One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase high-performance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C(8) column (150 mm x 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.     

Rapid method for evaluating reversed-phase high-performance liquid chromatography column stability

A procedure is presented for the rapid evaluation of HPLC stationary phase stability at pH 8.4 or 10.1 using a temperature of 60 degrees C. Mobile phase (MeOH-0.1 mol l(-1) aqueous NaHCO3, 50:50, v/v) is continuously passed through the column with periodic injections of a test solution until the several chromatographic parameters of the resulting chromatograms are degraded. The tests were applied to several commercial and laboratory-made stationary phases. After degradation two of these phases, one commercial and one laboratory-made, were examined by elemental analysis and scanning electron microscopy to elucidate the degradation process.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Analysis and purification of toxic peptides from cyanobacteria by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable chemical analysis method for microcystins (cyanoginosins) has been studied. Three different mobile phases for high-performance liquid chromatography were selected and optimized. Also the adsorptive powers of three commercially available C18 cartridges were compared and the results successfully applied to the clean up of three of the toxins. Finally a total system for the analysis and isolation of microcystins was established.     

Biologic activity of human chorionic gonadotropin following reversed-phase high-performance liquid chromatography

Human chorionic gonadotropin (hCG) was analyzed by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases previously described in the literature, as well as newly developed solvent systems. Fractions of hCG collected following reversed-phase HPLC were bioassayed by activation of adenylate cyclase to determine their biologic potencies. hCG retained only 10-60% of its biologic activity following reversed-phase HPLC, depending on the chromatographic conditions employed. A portion of the reduced biologic activity was attributed to dissociation of the alpha- and beta-subunits of hCG at the low pH of the mobile phases, since neutralization of the pH prior to lyophilization and bioassay increased the biologic potency of the chromatographed hormone. The remaining loss in biologic activity is presumably due to organic solvent denaturation.     

Determination of cortisol in human plasma by reversed-phase high-performance liquid chromatography

A method is described for the measurement of cortisol in human plasma using 45% aqueous methanol eluent on a 120 mm x 4.5 mm I.D. Hypersil octadecylsilane column with UV detection at 239 nm after a simple dichloromethane extraction and evaporation with a prednisone internal standard. The sample preparation time and chromatography time are each about 15 min and linear correlations have been obtained with plasma samples assayed by the Mattingly fluorimetric technique and a commercial-kit competitive protein binding method. Concentration down to 30 nmol/l may be measured and the method can be used when fluorimetry is invalidated by interference, particularly from spironolactone.     

Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR-RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR-RBP concentrations determined by this method are highly correlated to holo-TTR-RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR-RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR-RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Rapid method based on reversed-phase high-performance liquid chromatography for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides.

Reversed-phase high-performance liquid chromatography was applied to isolate myelin basic protein from human brain, followed by separation of proteolytic peptides thereof on the same chromatographic system. Brain tissue was delipidated under conditions that keep copurifying proteases inactive. The crude brain protein fraction was applied directly to a C4 column. The homogeneous protein obtained in this way was digested with thrombin and endoproteinase Lys-C in order to produce short defined myelin basic protein peptides. The purified peptides were used to determine the antigen fine specificity of myelin basic protein recognizing T lymphocyte lines isolated from multiple sclerosis patients.     

Microwave-immobilized polybutadiene stationary phase for reversed-phase high-performance liquid chromatography

Polybutadiene (PBD) has been immobilized on high-performance liquid chromatography (HPLC) silica by microwave radiation at various power levels (52-663 W) and actuation times (3-60 min). Columns prepared from these reversed-phase HPLC materials, as well as from similar non-irradiated materials, were tested with standard sample mixtures and characterized by elemental analysis (%C) and infrared spectroscopy. A microwave irradiation of 20 min at 663 W gives a layer of immobilized PBD that presented good performance. Longer irradiation times give thicker immobilized layers having less favorable chromatographic properties.     

Influence of hydrolysis, purification, and calibration method on furosine determination using Ion-pair reversed-phase high-performance liquid chromatography

The influence of HCI concentration (6M, 8M, and 10M) and the ratio of sample protein to acid (1 or 5 mg of protein per mL of acid) on furosine formation during sample hydrolysis is studied. The conditions that maximize furosine formation are 10M HCI in the ratio of 1 mg of protein to 1 mL of acid. Purification of the hydrolysate by solid-phase extraction is also considered by examining the effect of hydrolysate volume and volume of 3M HCI used to elute the furosine. Furosine quantitation is carried out using the standard additions and external standard methods. The results indicate that there is no interference by the sample matrix and that external calibration is adequate.     

Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.     

Chemometric classification of reversed-phase high-performance liquid chromatography columns

Cluster analysis and principal components analysis have been used to classify nine octadecyl (C18) high-performance liquid chromatography (HPLC) columns into three general groups displaying similar chromatographic behaviour. Principal components analysis was also able to identify the key test compounds on which the classification was most highly dependent. These identifications agreed with the classification and test compound selection by an HPLC specialist. In addition, the chemometric techniques can easily be extended to many more columns and test measurements than could be conveniently examined by a human expert.     

Usage of nonporous polymeric particles for reversed-phase high-performance liquid chromatography of oxidized and deamidated forms of recombinant human growth hormone

In this work, a C(18) reversed-phase column with nonporous polymeric 2.5- micro m particles is utilized to initially test the analysis of oxidized and deamidated human growth hormone (hGH). Phosphate buffer (pH 7.5) with 24% 1-propanol was used for elution. This quick method (analysis time is 20 min) gave a selectivity, as judged by the number of detected peaks, and resolution of hGH variants that is better than many methods in which porous silica particle columns are used. Only mixtures of oxidized and deamidated hGH are analyzed, and no characterization of the peaks is performed. The results indicate that C(18) nonporous polymeric column material is a promising alternative for the chromatographic separation of several hGH variants.