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.

Digital light processing 3D printing of microfluidic devices targeting high-pressure liquid-phase separations.

This paper focuses on 3D printing using digital light processing (DLP) to create microchannel devices with inner diameters of 100, 200, and 500 µm and cater flow-through applications within the realm of analytical chemistry, in particular high-pressure liquid chromatographic separations. Effects of layer thickness and exposure time on channel dimensions and surface roughness were systematically investigated. Utilizing a commercially accessible 3D printer and acrylate resin formulation, we fabricated 100-500 µm i.d. squared and circular channel designs minimizing average surface roughness (< 20%) by applying a 20-µm layer thickness and exposure times ranging from 1.1 to 0.7 s. Pressure resistance was measured by encasing microdevices in an aluminum chip holder that integrated flat-bottom polyetheretherketon (PEEK) nanoports allowing to establish the micro-to-macro interface to the HPLC instrument. After thermal post-curing and finetuning the clamping force of the chip holder, a maximum pressure resistance of 650 bar (1.5% RSD) was reached (n = 3). A polymer monolithic support structure was successfully synthesized in situ with the confines of a 500 µm i.d. 3D printed microchannel. A proof-of-concept of a reversed-phase chromatographic gradient separation of intact proteins is demonstrated using an aqueous-organic mobile-phase with isopropanol as organic modifier.

Minimize Precolumn Band Broadening with Immiscible Solvent Sandwich Injection.

We investigated the possibility of reducing the effect of precolumn band broadening (PreCBB) by sandwiching the sample between two small plugs of an immiscible liquid. It has been found that in cases of severe PreCBB, improvements in peak efficiency can amount up to 20 times for the early-eluting compounds. For smaller degrees of PreCBB, the gain on the efficiency of early-eluting compounds is smaller (order of 50%), yet it is still significant. It has been verified that the presence of the immiscible fluid sandwich does not affect the repeatability of the analysis nor the linearity of the calibration curves used for analyte quantitation. It is also shown that the main effect of the two sandwich plugs is the minimization of the dispersion in the precolumn transfer tubing itself, which makes the method fundamentally different from pure on-column focusing methods such as the performance optimizing injection sequence (POISe) method. It is further demonstrated that both halves of the sandwich are needed, since the beneficial effect is clearly much smaller when only one plug is present. A drawback of the method is that some of the late-eluting peaks are slightly adversely affected by the presence of the sandwich liquid in the case where 127 µm i.d. tubing was used. The mechanism for this peak deterioration effect is at present still unclear but only occurs under gradient conditions and is clearly linked to the size of the sandwich plugs (the smaller the plugs, the smaller the adverse effect) and the internal diameter of the tubing used between the injection valve and the column.

Removal of Low Trace ppb-Level Perfluorooctanesulfonic Acid (PFOS) with ZIF-8 Coatings Involving Adsorbent Degradation.

For the first time, low trace-level removal of perfluorooctanesulfonic acid (PFOS), i.e., 20-500 µg/L (ppb), from aqueous solutions using zeolitic imidazolate framework-8 (ZIF-8)-coated copper sheet (ZIF-8@Cu) composite is reported here. In comparison with different commercial activated carbon (AC) and all-silica zeolites, the composite showed the highest removal rate of 98%, which remained consistent over a wide range of concentrations. Additionally, no adsorbent leaching from the composite was noticed, which eradicated pre-analysis steps such as filtration and centrifugation, unless needed for other adsorbents studied here. The composite displayed fast uptake with saturation reaching within 4 h, irrespective of the initial concentration. However, the morphological and structural characterization revealed surface degradation of ZIF-8 crystals, along with a decline in the crystal size. The adsorption of PFOS on ZIF-8 crystals was linked to chemisorption, as the surface degradation surges with an increase in PFOS concentration or with cyclic exposure at low concentrations. Methanol seemingly removed surface debris (partially), thus providing access to ZIF-8 beneath the surface debris. Overall, the findings demonstrate that at low trace ppb-level PFOS concentrations ZIF-8 can be considered as a possible candidate for PFOS removal, even though it suffers slow surface degradation, it also removes efficiently PFOS molecules from aqueous solutions.

Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography.

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.] = 0.9%), peak widths (c.v. = 4.7%), and column pressures (c.v. = 3.1%) across the batches.

Design Guidelines and Kinetic Performance Limits for Spatial Comprehensive Three-Dimensional Chromatography for the Analysis of Intact Proteins.

The design aspects of microfluidic chips for spatial three-dimensional chromatography featuring an interconnected channel network and targeting protein analysis are discussed, and the corresponding kinetic performance limits have been established using a Pareto-optimality approach. The pros and cons to integrate different separation mechanisms (IEF, CE, SEC, RPLC, HILIC, HIC, and IEX) are discussed considering development stages in the spatial domain (xLC) in the first and second dimension and time domain (tLC) for the third dimension. Based on Pareto-optimization, we discuss the considerations of the channel length, particle diameter, and the effect of number of second- and third-dimension channels on the resulting peak capacity of a spatial xIEF × xSEC × tRPLC device. Novel equations are proposed to determine the peak capacity in xSEC and to account for sample modulation affected by the number of second- and third-dimension channels. The corresponding Pareto fronts have been constructed demonstrating the resolving power, in terms of peak capacity and analysis time, considering current state-of-the-art prototyping methodologies. A microfluidic spatial prototype chip with an integrated channel layout (64 2D and 4096 3D channels) has been created, which has the potential to yield a peak capacity of 32,600 within only 44 min of the total analysis time, by implementing xIEF × xSEC × tRPLC separation stages.

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins.

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.

Prototyping of a Microfluidic Modulator Chip and Its Application in Heart-Cut Strong-Cation-Exchange-Reversed-Phase Liquid Chromatography Coupled to Nanoelectrospray Mass Spectrometry for Targeted Proteomics.

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.

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).

Resolving power in liquid chromatography: A trade-off between efficiency and analysis time.

This review describes chromatographic dispersion and different plate-height models frequently used to assess the chromatographic performance of ultra-high-pressure liquid chromatography column technology. Furthermore, different performance indices, including the resolution, the separation impedance, and kinetic plots are discussed allowing to quantify and visualize the resolving power in liquid chromatography. The construction of kinetic plots is explained, and different visualization approaches are highlighted. Finally, key instrument and column-technology developments to advance the kinetic performance limits are discussed and selected state-of-the-art applications are highlighted.

Guidelines for tuning the macropore structure of monolithic columns for high-performance liquid chromatography.

The ability to control the external porosity and to tune the dimensions of the macropore size on multiple length scales provides the possibility of tailoring the monolithic support structure towards separation performance. This paper discusses the properties of conventional polymer-monolithic stationary phases and its limitations regarding the effects of morphology on kinetic performance. Furthermore, guidelines to improve the macropore structure are discussed. The optimal monolithic macropore structure is characterized by high external porosity (while maintaining ultra-high-pressure stability), high structure homogeneity, polymer globule clusters in the submicron range, and macropores with a diameter tuned toward speed (small diameter in the 100-500 nm range using short beds) or efficiency (larger macropores in the range of 500 nm-1 µm allowing the use of longer column formats). Finally, promising approaches to control the morphology are discussed.

Advances in native high-performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products.

The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state-of-the-art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid-chromatographic modes that are discussed include aqueous size-exclusion chromatography, hydrophobic interaction chromatography, and ion-exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid-chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody-drug conjugates is discussed.

Generic approach to the method development of intact protein separations using hydrophobic interaction chromatography.

We describe a liquid chromatography method development approach for the separation of intact proteins using hydrophobic interaction chromatography. First, protein retention was determined as function of the salt concentration by isocratic measurements and modeled using linear regression. The error between measured and predicted retention factors was studied while varying gradient time (between 15 and 120 min) and gradient starting conditions, and ranged between 2 and 15%. To reduce the time needed to develop optimized gradient methods for hydrophobic interaction chromatography separations, retention-time estimations were also assessed based on two gradient scouting runs, resulting in significantly improved retention-time predictions (average error < 2.5%) when varying gradient time. When starting the scouting gradient at lower salt concentrations (stronger eluent), retention time prediction became inaccurate in contrast to predictions based on isocratic runs. Application of three scouting runs and a nonlinear model, incorporating the effects of gradient duration and mobile-phase composition at the start of the gradient, provides accurate results (improved fitting compared to the linear solvent-strength model) with an average error of 1.0% and maximum deviation of -8.3%. Finally, gradient scouting runs and retention-time modeling have been applied for the optimization of a critical-pair protein isoform separation encountered in a biotechnological sample.

Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica Columns.

We report on a study wherein we synthesized TMOS-based silica monolithic skeletons in capillaries with an i.d. of 5 and 10 µm to produce skeleton structures with very low capillary-to-domain size aspect-ratios. These structures include the absolute minimal aspect-ratio case of a monolithic structure whose cross-section only contains a single node point. With domain-sized based reduced plate heights running as low as hmin = 1.3-1.5 for retained coumarin dyes providing a retention factor of k = 0.6-1.0, the study confirms the classic observation that ultralow aspect ratio columns generate a markedly lower dispersion than columns with a larger aspect ratio made in the past by Knox, Jorgenson, and Kennedy for the packed bed of spheres, but now for silica monoliths. The course of the reduced van Deemter curves, and more specifically the ratio of A-term versus C-term band broadening, could be interpreted in terms of the width and persistence length of the velocity bias zones in the columns. Considering the overall kinetic performance, it is found that the two best performing structures are also the structures with the lowest number of domains or node points, that is, with the lowest capillary-to-domain size aspect-ratio and, hence, resembling closest to the open-tubular format, which remains confirmed as the column format with the best kinetic performance. This is quantified by the fact that the minimal impedance values (order of Emin = 100) of the best performing ultralow aspect ratio monolithic columns are still significantly larger than the Emin values for the reference open-tubular columns (order of Emin = 15-20).

Very High Efficiency Porous Silica Layer Open-Tubular Capillary Columns Produced via in-Column Sol-Gel Processing.

It is demonstrated that 5 µm i.d. capillaries can be coated with mesoporous silica layers up to 550 nm thickness. All the columns produced using in-column sol-gel synthesis with tetramethoxysilane provide plate height curves that closely follow the Golay-Aris theory. In 60 cm long columns, efficiencies as high as N = 150 000 and N = 120 000 were obtained, respectively, for a 300 and 550 nm thick porous layer. An excellent retention and plate height reproducibility was obtained when the recipes were subsequently applied to produce very long (1.9 and 2.5 m) capillaries. These columns produced efficiencies up to N = 600 000 plates for a retained and around N = 1 000 000 plates for an unretained component. Given the good reproducibility on the long capillaries, and considering that mesoporous silica is still the preferred support for LC, it is believed the present study could spur a renewed interest in open-tubular LC.

Applicability of linear and nonlinear retention-time models for reversed-phase liquid chromatography separations of small molecules, peptides, and intact proteins.

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).

Aqueous size-exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency.

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 µm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 µm particles are discussed.

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography.

Poly(styrene-co-divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free-radical copolymerization in capillary columns. The morphology was investigated at the meso- and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 µm, respectively. Subsequently, nano-liquid chromatography experiments were performed on 200 µm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small-domain-size monoliths feature a relatively narrow macropore-size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy-dispersion contribution to band broadening. The small-domain size monolith also has a relatively steep mass-transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas-adsorption techniques combined with the non-local-density-function-theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene-based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.