Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
1999-08-06
2001-08-07
Fortuna, Ana (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S651000, C210S652000, C204S522000, C204S527000, C204S535000, C204S276000, C435S173100
Reexamination Certificate
active
06270672
ABSTRACT:
The invention relates to a method for removing pathogens from biological liquids and an apparatus for performing such a method.
The risk of transmission of viruses by blood products is known. There is comprehensive literature available dealing with the inactivation of infectious agents by various methods. These methods include the treatment of biological and pharmaceutical products with chemical substances (e.g. with detergents, solvents etc. or combinations thereof (EP 0 050 061 A, EP 0 131 740 A)), heating steps (e.g. heating in an aqueous solution in the presence of stabilizing agents, heating in the dry state and heating in the solid wet state) and physical methods (e.g. photoinactivation or nanofiltration).
Especially nanofiltration is a very promising technique due to its efficacy especially for industrial production methods.
However, there are several problems connected with nanofiltration: First, due to the small pore sizes it is often difficult to filter biological liquids through a nanofilter. Therefore, biological liquids frequently have to be diluted before and concentrated again after a nanofiltration.
Moreover, nanofilters have varying performance abilities (due to the method of production). Generally nanofilters are no absolute filters, i.e. their cut-off value is not absolute and an unwanted passage of (small) viruses through the filters frequently occurs (see, for example Eibl et al, Biologicals 24(3), 285-287 (1996), Manabe Dev. Biol. Stand. 88, 81-90 (1996) or O'Grady et al., Dev. Biol. Stand 88, 319-326 (1996)).
In order to accelerate the nanofiltration step, especially on a large scale, pressure is applied to facilitate the passage of biological solutions through the filter. Such a pressurizing, however is connected with a risk of breaking of the filter and may enhance a passage of viruses through the filters. Alternatively, if ultrafiltration means are used, an enhanced overflow rate is applied to facilitate membrane passage of proteins contained in the biological liquid (see, for example Di Leo et al., Biologicals 21(3), 287-296 (1993) and Di Leo et al., Biologicals 21(3), 275-286 (1993)).
Due to the sheer forces caused by these additional measures in the filtration steps, viruses or virus components may change their size which allow the passage of the filtration membranes despite the cut-off range which theoretically would not allow a virus passage. Such sheer forces, however, lead to detection of viruses in the filtrate due to change the shape of the virus particles during membrane passage.
It is an object of the present invention to provide an improved method for removing pathogens from biological liquids, especially proteinaceous solutions, with separation means.
It is a further object of the invention to provide a nanofiltration method which is more reliable especially with respect to the definiteness of virus cut-off than the nanofiltration techniques of the prior art. Furthermore, a nanofiltration method suitable for the production of biological products on large scale shall be provided with allows virus reduction without significant loss of protein yield in an industrially applicable method.
These objects are solved by a method for removing pathogens from biological liquids, said biological liquids containing at least one pharmaceutically active molecule, said method comprising the steps of
providing a biological liquid, wherein pathogens are potentially present, in an apparatus comprising an anode and a kathode and a separation means suitable for separating said pathogens from said pharmaceutically active molecule, said separation means being positioned between said anode and said kathode,
applying current between said anode and said kathode, thereby causing one of said pathogens or said pharmaceutically active molecule to pass said separation means and
recovering said pharmaceutically active molecule in a form being essentially free of said pathogens.
Essentially free has to be understood in that more than 99% of the contaminants present in the starting material are removed. Preferred that more than 99,9% and most preferred that more than 99,99% of the pathogens present in the starting material are removed.
The method according to the present invention allows a safe and relieable method for removing pathogens from biological liquids, thereby preventing the necessity of pressure or fast overflow. Moreover, due to the application of electric current it is possible to remove the pathogens from the pharmaceutically active substance not only based on size but also based on relative charge differences.
Biological liquids are liquids that are obtained from biological sources, for instance body liquids such as blood or liquids derived from cell culturing, especially culturing of recombinant cells. All such biological liquids have a certain risk of being contaminated with infectious agents, especially viruses, which should not be present in the final product containing the pharmaceutically active compound.
Such a risk of transmission of viruses is especially given and described for blood products. Under blood products, products of human and animal blood or plasma are understood which are intended for therapeutic, prophylactic or diagnostic applications. Such products may contain proteins, such as enzymes, proenzymes, coagulation factors, enzyme inhibitors, immunoglobulins, albumin, plasminogen, fibrinogen and fibronectin.
The separation means to be used according to the present invention can be any means suitable for separating the viral pathogen from the pharmaceutically active molecule, especially filtration means. According to the present invention a nanofilter preventing viruses from passing through the filter is preferred, for example Millipore PTMK 300 kD Polysulfor, Planova (Asahi) or Viresolve. Such filter means should have a cut-off range of a defined value as applied in current nanofiltration methods. The filter devides the apparatus used for performing the present invention (the filtration chamber) into two compartments. Usually in the first compartment the biological liquid (potentially infected with viral pathogens) is provided, the second compartment may be filled with a suitable puffer.
It is further preferred to use ultrafiltration membranes as a filtration means.
The term pathogen shall for the present invention not only include viruses but also other pathogens with a size comparable to viruses, such as prion pathogens, such as the BSE pathogen or the scrapie pathogen.
Especially for viral contaminations which have a peculiar structure or are very difficult to inactivate by other methods of virus inactivation or virus depletion the present method is very effective.
The method according to the present invention may be performed in a variety of modes depending on the structure and charge of the pathogen relative to the pharmaceutically active molecule. If for example the pathogen is uncharged, it retains in the compartment where it has been introduced due to its lack of electrophoretic mobility. A charged pharmaceutically active molecule, such as a macromolecule, especially a protein, is affected by the applied current and is forced to pass the filtration membrane. It is of course evident that the positioning of the kathode and anode has to be adapted to the biological liquid and to the system of pathogen/pharmaceutically active substance in order to allow an effective separation. This, however, is routine work for the skilled man in the art. The filtration membrane which may be in principle an ultrafilter prevents that the (uncharged) pathogen passes the filter by diffusion.
If the pharmaceutically active substance and the pathogen are equally charged and both have an electrophoretical mobility into the same direction, a filter with a suitable cut-off between the size of the pathogen and the size of the pharmaceutically active molecule prevents the passage of the larger of the two (in most cases: the viral pathogen) from passaging the membrane. Since the sheer forces present in the system according to the present invention are rela
Mattes Erwin
Schwarz Hans Peter
Turecek Peter
Baxter Aktiengesellschaft
Fortuna Ana
Oppenheimer Wolff & Donnelly LLP
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