Chemistry: analytical and immunological testing – Including chromatography
Reexamination Certificate
1998-11-10
2002-04-16
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Including chromatography
C436S086000, C436S088000, C530S412000, C530S413000, C530S362000, C530S363000, C530S364000
Reexamination Certificate
active
06372510
ABSTRACT:
The invention relates to the sphere of removing unconventional transmissible agents (UCTA). More specifically, the invention relates to a process for removing UCTA from a protein solution.
The unconventional transmissible agents (UCTA), which include prions, are at the root of the subacute transmissible spongiform encephalopathies, which are slow degenerative diseases of the central nervous system whose development is always fatal; the agents which are responsible for these diseases have still not been identified.
These subacute transmissible spongiform encephalopathies affect both man and animals: → in man: kuru, Creutzfelt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia and, for certain people, Alpers disease. → in animals: natural sheep scrapie, transmissible mink encephalopathy, chronic wasting syndrome of wild ruminants in captivity and in the wild, and bovine spongiform encephalopathy.
The problem of removing UCTA from products of biological origin poses very great difficulties and is currently very poorly resolved, apart from a few exceptions.
The methods of inactivation which have been identified to date, such as treating with soda or Javelle water, or heating to temperatures higher than 130° C., cannot be applied to most biological substances, which are too fragile to withstand such physicochemical conditions.
While separation methods aimed at removing UCTA can in theory be the same as those which are used for proteins in general, i.e. filtration, selective adsorption, chromatography and ultracentrifugation, they meet with a very great difficulty. These methods are not nowadays used for removing traditional viruses since part of the infectivity always remains, even if this part is only a minimum but still sufficient to transmit the disease after the product has been injected. The inactivation methods are rightly preferred to them. The transmissible character of prions is as powerful as that of most viruses, and no chromatographic methods are currently available which are known to remove, with certainty, the last traces which may contaminate a complex protein solution. Filtration methods have been suggested for this purpose and have demonstrated their efficacy in clearing solutions of proteins of a molecular weight of less than 100 kilodaltons. Beyond that, these screening methods come up against the following difficulty: the size of the pores selected for stopping prions also stops large proteins, either because the protein has a mean hydrated diameter which is greater than that of the pore or because adsorption phenomena complicate filtration of the protein. These problems are met with, in particular, in the purification of animal sera which are used in cell culture methods for the purpose of preparing vaccines or drugs.
Similarly, when albumin is extracted from a biological material, whether human or animal, and when this albumin is intended for a therapeutic use, it is essential to treat the albumin in order to remove from it the unconventional transmissible agents which might possibly be present.
A process for purifying albumin from human placentas which affords all the guarantees of safety with regard to possible viral contamination is known from the article “Viral validation of the manufacturing process of high purity albumin from placentas”, which was published in Brown F. (ed.): Virological Safety Aspects of Plasma Derivatives Dev. Bio Stand. Basel, Karger, 1993, vol. 81, pp. 237-244. As was mentioned in the article entitled “Safety of placental blood derivatives”, which was published on Jan. 15 1994 in Vol. 343 of The Lancet, this process has been regarded as being particularly suitable for removing prions. This is because the purification steps carried out in this process include, in particular, three steps, i.e. FC1, FC2 and Hcol, of precipitation with alcohol in acid medium, combined with filtrations. Since prions have a tendency to coprecipitate with denatured proteins, these three steps, which involve the removal of substantial quantities of protein precipitates, are particularly appropriate for removing prions.
However, even if it is well suited for purifying placental albumin, this process is in no way appropriate for purifying all protein solutions, particularly not solutions of albumin of plasma origin. For example, the precipitation step FC1 is a step whose implementation conditions are such that haemoglobin is itself denatured and precipitated specifically; the quantity of haemoglobin is very low in plasma; this step would not therefore have the same characteristics, and albumin itself, in particular, would be exposed to denaturation by the chemical agent.
It is therefore desirable to have available a process for removing unconventional transmissible agents from a protein solution without altering the properties of the proteins which are present in the solution in any noteworthy manner.
The object of the invention is to propose such a process which is suitable for treating an aqueous protein solution, whether the solution be purified or not, in particular an albumin solution, an animal serum which is intended to be used for carrying out cell culture, a preparation of immunoglobulins of serum origin, or a preparation of factors which are involved in coagulation.
Another object of the invention is to propose a process which is capable of being employed industrially.
In order to achieve these objects, the present invention relates to a process for removing unconventional transmissible agents from an aqueous protein solution, characterized in that the process essentially consists in treating the protein solution by simultaneous electrostatic and hydrophobic adsorption chromatography and collecting the filtrate.
According to one embodiment of the invention, the protein solution is treated by electrostatic and hydrophobic chromatography in two steps, with one step being carried out at acid pH and the other step being carried out at approximately neutral pH.
According to one particular embodiment, the step at acid pH is carried out before the step at approximately neutral pH.
According to another embodiment of the invention, the protein solution is additionally treated by anion exchange chromatography.
According to another embodiment, the process of the invention additionally consists in carrying out a preliminary filtration of the protein solution using a filter whose cut-off threshold is approximately equal to 0.2 &mgr;m. In this way, the chromatography columns are protected from any blockage or bacterial contamination.
The present invention will be better understood from reading the detailed description which will now follow.
The protein solution from which any UCTA which may be present should be removed may have a variety of origins. It may be an unpurified protein solution which can be contaminated with prions or a purified solution, which may or may not originate from serum, such as preparations of immunoglobulins of serum origin and preparations which contain coagulation factors. Specifically, the protein solution may be an albumin solution of plasma origin which is obtained by a method which includes the Cohn fractionation technique. In all instances, the protein solution to be treated is an aqueous solution.
The gel which is used for carrying out the electrostatic and hydrophobic adsorption chromatography according to the invention is a macroporous gel which possesses functional groups, such as cross-linked products of diethylaminopolystyrene or diethylaminopolyphenyl, or any other combination of positive charge-carrying radicals and hydrophobic molecules linked to an inert matrix (for example silica, cellulose, sepharose or acrylamide). DEA-Spherosil/LS®, which is supplied by BIOSEPRA (USA, the address of whose office in France is 35 avenue Jean Jaurés-92390 VILLENEUVE LA GARENNE), is preferably used.
The column support may be equilibrated with standard buffers, for example sodium phosphate buffers, to which sodium chloride is preferably added.
In the specific case in which this chromatography is carrie
Grandgeorge Michel
Labatut René
Rouzioux Jean-Marc
Tayot Jean-Louis
Veron Jean-Luc
Burns Doane Swecker & Mathis L.L.P.
Pasteur Merieux Serum et Vaccins
Wallenhorst Maureen M.
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