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Biomedical subjects

E Pungor

Publications and source records attributed to E Pungor.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of recombinant adenoviruses using an integrated microfluidic chip-based system.

The use of recombinant virus for gene therapy requires rigorous quality control methods to ensure that the viral vector preparations are functional and safe. A viral identity test is performed in which the viral payload, or transgene, is PCR amplified, followed by digestion with restriction enzymes that yields a characteristic "fingerprint." These DNA fragments are typically analyzed by agarose gel electrophoresis. The ethidium bromide-stained gels are photographed or scanned and the results are sufficient for a qualitative or semiquantitative identity confirmation of the viral product. We have investigated the use of an integrated microfluidic chip-based system as a new tool in the quality control testing of a recombinant, adenoviral, gene therapy product. The chip-based method was found to be very sensitive, requiring 100-fold less sample and only one-third the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.

Adenoviruses, Human↗
PubMed2000

Capillary zone electrophoresis of a recombinant adenovirus.

Adenovirus preparations are used as vectors in a number of gene therapy clinical development programs. The success of commercial production of adenovirus will strongly depend on the development of methods to define the recombinant virus product by analysis as opposed to being defined by the manufacturing process. While most analytical techniques examine portions of the virus, e.g. proteins or DNA, ion-exchange chromatography has been used to separate intact virus at low efficiency. A free zone capillary electrophoretic method was developed for high-efficiency separations of adenovirus 5. Experimental conditions such as buffer pH and concentration were explored which produced a high-efficiency separation in less than 20 min. The virus band was identified by collection of CE fractions and examination using a cell based assay. Initially, a single virus peak is found in fresh virus samples. After as little as one freeze-thaw in 1 x phosphate-buffered saline with 2% sucrose, the active virus migrates as a regular series of peaks. The nature of the virus modification leading to the differing electrophoretic mobilities is presently under investigation.

Adenoviridae↗
PubMed1999

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.

Adenoviruses, Human↗
PubMed1998

Affinity binding of a vampire bat plasminogen activator to SEC resins.

DSPAalpha1 is a recombinant form of the vampire bat plasminogen activator which we have produced in mammalian cell culture. During the development of a recovery process for DSPAalpha1 we observed an unexpected binding interaction between this protein and several types of gel filtration chromatography resins. Under typical operating conditions using neutral pH buffers, we found that DSPA flows through the sizing resin and is fractionated, as expected, according to its molecular size. However, DSPA applied under certain acidic conditions (<pH 3) binds tightly to the Sephacryl series of resins. The protein is not released until solvent conditions are changed, specifically the pH is raised above 3. From the results presented we conclude that this unexpected interaction with the gel filtration media is not simply an ion exchange nor a hydrophobic interaction, but rather a more complex, mixed mode "affinity" like binding. Several structural features of the DSPA protein which may be involved in this unique binding have been examined, including binding after inactivation of its active site, chemical deglycosylation, chemical denaturation, or limited proteolysis. The "affinity" interactions persist despite these treatments and lead us to conclude that there may be a unique peptide sequence(s) within the protein which is responsible for the binding interaction to Sephacryl resins.

Acrylic Resins↗
PubMed1998

A novel sensitive and selective bioassay for human type I interferons.

We describe a novel MxA gene-induction assay for type I interferons (IFN-alpha and IFN-beta) based on the specific induction of the MxA gene in cultured human cells. Accumulated intracellular MxA protein is determined by immunologic measurement by a rapid method using commercially available materials. IFN activity can be measured accurately over a concentration range of 0.1-30 IU/ml. In contrast, type II IFN and other cytokines are not significantly detected. The MxA-induction assay has advantages in terms of specificity, reliability, and sensitivity over other methods for assaying type I IFN. It has also been adapted and validated for measuring the titers of anti-IFN-beta neutralizing antibodies in human sera.

Antiviral Agents↗
PubMed1998

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Antigen-Antibody Reactions↗
PubMed1998

Application of new analytical technology to the production of a "well-characterized biological".

The role of new analytical technology in the development of the concept of a "Well Characterized Biological" is to provide suitable methodology that allows the characterization of even the most complex protein sample so that a consistent manufacturing process can be established. Glycoproteins are among the most challenging of products to characterize because of extreme sample microheterogeneity due to the carbohydrate moieties. As an example of the appropriate use of new analytical technology this review will examine the steps necessary to demonstrate that a glycoprotein is a "Well Characterized Biological". The key to characterization of complex protein samples lies in the use of appropriate combinations of the different methods that analyse the sample from substantially orthogonal and independent directions. An important advantage of capillary electrophoresis (HPCE) in this application is the complementarily of the technique with reversed phase HPLC (RPLC). Thus mixtures of variants of a polypeptide that are difficult to separate by RPLC can often be readily resolved by HPCE. Both separation techniques are well suited to the analysis of peptide maps, although RPLC is particularly powerful when used in combination with on-line electrospray mass spectrometry (ESI-MS) which allows for the effective ionization and detection of even high MW glycopeptides. In this sense the ESI-MS is an ideal detector for on-line mass detection after a RPLC separation of medium MW fragments (300 to 6000 emu) that are typically present in an enzyme digest of a protein. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) is a valuable technique for off-line characterization of CE fractions due to the high sensitivity of the method and its tolerance of samples with moderate levels of salt. The development of an effective protocol for the analysis of glycoform populations of intact glycoproteins by a combination of HPCE and off-line MALDI-TOFMS wil be demonstrated by the successful analysis of two highly heterogeneous glycoproteins, ovalbumin and Desmodus Salivary Plasminogen Activator (DSPA).

Animals↗
PubMed1997

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.

Animals↗
PubMed1996

Application of multidimensional affinity high-performance liquid chromatography and electrospray ionization liquid chromatography-mass spectrometry to the characterization of glycosylation in single-chain plasminogen activator. Initial results.

Preliminary results are presented using a combination of affinity chromatography, reversed-phase HPLC and electrospray ionization mass spectrometry to produced peptide maps for N-linked, O-linked and non-glycosylated peptides from an endoproteinase LysC digest of DSPA alpha 1, a recombinant DNA derived glycoprotein. Although the system was used to identify a number of major N-linked structures, notably complex biantennary structures attached to asparagine 362, no O-linked glycopeptides from the possible 4 attachment sites were identified. The system did, however, demonstrate the feasibility of the approach and the applicability of the instrumental system.

Carbohydrate Sequence↗
PubMed1996

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Amidohydrolases↗
PubMed1995

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator.

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.

Amino Acid Sequence↗
PubMed1991

Local depletion of monoamines induced with in vivo voltammetry in the cat brain.

A significant depletion of the electroactive monoamines and their metabolites in the vicinity of a carbon fiber microelectrode may be induced by in vivo staircase voltammetry in the brain, even if the duration of the voltammetric scans is relatively short (approximately 5 s). The variation of this depletion was determined in the extracellular fluid of the cat thalamus at different durations of the pauses separating consecutive measurements. Pauses not shorter than 5 min ensured a nearly full relaxation, so that at the beginning of each subsequent scan a virtually undisturbed environment surrounded the electrode. With pauses shorter than 5 min, it is still possible to monitor major changes in the monoamine concentration. Staircase scans separated with 45 s pauses, for example, were suitable to study the increase in monoamine levels after administration of reserpine, and release phenomena stimulated with KCl were monitored with frequently repeated voltammetric pulses. The electrochemically induced depletion, on the other hand, can be used for characterizing the dynamics of mass transport in the studied brain structure. This was demonstrated with staircase voltammetry alternated with pauses of 1-100 s, and with quasi-chronoamperometry. In vivo brain voltammetry is generally used for monitoring extracellular monoamine (including dopamine) levels. These may be significantly altered by the voltammetric measurement itself through depletion in the vicinity of the electrode. This effect can be minimized with appropriate selection of sampling intervals and other parameters of staircase voltammetry. Conversely, depletion and the following relaxation can be used for determining dynamic characteristics of the studied brain structure which would be difficult to obtain otherwise.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1989

Electrochemical calibration of in vivo brain dialysis samplers.

Measurement of state-related changes in the concentration of an endogenous substance using the in vivo dialysis technique requires a definition of the factors which relate the concentration detected in the outflowing perfusate to the concentration actually occurring in the extracellular space in which the sampler is located. In determine these factors, a rapid and highly accurate detector system is required which measures the concentration recovery in the perfusate and the dead space of the entire sampling system. To reduce the limitations of the microdialysis technique, two electrochemical microcells were developed for calibration of dialysis probes by computerized voltammetry. The electrochemically calibrated samplers were implanted into freely moving cats to measure concentration changes of monoamine metabolites in synchrony with sleep stages identified by polygraphy in order to demonstrate the applicability of electrochemical calibration in dialysis methods used in behavioral investigations.

Animals↗
PubMed1984

Determination of enzyme activity in biological fluids by means of electrochemical oxidation of NADH at a modified glassy carbon electrode.

This amperometric technique for the determination of enzyme activity is based on detecting a decrease in the concentration of the NADH co-factor of the enzyme reaction. A glassy carbon electrode, modified by adsorption of Mg2+ and NADH, is used to measure the anodic peak current that corresponds to the oxidation of NADH. We found no significant difference between the enzyme activity of lactate dehydrogenase (E.C.1.1.1.27) preparations as measured by the above amperometric technique and by a spectrophotometric method.

Electrochemistry↗