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• Theoretical evaluation of protein separations by simultaneous independent pH & NaCl gradients. • Experimental demonstration of two dimensional LC pH & NaCl gradients. • Isocratic [NaCl] in pH gradients and vice versa vary MAb isoform retention times in WCX LC. • Separation of proteins by uncoupled pH and [NaCl] gradients enhances & improves resolution. • No one single set of uncoupled salt and pH gradients optimizes the resolution of all MAb isoforms. In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG 1 (HIgG 1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins. [ABSTRACT FROM AUTHOR]

In this work the relation between physical and chemical resin characteristics and the total amount of adsorbed protein (saturation capacity) for ion-exchange resins is discussed. Eleven different packing materials with a sulfo-functionalization and one multimodal resin were analyzed in terms of their porosity, pore size distribution, ligand density and binding capacity. By specifying the ligand density and binding capacity by the total and accessible surface area, two different groups of resins were identified: Below a ligand density of approx. 2.5 μmol/m 2 area the ligand density controls the saturation capacity, while above this limit the accessible surface area becomes the limiting factor. This results in a maximum protein uptake of around 2.5 mg/m 2 of accessible surface area. The obtained results allow estimating the saturation capacity from independent resin characteristics like the saturation capacity mainly depends on “library data” such as the accessible and total surface area and the charge density. Hence these results give an insight into the fundamentals of protein adsorption and help to find suitable resins, thus limiting the experimental effort in early process development stages. [ABSTRACT FROM AUTHOR]

A monoclonal antibody exhibits a two- or three-peak elution behavior when loaded on the CEX resin POROS XS and eluted with a salt gradient. Two peaks are observed without a hold step while a third more strongly retained peak becomes noticeable with a hold time as low as 10 min. As the hold time is increased further, the first peak gradually disappears, the second peak initially increases and then decreases, and the third peak continuously increases. Dynamic light scattering shows that the third peak contains significant levels of aggregates formed in the column. Circular dichroism, HX-MS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HX-MS analysis indicate that the aggregates derive from an unfolded intermediate that is slowly formed while the protein is bound to the resin. Aggregate formation does not occur on a different CEX resin, Nuvia HR-S, with similar particle size but with a more homogenous structure or when the sodium acetate load buffer is replaced with arginine acetate. The two early eluting peaks observed for POROS XS regardless of hold time are shown to comprise exclusively monomeric species. A set of biophysical measurements as well as mechanistic modeling support the hypothesis that these two peaks form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics. [ABSTRACT FROM AUTHOR]

The mixed-mode (C18/strong cation exchange-SCX) solid-phase microextraction (SPME) fiber has recently been shown to have increased sensitivity for ionic compounds compared to more conventional sampler coatings such as polyacrylate and polydimethylsiloxane (PDMS). However, data for structurally diverse compounds to this (prototype) sampler coating are too limited to define its structural limitations. We determined C18/SCX fiber partitioning coefficients of nineteen cationic structures without hydrogen bonding capacity besides the charged group, stretching over a wide hydrophobicity range (including amphetamine, amitriptyline, promazine, chlorpromazine, triflupromazine, difenzoquat), and eight basic pharmaceutical and illicit drugs (pKa > 8.86) with additional hydrogen bonding moieties (MDMA, atenolol, alprenolol, metoprolol, morphine, nicotine, tramadol, verapamil). In addition, sorption data for three neutral benzodiazepines (diazepam, temazepam, and oxazepam) and the anionic NSAID diclofenac were collected to determine the efficiency to sample non-basic drugs. All tested compounds showed nonlinear isotherms above 1 mmol/L coating, and linear isotherms below 1 mmol/L. The affinity for C18/SCX-SPME for tested organic cations without H bond capacities increased with longer alkyl chains, ranging from logarithmic fiber-water distribution coefficients (log D fw ) of 1.8 (benzylamine) to 5.8 (triflupromazine). Amines smaller than benzylamine may thus have limited detection levels, while cationic surfactants with alkyl chain lengths >12 carbon atoms may sorb too strong to the C18/SCX sampler which hampers calibration of the fiber-water relationship in the linear range. The log D fw for these simple cation structures closely correlates with the octanol-water partition coefficient of the neutral form (K ow,N ), and decreases with increased branching and presence of multiple aromatic rings. Oxygen moieties in organic cations decreased the affinity for C18/SCX-SPME. Log D fw values of neutral benzodiazepines were an order of magnitude higher than their log K ow,N . Results for anionic diclofenac species (log K ow,N 4.5, pKa 4.0, log D fw 2.9) indicate that the C18-SCX fiber might also be useful for sampling of organic anions. This data supports our theory that C18-based coatings are able to sorb ionized compounds through adsorption and demonstrates the applicability of C18-based SPME in the measurement of freely dissolved concentrations of a wide range of ionizable compounds. [ABSTRACT FROM AUTHOR]

Present study aimed at graphene surface modification to achieve selective analyte binding in dispersive solid phase extraction. Magnetic graphene – polystyrene sulfonic acid (MG-PSS) cation exchange nano-composite was prepared by non-covalent wrapping method. Composite was characterized by FT-IR and zeta potential. Material exhibited good dispersion in water and high exchange capacity of 1.97 ± 0.16 mM g −1 . Prepared nano-sorbent was then exploited for the cation exchange extraction and gas chromatography mass spectrometric analysis of Chemical Weapons Convention relevant aminoalcohols and ethanolamines from aqueous samples. Extraction parameters such as sorbent amount, extraction time, desorption conditions and sample pH were optimized and effect of common matrix interferences such as polyethylene glycol and metal salts was also studied. Three milligram of sorbent per mL of sample with 20 min of extraction time at room temperature afforded 70–81% recoveries of the selected analytes spiked at concentration level of 1 μg mL −1 . Method showed good linearity in the studied range with r 2 ≥ 0.993. The limits of detection and limits of quantification ranged from 23 to 54 ng mL −1 and 72 to 147 ng mL −1 , respectively. The relative standard deviation for intra- and inter-day precision ranged from 4.6 to 10.2% and 7.4 to 14.8% respectively. Applicability of the method to different environmental samples as well as the proficiency tests conducted by the Organization for the Prohibition of Chemical Weapons (OPCW) was also ascertained. [ABSTRACT FROM AUTHOR]

A glycosylated IgG2 monoclonal antibody exhibits a two-peak elution behavior when loaded on a strong cation exchange column and eluted with either a linear salt gradient or two salt steps at increasing salt concentrations. The two-peak behavior is more pronounced for conditions where the initial antibody binding is stronger, i.e. at lower pH and buffer concentration, where the hold time prior to elution is longer, where the protein mass load is lower, and where the load flow rate is higher. The effect is also dependent on the resin type, being prominent for the polymer-functionalized resin Fractogel EMD SO3- and virtually absent for a macroporous resin with similar backbone but no grafted polymers. Size exclusion chromatography and dynamic light scattering show that the early eluting peak consists exclusively of the native monomeric species while the late eluting peak is a mixture of monomeric and aggregated species. Batch adsorption/desorption experiments show that the bound protein can be desorbed in two steps, with a fraction desorbed in 0.33M NaCl, corresponding to native monomer, and a second fraction desorbed in 1M NaCl. The latter fraction decreases with protein mass load and becomes almost negligible when the resin is initially completely saturated with protein. Confocal laser scanning microscopy showed that the two-peak elution/desorption behavior is related to the unique kinetics of protein binding in the Fractogel resin. Following partial loading of the resin, the bound protein migrates toward the center of the particles during a hold step and is redistributed across the particle volume attaining low local bound protein concentrations. For these conditions the protein is apparently destabilized forming a strongly-bound unfolded intermediate that, in turn, generates aggregates upon elution in high salt. [ABSTRACT FROM AUTHOR]

Hydrogen-deuterium exchange mass spectrometry (HX-MS) with proteolytic fragmentation is used to determine patterns of unfolding, as measured by increased solvent exposure, with peptide-level resolution for a glycosylated monoclonal antibody both when eluted from a tentacle-type cation exchange column (Fractogel EMD SO3-) and while bound to the resin. Two peaks are obtained when the bound protein is eluted with either a NaCl gradient or with two NaCl steps at increasing concentration. The first, early eluting peak contains only monomeric species whose structure is consistent with the native monomer. The second, late eluting peak contains a mixture of monomeric and aggregated species. The monomeric species in this mixture is also found to have a structure consistent with that of the native mAb, showing no evidence of increased solvent exposure. The aggregated species show instead significant unfolding in areas of the protein structure contained within the Fc region. The same peptides that exhibit the greatest level of solvent exposure in the aggregated species are also found in the fraction of protein that elutes from the resin only at high salt concentration, indicating that the aggregates are formed when the strongly-bound unfolded intermediate is desorbed at high salt. There is no evidence that the unfolded intermediate, formed while the protein is bound on the resin, is present in any of the eluted fractions indicating that, upon desorption from the resin, the intermediate either quickly refolds or forms aggregates which end-up co-eluting with the refolded protein at high salt concentrations. [ABSTRACT FROM AUTHOR]

Abstract: 4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by 13C cross polarization magic angle spinning (13C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, l-arginine, l-phenylalanine, and l-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation. [Copyright &y& Elsevier]

Abstract: A 7-μm polyacrylate (PA) coated fiber was successfully employed to determine freely dissolved concentrations of cationic surfactants by solid-phase microextraction (SPME) and utilizing the capability of the PA-coating to sorb organic cations via ion-exchange at carboxylic groups. Measured fiber–water partitioning coefficients (K fw) were constant below a fiber loading of 2mmol per liter polyacrylate, allowing for simple and accurate analysis in a concentration range that is relevant from a risk assessment point of view. Ion-exchange was confirmed to be the main sorption mechanism because of a decreasing K fw with either higher CaCl2 concentrations or lower pH, and maximum fiber uptake at the polyacrylate cation-exchange capacity (CEC, at 30mmol/L PA). Fiber–water sorption isotherms were established in various aqueous media in toxicological relevant concentrations. The developed SPME method has a high potential for application in ecotoxicological studies, as demonstrated in sorption studies with humic acid in different electrolyte solutions at aqueous concentrations down to the sub nM range. Cationic surfactant sorption affinities for humic acid also depend on medium composition but are orders of magnitude higher than to the PA fiber on a sorbent weight basis. [Copyright &y& Elsevier]

Abstract: A2E, known to be involved in the pathogenesis of age-related macular degeneration (AMD), is one of the major compounds that accumulate as fluorescent pigments in retinal pigment epithelial (RPE) cells with age and in some retinal disorders. While the biomimetic synthesis of A2E and its cis-isomer, iso-A2E is as simple as ‘one-pot’ reaction, the purification of these amphiphillic compounds has been a bottleneck for the mass production of these pathophysiologically important eye pigments. In order to provide a new method of rapid purification of A2E and iso-A2E, we employed a cation exchange resin for the separation of these pigments from crude reaction mixture. The reaction mixture was loaded on a weak acid resin and was eluted with 80% methanol with sodium hydroxide (pH 12), 100% methanol, and 100% methanol with 0.1% trifluoroacetic acid (TFA) in sequence. A2E and isoA2E were eluted only with 100% methanol solution containing TFA. Most of unreacted starting materials and intermediates were removed with 80% methanol containing sodium hydroxide. The new method can be used as a relatively simple and economic way to purify A2E and iso-A2E compared to conventional HPLC technique. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate functional increments of ion exchange type ligands, which may improve the performance of mixed-modal ligands for antibody capture out of feed solutions with pH above 6.0 and containing sodium chloride concentrations of 150mM and higher. For this purpose several functional groups such as sulfonyl, sulfanyl, amide, methoxy, short alkyl and aromatic moieties were tested in combination with a strong sulfonic acid and/or a weak carboxylic acid group. Therefore a series of ligands were synthesized and subsequently coupled onto epoxide activated Fractogel® EMD. In the first instance, all materials were tested by static binding capacity measurements (SBC) under test conditions, comprising a wide variety of different sodium chloride concentrations and differing pH values ranging from 4.5 to 7.5. From these preliminary experiment it was found that especially the aromatic groups improved the binding of human immunoglobulin G (h-IgG) under isotonic conditions, while other increments, e.g. thiophilic or amide groups, were not able to increase the capacity significantly. Taking the SBC results into account, the most promising materials were investigated under dynamic binding conditions (DBC) with a reduced selection of test conditions (pH 5.5, 6.5 and 7.4 at 75 and 150mM NaCl). N-benzoyl-homocysteine (material J) and 3,5-dimethoxybenzoyl-homocysteine (material K) showed 100% DBCs of 37mg/mL and 32mg/mL in the presence of 75mM NaCl and pH 6.5. Material L carrying mercaptobenzoic acid as a ligand and tested with the same solution provided a 100% DBC of 68mg/mL. The influence of Pluronic F68 in a mock feed solution as well as in cell culture supernatant was investigated with the best performing bio-affinity type adsorbent, material L. For the real sample feed subsequent SDS-PAGE was conducted for the collected fractions. [Copyright &y& Elsevier]

Abstract: Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO3 Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3mS/cm. HMW binding capacity was optimized at 5mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb''s commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes. [Copyright &y& Elsevier]

Abstract: Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900plates/m for peptides and 71,000plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195h. A cytochrome C digest was also separated using a linear gradient of sodium chloride. [Copyright &y& Elsevier]

Abstract: A mixed-mode reversed-phase/weak cation exchange (RP/WCX) phase has been developed by introducing a small amount of carboxylate functionality into a hydrophobic hyper-crosslinked (HC) platform. This silica-based HC platform was designed to form an extensive polystyrene network completely confined to the particle''s surface. The fully connected polymer network prevents the loss of bonded phase, which leads to superior hydrolytic stability of the new phase when compared to conventional silica-based phases. Compared to previously introduced HC phases the added carboxylic groups impart a new weak cation exchange selectivity to the base hydrophobic HC platform. The phase thus prepared shows a mixed-mode retention mechanism, allowing for both neutral organic compounds and bases of a wide polarity range to be simultaneously separated on the same phase under the same conditions. In addition, the new phase offers the flexibility that gradients in organic modifier, pH or ionic competitors can be used to affect the separation of a wide range of solutes. Moreover, the inherent weak acid cation exchange groups allow formic and acetic acid buffers to be used as eluents thereby avoiding the mass spectrometric ionization suppression problems concomitant to the use of non-volatile additives such as strong amine modifiers (e.g. triethylamine) or salts (e.g. NaCl) to elute basic solutes from the strong cation exchange phase which was previously developed in this lab. The use of the new phase for achieving strong retention of rather hydrophilic neurotransmitters and drugs of abuse without the need for ion pairing agents is demonstrated. [Copyright &y& Elsevier]

Abstract: This work provides a broad survey of binding and elution behavior of proteins on strong cation exchangers. Four proteins comprising two monoclonal antibodies, lysozyme, and cytochrome c were used as models in the investigation. Seven chromatography resins with different base matrices were compared. Dynamic binding capacity as a function of salt concentration was examined for a monoclonal antibody and lysozyme. Elution behavior as a function of gradient slope was modeled to determine the characteristic charge, essentially a measure of the number of sites involved in binding, for each protein on each resin. Trends with respect to dynamic binding capacity and elution behavior are analyzed and discussed. [Copyright &y& Elsevier]

Abstract: A novel type of silica-based sulfonate-modified reversed phase (−SO3-HC-C8) has been synthesized; it is based on a newly developed acid stable “hyper-crosslinked” C8 derivatized reversed phase, denoted HC-C8. The −SO3-HC-C8 phases containing controlled amounts of sulfonyl groups were made by sulfonating the aromatic hyper-crosslinked network of the HC-C8 phase at different temperatures. The −SO3-HC-C8 phases are only slightly less hydrophobic than the parent HC-C8 phase. The added sulfonyl groups provide a unique strong cation-exchange selectivity to the hydrophobic hyper-crosslinked substrate as indicated by the very large C coefficient as shown through Snyder''s hydrophobic subtraction reversed-phase characterization method. This cation-exchange activity clearly distinguishes the sulfonated phase from all other reversed phases as confirmed by the very high values of Snyder''s column comparison function F s. In addition, as was found in previous studies of silica-based and zirconia-based reversed phases, a strong correlation between the cation-exchange interaction and hydrophobic interaction was observed for these sulfonated phases in studies of the retention of cationic solutes. The overall chromatographic selectivity of these −SO3-HC-C8 phases is greatly enhanced by its high hydrophobicity through a “hydrophobically assisted” ion-exchange retention process. [Copyright &y& Elsevier]

Abstract: A weak cation-exchange monolithic column was prepared by modifying the GMA-EDMA (glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths with ethylenediamine and monochloracetic acid. The properties of the column were investigated; the column exhibited the ability of low backpressure and fast analysis. Using this monolithic column, trace doxazosin in human serum albumin (HSA) solution and plasma samples has been on-line tested, the extraction efficiency and the maximum loading capacity of the monolithic column were obtained. The results showed that the monolithic column could realize deproteinization and trace drug enrichment simultaneously in the HSA solution and human plasma, which provided a simple, cheap, effective and friendly to environment method for assaying drugs in the blood. [Copyright &y& Elsevier]

Abstract: Simulated moving bed (SMB) technology is a continuous chromatographic technique proven to have many advantages compared to conventional batch chromatography, such as: raised productivity and product concentration, reduced buffer consumption as well as more efficient use of raw material. In this study a 20 column SMB process for the separation of lactoperoxidase and lactoferrin from whey protein concentrate (WPC) was developed. A simplified approach with data from a single column experiment was used when designing the process. The SMB process data were compared to a theoretical scale-up of the breakthrough experiment reflecting the same 20 column set-up run in non-moving bed mode. The outcome of the comparison is a 48% raise in productivity, a 4.3 times decrease in buffer consumption, 6.5 times raise in target protein concentration with a raw material utilization which is slightly better for the SMB process. [Copyright &y& Elsevier]

A cation exchange retention mechanism concomitant with the well-known hydrophobic partition mechanism in a polymeric column has been observed and investigated. This exchange process is attributed to ionization of some acidic sites present in the polymer column at basic mobile phase pH values. Several drugs of different basicity have been chromatographed on a polymeric PLRP-S column with methanol-water and acetonitrile-water mobile phases. The cation exchange between the protonated basic drug and the buffer cations (Na+, K+ and BuNH4+) is observed at the pH range where the protonated drug and the ionized sites of the column coexist. This process produces a shift of the retention versus pH plot of the base to pH values lower than those expected from the pKa of the base as well as a maximum in the plot at basic pH values. These effects are more pronounced for acetonitrile-water mobile phases. [Copyright &y& Elsevier]

The chromatographic behavior of a monoclonal antibody (mAb) that exhibits a pronounced two-peak elution behavior is studied for a range of strong cation exchange resins and with varying load buffer pH and composition. Six stationary phases are considered, including two tentacle-type resins (Fractogel EMD SO3-(M) and Eshmuno S), a resin with grafted polymeric surface extenders (Nuvia S), a resin with a bimodal pore size distribution (POROS HS 50), and two macroporous resins without polymer grafts (Source 30S and UNOsphere Rapid S). The two-peak elution behavior is very pronounced for the tentacle and polymer-grafted resins and for POROS HS 50, but is essentially absent for the two macroporous resins. The extent of this behavior decreases as the buffer pH and concentration increase and, consequently, mAb binding becomes weaker. Replacing sodium with arginine as the buffer counterion, which is expected to decrease the mAb binding strength, nearly completely eliminates the two-peak behavior, while replacing sodium with tetra-n-butylammonium hydroxide, which is expected to increase the mAb binding strength, dramatically exacerbate the effect. As shown by hydrogen-deuterium exchange mass spectrometry (HX-MS), the two-peak elution behavior is related to conformational changes that occur when the mAb binds. These changes result in increased solvent exposure of specific peptides in the Fc-region for either the Fractogel or the Nuvia resin. No significant conformational changes were seen by HX-MS when the mAb was bound to the UNOsphere resin or on the Fractogel resin when arginine was used in lieu of sodium as the load buffer counterion. Experiments with two additional mAbs on the Fractogel resin show that the two-peak elution behavior is dependent on the particular antibody. Circular dichroism suggests that the propensity of different mAbs to either precipitate directly or to form stabilizing intermolecular structures upon exposure to thermal stress can be related to their chromatographic behaviors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A glycosylated IgG2 monoclonal antibody exhibits a two-peak elution behavior when loaded on a strong cation exchange column and eluted with either a linear salt gradient or two salt steps at increasing salt concentrations. The two-peak behavior is more pronounced for conditions where the initial antibody binding is stronger, i.e. at lower pH and buffer concentration, where the hold time prior to elution is longer, where the protein mass load is lower, and where the load flow rate is higher. The effect is also dependent on the resin type, being prominent for the polymer-functionalized resin Fractogel EMD SO₃(-) and virtually absent for a macroporous resin with similar backbone but no grafted polymers. Size exclusion chromatography and dynamic light scattering show that the early eluting peak consists exclusively of the native monomeric species while the late eluting peak is a mixture of monomeric and aggregated species. Batch adsorption/desorption experiments show that the bound protein can be desorbed in two steps, with a fraction desorbed in 0.33 M NaCl, corresponding to native monomer, and a second fraction desorbed in 1M NaCl. The latter fraction decreases with protein mass load and becomes almost negligible when the resin is initially completely saturated with protein. Confocal laser scanning microscopy showed that the two-peak elution/desorption behavior is related to the unique kinetics of protein binding in the Fractogel resin. Following partial loading of the resin, the bound protein migrates toward the center of the particles during a hold step and is redistributed across the particle volume attaining low local bound protein concentrations. For these conditions the protein is apparently destabilized forming a strongly-bound unfolded intermediate that, in turn, generates aggregates upon elution in high salt., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hydrogen-deuterium exchange mass spectrometry (HX-MS) with proteolytic fragmentation is used to determine patterns of unfolding, as measured by increased solvent exposure, with peptide-level resolution for a glycosylated monoclonal antibody both when eluted from a tentacle-type cation exchange column (Fractogel EMD SO3-) and while bound to the resin. Two peaks are obtained when the bound protein is eluted with either a NaCl gradient or with two NaCl steps at increasing concentration. The first, early eluting peak contains only monomeric species whose structure is consistent with the native monomer. The second, late eluting peak contains a mixture of monomeric and aggregated species. The monomeric species in this mixture is also found to have a structure consistent with that of the native mAb, showing no evidence of increased solvent exposure. The aggregated species show instead significant unfolding in areas of the protein structure contained within the Fc region. The same peptides that exhibit the greatest level of solvent exposure in the aggregated species are also found in the fraction of protein that elutes from the resin only at high salt concentration, indicating that the aggregates are formed when the strongly-bound unfolded intermediate is desorbed at high salt. There is no evidence that the unfolded intermediate, formed while the protein is bound on the resin, is present in any of the eluted fractions indicating that, upon desorption from the resin, the intermediate either quickly refolds or forms aggregates which end-up co-eluting with the refolded protein at high salt concentrations., (Copyright © 2014 Elsevier B.V. All rights reserved.)