Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind^® D membrane adsorber and HiTrap™ DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind^® D membrane at equivalent volumetric throughput and higher capacities than the HiTrap™ DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap™ DEAE FF resin column and was the same for the new AEX membrane and Sartobind^® D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.     

One‐step refolding and purification of recombinant human tumor necrosis factor‐α (rhTNF‐α) using ion‐exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic‐based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor‐α (rhTNF‐α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF‐α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF‐α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10⁸ IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF‐α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF‐α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.     

批量离子交换层析和凝胶过滤层析在类人胶原蛋白I纯化中的应用

研究了利用批量离子交换层析与凝胶过滤层析相结合的方法纯化类人胶原蛋白I的最佳条件。分别考察了不同离子交换树脂、缓冲溶液pH、离子强度、进料蛋白浓度对批量离子交换的影响,以及不同凝胶过滤介质对凝胶过滤层析的影响。结果表明,采用阴离子交换树脂DEAE52吸附杂蛋白,缓冲液pH为7.0,NaCl浓度为0.2mol/L,进料蛋白浓度为40mg/mL,并采用凝胶过滤介质SephadexG-100进一步纯化后,类人胶原蛋白I的纯度可达到98.2%,总纯化倍数为3.1,总回收率为80.6%。SDS-PAGE分子量分析和N末端氨基酸序列分析均表明纯化后的类人胶原蛋白I与基因设计一致。     

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N^pro autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO₃⁻, UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris–HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO₃⁻, refolding could be performed at column loadings of 50 mg fusion protein/ml gel, resulting in a final eluate concentration of around 10–15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.     

Chromatographic purification of human α1 proteinase inhibitor from dissolved Cohn fraction IV-1 paste

A novel chromatographic process for purification of α₁ proteinase inhibitor (α₁-PI) from Cohn fraction IV-1 paste is described. This process has been successfully scaled up to 50-l columns. It involves DEAE chromatography, sulfopropyl (S) cation chromatography, tri-n-butyl phosphate (TNBP)–cholate treatment, a second S cation chromatography, freeze–drying and dry-heat. The process has been optimized for purity, yield, lipid removal, chemical usage and water consumption. Filtration after TNBP–cholate treatment plays a key role in ensuring a low lipid content in the final product. Pre-equilibration with high salt buffer is necessary to reduce the water consumption significantly during the ion-exchange chromatography equilibration step. The final product is approximately 95% pure by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with a 64% to 70% yield from IV-1 paste.     

Separation of influenza virus‐like particles from baculovirus by polymer‐grafted anion exchanger

The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 10⁹ particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles.     

Efficient separation of homologous α-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography

Transgenic bovine milk could be a rich source of recombinant human proteins. However, the co-presence of bovine and human homologous proteins can be a challenge for product purification. In this study, the average surface hydrophobicity and electric potential of human α-lactalbumin (HLA) and bovine α-lactalbumin (BLA) were analyzed and compared through the exposure area calculation of different amino acids. Based on the analysis, calcium independent hydrophobic interaction chromatography was selected for separation of recombinant human α-lactalbumin (rHLA) from BLA in transgenic bovine milk. The operating conditions for the best separation of two proteins were predicted by fluorescence data. Three commercially available HIC resins (Butyl Sepharose 4 FF, Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF) were compared. The transgenic milk was skimmed and treated by pH adjustment to remove a large quantity of casein protein. The supernatant was loaded on the hydrophobic interaction chromatographic matrix. The correct elution fraction was further treated with gel filtration chromatography. The overall recovery of rHLA was up to 67.1% with the purity greater than 95%. Circular dichroism spectroscopy (CD) and mass spectrogram (MS) confirmed the native state and glycosylated form of the purified rHLA.     

A novel stationary phase derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed protein chromatography

Using agarose coated gigaporous polystyrene microspheres as a base support, a novel anion exchanger (DEAE-AP) has been developed after functionalization with diethylaminoethyl chloride. The gigaporous structure, static adsorption behavior, and chromatographic properties of DEAE-AP medium were characterized and compared with those of commercially available resin DEAE Sepharose Fast Flow (DEAE-FF). The results implied that there existed some through pores in DEAE-AP microspheres, which effectively reduced resistance to stagnant mobile phase mass transfer by inducing convective flow of mobile phase in the gigapores of medium. As a consequence, the column packed with DEAE-AP exhibited low column backpressure, high column efficiency, high dynamic binding capacity and high protein resolution at high flow velocity up to 2600 cm/h. In conclusion, all the results suggested that the gigaporous absorbent is promising for high-speed protein chromatography.     

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。     

Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by ion exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Application of liquid chromatography to protein refolding is an exciting step forward for this field. In this work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography (IEC) with a Q Sepharose FF column. Several chromatographic parameters affecting the refolding yield of the denatured/reduced rhG-CSF, such as the urea concentration, pH value, concentration and ratio of reduced/oxidized glutathione in the mobile phase, as well as the flow rate of the mobile phase, were investigated in detail and indicated that the urea concentration and the pH value were of great importance. At the optimal conditions, the renatured and purified rhG-CSF was found to have a specific bioactivity of 3.0 x 10(8) IU/mg, a purity of 96%, and a mass recovery of 49%. Compared with the usual dilution method, the IEC method developed here is more effective for rhG-CSF refolding in terms of specific bioactivity and mass recovery.     

Production of Influenza Virus HA1 Harboring Native-Like Epitopes by <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.     

Comparison of agarose and dextran-grafted agarose strong ion exchangers for the separation of protein aggregates

The control of aggregate levels in recombinant protein based drugs is a primary concern during process development and manufacture. In recent years, a novel class of dextran-grafted ion exchange matrices has gained popularity for process scale protein purification due to increased mass transfer rates and higher dynamic binding capacity compared to conventional matrices. Using bovine serum albumin and a monoclonal antibody as model proteins, we studied Sepharose FF and Sepharose XL ion exchangers for the separation of protein aggregates. Experimental results comparing linear gradient elution, stepwise elution, and flow-through chromatography for aggregate separation are described. Differences in performance for the various ion exchangers are discussed and modeled. Strategies for the optimization of protein aggregate separation are provided.     

Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin

Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 10¹⁰ viral particles/mL to at least 1.64 × 10¹¹ viral particles/mL, the limit of detection was found to be 2.56 × 10⁹ virus particles/mL and the limit of quantification was 5.12 × 10⁹ virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.     

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.     

Interaction of soybean agglutinin with leukemic T-cells and its use for their in vitro separation from normal lymphocytes by lectin-affinity chromatography

A procedure for separation of leukemic T-cells from normal lymphocytes, using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently coupled with soybean agglutinin (SBA), then served as an affinity probe for fractionation of mixture of normal lymphocytes and leukemic cells. Leukemic cell lines, derived from acute lymphoblastic leukemia (Jurkat, MOLT-4, RPMI-8402), were tested. The elution of normal lymphocytes was carried out by PBS(-). The leukemic T-cells, interacting with SBA, were removed by N-acetyl-D-galactosamine or low-concentration acetic acid. The type and viability of the separated cell fractions were analyzed by flow cytometry and fluorescent microscopy, using adequate fluorescent antibodies. The interaction of leukemic T-cells with free SBA, as well as with SBA-conjugated Sepharose beads, was examined fluorimetrically and visualized by fluorescent microscopy, using FITC-SBA as a marker. The rate of cell elution on SBA-affinity column decreased in order: normal > leukemic T-cells. Both normal lymphocytes and leukemic T-cells were removed in a mixture from SBA-free Sepharose 6MB by PBS(-) and were not fractionated discretely. The leukemic T-cells specifically interacted with SBA as well as with SBA-affinity adsorbent. In contrast, the normal lymphocytes did not interact with free SBA as well as with SBA-conjugated Sepharose beads in the concentrations applied. The method potentially combines a discrete cell fractionation with manifestation of a specific target cytotoxicity of SBA against leukemic T-cells, without any influence on normal lymphocytes.     

GC/MS-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza A virus

Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.     

Isolation of beta-glucan from the cell wall of Saccharomyces cerevisiae

Beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae (S. cerevisiae), has been found to enhance immune functions. At present study, we developed an optimal procedure to extract and purify beta-glucan. At first, yeast cells were grown in sabouraud dextrose agar and then cultured in yeast extract-peptone-glucose (YPG) broth. After incubation, cells were harvested, washed and disrupted by means of sonication method. The obtained cell walls were used to prepare alkali-soluble beta-glucan (glucan-S1). In this regard, 2% sodium hydroxide (NaOH) and 3% acetic acid were used in alkaline-acid extraction, respectively. This preparation contained 2.4% protein. In the next step, DEAE sephacel chromatography was used to remove remaining proteins (glucan-S2). Subsequently this preparation was applied into concanavalin-A sepharose column to remove manann. Finally, beta-glucan free of mannoprotein complexes was prepared (glucan-S3).     

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.