Ionic Capacities Research Articles

Green polyampholytic ionic scavengers as an alternative to crude oil derived chelating resins for removal of toxic metals from aqueous solutions

A group of six amino-hypophosphite polyampholytes (AHPs) was obtained and investigated for potential scavenging of four toxic metal ions – copper(II), cobalt(II), nickel(II), and lead(II). The materials were synthesised with a convenient green one-pot reaction from simple building blocks, including polyalkylenepolyamine, paraformaldehyde, and hypophosphorous acid. The AHPs are produced in a process that is greener and simpler than conventional chelating scavengers, which are mostly crude oil derived materials and difficult to dispose of in a sustainable way. The materials were characterised with FTIR spectra and tested for removal of four different toxic metal ions. It was found that all of the AHPs obtained possess ion scavenging properties in a wide pH range (2.0–8.0). Some AHPs exert a certain selectivity towards specific metal ions. The highest values of the ionic capacities derived from cation removal experiments vary in the range 0.7–2.5 mmol Me2+·g−1 of acidic AHP and 1–4 mmol Me2+·g−1 of basic AHP. These features suggest their potential application for toxic metal ion removal processes in a wide spectrum of wastewater types.

Evaluation of Strong Cation Ion-Exchange Resin Cost Efficiency in Manufacturing Applications-A Case Study.

The effective ionic capacities of strong cation ion-exchange resins were investigated and compared using conditions similar to those found in white goods, in order to establish behavioral differences between commercial products and evaluate their capacity in a broader business context. Nine different products of equivalent TDS (Technical Data Sheet) capacity were observed to examine their differences in approximately real-life conditions. For a broader context of applicability analysis, besides the absolute ionic operating capacity, the following additional factors were included in the evaluation: the standard deviation in the resins’ performances and their relative prices. A complete method for material applicability evaluation was hereby proposed and shown to offer cost factor benefits of up to 21.1% within the range of products examined, in comparison to a cost-only evaluation for equivalent materials.

Streamlined process development procedure incorporating the selection of various stationary phase types established in a mAb aggregate reduction study with different mixed mode ligands.

In biopharmaceutical process development time, cost and reliability are the relevant keywords. During the development of chromatographic processes these targets are challenged by many possible scaffolds, ligands and process parameters. The common response to this diversity is the establishment of platform processes in the development of chromatographic unit operations. However, while developing a platform library to simplify and accelerate chromatographic processes, the potential combination of scaffold, ligands and process parameters need to be characterized. This challenge is addressed in a case study on novel mixed mode (MM) adsorber for the removal of monoclonal antibody (mAb) aggregates. We propose a rigorous strategy to reduce the various experimental design space resulting from possible combinations in scaffolds, backbones and ligands. This strategy is based on theoretical considerations, identification of adsorber selectivity and capacity for the identification of a suitable membrane system. For this system, each potential MM membrane adsorber candidate is investigated in its high molecular weight species reduction potential for a given mAb feed stream and referenced to the performance of Capto™ Adhere. The introduced strategy can reduce the developmental effort in an early stage from three to two possible stationary phases. Thereafter, initial examinations at different ionic capacities enlighten one favorable stationary phase. Finalizing the development strategy procedure by studying five different MM ligands by HTS and confirming the study with a 2-3 MV higher dynamic breakthrough capacity in benchtop experiments and provides an insight in the benefits of a living process platform library.

Microhole‐voltammograms Controlled by Solution Reservoir at Cationic and Anionic Ion Exchange Membranes

AbstractMicrohole‐voltammetry in which ions pass through a hole by electric migration exhibits rectified current‐voltage curves. The rectification ratio, being of the high conductance to the low one, seems to depend on properties of ion exchange membranes. Our experiments at a cationic and an anionic membrane showed that the ratios for both the membranes were similar in spite of large difference in ionic capacities. The ratio was controlled by geometry of the reservoir of the solution rather than hole geometry and properties of membranes. Our model was composed of resistances of the hole, the membranes and the reservoir in series.

Increasing immunoglobulin G adsorption in dextran-grafted protein A gels.

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high‐capacity polymer‐grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD‐grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD‐grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high‐capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300–350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD‐grafted protein A gels than non‐grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.

Protein adsorption to poly(2-aminoethyl methacrylate)-grafted Sepharose gel: Effects of chain length and charge density

Grafting functional polymer chains onto porous resins has been found to drastically increase both adsorption capacity and uptake rate in protein chromatography. In this work, 2-aminoethyl methacrylate (AEM) was used for grafting onto Sepharose FF gel, and six anion-exchangers of different polyAEM (pAEM) chain lengths (ionic capacities, ICs), FF-pAEM, were obtained for protein adsorption and chromatography. It was found that protein adsorption capacity (qm) increased with increasing pAEM chain length, but the uptake rate, represented by the ratio of effective pore diffusivity to the free solution diffusivity (De/D0), showed an up-down trend, reaching a peak value (De/D0=0.55) at an IC of 313 mmol/L. Partial charge neutralization of the AEM-grafted resin of the highest IC (513 mmol/L) by reaction with sodium acetate produced three charge-reduced resins, FF-pAEM513-R. With reducing the charge density, the adsorption capacity kept unchanged and then decreased, but the uptake rate monotonically increased, reaching a maximum (about 2-fold increase) at a residual IC of 263 mmol/L. It is notable that, at the same IC, the charge-reduced resin (FF-pAEM513-R) presented similar or even higher values of qm and De/D0 than its FF-pAEM counterpart. Particularly, at the same IC of 263 mmol/L, a ~50% enhancement of De/D0 was observed. Both adsorption capacity and uptake rate in the charge-reduced resin with a residual IC of 339 mmo/L (FF-pAEM513-R339) decreased more sharply with increasing NaCl concentration by comparison with FF-pAEM513, indicating its increased salt-sensitivity than FF-pAEM513. That is, charge reduction on the AEM-grafted resin could accelerate protein uptake at 0 mmol/L NaCl but decrease salt tolerance. Column breakthrough experiments showed that FF-pAEM513-R339 was favorable for high flow rate protein chromatography at low NaCl concentration (0 mmol/L), whereas FF-pAEM513 was a good choice in a wide range of salt concentrations at low flow rate. This research proved the excellent protein chromatography performance of the AEM-based anion-exchangers.

Development of poly(methacrylate)-grafted Sepharose FF for cation-exchange chromatography of proteins

Polymer-grafted media have been a focus of recent development for ion exchange chromatography (IEC) because of their capacity and uptake kinetics that can lead to high dynamic capacity in protein purification. This work is devoted to developing novel cation exchangers of high adsorption performance by grafting polymerization of sodium methacrylate (MA) onto a commercial agarose gel Sepharose FF (FF). Five polyMA (pMA)-grafted FF gels were prepared with the same grafting density but different chain lengths (i.e., different ionic capacities, ICs), and named as FF-pMA-IC (IC denotes IC value in mmol/L). The effects of chain length (IC) and ionic strength (IS) on protein adsorption and chromatographic behaviors were examined using lysozyme (at pH 8.0) and γ-globulin (at pH 5.0) as model proteins. It was found that lysozyme adsorption capacity increased with increasing IC till reaching a plateau (390 mg/mL) over IC=540 mmol/L (FF-pMA-540), while there was an optimum IC (320 mmol/L, FF-pMA-320) at which γ-globulin adsorption capacity reached the highest (208 mg/mL). With increasing chain length (IC), the uptake rates of both the proteins presented decreasing trends due to the steric hindrance caused by the polymer chains. At the same IC, however, the uptake rate of lysozyme was much higher than that of γ-globulin because of the different sizes of the two proteins. Increasing salt concentration obviously promoted the uptake rates of the proteins, which led to the increase of dynamic binding capacities (DBCs) in different salt concentration ranges. The DBC value of lysozyme on FF-pMA-540 kept as high as 108-198 mg/mL in the salt concentration range of 0-150 mmol/L, and the DBC of γ-globulin on FF-pMA-320 increased to 27 mg/mL with increasing salt concentration from 100 mmol/L. This work clearly indicated the presence of optimal IC values (chain lengths) for different sized proteins, and IS was also crucial for reaching a high DBC for a specific protein. The findings provided insight into the selection of FF-pMA-n gels and operational conditions (e.g., IS) for the purification of a target protein of defined size.

Implications from the grafting density and ionic capacity effects on protein adsorption to poly (N,N-dimethylaminopropyl acrylamide)-grafted sepharose FF

Our previous study reported a novel protein ion-exchanger, N,N-dimethylaminopropyl acrylamide (DMAPAA)-grafted Sepharose FF, of very high protein adsorption capacity and uptake rate. To gain new insight into the polymer grafting effects on protein adsorption and chromatography, 18 DMAPAA-grafted anion exchangers of four grafting densities and various chain lengths (i.e., ionic capacities, ICs) were prepared and named as FF-Brx-pDMAPAAn (x represents grafting density and n stands for IC in mmol/L). It was found that an optimal IC existed at each grafting density to present a maximal capacity. Of the four optimal-IC resins, FF-Br3-pDMAPAA587 showed the highest capacity (377 mg/mL), but the protein uptake rate, defined as the ratio of effective pore diffusivity to free diffusivity (De/D0), was smaller than the resin of a similar ionic capacity but lower grafting density (FF-Br2-pDMAPAA592). Consequently, the dynamic binding capacity (DBC) of FF-Br2-pDMAPAA592 was higher than that of FF-Br3-pDMAPAA587 at flow velocities higher than 450 cm/h. Moreover, FF-Br3-pDMAPAA587 showed higher adsorption capacity than FF-Br2-pDMAPAA592 at 0−150 mmol/L NaCl, leading to its higher DBC values as compared to FF-Br2-pDMAPAA592 in the same salt concentration range. Taken together, DMAPAA-grafted resins of different grafting densities would be useful for purifying feedstocks of different ionic strengths.

Protein cation exchangers derived by charge reversal from poly(ethylenimine)-Sepharose FF: Comparisons between two derivatization routes

It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600 mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700 mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394 mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589 mmol/L) than PEI-L700-CRn (463 mmol/L) in which De/D0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192 mg/mL) is 33% higher than that of PEI-L700-CR680 (144 mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose.

Poly(N,N-dimethylaminopropyl acrylamide)-grafted Sepharose FF: A new anion exchanger of very high capacity and uptake rate for protein chromatography

Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65–831 mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (qm) increased rapidly from 91 mg/mL at IC = 65 mmol/L till reaching a peak value (368 mg/mL) at IC = 458 mmol/L and then decreased slightly with further increasing IC (chain length) to 831 mmol/L. The uptake rate, De/D0, defined as the ratio of effective diffusion coefficient (De) to free diffusivity of the protein (D0), also presented similar changes as a function of IC. Namely, De/D0 increased from 0.25 to 2.45 in the IC range of 65 to 458 mmol/L, kept almost unchanged from 458 to 573 mmol/L and then decreased slightly with further increase of IC to 831 mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high qm and De/D0 values (368 mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The qm and De/D0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230 mg/mL and 2.3, respectively, at salt concentrations up to 100 mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180–220 mg/mL in the superficial flow velocity rang of 150–1350 cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography.

Grafting glycidyl methacrylate-iminodiacetic acid conjugate to Sepharose FF for fabrication of high-capacity protein cation exchangers

This work was designed to acquire novel high-capacity cation exchangers by grafting glycidyl methacrylate (GMA)-iminodiacetic acid (IDA) conjugate (GMA-IDA) to Sepharose FF. Six GMA-IDA-grafted resins of the same grafting density but different chain lengths (ionic capacities, ICs) were synthesized and denoted as FF-Br2-pG-In (n denotes for IC value). Lysozyme adsorption and chromatography were studied to evaluate the cation exchangers. It was found that resins of moderate IC values (FF-Br2-pG-I480 and FF-Br2-pG-I590) afforded lysozyme adsorption capacities higher than 300 mg/mL at 50 and 100 mmol/L NaCl, over three times higher than the commercial resin CM Sepharose FF. Beside, the uptake rate (De/D0) of lysozyme decreased with increasing IC at 50 mmol/L NaCl because of the increased polymer chain length that limited surface diffusion of the bound protein. Protein adsorption capacities of FF-Br2-pG-I480 and FF-Br2-pG-I590 kept higher than that of CM Sepharose FF in the salt concentration range of 0–150 mmol/L, and the uptake rates on the two resins increased with increasing salt concentration. Therefore, the dynamic binding capacities of them were remarkably higher than that of CM Sepharose FF and kept over 70 mg/mL at the salt concentration up to 150 mmol/L. The results demonstrated that GMA-IDA-grafted resins are promising for use as high-capacity protein chromatography.

High uptake rate and extremely salt-tolerant behavior of protein adsorption to 900 kDa poly(allylamine)-modified Sepharose FF

This work reports poly(allylamine) of 900 kDa (PAA-L) as a polymeric ligand for protein ion-exchange chromatography. PAA-L-modified Sepharose FF with 9 ionic capacities (ICs) from 80 to 880 mmol/L were prepared and bovine serum albumin adsorption and chromatography were investigated. With increasing IC, protein adsorption capacity increased somewhat in the IC range of 80–420 mmol/L, and then increased twice in the IC range of 420–740 mmol/L, and finally kept unchanged till 880 mmol/L; the uptake rate (De/D0) increased about 14 times (from 0.029 to 0.42) in the IC range of 420–700 mmol/L, and finally kept almost unchanged till 880 mmol/L. The maximum adsorption capacity and uptake rate of the resins reached 336 mg/mL and 0.42, which were 24% and 2.5 times respectively larger than those of 17.5 kDa PAA-grafted resins. The selected PAA-L resin of IC = 740 mmol/L was salt-tolerant up to 750 mmol/L NaCl. The dynamic binding capacity (DBC) of this resin showed a maximum of 142 mg/mL at 350 mmol/L NaCl, and kept over 90 mg/mL in the range of 150–500 mmol/L NaCl. Linear gradient elution on the column of this resin proved high protein recovery (>97%) and stability of the column. Taken together, the results indicate that the PAA-L resin was a promising anion exchanger for high-performance protein chromatography for use at extended ionic strengths.

Implications from protein adsorption onto anion- and cation-exchangers derivatized by modification of poly(ethylenimine)-Sepharose FF with succinic anhydride

Poly(ethylenimine) (PEI)-grafted Sepharose FF with an ionic capacity (IC) of 740 mmol/L (FF-PEI-L740) was modified with succinic anhydride, and six mix-charged resins of different ionic capacities (ICs) were synthesized [denoted as “Am-Cn” according to their anion exchange groups (amino groups) and cation exchange groups (carboxyl groups)]. Protein adsorption behaviors were investigated using bovine serum albumin (BSA) and lysozyme (LZM) as model proteins. The modification led to the net positive charge decrease to approximately zero (A320-C270), and then reversed the net charge to negative till the maximum negatively charge resin A20-C970. Adsorption capacity for BSA decreased from 205 ± 5 mg/mL (FF-PEI-L740) to 65 ± 3 mg/mL (A320-C270), but increased, for LZM, from 31 ± 1 mg/mL (A320-C270) to 281 ± 13 mg/mL (A20-C970) with increasing net negative charge. The uptake rate of BSA or LZM decreased with decreasing net positive charge or increasing net negative charge due to the decreased flexibility of the PEI chains brought from the increased electrostatic attraction or repulsion and/or tight binding due to the high net charge density. Effect of ionic strength (IS) showed that the resins with high net charge densities exhibited high adsorption capacities at high IS. Besides, dynamic binding capacity values achieved as high as 55 mg/mL for BSA and 143 mg/mL for LZM. Chromatographic experiments demonstrated favorable elution of bound proteins at NaCl lower than 370 mmol/L.

Alginate-grafted Sepharose FF: A novel polymeric ligand-based cation exchanger for high-capacity protein chromatography

This work was designed to acquire a new high-capacity cation-exchange chromatography resin of protein by modification of Sepharose FF gel with sodium alginate. Three cation exchangers of different ionic capacities (ICs, 140–240mmol/L) were synthesized, and the alginate-based resins, denoted as Alg-FFs, were studied for lysozyme (Lys) adsorption and chromatography. It was found that the adsorption capacity and effective protein diffusivity of Lys increased with increasing alginate-grafting density or the IC value. The resin with the highest IC value (240mmol/L), Alg-FF-240, was compared with a commercial cation exchanger, CM Sepharose FF. It revealed that both protein adsorption capacity and uptake rate of Alg-FF-240 were remarkably higher than CM Sepharose FF at NaCl concentrations lower than 100mmol/L, so the dynamic binding capacity (DBC) of Alg-FF-240 was much higher than CM Sepharose FF at the salt concentration range. Moreover, the DBC of Alg-FF-240 maintained high level at the flow rate up to 1350cm/h, and at the highest flow rate studied, the DBC of Alg-FF-240 for Lys was about 1.7 times higher than that on CM Sepharose FF. Salt-gradient elution revealed similar protein elution behaviors on the Alg-FF-240 and CM Sepharose FF columns. Altogether, the findings in this research indicate that alginate-based cation exchangers deserve further development for high-performance protein chromatography.

Characterization of poly(allylamine) as a polymeric ligand for ion-exchange protein chromatography

This work reports poly(allylamine) (PAA), as a polymeric ion-exchange ligand for protein chromatography. Sepharose FF was modified with PAA, and six anion exchangers with ionic capacities (ICs) from 165 to 618mmol/L were prepared. Inverse size exclusion chromatography, adsorption equilibrium, uptake kinetics and column elution were performed. It was found that both the adsorption capacity and effective diffusivity maintained low values in the IC range of 165–373mmol/L, but they started to increase beyond 373mmol/L, and increased by 80% and 23 times, respectively, when the IC reached 618mmol/L. Interestingly, a drastic decrease of pore size was observed around the IC of 373mmol/L. The results suggest that the PAA chains played an important role in protein adsorption by altering the inner pore structure of the gels. It is considered that, PAA chains turn from inextensible states with multipoint-grafting on the pore surface at low coupling densities (IC<373mmol/L) to closer, extended and flexible grafting states with less coupling points at higher coupling densities (IC>373mmol/L). These characters of the grafted chains at higher IC values benefit in protein adsorption by three-dimensional binding and encouraged the happening of “chain delivery” of bound proteins on the chains. Besides, the ion exchangers showed favorable adsorption and uptake properties in a wide ionic strength range, 0–500mmol/L NaCl, indicating much better salt tolerance feature than the so-far reported ion exchangers. Moreover, a mild condition of pH 5.0 offered effective recovery of bound proteins in elution chromatography. The results indicate that the PAA-based anion exchanger of a high IC value is promising for high-capacity protein chromatography dealing with feedstock of a wide range of ionic strengths.

Mechanistic evaluation of virus clearance by depth filtration.

Virus clearance by depth filtration has not been well-understood mechanistically due to lack of quantitative data on filter charge characteristics and absence of systematic studies. It is generally believed that both electrostatic interactions and sized based mechanical entrapment contribute to virus clearance by depth filtration. In order to establish whether the effectiveness of virus clearance correlates with the charge characteristics of a given depth filter, a counter-ion displacement technique was employed to determine the ionic capacity for several depth filters. Two depth filters (Millipore B1HC and X0HC) with significant differences in ionic capacities were selected and evaluated for their ability to eliminate viruses. The high ionic capacity X0HC filter showed complete porcine parvovirus (PPV) clearance (eliminating the spiked viruses to below the limit of detection) under low conductivity conditions (≤2.5 mS/cm), achieving a log10 reduction factor (LRF) of > 4.8. On the other hand, the low ionic capacity B1HC filter achieved only ∼2.1-3.0 LRF of PPV clearance under the same conditions. These results indicate that parvovirus clearance by these two depth filters are mainly achieved via electrostatic interactions between the filters and PPV. When much larger xenotropic murine leukemia virus (XMuLV) was used as the model virus, complete retrovirus clearance was obtained under all conditions evaluated for both depth filters, suggesting the involvement of mechanisms other than just electrostatic interactions in XMuLV clearance.

Preparation and characterization of grafted cellulosic fibers and their applications in protein purification

Fibrous adsorbents were subjected either to chemical treatment or gamma irradiation to introduce epoxide groups onto their cellulosic backbone using glycidyl methacrylate. These epoxide moieties were modified to have diethylaminoethanol (DEAE) as well as quaternary ammonium (Q) functionalities. The resulting anion-exchange adsorbents were characterized by their FTIR spectra and ionic capacities. The fiber-based adsorbent systems showed similar packing efficiency to the commercially available adsorbents, where the Péclet number values were ⩾60, suggesting near-plug-flow conditions. The total ionic capacities obtained for these chemically grafted adsorbents were ca. 400mmol/L. These adsorbents showed dynamic binding capacities (DBC) of ca. 48mg/mL for bovine serum albumin (BSA). Protein binding capacities obtained from chemical grafting initiation techniques were 18-fold-higher than radiation-induced techniques. The advantage of these adsorbents lies in their high operational flow rates while maintaining their high binding capacities.

Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics

To explore the details of protein uptake to polymer-grafted ion exchangers, Sepharose FF was modified with poly(ethylenimine) (PEI) to prepare anion exchanger of 10 different ionic capacities (ICs, 100–1220mmol/L). Adsorption equilibria and kinetics of bovine serum albumin (BSA) were then studied. It is found that ionic capacity, i.e., the coupling density of PEI, had significant effect on both adsorption capacity (qm) and effective protein diffusivity (De). With increasing ionic capacity, the qm value increased rapidly at IC<260mmol/L and then increased slowly till reaching a plateau at IC=600mmol/L. In the IC range of 100–600mmol/L, however, the De values kept at a low level (De/D0<0.07); it first decreased from 0.05±0.01 at IC=100mmol/L to 0.01±0.01 at IC=260mmol/L and then increased to 0.06±0.01 at IC=600mmol/L. Thereafter, sharp increases of the qm and De values [36% (from 201 to 273mg/mL) and 670% (from 0.06±0.01 to 0.49±0.04), respectively] were observed in the narrow range of IC from 600 to 740mmol/L. Finally, at IC>740mmol/L, the qm value decreased significantly while the De value increased moderately with increasing the IC. The results indicate that PEI chains played an important role in protein adsorption and transport. In brief, there was a critical IC (cIC) or PEI chain density, above which protein adsorption and transport behaviors changed drastically. The cIC was identified to be about 600mmol/L. Estimation of PEI grafting-layer thickness suggests that PEI chains formed an extended three-dimensional grafting-layer at IC>cIC, which provided high flexibility as well as accessibility of the chains for protein binding. Therefore, at IC>cIC, the adjacent PEI chains became close and flexible enough, leading to facilitated transport of adsorbed protein molecules by the interactions of neighboring chains mediated by the bound molecules. It is regarded as “chain delivery” effect. At the same time, improved accessibility of binding sites led the significant increase of binding capacity. The decrease of qm value at IC>740mmol/L is considered due to the decrease of effective porosity. The research has thus provided new insight into protein adsorption and transport in polymer-grafted ion-exchange media.

Protein adsorption to poly(ethylenimine)-modified Sepharose FF: II. Effect of ionic strength

In Part I of this work, we have studied the effect of ionic capacity (IC) on bovine serum albumin (BSA) adsorption equilibria and kinetics to poly(ethylenimine) (PEI)-grafted Sepharose FF, and found a critical IC (cIC, 600mmol/L), above which both protein capacity and uptake rate increased drastically. In this work, five PEI-Sepharose FF resins of typical ICs reported earlier were selected to explore the effect of ionic strength (IS) on the adsorption equilibria and kinetics of BSA. Commercially available DEAE (IC=160mmol/L) and Q Sepharose FF (IC=269mmol/L) resins were used for comparisons. It is found that at similar ionic capacities, protein adsorption capacities on both the PEI-Sepharose FF resins and the commercial resins decreased with increasing IS, but on the capacity sensitivity to salt concentration, the former was lower than the latter. In addition, the effective diffusivities (De) of the former were smaller than the latter in the entire IS range studied. The low IS sensitivity of adsorption capacity of the PEI-Sepharose FF resins could be interpreted by the increase of pore accessibility with increasing IS; the smaller De values in the PEI-Sepharose FF resins were considered due to the lack of surface diffusion in the PEI-Sepharose FF resins of low PEI densities. For the PEI-Sepharose FF resins of high ICs (520, 740 and 1220mmol/L), both protein capacity and De values increased first and then decreased with increasing IS. The increasing trend of protein capacity in the low IS range was considered due to the increase of accessible pores for BSA. The rise–fall trend of De was attributed to the dependencies of the “chain delivery” effect on protein capacity and binding strength, both of which are related to IS. Moreover, the IS sensitivity of the De for the resins of ICs>cIC (740 and 1220mmol/L) was much higher than those of ICs<cIC, further proving that the “chain delivery” effect in PEI layer did contribute significantly to the overall mass transfer at IC>cIC. Furthermore, the two PEI-Sepharose FF resins of ICs>cIC kept high adsorption capacities and De values up to 200–300mmol/L NaCl. Therefore, the operating IS ranges for these two PEI-Sepharose FF resins can be much broader than the traditional ion-exchange media.

Effect of ionic capacity on dynamic adsorption behavior of protein in ion-exchange electrochromatography

Four anion-exchangers of different ion-exchange capacities (21–129 μmol/mL) were prepared by coupling diethylaminoethyl to Sepharose 6FF. The adsorbents were used to study the dynamic protein adsorption in ion-exchange electrochromatography with an oscillatory transverse electric field perpendicular to the mobile-phase flow (IEEC). The static adsorption capacity of bovine serum albumin (BSA) increased from 67 to 186 mg/mL in the ionic capacity range, but the effective pore diffusion coefficient of the protein decreased from 10.9 × 10 −12 to 1.85 × 10 −12 m 2/s with increasing the ionic capacity due to the hindrance effect of the bound protein molecules at the pore entrance. So, the dynamic binding capacity (DBC) of protein in ion-exchange chromatography decreased with increasing the ionic capacity. By applying an electric field of 30 mA, the DBC in IEEC packed with ion-exchangers of high ionic capacities (53 and 129 μmol/mL) increased significantly (over 30% and 100%, respectively). This was because the high surface charge density led to high electroosmotic flow that enhanced intraparticle mass transfer in IEEC. In comparison, the DBC in IEEC packed with ion-exchangers of low ionic capacities (21 and 35 μmol/mL) increased only slightly (ca. 10%) under the same condition. The results indicated the minor effect of electrophoretic mobility on the intraparticle mass transfer. Hence, it is beneficial to use ion-exchangers of high ionic capacity for high-capacity purification of proteins by IEEC.