Fouling of virus filtration membranes by monoclonal antibody feeds with low aggregate content.

Membrane fouling by monoclonal antibodies (mAbs) is one of the main challenges in virus-filtration processes. Previous publications attributed membrane fouling to the presence of mAb aggregates in the solution, which block the membrane pores. This fouling mechanism can be solved by a prefilter; however, it was shown that there are mAbs that severely foul the membranes (reduce permeability by 90% and more) even after prefiltering the aggregates, while other mAbs foul the membrane weakly (reduce permeability by ~10% and less). Unfortunately, the differences between the fouling- and the nonfouling mAbs have never been convincingly explained. To get a deeper insight on these differences, we measured the fouling of chemically modified Isoprene-Styrene-4-vinylpyridine (ISV) membranes (TeraPore Technologies) by 8 mAbs exhibiting different hydrophobicity and charge. The results show that mAb solutions with low concentration of aggregates foul ISV membranes via an adsorptive mechanism, and the adsorption is driven mainly by hydrophobic forces between the mAb and the membrane. The charge of the mAbs plays a secondary role in fouling. We want to emphasize that the conclusions pertain to ISV membranes; the insights presented in this paper can potentially be used to engineer new surface chemistries to mitigate fouling of other virus-filtration and/or ultrafiltration membranes.

High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N-Glycome of a Recombinant Human Erythropoietin.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry.

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 µg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

Microfluidic membrane suppressor module design and evaluation for capillary ion chromatography.

A microfluidic ion-suppression module for use in ion-exchange chromatography has been developed and evaluated. The device consists of an ion-exchange membrane clamped between two polymer chips featuring a 200×100µm (width×depth) eluent channel (l=60mm), and a 300×150µm regenerant channel (60mm), respectively. The suppression efficacy using a Nafion membrane was compared with that of a styrene-sulfonate grafted fluorinated ethylene propylene (FEP) membrane. The latter was found to outperform Nafion in terms of lowest attainable background signal (suppression efficacy) and dynamic suppression range. Increasing the suppressor temperature or the sulfuric acid regenerant concentration led to an extension of the operational suppression range, this however at the cost of an increased background signal due to enhanced diffusion, inducing sulfate bleed. Under optimized operating conditions, the microfluidic suppressor provided a dynamic capacity of 0.35µEq./min, being compatible with gradient separations applying up to 70mM KOH in combination with 400µm i.d. capillary columns operated at the optimal flow velocity. The applicability of the miniaturized suppressor is demonstrated for both isocratic and gradient separations of mixtures of inorganic anions. Band-broadening characteristics of the suppressor were optimized with respect to a commercial capillary hollow-fiber suppressor, yielding comparable overall system efficiency, e.g., 8500 plates for nitrate recorded on a 150mm long capillary column. A second chip device was also constructed, featuring suppression at both sides of the eluent flow path. This double-sided suppressor allowed to increase sample throughput and operate at eluent flow rates of 10µL/min, while maintaining efficient suppression characteristics.

Using contemporary liquid chromatography theory and technology to improve capillary gradient ion-exchange separations.

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 µm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion.

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.

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.

Reversed-phase monoliths prepared by UV polymerization of divinylbenzene.

Many different techniques have been developed to prepare monolithic materials specifically for chromatographic techniques. The two most popular polymerization techniques being thermal or via ultra violet (UV) light. Whereas thermal polymerization is easily employed for a whole variety of monomer and porogen systems, UV polymerization has been limited to methacrylate-based systems, and styrenic systems have been avoided due to their strong absorbance at low wavelengths. By careful consideration of wavelength, initiator and other system components, it was proven that reversed-phase columns for the separation of proteins and peptides can be prepared using divinylbenzene through UV initiation of 2-methyl-4'-(methylthio)-2-morpholinopropiophenone at a wavelength of 350 nm.