SP Sepharose XL Research Articles

Effects of Resin Architecture and Protein Size on Nanoscale Protein Distribution in Ion-Exchange Media.

Knowledge of the nanoscale distribution of proteins in chromatographic resins is critical to our mechanistic understanding of separations performance. However, the nano- to mesoscale architecture of these materials is challenging to characterize using conventional techniques. Small-angle neutron scattering was used to probe (1) the nano- to mesoscale structure of chromatographic media and (2) protein sorption in these media in situ with protein-scale resolution. In particular, we characterize the effect of the architecture of cellulose-based and traditional and dextran-modified agarose-based ion-exchange resins on the nanoscale distribution of a relatively small protein (lysozyme) and two larger proteins (lactoferrin and a monoclonal antibody) at different protein loadings. Traditional agarose-based resins (SP Sepharose FF) can be envisioned as comprising long, thin strands of helical resin material around which the proteins adsorb, while higher static capacities are achieved in dextran-modified resins (SP Sepharose XL and Capto S) due to protein partitioning into the increased effective binding volume provided by the dextran. While protein size is shown not to affect the underlying sorption behavior in agarose-based resins such as SP Sepharose FF and XL, it plays an important role in the cellulose-based S HyperCel and the more highly cross-linked agarose-based Capto S, where size-exclusion effects prevent larger proteins from binding to the base matrix resin strands. Based on the data, we propose that entropic partitioning effects such as depletion forces may drive the observed protein crowding. In general, these observations elucidate the structure and point to the mechanism of protein partitioning in different classes of chromatographic materials, providing guidance for optimizing their performance.

Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high-throughput small-scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two-step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two-step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1.

Cover Chem. Eng. Technol. 1/2012

AbstractMicroscopy Methods used to Elucidate Protein Adsorption and Transport Behavior on Novel Chromatographic MediaTop left: polarization anisotropy map for fluorescently labeled lysozyme in solution and adsorbed on SP Sepharose XL particles. Top right (courtesy of Dr. Harun Koku): SEM of Sepharose FF (left half) and Sepharose XL (right half), both with protein adsorbed. Bottom right: photobleaching of fluorescently labeled lysozyme adsorbed on SP Sepharose FF. Bottom left: confocal microscopy of simultaneous loading of fluorescently labeled lysozyme (green) and lactoferrin (red) on SP Sepharose FF.DOI: 10.1002/ceat.201100304B. D. Bowes, S. J. Traylor, S. M. Timmick, K. J. Czymmek, A. M. Lenhoff*Insights into Protein Sorption and Desorption on Dextran‐Modified Ion‐Exchange MediaChem. Ing. Technol. 2012, 35 (1), 91–101

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

Abstract 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.

Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-β, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-β products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-β was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-β with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose XL column. The two major protein species separated through a SOURCE 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-β with minimal inhibitory concentrations of 8-64 μg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-β, and that expressed hPAB-β in P. pastoris may be a potential antimicrobial agent against MRSA infection.

Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents

Chromatography is the most widely used technique for the purification of biopharmaceuticals in the biotech industry. Surprisingly, process development is often still based on empirical studies or experience; recently high-throughput screening stations are employed to minimize development time and to improve screening quality. Still, experimental effort remains high and a more detailed understanding of adsorption mechanisms on a molecular level underlying chromatographic separation could help in the future to select and design chromatography steps in silico. In this study, we focused on the elucidation of protein orientation upon adsorption onto a chromatographic resin. We identified two characteristic binding sites of lysozyme on SP Sepharose Fast Flow and one multipoint interaction of lysozyme with SP Sepharose XL. Increasing ionic strength did not significantly influence the binding, whereas changes in the mobile phase pH led to a re-orientation on SP Sepharose FF. This phenomenon agrees well with theoretical considerations, including a detailed description of the surface charge distribution with changing pH and linear elution experiments, giving an idea why proteins are often retained on ion-exchange materials beyond their isoelectric point.

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.

Stationary phase effects on the dynamic affinity of low-molecular-mass displacers

In this paper, the selectivity of a variety of cation-exchange stationary phases was investigated using a homologous series of displacer molecules based on pentaerythritol. These displacers were derived from pentaerythritol and contained either four trimethyl ammonium groups [pentaerythrityl-(trimethylammonium chloride) 4, PE(TMA) 4], benzene rings [pentaerythrityl-(benzyl dimethylammonium chloride) 4, PE(DMABzCl) 4], heptyl groups [pentaerythrityl-(heptyl dimethylammonium iodide) 4, PE(DMAHepI) 4] or cyclohexyl groups [pentaerythrityl-(cyclohexyl dimethylammonium iodide) 4, PE(DMACyI) 4]. This series enabled us to probe the secondary interactions that can play a role in the affinity of low-molecular-mass displacers for different stationary phases. The relative affinities of these displacers were examined using a displacer ranking plot based on the steric mass action (SMA) isotherm model. While hydrophobicity and aromaticity played important roles in generating the affinity to the hydrophilized polystyrene–divinylbenzene (Source 15S) and polymethacrylate-based (Toyopearl 650M) resins, these secondary interactions had a minimal impact on the selectivity in agarose resins coated with dextran (SP Sepharose XL), “gel in a shell” (S Ceramic HyperD F), and monolithic (Bio-Rad Uno S6) cation-exchange materials. Further, the results with a tentacular stationary phase (Fractogel EMD) suggest that the alkyl chains on PE(DMAHepI) 4 play an important role in increasing the affinity, possibly because of strong interactions between the alkyl moiety and the polymer matrix as well as between the charged groups and the polyelectrolyte tentacles. The results of this study provide insight into the design of high affinity, low-molecular-mass displacers for different cation-exchange stationary phase materials.

Visualising intraparticle protein transport in porous adsorbents by confocal microscopy

Confocal scanning microscopy was used to study protein uptake to porous adsorbents during batch experiments in a finite bath. By coupling of a fluorescent dye to the protein molecules the penetration of single adsorbent particles at different times during batch uptake could be observed visually. Intensity profiles of the protein distribution within a single particle were obtained by horizontal scanning. After integration of the profiles the overall fluorescence within a bead could be calculated. Relating the overall fluorescence at different incubation times to the value at equilibrium allowed the construction of the fractional approach to equilibrium versus time. These data were compared to uptake curves, which had been obtained by measurements of the protein concentration in the supernatant and an excellent agreement of the curves was detected. The procedure was performed for two different proteins (lysozyme and human IgG) on two different media for protein adsorption (SP Sepharose Fast Flow and SP Sepharose XL; Pharmacia Biotech) and in all cases it could be shown, that the results from the direct measurements by confocal microscopy correspond very well to the data obtained from the indirect measurements in the fluid phase.