Removal of viruses from protein solutions by ultrafiltration

Details Removal of viruses from protein solutions by ultrafiltration Removal of viruses from protein solutions by ultrafiltration

1996-02-07

2002-05-21

Wortman, Donna C. (Department: 1648)

Chemistry: analytical and immunological testing

Involving immune complex formed in liquid phase

Separation of immune complex from unbound antigen or antibody

C436S536000, C435S005000, C210S651000, C604S005020

Reexamination Certificate

active

06391657

ABSTRACT:

The invention relates to the removal of viruses from aqueous solutions, as a rule protein solutions, by ultrafiltration. This entails the viruses to be removed being increased in size by incubation with high molecular weight ligands binding thereto, preferably specific antibodies, so that, on the one hand, the separation effect is improved and, on the other hand, a larger pore diameter which can now be chosen for the filters used also makes it possible for smaller viruses to be separated from larger protein molecules present in protein solutions, and, where appropriate, the filtration rate is increased.
Proteins purified and concentrated from human plasma are used for therapy and prophylaxis of human diseases. These products are prepared from plasma pools consisting of about 10,000 individual donations. Since some of these donations may potentially be contaminated with human pathogenic viruses such as HIV-1/2, hepatitis B virus, hepatitis C virus and other viruses, there is the possibility of infection being caused by administration of the plasma proteins. In order to minimize this contamination hazard, donations are obtained only from healthy donors who are additionally tested for infection markers (antibodies against HIV 1 and HIV 2, HBsAg, antibodies against HCV and elevated liver function test results (ALT)); positive donations are rejected and not used for obtaining plasma proteins. The purification and concentration steps used in the industrial preparation of plasma proteins and, in particular, steps specifically introduced into the production to eliminate and/or inactivate viruses lead to plasma proteins with a very high safety standard.
In order to increase the safety of plasma proteins even further, there have been investigations of the use of filtration methods, for example dead-end and tangential flow filtration, in order to eliminate any viruses present in the protein solution. Filter units for eliminating viruses are produced by various companies (DiLeo, A. J. et al. Biologicals 21, 275-286 (1993); DiLeo A. J. et al. Biologicals 21, 287-296 (1993); Burnout, T. et al., Vox Sang. 67, 132-138 (1994)). Thus, for example, Asahi Chemical Industry Co. Ltd., Tokyo, Japan, produces filter units stating a defined (average) pore size, while, for example, Millipore Corp., Bedford, Mass., USA, produces filter units stating a nominal molecular weight cut off.
It has emerged from our investigations that viruses are held back at different rates by filters with different pore sizes, depending on the diameter of said viruses (HIV: 80-100 nm: HCV: 40-60 nm; HBV: 40-45 nm; picornaviruses: 24-30 nm; parvoviruses: 18-25 nm): (I) filters with an average pore diameter of 75 nm essentially retain HIV, while the other specified viruses are found in the filtrate; (II) filters with an average pore diameter of 35 nm retain HIV completely and HCV and HBV to a large extent, while, for example, picornaviruses and parvoviruses are found in the filtrate; (III) filters with an average pore diameter of 15 nm retain HIV, HCV and HBV and, to a large extent, for example picornaviruses and parvoviruses. Filtration through a filter with an average pore diameter of 15 nm leads to a general increase in the virus safety of plasma proteins. However, since most plasma proteins have such a high molecular weight that they cannot be filtered through a 15 nm filter, i.e. are likewise held back, only filters with an average pore diameter of 35 nm (or a nominal molecular weight cut off of 70,000 D to 100,000 D) are suitable for filtering most plasma proteins, but these do not remove to an adequate extent at least picornaviruses (such as, for example, hepatitis A virus) and parvoviruses (such as the human pathogenic parvovirus B 19) from plasma proteins.
The object therefore was to achieve an adequate, i.e. complete, retention even of small viruses by filtration, and to make filtration methods also applicable to those proteins which resemble simply in terms of their size a small virus. In addition, it was intended to increase the filtration rate as far as possible.
The object is achieved by the present invention in that the viruses to be removed are increased in size by binding to high molecular weight ligands, preferably specific antibodies, particularly preferably monoclonal antibodies, in principle of all subclasses, but preferably subclass IgG or IgM or parts thereof still capable of binding, which are, where appropriate, modified or enlarged by genetic engineering, to such an extent that they can be held back by filtration. The increase in size can also be achieved by aggregate formation. It is in fact possible with this method to separate relatively large proteins such as factor VIII or von Willebrand factor from such viruses of increased size by filtration, it now being possible to choose the pore size such that the proteins pass through and the viruses of increased size are held back. It is moreover possible by choice of a larger pore width, which is now possible, to increase the filtration rate. In the most general form, the present invention makes it possible to increase the size of any constituents of an aqueous solution by binding to high molecular weight ligands to such an extent that separation is then possible from the now smaller constituents in a filtration step.


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Anthony J. DiLeo et al., “Size Exclusion Removal of Model Mammalian Viruses Using a Unique Membrane System, Part II: Module Qualification and Process Simulation,” Biologicals 21: 287-296 (1993).
M. Burnouf-Radosevich et al, “Nanofiltration, a New Specific Virus Elimination Method Applied to High-Purity Factor IX and XI Concentrates,” Vox Sang 67: 132-138 (1994).
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