Biomedical subjects

D Josic

Publications and source records attributed to D Josic.

24 records · Page 2Linked to original sources

PubMed1994

Purification of human tumour necrosis factor by membrane chromatography.

The recombinant human tumour necrosis factor alpha from an extract of Escherichia coli was enriched to homogeneity according to specific activity and sodium dodecyl sulphate-polyacrylamide gel electrophoresis by purification using anion-exchange HPLC and hydrophobic interaction HPLC. Parallel experiments with the same separation methods, but carried out with membrane chromatography on compact discs, gave similar results in terms of yield and purity of the product. The active form of the protein is a trimer. The second isolation step, hydrophobic interaction chromatography, causes dissociation of the trimer into monomers and a partial loss of the biological activity of the protein. The phenomenon occurs on both the column and the disc. This in turn indicates strongly that the dissociation of the protein is a consequence of interaction between the samples and the hydrophobic ligand, and is not caused by non-specific interaction with the matrix.

Blotting, Western↗

PubMed1994

Extraction of Triton X-100 and its determination in virus-inactivated human plasma by the solvent--detergent method.

For inactivation of lipid-enveloped viruses during the production of fresh frozen and lyophilized human plasma, the solvent-detergent method was applied. In this process, the solvent tri-n-butyl phosphate is removed by extraction with castor oil. The removal of the non-ionic detergent Triton X-100 is performed by solid-phase extraction using reversed-phase supports. For this purpose, different polymer- and silica-based supports were tested. The highest capacity for Triton X-100 was achieved with C18 silica gels. These supports can bind more than 0.1 ml of Triton X-100 per ml of support. None of the proteins, e.g., clotting factors, bind to the support and therefore they pass through the column and their biological activity is hardly affected. The determination of detergent during the production process was also studied. The application of special columns allowing direct sample injection was introduced. This is a simple method for the rapid in-process determination of Triton X-100 in human plasma by reversed-phase chromatography under isocratic conditions. Using the method developed here, less than 1.0 ppm of Triton X-100 can be detected in less than 12 min without any sample pretreatment.

Blood↗

PubMed1994

A solvent I detergent treated, pasteurised and highly purified factor VIII concentrate.

In an assessment of the risks of virus transmission by clotting concentrates it is clear that the currently practised procedures for virus inactivation are not equally effective against all types of viruses; neither a pasteurisation nor the solvent detergent (SID) process alone are adequate enough to inactivate viruses that are strongly resistant to heat and organic solvents. In this context, human parvovirus B19 and hepatitis A virus (HAV) are of particular concern. In order to improve this situation which still poses a risk to the haemophiliac patients, a more effective pasteurisation process has been developed that could be easily applied to an already well established factor VIII (FVIII) process in addition to the SID-treatment. Experiments using temperatures above 60 degrees C were performed prompted by two recent publications, which demonstrate that HAV becomes instable at temperatures exceeding 62 degrees C. It is the purpose of this paper to present the following progress: achievement of a pasteurisation procedure for FVIII at 63 degrees C for 10 h with no discernible change in the structure of the factor VIII/von Willebrand factor (FVIII/VWF) complex owing to a newly developed composition of stabilizers; application of this pasteurisation procedure to a purified FVIII fraction that has already been submitted to a SID-treatment: Doing so, two independent virus inactivation steps are performed as previously recommended by the International Association of Biological Standardization (IABS). Introduction of a second purification step on an anion exchange resin, achieving an additional virus reduction over the presently manufactured FVIII preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Protein Electrophoresis↗

PubMed1986

Regulation of intracellular iron distribution in K562 human erythroleukemia cells.

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.

Biological Transport↗

PubMed1986

Quantitative determination of intracellular, ferritin-associated radioactive iron by high-performance liquid chromatography and immunoprecipitation.

Antibodies raised against ferritin preparations of diverse origin provide an uncertain reagent for quantitation of the ferritin present in specific cell lysates. Utilizing K562 cells, a human leukemic cell line, techniques are described to resolve and to quantitate the ferritin-bound cytosolic iron. Processing the cell lysates by HPLC employing an anion-exchange or hydrophobic interaction column resulted in recovery of a single, ferritin-containing radioactive peak widely separated from the bulk of the non-ferritin-bound iron. Comparison of the yield obtained by chromatography with that by immunoprecipitation confirmed both the specificity and the quantitation of the antibody technique.

Binding Sites↗

PubMedDate not supplied

Continuous removal of protein aggregates by annular chromatography.

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.

Chromatography, Gel↗