DE102005037824A1 - Reduction of virus infection in a biological process comprises using chemical and/or physical methods that possess no or only minimal virus inactivation capacity and exposing the pre-treated process to shearing stress to clear inactivation - Google Patents

Abstract

Procedure for the reduction of virus infection in a biological manufacturing process comprises subjecting chemical and/or physical methods that possess no or only minimal virus inactivation capacity and exposing the pre-treated biological process to mechanical shearing stress that leads to clear virus inactivation.

Description

The The invention relates to a method for reducing the infectivity of viruses in the manufacturing process of biologics, being chemical and / or physical Methods which as such have no or only minimal virus inactivation capacity combined with the application of mechanical shear forces.

The Virus safety of biologics relies on a multifactorial Approach with (1) Selection of starting material with no or minimal Content of infectious Viruses, (2) testing of product intermediates and (3) manufacturing process with procedural steps that inactivate and / or remove viruses. Biologics are or contain therapeutic proteins (active ingredients) the, e.g. from blood or plasma, urine or other biological fluids of animal or human origin, from in vitro cultures of Cells of animal or human origin or from in vivo cultures of cells or organs and tissues animal and human Origin have been obtained. Many process steps with high capacity Inactivating and / or removing viruses are known, e.g. Virus inactivation by increased Temperature or by chemicals, in particular the lipid envelope of Destroy viruses. Pasteurization (heat treatment in a (stabilized) solution at usually 60 ° C over 10 hours; Chandra et al. Effectiveness of alternative treatments for reducing potential viral contamination from plasma-derived products - thrombosis Research 2002; 105: 391-400), steam treatment (Kreil et al., West Nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on virus data, transfusion 43, 2003, 1023-1028) and dry heating (Roberts and Hart, Comparison of the inactivation of canine and bovine parvovirus by freeze drying and dry-heat treatment in two high purity factor VIII concentrates, Biologicals 28, 2000, 185-188) leads to effective inactivation of viruses. The solvent / detergent (SD) method (Horowitz et al., Inactivation of viruses in labile blood derivatives. Disruption of lipid-enveloped viruses by tri (n-butyl) phosphate detergent Combinations. Transfusion 25, 1985, 516-522) as well as the treatment of product intermediates with caprylate (Lebing et al., Properties of a new intravenous immunoglobulin produced by virus inactivation with caprylates and column chromatography, Vox Sanguinis 84, 2003, 193-201) leads to a effective inactivation of enveloped Viruses. In addition, other methods such as irradiation (UV-C or Gamma irradiation) or the use of nucleic acid-intercalating substances for use. Besides these examples of methods for virus inactivation find action Application that removes viruses, e.g. Virus filtration. in this connection Viruses are sized according to their size by small pore filter materials retained and plasma proteins are collected in the filtrate.

Since virus reduction methods are influenced by the physicochemical properties of viruses, eg the SD method is effective only against enveloped viruses and irradiation especially against viruses with single-stranded nucleic acid, according to the guidelines of the authorities (eg CPMP / BWP / 269/95) frequently 2 Procedural steps for virus reduction integrated in the production process of biologicals to inactivate and / or remove a wide range of viruses such as enveloped and non-enveloped viruses. Thus, for example, the combination SD method and dry heating is a widely used method in plasma derivatives (Dichtelmüller et al., Improvement of virus safety of an S / D-treated factor VIII concentrate by additional dry heat treatment at 100 degrees C, Biologicals 24, 1996 , 125-30). The combination SD method and virus filtration is also a way to inactivate and remove a wide range of viruses ( EP1161958A ).

The reliable Removal of viruses is dependent from the properties of the viruses and the active ingredients of the Biologics. Since many plasma protein molecules are similar in size to small viruses virus removal by filtration on these plasma proteins not possible, because the proteins are also retained by the filter. Therefore Small (relative to viruses) protein molecules can be relatively Simply separate from viruses by filtration, but not larger protein molecules, the the same or even larger diameter have like little viruses.

In the patent EP 0 727 226 B1 / US 6,391,657 B1 It has been described that the diameter of viruses can be increased by binding to antibodies so as to achieve a separation of viruses and larger protein molecules by filtration. However, the addition of antibodies to complex viruses is not always possible because (1) antibodies may interfere with the further production process of biologics or (2) should not be included in the product or (3) antibodies against viruses, including new emerging are not available in sufficient quality and quantity to be added to the production process of biologics.

As mentioned the SD method is very effective against enveloped viruses. It showed however, the detergent (e.g., Tween 80) alone will only a negligible one Virus inactivation leads (Horowitz, B. - Curr Stud Hematol Blood Transfus 1989; 56: 83-96), making a solver like TNBP (tris-N-butyl-phosphate) for one effective virus inactivation is mandatory. Basically, should but with Biologika solubilizers avoid as little toxic reagent as possible To have product intermediate, which is then expensive to remove, or no reaction of the solubilizer with substances of the product intermediates.

Surprisingly It has now been found that the sole addition of a detergent without solubilizers in a concentration that does not inactivate as such viruses, to a significantly increased Reduction of viral infectivity leads to virus filtration, even with filters with a pore size that matches the size of the virus even exceed can.

It could be shown that chemical or physical methods that lead to a destabilization of the virus structure led to an increased virus reduction when shear forces act on the thus destabilized viruses. This result also occurs at concentrations or intensities which alone result in little or no viral inactivation. Examples of these methods for destabilizing viruses are amphiphilic substances such as nonionic, cationic, anionic and amphoteric surfactants and phospholipids, alcohols, fatty acids, chaotropic substances such as urea, thiocyanate, guanidinium hydrochloride, chelating agents such as EDTA, high ionic strength such as high concentrations of cations eg Na ⁺ and Ca ²⁺ and anions such as Cl ^- and SO ₄ ^2- , (ammonium sulphate), pH and elevated temperature. Shear forces which can inactivate viruses in the presence of destabilizing agents or processes occur, for example, in filtration (depth filter, sterile filter and virus / nanofilter) and chromatography (gel filtration, IEX, HIC, affinity chromatography, membrane chromatography), but also in the known homogenization methods, such as ultrasound or mechanical homogenizers as UltraTurrax® ^® (IKA Werke, Janke & Kunkel GmbH & Co KG, Staufen, Germany), high pressure homogenizer or ball mills and the like on. Viruses inactivated by the method described are detected by NAT / PCR (Nucleic Acid Amplification Technique / Polymerase Chain Reaction). The destabilization and application of the shear forces can occur virtually simultaneously or sequentially.

preferably, Amphiphilic substances, in particular surfactants, are used in Concentrations of at most 0.1% (w / v), preferably at most 0.05%, more preferably 0.001-0.05%, most preferably 0.001-0.02%.

in the Comparison of infectiousness data and NAT / PCR data is shown to be infectious by the method described stronger is reduced as viral nucleic acids. Digestion of aliquots of the starting material with DNase in DNA viruses and RNase in RNA Viruses and subsequent NAT / PCR show that the described method destroys virus particles and thus leads to a significant reduction of infectivity in the filtrate.

Significant for the purposes of this application means an increase in the virus reduction factor of at least 1 log ₁₀ , preferably 2 log ₁₀ , more preferably at least 3 log ₁₀ .

The The following example is intended to explain the invention, in no way against it limit.

Bovines viral diarrhea virus (BVDV), an enveloped virus of the family Flaviviridae, with a diameter of about 40 to 60 nm became more physiological Saline (0.9% (w / v) NaCl pH 7.2). To this virusversetzten solution was Tween 80 stock solution (final concentration of Tween 80 was 0.02%). Samples were treated by a virus filter with a nominal pore size of 75 nm (Planova75N, Asahi-Kasei) according to the manufacturer's instructions filtered. The results (Table 1) show that BVDV is not significant retained by the filter becomes; the addition of Tween 80 in a concentration that does not cause virus inactivation caused (see level control), however, led to a significant Reduction of infectivity in the filtrate.

Table 1: Increased virus reduction by filtration after addition of polysorbate 80

Claims (2)

A method for reducing the infectivity of viruses in the production process of biologics, characterized in that the biologics 1) are subjected to chemical and / or physical methods which as such have no or only minimal virus inactivation capacity, and 2) these biologics pretreated in this way are subjected to mechanical shearing forces lead to a significant virus inactivation. Method according to claim 1, characterized in that the biologics 1) with so much Surfactant can be added that a final concentration of at most 0.1 (w / v) is achieved, and 2) pretreated this way Biologics of a filtration through a filter with a pore size, the corresponds approximately to the virus size, get abandoned.