Comparison of chromatographic ion-exchange resins. III. Strong cation-exchange resins

A comparative study was performed on strong cation-exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and SEM pictures of chromatographic resins. The resins tested included: SP Sepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source 30S, TSKGel SP-5PW-HR20, and Toyopearl SP 650c. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high pI. An unexpected binding at pH 7.5 of anti-FVII Mab with pI < 7.5 was observed on several resins. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow rate of soft resins compared to resins for medium and high-pressure operation. Determination of particle size distribution by two independent methods, Coulter counting and SEM, was in very good agreement. The mono-dispersed nature of Source 30S was confirmed. Binding to cation-exchange resins as a function of ionic strength varies depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Toyopearl SP 550c and Poros 50 HS, while binding and elution at low salt concentration may be performed with Toyopearl SP 650c. A very high binding capacity was obtained with SP Sepharose XL. Comparison of static capacity and dynamic capacity at 10% break-through shows in general approximately 50-80% utilisation of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by others. The results of this study may be used for selection of resins for testing in process development. The validity of experiments and results with model proteins were tested using human insulin precursor in pure state and in real feed-stock on Toyopearl SP 550c, SP Sepharose BB, and Toyopearl SP 650c. Results showed good agreement with experiments with model proteins.     

Comparison of chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and heparin resins

A comparative study was performed on heparin resins and strong and weak cation exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy pictures of chromatographic resins. The resins tested include: Heparin Sepharose FF, SP Sepharose FF, CM Sepharose FF, Heparin Toyopearl 650 m, SP Toyopearl 650 m, CM Toyopearl 650 m, Ceramic Heparin HyperD M, Ceramic S HyperD 20, and Ceramic CM HyperD F. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high isoelectric point (pI), though some decrease of retention with increasing pH was observed for CM Ceramic HyperD F and S Ceramic HyperD 20. Binding of anti-FVII Mab with pI < 7.5 was observed on several resins at pH 7.5. Efficiency results show the expected trend of increasing dependence of the plate height with increasing flow rate of Ceramic HyperD resins followed by Toyopearl 650 m resins and the highest flow dependence of the Sepharose FF resins corresponding to their pressure resistance. Determination of particle size distribution by two independent methods, coulter counting and SEM, was in good agreement. Binding strength of cation-exchange resins as a function of ionic strength varies depending on the protein. Binding and elution at high salt concentration may be performed with Ceramic HyperD resins, while binding and elution at low salt concentration may be performed with model proteins on heparin resins. Employing proteins with specific affinity for heparin, a much stronger binding is observed, however, some cation exchangers may still be good substitutions for heparin resins. Dynamic capacity at 10% breakthrough compared to static capacity measurements and dynamic capacity displays that approximately 40-80% of the total available capacity is utilized during chromatographic operation depending on flow rate. A general good agreement was obtained between results of this study and data obtained by others. Results of this study may be used in the selection of resins for testing during protein purification process development.     

Comparison of chromatographic ion-exchange resins. I. Strong anion-exchange resins

A comparative study has been undertaken on various strong anion-exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, and dynamic capacity of the chromatographic resins. The resins tested included: Macro-Prep 25Q, TSK-Gel Q-5PW-HR, Poros QE/M, Q Sepharose FF, Q HyperD 20, Q Zirconia, Source 30Q, Fractogel EMD TMAE 650s, and Express-Ion Q. Testing was performed with five different proteins: Anti-FVII Mab (IgG), aprotinin, BSA, lipolase, and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with low pI. No direct link between pH dependence on retention and titration curves of the different resins was observed. Efficiency results show the expected trend of lower dependence of the plate height with increasing flow-rate of resins for medium and high pressure operation compared to the soft resins. Binding to the anion-exchange resins as a function of ionic strength may vary depending on the specific protein. Generally, binding and elution at a high salt concentration may be performed with Poros QE/M or Macro-Prep 25Q, while binding and elution at low salt concentration may be done with TSK-Gel Q-5PW. Dynamic capacities are strongly dependent on the specific protein employed and for some resins dependent on the flow-rate. A general good agreement was obtained between this study and data obtained by suppliers for the dynamic capacity. The results of this study may be used for selection of resins for testing in process development, however, the data does not tell anything about specific selectivity differences or resolution between a target protein and a given impurity. None of the resins studied here should be regarded as good or bad, but more or less suitable for a specific purpose, and only testing for the specific application will determine which one is the optimal resin.     

Comparison of chromatographic ion-exchange resins VI. Weak anion-exchange resins

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.     

Comparison of chromatographic ion-exchange resins V. Strong and weak cation-exchange resins

Strong and weak cation-exchangers were compared for a number of chromatographic parameters, i.e. pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy (SEM) pictures. Chromatographic resins investigated were Fractogel EMD SO3- (M), Fractogel EMD SE Hicap (M), Fractogel EMD COO- (M), MacroPrep 25S, MacroPrep High S, MacroPrep CM, CM HyperZ, and Matrex Cellufine C-500. Testing was done with three proteins: Anti-FVII Mab (IgG), aprotinin, and lysozyme. For lysozyme and aprotinin with pI above experimental pH, dependence of pH on retention was generally low, though some pronounced decrease of retention with increasing pH was observed for CM HyperZ. For Anti-FVII Mab with pI<7.5, binding was observed on several resins at pH 7.5. Efficiency results present the expected trend of increasing dependence of plate height as a function of increasing flow rate, and the highest flow dependence was observed for Fractogel EMD COO-. Particle size distribution was determined by two independent methods, coulter counting and SEM pictures, with fair agreement. Binding strength data of cation-exchange resins as a function of ionic strength depends on the protein, but binding and elution at high salt concentration may in general be performed with MacroPrep resins. Comparison of dynamic capacity data at 10% break-through and static capacity measurements shows that a very diverse utilization of approximately 25-90% of the total available capacity is employed during chromatographic operation. The effect of competitive binding from yeast fermentation components on dynamic binding capacity of aprotinin was studied showing a significant decrease in binding capacity. Sepharose FF, Toyopearl 650 M, and Ceramic HyperD F strong and weak cation-exchange resins were included in this study. Resins with good pure aprotinin capacity also performed well for aprotinin in fermentation broth, but the highest relative capacity was obtained with MacroPrep High S having a fairly low pure component dynamic capacity. Results of this paper may be used in the selection of resins for further testing in biopharmaceutical protein purification process development.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

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

Comparison between mass transfer properties of weak-anion-exchange resins with graft-functionalized polymer layers and traditional ungrafted resins

Glycidylmethacrylate was grafted to Toyopearl HW-65M and subsequently modified with diethylamine to obtain a weak anion exchanger. The degree of grafting was varied from 11 to 50%. The binding capacity for bovine serum albumin was 11 mg/ml for the lowest degree of grafting and 97 mg/ml for the highest degree of grafting. The maximum binding capacity was observed at 27% degree of grafting. The mass transfer properties of the grafted resins and an ungrafted resin(Toyopearl DEAE 650M) were investigated assuming rectangular isotherms. Simple models for reaction kinetics, pore- and surface diffusion and film diffusion were used to describe the concentration-time data in batch mode. The data were best fitted by a pore diffusion model. The estimated pore diffusion coefficients (D(P)) for bovine serum albumin were fitted by a polynome to the degree of grafting with an maximum value at 27% of D(P) = 1.95-10(-11) m2/s. Compared to published data of other ungrafted resins and to the molecular diffusion coefficient of bovine serum albumin in free solution of D(P) = 5.6 10(-11) m2/s, the diffusion in grafted layers seems to be accelerated. The breakthrough curves for columns packed with various resins showed a decrease in sharpness with increasing degree of grafting which could not be described by a simple pore diffusion model using the calculated transport coefficients from batch mode. The shape of the breakthrough curves could be well described by a combined film and pore diffusion model. For the ungrafted Toyopearl DEAE 650M resin the breakthrough curve is more favorable and the influence of film diffusion to the mass transfer is reduced. It can be concluded that grafting will increase the capacity and the pore diffusion in batch mode but in column operation the grafting layer has a film resistance which plays an important role in the overall mass transfer.     

Synthesis of high-capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab' fragments for isolation of proteins

Two methods for synthesizing high-capacity immunoaffinity sorbents on Sepharose and Separon HEMA E-1000 are described. The first is the oriented immobilization of monovalent immunoglobulin Fab fragments on a maleimide derivative of Sepharose via the formation of a covalent bond between the SH group of the Fab fragment at the C-terminus of the molecule and the maleimide covalently coupled to Sepharose. The second method is based on the oxidation of the immunoglobulin carbohydrate component, located in the Fc fragment, by periodate with subsequent immobilization of the derivatives on hydrazide derivatives of Sepharose or Separon. Sorbents for the isolation of monoclonal antibodies from the culture supernatants and the elongation factor EF-G from a crude extract of Escherichia coli cells were obtained. These sorbents are characterized by a high capacity, minimal non-specific sorption and high stability.     

Impact of ionic strength on adsorption capacity of chromatographic particles employed in separation of monoclonal antibodies

The influence of ionic strength on the adsorption capacity of seven commercial adsorbents used in downstream processing of monoclonal antibodies was examined. Affinity (MabSelect, Poros 50A High Capacity, ProSep-vA High Capacity), hydrophobic charge-induction (MEP HyperCel), and cation exchange adsorbents (FractoGel EMD SE Hicap (M), SP Sepharose Fast Flow, Ceramic HyperD F) were used to study the adsorption of polyclonal human immunoglobulin G at optimal pH values. The ionic strength, adjusted by sodium chloride concentrations in the range of 0–225 mM, strongly decreased the adsorption capacity of the cation exchangers. Equilibrium data were described in the form of the dependence of the ratio of protein concentrations in the solid and liquid phases on the total concentration of cation counter ions. They were successfully fitted and interpreted through a stoichiometric ion-exchange model.     

Adsorption of plasmid DNA on anion exchange chromatography media

Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 A 30 microm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of self-assembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.     

Affinity resins for the purification of opioid-binding materials

Two new affinity resins for the purification of opioid-binding materials were prepared. One was AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin and the other was AF-Amino Toyopearl with [D-Ala2, Leu5]enkephalin. Solubilized-opioid receptors from rat brain were treated with these affinity resins and the materials with opioid-binding activities were purified. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the purified materials showed one major band with a molecular weight of 62000-64000. The results suggested that the prepared resins are useful tools for the purification of opioid receptors.     

Protein adsorption and transport in dextran-modified ion-exchange media. II. Intraparticle uptake and column breakthrough

Protein transport behavior was compared for the traditional SP Sepharose Fast Flow and the dextran-modified SP Sepharose XL and Capto S resins. Examination of the dynamic binding capacities (DBCs) revealed a fundamental difference in the balance between transport and equilibrium capacity limitations when comparing the two resin classes, as reflected by differences in the locations of the maximum DBCs as a function of salt. In order to quantitatively compare transport behavior, confocal microscopy and batch uptake experiments were used to obtain estimates of intraparticle protein diffusivities. For the traditional particle, such diffusivity estimates could be used to predict column breakthrough behavior accurately. However, for the dextran-modified media, neither the pore- nor the homogeneous-diffusion model was adequate, as experimental dynamic binding capacities were consistently lower than predicted. In examining the shapes of breakthrough curves, it was apparent that the model predictions failed to capture two features observed for the dextran-modified media, but never seen for the traditional resin. Comparison of estimated effective pore diffusivities from confocal microscopy and batch uptake experiments revealed a discrepancy that led to the hypothesis that protein uptake in the dextran-modified resins could occur with a shrinking-core-like sharp uptake front, but with incomplete saturation. The reason for the incomplete saturation is speculated to be that protein initially fills the dextran layer with inefficient packing, but can rearrange over time to accommodate more protein. A conceptual model was developed to account for the partial shrinking-core uptake to test whether the physical intuition led to predictions consistent with experimental behavior. The model could correctly reproduce the two unique features of the breakthrough curves and, in sample applications, parameters found from the fit of one breakthrough curve could be used to adequately match breakthrough at a different flow rate or batch uptake behavior.     

A comparison of various commercially-available liquid chromatographic supports for immobilization of enzymes and immunoglobulins

A number of commercially-available, activated supports were evaluated and compared for the immobilization of enzymes (alkaline phosphatase, glucose oxidase and peroxidase) and human immunoglobulin G (IgG). The supports studied included pressure-stable, epoxy-activated acrylate-based supports (Separon HEMA 1000 and Eupergit C); agarosebased, epoxy-, cyanogen bromide-, glutaraldehyde- or N-hydroxysuccimide-activated supports (epoxy-activated or cyanogen bromide-activated Sepharose, ACT-Ultrogel AcA 22, Reacti-Gel 6X and Affi-Gel 10); and glass bead-based, activated supports (CDI- and NHS-Glycophase). As expected, the pH required for maximum protein immobilization and retention of activity varied with both protein and support. For example, the amount of alkaline phosphatase coupled was maximum at pH 3 or 5 for most supports, but retention of activity was greatest for immobilization at pH 7, 9 or 11. Glucose oxidase and peroxidase coupling and activity retention in general showed less variation in optimal coupling pH. Coupling of IgG and retention of anti-IgG binding activity were both optimal at a coupling pH of 9 or 11. The Separon HEMA-IgG support made in these studies was also utilized for rapid h.p.l.c, purification of anti-IgG from serum.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.     

Protein adsorption equilibrium and kinetics in multimodal cation exchange resins

Protein adsorption equilibria and kinetics are obtained experimentally for two multimodal cation exchange resins—Nuvia cPrime, which is based on a polymeric matrix, and Capto MMC, which is based on an agarose matrix. In both resins, the ligand contains a phenyl group, a carboxyl group, and a peptide bond but with a different arrangement. Transmission electron microscopy and inverse size exclusion chromatography indicate a bimodal distribution of pores in Nuvia cPrime, including small pores with 10 nm radius and pores larger than 400 nm, and a monodispersed distribution of pores in Capto MMC, averaging 32 nm in radius. Potentiometric titration curves show similar buffering ranges and pK _a values for the ligands in both resins and a slightly higher ligand density for Nuvia cPrime. Equilibrium binding capacities for lysozyme and a monoclonal antibody (mAb) are also similar for both resins at comparable pH and salt concentrations, although Capto MMC shows a weaker dependence on salt concentration as a result of its more hydrophobic character. The main difference is the adsorption kinetics of the mAb, which is the larger of the two proteins studied. For both resins, as shown by means of confocal laser scanning miscopy, the adsorption kinetics is controlled by pore diffusion. Capto MMC with its smaller pores has a slower rate of mass transfer than Nuvia cPrime. As a result, for the mAb, much higher column dynamic binding capacities are obtained for Nuvia cPrime than for Capto MMC.     

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.     

Isolating the whole complex of target proteins of FK506 using affinity resins from novel solid phases

The development of novel solid phases enabled us to create affinity resins that could be used to isolate the whole complex of target proteins responsible for the immunosuppressive effects of FK506 from rat brain lysate, whereas the affinity resins from commercially available matrices could not achieve this isolation. The results illustrate the enhanced effectiveness of the affinity resin made from this novel material at identifying the target protein of the bioactive compound compared to resins made from the well-known materials Affigel or Toyopearl. This effectiveness arises because the novel material is hydrophilic enough to reduce nonspecific binding proteins and because it has a higher density of ligands that capture the nonubiquitous target protein. Electronic Supplementary Material Supplementary material is available for this article at     

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes

Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 °C and stable between pH 7.0 and 10.0. SOMP was stable below 45 °C and Ca²⁺ increased its thermostability. Ca²⁺ enhanced while Co²⁺, Cu²⁺, Zn²⁺, Mn²⁺, and Fe²⁺ inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H₂O₂. The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.     

Effect of pH changes on water release values in hydrophobic interaction chromatographic systems

The effect on pH on protein binding in HIC systems was investigated. Isocratic experiments were carried out to determine the capacity factors of various proteins as a function of temperature, pH and salt type. This paper presents a framework based on the Maxwell linkage function for estimating the number of released water molecules during the adsorption/desorption process due to a change of buffer pH. This approach also enables one to predict the effect of pH change on the water released values upon binding at any temperature condition. The results indicate that the total number of released water molecules (delta nu) for a pH change increased more on aromatic surfaces (phenyl Sepharose) than on aliphatic resins (butyl Sepharose). The results also indicate that the total number of released water molecules (deltanu) for a pH change increased with salt concentration and when changing from chaotropic to kosmotropic salts. The (deltanu) values also increased as the buffer pH approached the protein's pI, and decreased away from its pI. This work helps to establish a framework for the investigation of pH effects on protein selectivity in HIC systems.     

Removal of iodide from aqueous solutions by polyethylenimine-epichlorohydrin resins

The iodide removal from aqueous solutions (initial I^?-concentration: 300–5,000 mg/L) by a low and a high molecular weight polyethylenimine-epichlorohydrin resin was investigated both in absence and presence of background electrolytes (NaCl and Na₂SO₄, ionic strength due to background electrolyte 0.1 M) using a batch technique, ¹³¹I as radioactive tracer and high-resolution γ-ray spectrometry. The experiments in absence of background electrolyte were performed using solutions of initial pH 3 and 7, whereas those in presence using solutions of initial pH 3. The results, which demonstrated the high iodide-removal efficiency of both resins, were modeled using the Langmuir and Freundlich isotherm equations. The experimental data were better reproduced using the Langmuir equation. Using this equation maximum sorption capacity values (Q _max) of 638.8 and 603.3 mg/g were obtained for the high molecular weight resin from solutions of initial pH 3 and 7 respectively, whereas the corresponding values for the low molecular weight one were slightly lower (552.4 and 507.5 mg/g respectively). The iodide uptake by the resins strongly depended on the presence of competing anions and especially of sulfates. The examination of sections of the I-loaded resins grains by scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) revealed that iodine was evenly distributed throughout the bulk of the resins and not only bound to their surface.