CA2228031A1

CA2228031A1 - Doubly virus-inactivated human blood plasma, blood plasma derivatives produced therefrom and method for their production

Info

Publication number: CA2228031A1
Application number: CA002228031A
Authority: CA
Inventors: Annick Faucompre; Gunter Wober
Original assignee: Individual
Current assignee: BLUTSPENDEDIENST DER DRK-LANDESVERBANDE NORDRHEIN und WESTFALEN-LIPPE GmbH
Priority date: 1995-08-01
Filing date: 1995-11-03
Publication date: 1997-02-13
Also published as: EP0841954B1; EP0841954A1; WO1997004815A1; NO980275D0; DE59506747D1; DE19528221C2; NO311662B1; DK0841954T3; AU3846595A; GR3031779T3; ES2137544T3; NO980275L; DE19528221A1; ATE183928T1

Abstract

The invention concerns a method of producing a virally safe therapeutic biological preparation from human plasma using a detergent and retaining at least 50 % of the biological activity, the detergent being removed at the end of the method. Non-ionic surfactants, whose molecular structure comprises a hydrophobic part consisting of a linear aliphatic chain of between 8 and 10 carbon atoms, are used as detergents. The detergent treatment can be followed by a heat treatment, optionally using conventional stabilizers.

Description

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nF~RTVA'rTVP~ PROI:~UC~n T~R~ROM ,~n ~ l~OD FOR T~TR.
PROnUCTTON
The present invention concerns, first, human blood plasma wherein any viruses have been twice inactivated, second, derivatives of such plasma, and, third, a method, employing cletergent, of preparing a virally innocuous biological preparation from the plasma. The preparation will retain at ]east 50 % of the plasma's original activity. The method comprises treating the plasma with a detergent that is removed at the end.
"Biological preparations" are to be understood herein as products of human blood or plasma intended for therapeutic, prophylactic, or diagnostic purposes. Among such products are enzymes and pro-enzymes, coagulation factors for instance, enzyme inhibitors, immunoglobulins, albumin, p:Lasminogen, fibrinogen, and plasma itself. When obtained from infected donors, these preparations can contain viruses that have to be removed or inactivated.
Since a great deal of the patent and scientific literature describes methods of virus inactivation, only a few particularly relevant examples will be cited as background herein.
European Patent No. 0 050 061 (inventor: Shanbrom) describes a method of decreasing or eliminating viral infectiousness in biological products. An amphiphil is added at one point. The products are treated by leaving them for an extended period, at least 30 minutes, in contact with a solution or suspension of 0.25 to 10 ~ by weight of the non-denaturing amphiphil. The amphiphil is then removed, taking any pyrogenic substances along with it. This treatment i~s intended to inactivate membrane-encapsulated hepatitis-B and-non-A non-~, hepatitis viruses.
European Patent No. 0 131 740 (inventors: Neurath and Horowitz) describes an improvement in the method described in European Patent 0 050 061. This method is intended for rendering essentially free of lipid-encapsulated viruses a composition that contains a labile protein, without, however, substantially denaturing the protein. The method comprises contacting the composition with enough di- or tri-alkyl phosphate to be effective long enough in the presence of a dissolution promotor in the form of a tenside to render the composition free of lipid-containing viruses but without substantia]ly denaturing the protein. The method is usually carried out: at 0 to 70 ~ C. The labile proteins must be stabilized when the solution is heated.
Treatment with detergents generally has the drawback that they are targeted only against membrane-encapsulated viruses. When the concentration of the detergent is higher than the micelle-forming concentration (critical micelle concentration, "CMC"), lipid-containing membranes are solubilizecl and the virus inactivated. Viruses like the hepatitis-~ virus that inherently have no lipid-containing membrane, on the other hand, are released from any vesicles by deterger~t treatment and accordingly activated instead of inactivatecl.
The af'oresaid methods also absolutely require the ~
addition of a virucidal chemical like tri-n-butyl phosphate ~
to attain lull effectiveness (B. Horowitz et al, Tr~n~fllc;on 25 [1985], 516-22).
Also known from the literature is that the Vaccinia virus, one of the pox viruses, is, although a membrane-encapsulated virus, surprisingly unaffected by the Neurath-Horowitz solvent-detergent method (H. Harz et al, "Model Virus Studies of Solvent/Detergent Treatment used in the Production of High-Potency Factor VIII", Posters, University of Sterling, near Scotblood, 1992). These authors suspect that the treatment is too gentle for the Vaccinia virus's membrane.
There is accordingly a need for a method that basically incorporates the proven technology of detergent treatment followed o] preceded by heating and that will inactivate not only membrane-unencapsulated but also membrane-encapsulated viruses while employing detergents that are relatively easy to remove subsequent to treatment.
This object is attained in accordance with the present invention in a method of the aforesaid genus characterized in that the detergent is a non-ionic detergent with a hydrophobic component comprising a linear aliphatic chaln of 8 to 10 carbon atoms in the molecule.

Typical examples of such non-ionic detergents are tri-ethylene-glycol mono-octyl ether, l-0-n-octyl B-glucopyranoside, N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)~-N-methyl octanamide, i-thio-n-octyl B-D-glucopyranoside, and 6-0-(N-heptylcarbamoyl)-methyl B-D-glucopyranoside.
These substances are commercially available. N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl octanamide for instance ic sold as "Mega-8" and 6-O-(N-heptylcarbamoyl)-methyl B-D-glucopyranoside as "Hekamec".
In a typical treatment the concentration of the detergent is between 5 g/l and 20 g/l and it is allowed to act for 5 t:o 25 and preferably lO to 20 minutes at 20 to 30 and preferably 25 ~ C.
The treatment obviously lasts substantially less than 30 minutes. The treatment and action can be abruptly terminated by diluting the detergent with water to below the CMC, discontinuing its action.
The detergent can then be removed from the solution by dialysis or diafiltration.
The detergent treatment can be combined with pasteuriza1ion (heating). Much of the literature addresses the inactivation of infectious agents in blood products with heat. Examples of such procedures include heating the products in an aqueous solution in the presence of stabilizer" heating the dry products, and heating them solid but moist.

Such methods are known and are not in themselves being claimed as patentworthy herein. One of skill in the art will be aware that a heating approach should always be employed when it bot:h limits the preparation's potential residual infectiousness and extensively maintains its biological activity.
Heating the blood products will attack and de-activate both membrane-encapsulated and non membrane-encapsulated vlruses .
Although the detergent treatment is extensively effective and inactivates pox viruses in particular, the sensitive plasma proteins will be extensively spared. Adding toxic chemicals (e.g. TNBP) is unnecessary. The toxicity of TNBP can be long-term, and the substance should be strictly avoided.
Although the inactivating chemicals must of course be removed,-doing so does not take as long as it does at the state of the art. They can for example be removed with C18 resin (European Patent No. 366 946 Bl, inventor: Bonome).
It is of advantage to add a solution of such in-themselves known stabilizers as ~-amino acids, sucrose, and sorbitol during the heat treatment. The treatment itself will be carried out at 50 to 65~ C and usually for several hours. The solution is then diluted to decrease viscosity and the stabilizers eliminated by dialysis or a similar process.

The method will now be specified with reference to several examples.

Exam~le 1 Frozen plasma obtained for example by separating the cells from donated whole blood or by plasmapheresis is thawed at 20 ~ C, its pH adjusted if necessary to 7.30, and treated wit:h a stabilizer solution (2 ml per 100 ml of plasma). The stabilizer solution itself contains 3.7 g of calcium ch]oride (CaCl2 ~ H20), 15.0 g of arginine, 15.0 g of lysine, and 18.0 g of sodium citrate (C6HsNa307 5-5 H20) at a pH of 7.30 per 100 ml of water. Next, 3 g of glycine and 600 g of sucrose per liter of plasma are added. The solution is weighed and combined with the same weight of sorbitol. EIeat treatment is conducted for 10 hours at 62 +
0.5 ~ C. The solution is diluted with dialysis buffer to decrease i1_s viscosity, and the stabilizers eliminated by dialysis, with hollow-fiber cartridges or by cross-flow filtration over membranes. The buffer contains 0.74 g of calcium chLoride (CaCl2 H20), 3.0 g of arginine, 5.0 g of lysine, 3.l~ g of sodium citrate (C6H5Na30~ 5.5 H20), 3.0 g of glycine, and 5.84 g of sodium chloride (NaCl). The plasma proteins retain at least 50 % of their pre heat-treatment biological activity--fibrinogen, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, AT III, plasminogen, Protein C, Protein S, -and a2-plasmin inhibitor. The presence of activated coagulation factors is ruled out by two sensitive tests (non-activated partial thromboplastin time and thrombin~
formation time--C.W. Powse et al, Thrombosis Haemostastis, Stuttgart, 42, 1355-1367, 1979) In a separate experiment, 1 ml of a hepatitis-A virus suspension is added to 50 ml of the solution at room temperature once all the stabilizers have been added, and a sample extracted to determine the virus titer. The temperature of the solution is then uniformly increased to 62 ~ C for two hours and maintained at that temperature for ten hours. This procedure simulates production conditions.
Dialysis is followed by extracting another sample to determine the virus titer of the treated batch. The titer of the reference sample is 104-9, and no virus is found in the second sample.

Example 2 As in Example 1, a Sindbis-virus suspension is added at room temperature to a batch containing all the stabilizers and a samp~le extracted to determine the virus titer. A
second sample is extracted subsequent to heat treatment and dialysis as described with reference to Example 1. The titer of the reference sample is 1056, and no virus can be found in the second sample.

Example 3 PlasmaL treated and dialyzed as described with reference to Example 1 is treated with l-0-n-octyl B-glucopyranoside (10 g per ] of plasma) and slowly stirred for 20 minutes at -25 ~ C. The! solution is then diluted with dialysis buffer (as in Example 1) to a ratio of 1:3 by volume and the detergent removed by dialysis or diafiltration. The aforesaid plasma proteins retain at least 50 % of their pre heat-treatment biological activity.
In a ~;eparate experiment, 1 ml of a suspension of Myxoma virus is added to 50 ml of plasma and a sample extracted to determine the virus titer. Detergent is added, and the batch incubated for 15 minutes at 25 ~ C and diluted with dialysis buffer to a ratio of 1:10 by volume. The detergent is eliminated by dialysis or diafiltration and a plasma sample extracted to determine the virus titer. The titer of the reference sample is 1062 and that of the second sample 10~9.

~xample 4 The same quantities of salts and amino acids as those employed for the dialysis buffer in Example 1 are added to 1 1 of untreated plasma. A solution of 15 g of tri-ethylene-glycol mono-octyl ether per liter is added and the batch incubated for 10 minutes at 25 ~ C. The batch is diluted with dialysis buffer to a ratio of 1:5 by volume and the detergent eliminated by dialysis or diafiltration. The stabilizers for the heat treatment are added as described in Example 1 and the same treatment carried out. The batch.is diluted ancl the stabilizers eliminated by diaiysis or ' - -diafiltratiLon. The aforesaid plasma proteins retain at least 50 % of the biological activity of the untreated plasma.

,Example ~
The same quantities of salts and amino acids as those employed for the dialysis buffer in Example 1 are added to 1 1 of untreated plasma followed by 1 ml of a suspension of Pseudorabies virus. A sample is extracted to determine the virus titer. Next, 0.5 g of i-thio-n-octyl B-glucopyranoside is added and the batch incubated for 15 minutes at room temperaturle. The batch is diluted to a ratio of 1;10 by volume and the detergent eliminated by dialysis or diafiltration. Another sample is extracted to determine the virus titer. The titer of the reference sample is 1058, and no virus can be found in the second sample.

Example 6 By a procedure similar to that described in Example 5, salts, amino acids, and 1 ml of bovine viral-diarrhea virus (BVDV) are added to 50 ml of plasma. A sample is extracted to determine the virus titer. Next, 0.4 g of N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl octanamide are added and the batch incubated for 10 minutes at 25 ~ C. The batch is diluted to a ratio of 1:10 by volume and the detergent eliminated by dialysis or diafiltration. Another sample,is extracted t:o determine the virus titer. The titer of the - -reference sample is lO[ille9ible], and no virus can be found in the second sample.
Twice inactivated plasma is in itself a valuable therapeutic and very safe from the transmission of viruses from blood. Furthermore, plasma derivatives of equal safety can be iso]ated by in-themselves known procedures. The procedure is of particular advantage because the complicated logistics of spatially separating the intermediate steps of plasma frac~tionation before and after virus inactivation is unnecessary. The following examples specify the procedure.

le 7 Detergent-treated or twice virus-inactivated plasma is frozen, thawed to 4 ~ C to separate a cryoprecipitate, and centrifuged at the same temperature. The cryoprecipitate is washed with ice-cold buffer to harvest the fibrin glue's Component L (German Pharmacopeia 10, 3, Supplement). The detergent l_reatment or the sequential detergent-plus-heat treatment can also be carried out on the purified preparation.

. CA 02228031 1998-01-27 Example ~
Factor ~II from a Willebrand concentrate is purified from a solution of the cryoprecipitate prepared as described in Example 7 by immuno-affinity chromatography (European Patent No. 122 209) or ion-exchange chromatography (German Patent No. 1 184 782) for example. The detergent treatment or the sequential detergent-plus-heat treatment can also be carried out on the purified preparation. The detergent can also be eliminated during the chromatography.

Example 9 The prothrombin-complex factors, Factor II, Factor VII, Factor IX, and Factor X are harvested from a low-cryoprecipitate plasma by ion-exchange chromatography (J.
Heystek et al, YQX Sanguinis 25, 113-23, 1975). The detergent t:reatment or the sequential detergent-plus-heat treatment can also be carried out on the purified preparation. The detergent can also be eliminated during the chromatography.

~xample 1~
To dec:rease the risk of thrombosis, hemophilia B is preferably treated with high-purity Factor IX. A prothrombin complex obt:ained as described in Example 9 can for example be purifiecl by affinity chromatography (C. Michalski et al, Vox Sanguinis 55, 202-21, 1988 and F.A. Feldmann et al, , CA 02228031 1998-01-27 Rloo~ Co~ t;on ~n~ F;hr;nolys;s 5, 939-48, 1994). The detergent treatment or the secluential detergent-plus-heat treatment can also be carried out on the purified preparation. The detergent can also be eliminated during the-chromatography.

Example 1l Antithrombin III can be purified by affinity chromatography from a plasma once the cryoprecipitate and prothrombin complex have been separated (R. Rosenberg & P.S.
Damus, Journal ~ Biolog;cal Chemistry 248, 6490-6505). The detergent treatment or the secIuential detergent-plus-heat treatment can also be carried out on the purified preparation. The detergent can also be eliminated during the chromatogra,phy.

le 1~
Immunoglobulin G can be purified by any version or modification of the cold-alcohol precipitation method from a plasma from which the cryoprecipitate and optionally other plasma prot:eins have been separated (E.J. Cohn et al, Journal of the American Chemical Society 68, 459-75, 1946 and P. Kist:ler and H. Nitschmann, Vox Sanguinis 7, 414-24, 1962). The detergent treatment or detergent-plus-heat treatment can also be carried out on the purified . CA 02228031 1998-01-27 preparation. The detergent can also be eliminated by precipitating the immunoglobulin.

Claims

1. Method of preparing a virally innocuous therapeutic biological preparation from human plasma with a detergent, whereby the preparation retains at least 50 % of the plasma's original activity and whereby the detergent is removed at the end, characterized in that the detergent is a non-ionic tenside with a hydrophobic component comprising a linear aliphatic chain of 8 to 10 carbon atoms in the molecule.

2. Method as in Claim 1, characterized in that the detergent treatment can be followed by a heat treatment, optionally employing conventional stabilizers.

3. Method as in Claim 1 or 2, characterized in that the detergent is allowed to act for 5 to 25 and preferably for 10 to 20 minutes.

4. Method as in Claim 3, characterized in that, once the action is complete, it is discontinued by diluting the detergent to below the critical micelle concentration (CMC).

5. Method as in Claims 1 through 4, characterized in that the concentration of the detergent is between 5 and 20 g per liter of plasma.

6. Method as in Claims 1 through 5, characterized in that the detergent treatment is carried out at 20 to 30 and preferably at 25 ° C.

7. Method as in at least one of the foregoing claims, characterized in that the detergent is eliminated at the end of the treatment by dialysis or diafiltration.

8. Method as in one of the foregoing claims, characterized in that the detergent treatment and optional heat treatment can be carried out in any order.

9. Twice virus-inactivated human blood plasma and blood-plasma derivatives prepared therefrom, prepared as recited in one of the foregoing claims.