Mechanistic modeling of minute virus of mice surrogate removal by anion exchange chromatography in micro scale.

Biopharmaceutical products are often produced in Chinese hamster ovary (CHO) cell cultures that are vulnerable to virus infections. Therefore, it is a regulatory requirement that downstream purification steps for biopharmaceuticals can remove viruses from feedstocks. Anion exchange chromatography (AEX) is one of the downstream unit operations that is most frequently used for this purpose and claimed for its capability to remove viruses. However, the impact of various process parameters on virus removal by AEX is still not fully understood. Mechanistic modeling could be a promising way to approach this gap, as these models require comparatively few experiments for calibration. This makes them a valuable tool to improve understanding of viral clearance, especially since virus spiking studies are costly and time consuming. In this study, we present how the virus clearance of a MVM mock virus particle by Q Sepharose FF resin can be described by mechanistic modeling. A lumped kinetic model was combined with a steric mass action model and calibrated at micro scale using three linear gradient experiments and an incremental step elution gradient. The model was subsequently verified for its capability to predict the effect of different sodium chloride concentrations, as well as residence times, on virus clearance and was in good agreement with the LRVs of the verification runs. Overall, models like this could enhance the mechanistic understanding of viral clearance mechanisms and thereby contribute to the development of more efficient and safer biopharmaceutical downstream processes.

A batch screening technique for the calculation of chromatographic separability.

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Perfluoroalkyl acid adsorption by styrenic ß-cyclodextrin polymers, anion-exchange resins, and activated carbon is inhibited by matrix constituents in different ways.

Perfluoroalkyl acids (PFAAs) are ubiquitous environmental contaminants of global concern, and adsorption processes are the most widely used technologies to remove PFAAs from water. However, there remains little data on the ways that specific water matrix constituents inhibit the adsorption of PFAAs on different adsorbents. In this study, we evaluated the adsorption of 13 PFAAs on two styrene-functionalized ß-cyclodextrin (StyDex) polymers, an activated carbon (AC), and an anion-exchange resin (AER) in the absence and presence of specific water matrix constituents (16 unique water matrices) in batch experiments. All four adsorbents exhibited some extent of adsorption inhibition in the presence of inorganic ions and/or humic acid (HA) added as a surrogate for natural organic matter. Two PFAAs (C5-C6 perfluorocarboxylic acids (PFCAs)) were found to exhibit relatively weak adsorption and five PFAAs (C6-C8 perfluorosulfonic acids (PFSAs) and C9-C10 PFCAs) were found to exhibit relatively strong adsorption on all four adsorbents across all matrices. Adsorption inhibition was the greatest in the presence of Ca2+ (direct site competition) and HA (direct site competition and pore blockage) for AC, NO3- (direct site competition) and Ca2+ (chemical complexation) for the AER, and SO42- (compression of the double layer) for the StyDex polymers. The pattern of adsorption inhibition of both StyDex polymers were similar to each other but different from AC and AER, which demonstrates the distinctive PFAA adsorption mechanism on StyDex polymers. The unique performance of each type of adsorbent confirms unique adsorption mechanisms that result in unique patterns of adsorption inhibition in the presence of matrix constituents. These insights could be used to develop models to predict the performance of these adsorbents in real water matrices and afford rational selection of adsorbents based on water chemistry for specific applications.

Sequential diethylaminoethyl dextran-grafting and diethylaminoethyl modification improve the adsorption performance of anion exchangers.

Ion exchangers with high adsorption capacity, fast mass transfer, and high salt-tolerance synchronously are highly desired for high-performance protein purification. Here, we propose a sequential diethylaminoethyl dextran-grafting and diethylaminoethyl chloride modification strategy to achieve high-performance anion exchangers. The advantages of the double-modification strategy lie in: (1) the introduction of diethylaminoethyl in the second modification has no diffusion limitation due to the small molecular size, thus a high ionic capacity; (2) the grafting ligands not only provide three-dimensional adsorption space for high adsorption capacitybut alsofacilitate surface diffusion of protein by chain delivery. The maximum adsorption capacity of the obtained anion exchangers for bovine serum albumin reaches 333 mg/mL, the ratio of effective pore diffusivity (De) to free solution diffusivity (D0) reaches 0.69, and the adsorption amount reaches 97 mg/mL even in 100 mmol/L NaCl concentration,. All these results demonstrate the proposed sequential modification strategy are promising for the preparation of high-performance ion exchangers.

Remove legacy perfluoroalkyl acids and emerging per- and polyfluoroalkyl ether acids by single-use and regenerable anion exchange resins: Rapid small-scale column tests and model fits.

Rapid small-scale column tests (RSSCT) are used to study the removal of per- and polyfluoroalkyl substances (PFAS) for drinking water treatment by ion exchange. Breakthroughs of 15 emerging per- and perfluoroalkyl ether acids and six legacy perfluoroalkyl acid analogs are studied using a single-use PFAS-selective anion exchange resin (AER1) and a regenerable, generic anion exchange resin (AER2). The Bohart-Adams model was used to describe and predict breakthrough, with the modeled results reasonably aligned with RSSCT results in most cases, enabling shorter RSSCT duration for future applications. AER1 exhibited high uptake capacity with no breakthrough for 11 of the 21 tested PFAS during the 144,175 BV continuous operation, allowing compliance with the new National Primary Drinking Water Regulation in many application scenarios. AER2 exhibited much faster breakthroughs for most PFAS and is not a promising option for drinking water treatment. However, the summed PFAS capacity via model fit and total PFAS adsorbed via measurement were only <0.01 % of both resin capacities at full breakthrough, suggesting PFAS could only occupy a tiny portion of the ion exchange sites even for the PFAS-selective AER1. Ether group insertion in the PFAS group leads to later breakthrough, and linear isomers were better captured by the resins than the branched isomers. Overall, PFAS uptake capacity increases and kinetics decrease when the PFAS molecular volume increases. Regeneration using 10 % NaCl solutions partially released PFAS from AER2 but not from AER1, with more short-chain PFAS released than long-chain ones. Ether group insertion decreased the PFAS recoveries during the regeneration of AER2. The regenerated resins showed much faster breakthroughs than the pristine resins, making them unfavorable for drinking water treatment applications. Adsorption displacement of short-chain PFAS by long-chain PFAS was observed in pristine AER1, and post-regeneration leaching occurred for both resins, both phenomena making the resins a possible PFAS source in long-term use.

Important properties of anion exchange resins for efficient removal of PFOS and PFOA from groundwater.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) present in various water sources have raised a serious concern on their health risk worldwide. Anion exchange is known to be one of the effective treatment methods but the resin properties suitable for theses contaminants have not been fully understood. We examined four commercially available anion exchange resins with different properties (DIAION™ PA312, HPA25M, UBA120, and WA30) and one polymer-based adsorbent (HP20), for their PFOA and PFOS removal in the batch experiment. All or a part of the selected resins were further characterized for their functional group, surface morphology and pore size distribution. The 72 h batch experiment with the 100 mg/L PFOA or PFOS in the laboratory pure water matrix showed a superior capacity of the strong base anion exchange resins, the porous-type HPA25M and PA312, and the gel-type UBA120, for PFOA removal (92.6-97.9%). Among those resins, the high porous HPA25M was suggested most effective due to its remarkably high reaction rate and effectiveness to PFOS (99.9%). In the groundwater matrix, however, the performance of the those anion exchange resins was generally suppressed, causing up to 71% decrease in their removal rates. The least matrix impact was observed for PFOS removal by HPA25M, which indicated the resin's high selectivity to the contaminant. The physiochemical analysis indicated that the presence of relatively large pores (1 nm-10 nm) over HPA25M played an important role in the PFAS removal.

Resin structure impacts two-component protein adsorption and separation in anion exchange chromatography.

The influence of the resin structure, on the competitive binding and separation of a two-component protein mixture with anion exchange resins is evaluated using conalbumin and green fluorescent protein as a model system. Two macroporous resins, one with large open pores and one with smaller pores, are compared to a resin with grafted polymers. Investigations include measurements of single and two-component isotherms, batch uptake kinetics and two-component column breakthrough. On both macroporous resins, the weaker binding protein, conalbumin, is displaced by the stronger binding green fluorescent protein. For the large pore resin, this results in a pronounced overshoot and efficient separation by frontal chromatography. The polymer-grafted resin exhibits superior capacity and kinetics for one-component adsorption, but is unable to achieve separation due to strongly hindered counter-diffusion. Intermediate separation efficiency is obtained with the smaller pore resin. Confocal laser scanning microscopy provides a mechanistic explanation of the underlying intra-particle diffusional phenomena revealing whether unhindered counter-diffusion of the displaced protein can occur or not. This study demonstrates that the resin's intra-particle structure and its effects on diffusional transport are crucial for an efficient separation process. The novelty of this work lies in its comprehensive nature which includes examples of the three most commonly used resin structures: a small pore agarose matrix, a large-pore polymeric matrix, and a polymer grafted resin. Comparison of the protein adsorption properties of these materials provides valuable clues about advantages and disadvantages of each for anion exchange chromatography applications.

Resin and loading condition screening for effective and robust byproducts removal by anion exchange chromatography: A case study.

In downstream processing of protein therapeutics, ion exchange (IEX) chromatography is a powerful tool for removing byproducts whose isoelectric point (pI) is appreciably different from that of the product. Although in theory for a given case cation exchange (CEX) and anion exchange (AEX) chromatography should be equally effective for separation, in reality they may show different effectiveness. In the current work, with a case study, we demonstrated that AEX is more effective than CEX chromatography at removing the associated byproducts. In addition, we screened AEX resins and loading conditions to achieve best separation. Finally, we demonstrated that effective separation was achieved with the selected resin/condition, and chromatography performance was comparable between runs conducted at low and high load densities, suggesting that the developed process was relatively robust. The procedure described in this work can be used as a general approach for selecting resin and loading condition that allow for effective and robust removal of byproduct that binds weaker than the product to the selected type of column.

Release regularity and cleaning measures of magnetic anion exchange resin during application.

Anion exchange resin is responsible for removing harmful anionic contaminants in drinking water treatment, but it may become a significant source of precursors for disinfection byproducts (DBPs) by shedding material during application without proper pretreatment. Batch contact experiments were performed to investigate the dissolution of magnetic anion exchange resins and their contribution to organics and DBPs. Dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) released from the resin were highly correlated with the dissolution conditions (contact time and pH), in which 0.7 mg/L DOC and 0.18 mg/L DON were distributed at exposure time of 2 h and pH 7. The formation potential of four DBPs in the shedding fraction was also revealed that trichloromethane (TCM), dichloroacetonitrile (DCAN), nitrosodimethylamine (NDMA), and dichloroacetamide (DCAcAm) concentrations could reach 21.4, 5.1, 12.1 µg/L, and 69.6 ng/L, respectively. Furthermore, the hydrophobic DOC that preferred to detach from the resin mainly originated from the residues of crosslinkers (divinylbenzene) and porogenic agents (straight-chain alkanes) detected by LC-OCD and GC-MS. Nevertheless, pre-cleaning inhibited the leaching of the resin, among which acid-base and ethanol treatments significantly lowered the concentration of leached organics, and formation potential of DBPs (TCM, DCAN, and DCAcAm) below 5 µg/L and NDMA dropped to 10 ng/L.

Isotherm model discrimination for multimodal chromatography using mechanistic models derived from high-throughput batch isotherm data.

In this work, we have examined an array of isotherm formalisms and characterized them based on their relative complexities and predictive abilities with multimodal chromatography. The set of isotherm models studied were all based on the stoichiometric displacement framework, with considerations for electrostatic interactions, hydrophobic interactions, and thermodynamic activities. Isotherm parameters for each model were first determined through twenty repeated fits to a set of mAb - Capto MMC batch isotherm data spanning a range of loading, ionic strength, and pH as well as a set of mAb - Capto Adhere batch data at constant pH. The batch isotherm data were used in two ways-spanning the full range of loading or consisting of only the high concentration data points. Predictive ability was defined through the model's capacity to capture prominent changes in salt gradient elution behavior with respect to pH for Capto MMC or unique elution patterns and yield losses with respect to gradient slope for Capto Adhere. In both cases, model performance was quantified using a scoring metric based on agreement in peak characteristics for column predictions and accuracy of fit for the batch data. These scores were evaluated for all twenty isotherm fits and their corresponding column predictions, thereby producing a statistical distribution of model performances. Model complexity (number of isotherm parameters) was then considered through use of the Akaike information criterion (AIC) calculated from the score distributions. While model performance for Capto MMC benefitted substantially from removal of low protein concentration data, this was not the case for Capto Adhere; this difference was likely due to the qualitatively different shapes of the isotherms between the two resins. Surprisingly, the top-performing (high accuracy with minimal number of parameters) isotherm model was the same for both resins. The extended steric mass action (SMA) isotherm (containing both protein-salt and protein-protein activity terms) accurately captured both the pH-dependent elution behavior for Capto MMC as well as loss in protein recovery with increasing gradient slope for Capto Adhere. In addition, this isotherm model achieved the highest median score in both resin systems, despite it lacking any explicit hydrophobic stoichiometric terms. The more complex isotherm models, which explicitly accounted for both electrostatic and hydrophobic interaction stoichiometries, were ill-suited for Capto MMC and had lower AIC model likelihoods for Capto Adhere due to their increased complexity. Interestingly, the ability of the extended SMA isotherm to predict the Capto Adhere results was largely due to the protein-salt activity coefficient, as determined via isotherm parameter sensitivity analyses. Further, parametric studies on this parameter demonstrated that it had a major impact on both binding affinity and elution behavior, therein fully capturing the impact of hydrophobic interactions. In summary, we were able to determine the isotherm formalisms most capable of consistently predicting a wide range of column behavior for both a multimodal cation-exchange and multimodal anion-exchange resin with high accuracy, while containing a minimized set of model parameters.

Novel chromatographic purification of succinic acid from whey fermentation broth by anionic exchange resins.

Replacement of the petroleum-based refineries with the biorefinery is regarded as an essential step towards a "zero" waste (circular) economy. Biobased succinic acid (SA) is listed by the United States Department of Energy among the top ten chemicals with the potential to replace chemicals from petroleum synthesis with renewable sources. Purification of bio-based succinic acid from fermentation by-products such as alcohols, formic acid, acetic acid and lactic is a major drawback of fermentative SA production. This study addresses this issue through a novel chromatographic separation using three distinct anionic resins: Amberlite IRA958 Cl (strong base anion exchange resin), Amberlite HPR 900 OH (strong base anion exchange resin) and Amberlyst A21 (week base anion exchange resin). The influence of process variables such as flow rate (0.18 BV/h, 0.42 BV/h and 0.84 BV/h), eluent concentration (1%, 5% and 10% HCl) and temperature (20, 30 and 40 °C) were investigated. The results indicated SA separation efficiency of 76.1%, 69.3% and 81.2% for Amberlyst A21, Amberlite HPR 900 OH and Amberlite IRA958 Cl, respectively. As the regenerant HCl concentration increased from 1 to 10%, calculated succinic acid separation efficiencies decreased from 80.3 to 70.7%. Notably, as the regenerant strength increased from 1 to 10%, the total amount of organic acids desorbed from the resin sharply increased. At operation temperatures of 20, 30 and 40 °C, SA separation efficacies were 81.2%, 73.9% and 76.4%, respectively. The insights from this study will be of great value in design of chromatographic separation systems for organic acids.

Comparative investigation of PFAS adsorption onto activated carbon and anion exchange resins during long-term operation of a pilot treatment plant.

Widespread contamination of groundwater with per- and polyfluoroalkyl substances (PFAS) has required drinking water producers to quickly adopt practical and efficacious treatments to limit human exposure and deleterious health outcomes. This pilot-scale study comparatively investigated PFAS adsorption behaviors in granular activated carbon (GAC) and two strong-base gel anion exchange resin (AER) columns operated in parallel over a 441-day period to treat contaminated groundwater dominated by short-chain perfluorocarboxylic acids (PFCA). Highly-resolved breakthrough profiles of homologous series of 2-8 CF2 PFCA and perfluorosulfonic acids (PFSA), including ultrashort-chain compounds and branched isomers, were measured to elucidate adsorption trends. Sample ports at intermediate bed depths could predict 50% breakthrough of compounds on an accelerated basis, but lower empty bed contact times led to conservative estimates of initial breakthrough. Homologous PFAS series displayed linear (GAC) and log-linear (AER) relationships between chain-length and breakthrough, independent of initial concentration. AERs generally outperformed GAC on a normalized bed volume basis, and this advantage widened with increasing PFAS chain-length. As designed, all treatments would have short full-scale service times (≤142 days for GAC; ≤61 days for AERs) before initial breakthrough of short-chain (2-4 CF2) PFCA. However, AER displayed far longer breakthrough times for PFSA compared to GAC (>3× treatment time), and breakthrough was not observed for PFSA with >4 CF2 in AERs. GAC had a finite molar adsorption capacity for total PFAS, leading to a stoichiometric replacement of short-chain PFCA by PFSA and longer-chain PFCA over time. AERs quickly reached a finite adsorption capacity for PFCA, but they showed substantially greater selectivity for PFSA whose capacity was not reached within the duration of the pilot. Breakthrough characteristics of keto- and unsaturated-PFSA, identified in the groundwater by suspect screening, were also evaluated in absence of reference standards. Modified PFAS structures (branched, keto-, unsaturated-) broke through faster than linear and unmodified perfluorinated structures with equal degrees of fluorination, and the effects were more pronounced in GAC compared to AERs. The results highlight that the design of robust PFAS treatment systems should consider facets beyond current PFAS targets including operational complexities and impacts of unregulated and unmonitored co-contaminants.

The synthesis of thermostable, strongly basic Anion-Exchange resins using Cross-Linked biguanide and its application in the extraction of Sodium copper chlorophyllin.

Commercially available, strongly basic anion-exchange resins with quaternary ammonium groups have been widely used in the purification of natural plant extracts. However, under the condition of high temperature (greater than 60 °C), these resins could not be used for long periods because of the Hofmann degradation of the strongly basic groups. In this work, the synthesis of novel, thermally stable, strongly basic resins, which has a cross-link biguanide structure, was reported. The mechanism of thermal degradation was investigated, and the result indicated that not only the stability of the functional group but also the link mode between the functional group and the resin matrix should influence the thermal stability of the resin. In our experiment, the PDG2 resin was selected to separate sodium copper chlorophyllin (SCC), a type of edible pigment derived from plants, due to its optimal thermal stability and adsorption capacity. The adsorption mechanism and thermodynamics of PDG2 were also investigated. The results demonstrated that the main adsorption affinity of PDG2 toward SCC was due to the synergistic effects of the hydrophobic and ionic interactions, and the rise in temperature will benefit the adsorption equilibrium, which differed from the equilibrium for lutein. Therefore, under suitable gradient desorption conditions, a high-purity SCC extract was prepared. After eight cycles, the adsorption capacity of the PDG2 remained constant and reproducible at a high temperature (70 °C).

Pilot study comparison of regenerable and emerging single-use anion exchange resins for treatment of groundwater contaminated by per- and polyfluoroalkyl substances (PFASs).

This study reports the results of an 8-month pilot study comparing both regenerable and emerging single-use anion exchange resins (AERs) for treatment of per- and polyfluoroalkyl substances (PFASs) at a source zone impacted by historical use of aqueous film-forming foam (AFFF). Two regenerable (Purolite A860 and A520E) and three single-use (Purolite PFA694E, Calgon CalRes 2301, and Dowex PSR2+) AERs were tested in parallel, collecting effluent samples after treatment for 30-sec and 2-min total empty bed contact time (EBCT). Results demonstrate that single-use AERs significantly outperform regenerable resins, particularly for treatment of long-chain perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs). No detectable concentrations of ≥C7 PFCAs or PFSAs were observed within 150,000 bed volumes (BVs) after treatment with the single-use resins (2-min EBCT). Analysis of effluent samples following 30-sec EBCT treatment shows that even the shortest-chain PFSAs do not reach 50% breakthrough within the first 350,000 BVs, though differences in removal of short-chain PFCAs was less dramatic. The regenerable polyacrylic A860 resin performed very poorly compared to all polystyrene resins, with >90% breakthrough of all PFASs occurring within 10,000 BVs. The greater affinity of polystyrene resins is attributed to increased hydrophobic interactions in addition to electrostatic ion exchange. Analysis of breakthrough profiles reveals empirical correlation with ion exchange affinity coefficients (logKex) measured in batch experiments. Postmortem analysis of PFASs extracted from spent resins revealed chromatographic elution behavior and competition among PFASs for adsorption to the resins. PFSAs and long-chain PFCAs were preferentially adsorbed to earlier sections in the AER columns, whereas short-chain PFCAs were competitively displaced towards the later sections of the columns and into the effluent, consistent with effluent concentrations of the latter structures exceeding influent values. These results provide insights into the mechanisms that govern PFAS adsorption to AERs in real multisolute groundwater matrices and support findings from other diverse sites regarding PFAS affinity, elution behavior, and competition for exchange sites.

Bioregeneration of sulfate-laden anion exchange resin.

Ion exchange technology removes ionic compounds from waters effectively but treatment of the spent regenerant is expensive. The bioregeneration of sulfate-laden strong base anion exchange resin was successfully tested using both pure and mixed sulfate-reducing bacterial cultures. The resin was first used for removal of sulfate from neutral (pH 6.7 ± 0.5) synthetic sodium sulfate solutions, after which the spent resin was regenerated by incubating with a viable sulfate-reducing bacterial culture in batch and column modes. In the batch bioregeneration tests, the achieved bioregeneration was 36-95% of the original capacity of the fresh resin (112 mg SO42-/g) and it increased with regeneration time (1-14 days). The capacity achieved in the column tests during 24 hours of bioregeneration was 107 mg SO42-/g after the first regeneration cycle. During the bioregeneration, sulfate was mainly reduced by the sulfate-reducing bacteria (approx. 60%), but part of it was only detached from the resins (approx. 30%). The resin-attached sulfate was most likely replaced with ions present in the liquid sulfate-reducing bacterial culture (e.g., HCO3-, HS-, and Cl-). During the subsequent exhaustion cycles with the bioregenerated resin, the pH of the treated sodium sulfate solution increased from the original 6.7 ± 0.5 to around 9. The study showed that biological sulfate reduction could be used for sulfate removal in combination with ion exchange, and that the exhausted ion exchange resins could be regenerated using a liquid sulfate-reducing bacterial culture without producing any brine.

A dual grafted fluorinated hydrocarbon amine weak anion exchange resin polymer for adsorption of perfluorooctanoic acid from water.

Perfluorooctanoic acid (PFOA) is a persistent and recalcitrant organic contaminant of exceptional environmental concern, and its removal from water has increasingly attracted global attention due to its wide distribution and strong bioaccumulation. Adsorption is considered an effective technique for PFOA removal and more efficient PFOA sorbents are still of interest. This study developed a dual grafted fluorinated hydrocarbon amine weak anion exchange (WAX) polymeric resin (Sepra-WAX-KelF-PEI) for PFOA removal from water. This polymer was synthesized by a two-step amine grafting reaction procedure involving first the reaction of the Sepra-WAX hydrocarbon polymer with poly(vinylidinefluoride-chlorotrifluoroethylene) (Kel-F 800) and then a second reaction with polyethyleneimine (PEI). Characterization of the synthesized polymers was performed using scanning electron microscopy and elemental analysis (F and Cl) by energy dispersive X-ray spectroscopy. The PFOA adsorption performance evaluations were conducted by packed column flow analyses with on-line detection. The results show the breakthrough of the Sepra-WAX-KelF-PEI synthesized with optimum stoichiometry was two times better than the starting anion exchange polymer Sepra-WAX, and six times better than powdered activated carbon, when using the same column size. The adsorption mechanisms of this novel adsorbent including hydrophobic interaction and electrostatic interaction were also clarified in this study. The adsorption kinetic parameters of the two optimum synthesized sorbents were determined using the Thomas model, the Yoon-Nelson model, and batch isotherm studies, and compared with those found with activated carbon and the starting WAX resin. Good agreement of the batch isotherm and column studies with respect to adsorption capacities trends between all three polymers (Sepra-WAX, Sepra-WAX-KelF, and Sepra-WAX-KelF-PEI) were noted.

Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC.

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several "high-energy" diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.

Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies.

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.

High-capacity multimodal anion-exchange membranes for polishing of therapeutic proteins.

This contribution reports on a study using Purexa™-MQ multimodal anion-exchange (AEX) membranes for protein polishing at elevated solution conductivities. Dynamic binding capacities (DBC10 ) of bovine serum albumin (BSA), human immunoglobulins, and salmon sperm DNA (ss-DNA) are reported for various salt types, salt concentrations, flowrates, and pH. Using 1 mg/ml BSA, DBC10 values for Purexa™-MQ were >90 mg/ml at conductivities up to 15 mS/cm. The membranes maintained a high, salt-tolerant BSA DBC10 of 89.8 ± 2.7 (SD) over the course of 100 bind-elute cycles. Polishing studies with acidic and basic monoclonal antibodies at >2 kg/L loads showed that Purexa™-MQ had higher clearance of host cell proteins and aggregate species at high conductivity (13 mS/cm) and in the presence of phosphate than other commercial AEX media. Purexa™-MQ also had a high ss-DNA DBC10 of 50 mg/ml at conductivities up to 15 mS/cm, markedly outperforming other commercial products. In addition to the effectiveness of Purexa™-MQ for protein polishing at elevated solution conductivities, its unusually high binding capacity for ss-DNA indicates potential applications for plasmid DNA purification.