Method for inactivating prions

0001The present invention relates to a method of inactivating prions and viruses in biological and biogenic materials, particularly in a suitable starting material for albumin, plasma or serum, under Using a chaotropic reagent.

0002intended by the method according to the present invention Thus in the preparation of proteins, especially serum albumins, any existing prions u. Viruses reliable are inactivated.

0003In the production of proteins from animal (or human) material is always the risk of contamination given with infectious particles, which in the starting material were present. A tragic example of the recent past provides many people with HIV from the infection, the Agents of AIDS, is represented by the administration of Blood derivatives at z. B. hemophiliacs done. This happened especially before the discovery of the virus causing AIDS; since then done monitoring of blood donors, the so-called "screening", and also looking for the manufacturers procedures that may still occurring in the donor blood HIV virus during Cleaning of serum proteins and the final packaging of the Therapeutics eliminate.

0004The more known about the nature of the pathogen of a disease is, the easier and faster method for its Destruction are developed. For the (historically) first infectious particles, the bacteria, there are therefore recognized for decades procedure, all of the state art include (steam sterilization, dry heat sterilization, Pasteurization, sterile filtration, ethylene oxide, Strahlungsinaktivierung, etc.).

0005For the virus, another group of pathogens that until 20th century was discovered, there are also methods for elimination, but these are specific to the type tune the pathogen. While for some viruses a pH-lowering suffice to 4 to complete destruction, are other types only by organic solvents in high concentration inactivated.

0006For several years now a new group of appear Pathogens that even the central dogma of molecular biology seems to call into question increasingly in scientific publications: the prion. sometimes also known as unconventional viruses or "slow virus". About their material nature reigns nocht disagreement: while some of the scientists they for extremely small viruses, ie, a small piece of nucleic acid with some envelope proteins, holds, attracts an increasingly larger part of the lack of detection of any nucleic acid and from many other findings the revolutionary conclusion, or that this is or infectious protein without DNA RNA acts. This is the first contradiction to the dogma that the duplication of infectious particles nucleic acids (Genetic material) are necessary.

0007Prevails over the nature of prions still unclear, yet some diseases are known, people or Animals infected and against which there is no treatment options, ie are always fatal. Since it is hiebei are infectious diseases whose incubation period is up to 30 years, is the unease among researchers and Manufacturers of drugs is very high, but also increasingly the consumer: One of these diseases is in fact BSE (Bovine Spongiphorme encephalopathies), the enigmatic British cattle disease. Another very similar Disease is SCRAPIE in sheep and goats (perhaps the origin of the current BSE epidemic).

0008In humans there are KURU, a disease that occurs in ritual cannibals in Papua New Guinea, the Jakob Creutzfeldt Syndrome and the Gerstmann syndrome. However, there are striking similarities with the Alzheimer 's disease, and thus would be the group of exotic diseases (with probabilities of occurrence of 1: 1 million) leave.

0009There is evidence that BSE to the consumption of meat meal from rendering plants, which also Scrapie suffering sheep discarded, in England was an epidemic. Unfortunately, there is also a correlation in humans: in the production of human growth hormone from human pituitaries (from autopsy material) arrived pathogen of Creutzfeldt-Jakob syndrome in a batch of a manufacturer; Several cases of this Disease have already occurred in recipients of this hormone batch, namely among young people, although they otherwise occurs in people over the 50th

0010This may suffice to show the potential danger resulting from this new form of circle of diseases comes. This is particularly the assessment of the threat as well as the treatment by the low knowledge of these new pathogens, prions: it seems that they have little or no nucleic acid and that their prion protein from a host gene encoding and is later modified pathogen. Moreover, prions cause no immune response as Antibody formation, which greatly complicates the diagnosis.

0011If one tries to destroy prions, the result is a further problem: they are extremely resistant against all physical and chemical methods; so far it is only known that they concentrated by treatment with Mineral acids or alkalis, best at high temperature, by treatment with hypochlorite solution or are eliminated by temperatures of over 150 ° C; it is clear that in such extreme conditions, any biological destroying agent immediately, or at least make them ineffective.

0012Previously, attempts to obtain the raw material from areas where the above-mentioned diseases have not yet occurred to produce prion free biological materials. However, considering the extremely long Incubation period of up to 30 years, this approach seems very dangerous. By screening of the starting material Prions can not be determined reliably, because - as already mentioned - no immune response occurs. Since the biological test (injection of the material in question in the brains of mice, waiting for pathological Behavior, animal death, confirmed by molecular biological and histological findings as well as recent Injection in Mäusegerhirne) lasts approximately 14 months and is extremely costly, it is not for the control of Starting material or for the batch testing of finished drug, it can only to validate new manufacturing process serve (after addition of prions). This must be done in special laboratories where the test animals biggest security be kept and studied (P 3 zones). It is therefore an object of the present invention, a method create, the possibly existing prions in biological or biogenic material reliably eliminated without this material destroy, and all known groups of viruses destroyed.

0013This object is achieved by a method of the aforementioned Art inventively achieved in that the biological or biogenic material - optionally after dilution with pyrogen, sterile water - adjusted to a pH of about 6.5 and about 1 g / l of a detergent, in particular of an anionic Detergent, and then adding this material with stirring slowly heated to about 70 ° C and at this temperature at least 15, preferably about 30 minutes further stirring, after which it rapidly cooled, acidified to a pH of 4 to 4.2, and the precipitated Globulins are separated, and that putting it in the detergent / heat treatment with a chaotropic treated reagent at least 12, preferably 18 hours.

0014It has namely surprisingly in the present invention found that prions together by moderate heating with prolonged treatment with a chaotropic reagent latter at normal room temperature (20-25 ° C) can be eliminated. By such treatment can be biological materials such as albumin does not interfere. Even viruses and bacteria are thus reliably eliminated.

0015As chaotropic reagent is preferably urea in a Concentration 6-8 molar, based on the total amount, or Sodium thiocyanate in a concentration from 1 to 2 molar, based on the total amount, are used. It can after treatment by dialysis, diafiltration or gel permeation chromatography be removed.

0016As a detergent, an alkyl sulfate or a derivative thereof, preferably Sodium lauryl sulfate, and / or a sarcosinate, preferably Sodium (N-lauroylsarcosinate) and / or an alkyl sulfonate or a derivative thereof can be used. The pH is one expediently by adding a diluted hydrochloric acid.

0017Preferably used in addition to the detergent 8-10 Vol .-% Methyl or ethyl alcohol to.

0018In the context of the present invention is bovine serum albumin (BSA) was chosen as an example because it in large quantities is used, the source of raw materials "beef" by BSE risk and because also the human analogues, Human serum Albumin (HSA), a very important therapeutic is. The The method can be directly without modification to the HSA recovery transfer; animal or human to other proteins Origin, it can be modified.

0019The inventive method - applied to BSA - can be summarized as follows:

0020The plasma or serum from the BSA or HSA is recovered, after dilution with water, change of pH to 6.5 and the addition of 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 touched by other analogs. It is then cooled rapidly, the pH to 4.0 to 4.2 set, the precipitated globulins are removed and the clear solution is neutralized. Thereafter, a strong chaotropic reagent in a high concentration added, preferably 6 - 8 molar urea or 1-2 molar Sodium thiocyanate, the solution 16 hours at room temperature left and then removed, the reagent, preferably by chromatography, as well as dialysis or diafiltration are possible. The desalted solution is then concentrated, optionally provided with additives, and then either sterilized and sterile bottled or dried, in particular by freeze-drying. A thermal treatment of Solution ( "pasteurization" in the presence of stabilizers) or the powder (steam injection) can be connected.

0021Instead serum or plasma, any other raw material, where albumin is to be used as Placentalblut, Fractions of plasma fractionation according to Cohn, etc.

0022Efficacy was in the biological model by adding of scrapie prions to the starting plasma and injecting the Albumin solution detected in the brains of mice, as well as by the addition of high concentrations conventional viruses such as Bovine Viral Diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), parainfluenza 3, FMD, maedi visna virus and Parapox sheep virus (ORF). The thus treated albumin solutions were autosterile, proving the destruction of bacteria.

0023Specifically as follows has been taken: 3 g of dried bovine plasma (corresponding to 2 g of protein) were dissolved in 35 ml distilled, sterile Water. 4 ml absolutiertes ethanol were added. The pH was adjusted with dilute hydrochloric acid to 6.5. 45 mg (= 0.1%) of sodium lauryl sulfate (at least 95% pure) were dissolved. In total, there were 44 mL total.

0024Among the necessary safety precautions (P 3 laboratory, Laminar Flow Hood ...) were dispersed 225 ul of 20% Scrapie brain homogenate (titer: 2:10⁹ LD₅₀/ G) was added and homogenized, or there were viruses added, are given the nature and concentration in Tab. 1. The solution was placed in a water bath at 70 ° C; by 10 min was also continued the contents 70 ° C, then the treatment was continued for 30 min. Every 10 minutes was minutes stirred 1 using a magnetic stirrer. After the end of incubation period, the sample was placed on ice and cooled. The cold solution was contacted with 200 ul of hydrochloric acid to pH 4.2 and again homogenized.

0025The removal of the precipitated globulins was carried out at 6000 revolutions per minute (corresponding to 4000 g) at 4 ° C for 10 min. The clear supernatant was recovered by decanting; he was pure bovine albumin solution, volume: 20.5 ml.

0026This solution was neutralized by adding 250 microliters of a sodium hydroxide solution (pH 7.0). Thereafter, 15 were g of urea was added, whereby the volume to 30 ml enlarged, so that a concentration of 8 molar revealed. This solution was left at room temperature (21 ° C) for 16 hours.

0027For the removal of the urea, the method of gel permeation chromatography was used:<sl><li>15 g of Sephadex G 50 (Fa. Pharmacia, Uppsala) were heated in 500 ml of sterile distilled water and Allowed to stand overnight. The swollen gel would in a acrylic column of 5 cm diameter and 30 cm height filled and washed with sterile distilled water (200 ml). , The flow rate was 7.5 ml / min.</li><li>30 ml of the above solution was applied, followed by 100 ml of water washed; the fractions were combined as follows:. 50 + 40 ml After no more protein was observed in the leachate. The 90 ml were lyophilized and yielded 0.7 g of bovine serum albumin.</li></sl>

0028Alternatively, the urea was in some experiments by diafiltration in an Amicon S-1 module, cut-off 10,000 d. Away. It was least with about 1000 ml. diafiltered water and then concentrated to about 50 ml, then lyophilized. Biological Testing for scrapie / BSE:

0029This albumin was dissolved in 3 ml of physiological saline solution and in aliquots of 20 .mu.l mice injected into each one hemisphere. Total number of mice: 136. Dieverwendeten mice were from strain C57 / B16.

0030As a positive control, a dilution series of scrapie inoculum (served as 2.10⁹ LD₅₀/ G), 8 dilution steps. As a negative control which has been, however, prepared as above without scrapie inoculum was bovine serum albumin, was.

0031All inoculations were performed on 12 animals each, ie. In 12x repetition

0032As a result, it was found that the titer in the positive control could be confirmed, while not in the negative control still in the experimental group, the disease or deaths occurred.

0033Biological testing in the case of conventional viruses:

0034The BSA was dissolved in cell culture medium. This medium was used as a culture medium for mammalian cells can serve as host cells for each virus (see Table 1). Positive (virus stock) and negative (only Medium) trials and tests on nonspecific cytopathogenicity albumin completed these tests.

0035The kill rates are given in Table 1 and were limited in all cases by the Ausgangstiter.<tables><table><tgroup cols="5"><tbody><row><entry align="left">virus</entry><entry align="left">Ausgangskonz. TC1D</entry><entry align="left">host cell</entry><entry align="left">Survivor. viruses</entry><entry align="left">Abtötrate</entry></row><row><entry align="left">BVD strain "Singer", pass.9</entry><entry align="left">1x10^8th</entry><entry align="right" /><entry align="center">0</entry><entry align="right">2.5x10⁶</entry></row><row><entry align="left">IBR strain "ames", pass.18</entry><entry align="left">1,8x10⁶</entry><entry align="right" /><entry align="center">0</entry><entry align="right">7 x 10⁴</entry></row><row><entry align="left">PI 3 strain "Freistadt" pass.78</entry><entry align="left">80 HTH units</entry><entry align="right" /><entry align="center">0</entry><entry align="right">-</entry></row><row><entry align="left">MKS O 1 BFS 1860, pass.5</entry><entry align="left">2 x 10⁷</entry><entry align="right" /><entry align="center">0</entry><entry align="right">1 x 10⁶</entry></row><row><entry align="left">MVV (ATCC VR-779)</entry><entry align="left">1.2x10⁹</entry><entry align="left">WSCP</entry><entry align="center">0</entry><entry align="right">2,9x10⁶</entry></row><row><entry align="left">ORF (Dept.of Path., Univ. Glasgow)</entry><entry align="left">2x10⁹</entry><entry align="left">PAL 6</entry><entry align="center">0</entry><entry align="right">3,4x10⁶</entry></row></tbody></tgroup></table></tables>

0036The equivalence of N-lauroyl sarcosinate with lauryl sulfate as a detergent in the preparation of albumin from Plasma was shown in a similar experiment, but without the addition of infectious material; measured Parameters were yield and purity of protein (both detergents than 98%).

0037The equivalence of urea and sodium thiocyanate as the chaotropic reagents is in the treatment of Ribonucleoprotein particles of influenza virus (a model for nucleic acid-protein interactions) and the abolition of affinity bonds such. B. at Gelatin-fibronectin, tested and proven.