Inactivation of viruses in labile protein-containing compositions using fatty acids
Inactivation of viruses in labile protein-containing compositions using fatty acids
There is disclosed a process for rendering a labile protein-containing composition, substantially free of lipid-containing viruses without incurring substantial protein denaturation comprising contacting said composition with an effective amount of a fatty acid or a soluble ester, alcohol or a salt thereof for a sufficient period of time to inactivate virus contained therein.
Inventors: Horowitz; Bernard (New Rochelle, NY)
Assignee: New York Blood Center, Inc. (New York, NY)
[*] Notice: The portion of the term of this patent subsequent to September 23, 2003 has been disclaimed.
Family ID: 25372050
Appl. No.: 06/878,446
Filed: June 25, 1986
Field of the Invention
This invention relates to undenatured virus-free biologically active protein-containing compositions. More especially, this invention relates to the inactivation of viruses, especially lipid coated viruses, e.g., hepatitis B, in human blood, blood components, blood plasma or any fraction, concentrate or derivative thereof containing blood proteins or non-blood sources including normal or cancer cells, the exudate from cancer or normal cells grown in cultures, hybridomas, in products from gene splicing (DNA), etc., by the use of a fatty acid or an ester or salt thereof or by the use of a long chain unsaturated monoglyceride.
In particular, this invention relates to blood plasma or other plasma protein-containing compositions which are rendered substantially free of hepatitis B and/or non-A and non-B hepatitis or other viral infectivity, such blood plasma or fractions thereof having valuable labile proteins, such as, for example, factor VIII, by using fatty acids or long chain unsaturated monoglycerides.
What is claimed is:
1. A process for rendering a labile protein-containing composition substantially free of lipid-containing viruses without incurring substantial protein denaturation comprising contacting said composition with an effective amount of fatty acid having at least 16 carbon atoms or a salt or a monoglyceride thereof for a sufficient period of time and at a temperature of 0.degree. C. to 37.degree. C.
2. A process according to claim 1, wherein the fatty acid is unsaturated.
3. A process according to claim 1, wherein the fatty acid has 16 to 20 carbon atoms.
4. A process according to claim 1, wherein the fatty acid has at least one double bond in the cis configuration
5. A process according to claim 1, wherein said fatty acid is selected from the group consisting of 11-eicosenoic acid, arachidonic acid, linoleic acid, linolenic acid, palmitoleic acid, elaidic acid, linolenic acid, gamma-linolenic acid, palmitic acid and arachidic acid.
6. A process according to claim 1, wherein said salt is an alkali or alkaline earth metal salt.
7. A process according to claim 1, wherein said contacting is conducted in the presence of a wetting agent.
8. A process according to claim 7, wherein said wetting agent is a non-ionic detergent.
9. A process according to claim 7, wherein said wetting agent is added to said blood composition prior to contacting said blood product with said fatty acid or salt or monoglyceride thereof.
10. A process according to claim 7, wherein said wetting agent is added simultaneously with said fatty acid or salt or monoglyceride thereof to said blood composition.
SUMMARY OF THE INVENTION
The present invention is directed to achieving three goals, namely, (1) a safe, (2) viral inactivated protein-containing composition, i.e., a blood product containing a labile protein, (3) without incurring substantial protein denaturation. As shown above, these three goals are not necessarily compatible since, for example, beta-propiolactone inactivates viral infectivity, but is unsafe and substances such as formaldehyde inactivate viruses, but also substantially denaturate the valuable plasma proteins, for example, factor VIII.
It, therefore, became desirable to provide a process for obtaining protein-containing compositions which does not substantially denature the valuable protein components therein and which does not entail the use of a proven carcinogenic agent. More especially, it is desirable to provide blood protein-containing compositions in which substantially all of the hepatitis viruses and other viruses present are inactivated and in which denatured protein such as factor VIII account for only a small amount of the total amount of these proteins in the blood protein-containing composition.
It is a further object to provide products from cancer or normal cells or from fermentation processes following gene insertion which are substatially free of virus, especially lipid-containing viruses.
It has now been discovered, quite surprisingly, that while most of the viral inactivating agents denature factor VIII and other valuable blood plasma proteins, that not all viral inactivating agents have such effect. It has been discovered that a labile protein-containing composition, e.g., blood cell proteins, blood plasma, a blood plasma fractionation precipitate, a blood plasma fractionation supernatant, cryoprecipitate, cryosupernatant, or portion or derivative thereof or serum or a non-blood product produced from normal or cancerous cells (e.g., via recombinant DNA technology) is contacted for a sufficient period of time with a fatty acid, or a soluble ester, alcohol or a salt thereof, e.g., an alkali or alkaline earth metal salt thereof that lipid-containing viruses such as the hepatitis viruses present in the composition are virtually entirely inactivated without substantial denaturation of proteins contained therein. By contacting a blood protein mixture or concentrate thereof or fraction thereof with a fatty acid or a C.sub.1-4 alkyl ester thereof, or alcohol thereof or a salt thereof, e.g., an alkali or alkaline earth metal salt thereof, or with a long chain unsaturated monoglyceride, hepatitis viruses can be substantially inactivated, e.g., to an inactivation of greater than 4 logs, while realizing a yield of protein activity to total protein of at least 60%.
By such procedures there is provided a labile protein-containing composition, for example, a blood protein-containing such as mammalian blood cell derivative (e.g., hemoglobin, alpha-interferon, T-cell growth factor, platelet-derived growth factor, etc.), plasminogen activator, blood plasma, blood plasma fraction, blood plasma precipitate (e.g., cryoprecipitate, ethanol supernatant or polyethylene glycol supernatant), characterized by the presence of one or more blood proteins, such as labile blood factor VIII having a total yield or protein activity to total protein of at least 60%, preferably at least 70%, said blood protein-containing composition having greatly reduced or virtually no hepatitis viruses. Virus in a serum is determined by infectivity titrations.
By the inactivation procedure of the invention, most, if not virtually all, of the hepatitis viruses contained therein are inactivated. The method for determining infectivity levels by in vivo chimpanzees is discussed by Prince, A.M., Stephen, W., Brotman, B. and van den Ende, M.C., "Evaluation of the Efect of Beta-propiolactone/Ultraviolet Irradiation (BPL/UV) Treatment of Source Plasma on Hepatitis Transmission by Factor IX Complex in Chimpanzees", Thrombosis and Haemostasis, 44, 138-142, 1980.
The hepatitis virus is inactivated by treatment with a fatty acid, preferably an unsaturated fatty acid, or a soluble ester, alcohol as described herein, and is not inactivated because of inclusion in the plasma of antibodies which bind with the hepatitis viruses and form immune complexes.
Inactivation of virus is obtained to the extent of at least "4 logs", i.e., virus in a serum is totally inactivated to the extent determined by infectivity studies where that virus is present in the untreated serum in such a concentration that even after dilution to 10.sup.4, viral activity can be measured.
The fatty acid, or a soluble ester, alcohol or salt thereof is preferably employed in a concentration of a least 0.02 weight percent, generally 0.025 to 0.1 weight percent. Concentrations higher than 0.4 weight percent do not appear to provide improved virus inactivation as at the lower concentrations of 0.02 to 0.4, especially about 0.035 the degree of viral inactivity is such that virus is undetectable or substantially undetectable.
The fatty acid or a soluble ester, alcohol or salt thereof treatment is effected for up to about 24 hours. However, shorter treatment periods are preferred. For AHF, treatment is conducted for about up to 5 hours as longer periods of treatment, e.g., 6 hours decrease the yield of labile protein, at least in the case of AHF when concentrations of the fatty acid, or an ester, alcohol or salt thereof of 0.1 weight percent are employed.