Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Albumin
Patent
1995-10-30
1998-10-06
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Albumin
530380, 530392, 530393, 530394, 530414, 530418, 530419, 530422, 530427, C07K 100, A61K 3514
Patent
active
058177655
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a process for reduction of virus inactivating chemicals and/or detergents in an aqueous composition containing a water-soluble plasma protein. By selecting a suitable combination of temperature and concentration above 0.5M of a salt with a high salting out effect according to the Hofmeister series, vesicles containing the virus inactivating chemical and/or detergent are formed. These vesicles are removed from the aqueous phase, e.g. by phase separation or filtration, and the protein thereafter isolated from the aqueous phase. When the aqueous phase comprises a salt of citrate or sulphate in a concentration above 1M at room temperature, the reduction of virus inactivating chemical or detergent can be as high as 2000 times or more, giving a final concentration below 5 ppm.
BACKGROUND OF THE INVENTION
The inactivation of virus in blood products such as factor VIII, albumin, factor IX and antithrombin is a known problem for manufacturers. This has normally been solved by heating. If the product is not inactivated by such a process, adding of virus inactivating chemicals may be used. In this case, however, there is a need for removing the added chemicals before use of the medical products.
EP-A-0 218 090 (Miles Laboratories) relates to a process for separating and recovering proteins or nucleic acids, from an aqueous system also containing a component having the ability to create two liquid phases by use of salt partitioning technology. In this process, a polymer and a water soluble salt, such as potassium or sodium phosphate or ammonium sulphate, are added to the protein or nucleic acid, whereupon the resulting solution is left to separate. Information about virus inactivating chemicals or detergents is lacking, as is information about techniques to reduce the concentration of such compounds to a pharmaceutically acceptable level.
EP-A-0 239 859 (New York Blood Center) relates to a method for removing lipid soluble process chemicals, such as virus inactivating solvents and/or detergents, from biological materials, by contacting the biological material with an oil extract, agitating the resultant mixture, separating out an upper phase and a lower phase by sedimentation or centrifugation, and decanting the upper phase containing oil and extracted process chemicals. There is no information about addition of organic or inorganic salts with a high salting out effect or the possible advantage derived by their presence. The lack of these salts in the aqueous phase necessitates a complicated process with repeated extraction steps to arrive at a sufficiently low level of the virus inactivating solvents and/or detergents.
Another method is disclosed in EP-A-0 131 740 (New York Blood Center), in which a protein-containing composition is virus inactivated by contacting the composition with di- or trialkylphosphate, preferably in the presence of a non-ionic detergent. According to this disclosure, the di- or trialkylphosphate is removed by precipitation of the protein with glycine and sodium chloride. The detergent can be removed by several steps, chosen among diafiltration, adsorption on chromatographic supports and precipitation.
These methods may be laborious, time consuming and may often not give a satisfying reduction of detergents and virus inactivating chemicals.
From an article by R. A. Ramelmeier et al, Bioseparation 2; 315-324, 1991, it is known that a hydrophobic enzyme can be purified from fermentation broths in a detergent based aqueous two-phase system by variation of e.g. temperature and salt concentration. No higher salt concentration than 0.2M was used. The enzyme is recovered in the detergent phase and can be recovered to 80-90%.
DESCRIPTION OF THE INVENTION
We have now surprisingly found that when the concentration of certain salts is increased to above 0.5M in a composition containing a plasma protein (either fractionated from plasma or recombinant produced), a virus inactivating chemical, preferably tri-n-butyl phosphate (TNBP), and/or a detergent, preferably a TRITON.RT
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Isaksson Sven
Winge Stefan
Mohamed Abdel A.
Pharmacia & Upjohn Aktiebolag
Tsang Cecilia J.
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