Literature | MOLNÁR-INSTITUTE

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 Chromatography A · 1998

Maintaining fixed band spacing when changing column dimensions in gradient elution

J. W Dolan, L. R Snyder

In gradient elution separations, it may be required to change either column length (to increase resolution or shorten run time) or column diameter (for an increase in sensitivity or for preparative separations). In either of these changes of column dimensions, it is usually desired to maintain the same relative band spacing (selectivity), so as to increase resolution in proportion to (column plate number)1/2 when increasing column length, or to maintain constant resolution when changing column diameter. A general rule for avoiding changes in band spacing in these situations is to maintain the quantity [(gradient time)×(flow-rate)/(column volume)] constant, while holding the initial and final gradient mobile phase compositions (%B) fixed. This rule is only valid as long as the equipment hold-up volume (dwell volume) is negligible, or if all sample components are strongly retained at the start of the gradient. When neither of the latter conditions apply, then significant changes in band spacing may result when changing column size. Rules are presented for recognizing this potential problem for a given sample/HPLC-equipment combination, and adjustments in separation conditions that can avoid this problem are discussed. Changes in band spacing as a result of change in column size are of special concern when developing procedures for preparative chromatography under gradient conditions.

Journal of Chromatography A · 1996

Combined use of temperature and solvent strength in reversed-phase gradient elution III. Selectivity for ionizable samples as a function of sample type and pH

P. L. Zhu, John W. Dolan, Lloyd R. Snyder, D. W. Hill, L. Van Heukelem, T. J. Waeghe

The ability of temperature and gradient steepness to change band spacing has been investigated for several ionizable samples that include 8 substituted benzoic acids, 9 substituted anilines, 22 basic drugs, 9 structurally-related herbicide impurities, 7 chlorophylls and 72 peptides and proteins. Mobile phase pH was also varied to determine the effect of sample ionization on temperature and gradient-steepness selectivity.

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.

TrAC Trends in Analytical Chemistry · 2017

Current challenges and future prospects in chromatographic method development for pharmaceutical research

Frederick T. Mattrey, Alexey A. Makarov, Erik L. Regalado, Frank Bernardoni, Margaret Figus, Michael B. Hicks, Jinjian Zheng, Lin Wang, Wes Schafer, Vincent Antonucci, Simon E. Hamilton, Kerstin Zawatzky, Christopher J. Welch

A survey of different strategies for chromatographic method development in pharmaceutical research and development is presented. Owing to the widespread utilization of chromatography within diverse areas of pharmaceutical research, a variety of strategies for method development have arisen. We survey the current state of the art, discuss recent trends and approaches and highlight future prospects and capability gaps.

Journal of Pharmaceutical and Biomedical Analysis · 2016

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

The goal of this work is to provide some recommendations for method development in HIC using monoclonal 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 software, 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%.

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.

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

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 Liquid Chromatography · 1987

Computer Simulation in HPLC Method Development. Reducing the Error of Predicted Retention Times

L. R. Snyder, M. A. Quarry

Computer simulation uses two experimental HPLC runs to allow prediction of sample retention as a function of mobile phase composition or gradient conditions. The general approach is rigorous, but it is assumed that reversed-phase retention obeys the relationship log k' = log kw - S 0. Small deviations in this relationship can lead to error in predicted retention times. We have studied this error empirically for several different reversed-phase systems. This provides a basis for partially correcting these errors, and suggests recommendations for avoiding significant errors during computer simulation.

Decades of chromatography research done with DryLab®.