Mass spectrometric analysis of recombinant human α-2 interferon

Mass spectrometry (MS) was used for the characterization of recombinant human α-2 interferon (α-2 IFN) produced in E. coli. After purification by monoclonal antibody affinity chromatography, α-2 IFN showed two major peaks in reversed-phase liquid chromatography (RP- LC). Each component was digested with trypsin and Staphylococcus aureus protease V8, separately or in tandem, and the peptide mixture was analysed by MS without further purification. The first peak corresponded to the 165 amino acid sequence of human α-2 IFN and the main component of the second peak was the acetylated Cys₁ α-2 IFN. It was also possible to verify by MS the location of the SS bonds in α-2 IFN and the occurrence of incorrect SS bridges in the products of some renaturation processes. The best renaturation process for obtaining a product without adducts or scrambling of disulphide bonds could be found by using RP-LC and fast atom bombardment MS.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.     

Molecular weight determination of recombinant interleukin 2 and interferon gamma by electrospray ionization mass spectroscopy.

The molecular weights of the recombinant proteins interleukin 2 and interferon gamma were determined by electrospray ionization (ESI) mass spectroscopy on the whole molecules. For interleukin 2 an average experimental mass of 15,549.4 u, was observed in excellent agreement with the theoretical average mass of 15,547.2 u. The mass observed for interferon gamma (16,908.4 u) agreed with the theoretical molecular weight of the monomeric species (16,907.3 u). By ESI mass spectrometry it was possible to determine the presence of proteins lacking one or two amino acids.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

Microcalorimetric study of virus infection; The effects of hyperthermia and 1b recombinant homo interferon on the infection process of BHK-21 cells by foot and mouth disease virus

The metabolic process of BHK-21 cell line infected by foot and mouth disease virus (FMDV) was determined by using LKB-2277 Bioactivity Monitor. The aim of the present study is to investigate the metabolic thermal power of the virus infection process of BHK-21 cells, the effects of combinational treatments of hyperthermia and 1b recombinant homo interferon on this process. In contrast to the metabolic thermal power of uninfected BHK-21 cells, the thermogenetic curves show that the energy metabolism mechanisms of BHK-21 cells were significantly changed by the virus infection process. The maximum thermal power decreased and the time needed to reach the maximal thermal power increased with the increasing concentration of interferon. The results also show that the infection process was thermosensitive. But no apparent synergetic effect of the combinational treatments of hyperthermia and interferon was observed. The present microcalorimetric results are in accordance with the cytomorphology observations.     

Analysis of biogenic carbonyl compounds in rainwater by stir bar sorptive extraction technique with chemical derivatization and gas chromatography‐mass spectrometry

Stir bar sorptive extraction is a powerful technique for the extraction and analysis of organic compounds in aqueous matrices. Carbonyl compounds are ubiquitous components in rainwater, however, it is a major challenge to accurately identify and sensitively quantify carbonyls from rainwater due to the complex matrix. A stir bar sorptive extraction technique was developed to efficiently extract carbonyls from aqueous samples following chemical derivatization by O‐(2,3,4,5,6‐pentafluorobenzyl) hydroxylamine hydrochloride. Several commercial stir bars in two sizes were used to simultaneously measure 29 carbonyls in aqueous samples with detection by gas chromatography with mass spectrometry. A 100 mL aqueous sample was extracted by stir bars and the analytes on stir bars were desorbed into a 2 mL solvent solution in an ultrasonic bath. The preconcentration Coefficient for different carbonyls varied between 30 and 45 times. The limits of detection of stir bar sorptive extraction with gas chromatography mass spectrometry for carbonyls (10–30 ng/L) were improved by ten times compared with other methods such as gas chromatography with electron capture detection and stir bar sorptive extraction with high‐performance liquid chromatography and mass spectrometry. The technique was used to determine carbonyls in rainwater samples collected in York, UK, and 20 carbonyl species were quantified including glyoxal, methylglyoxal, isobutenal, 2‐hydroxy ethanal.     

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen–antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.     

Rapid detection of perchlorate in groundwater using capillary electrophoresis

Summary Perchlorate is a groundwater contaminant originating from facilities that manufacture and test solid rocket fuel. A new technology, capillary electrophoresis, has the potential to measure perchlorate rapidly and inexpensively in water samples. With its speed and simplicity, this method would complement existing methods. The perchlorate anion is routinely detected in water samples using high performance ion exchange chromatography, a very sensitive yet time consuming and expensive method. In this work, the parameters for detection of perchlorate are optimized to permit detection of 0.400 mgL⁻¹ perchlorate in a standard solution. The usefulness of this technology is demonstrated for measuring perchlorate in several ground-water samples from the Western United States. The results demonstrate that CE can be used to rapidly screen environmental samples for perchlorate at intermediate to high levels (greater than 0.400 mgL⁻¹). This technique allows faster, easier screening of potential contamination sites and could complement the use of ion exchange chromatography for groundwater testing.     

Production, purification and characterization of recombinant human interferon gamma.

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.     

The errors in assembly of MuLV in interferon treated cells.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.     

Trace analysis of methanol in rat serum by headspace high resolution gas chromatography/selected ion monitoring

A high resolution headspace gas chromatography/selected ion monitoring analytical method has been developed for the quantitation of methanol in body fluids. The addition of tetradeuterated methanol as internal standard gives this method high accuracy and reproducibility. The high sensitivity of the mass spectrometer permits analysis of methanol in aqueous solutions at concentrations down to 0.012 mg/l. The analytical procedure described was used to measure serum methanol in rats given 5 mg/kg of this alcohol orally. The method proved suitable for analysis of endogenous levels of methanol and of variations close to endogenous levels.     

Determination of the Dissociation Constant and Stoichiometry of a Complex of the Protein Interferon α-2b with Cibacron Blue F3G-A

The nonlinear least-squares technique was used for determining the complexation parameters of the recombinant human cytokine interferon -2b with a pseudobiospecific ligand, Cibacron Blue F3G-A. The dissociation constant of the complex and the number of ligand–protein binding sites were found.     

Epitope affinity chromatography and biophysical studies of monoclonal antibodies and recombinant antibody fragments

Peptide epitope affinity chromatography is a powerful technique for the purification of antibodies. This study aims to demonstrate the versatility of the technique and to show how biophysical techniques such as circular dichroism (CD) and fluorescence quenching (FQ) can aid the rational design of affinity ligands and characterization of antibody-based reagents. The performance of a number of peptide ligands for the purification of a range of different antibodies and recombinant fragments is investigated by automated fast-protein liquid chromatography. Purified products are analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They are then radiolabelled and the immunoreactivity is determined. Ligands are analyzed for secondary structural characteristics by CD and for binding affinity by FQ. Finally, a study is performed to investigate the thermal stability of a recombinant antibody fragment by CD analysis. It is found that simple ligand modifications such as the introduction of a C-terminal cysteine residue can improve purification performance. The FQ studies show that the modified peptide has a higher affinity for antibody. The CD analysis shows that it has a tendency to dimerize because of the formation of disulfide bonds. The versatility of epitope affinity is demonstrated through the purification of a recombinant diabody (dbFv) and by the use of a separate peptide matrix for the purification of an unrelated antibody. All studies result in antibody preparations of high purity and immunoreactivity. The CD analysis of the dbFv shows that it is denatured at 37 degrees C and is therefore unsuitable as a targeting reagent for use in humans in its present form. It is concluded that epitope affinity chromatography coupled with biophysical analyses plays an important role in the production and characterization of antibody-based reagents for targeted diagnosis and therapy of human diseases.     

Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.     

Expression of Biologically Active Human Recombinant Interferon Alpha 2b in Human Breast Cancer Cell Line Bcap-37

Human interferon alpha 2b (IFNα-2b) is a pleiotropic cytokine used to treat various viral diseases and cancers. Conventionally, recombinant human IFNα-2b used in clinics was produced by prokaryotic expression system, which always lack of enough biological activity due to limitations on proper folding and post-translational modifications, so the eukaryotic expression system are becoming prevailing method for the production of recombinant proteins. In this study, human breast cancer cell Bcap-37 was firstly used as host for the expression of human IFNα-2b, with the expression vector pIRES2-IFN-EGFP, in which IFNα-2b gene is under the control of CMV promoter. The expression of recombinant IFNα-2b was detected by Western blot and ELISA. Results showed that the concentration of the secreted recombinant IFNα-2b in culture medium was 435.7 pg/mL/24 h. Biological activity of the recombinant IFNα-2b was assayed by detecting the expression of IFN-inducible genes, including MxA, OAS, PKR, and Caspase1 through QRT-PCR. Results demonstrated that recombinant IFNα-2b possess the biological activities. Compared to non-transgenic cells, the expression levels of the aforementioned four IFN-inducible genes were increased by 18.098-, 1.843-, 2.21-, and 3.066-folds, respectively. We got to a conclusion that the human breast cancer cell Bcap-37 could express bioactive recombinant IFNα-2b.     

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.     

Identification of N-terminal modification for recombinant monoclonal antibody light chain using partial reduction and quadrupole time-of-flight mass spectrometry

Since most recombinant monoclonal antibodies (mAbs) contain glutamic acid or glutamate at their N-terminus, cyclization of these residues to form pyroglutamate is an important degradation pathway that often occurs in therapeutic mAb development. In this work, a rapid method was developed to determine pyroglutamate at the N-terminus of mAb light chain by liquid chromatography coupled with electrospray ionization on a quadrupole time-of-flight mass spectrometer (QTOF). High levels of pyroglutamate were found at the N-terminus of the light chain of a typical recombinant mAb. The quantitative results were comparable to those obtained with a more conventional peptide mapping method. The direct method outlined here can be used to evaluate the impact of N-terminal cyclization during the processing of recombinant mAbs.     

Measurement of anions in ambient particulate matter by ion chromatography: A novel sample preparation technique and development of a generic uncertainty budget

A novel sample preparation technique for the measurement of anions in particulate matter by ion chromatography is presented. The technique uses the eluant from the ion chromatography analysis stage to perform the extraction of the anions from the particulate matter. The demonstrated advantages of the new technique are manifold: faster sample throughput, minimised sample preparation, and a matrixed-matched sample for analysis. The repeatability of the technique has been tested using nominally identical ambient particulate matter samples (sub-sampled from one large filter) and the uncertainty of the technique has been estimated. The uncertainty estimate is transferable to other similar methods used to measure ions in particulate matter. Real data obtained using the new method are also presented.     

Trace level perchlorate analysis by ion chromatography-mass spectrometry

Perchlorate is commonly used as an oxidant in solid fuel propellant for rockets and missiles. Recently perchlorate contamination was found in many aquifers associated with Colorado River and other sites. Perchlorate was also found at elevated level in crops that use contaminated water for irrigation. Ion chromatography with conductivity detection could be used to measure perchlorate levels in drinking and wastewaters as per United States Environmental Protection Agency method 314, but at lower levels and with complexity of the matrix there could be false positive and/or false negative. This study was done to demonstrate the detection of perchlorate with lower detection limit with high ionic matrix by ion chromatography-mass spectrometry.     

Viscosity,density, interfacial tension and compositional data for near critical mixtures of methane + butane and methane + decane systems at 310.95 K

Single- and two-phase binary mixtures of near critical methane + butane and methane + decane systems have been investigated at 310.95 K. A capillary tube viscometer was used to measure the viscosity and a high-pressure densitometer was employed for density measurements. The mixture was prepared gravimetrically and a direct sampling system was used to measure the composition of equilibrium phases in the two-phase region by gas chromatography. The meniscus height technique was used to generate the interfacial tension data.