Method for inactivating prions.

The present invention relates to a method for inactivating prions in a biological or biogenic material, in particular in a plasma or serum suitable as a starting material for albumin.

The method according to the present invention is intended to reliably inactivate any prions that may be present in the preparation of proteins, in particular serum albumin.

When proteins are made from animal (or human) material, there is always the risk of contamination with infectious particles that were present in the starting material. A tragic example of the recent past is the infection of many people with HIV viruses, the causative agents of AIDS, which occurred through the administration of blood derivatives to hemophiliacs, for example. This happened mainly before the discovery of the AIDS pathogen; Since then, blood donors have been monitored, known as screening, and the manufacturers are also looking for methods to eliminate any HIV viruses that may still appear in the donor blood during the cleaning of the serum proteins and the final packaging of the therapeutic agents.

The more we know about the nature of the causative agent of a disease, the easier and faster it is possible to develop methods of destroying it. For the (historically speaking) first infectious particles, the bacteria, there have been recognized methods for decades, all of which are state-of-the-art (steam sterilization, dry sterilization, pasteurization, sterile filtration, ethylene oxide, radiation inactivation, etc.).

For viruses, another group of pathogens that was only discovered in the 20th century, there are also methods of elimination, but these must be tailored more specifically to the type of pathogen. While for some viruses a pH value lowering to 4 is sufficient for complete destruction, other types are only inactivated by organic solvents in high concentrations.

For some years now, a new group of pathogens, which even seems to question the central dogma of molecular biology, has been appearing increasingly in scientific publications: the prions, sometimes referred to as unconventional viruses or slow viruses. There is still disagreement about their material nature: while some scientists use them for extremely small viruses, ie a small piece of nucleic acid with a few coat proteins holds, an increasingly larger part draws the revolutionary conclusion from the lack of evidence of any nucleic acid as well as from many other findings that it is an infectious protein without DNA or RNA. This would be the first contradiction to the dogma that nucleic acids (genetic material) are necessary for the replication of infectious particles.

If the nature of the prions is still unclear, some diseases are known that affect humans or animals and for which there are no treatment options, which are therefore always fatal. Since these are infectious diseases with an incubation period of up to 30 years, the unease among researchers and manufacturers of therapeutic agents is very high, but also increasingly among consumers: One of these diseases is BSE (Bovine Spongiphorm Encephalopathies), the enigmatic English cattle disease . Another very similar disease is SCRAPIE in sheep and goats (perhaps the origin of the current BSE epidemic).

In humans, it is KURU, a disease that occurs among ritual cannibals in Papua New Guinea, Jakob-Creutzfeldt Syndrome and Gerstmann-Sträussler Syndrome. However, there are striking similarities with Alzheimer's disease, and this would leave the group of exotic diseases (with a probability of occurrence of 1: 1 million).

There is evidence that BSE became epidemic in England through the feeding of animal meal from rendering plants which also disposed of scrapie-suffering sheep. Unfortunately, there is also an equivalent in humans: during the production of human growth hormone from human pituitary glands (from autopsy material), the pathogens of Creutzfeldt-Jakob syndrome ended up in a batch of a manufacturer; several cases of this disease have already occurred in recipients of this batch of hormones, namely in adolescents, although it usually occurs in people over 50.

This should suffice to point out the potential danger that arises from this new type of disease. The assessment of the risk, as well as the treatment, is made more difficult by the lack of knowledge of these new pathogens, the prions: it appears that they have little or no nucleic acid and that their prion protein is encoded by a host gene and later

AT 408 191 B is changed pathogenically. In addition, prions do not cause an immune response such as antibody formation, which makes diagnosis very difficult.

If one tries to destroy prions, another problem arises: they are extremely resistant to all physical and chemical methods; So far it is only known that they can be eliminated by treatment with concentrated mineral acids or alkalis, best at high temperature, by treatment with hypochlorite alkaline solution or by temperatures above 150 ° C .; it is clear that such extreme conditions will immediately destroy or at least render ineffective any biological therapeutic.

So far, attempts have been made to obtain the starting material from areas in which the above-mentioned epidemics have not yet occurred in order to produce prion-free biological material. However, if you consider the extremely long incubation period of up to 30 years, this approach seems very dangerous. By screening the starting material, prions cannot be reliably determined because - as already mentioned - no immune reaction occurs. Since the biological test (injection of the material in question into the brains of mice, waiting for pathological behavior, death of the animals, confirmation by molecular biological and histological findings as well as by re-injection into the mice's mind) approx. 14th Takes months and is extremely time-consuming, it is neither suitable for checking the starting material nor for batch checking of finished drugs; it can only be used to validate new manufacturing processes (after adding prions). This must be done under the greatest safety precautions in special laboratories in which the test animals are also kept and examined (P 3 zones).

It is therefore the object of the present invention to create a method which reliably eliminates any prions that may be present in biological or biogenic material without destroying this material.

This object is achieved according to the invention by a method of the type mentioned at the outset in that this material is treated with a chaotropic reagent for at least 12, preferably 18, hours.

It has been surprisingly found within the scope of the present invention that prions can be eliminated by prolonged treatment with a chaotropic reagent, specifically at normal room temperature (20-25 ° C.). Such treatment does not affect biological materials such as albumins.

The chaotropic reagent used is preferably urea in a concentration of 6-8 molar, based on the total amount, or sodium thiocyanate in a concentration of 1-2 molar, based on the total amount. It can be removed after treatment by dialysis, diafiltration or gel permeation chromatography.

In order to increase the reliability of the elimination of the prions, it is preferred that the biological or biogenic material is adjusted to a pH of about 6.5 and about 1 g / l of a detergent, in particular an anionic detergent, is added. An alkyl sulfate or a derivative thereof, preferably sodium lauryl sulfate, and / or a sarcosinate, preferably sodium (N-lauroyl sarcosinate) and / or an alkyl sulfate or a derivative thereof, can be used as the detergent. The pH is expediently adjusted by adding dilute hydrochloric acid.

Preferably, in addition to the detergent, 8-10% by volume of methyl or ethyl alcohol is added.

Finally, it is beneficial if, after adding the detergent and, if necessary, the methyl or ethyl alcohol, the biological or biogenic material is slowly heated to about 70 ° C. while stirring and stirring is continued at this temperature for at least 15, preferably about 30 minutes, after which it is quickly cools, acidified to a pH of 4 to 4.2 and separates the precipitated globulins.

In the context of the present invention, bovine serum albumin (BSA) is chosen as an example because it is used in large quantities, the raw material source cattle is endangered by BSE and also because the human analogue, human serum albumin (HSA), is a very important therapeutic agent. The method can be transferred directly to HSA extraction without modification; it may have to be modified for other proteins of animal or human origin.

AT 408 191 B

The method according to the invention - applied to BSA - can be summarized as follows:

The plasma or serum from which BSA or HSA is obtained is after dilution with water, changing the pH value to 6.5 and adding ethyl or methyl alcohol at a temperature of 70 ° C for 30 minutes in the presence of a strong surfactant , preferably sodium lauryl sulfate (dodecyl sulfate sodium salt) or an analog, sodium (N-lauroyl sarcosinate) or other analogs. It is then cooled rapidly, the pH is adjusted to 4.0 to 4.2, the precipitated globulins are removed and the clear solution is neutralized. A highly chaotropic reagent is then added in high concentration, preferably 6-8 molar urea or 1-2 molar sodium thiocyanate, the solution is left at room temperature for 16 hours and the reagent is then removed, preferably by chromatography, but dialysis or diafiltration are also possible. The desalinated solution is then concentrated, possibly provided with additives, and then either sterile-filtered and sterile bottled or dried, in particular by freeze-drying. A thermal post-treatment of the solution (pasteurization in the presence of stabilizers) or the powder (steam injection) can be added.

Instead of serum or plasma, any other starting material containing albumin can be used, for example placental blood, fractions of plasma fractionation according to Cohn, etc.

The effectiveness was demonstrated in the biological model by adding scrapie prions to the starting plasma and injecting the albumin solution into the brains of mice. In detail, the procedure was as follows:

g dried bovine plasma (corresponding to 2 g protein) were dissolved in 35 ml distilled, sterile water. 4 ml of absolute ethanol were added. The pH was adjusted to 6.5 with dilute hydrochloric acid. 45 mg (= 0.1%) sodium lauryl sulfate (at least 95% pure) were dissolved. The total volume was 44 ml.

Under the necessary safety precautions (P 3 laboratory, laminar flow hood ...), 225 μΐ of a 20% scrapie brain homogenate (titer: 2.10⁹ LD₅₀/ g) added and homogenized. The solution was placed in a water bath at 70 ° C; after 10 minutes the contents were also 70 ° C., after which the treatment was continued for a further 30 minutes. After 10 minutes in each case, the mixture was stirred for 1 minute using a magnetic stirrer. At the end of the incubation period, the sample was placed on ice and cooled. The cold solution was brought to pH 4.2 with 200 μl hydrochloric acid and homogenized again.

The precipitated globulins were removed at 6000 revolutions per minute (corresponds to 4000 g) for 10 minutes at 4 ° C. The clear supernatant was obtained by decanting; it was pure bovine albumin solution, volume: 20.5 ml.

This solution was neutralized by adding 250 μl of a sodium hydroxide solution (pH 7.0). Then 15 g of urea were added, whereby the volume increased to 30 ml, so that a concentration of 8 molar resulted. This solution was left at room temperature (21 ° C.) for 16 hours.

The method of gel permeation chromatography was used to remove the urea:

g of Sephadex G 50 (from Pharmacia, Uppsala) were heated in 500 ml of sterile distilled water and left to stand overnight. The swollen gel was filled into an acrylic glass column with a diameter of 5 cm and a height of 30 cm and washed with sterile distilled water (200 ml). The flow rate was 7.5 ml / min.

ml of the above solution were applied, followed by washing with 100 ml of water; the fractions were combined as follows: 50 + 40 ml. Thereafter, no more protein could be detected in the eluate. The 90 ml was lyophilized to give 0.7 g of bovine serum albumin.

This albumin was dissolved in 3 ml of physiological saline solution and injected into one hemisphere of the brain in aliquots of 20 μl mice. Total number of mice: 136. The mice used were of strain C57 / B16.

A dilution series of the scrapie inoculum served as a positive control (see ο., 2.10⁹ LD₅₀/ g), 8 dilution levels. Bovine serum albumin, which had been prepared as above, but without a scrapie inoculum, served as negative control.

All inoculations were carried out on 12 animals each, ie in 12-fold repetitions.

As a result, it was found that the titer in the positive control could be confirmed while

AT 408 191 B neither in the negative control nor in the test group the clinical picture or deaths occurred.

The equivalence of N-lauroyl sarcosinate with lauryl sulfate as detergent in the preparation of albumin from plasma was shown in a similar experiment, but without the addition of infectious material; the parameters measured were yield and protein purity (over 98% for both detergents).

The equivalence of urea and sodium thiocyanate as chaotropic reagents has been tested and proven in the treatment of ribonucleoprotein particles of influenza viruses (a model for nucleic acid-protein interactions) and in the breaking of affinity bonds, such as with gelatin-fibronectin.