RESUMO
A method for large scale isolation of alpha 1-proteinase inhibitor (alpha 1-PI) is described. This method employs waste Cohn Fraction IV-1 as the starting material and involves fractional precipitation with polyethylene glycol followed by ion exchange chromatography on diethylaminoethanol (DEAE)-Sepharose. The process also incorporates a ten hour, at 60 degrees C, heat-treatment step to reduce or eliminate the risk of transmission of viral disease. The final product, having a purity of approximately 60%, is freeze-dried. This preparation behaves almost identically to the alpha 1-PI in plasma and is suitable for replacement therapy in hereditary emphysema.
Assuntos
alfa 1-Antitripsina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Eletroforese em Acetato de Celulose , Humanos , Métodos , alfa 1-Antitripsina/análiseRESUMO
IgG was isolated from plasma at low temperatures using the Cohn fractionation method, and then processed to three different products in order to evaluate how chemical and physical manipulations affect antibodies. Evaluation of the antibodies was done by measuring their ability to bind human immunodeficiency virus, cytomegalovirus, Herpes simplex virus, and rubella virus. In addition, the IgG products were compared by crossed immunoelectrophoresis, and circular dichroism and ultra violet spectroscopy. It was found that exposure of purified IgG to physiological pH altered the molecular conformation of IgG and induced various degrees of irreversible loss in antibody binding to viruses. These observations indicate that more efficacious antibodies may be obtained for clinical use if isolation at their isoelectric point is avoided.
Assuntos
Imunoglobulina G/isolamento & purificação , Precipitação Química/métodos , Liofilização/métodos , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Ultrafiltração/métodos , Vírus/imunologiaRESUMO
The first purified human immunoglobulin G (IgG) preparation used clinically was immune serum globulin (ISG), which was prepared in the 1940s by E. J. Cohn's group. It was originally formulated in water with 0.3 M glycine at pH 6.8 and was 70%-80% monomeric. ISG was safe when given intramuscularly and efficacious for measles and hepatitis prophylaxis. The next generation of purified IgG began in the 1960s with chemically modified preparations suitable for intravenous administration. The first such IgG intravenous preparation (IGIV) in the United States was IGIV pH 6.8 (Gamimune, Cutter Biological), in which the anticomplement activity found in ISG was removed by reduction and alkylation of disulfide bridges. This product was originally formulated as a 5% IgG solution in water (pH 6.8) with 0.2 M glycine in 10% maltose for stabilization. It remained stable for at least 2.5 years at 5 degrees C, was 80%-90% monomeric, had virtually no anticomplement activity, was safe given intravenously, and was efficacious for prophylaxis in agammaglobulinemic patients. A third generation of purified IgG has since been developed; IGIV pH 4.25, (Gamimune N, Cutter Biological), which was isolated by the Cohn method from human plasma and is safe for intravenous use, is a 5% solution of IgG in water (pH 4.25) with 10% maltose. The product is greater than 99% IgG, greater than 95% monomeric, and has greater than 90% less anticomplement activity than ISG.