Process for separating HIV from a fluid

Details Process for separating HIV from a fluid Process for separating HIV from a fluid

1999-06-21

2001-06-05

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

Inactivation or attenuation; producing viral subunits

C435S235100, C435S236000, C435S283100, C435S286500, C422S028000, C422S030000, C422S044000

Reexamination Certificate

active

06242239

ABSTRACT:

The invention relates to a process for separating human immunodeficiency virus or viruses (HIV) from a fluid, in particular from blood, blood plasma or blood serum. The process can be carried out both for the preparation of HIV-free blood donations and therapeutically for the reduction of the virus load in the blood by means of a blood lavage under the conditions of an extracorporeal blood circulation.
The invention is moreover directed at a filter which is suitable for the separation of HIV from a fluid.
It is known that the removal of HIV from all sorts of biological fluids, but especially from blood, blood plasma or blood serum, is an important prerequisite for its risk-free use for all sorts of medical purposes. Numerous processes have therefore already been proposed using which removal of HIV from biological fluids should be achieved. Thus, in the international Patent Application WO 97/07674, a process has been proposed using which HIV can be removed from biological fluids or inactivated by treating it with certain ethylenimine oligomers. It is important in this case that other constituents of the blood, in particular the cellular constituents, especially the erythrocytes, are not damaged by a treatment of this type and the removal of the HIV can be carried out in a simple manner and short period of time in order that sufficiently large amounts of purified blood can be obtained in an economically justifiable process.
It has now been found that these requirements can be fulfilled in an outstanding manner by a process if the C1 inhibitor is employed for the removal of the HIV from biological fluids.
The C1 inhibitor, also called C1 esterase inhibitor, is a protein which is present in the blood and is the main inhibitor of the classical pathway of the complement system and of the contact system. The C1 inhibitor can inhibit the activated form of factor XII and of kallikrein (Schapira M. et al., 1985,
Complement
2: 111; Davis A. E., 1988,
Ann Rev Immunol
6: 595; Sim R. B. et al., 1979,
FEBS Left
97: 111; De Agostini A. et al., 1984,
J Clin Invest
73: 1542; Pixley R. A. et al., 1985,
J Biol Chem
206: 1723; Schapira M. et al., 1982,
J Clin Invest
69: 462; Van der Graaf F. et al., 1983
J Clin Invest
71: 149; Harpel P. C. et al., 1975,
J Clin Invest
55: 593). The C1 inhibitor thus regulates the activities of two plasma cascades, namely the complement system and the contact system, by which biologically active peptides are produced. The C1 inhibitor is therefore also an important regulator of the inflammatory system. Moreover, the C1 inhibitor inhibits activated factor XI (Meijers J. C. M. et al., 1988,
Biochemistry
27: 959; Wuillemin W. A. et al., 1995,
Blood
85: 1517). It follows from this that the C1 inhibitor can be considered as a coagulation inhibitor. The tissue plasminogen activator and plasmin are also inhibited to a certain extent by the C1 inhibitor, although that is not its main function (Harpel P. C. et al., 1975,
J Clin Invest
55: 149; Booth N. A. et al., 1987
Blood
69: 1600).
The C1 inhibitor is obtained from plasma by purification to a considerable extent and utilized for clinical applications, in particular in the treatment of hereditary angioedema, a disorder which is caused by a genetically related deficiency of the C1 inhibitor. Moreover, it has already been described that good therapeutic results were achieved by administration of the C1 inhibitor in systemic inflammations [International Patent Application WO 92/22320 (Genentech Inc.)], in severe burns, pancreatitis, bone marrow transplants, cytokine therapy and during use in extracorporeal blood circulations [DE-A4 227 762 (Behringwerke AG)].
The complete genomic and the cDNA which codes for the C1 inhibitor has already been cloned (Bock S. C. et al., 1986
Biochemistry
25: 4292; Carter P. E. et al., 1988,
Eur J Biochem
173: 163). Various variants of the recombinant C1 inhibitor with amino acid mutations in the P1 and the P3 and/or P5 positions of the reactive center and variants which were isolated from patients with a hereditary angioedema have already been prepared recombinantly (Eldering E. et al., 1988,
J Biol Chem
263: 11776; Eldering E. et al., 1993,
J Biol Chem
267: 7013; Eldering E. et al., 1993,
J Clin Invest
91: 1035; U.S. Pat. No. 5,622,930; Davis A. E. et al., 1992,
Nature Genetics
1: 354; Eldering E. et al., 1995,
J Biol Chem
270: 2579; Verpy et al., 1995,
J Clin Invest
95: 350).
The C1 inhibitor belongs to the large family of serine proteinase inhibitors which are also called serpines (Travis J. et al., 1983,
Ann Rev Biochem
52: 655; Carrel R. W. et al., 1985,
Trends Bioch Sci
10: 20). On SDS-polyacrylamide gels, the C1 inhibitor exhibits a molecular weight of approximately 105 kD. Its plasma concentration is approximately 270 mg/l (Schapira M. et al., 1985,
Complement
2: 111; Nuijens J. H. et al., 1989,
J Clin Invest
84: 443). The C1 inhibitor is a protein whose plasma level can increase up to twofold in uncomplicated infections and other inflammations (Kalter E. S. et al., 1985,
J Infect Dis
151: 1019). The increased formation of the C1 inhibitor in inflammations probably serves for the protection of the body against the harmful effects of the intravascular activation of the complement system and of the contact system during the acute reactions.
The serpines react as inhibitors by formation of bimolecular complexes with the proteinase to be inhibited. In these complexes, the active center of the proteinase is bound by the active center of the serpine and thus inactive (Travis J. et al., 1983,
Ann Rev Biochem
52: 655). The serpines react specifically with certain proteinases, this specificity being determined by the amino acid sequence of the reactive center.
The abovementioned varied actions of the C1 inhibitor did not, however, give any indication of its strong affinity for HIV and in particular did not suggest that separation of HIV from biological fluids such as blood, blood plasma or blood serum is possible with the aid of the C1 inhibitor. It was therefore a very unexpected finding that HIV binds to the C1 inhibitor and can thereby be separated from mixtures which contain HIV with the aid of the processes below.
The invention relates to a process for separating HIV from a fluid such as blood, blood plasma or blood serum, in which the HIV is bound to a C1 esterase inhibitor immobilized on a support material. This process is expediently carried out such that the C1 esterase inhibitor is bonded to an inert matrix which can be employed in affinity chromatography, by means of which the biological fluid to be freed of the HIV is added in a procedure customary in column chromatography.
Suitable matrices on which the C1 inhibitor is immobilized include dextrans, polyacrylamides and agarose, but other supports customarily employed in affinity chromatography can also be used for the process according to the invention. As a result, HIV-free blood donations can be obtained. However, the virus load in the blood can also be therapeutically reduced if HIV is absorbed on a matrix impregnated with a C1 inhibitor by means of an extracorporeal blood lavage before or during chemotherapy.
A particularly effective and rapid separation of the HIV can be achieved according to the invention if the fluid containing the HIV is filtered through a fiber material which is impregnated with the C1 esterase inhibitor. For this, a filter has proven suitable which consists of a container in which is packed a fiber material which is impregnated with the C1 esterase inhibitor. The fiber material can either consist of fibers which are interwoven or entangled with one another or can be present in the form of a woven or web-like material. A particularly effective and rapid filtration can in this case be achieved using a filter which consists of fibers impregnated with the C1 esterase inhibitor which have an average diameter of less than 10 mm, preferably of 0.3 to 3 mm, and a bulk density of 0.15 to 0.5 g/cm
3
and an average fiber spacing of 0.5 to 0.7 mm

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