Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography

This work examines the relationship between the physical properties of agarose and dextran-grafted agarose cation exchangers and protein adsorption equilibrium and rates. Four different sulfopropyl (SP) matrices were synthesized using a neutral agarose base material--two based on a short ligand chemistry and two obtained by grafting 10 and 40kDa dextran polymers. The pore accessibility, determined by inverse size exclusion chromatography (iSEC) with dextran probes, decreases dramatically as a result of the combined effects of crosslinking, dextran grafting, and the introduction of ionic ligands, with pore radii decreasing from 19nm for the base matrix to 6.1nm for the 40kDa dextran-grafted SP-matrix. In spite of this reduction, while the adsorption isotherms were similar, protein uptake rates were greatly increased with the dextran-grafted SP-matrices, compared to SP-matrices based on the short ligand chemistry. The effective pore diffusivities were 4-10 times higher than free solution diffusivity for the dextran-grafted matrices, indicating that the charged dextran grafts result in enhanced protein mass transfer rates.     

Two-component protein adsorption to the cation exchanger S Sepharose FF

A model system, consisting of bovine serum albumin, lysozyme and the cation exchanger S Sepharose FF, was used to investigate multicomponent protein adsorption to ion exchangers. Two models, one based on a complete absence of competition between adsorbing molecules and the other a competitive model, based on the assumption that all adsorption sites are available to both proteins, have been compared to experimental results. Evidence for competitive adsorption was seen in experiments in which breakthrough curves and the profiles of adsorbed proteins in packed beds were determined. However, although the results for packed-bed experiments were more closely predicted by the fully competitive model, some discrepancies were found suggesting that when considering multicomponent protein adsorption to ion exchangers it may also be necessary to take account of factors such as the molecular size of the adsorbing proteins and any potential inter-protein interactions, factors which may hinder the development of a general model of multicomponent protein adsorption to ion exchangers.     

Modeling of salt and pH gradient elution in ion‐exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion‐exchange columns is a well‐established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO₃ (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well‐characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.     

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.     

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

Chemical characterisation of different separation media based on agarose by static time-of-flight secondary ion mass spectrometry

In this paper, the novel application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for qualitative and semi-quantitative investigation of the surface chemistry of separation media based on beaded agarose is reported. Five different media were studied: DEAE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, Phenyl Sepharose Fast Flow at ligand densities between 7 and 33% (w/w) and the base matrix Sepharose 6 Fast Flow. The obtained TOF-SIMS spectra reveal significant chemical information regarding the ligands (DEAE, Q, SP and Phenyl) which are covalently attached to the agarose-based matrix Sepharose 6 Fast Flow. For the anion-exchange media (DEAE and Q Sepharose Fast Flow), the positive TOF-SIMS spectra yielded several strong characteristic fragment peaks from the amine ligands. Structural information was obtained, e.g. from the peak at m/z 173.20, originating from the ion structure [(C2H5)2NCH2CH2NH(C2H5)2l+, which shows that the ligand in DEAE Sepharose Fast Flow is composed of both tertiary and quaternary amines. The positive spectrum of Phenyl Sepharose Fast Flow contained major fragments both from the base matrix and the ligand. The cation-exchanger (SP Sepharose Fast Flow) gave rise to a positive spectrum resembling that of the base matrix (Sepharose 6 Fast Flow) but with a different intensity pattern of the matrix fragments. In addition, peaks with low intensity at m/z 109.94, 125.94 and 139.95 corresponding to Na2SO2+, Na2SO3+ and Na2SO3CH2+, respectively, were observed. The positive TOF-SIMS spectrum of Sepharose 6 Fast Flow contains a large number of fragments in the mass range up to m/z 200 identified as CxHyOz and CxHy structures. The results clearly show that positive TOF-SIMS spectra of different media based on Sepharose 6 Fast Flow are strongly influenced by the ligand coupled to the matrix. The negative TOF-SIMS spectra contained several ligand-specific, characteristic peaks for the cation-exchanger, having sulphonate as the ion-exchange group. Negative fragments such as S-, SO-, SO2-, SO3-, C2H3SO3-, C3H5SO3- and OC3H5SO3- were observed. Phenyl Sepharose Fast Flow, which has an uncharged group (Phenyl) coupled to the agarose matrix yielded one ligand-related peak corresponding to the C6H5O- fragment. DEAE and Q ligands could only be identified by the appearance of the fragments CN- and CNO- in the negative spectrum. However, a strong peak corresponding to the counter ion (Cl-) was observed. TOF-SIMS analysis can also be used for the investigation of residues from the coupling procedure that bonds the ligands to the matrix. One example is the observation of bromine peaks in the negative spectrum of Q Sepharose Fast Flow. Furthermore, it has also been shown that different ligand concentrations of Phenyl Sepharose Fast Flow can easily be detected by TOF-SIMS analysis. Information regarding the difference between the ligand density on the surface of the beads and in the bulk can also be obtained. However, spectra registered on the outermost surface and on the pore surface (crushed beads) of DEAE Sepharose Fast Flow clearly show that the agarose and the DEAE groups are homogeneously distributed in the beads.     

Binding and elution behavior of proteins on strong cation exchangers

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

Comparison of Sepharose CL-2B and CL-4B Matrices in Purification of the Hepatitis B Surface Antigen by Affinity Chromatography

Sepharose CL-4B and Sepharose CL-2B with immobilized CB.Hep-1 monoclonal antibody were compared for studying the matrix efficiency in the immunopurification of the recombinant hepatitis B surface antigen (rHBsAg). The elution capacities of both matrices were similar over eight chromatographic cycles, significant differences were only observed in the first purification cycles. A high percentage of the adsorbed rHBsAg was not eluted from both matrices. The rHBsAg purity was not affected by matrix characteristics (pore size and percentage of agarose) and differences between Sepharose CL-4B and Sepharose CL-2B do not provoke differences in the antibody released from the matrices under defined experimental conditions.     

Comparison of chromatographic ion-exchange resins. II. More strong anion-exchange resins

A comparative study was performed on strong anion exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included Q Sepharose XL, UNO Q-1, Poros 50 HQ, Toyopearl QAE 550c, Separon HemaBio 1000Q, Q-Cellthru Bigbeads Plus, Q Sepharose HP and Toyopearl SuperQ 650s. Testing was performed with five different proteins: anti-Factor VII monoclonal antibody (immunoglobulin G), aprotinin, bovine serum albumin, lipolase and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with a low isoelectric point (pl). An unexpected binding at pH 7-8 of aprotinin with pI >11 was observed on Separon HemaBio 1000Q. No link between pH dependence on retention and titration curves of the different resins was observed. 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. No or a very small difference in particle size distribution was obtained between new and used resins. Binding to anion-exchange resins as a function of ionic strength varies to some extent depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Q Sepharose XL, Toyopearl QAE 550c, Q Sepharose HP and Poros 50 HQ, while binding and elution at low salt concentration may be performed with Q-Cellthru Bigbeads Plus. A very high binding capacity was obtained with Q Sepharose XL. Comparison of static capacity and dynamic capacity at 10% breakthrough shows approx. 50-80% utilization of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by the suppliers. The results of this study may be used for selection of resins for testing in process development.     

Physicochemical basis of the ion-exchange separation of gold cyanide complexes

The mechanism of the separation of gold cyanide complexes is discussed, along with ion exchanger selection, selectivity, elution, and industrial applications. The ion-exchange mechanism for the sorption of gold cyanide complexes is established, and a criterion is suggested for selecting the anion exchanger for their extraction (specifically, the pK _a of the anion exchanger). The selectivity of the sorption of gold cyanide complexes by anion exchangers with rarely distributed ionogenic groups is demonstrated. A procedure for the elution of gold cyanide complexes using alkaline solutions is developed.     

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.     

Investigation of the resistance of some naturally occurring and synthetic inorganic ion exchangers against gamma radiation

The effect of various doses of gamma radiation on the ion-exchange capacity, distribution coefficient values, elution behaviour, physical effect, pH titration and infrared spectra of some synthetic inorganic ion exchangers, namely the cerium substituted phosphates; and naturally occurring inorganic ion exchangers, zeolites from different parts of Iran, have been studied systematically.No significant change has been observed in the ion-exchange capacity (with the exception of CeP(Na), CeP(Di·Na) and zeolite 5 (deposits of arababad talas)), elution behaviour, physical effect, chemical stability and the infrared spectra of the synthetic ion exchangers irradiated upto a total dose of 200 kGy, while a change has been observed in the pH-titration and distribution behaviour. The increase in pH is sharper for irradiated samples with divalent cations than for the normal samples. Furthermore, the K_d values, and hence the selectivity towards certain cations increase with the total dose absorbed, reaching its optimum selectivity with the dose of 50–100 kGy. The natural zeolites chosen for these studies, show, similar pattern to those of synthetic ion exchangers, and in some cases an extremely high selectivity toward certain cations, like Be^II. These make, zeolites, which are naturally occurring ion exchangers more viable economically, and extremely useful alternative in this industry.     

Methods for Preparing High Performance Stationary Phases for Anion-Exchange Chromatography

The development of new high-performance anion-exchange stationary phases is one of the priority tasks of modern ion chromatography (IC). Most of the research in this field is aimed at improving the selectivity of anion exchangers and increasing the sensitivity, efficiency, and rapidity of the analysis. The selectivity of the stationary phases is largely determined by the chemistry of the adsorbent surface; therefore, it is particularly important to understand the relationship between the structure of the functional layer of the anion exchanger and its chromatographic properties. This review is devoted to methods for modifying polymer matrices in order to obtain highly efficient anion exchangers for the separation of inorganic and organic anions.     

Tris(hydroxymethyl)aminomethane-functionalized agarose particles: parameters affecting the binding of bovine serum albumin

A new protein adsorbent is introduced based on the coupling of the common buffer ion, tris(hydroxymethyl)aminomethane, to the agarose gel Sepharose HP from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. The article describes the synthesis of the new adsorbent and the use of BSA as a model in a binding study. By optimization of the coupling procedure, a maximum ligand density of 63.5 μmol/mL gel could be obtained. Adsorption equilibria were investigated in the pH range 5.0-8.0 and at salt concentrations of 0-0.4 mol/L. Binding of BSA under different conditions indicated that both electrostatic interaction and hydrogen bonding were involved in the adsorption process where the former played a major role.     

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.     

Ion-exchange high-performance liquid chromatography of nucleotides and polypeptides on new types of ion-exchange sorbents, based on polystyrene-coated silicas.

A novel type of ion exchanger was prepared by multipoint covalent binding of polystyrene chains onto the surface of porous silica followed by polymer-analogous modification of the bonded layer. Both anion and cation exchangers were synthesized and examined in the separation of nucleotides and proteins. Rapid and efficient separation of basic polypeptides on strong anion exchangers and that of acidic polypeptides on strong cation exchangers could be achieved. For the separation of complete mixtures of polypeptides the application of zwitter-ionic ion exchangers can be recommended.     

Comparison of separation efficiency of spheroid cation exchangers for lanthanides and transplutonium elements

The advantages of fine spheroid cation exchangers /Ostion, Aminex/ were made use of in the separation of lanthanides and transplutonium elements by low-pressure elution.     

阴离子交换和羟基磷灰石色谱从螺旋藻中提纯藻胆蛋白的研究

本文采用离子交换层析和羟基磷灰石吸附层析技术从螺旋藻中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较 ＡＮＸ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗ（ｈｉｇｈ ｓｕｂ／ｌｏｗ ｓｕｂ）、 ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ和 Ｑ Ｓａｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ等阴离子交换树脂的动态吸附容量以及目标产品的纯度,选用 ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ作为层析介质．对离子交换的产物进行了电泳分析,藻蓝蛋白和别藻蓝蛋白的等电点接近,电迁移速率相似。采用羟基磷灰石吸附技术对藻胆蛋白混合物进一步分离纯化,分别得到了藻蓝蛋白和别藻蓝蛋白的纯品,经等电聚焦实验验证显示为均一组成。     

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.