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Biomedical subjects

H Schwinn

Publications and source records attributed to H Schwinn.

33 records · Page 2Linked to original sources
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↗
PubMed1993

Isolation of plasma proteins from the clotting cascade by heparin affinity chromatography.

The use of heparin affinity chromatography for the isolation of plasma proteins from the clotting cascade is described. The separation is carried out with heparin agarose and, in parallel operations, with different rigid gels on a polymer base. The quality of the separation and the reproducibility of the results were investigated and the stability of the materials at high pH was tested. The affinity supports were used for the isolation of antithrombin III from human plasma and for the separation of factor IX from factor X, after partial purification by anion-exchange chromatography. The isolation of antithrombin III from human plasma served as a model. The non-specific bindings were investigated, together with the resistance of the support when treated with 0.2 and 0.5 M sodium hydroxide. Heparin agarose has low non-specific bindings, but it cannot be exposed to high pH. The supports on a polymer base are resistant to high pH, up to 13.7. However, they may remain slightly hydrophobic, and the hydrophobicity of the matrix leads to an increase in non-specific bindings. When antithrombin III is isolated, the non-specific bindings result in contamination of the final product. The lack of resistance of the matrix at high pH causes a weaker binding of antithrombin III, and the product is eluted at lower and lower sodium chloride concentrations. The results can be indicative of the behaviour of the support in the separation of factor IX from factor X. High non-specific bindings will lead to contamination of the factor IX product and consequently to low specific activity. Insufficient resistance of the support at high pH will result in failure to separate the two clotting factors satisfactorily. The separation can be monitored by heparin high-performance membrane affinity chromatography (HPMAC). Contamination of the sample, which occur in sodium dodecyl sulphate-polyacrylamide gel electrophoresis are detected within minutes by fast heparin HPMAC.

Antithrombin III↗
PubMed1992

Manufacture and in vitro characterization of a solvent/detergent-treated human plasma.

We have developed a modified solvent/detergent (S/D) treatment to inactivate viruses in human plasma using 1% w/w final concentration of tri(n-butyl) phosphate (TNBP) and Triton X-100 and an incubation period of 4 h at 30 degrees C. The procedure inactivates > or = 10(6) chimpanzee-infectious doses (CID50) of HBV, > or = 10(5) CID50 of HCV, and > or = 10(6.2) tissue culture infectious doses (TCID50) of HIV. After virus inactivation, eleven plasma batches were lyophilized and 12 batches were deep-frozen until further use. The batches were characterized by extensive laboratory tests including measurement of clotting factors I-XIII, von Willebrand factor, plasminogen, inhibitors of blood coagulation and fibrinolysis, and other clinically important plasma proteins. All parameters were determined before and after S/D treatment. Twelve conventional single donor plasma units served as control. There were no marked losses of activities of clotting factors, antithrombin III, protein C, plasminogen, and C1-esterase inhibitor due to treatment. After the S/D step, the levels of these parameters were within the normal range in all batches. The same holds true for total protein, immunoglobulins, albumin, complement factors C3 and C4, haptoglobin, hemopexin, caeruloplasmin, alpha 1-antitrypsin, and pH. Protein S and alpha 2-antiplasmin activities decreased by about 50% and were frequently found to be slightly below the lower limit of the respective normal range after treatment. The interindividual variations of all proteins analysed were significantly lower than in the single donor plasma units. The S/D procedure did not lead to increases of markers indicating activation of hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗
PubMed1991

Removal of parvovirus B19 from contaminated factor VIII during fractionation.

A solution of pooled cryo-precipitate for preparing factor VIII (F VIII) by the solvent/detergent method was contaminated experimentally with parvovirus B19-positive plasma to evaluate virus reduction achieved by the final steps of the F VIII production process. Virus reduction was at least 2 logs of the total amount of B19 virus added to the pooled cryo-precipitate. The major amount of B19 virus was detected in the solution used to regenerate the F VIII-selective anion exchange chromatography column. A few viral particles were detected in the final F VIII concentrate before filtration but these were aggregates and were removed by filtration, and in the solution used to regenerate the anion exchange resin. It is not known whether the residual viral DNA present in the final product represents infectious or inactivated particles.

Blood Preservation↗
PubMed1989

Progress in purification of virus-inactivated factor VIII concentrates. Three generations of solvent/detergent treated plasma derivatives.

A production process of a newly developed highly purified and virus-inactivated Factor (F) VIII-concentrate (Octa V.I. and Octavi) is presented. Taking advantage of a selective resin matrix and the solvent/detergent procedure for virus inactivation--known not to denaturate proteins--a product of a specific activity greater than or equal to 100 IU F VIII/mg could be developed in the final container without the use of an immuno-affinity adsorption step. The main steps of the procedure are: Pooled cryoprecipitate is extracted, the extract is cleared from fibrinogen at + 10 degrees C and virus-inactivated at + 28 degrees C after addition of tributyl-phosphate (TNBP) and detergent. Thereafter the extract is brought in contact to a F VIII-selective anion exchange resin using a chromatographic column. TnBP and the detergent are removed by an extensive washing process and the F VIII-activity is concentrated in a fraction, ready for filling, by means of a cascade of wahing- and elution-buffers. The product is free from coagulable protein and gamma-globulins. The F VIIIC: Ag/F VIII:C-ratio is about unity, suggesting the F VIII-molecule remained in its native state. The development of highly purified F VIII concentrate is based on two previous products of lesser purity (spec. activity of about 1 and 10 IU/mg). The evolution is shown by a comparison of detailed analytical data.

Chromatography, Ion Exchange↗
PubMed1981

[Factor VIII concentrate, highly purified and heated in solution (author's transl)].

A process is described to produce a highly purified factor VIII concentrate heated in solution. Pooled cryoprecipitate from citrated plasma is adsorbed on aluminum hydroxide gel. The fibrinogen is removed by heat denaturation in the presence of glycine; factor VIII is precipitated with sodium chloride from the supernatant. The precipitate is dissolved in a saccharose/glycine solution and heated at 60 degrees C for 10 h. The factor VIII is then separated by further precipitation with sodium chloride, the precipitate dissolved, dialysed and sterilized by filtration. The factor VIII concentrate contains approximately 6 units F VIII:C per mg protein. the ratio of F VIII R:Ag/F VIII:C is 3. The product is free from coagulable protein and gamma-globulins. The efficacy of the heating step in the reduction of the hepatitis B-infectious titre was proved in chimpanzees. For this purpose, hepatitis B virus was added to the pooled cryoprecipitate.

Drug Stability↗
PubMed1980

[Influence of phospholipids and their combination with prothrombin complex and calcium ions on coagulation in mice and rabbits both normal and affected by acetylsalicylic acid (author's transl)].

The influence of phospholipids and their combination with prothrombin and calcium on coagulation both normal and affected by acetylsalicylic acid (ASA) was investigated by determination of the bleeding time of mice, thrombelastographic (TEG) measurements and counting of thrombocytes of rabbits. Bleeding times and times of the TEG were prolonged after oral application of ASA, while thrombus stability was not altered. The number of thrombocytes decreased. Bleeding- and TEG-times normalized after injection of phospholipids. This effect was increased and of longer duration when phospholipids were combined with calcium and prothrombin. Decrease of the number of thrombocytes caused by pretreatment with ASA normalized only after injection of phospholipids together with prothrombin and calcium. The number of thrombocytes was not negatively influenced by the therapy in animals with normal coagulation.

Animals↗
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↗