of oligonucleotide-based therapeutics.

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.

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 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.

CRC Press · 2021

Advances in Chromatography: Volume 57

Nelu Grinberg, Peter W. Carr

Chromatographia · 1987

Computer simulation as a means of developing an optimized reversed-phase gradient-elution separation

J. W. Dolan, L. R. Snyder, M. A. Quarry

A personal-computer program (DryLab G) is described for the simulation of reversed-phase gradient-elution separations. After two experimental gradient runs are carried out initially, this program allows the user to develop a final separation by varying gradient conditions (gradient time, initial and final %-organic in the mobile phase, gradient shape), column dimensions, flowrate and particle size. This approach takes advantage of “solvent-strength selectivity”, as reported recently [28] for isocratic separations. Method development using this procedure can result in better separations with much less effort. Examples of its validation and application are presented.

Analytical Chemistry · 1983

Gradient elution in reversed-phase HPLC-separation of macromolecules

L. R. Snyder, M. A. Stadalius, M. A. Quarry

Journal of Chromatography · 1985

Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography: Verification of band width relationships for acetonitrile-water mobile phases

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

A previously reported model describes retention and band width as functions of experimental conditions, for the reversed-phase gradient elution separation of peptides and proteins. The model begins by relating separation in gradient elution to corresponding separations by isocratic elution (same high-performance liquid chromatographic system). The use of certain semi-empirical relationships then allows band width to be predicted for samples for which isocratic data are unavailable. This in turn allows the prediction of band width, peak capacity and relative peak height (or peak volume) as a function of experimental conditions such as column dimensions, gradient conditions, mobile phase flow-rate, sample molecular weight, etc. The next paper [J. Chromatogr., 327 (1985) 93] demonstrates the practical value of this approach for optimizing the separation of peptide and protein mixtures. In the present paper it is first shown that isocratic data for desamido-insulin (6000 daltons) allow the accurate prediction of band widths as a function of experimental conditions in related gradient separations. It is further shown that the present model accurately predicts how band widths vary with experimental conditions for peptides and proteins having molecular weights in the range 600–80 000 daltons.

Journal of Chromatography A · 2013

Intergroup cross-comparison for the evaluation of data-interchangeability from various chromatographic tests

Chamseddin Chamseddin, Imre Molnár, Thomas Jira

The recently dramatic increase in the available choices of reversed-phase columns could be an advantage of this mode of separation. However, due to the insufficiency of available information in terms of the exact functionality of these phases and the similarities and differences between these newly introduced and conventional reversed-phase columns, it is now somehow problematic to determine which could be the best column for a given analytical problem. There is no single column that will give us a good separation for all applications. As a result, there have been several attempts to develop testing strategies to characterize column chemistries. In this study three of the most widely used and acceptable approaches for the characterization of reversed-phase columns, which are Tanaka, United States Pharmacopeia (USP), and Snyder–Dolan, are systemically applied to investigate the chromatographic properties of calixarene- and resorcinarene-bonded stationary phases, polar-embedded and polar-endcapped stationary phases, phenyl and ether-linked phenyl with the presence of conventional alkyl-bonded phases (octyl- and octadecylsilane). Although all column classification systems aim to evaluate “more or less” the same characteristics, each system uses different test mixtures in different chromatographic conditions. It is therefore very important to evaluate the similarities and differences in the resulted “column parameters” and the possible interchangeability of them. The results of this comparative study show that the used parameters of Tanaka and of Snyder–Dolan have in many cases a good to very good correlation. The USP approach, which is based on single run, is related to Tanaka and Snyder–Dolan only in terms of hydrophobic characters, and no relation could establish in the other parameters. The hydrophobic-subtraction model could be extended to describe the ligand–solute interactions of calixarene- and resorcinarene-bonded stationary phases, which are belonging to reversed phase material. However they show, depending on the analytes, some additional interactions, since their steric, polar and ionic properties are different compared to those of conventional alkyl-bonded phases.

Journal of Pharmaceutical and Biomedical Analysis · 2013

Comparison of supercritical fluid chromatography and reverse phase liquid chromatography for the impurity profiling of the antiretroviral drugs lamivudine/BMS-986001/efavirenz in a combination tablet

A. J. Alexander, L. Zhang, T. F. Hooker, F. P. Tomasella

Dual and triple combinations of antiretroviral drugs are a cornerstone of human immunodeficiency virus type 1 (HIV-1) treatment. Supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) methods have been developed for the impurity profiling of a prototype combination tablet containing three such drugs: lamivudine, BMS-986001 and efavirenz. Separation by SFC was achieved using a Princeton 2-ethyl pyridine stationary phase and a mobile phase B consisting of methanol with 10mM ammonium acetate and 0.1% isopropyl amine. This combination of mobile phase additives was required for both the separation of minor components and to minimize peak tailing of the active pharmaceutical ingredients (APIs). Separation by RPLC was achieved using a Discovery HSF5 stationary phase and a mobile phase consisting of 10mM ammonium acetate, pH 5.5 and methanol. Mobile phase gradient elution was employed in each case to elute components with a wide range of polarities. Both these methods were found to have advantages and disadvantages. Out of the three APIs and 13 possible impurity/degradation products selected, all were resolved by RPLC. By SFC, 15 peaks were resolved with one co-eluting pair and a high degree of orthogonality was achieved relative to RPLC. A more even distribution of peaks across the separation space, a non-sloping baseline and fewer system peaks were significant advantages associated with the SFC method. Particular attention had to be paid to optimizing the reverse phase diluent strength/initial mobile phase composition to avoid distortion of the peak shapes for early eluting components. This was not an issue with SFC, as the diluent of choice (methanol) was also the solvent of choice (in combination with ≤20% water) for the dissolution of the triple combination tablet. As with RPLC, SFC was found to exhibit the required sensitivity for successful quantitation of potential impurities/degradation products at the 0.05–0.1 area% level.

Wiley · 2010

Equipment

Lloyd R. Snyder, J. J. Kirkland, John W. Dolan

Journal of Chromatography A · 1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: I. Peak capacity limitations

J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, T. J. Waeghe

The separation of samples that contain more than 15 to 20 analytes (n>15–20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8≤n≤48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

Journal of Pharmaceutical and Biomedical Analysis · 2012

Analysis of recombinant monoclonal antibodies by RPLC: Toward a generic method development approach

Szabolcs Fekete, Serge Rudaz, Jenő Fekete, Davy Guillarme

Monoclonal antibodies (mAbs) are an emerging class of therapeutic agents that have recently gained importance. To attain acceptable kinetic performance with mAbs in reversed phase liquid chromatography, there is a need to work with the last generation of wide-pore sub-2μm fully porous or core–shell particles stationary phases. In addition, temperature in the range 60–90°C was found to be mandatory to limit adsorption phenomenon of mAbs and their fragments. A generic method development strategy was proposed to account for the selectivity, efficiency, recovery, and the possible thermal degradation. This study also demonstrated that the gradient steepness and temperature cannot be optimized using van’t Hoff type linear models. Similarly, the common linear solvent strength model also generated some error in predicting the retention times. In contrast, when quadratic models were employed, the prediction accuracy of retention times was found to be excellent (relative error between 0.5 and 1%) using a reasonable number of experiments (9 or 6 experiments for optimization of gradient time and temperature, which requires between 6 and 8h). Two separations of mAbs fragments were performed to demonstrate the reliability of the quadratic approach.

Journal of Chromatography A · 1994

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples: I. Optimizing the separation of a growth hormone tryptic digest

W. S. Hancock, Rosanne C. Chloupek, J. J. Kirkland, L. R. Snyder

Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested tor varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.

Analyst · 2016

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

Eva Tyteca, Jean-Luc Veuthey, Gert Desmet, Davy Guillarme, Szabolcs Fekete

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 interaction 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.

Journal of Chromatography A · 2015

Possibilities of retention prediction in fast gradient liquid chromatography. Part 3: Short silica monolithic columns

Pavel Jandera, Tomáš Hájek

We studied possibilities of prediction of the gradient elution data for alkylbenzenes, flavones and phenolic acids on two short octadecyl silica gel monolithic columns, namely a Chromolith Flash C18, 25×4.6mm, and a “new generation” Chromolith High Resolution C18, 50×4.6mm, in fast 1−2min gradients. With fixed short gradient times and varying gradient ranges of acetonitrile concentration in water, high flow rates of the mobile phase (3−5mL/min) could be used. The gradient elution data were predicted from four gradient models based on two-parameter and three-parameter isocratic retention equations. Various gradient retention models can be used for prediction of chromatograms and optimization of separation within a fixed gradient time. A two-parameter log−log model introduced in 1974 and a three-parameter model introduced in 1980 provided slightly more accurate prediction than the Linear Solvent Strength (LSS) semi-logarithmic two-parameter model, most frequently used in reversed-phase LC. A three-parameter model introduced in 1978 provided slightly improved accuracy of prediction of gradient data with respect to two-parameter models, in contrast to another, more recent three-parameter empirical model introduced in 2010 (which failed for gradients starting at a non-zero concentration of acetonitrile). Both a longer (5cm) and more efficient Chromolith HR column and a shorter (2.5cm) slightly less efficient Chromolith Flash column provide useful separations in fast gradients (1−2min) at high flow rates (3.5−5mL/min), especially in second dimension of two-dimensional LC×LC, in combination with HILIC separation on monolithic microcolumn in D1.