Search PubMed⌕ Search

Biomedical subjects

D Josic

Publications and source records attributed to D Josic.

At least 19 recordsLinked to original sources
PubMed2003

Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography, static and dynamic light scattering.

Large aggregates (Mr: 10(6)-10(7) g/mol) of human immunoglobulins are present in extremely small concentrations in IgG preparations (<0.1%). Traces of large protein aggregates cannot be determined by conventional size-exclusion chromatography (SEC) using UV detection due to limitations in sensitivity. The conventional analysis of IgG by SEC is limited to dimers and oligomers. Using light scattering it is possible to determine significant differences concerning the aggregate composition and the extent of protein aggregation in samples of different process steps. Two different pilot preparations were analyzed by SEC with UV and static light scattering detection and compared to dynamic light scattering in the batch mode. The change of large aggregates could be monitored and data were corroborated by dynamic light scattering.

Chromatography, Gel↗
PubMed2002

Molecular defects in coagulation Factor VIII and their impact on Factor VIII function.

Molecular defects in Factor VIII (FVIII), such as haemophilia A-related mutations or denaturative conformational changes, may affect the stability of FVIII as well as its interactions with physiological activators, von Willebrand Factor, phospholipid, or conformationally sensitive antibodies. We summarize the contemporary assays which allow identification of impaired functional interactions of FVIII that cause a reduction or loss of its cofactor activity and/or increased immunogenicity. These assays can potentially be used for detection of molecular defects in FVIII and elucidation of the function impaired by these defects.

Blood Coagulation↗
PubMed2001

Molecular modifications in factor VIII concentrates produced from different plasma pools.

We defined the main cause for the increased immunogenicity of the commercial factor VIII (fVIII) plasma-derived concentrates reported to induce formation of inhibitory antibodies in haemophilia A patients in Germany and Belgium. Formation of these antibodies directed against the C2 domain of fVIII was previously attributed to the use of solvent/detergent (S/D) treatment and pasteurisation for virus inactivation of fVIII concentrate. Since fVIII concentrates associated with increased immunogenicity were prepared from plasma pools characterised by elevated levels of coagulation markers, we examined whether the plasma source or S/D treatment and pasteurisation are responsible for structural changes within the C2 domain causing its abnormal immunogenicity. We found that samples of fVIII concentrate that originated from the abnormal plasma pool had a reduced ability to bind to phospholipid and conformationally sensitive anti-C2 domain antibodies, this effect being mostly pronounced in the samples that underwent both S/D treatment and pasteurisation. Thus, our study suggests that insufficient quality of the starting plasma pools is the major factor determining the structural alterations in the C2 domain of fVIII, whereas combination of S/D treatment and pasteurisation aggravates these changes.

Antithrombin III↗
PubMed2001

Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion.

Monoliths are considered as a novel generation of stationary phases. They were applied for capillary electrochromatography and liquid chromatography exploiting every action principle such as ion-exchange, affinity recognition, reversed-phase, and hydrophobic interaction. The fast separation was explained by convective transport of the solutes through the bed. The contribution of this mode of transport is similarly explained as done for the beds packed with particles with gigapores. For monolithic beds, the concept of an ultrashort bed was frequently used. This mode of operation allows very short separation time. In many cases a gradient elution is necessary to achieve separation. Examples of applications for protein and polynucleotide separation performed on monoliths are given. Enzymatic conversion was described showing the examples of several immobilzed enzymes.

Enzymes↗
PubMed2001

Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements.

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 microl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.

Biosensing Techniques↗
PubMed2001

Vitronectin in clotting factor IX concentrates.

Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here.

Blood Coagulation Factors↗
PubMed1999

Effect of von Willebrand Factor and its proteolytic fragments on kinetics of interaction between the light and heavy chains of human factor VIII.

It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (k(on)) for this interaction. The value of the dissociation rate constant (k(off)) for LCh/HCh complex was lower in the presence of Mn2+ (k(off) 4.6x 10(-6) s(-1)) than Ca2+ (k(off) 8.4 x 10(-6) s(-1)) but in both cases vWF had no effect on k(off). This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the k(off) value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1-1365), but not small monomeric N-terminal fragment SPIII-T4 (1-272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of k(on) and did not alter the k(off) value.

Binding Sites↗
PubMed1999

Use of surface plasmon resonance for studies of protein-protein and protein-phospholipid membrane interactions. Application to the binding of factor VIII to von Willebrand factor and to phosphatidylserine-containing membranes.

The surface plasmon resonance phenomenon is used for real time measurements of protein-protein and protein-membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor VIII (fVIII) with von Willebrand factor (vWf) and phospholipid have been developed. These interactions of fVIII are required for maintenance of fVIII concentration in circulation and for the assembly of the functional factor Xase complex, respectively. With these binding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% phosphatidylserine (PS) and 4% PS was examined. The finding that Kd of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), indicates that the heavy chain (HCh) is required for the maximal affinity of fVIII for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles are similar to that of fVIII, indicating that LCh is solely responsible for these interactions. It was also examined how removal of the acidic region affects the binding affinity of the remaining part of LCh for vWf and phospholipid. It was demonstrated that the loss of the LCh acidic region upon thrombin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (k(off) value) and a 165 and 1500-fold increase in the Kd value of the binding of fVIII fragment A3-C1-C2 to vWf compared to that of LCh and fVIII, respectively. In contrast, the binding affinity of A3-C1-C2 for PS-containing membranes was 8-11-fold higher than that of LCh. Possible conformational change(s) in C2 domain upon removal of the acidic region were studied using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3-C1-C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indicates that these conformational changes do occur.

Antibodies, Monoclonal↗
PubMed1999

Use of high-resolution techniques for the characterization of clotting factor VIII.

Dealing with the structural characterization of clotting factor VIII (FVIII) requires the application of several high-resolution analytical techniques. Besides the analytical point of view, a detailed knowledge of FVIII structure, production and therapeutic application is necessary. This review gives an overview of most of the currently applied analytical methods and how they deal with the complex analytical problem, investigating FVIII in a sample matrix containing large amounts of accompanying plasma proteins.

Factor VIII↗
PubMed1999

Matrix-assisted laser desorption/ionization time-of-flight and nano-electrospray ionization ion trap mass spectrometric characterization of 1-cyano-2-substituted-benz[f]isoindole derivatives of peptides for fluorescence detection.

A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both [M + H]+ ions by MALDI and both [M + H]+ and [M + 2H]2+ ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN]+ ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.

Fluorescent Dyes↗
PubMed1998

Peptide affinity chromatography of human clotting factor VIII. Screening of the vWF-binding domain.

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.

Amino Acid Sequence↗
PubMed1998

High-performance capillary electrophoresis for in-process control in the production of antithrombin III and human clotting factor IX.

Antithrombin III (ATIII) and factor IX (FIX), two proteins from the clotting cascade, were investigated in parallel experiments, using capillary gel electrophoresis and capillary isoelectric focusing. The results from these experiments were compared with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and slab gel isoelectric focusing. In the case of ATIII, capillary gel sieving showed comparable results to SDS-PAGE with the added advantage of the shorter time required for analysis. By optimizing capillary isoelectric focusing (cIEF), a separation of the ATIII isoforms was achieved. In the case of FIX, capillary gel electrophoresis (SDS-CE) of a FIX preparation gave similar results to those obtained by size-exclusion high-performance liquid chromatography and SDS-PAGE, but turned out to be less sensitive in detecting protein impurities at low concentrations. The microheterogeneity of this protein was shown by using cIEF.

Antithrombin III↗
PubMed1997

Pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration.

Amlodipine, 3-ethyl 5-methyl-2-[(2-aminoethoxymethyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methy l-3,5-pyridinedicarboxylate, is a chiral calcium antagonist, currently on the market and in therapeutic use as a racemate. The pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration to healthy male human volunteers together with comparative administration of the racemic mixture of both enantiomers were studied. Plasma levels were studied as a function of time and assayed using an enantioselective chromatographic method (coupled chiral and achiral HPLC) with on-line solid-phase extraction and UV absorbance detection. The method was validated separately for the R-(+)- and S-(-)-enantiomer, respectively. Results of the study indicate that the pharmacokinetic behaviour of R-(+)- and S-(-)-amlodipine after single enantiomer administration is comparable to that of each enantiomer after administration of the racemate. No racemization occurs in vivo in human plasma after single enantiomer administration.

Administration, Oral↗
PubMed1995

Improved virus safety and purity of a chromatographically produced factor IX concentrate by nanofiltration.

A virus removal system based on tangential flow filtration was introduced into a Factor IX production process. Beside the intended virus reduction potency of filter membranes, an additional purification effect could be achieved. This purification effect was evaluated in detail by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion HPLC. High-molecular-mass impurities were retained by the membrane, thus increasing the specific activity of the product.

Blood-Borne Pathogens↗
PubMed1995

Development and biochemical characterization of a double-virus-inactivated factor VIII preparation.

This paper describes the development of a double-virus-inactivated preparation. The original goal was to inactivate and/or remove parvovirus from plasma-derived factor VIII. More recently, attention has also been focused on the controversial issue of transmission of the non-lipid-coated hepatitis A virus by preparations of factor VIII which have been solvent/detergent-treated and purified by ion-exchange chromatography.

Antiviral Agents↗
PubMed1994

Purification of factor VIII and von Willebrand factor from human plasma by anion-exchange chromatography.

Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chromatographic procedures. After precipitation, the first step in virus inactivation is achieved through the effect of a non-ionic detergent such as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By subsequent anion-exchange chromatography, a highly enriched product is isolated, consisting of a complex formed by factor VIII and von Willebrand factor (FVIII-vWF). This treatment also removes the virus-inactivating reagents to quantities in the low ppm range. The second step in virus inactivation is aimed specifically at the non-enveloped viruses and consists of pasteurization at temperatures higher than 60 degrees C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteurisation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently removed by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows the destruction of both lipid-enveloped and non-enveloped viruses. During the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding calcium ions. Subsequently both glycoproteins from this complex are separated from one another by further anion-exchange chromatography.

Biocompatible Materials↗
PubMed1994

Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies.

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

Animals↗