Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2001-04-27
2002-10-22
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S180000, C424S530000, C210S656000, C530S387100, C530S412000, C530S414000, C530S415000, C530S417000, C530S418000, C530S422000
Reexamination Certificate
active
06468733
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns a method for the removal and inactivation of viruses from biological preparations.
BACKGROUND OF THE INVENTION
Biologically derived liquid preparations such as blood and plasma preparations are used as raw materials from which a plurality of biologically useful compounds can be purified. Examples of such compounds include immunoglobulin, factor VIII, albumin, &agr; 1 anti trypsine, Factor IX, factor XI, PPSB, fibrinogen, and thrombin (prothrombin). In addition, various biological products such as hormones, growth factors, enzymes and ligands are isolated from biological preparations obtained from cell cultures.
The cells used in the production of these useful materials may be either wild-type cells from various animal sources, or alternatively, genetically engineered prokaryotic or eukaryotic cells. Where the biological materials obtained from these liquid preparations are to be administered to humans for therapeutic purposes, in particular by intravenous administration, the sterility of the preparation is of major concern. Thus, great efforts are invested in the inactivation of viruses, such as hepatitis viruses and HIV viruses, which may be present in these preparations.
Lipid coated viruses are effectively inactivated by treatment with non-ionic biocompatible solvents and detergents. Methods for virus inactivation by solvent-detergent applications are described, for example, in EP 0131740. However, non-lipid coated viruses cannot be inactivated by solvent-detergent treatments, thus, other inactivation methodologies have to be used for their inactivation. These include the application of heat (pasteurization), the application of irradiation such as short ultra violet light (UVC) or gamma radiation, as well as eliminating by physical means, e.g., the filtration of the preparation through very narrow filter holes so as to remove viruses by size exclusion (nanofiltration).
Noreen et al., (
Biologicals,
26:321-329 1998) examines the use of the Memberg Microporous membrane hollow fibers (Planova) 35 mn filters to reduce potential loads of both non-enveloped and enveloped viruses, prior to the solvent detergent treatment in a 7% IVIg solution. In the above study, nanofiltration was validated for the removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhea virus (BVDV), Feline calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The study showed a complete reduction (to the limit of detection assay) of all viruses larger than 35 nm. Interestingly, even smaller viruses such as EMC and HAV were at least partially removed by this method of filtration.
These studies led to the use of nanofiltration for the removal of viruses from biological preparations. However, in cases where it was desired to combine both viral inactivation by the solvent-detergent method, (in order to inactivate lipid-coated viruses) as well as nanofiltration elimination (for the size exclusion and hence removal of non-lipid coated viruses), it was discovered that the nanofiltration step had to proceed the solvent-detergent application, particularly, where the solvent-detergent was to be removed by oil extraction and C-18 reverse phase resin. The reason for this was that after extraction of the solvent-detergent, there are always traces of small oil droplets, as well as residues of solvent-detergent, which bind to the hydrophilic part of the resin. Moreover, some of the proteins purified from the biological preparation may be modified during the purification process, and the altered proteins can form dimers and polymers which change in the hydrophobicity of the altered protein. These traces of oil droplets, protein dimer aggregates and the mixture of oil and protein residues tend to block the small holes of the nanofilter, thus considerably increasing the time of the filtration, requiring the frequent replacement of expensive filters, and generally decreasing the yield of the product.
The residues of oil droplets (contaminants), which tend to block nanofilters, were removed by using either a chromatography mechanism of molecular exclusion or by hydrophobic chromatography. However, none of the conventional methods for the removal of solvent detergent has yet addressed the issue of dimerization or aggregation caused by the solvent detergent, and this problem still remains. Removal of the solvent-detergents from biological liquid preparations, such as immunoglobulin preparations, is generally carried out by using gel chromatography. U.S. Pat. No. 5,094,960 and U.S. Pat. No. 5,648,472 concern the removal of solvent-detergent from immunoglobulin preparations without using gel reverse phase (hydrophobic) chromatography.
Two other patents have been granted to processes which indicate that the removal of solvent detergent affects the stability of a liquid IVIg product (U.S. Pat. No. 5,094,960, U.S. Pat. No. 4,789,545 and U.S. Pat. No. 5,648,472). G. Werner and P. Selosse (U.S. Pat. No. 5,648,472) describe a process for preparing envelope virus-inactivated immunoglobulin solutions suitable for intravenous application, comprising treating the immunoglobulin with TnBP and/or Tritonx100™ (octylphenol ethylene oxide condensate; CAS 9002-93-1), followed by an extraction using biologically compatible vegetable oil, when TnBP and/or Tritonx100™ and vegetable oil are subsequently removed by solid-phase extraction on hydrophobic materials. This indicated that the combination of vegetable oil extraction of IVIg followed by chromatography through a hydrophobic column produces a very stable liquid solution of immunoglobulins, even at elevated temperature. This patent claimed that IVIg preparation is prepared in accordance to two previous patents:
Bonomo, 1992, (U.S. Pat. No. 5094960) teaches the use of solid phase extraction and Bulk C-18 packing from Waters, Inc. as a preferred resin. Woods 1986 (U.S. Pat. No. 4,789,545), features the removal of solvent detergent by naturally occurring oils, however the products obtained by this method resulted in the unstable preparation of intravenous immunoglobulins.
Guerrier et al (
Journal of Chromatography B
664:119-125 (1995)) describes a specific sorbent which is intended to remove solvent-detergent mixtures from virus-inactivated biological fluids. The solvent-detergent removal (SDR) HyperD™ sorbents were supplied by Biosepra (Ceroy-Saint-Christophe, France). This chromatographic packing was made of silica beads in which the pore volume was filled with a three-dimensional cross-linked hydrophobic acrylic polymer.
SUMMARY OF THE INVENTION
The present invention is based on the finding that a method for the inactivation of viruses present in biological preparations comprising first treating the preparation by solvent-detergent, after which the solvent-detergent is extracted by the use of vegetable oil, in a combination with/without solid phase extraction (specifically using bulk C18 as recommended by U.S. Pat. No. 5,648,472) resulted in an immunoglobulin liquid preparation which was very difficult to pass through the Planova ™Filter 35 nm. Thus, it was realized that such a procedure was unsuitable for the efficient inactivation of viruses.
In accordance with the present invention, it was surprisingly discovered that the combination of solvent-detergent treatment, followed by chromatography utilizing SDR HyperD™ (ion-exchange resin) from Biosepra, facilitated the passage of the IVIg at a high flow rate through Planova Filters 35 nm, and resulted in a liquid preparation devoid of active viruses, while featuring a very high yield. In accordance with the present invention, it was further found that the problem of dimerization of immunoglobulins can be partially solved by reducing the pH to 4.0 before the nanofiltration step.
Thus, the present invention concerns a method for the inactivation of viruses present in a liquid preparation, comprising the steps of:
(i)
Bar Liliana
Nur Israel
Browdy and Neimark , P.L.L.C.
Omrix Biopharmaceuticals Inc.
Stucker Jeffrey
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