Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography.

Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.

New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization.

Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.

Ultra-High-Pressure Ion Chromatography with Suppressed Conductivity Detection at 70 MPa Using Columns Packed with 2.5 µm Anion-Exchange Particles.

The use of ultrahigh pressures in combination with columns packed with 2.5 µm microporous and supermacroporous (perfusive) stationary phase particles coated with nanobeads has been successfully explored in ion chromatography with online eluent generation and suppressed conductivity detection. Isocratic separations of inorganic anions and organic acids yielding reduced plate heights as low as 2.1 were achieved, corresponding to efficiencies up to 190000 plates/m, using an optimized system configuration with respect to injection parameters, considering volume and mass loadability, and extra-column dispersion. Viscous-heating effects have been assessed for PEEK-lined stainless steel columns operated at 70 MPa, and effects of thermal gradients on separation efficiency and retention are demonstrated. Whereas the PEEK-lined column hardware acts to some extent as an insulator, a 10% increase in plate number could be obtained when applying a still-air column oven configuration. In the forced-air mode, an increase in retention was observed for polyvalent ions. Finally, the kinetic performance limits of ultrahigh-pressure ion chromatography applying 2.5 µm particle-packed columns operated at 70 MPa were compared to conventional ion-chromatography technology using columns packed with 4 µm particles operated at a maximum pressure of 35 MPa. Downscaling the particle size and increasing the operating pressure led to a maximum time gain with a factor of 3.4, without compromising separation efficiency (N = 10000).

Retention prediction using quantitative structure-retention relationships combined with the hydrophobic subtraction model in reversed-phase liquid chromatography.

The hydrophobic subtraction model (HSM) combined with quantitative structure-retention relationships (QSRR) methodology was utilized to predict retention times in reversed-phase liquid chromatography (RPLC). A selection of new analytes and new RPLC columns that had never been used in the QSRR modeling process were used to verify the proposed approach. This work is designed to facilitate early prediction of co-elution of analytes in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR models were constructed through partial least squares regression combined with a genetic algorithm (GA-PLS) which was employed as a feature selection method to choose the most informative molecular descriptors calculated using VolSurf+ software. The analyte hydrophobicity coefficient of the HSM was predicted for subsequent calculation of retention. Clustering approaches based on the local compound type and the local second dominant interaction were investigated to select the most appropriate training set of analytes from a larger database. Predicted retention times of five new compounds on five new RPLC C18 columns were compared with their measured retention times with percentage root-mean-square errors of 15.4 and 24.7 for the local compound type and local second dominant interaction clustering methods, respectively.

Retention Index Prediction Using Quantitative Structure-Retention Relationships for Improving Structure Identification in Nontargeted Metabolomics.

Structure identification in nontargeted metabolomics based on liquid-chromatography coupled to mass spectrometry (LC-MS) remains a significant challenge. Quantitative structure-retention relationship (QSRR) modeling is a technique capable of accelerating the structure identification of metabolites by predicting their retention, allowing false positives to be eliminated during the interpretation of metabolomics data. In this work, 191 compounds were grouped according to molecular weight and a QSRR study was carried out on the 34 resulting groups to eliminate false positives. Partial least squares (PLS) regression combined with a Genetic algorithm (GA) was applied to construct the linear QSRR models based on a variety of VolSurf+ molecular descriptors. A novel dual-filtering approach, which combines Tanimoto similarity (TS) searching as the primary filter and retention index (RI) similarity clustering as the secondary filter, was utilized to select compounds in training sets to derive the QSRR models yielding R2 of 0.8512 and an average root mean square error in prediction (RMSEP) of 8.45%. With a retention index filter expressed as ±2 standard deviations (SD) of the error, representative compounds were predicted with >91% accuracy, and for 53% of the groups (18/34), at least one false positive compound could be eliminated. The proposed strategy can thus narrow down the number of false positives to be assessed in nontargeted metabolomics.

Benchmarking of Computational Methods for Creation of Retention Models in Quantitative Structure-Retention Relationships Studies.

Quantitative structure-retention relationship (QSRR) models are powerful techniques for the prediction of retention times of analytes, where chromatographic retention parameters are correlated with molecular descriptors encoding chemical structures of analytes. Many QSRR models contain geometrical descriptors derived from the three-dimensional (3D) spatial coordinates of computationally predicted structures for the analytes. Therefore, it is sensible to calculate these structures correctly, as any error is likely to carry over to the resulting QSRR models. This study compares molecular modeling, semiempirical, and density functional methods (both B3LYP and M06) for structure optimization. Each of the calculations was performed in a vacuum, then repeated with solvent corrections for both acetonitrile and water. We also compared Natural Bond Orbital analysis with the Mulliken charge calculation method. The comparison of the examined computational methods for structure calculation shows that, possibly due to the error inherent in descriptor creation methods, a quick and inexpensive molecular modeling method of structure determination gives similar results to experiments where structures are optimized using an expensive and time-consuming level of computational theory. Also, for structures with low flexibility, vacuum or gas phase calculations are found to be as effective as those calculations with solvent corrections added.

Universal ion chromatography method for anions in active pharmaceutical ingredients enabled by computer-assisted separation modeling.

Ion Chromatography (IC) is one of the most widely used methods for analyzing ionic species in pharmaceutical samples. A universal IC method that can separate a wide range of different analytes is highly desired as it can save a lot of time for method development and validation processes. Herein we report the development of a universal method for anions in active pharmaceutical ingredients (APIs) using computer-assisted chromatography modeling tools. We have screened three different IC columns (Dionex IonPac AS28-Fast 4 µm, AS19 4 µm and AS11-HC 4 µm) to determine the best suitable column for universal IC method development. A universal IC method was then developed using an AS11-HC 4 µm column to separate 31 most common anionic substances in 36 mins. This method was optimized using LC Simulator and a model which precisely predicts the retention behavior of 31 anions was established. This model demonstrated an excellent match between predicted and experimental analyte retention time (R2 =0.999). To validate this universal IC method, we have studied the stability of sulfite and sulfide analytes in ambient conditions. The method was then validated for a subset of 29 anions using water and organic solvent/water binary solvents as diluents for commercial APIs. This universal IC method provides an efficient and simple way to separate and analyze common anions in APIs. In addition, the method development process combined with LC simulator modeling can be effectively used as a starting point during method development for other ions beyond those investigated in this study.

Hybrid grafted and hyperbranched anion exchangers for ion chromatography.

Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.

Capillary ion chromatography at high pressure and temperature.

The application of high pressure and temperature in ion chromatography (IC) can significantly improve the efficiency and reduce the analysis time. In this work, the kinetic-performance limits of capillary IC columns with inner diameters of 400 µm packed with 4 and 7 µm macroporous anion-exchange particles were investigated employing a capillary ion-exchange instrument allowing column pressures up to 34 MPa and column temperatures up to 80 °C. Plate heights below 10 µm could be realized using capillary columns packed with 4 µm particles. Compared to conventional IC using 7 µm particles and pressures up to 21 MPa, a 40% improvement in plate number could be achieved when working at the kinetic performance limits at 34 MPa and using columns packed with 4 µm particles. Using coupled columns with a total length of 400 mm, a mixture of seven anions was separated within 7.5 min while yielding 20,000 plates. Increasing the temperature improved the performance limits when operating in the C-term region (for fast IC separation using columns <75 cm). Temperature also affected the retention properties and hence the selectivity. At higher temperature, retention for monovalent ions was mainly governed by ion diameter. An increase in retention with temperature was observed for small ions, and there was a decrease for ions having a larger diameter. The retention factor for divalent and trivalent anions increased with temperature.

Performance evaluation of ion-exchange chromatography in capillary format.

The performance of a recently introduced capillary ion-exchange chromatography system was explored. Experiments were conducted in isocratic mode with a commercial capillary anion-exchange column (id = 0.4 mm, L = 15 cm) using a five-anion standard mixture. The achieved results were compared to the performance of a standard bore ion-exchange system (id = 4 mm, L = 15 cm), which was considered as a reference. The first-generation capillary columns exhibited a minimal reduced plate-height value below two witnessing a good packing quality and system performance. However, compared to the standard bore system the capillary system displayed an increased apparent C-term which could be due to a difference in packing morphology and/or possible external band-broadening contributions. For fast separations, the standard bore system outperformed the capillary system, while for complex separations both systems performed nearly equally well. In addition, the retention characteristics of the capillary system were investigated. To illustrate the suitability of the capillary system, the analysis of real-world water samples originating from two local Belgian rivers was demonstrated.

Ion chromatography on carbon clad zirconia modified by diazonium chemistry and functionalized latex particles.

This work explores the potential of 3 µm carbon coated zirconia particles as a stationary phase for ion chromatography for the separation of organic acids and inorganic ions. A 4-phenylsulfonic acid functionality is introduced onto the carbon surface by reducing 4-phenylsulfonic acid diazonium chloride with borohydride in the presence of carbon clad zirconia particles. The elemental sulfur analysis gave 132 µeq-SO(3)H/g carbon clad zirconia and 2% S atomic concentration by XPS analysis. The -SO(3)(-) groups serve as electrostatic anchors for latex nanoparticles bearing quaternary triethylamine functional groups. The agglomeration step in 5 × 0.4 cm i.d. columns converts the packed particles into an anion exchanger. The breakthrough curves with nitrate indicate a capacity of 3 µeq/column. Separation of common organic acids and inorganic ions using carbonate eluent and suppressed conductivity detection yield plate heights (H) of 0.023-0.05 mm.

Preparation of ion exchange columns with longitudinal stationary phase gradients.

Preparation of columns using electrostatic attachment of anion exchange latex particles with charge density gradients is demonstrated. When such columns are oriented with the highest charge density at the column outlet, the chromatographic performance at low linear velocity is enhanced. When multiple successive charge density gradients are prepared along the length of the column with the highest capacity oriented at the inlet end of the column, significant improvement in chromatographic performance is observed during gradient elution chromatography.

HILIC behavior of a reversed-phase/cation-exchange/anion-exchange trimode column.

The hydrophilic interaction liquid chromatography behavior of a novel trimodal stationary phase with reversed-phase/cation-exchange/anion-exchange characteristics was studied. This material is based on nanopolymer silica hybrid technology and provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It is constructed with high-purity porous spherical silica particles coated with charged organic polymer nano-beads (d(p) approximately 100 nm). The inner-pore area of the silica gel is covalently modified with an organic layer that provides both reversed-phase and weak anion-exchange properties. The outer surface is modified with strong cation-exchange functionality. This chemistry ensures distinctive spatial separation of the anion-exchange and cation-exchange regions, which allows both retention mechanisms to function simultaneously and provides more flexibility in selectivity control. The new phase exhibits hydrophilic interaction liquid chromatography characteristics: at high acetonitrile levels, the retention for charged analytes is governed by an ion-exchange process, accompanied by hydrophilic interaction (partitioning mechanism). This feature provides good flexibility in pharmaceutical method development such as simultaneous separation of hydrophilic drug molecule and its counterions (e.g. penicillin G potassium salt).

Localised quantitative structure-retention relationship modelling for rapid method development in reversed-phase high performance liquid chromatography.

Quantitative structure-retention relationships (QSRR) predicting the values of solute "hydrophobicity" coefficient η' in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol-water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η' values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η' values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η' values of the models and the stationary phase "hydrophobicity" parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η' cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.

Packing procedures for high efficiency, short ion-exchange columns for rapid separation of inorganic anions.

An optimised packing procedure for the production of high efficiency, short, particle-packed ion-exchange columns is reported. Slurry-packing techniques were applied to a series of interconnected short columns, with the columns situated intermediate between the inlet and outlet ends of the series being used for separations. The fast separation and determination of inorganic anions was achieved using short (4mm ID, 30mm long) columns packed with Dionex AS20 high-capacity anion-exchange stationary phase. Seven inorganic anions (bromate, chloride, chlorate, nitrate, sulfate, chromate and perchlorate) are separated in 2.6min using a hydroxide gradient and a flow-rate of 1.8mL/min (total analysis time including re-equilibration was 3.5min). Under isocratic conditions, the home-packed columns exhibited efficiency values of 43,000N/m for chloride at a flow-rate of 0.3mL/min, compared to 54,000N/m for a commercial 250mm AS20 column at the same flow-rate. However, the short columns gave approximately a threefold higher sample throughput. The short, home-packed columns could be produced reproducibly and gave consistent performance over extended periods of usage.

Column selection for comprehensive multidimensional ion chromatography.

This paper discusses the selection of ion chromatography (IC) columns for use in comprehensive multidimensional ion chromatography (IC x IC). First, a single number was determined for a wide range of anions (one number for each anion) using the linear solvent strength model. These numbers were then used to compare the column selectivity characteristics for five different columns. Principal component analysis was used to illustrate selectivity differences between columns. Dionex AS16 and AS20 columns were selected for use in the development of an IC x IC method for the separation of ten anions. To achieve the required speed of analysis in both the first and second separation dimensions, custom column lengths were packed in-house. The use of an eluent suppressor between the first and second columns permits a relatively low flow ratio regime of only <1:20 in the first and second dimensions, respectively, which reduces dilution effects common in comprehensive multidimensional LC. Selection of the second dimension eluent conditions was aided by the development of a spreadsheet based on the linear solvent strength model.

Separation of inorganic anions on a high capacity porous polymeric monolithic column and application to direct determination of anions in seawater.

A commercially available 4.6 mm id x 50 mm polymethacrylate-based monolithic strong anion exchange column (ProSwift SAX-1S) designed for the separation of proteins has been successfully used to separate small inorganic anions in the presence of a seawater sample matrix. Using a hydroxide eluent with suppressed conductivity detection the ion exchange capacity of this column declined over time; however, using KCl as the eluent, the column performance was stable with a capacity of 530 microequiv. for nitrate. The optimum conditions for the separation of iodate, bromate, nitrite, bromide and nitrate were assessed by constructing van Deemter plots using 1.00 and 0.100 M KCl. Efficiencies of up to 26 700 plates/m were recorded using 1.00 M KCl, at a flow rate of 0.20 mL/min but iodate was not baseline resolved from the void peak. By reducing the concentration of the eluent to 0.100 M, efficiencies of up to 39 900 plates/m could be obtained at 0.35 mL/min. By employing a linear gradient ranging from 0.05 to 1.00 M KCl the ions dissolved in distilled water or a salt water matrix could be baseline separated in less than 3 min at a flow rate of 2.50 mL/min.

Novel method for manipulation of anion-exchange selectivity by derivatizing hydroxyl groups in the proximity of quaternary nitrogen ion-exchange sites with glycidol.

A general approach for the manipulation of anion-exchange selectivity through derivatization of hydroxyl groups adjacent to quaternary anion-exchange sites with glycidol is described. Repetitive reactions with glycidol result in dramatic shifts in the retention of divalent anions. Unique selectivities are observed for specific divalent species resulting in shifts in elution order. Modification of anion-exchange materials with glycidol has a small effect on the selectivity of monovalent anions, but in some cases, significant shifts in selectivity are observed. Use of the synthetic approach for modification of commercially available ion-exchange materials is demonstrated.

Electrostatically attached highly branched anion-exchange phases derived from diamines and diepoxides.

A new synthesis approach for the preparation of highly branched anion exchange materials utilizing diamine and diepoxide reagents is described. Unlike previously reported condensation polymers prepared from primary amine and diepoxide reagents, anion exchange polymers prepared from diamines and diepoxide reagents exhibit exceptionally low affinity for polyvalent ions. Use of anion-exchange materials synthesized utilizing this new synthetic method for the analysis of common inorganic anions is demonstrated.