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.     

Characterization of human progesterone receptor by high performance hydrophobic interaction chromatography

High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl-5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low-salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high-salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.     

Scale-up of recombinant protein purification by hydrophobic interaction chromatography

The scale-up of hydrophobic interaction chromatography is described. Human recombinant superoxide dismutase was used as a model. The scale-up was performed by keeping the height to diameter (H/D) ratio of the column constant. The success of scale-up was evaluated by reversed-phase high-performance liquid chromatography of the eluted material. The wrong H/D ratio causes decreased resolution.     

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。     

Rapid and efficient purification of positron emission tomography probes by hydrophilic interaction chromatography

A rapid and efficient preparative high-performance liquid chromatographic procedure was established to purify short-lived positron emission tomography radio-probes. This method is based on hydrophilic interaction chromatography utilizing a semi-preparative silica column (10 mm I.D.) and a high volatile organic mobile phase (>90% acetonitrile). In nine different radiopharmaceuticals studied, six compounds could be separated from the unlabeled precursor with good resolution and faster elution than its precursor. These characteristics enabled significant shortening of the separation and evaporation processes in the manufacture of short-lived radiopharmaceuticals. Several ¹¹C-radiopharmaceuticals could be prepared within one half-life of carbon-11 (20.4 min), including radiosynthesis, purification and formulation steps with sufficient radiochemical/chemical purity and high levels of radioactivity/specific radioactivity.     

Determination and purification of metallothioneins by high performance liquid chromatography.

A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein-I (MT-I) and metallothionein-II (MT-II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT-I or MT-II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT-I and MT-II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd-injected samples and 60Co-irradiated samples. A detection limit of 5 micrograms/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.     

Purification of labelled antibodies by hydrophobic interaction chromatography

In order to improve the sensitivity of immunometric assays, a chromatographic technique was developed that virtually eliminates components causing non-specific background. Labelled antibodies were applied to a phenyl-Sepharose column in physiological buffer. When labelled anitbodies were purified by this technique, the non-specific background of various time-resolved immunofluorometric assays was reduced 3- to 10-fold and was very close to the instrument background. The assay sensitivity was simultaneously increased by a factor of 2 to 16. This purification method might be used to improve the results of immunometric assays in general.     

Rapid purification yielding highly active 17 beta-hydroxysteroid dehydrogenase: application of hydrophobic interaction and affinity fast protein liquid chromatography.

Homogeneous human placental 17 beta-hydroxysteroid dehydrogenase was obtained by a procedure consisting of two fast protein liquid chromatographic (FPLC) steps using Phenyl-Sepharose hydrophobic interaction and Blue-Sepharose affinity columns. In the first chromatography, the enzyme eluted only when an additional decrease in ionic strength was inserted after the ammonium sulphate concentration had reached zero, thus enhancing the separation. In the affinity chromatography, separation of contaminating proteins occurred at different stages of loading and washing. The specific elution of the enzyme by the co-factor NADP+ is very efficient in obtaining a homogeneous preparation in high yield. The rapidity of FPLC was further increased by a maximum simplification of the intermediate steps, and the whole procedure lasted only two days. This preparation has a yield of more than 50% and a high specific activity, catalysing the formation of 7.9 mumol of estrone from estradiol per minute at pH 9.2 and 23 degrees C. It has an apparent molecular mass of 35,000. This provides an efficient candidate for the purification of other membrane-associated proteins.     

Rapid analysis of opium alkaloids by ion-pair high performance liquid chromatography

A new procedure for the rapid analysis of four major opium alkaloids is described using ion-pair, reversedphase high performance liquid chromatography and recently developed radial compression column technology. The solvents were aqueous 10mM potassium perchlorate with 5.0mM n-butylamine (pH 3.0) and pure acetonitrile. A gradient was run for five minutes from 10% to 70% acetonitrile and held at that level for two minutes. Flow rates were 3 ml/min. Two column packing materials were compared with this solvent system: standard C-18 packing and a fully-end-capped C-18 material. Results obtained with the fully-end-capped C-18 packing and the two solvent ion-pair gradient showed elution times under six minutes, with greater sensitivity (<50 nanograms) than previous systems.     

鸡蛋中三聚氰胺的液相色谱快速测定

建立了鸡蛋中三聚氰胺的液相色谱快速检测方法。样品经1%三氯乙酸溶液超声提取,磺基水杨酸和乙腈沉淀蛋白,高速离心分离,上清液过滤后直接由高效液相色谱分析。在优化的条件下,样品的色谱分析时间仅为10 min;三聚氰胺标准品在0.05～20 mg/L范围内,线性相关系数为0.9999;方法定量限为0.10 mg/kg (S/N＞10);1.0,2.0,4.0,6.0 mg/kg 4个加标水平测试,回收率在94.2%～107%之间;6次平行测试的相对标准偏差(RSD)为1.53%～2.06%。该方法前处理简单、分析速度快、灵敏度高,满足鸡蛋中三聚氰胺检验工作的快速测定要求。     

Evaluation of hydrophobic interaction between acidic drugs and bovine serum albumin by reversed-phase high-performance liquid chromatography

The contribution of hydrophobic interaction to the protein binding of acidic drugs has been evaluated in terms of a new hydrophobic index (r-value), defined as the slope of the log-log plots of capacity factor vs. reciprocal of methanol concentration in an aqueous binary mobile phase, measured by the reversed-phase high-performance liquid chromatography. The logarithms of the binding constants (log K1) of the selected acidic drugs and the related aromatic carboxylic acids indicated linear relationship with their r-values, suggesting that the effect of hydrophobicity on protein binding can be explained similarly to that on the retention onto the reversed-phase stationary ligand.     

Enrichment of low-copy-number gene products by hydrophobic interaction chromatography

Enrichment of proteins in solution is the goal of a purification process and often a scientific challenge. We investigated the capacity of hydrophobic interaction chromatography to enrich proteins, potential candidates for novel drug targets. The soluble protein fraction of Haemophilus influenzae was fractionated over a TSK Phenyl column and the proteins resolved were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Approximately 150 proteins, bound to the column, were identified, 30 for the first time. Most of the proteins enriched by hydrophobic interaction chromatography were represented by major spots, so that an enrichment of low-copy-number gene products was only partially achieved. The proteins enriched by this chromatographic approach belong to various protein classes, including enzymes, ribosomal proteins and proteins with as yet unknown functions. The results include two-dimensional maps and a list of the proteins enriched by hydrophobic interaction chromatography.     

Purification of125I-labelled aprotinin by hydrophobic interaction chromatography

Summary ¹²⁵I-aprotinin has been prepared and purified by affinity chromatography in combination with hydrophobic interaction chromatography (HIC). The method allows the preparation of the labelled inhibitor practically carrier-free, in saline solution without organic modifier and the isolated compound retained inhibition activity. Recovery of radioactive material was virtually quantitative, without substantial accumulation of radioactivity on the column. Compared with currently used procedures, the method is more reliable because the labelled polypeptide is collected by following its specific chromatographic signal.     

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Purification of phospholipase C by hydrophobic interaction affinity chromatography.

A simple procedure is described for the purification of phosphatidylcholine-hydrolyzing phospholipase C(PLC). Lecithin, the substrate for PLC, was ligated hydrophobically to octyl-Sepharose in 2 M (NH4)2SO4. The washed lecithin-conjugated resin was then used to purify PLC from crude preparations by affinity chromatography. PLC binds to the lecithin moiety in the presence of Zn2+ and is eluted with an acidic buffer containing EDTA. PLC activity was recovered in the eluate. Both sodium dodecyl sulphate polyacrylamide gel electrophoresis and pI electrofocusing showed that the eluate contained a single monomeric protein with an apparent molecular mass of 66 kDa and a pI of 5.5.     

A theory of protein-resin interaction in hydrophobic interaction chromatography

Docking simulations were performed in order to investigate surface area of interaction between several ribonucleases and a reduced model for the hydrophobic moiety used in Phenyl Sepharose using the program AutoDock 3.0. For each ribonucelase, 80 independent simulations with populations consisting of 100 random structures were performed and from these the most probable docked protein-ligand conformations were obtained. A new methodology was used to select the most probable conformations, based on qualitative and quantitative considerations. The interacting amino acids in each protein were identified. The average surface hydrophobicity of the interfacial zone (local hydrophobicity, LH) was determined. The LH showed a high correlation level (r2 = 0.99) with the "hydrophobic contact area" (HCA) experimentally determined for the different ribonucleases as well as with the dimensionless retention time (r2 = 0.90). This study allowed us to identify the zones on the protein surface most probably involved in protein retention in HIC, without tedious experimental work. Given the good correlation level obtained, this new methodology may constitute a novel approach that could be used to predict protein behavior in HIC.     

Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography

Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.