DNA removal on different chromatography supports used for EPO purification by spike studies

Chromatographic techniques are used in the purification step of human recombinant erythropoietin production process to obtain a reliable product with high purity. Anion-exchange chromatography supports have proved high efficient in removing contaminants such as DNA. For that reason, the DNA removal was determined by spike studies, on three anion-exchange chromatographic supports: gel, membrane, and monolithic column, which is used in intermediate purification stage. This study showed that membrane and monolith columns have very good results in the removal of contaminants at this step. Log removal values (LRV) greater than 3.5 were obtained from DNA spike clearance studies. Monolithic column was determined as the best technological proposal, with more than 4 LRV, 7.72?mg DNA per milliliter of adsorbent and 85% protein recovery in nonspike run. The results of this study may be used as a guide in the selection of commercially available chromatography supports for intermediate purification steps in recombinant protein production.     

Impact of multiple re-use of anion-exchange chromatography media on virus removal

We evaluated viral clearance in multiply-cycled anion-exchange media run in flow-through mode. We found that anion-exchange columns do not lose viral clearance capacity after extensive re-use, if they are cleaned with recommended buffers that do not chemically degrade the media. In contrast, anion-exchange (AEX) columns that are not cleaned or are cleaned with buffers that chemically degrade the media lost viral clearance capacity after extended use. In these cases, other performance attributes that changed at the same time were increased band spreading, decreased DNA clearance and accumulating backpressure that prevented re-use past 80-120 cycles. Thus, our data suggests that flow through mode anion-exchange columns that are cleaned with recommended cleaning buffers, and periodically monitored for band spreading, DNA clearance and/or backpressure need not be re-evaluated for viral clearance at the end of the validated media lifetime.     

Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography

In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na₂SO₄ with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.     

Robustness of virus removal by protein A chromatography is independent of media lifetime

The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to na?ve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age.     

Real-time control of antibody loading during protein A affinity chromatography using an on-line assay.

We show that an on-line chromatographic assay can reliably control antibody loading in real-time during protein A affinity chromatography purification of a recombinant antibody from clarified Chinese hamster ovary cell culture fluid. The on-line assay directly sampled preparative column effluent and provided real-time measurement of antibody breakthrough during loading. The on-line assay used protein A immobilized on perfusion chromatography media, equilibrated with phosphate-buffered saline at pH 7.2 and eluted with phosphate-buffered saline at pH 2.2. The assay reliably ended loading at 1% breakthrough with minimal yield loss. Reproducible yield and purity were obtained over 23 consecutive cycles. Yield remained constant while breakthrough capacity and the antibody concentration in the load changed.     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.     

Mechanically stable porous cellulose media for affinity purification of family 9 cellulose-binding module-tagged fusion proteins

A mechanically stable cellulose-based chromatography media was synthesized to permit inexpensive affinity purification of recombinant proteins containing the family 9 carbohydrate-binding module (CBM9) fused to either the N- or C-terminus of the target protein. A second-order response surface model was used to identify optimal concentrations of the primary reactants, epichlorohydrin and dimethyl sulfoxide (DMSO), required to cross-link the starting material, Perloza MT100, a compressible but inexpensive cellulose-based chromatography resin. This resulted in a mechanically stable cross-linked affinity chromatography media capable of operating at an order-of-magnitude higher linear velocity than permitted by unmodified MT100. Moments and Van Deemter analyses were used to show that rates of solute mass transfer within the column are largely unaffected by the cross-linking reaction, while the binding capacity decreased by 20% to 7.1 micromol of protein/g resin, a value superior to most commercial affinity chromatography media. In sharp contrast to MT100, the mechanical stability and purification performance of the cross-linked media are not diminished by scale-up or repeated column use.     

Comparison of protein A affinity sorbents III. Life time study

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.     

Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry

In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30 mM Tris-HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris-HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Evaluation of protein-A chromatography media

In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A and 1000 A. Adsorption equilibrium data of human immunoglobulins on these media were measured via a batch technique and correlated using the Langmuir isotherm model. A larger static capacity was found for the smaller pore size material, which is probably a result of the larger specific surface area and associated higher ligand concentration. The protein uptake kinetics were also obtained via a stirred tank experiment using different initial protein concentrations. A surface layer model was used to represent the protein uptake by the media and to estimate values of a concentration-independent effective diffusivity within the particle. Experimental breakthrough curves were also obtained from packed beds operated under different conditions. Calculated breakthrough profiles were found to be in good agreement with the experimental results. Experimental breakthrough data were used to determine the dependence of the dynamic capacity of the media as a function of the fluid residence time. A larger dynamic capacity was also found for the smaller pore size media. The permeability of large scale packed beds was also reported and used in conjunction with the dynamic capacity to calculate the process production rate.     

Rapid purification of arrowhead proteinase inhibitors by high performance hydrophobic interaction chromatography on a PEG bonded phase column

A new hydrophobic interaction HPLC column is used for the rapid purification of proteinase inhibitors isolated from arrowhead. The inhibitors, partially purified by DEAE-cellulose column chromatography, are resolved into three components with a mobile phase gradient of decreasing salt concentration from 1.1 M ammonium sulfate in 0.01 M phosphate buffer to phosphate buffer alone. This new HPLC column is found to be very useful for rapid, semipreparative purification of hydrophobic protein and sample loading of up to 1.6 mg of inhibitors can be fully resolved on an analytical column.     

Influence of column design on process-scale ion-exchange chromatography

The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using Whatman Express-Ion Exchanger Q. A 16 1 Side-Pack column and a 24 1 IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 1 feedstock containing 5-7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5 M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.     

超大孔离子交换制备色谱分离纯化高活性凝血因子VIII

建立了一条从人血浆中分离高活性凝血因子VIII(FVIII)的纯化工艺。基于FVIII和介质孔径的尺度比及其对蛋白质活性影响的分析,设计了以超大孔离子交换制备色谱为核心步骤的新型分离纯化工艺。分别进行超大孔离子交换色谱与传统离子交换色谱的条件优化,并对优化工艺所得产品进行了活性检测(底物显色法)和纯度检测(高效凝胶过滤和凝胶电泳)。结果表明,超大孔介质结构不但可以有效地保护蛋白质大分子结构,而且能够大幅度地提高制备色谱的传质速率,从而得到具有高凝血活性的FVIII产品。FVIII在超大孔制备色谱过程中的回收率(85%)比传统离子交换制备色谱高4～5倍,产品比活高达154 IU/mg。此外,还研究了超大孔介质的再生程序,采用5个柱体积的1 mol/L NaOH低流速清洗色谱柱,保证了色谱工艺的稳定性。本纯化工艺步骤简单,重现性好,易于放大生产。     

Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods

Antifreeze proteins (AFPs) are known as thermal hysteresis proteins, which can depress the freezing points of the solution by noncolligative effects, but do not affect the melting points. Although some AFPs have been found in some plants, the identity of most proteins remains unclear, owing to insufficient quantity and quality to characterize them. In this report, we describe the purification of an AFP from the winter leaves of Ammopiptanthus mongolicus using a combination of column chromatography and gel electrophoresis. After homogenization in ascorbate-acid-containing Tris buffers (pH 7.4) the soluble proteins are captured by (diethylamino)ethyl-cellulose 52 material. An elution with 0.1-0.3M KCl leads to a crude active fraction. The crude fraction is further purified on a Superdex 75 prep-grade column and finally a Poros 20HP2 column. A complex, consisting of two proteins with relative molecular masses of 34,700 and 37,100, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, is obtained by this protein purification protocol. The recovery of two proteins from the gel is carried out by electrophoresis. The purified protein, with a molecular mass of 37,100, shows thermal hysteresis activity (THA) and can modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24 degrees C at the concentration of 5 mg/mL.     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

Complimentary Role of Micellar Electrokinetic Capillary Chromatography and High Performance Liquid Chromatography in the Separation of Plant Phenolics

Abstract

A comparison was made between the efficiency of micellar electrokinetic capillary chromatography (MECC) and reversed phase high performance liquid chromatography (RP-HPLC) to separate a mixture of plant phenolics. Of a range of buffers 6 mM borate/10 mM phosphate/100 mM SDS at pH 8.5 was the most effective in separating a complex mixture of phenolics using MECC. Using this buffer the elution order and resolution was different from that obtained by HPLC using a reversed phase C18 column. These results illustrate how MECC and RP-HPLC are complimentary when examining complex mixtures such as those obtained from plant extracts. MECC using these conditions was applied to the examination of phenolics from leaf tissue of Eucalyptus margmata (jarrah).     

Analysis of aminofluorescein-fatty acid derivatives by capillary electrophoresis with laser-induced fluorescence detection at the attomole level: application to mycobacterial fatty acids

In the present work, a new method of purification for antithrombin was developed using an expanded bed chromatography technique. Milk fat was removed by centrifugation and caseins were precipitated selectively by addition of zinc chloride. Crude skim milk was then directly fed to an expanded bed column containing the ion-exchange matrix. The use of a cation-exchanger (P-11) resulted in 100% adsorption and 13% recovery whereas the use of an anion-exchanger (DE-52) resulted in 100% adsorption and 84% recovery and up to five-fold purification of antithrombin. The buffer, 25 mM Tris-HCl pH 8.0; the eluting agent, 2 M (NH4)2SO4; and 100% expansion of settled bed were determined to be the optimum conditions for the purification of antithrombin by ion-exchange expanded bed chromatography. A comparison of column diameters revealed that the elution yields increase by two-fold while the column diameter increases from 1 to 2.5 cm. However, antithrombin III was concentrated to a higher degree by using the column with an internal diameter of 1 cm.     

Analysis and modeling of fixed bed column operations on As(V) removal by adsorption onto iron oxide-coated cement (IOCC)

Fixed-bed column studies were undertaken to evaluate the performance of iron oxide-coated cement (IOCC) in removing As(V), from aqueous environment, under varying operating conditions. Investigations revealed IOCC to be a very efficient media for the removal of As(V) from water. The Bohart and Adams sorption model was employed to determine characteristic parameters such as depth of exchange zone, time required for exchange zone to move its own height, adsorption rate, and adsorption capacity, useful for process design. The IOCC media effectively removed As(V) from a 2 mg/L solution with a maximum adsorption capacity of 505.3 mg/L. Predicted data points obtained using a Freundlich isotherm model were compared against experimental data points determined in the dynamic process. The effects of important design parameters such as flow rate and influent concentration on the As(V) breakthrough curves were also investigated. The bed depth service time (BDST) model was extended to the prediction of service times of columns operated at different flow rates and influent concentrations and these predicted values were compared with the experimental values. The columns were regenerated by eluting the bound As(V) with 10% NaOH after the adsorption studies.