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Biomedical subjects

E Sawatani

Publications and source records attributed to E Sawatani.

9 recordsLinked to original sources
PubMed2001

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology.

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or =99% purity, a yield of 25.0 +/- 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60 degrees C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37 degrees C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

Agglutination Tests↗
PubMed2000

High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography.

In order to obtain intravenous immunoglobulin G (iv IgG) of high quality from F-I+II+III or F-II+III pastes prepared by the Cohn method, we developed a chromatography process using ion exchange gels, Q-Sepharose FF and CM-Sepharose FF, and Sephacryl S-300 gel filtration. Viral inactivation was performed by incubating the preparation with pepsin at pH 4.0 at 35 degrees C for 18 h. The characteristics of 28 batches produced by us were: yield 4.3 +/- 0.2 g/l plasma, i.e., a recovery of 39.1 +/- 1.8%; IgG subclasses distribution: IgG1 = 58.4%, IgG2 = 34.8%, IgG3 = 4.5% and IgG4 = 2. 3%; IgG size distribution was 98.4% monomers, 1.2% dimers and 0.4% polymers and protein aggregates; anticomplement activity was less than 0.5 CH50/mg IgG, and prekallikrein activator activity (PKA) was less than 5 IU/ml. These characteristics satisfied the requirements of the European Pharmacopoea edition, and the regulations of the Brazilian Health Ministry (M.S. Portaria No. 2, 30/10/1998).

Chromatography, Liquid↗
PubMed1998

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use.

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37 degrees C for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8 degrees C. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Chromatography↗
PubMed1998

Purification of human albumin by the combination of the method of Cohn with liquid chromatography.

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60 degrees C for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 +/- 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were < or = 5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.

Chemical Fractionation↗
PubMed1996

An alternative column chromatographic process for the production of human albumin.

We have modified a standard column chromatography method for the preparation of albumin from human plasma. The proposed method utilizes Sephadex G-25, euglobulin precipitation, DEAE-Sepharose, ethanol heat treatment and Sephacryl S-200 HR. The procedure differs from that normally used by the introduction of Schneider's ethanol/heat treatment step and the elimination of a CM-Sepharose step, after the DEAE-Sepharose step. The proposed method was used to produce 15 batches of albumin, 100 liters of plasma per batch. Purity of more than 99% was obtained at an average yield of 26.5 g/l plasma for the albumin produced. All batches presented 99.2 to 99.9% monomer and 0.1 to 0.8% dimer, with no larger polymers detected. The utilization of the final gel filtration step eliminated highly polymerized albumin usually obtained during the process. The advantages of the proposed method are reduction in the overall process time from 6 to 5 days, elimination of the CM-Sepharose step and the reduction of 6 to 4 columns in series for the Sephacryl S-200 HR step.

Chemical Fractionation↗