Miles Laboratories, Inc.

Miles Inc.

Cutter Laboratories, Inc.

Gautam Mitra

Milton M Mozen

George B Dove

John L Lundblad

Michael H Coan

Paul K Ng

Shohachi Wada

Melvin F Wong

James W Bloom

Robert E Jordan

Robert E Louie

The effects of Cohn-Oncley cold alcohol fractionation on infectivity of two prototype retroviruses, mouse xenotropic type C and the LAV strain of AIDS retrovirus result in a significant reduction in retrovirus titer. At least a 100,000-fold reduction of LAV was obtained when fractionating plasma to fraction II. Adjustment to pH 4.0 at filtrate III was as effective for viral inactivation as was precipitation of fraction II from filtrate III with 25% ethanol. Liquid immunoglobulin preparations spiked with LAV and incubated at 27.degree. C. for 3 days resulted in an additional 1,000-10,000-fold reduction of LAV titer. Our results support the clinical and epidemiological evidence that therapeutic immunoglobulin preparations prepared by Cohn-Oncley cold ethanol processing (at least about 18% v/v alcohol, pH.ltoreq.5.4) and then stored at a pH equal to or less than about 5.4 and a temperature of at least 27.degree. C. for at least 3 days, do not transmit retroviruses such as the LAV strain of the AIDS virus. Similar fractionation followed by storage at a higher pH (6.8) followed by a storage at a higher temperature (45.degree. C.) for at least 8 hours also appears effective in avoiding transmission of the retroviruses.

Preparation of human immunoglobulin free of hepatitis C

Antibodies, including monoclonal antibodies (Mabs), can be made substantially free of infectious viruses by storing them in a liquid state at conditions of pH, temperature and time sufficient to inactive substantially all infectious viruses. Preferred inactivation methods involve use of a pH equal to or less than about 4.0 at a temperature of at least about 5.degree. C. for at least about 16 hours.

Preparation of virus-free antibodies

An essentially pure and stablized antibody preparation comprising IgM antibodies having a purity greater than about 98% by weight and a nucleic acid content of less than about 200 pg per mg IgM. In one embodiment IgM antibodies from a monoclonal source are subjected to ion exchange and size exclusion chromatography at an alkaline pH to yield a purified IgM having a nucleic acid content of less than 10 pg/mg IgM, preferably less than about 4 pg/mg IgM. A highly purified and stabilized preparation of anti Pseudomonas aeruginosa antibodies is disclosed. The removal of nucleic acids is assured by subjecting the antibody source to at least one and preferably two low pH precipitation steps. In a very preferred embodiment, ion exchange and/or size exclusion chromatography is used to remove any residual nucleic acids.

Removal of nucleic acids from monoclonal antibody preparations

Antibody preparation purified using immobilized protein A and yet substantially free of protein A that may have solubilized during the purificaiton process. The antibodies include less than 15 ng protein A per mg of antibody, preferably less than 1 ng/mg. Low protein A content is obtained by first contacting the antibodies and solubilized protein A with an ion exchange resin under conditions to adsorb both. The antibodies and protein A are then sequentially eluted under conditions of increasing ionic strength.

Removal of protein A from antibody preparations

Controlled enzymatic treatment may be used to selectively degrade undesirable contaminants to a size or charge range which can be more readily removed by subsequent separation steps. Treatment is especially useful for purifying rDNA or monoclonal antibody culture products by using nuclease enzyme treatment to degrade undesirable residual nucleic acids to a molecular size or charge range sufficiently different from the product to be purified so that this difference can be exploited in a subsequent purification step (e.g. precipitation, size exclusion chromatography or ion exchange chromatography). The nuclease enzyme treatment is done in the presence of a detergent.

Enzymatic purification process

Protein A is selectively isolated from an antibody--Protein A mixture by exposing the mixture to an anion exchange material under conditions sufficient to adsorb both components and then sequentially eluting the antibodies and protein A under conditions of increasing ionic strength. Resulting antibody preparations have less than about 15 ng of Protein A per mg of antibody.

Immune serum globulins (ISG) can be made substantially free of infectious retroviruses by storing the ISG in a liquid state at conditions of pH, temperature and time sufficient to inactivate substantially all infectious retroviruses. Preferred inactivation methods involve use of either of two specified storage conditions: (1) at a pH equal to or less than about 4.25 at a temperature of about 27.degree. C. for at least 3 days, or (2) at a pH equal to or less than about 6.8 at a temperature of about 45.degree. C. for at least about 8 hours.

Preparation of retrovirus-free immunoglobulins

Immune serum globulins (ISG) can be made substantially free of infectious retroviruses by preparing the ISG from human plasma using a cold ethanol plasma fractionation process at a pH equal to or less than 5.4 and then storing the ISG at either of two specified storage conditions: (1) at a pH equal to or less than about 4.25 at a temperature of about 27.degree. C. for at least 3 days, or (2) at a pH equal to or less than about 6.8 at a temperature of about 45.degree. C. for at least about 8 hours. pg,10

There is disclosed a process for producing a convalently bound heparin--antithrombin-III complex useful for anticoagulant therapy, a covalently bound heparin--antithrombin-III complex produced by the process, a composition thereof in a pharmaceutically acceptable carrier, and a method for preventing and treating thromboembolisms by administering to a human patient a therapeutically effective amount of the complex or preparation.

Covalently bound heparin--antithrombin-III complex

There is disclosed an improved method for separating and recovering proteins, particularly therapeutically active proteins, from an aqueous system also containing a component having the ability to create two liquid phases by use of salt partitioning technology. By the addition of water soluble inorganic salts to an aqueous system containing one or more therapeutically active proteins or nucleic acids, especially an aqueous system obtained from fractionation of a blood plasma fraction or from a tissue culture fluid resulting from a biotechnology production operation such as recombinant DNA and monoclonal antibody technologies, the aqueous system may be separated into two or more liquid phases. Such separated phases may be selectively enriched in components of the original aqueous system having differing solubility in the so-separated liquid phases.

Method of separating proteins from aqueous solutions

There is disclosed an improved process for producing high purity antihemophilic factor concentrate from an antihemophilic factor-containing dispersion or solution isolated from blood plasma or a blood plasma fraction, wherein the improvement is in carrying-out two consecutive precipitations using a combination of precipitants in each precipitation, first a combination of 1-4% by weight, based on weight of solution, of polyethylene glycol and 0.1-0.2 ml of 1-3%, based on weight of suspension, aluminum hydroxide suspension per gram of protein in the starting dispersion or solution, followed by a combination of added polyethylene glycol to provide a final concentration of 9-13% by weight, based on weight of the resulting solution, and 10-20% by weight of glycine, based on weight of the polyethylene glycol solution, and 10-20% by weight, based on weight of the polyethylene glycol solution, of sodium chloride.

Process for producing a high purity antihemophilic factor concentrate

There is disclosed a process for producing a covalently attached alpha-1-proteinase inhibitor - water soluble polymer complex useful for pulmonary emphysema therapy, a covalently attached alpha-1-proteinase inhibitor - water soluble polymer complex produced by the process, a composition thereof in a pharmaceutically acceptable carrier, and a method for treating pulmonary emphysema by administering to a human patient a therapeutically effective amount of the complex or preparation.

Covalently attached complex of alpha-1-proteinase inhibitor with a water

Compositions containing a concentrate of coagulation Factors II, VII, IX, and X are pasteurized without substantial loss of therapeutic activity by mixing the protein composition with a pasteurization-stabilizing amount of a polyol and a source of citrate ions prior to pasteurization. Pasteurized compositions containing a concentrate of coagulation Factors II, VII, IX, and X, which have heretofore been unattainable, can be prepared by the method of the invention.

Pasteurized therapeutically active blood coagulation factor concentrates

Novel antihemophilic factor concentrates and process for making the same are disclosed. An aqueous solution of anithemophilic factor proteins is obtained, preferably by solubilizing antihemophilic factor proteins from blood plasma cryoprecipitate in water. The aqueous solution is purified to remove unwanted protein, for example, by mixing the aqueous solution with aluminum hydroxide. Then, the aqueous solution is ultrafiltered to concentrate it, mixed with buffer and saline and adjusted to an acid pH, and freeze-dried.

Antihemophilic factor concentrate

This disclosure pertains to a concentrate for controlling bleeding in hemophilia, free of thrombin, heparin, thromboplastin activity, anticomplement activity, depressor activity, and activated Factor X, and containing coagulation Factors II, VII, IX, and X in non-activated form and containing no greater amounts of calcium-activated factors and Factor VIII Inhibitor Bypassing Activity substance than that found in concentrates of Factor II, VII, IX, and XI that have not been treated, respectively, with calcium or a material which would produce a Factor VIII Inhibitor Bypassing Activity substance in such concentrates and having a specific activity of at least about 1.5 Factor IX units per milligram of total protein and a Factor IX:Factor VII ratio of at least about 6:1, a Factor IX:Factor II ratio of about 1:1 to 2:1, and a Factor IX:NAPTT ratio of at least about 5:1 on a Units per milliliter basis, a Factor II Specific Activity of about 1.0-4.0 Units per milligram of total protein, and a Factor IX:ceruloplasmin ratio of at least about 25:1. In the method of the invention a blood fraction containing coagulation Factors II, VII, IX, and X is applied to an anion exchanger to adsorb the coagulation Factors, which are selectively eluted therefrom using an aqueous sodium chloride-citrate solution of increasing ionic strength. The eluate is treated to reduce its concentration of non-volatile salt and concentrated to yield the above product.

Growing community of inventors

Kensington, CA, United States of America

Gautam Mitra