High-capacity Cation Exchange Research Articles

New and efficient purification process for recombinant human insulin produced in Escherichia coli.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Moldable Strong Cation Exchange Polymer and Microchannel Fabrication.

We characterize a high-capacity cation exchange membrane (CEM) synthesized from an aqueous poly(vinyl alcohol) (PVA) solution and varying amounts of a water-soluble ionic monomer, sodium styrenesulfonate. Highly hydrophilic but water-insoluble transparent polymers with a range of ion exchange capacities (IECs) can be made; at the high end, the ion exchange capacities (IEC) is >2× that of the benchmark CEM, Nafion. The water uptake of the polymer (as moles of water/mole H+) is 5-10× greater than that of Nafion. Except at the highest IECs (where steeply increasing water sorption and resultant swelling outpaces IEC increase), the specific conductance increases with increasing IEC while the conductance anisotropy decreases. The material withstands repeated regeneration cycles and hour-long boiling in water or alcohols. The aqueous prepolymer mixture can be cast around a fine wire acting as a mandrel. Fabrication of an ion exchanger microchannel, capable of withstanding at least 300 psi, is demonstrated. We also discuss entirely novel considerations on ab initio limits of conductance of ion exchange membranes.

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.

Chromatin-directed clarification in cell culture fluid enables non-protein affinity antibody purification by tangential flow filtration integrated with high-capacity cation exchange chromatography

Traditional purification platforms for monoclonal antibodies (mAbs) rely heavily on 3–4 step chromatographic operations with protein A affinity chromatography as the essential antibody capture approach. In this study, we applied for the first time the established fatty acid-mediated chromatin-directed clarification to cell fermentation broth. An efficient chromatin-directed cell culture fluid (CCF) clarification process was developed to remove most host DNA and histones, as well as reduce non-histone host cell proteins (n-h HCPs) by 94.7%, with a 92.5% IgG recovery yield. The pre-removal of a large amount of host cell contaminants allows tangential flow filtration (TFF) to concentrate clarified cell culture supernatant (CCS) and perform buffer exchange. High-capacity cation exchange chromatography (CEX) was shown to effectively remove the remaining host impurities. This new antibody purification platform achieves an 86.7% antibody recovery yield with an end-product meeting all clinical requirements, greatly shortening the antibody purification process and improving the antibody production efficiency.

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.

Fabrication of high-capacity cation-exchangers for protein adsorption: Comparison of grafting-from and grafting-to approaches

In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharose FF (the thus resulting exchanger is referred as Sep-g-SPM), and another via the grafting-to approach, in which the polymer of SPM is directly coupled onto Sepharose FF (the thus resulting exchanger is called as Sep-pSPM). Protein adsorption on these two cation exchangers have been also investigated. At the same ligand density, Sep-g-SPM has a larger accessible pore radius and a smaller depth of polymer layer than SeppSPM, due to the controllable introduction of polymer chains with the regular distribution of the ligand. Therefore, high-capacity adsorption of lysozyme and γ-globulin could be achieved simultaneously in Sep-g-SPM with an ionic capacity (IC) of 308 mmol$L–1. However, Sep-pSPM has an irregular chain distribution and different architecture of polymer layer, which lead to more serious repulsive interaction to proteins, and thus Sep-pSPM has a lower adsorption capacity for γ-globulin than Sep-g-SPM with the similar IC. Moreover, the results from protein uptake experiments indicate that the facilitated transport of adsorbed γ-globulin occurs only in Sep-pSPM and depends on the architecture of polymer layers. Our research provides a clear clue for the development of high-performance protein chromatography.

A novel polymer-grafted cation exchanger for high-capacity protein chromatography: The role of polymer architecture

In this work, a branched polymer-grafted cation exchanger was synthesized on a Sepharose FF matrix via atom transfer radical polymerization using 3-sulfopropyl methacrylate potassium salt (SPM) as a functional monomer and 2-(2-bromo- isobutyryloxy)ethyl methacrylate as a branching monomer. The resulting branched poly(SPM)-grafted cation exchangers exhibit typical ionic exchange characteristics for lysozyme adsorption and the maximum adsorption capacity reach 450mg/mL on Sep-BrS-S12B2 whereas γ-globulin adsorption is more dependent on polymer architecture. By adjusting the ratio of the monomers, very high adsorption capacity for lysozyme and γ-globulin can be achieved in the more branched Sep-BrL-S4B3 while γ-globulin has a much higher effective diffusivity in Sep-BrL-S4B3. Experimental evidences show that the performance of polymer-grafted cation exchanger can be improved by regulating polymer architecture. The calorimetric results further indicate that counter-ions are released from polymer and proteins during protein adsorption. With an increase of the adsorption density, adsorbed proteins experience a change of molecular orientation along poly(SPM) chain. In Sep-BrL-S4B3, dynamic binding capacities reached 145mg/mL for lysozyme and 96mg/mL for γ-globulin, demonstrating that Sep-BrL-S4B3 is a promising type of novel high-capacity cation exchangers. This research gives clue to the design of high-capacity cation exchangers and offers insights into protein adsorption on polymer-grafted cation exchangers.

Polyacrylic acid (PAA)- surface grafted dense nanosilica spheres for ibuprofen delivery

In this work, a branched polymer-grafted cation exchanger was synthesized on a Sepharose FF matrix via atom transfer radical polymerization using 3-sulfopropyl methacrylate potassium salt (SPM) as a functional monomer and 2-(2-bromo- isobutyryloxy)ethyl methacrylate as a branching monomer. The resulting branched poly(SPM)-grafted cation exchangers exhibit typical ionic exchange characteristics for lysozyme adsorption and the maximum adsorption capacity reach 450 mg/mL on Sep-BrS-S12B2 whereas γ-globulin adsorption is more dependent on polymer architecture. By adjusting the ratio of the monomers, very high adsorption capacity for lysozyme and γ-globulin can be achieved in the more branched Sep-BrL-S4B3 while γ-globulin has a much higher effective diffusivity in Sep-BrL-S4B3. Experimental evidences show that the performance of polymer-grafted cation exchanger can be improved by regulating polymer architecture. The calorimetric results further indicate that counter-ions are released from polymer and proteins during protein adsorption. With an increase of the adsorption density, adsorbed proteins experience a change of molecular orientation along poly(SPM) chain. In Sep-BrL-S4B3, dynamic binding capacities reached 145 mg/mL for lysozyme and 96 mg/mL for γ-globulin, demonstrating that Sep-BrL-S4B3 is a promising type of novel high-capacity cation exchangers. This research gives clue to the design of high-capacity cation exchangers and offers insights into protein adsorption on polymer-grafted cation exchangers.

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.

Antimicrobial activity of buttermilk and lactoferrin peptide extracts on poultry pathogens.

Antibiotics are commonly used in poultry feed as growth promoters. This practice is questioned given the arising importance of antibiotic resistance. Antimicrobial peptides can be used as food additives for a potent alternative to synthetic or semi-synthetic antibiotics. The objective of this study was to develop a peptide production method based on membrane adsorption chromatography in order to produce extracts with antimicrobial activity against avian pathogens (Salmonella enterica var. Enteritidis, Salmonella enterica var. Typhimurium, and two Escherichia coli strains, O78:H80 and TK3 O1:K1) as well as Staphylococcus aureus. To achieve this, buttermilk powder and purified lactoferrin were digested with pepsin. The peptide extracts (<10 kDa) were fractionated depending on their charges through high-capacity cation-exchange and anion-exchange adsorptive membranes. The yields of cationic peptide extracts were 6·3 and 15·4% from buttermilk and lactoferrin total peptide extracts, respectively. Antimicrobial activity was assessed using the microdilution technique on microplates. Our results indicate that the buttermilk cationic peptide extracts were bactericidal at less than 5 mg/ml against the selected avian strains, with losses of 1·7 log CFU/ml (Salm. Typhimurium) to 3 log CFU/ml (E. coli O78:H80); viability decreased by 1·5 log CFU/ml for Staph. aureus, a Gram-positive bacterium. Anionic and non-adsorbed peptide extracts were inactive at 5 mg/ml. These results demonstrate that membrane adsorption chromatography is an effective way to prepare a cationic peptide extract from buttermilk that is active against avian pathogens.

Fabrication of high-capacity cation-exchangers for protein chromatography by atom transfer radical polymerization

This work reports the synthesis of novel cation-exchanger with controllable charge density and polymer chain length of poly(3-sulfopropyl methacrylate) (poly(SPM)) grafted via atom transfer radical polymerization onto Sepharose FF matrix. Polymer grafting provided a three-dimensional regular arrangement of the ligand and increased ionic capacity of the poly(SPM)-grafted cation-exchangers. The result showed that adsorption capacity for lysozyme enhanced greatly in poly(SPM)-grafted cation-exchangers whereas adsorption capacity for γ-globulin decreased dramatically. It can be attributed to the consequence of the competition between electrostatic interaction and repulsive excluded volume interaction of grafted polymer and protein. For lysozyme, protein adsorption on poly(SPM)-grafted cation exchangers was driven dominantly by electrostatic interaction. By constrast, the repulsive excluded volume interaction between grafted polymer and protein was remarkable in adsorption of γ-globulin on poly(SPM)-grafted cation exchangers. Moreover, the poly(SPM)-grafted cation exchangers exhibited great salt tolerance in protein adsorption and distinct intraparticle mass transfer properties. Finally, the results of dynamic binding capacity (DBC) for lysozyme showed that the poly(SPM)-grafted cation-exchangers had higher binding capacities than did the commercial SP Sepharose FF, and the maximal DBCs reached 192mg/mL at 50mmol/L NaCl. These results demonstrate great potential of poly(SPM)-grafted cation-exchangers for the large-scale purification of proteins.

Nanostructured biocompatible thermal/electrical stimuli-responsive biopolymer-doped polypyrrole for controlled release of chlorpromazine: Kinetics studies

Biocompatible nanostructured conductive heparin-doped polypyrrole film was fabricated and employed as a high-capacity cation exchanger for programmable release of neuroleptic drug, chlorpromazine (CPZ) with thermally and electrical dual-stimulation. Releasing behavior were studied at different applied potentials and temperatures by in-situ monitoring of UV absorbance measurements. Three mathematical models (Higuchi, Power, and Avrami equation) were employed to investigate kinetics of the release. Based on the obtained results, the Avrami model found to be more comprehensive than two other ones for mathematical description of electro-stimulated release of CPZ. A quantitative relationship between activation energy parameters (Ea, ΔG≠, ΔH≠, and ΔS≠) and release conditions (applied potential and temperature) has been developed and established to predict release rate constants at various applied conditions.

Evaluation of high‐capacity cation exchange chromatography for direct capture of monoclonal antibodies from high‐titer cell culture processes

Advances in molecular biology and cell culture technology have led to monoclonal antibody titers in excess of 10 g/L. Such an increase can pose concern to traditional antibody purification processes due to limitations in column hardware and binding capacity of Protein A resins. Recent development of high capacity cation exchangers can make cation exchange chromatography (CEX) a promising and economic alternative to Protein A capture. This work investigates the feasibility of using CEX for direct capture of monoclonal antibodies from high titer cell culture fluids. Two resin candidates were selected from seven newer generation cation exchangers for their higher binding capacity and selectivity. Two monoclonal antibodies with widely differing pI values were used to evaluate the capability of CEX as a platform capture step. Screening of loading pH and conductivity showed both resins to be capable of directly capturing both antibodies from undiluted cell culture fluid. At appropriate acidic pH range, product loading of over 65 g/L resin was achieved for both antibodies. A systematic design of experiment (DOE) approach was used to optimize the elution conditions for the CEX step. Elution pH showed the most significant impact on clearance of host cell proteins (HCPs). Under optimal conditions, HCP reduction factors in the range of 9-44 were achieved on the CEX step based on the pI of the antibody. Apart from comparing CEX directly to Protein A as the capture method, material from either modality was also processed through the subsequent polishing steps to compare product quality at the drug substance level. Process performance and product quality was found to be acceptable using the non-affinity based process scheme. The results shown here present a cheaper and higher capacity generic capture method for high-titer antibody processes.

Synthesis of high capacity cation exchangers from a low-grade Chinese natural zeolite

The Chinese natural zeolite, in which clinoptilolite coexists with quartz was treated hydrothermally with NaOH solutions, either with or without fusion with NaOH powder as pretreatment. Zeolite Na–P, Na–Y and analcime were identified as the reacted products, depending on the reaction conditions such as NaOH concentration, reaction time and hydrothermal temperature. The products were identified by X-ray diffraction, and characterized by Fourier transform IR and ICP. With hydrothermal treatment after fusion of natural zeolite with NaOH, high purity of zeolite Na–Y and Na–P can be selectively formed, their cation exchange capacity (CEC) are 275 and 355 meq/100 g respectively, which are greatly higher than that of the natural zeolite (97 meq/100 g). Furthermore, the ammonium removal by the synthetic zeolite Na–P in aqueous solution was also studied. The equilibrium isotherms have been got and the influence of other cations present in water upon the ammonia uptake suggested an order of preference Ca 2+ > K + > Mg 2+.

Breakthrough performance of high-capacity membrane adsorbers in protein chromatography

The breakthrough of lysozyme (HEWL) and bovine serum albumin (BSA) on high-capacity cation exchange membranes was studied (Sartobind-S; Sartorius AG, Germany). The ligand density of these membranes is increased significantly by anchoring sulfonic acid groups (SO 3H) to cross-linked polymer chains grafted onto the basic polymer resulting in a three-dimensional adsorbing layer. The static and dynamic protein binding behavior was characterized for two types of membranes with a different degree of modification. Static capacities were found to be increased up to 100-fold compared to a conventional modification with a direct coupling of the ligands to the stationary phase. A dimensionless treatment of the breakthrough zone in frontal analysis led to the identification of a rate-limiting diffusive mechanism resulting from slow transport of proteins into the three-dimensional adsorbing layer. A combination of pore diffusion and surface diffusion is well suited to explain the effect of the feed concentration upon the sharpness of the breakthrough curves. These results reveal that the common assumption of an ideal interaction between protein and ligand in membrane adsorption chromatography is not realistic for a theoretical description of these high-capacity membranes. The analysis of breakthrough performance forms the basis for an optimization of the membrane modification leading to a ‘useful’ ligand density with high capacities at enhanced mass transfer. The dimensionless analysis of fixed-bed adsorption is outlined for different rate limitations resulting from various mass transfer mechanisms and binding kinetics. This frame-work enables a comparison between the performance of both, membrane adsorbers and conventional gel beads, and an appropriate choice of operating conditions.

96/05962 High capacity cation exchanger by hydrothermal zeolitization of coal fly ash

96/05962 High capacity cation exchanger by hydrothermal zeolitization of coal fly ash

High capacity cation exchanger by hydrothermal zeolitization of coal fly ash

During; hydrothermal treatment in NaOH medium coal fly ash partially transformed to zeolite P and/or hydroxysodalite, while quartz slowly dissolved and mullite remained stable. Residual coal favored the formation of zeolite P. The relative proportion of the two zeolites could be changed by seeding. During fusion of fly ash with NaOH an endothermic reaction at 170–180°C occurred, resulting in formation of an unreported Na aluminosilicate with approximate composition Na 15Si 4Al 30 20 and major XIRD reflections at 4.793, 3.828, 3.000, 2.854, 2.578, and 2.524 Å. The product of fusion interacted with water giving aluminosilicate gel, which yielded zeolite P upon hydrothermal treatment at 100°C without aging and zeolite X after aging in water for 12 hours at room temperature. Cation exchange capacity was 420 and 400 meq/ 100 g for zeolite P and zeolite X respectively.

Studies on system performance and sensitivity in ion chromatography

Improvements in HPLC instrumentation, suppressor technology and ion-exchange columns have occurred over the past several years to the point where an ion chromatograph can now be configured for anion analysis using a considerable variety of hardware, suppressor and column combinations. A number of parameters, including hardware configuration, effect of temperature, column type and suppressor device, were studied with a view toward optimizing the performance of an ion chromatographic (IC) system. It was found that dual-piston, reciprocating pumps used with low-pressure pulse dampeners significantly reduced baseline noise for both suppressed and non-suppressed conductivity detector combinations, while column temperature control proved essential in order to achieve routine sub-ppb detectability in non-suppressed IC. In general, the use of suppressed IC resulted in lower detection limits than non-suppressed IC when using the same columns and hardware. Of the columns studied, the methacrylate-based HR column was found to give the best overall separation selectivity when using a carbonate-bicarbonate eluent and suppressed conductivity detection. The majority of column and suppressor combinations evaluated gave acceptable performance, although some gave less than satisfactory results. Also, some combinations resulted in lower than expected analytical results, particularly for chloride, when quantitated using single-point calibration. In addition to the use of commercially available suppressor devices, there is also a large number of high-capacity cation exchangers which can be used as suppressors and these columns, in some instances, may offer equivalent (or superior) performance compared to the commercial devices.

Sulfuric Acid Regeneration of High Capacity Cation Exchanger

Sulfuric Acid Regeneration of High Capacity Cation Exchanger