Method for inactivating prions.

0001The present invention relates to a method for inactivating prions, viruses and other infectious agents (eg bacteria) in biological or biogenic materials, in particular in a plasma or serum suitable as a starting material for albumin.

0002By the method according to the present invention, any prions, bacteria and the like that are present in the preparation of proteins, in particular serum albumin. Viruses can be reliably deactivated.

0003When producing proteins 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 are caused by the administration of blood derivatives to e.g. B. bleeders. This happened especially before the discovery of the AIDS pathogen; Since then, blood donors have been monitored, so-called "screening", and manufacturers are also looking for methods that can still eliminate HIV viruses that may still appear in the donor blood during the purification of the serum proteins and the final packaging of the therapeutic agents.

0004The more known about the nature of the pathogen of a disease, the easier and faster it is to develop methods for its destruction. For the (historically speaking) first infectious particles, the bacteria, there have therefore been recognized processes for decades, all of which belong to the state of the art (steam sterilization, dry sterilization, pasteurization, sterile filtration, ethylene oxide, radiation inactivation, etc.).

0005For viruses, another group of pathogens, which was only discovered in the 20th century, there are also methods of elimination, but these are more specific to the type of pathogen. While for some viruses a pH reduction to 4 is sufficient to completely destroy them, other species are only inactivated by organic solvents in high concentrations.

0006For some years now, a new group of pathogens, which even seems to question the central dogma of molecular biology, has appeared increasingly in scientific publications: the prions. sometimes referred to as unconventional viruses or "slow virus". There is still disagreement about their material nature: while some of the scientists use them for extremely small viruses, ie a small piece of nucleic acid with some coat proteins, an increasingly large part draws from the lack of detection of any nucleic acid, as well as from many other findings, the revolutionary conclusion 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 multiplication of infectious particles.

0007If 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 always result in death. Since these are infectious diseases with an incubation period of up to 30 years, the discomfort among researchers and manufacturers of therapeutic agents is very high, but also increasingly among consumers: One of these diseases is BSE (Bovine Spongiphorme Encephalopathies), the enigmatic English cattle disease . Another very similar disease is SCRAPIE in sheep and goats (perhaps the origin of the current BSE epidemic).

0008In humans, it is KURU, a disease that occurs in 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 circle of exotic diseases (with a probability of 1: 1 million).

0009There is evidence that BSE became epidemic in England by feeding animal meal from rendering plants, which also disposed of sheep suffering from scrapie. Unfortunately, there is also a correspondence in humans: in the production of human growth hormone from human pituitary glands (from autopsy material), pathogens of Creutzfeldt-Jakob syndrome entered a batch from a manufacturer; several cases of this disease have already occurred in recipients of this batch of hormones, namely in adolescents, although it otherwise occurs in people over 50.

0010This may be enough to point out the potential danger that comes from this new form of diseases. The assessment of the hazard 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 changed pathogenically. In addition, prions do not cause an immune response such as antibody formation, which makes diagnosis very difficult.

0011If you try to destroy prions, there is another problem: 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 bases, preferably at high temperature, by treatment with hypochlorite base or by temperatures above 150 ° C; it is clear that such extreme conditions destroy any biological therapeutic agent immediately, or at least render it ineffective.

0012So far, attempts have been made to obtain the raw material from areas where the above-mentioned epidemics have not yet occurred in order to produce prion-free biological material. However, considering the extremely long incubation period of up to 30 years, this procedure seems very dangerous. By screening the starting material, prions cannot be reliably determined because - as already mentioned - there is no immune reaction. 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, and by renewed injection into mouse brains) approx. 14 It takes months and is extremely time-consuming, it is neither suitable for the control of the starting material nor for the batch check 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).

0013It is therefore an object of the present invention to provide a method which reliably eliminates any prions present in biological or biogenic material without destroying this material and which also destroys all known groups of viruses.

0014This object is achieved according to the invention by a process of the type mentioned at the outset by treating this material with a chaotropic reagent for at least 12, preferably 18 hours.

0015It has been surprisingly found in the context of the present invention that prions can be eliminated by prolonged treatment with a chaotropic reagent, namely at normal room temperature (20-25 ° C.). Such a treatment biological materials such. B. Albumine not affected. Viruses and bacteria are also reliably eliminated.

0016The 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.

0017In order to increase the reliability of the elimination of the pathogens, it is preferred that the biological or biogenic material is adjusted to a pH of about 6.5 and about before the treatment with the chaotropic reagent - if necessary after dilution with pyrogen-free, sterile water 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 sulfonate or a derivative thereof can be used as detergent. The pH is expediently adjusted by adding dilute hydrochloric acid.

0018In addition to the detergent, 8-10% by volume of methyl or ethyl alcohol is preferably added.

0019Finally, it is advantageous if, after adding the detergent and, if appropriate, the methyl or ethyl alcohol, the biological or biogenic material is slowly heated to about 70 ° C. with stirring and stirred at this temperature for at least 15 more, preferably about 30 minutes, after which it is rapidly stirred cools, acidified to a pH of 4 to 4.2 and the precipitated globulins are separated off.

0020In 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 "bovine" is at risk from BSE and because the human analogue, human serum albumin (HSA), is also a very important therapeutic agent . The process can be transferred directly to HSA production without modification; it can be modified for other proteins of animal or human origin.

0021The method according to the invention - applied to BSA - can be summarized as follows:

0022The plasma or serum, from which BSA or HSA is obtained, is diluted with water, the pH is changed to 6.5 and ethyl or methyl alcohol is added 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. The mixture is then rapidly cooled, the pH is adjusted to 4.0 to 4.2, the globulins precipitated are removed and the clear solution is neutralized. A strongly 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 then the reagent is removed, preferably by chromatography, but dialysis or diafiltration are also possible. The desalted solution is then concentrated, possibly with additives, and then either sterile filtered and sterile filled or dried, in particular by freeze-drying. A thermal aftertreatment of the solution ("pasteurization" in the presence of stabilizers) or the powder (steam injection) can be connected.

0023Instead of serum or plasma, any other starting material containing albumin can be used, such as placental blood, fractions of the plasma fractionation according to Cohn, etc.

0024The 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, as well as by adding high concentrations of conventional viruses such as Bovine Viral Diarrhea (BVD), Infectious Bovine Rhinotracheitis (IBR), Parainfluenza 3 , Foot and mouth disease, Maedi-Visna virus and parapox virus of sheep (ORF). The albumin solutions treated in this way were autosterile, which demonstrates the destruction of bacteria.

0025The procedure was as follows:    3 g of dried beef plasma (corresponding to 2 g of protein) were dissolved in 35 ml of distilled, sterile water. 4 ml of absolute ethanol was 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.

0026Under the necessary safety precautions (P 3 laboratory, laminar flow hood ...) 225 µl of a 20% scrapie brain homogenate (titer: 2.10⁹ LD₅₀ / g) were added and homogenized, or viruses were added, the type of which and concentration are given in Table 1. The solution was placed in a water bath at 70 ° C; after 10 min the content was also 70 ° C, after which the treatment was continued for 30 min. After every 10 min, stirring was carried out for 1 min 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.

0027The precipitated globulins were removed at 6000 revolutions per minute (corresponds to 4000 g) at 4 ° C. for 10 minutes. The clear supernatant was obtained by decanting; it was pure bovine albumin solution, volume: 20.5 ml.

0028This solution was neutralized by adding 250 μl of a sodium hydroxide solution (pH 7.0). Then 15 g of urea were added, which increased the volume to 30 ml, resulting in a concentration of 8 molar. This solution was left at room temperature (21 ° C) for 16 hours.

0029The gel permeation chromatography method was used to remove the urea:    15 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 5 cm in diameter and 30 cm in height and washed with sterile distilled water (200 ml). The flow rate was 7.5 ml / min.

003030th ml of the above solution were applied, followed by washing with 100 ml of water; the fractions were combined as follows: 50 + 40 ml. After that, no protein was detectable in the eluate. The 90 ml was lyophilized to give 0.7 g bovine serum albumin.

0031Alternatively, in some experiments, the urea was diafiltered in an Amicon S-1 module, cut-off 10,000 d. away. It was distilled with about 1000 ml. Water diafiltered and then concentrated to about 50 ml, then lyophilized. Biological testing for scrapie / BSE:

0032This albumin was dissolved in 3 ml of physiological saline and injected in aliquots of 20 μl mice into one half of the brain. Total number of mice: 136. The mice used were from strain C57 / B16.

0033A dilution series of the scrapie inoculum (see above, 2.10⁹ LD₅₀ / g), 8 dilution stages, served as a positive control. Bovine serum albumin, which had been produced as above, but without a scrapie inoculum, served as a negative control.

0034All inoculations were carried out on 12 animals each, ie in 12 repetitions.

0035As a result, it was found that the titer could be confirmed in the positive control, while the clinical picture or deaths did not occur either in the negative control or in the test group.

0036Biological testing in the case of conventional viruses:

0037The BSA was dissolved in cell culture medium. This medium served as a nutrient medium for mammalian cells, which can serve as host cells for the respective virus (see Table 1).

0038Positive (virus stock solution) and negative (medium only) tests as well as tests for unspecific cytopathogenicity of albumin completed these tests.

0039The kill rates are given in Table 1 and were in all cases limited by the initial titers.<tables id="tabl0001" num="0001"><img file="EP0530173A2_D0001.tif" /></tables>

0040The equivalence of N-lauroyl sarcosinate with lauryl sulfate as detergent in the preparation of albumin from plasma was demonstrated in a similar experiment, but without the addition of infectious material; the measured parameters were yield and protein purity (over 98% for both detergents).

0041The equivalence of urea and sodium thiocyanate as chaotropic reagents has been demonstrated in the treatment of ribonucleoprotein particles by influenza viruses (a model for nucleic acid-protein interactions) and in the removal of affinity bonds such as e.g. B. with gelatin fibronectin, tried and proven.