Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.     

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.     

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.     

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.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations

The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.     

On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption

Protein refolding at high concentrations always leads to aggregation, which limits commercial application. An ion-exchange chromatography process with gradient changes in urea concentration and pH was developed to refold denatured lysozyme at high concentration. After adsorption of the denatured protein onto an ion-exchange medium, elution was carried out in combination with a gentle decrease in urea concentration and elevation of pH. Protein would gradually refold along the column with high activity yield. Denatured and reduced lysozyme at 40 mg/ml was loaded into a column filled with SP Sepharose Fast Flow, resulting in 95% activity recovery and 98% mass yield within a short period of time.     

Oscillatory electroosmosis-enhanced intra/inter-particle liquid transport and its primary applications in the preparative electrochromatography of proteins.

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE-Sepharose Fast Flow, and BSA on hydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale chromatography.     

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.     

Z(basic)--a novel purification tag for efficient protein recovery

A positively charged protein domain, Z(basic), can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic), was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic) tag in small and pilot-plant-scale downstream processing.     

Fabrication of Hard Dextran DEAE: Adsorption Equilibria of BSA

A hard dextran-DEAE ion exchanger (Hard Dextran DEAE) was developed. It is hard and keeps good properties of dextran-DEAE for protein separation. It is not compressed in a column and can be used in much wider range of flow rate in the column than the commercial hard gel, DEAE Sepharose Fast Flow made of agarose. The saturation capacity of BSA on Hard Dextran DEAE is about 1.7 times of that on DEAE Sepharose Fast Flow at pH 6.9. Equilibrium isotherm for adsorption of BSA depends on pH considerably. When pH 5.5, the equilibrium isotherm is correlated by the Langmuir equation. When pH 5.05, the isotherm is correlated by the Freundlich equation. The higher the concentration of NaCl is, the smaller the amount of BSA adsorbed. When the concentration of NaCl is higher than 100 mol m–3 at pH 6.9 and 50 mol m–3 at pH 4.8, BSA was not adsorbed on the resin. This may suggest that BSA is adsorbed by electrostatic attraction. About 100 mol m–3 NaCl aqueous solution can be used as an eluant of 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.     

Measurement of ligand distribution in individual adsorbent particles using confocal scanning laser microscopy and confocal micro-Raman spectroscopy

Two methods, confocal scanning laser microscopy and confocal micro-Raman spectroscopy were used to analyse the distribution of IgG antibodies immobilized on CNBr-activated agarose beads. In the first method the internal distribution profile of fluorescent labelled Protein A was used as an indirect measure of the distribution of IgG, while the second method detects vibrations originating from aromatic amino acids present in the immobilized antibodies. Both these methods indicate an homogeneous ligand distribution within IgG Sepharose 4 Fast Flow and IgG Sepharose 6 Fast Flow.     

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。     

Enrichment of proteinaceous materials on a strong cation-exchange diol silica restricted access material: protein-protein displacement and interaction effects

A study of size exclusion and enrichment of proteins employing strong cation-exchange diol silica restricted access material (SCX-RAM) under saturation conditions is presented. Experiments were carried out with bacitracin, protamine, ribonuclease, lysozyme and bovine serum albumin as individual proteinaceous analytes as well as comprehensive binary mixtures and with human urine samples. Protein size dependent capacity features of the SCX-RAM column was observed. Bacitracin demonstrated the highest capacity followed by protamine while adsorption capacities of both ribonuclease and lysozyme were found smaller by a factor of 10. Applying binary protein samples occurring displacement effects were apparent: proteins with strong cationic properties displaced those already adsorbed by the bonded cation-exchange ligands. Bacitracin was displaced in all binary mixture experiments in particular by protamine. Furthermore, the binary mixtures displayed increased adsorption for some proteins due to complex formation. Lysozyme and ribonuclease showed double capacity values when paired with bacitracin. Both phenomena, displacement and enhanced adsorption occurred in the saturated state and led to changes in the urine composition during sample preparation. Injecting urine samples the relative proportions of fractions changed from 4 up to more than 20 times, due to the differences of the protein adsorption capacities on the SCX-RAM column. Analysing urine samples the SCX-RAM column provided extensive long-term stability.     

Chromatographic behaviors of proteins on cation-exchange column

A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process.The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed.     

Determinants of protein retention characteristics on cation-exchange adsorbents.

There are currently a large number of commercially available strong and weak cation-exchange adsorbents for preparative protein purification, typically prepared by coupling charged ligands to a mechanically rigid porous bead. Because of the diverse chemical nature of the base matrix (carbohydrate, synthetic polymer, inorganic) and the coupling and ligand chemistry, cation-exchange adsorbents from different suppliers can differ substantially in chemical surface properties and physical structure. The differences in chemical properties can be in ionic capacity, hydrophobicity, the presence of hydrogen bond donors/acceptors, and the nature of the charged functional groups. In order to probe the effects of these factors on protein affinity, the isocratic retention of a set of model proteins was examined on a set of cation-exchange adsorbents to obtain a quantitative assessment of retention differences between adsorbents. Two adsorbent factors were found to be the dominant determinants of overall protein retention: the anion type and the adsorbent pore size distribution. Protein retention on strong cation-exchangers was found to be greater than that on corresponding weak cation-exchangers. Protein retention was increased on adsorbents with pore size distributions that include significant amounts of pore space with dimensions similar to those of the protein solute.     

Protein-labeling effects in confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) is being increasingly used for observing protein uptake in porous chromatography resins. Recent CLSM studies have revealed the possible existence of a nondiffusive protein transport mechanism. Observing protein uptake with CLSM requires labeling the protein with a fluorescent probe. This study examines the effect of the probe identity on the subsequent CLSM adsorption profiles. The adsorption of lysozyme conjugated with different fluorescent probes (Cy5, BODIPY FL, Atto 635, and Atto 520) on SP Sepharose Fast Flow was measured using CLSM and zonal chromatography experiments. Results from zonal chromatography show that the retention time of lysozyme-dye conjugates differ significantly from unlabeled lysozyme. The change in retention of lysozyme upon conjugation with a fluorescent probe is consistent with the difference in net charge between the lysozyme-dye conjugate and unlabeled lysozyme. The adsorption profiles measured by CLSM show significantly different behavior depending upon whether the lysozyme-dye conjugate is retained longer or shorter than the unlabeled lysozyme. These results strongly suggest that the lysozyme concentration overshoot observed in previous CLSM experiments is the result of displacement of weaker binding labeled lysozyme by stronger binding unlabeled lysozyme.     

Chromatography of human prothrombin from Nitschmann fraction III on DEAE Sepharose Fast Flow using axial and radial flow column

An axial column (3 x 2.6 cm) and a radial flow column (3.5 x 5 cm) packed with DEAE Sepharose Fast Flow media was evaluated for the separation of human prothrombin from Nitschmann fraction III. Under radial flow conditions, a sample flow rate up to 14 mL/min (approximately 18 bed vols/h) was achieved. Breakthrough capacity was determined and both columns had almost the same breakthrough capacity per mL media, indicating that the sample loading was independent of radial column geometry.