Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1997-03-27
1999-12-21
Wax, Robert A.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530383, 530412, 530415, A23J 100, A61K 3514
Patent
active
060050829
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for purifying recombinant coagulation factor VIII by loading an aqueous solution containing factor VIII onto a hydrophobic interaction chromatography (HIC) gel, wherein at least one surfactant is present in the aqueous solution and/or a buffer solution used to equilibrate the gel prior to loading the aqueous solution onto the gel. The presence of a surfactant when loading the solution containing factor VIII onto the HIC gel, makes it possible to efficiently separate the intact and active factor VIII molecules from molecules with structural deviations. With the present invention it is further possible to reduce the content of DNA and/or CHO cell contaminants considerably and retain the activity of the factor VIII product to a hitherto unknown extent. The invention further relates to an aqueous solution containing recombinant factor VIII which has been purified according to the present process and use of such an aqueous solution, for the manufacture of a medicament for administration to a patient having the symptoms of hemophilia. Also, the invention relates to a method for treatment of hemophilia by administration of a therapeutically effective amount of recombinant factor VIII which has been purified according to the present process.
BACKGROUND OF THE INVENTION
Hemophilia is an inherited disease which has been known for centuries, but it is only within the last four decades that it has been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10 000 live-born males. The disease is caused by strongly decreased level or absence of biologically active coagulation factor VIII (antihemophilic factor), which is a protein normally present in plasma. The clinical manifestation of hemophilia A is a strong bleeding tendency and before treatment with factor VIII concentrates was introduced, the mean age of the patients concerned was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has improved the situation for treatment of hemophilia patients considerably and offered them the possibility of living a normal life.
Until recently, therapeutic factor VIII concentrates have been prepared by fractionation of plasma. However, there are since some years methods available for production of factor VIII in cell culture using recombinant DNA techniques as reported in e.g. W. Wood et al, Nature, 312, p. 330-37 (1984) and EP-A-0 160 457.
Factor VIII concentrates derived from human plasma contain several fragmented fully active factor VIII forms as described by Andersson et al, Proc. Natl. Acad. Sci. USA, 83, p. 2979-83 (May 1986). The smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by metal ion(s). Reference is here made to EP-A-0 197 901.
Pharmacia AB of Stockholm, Sweden, has developed a recombinant factor VIII product which corresponds to the 170 kDa plasma factor VIII form in therapeutic factor VIII concentrates. The truncated recombinant factor VIII molecule is termed r-VIII SQ and is produced by Chinese Hamster Ovary (CHO) cells in a cell culture process in serum-free medium.
The structure and biochemistry of recombinant factor VIII products in geneal have been described by Kaufman in Trends in Biotechnology, 9 (1991) and Hematology, 63, p. 155-65 (1991). The structure and biochemistry of r-VIII SQ have been described in WO-A-9109122.
High-performance hydrophobic interaction chromatography (HIC) is a separation technique suitable for purifying proteins. The general characteristics and suitable conditions for carrying out a HIC step have been described by K-O Eriksson in Protein Purification; Principles, High Resolution Methods, and Applications, VCH Publishers, Inc., New York, p. 207-226 (1989). In this technique, proteins are eluted from relatively weak hydrophobic stationary phase
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Genetics Institute Inc.
Stole Einar
Wax Robert A.
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