FR2746030A1 - Elimination of unconventional transmissible agents - Google Patents

Abstract

Unconventional transmissible agents are removed fromaqueous protein solutions by simultaneous electrostatic adsorption chromatography and hydrophobic chromatography, and recovering the filtrate.

Description

PROCESS FOR ELIMINATING NON-COMMUNICABLE AGENTS
CONVENTIONAL OF AN ALBUMIN SOLUTION
The invention relates to the field of elimination of non-conventional transmissible agents (ATNC). More particularly, the invention relates to a method for removing ATNC from an albumin solution.

 Unconventional transmissible agents (CNTA), among which prions are the cause of transmissible subacute spongiform encephalopathies which are slow degenerative diseases of the central nervous system, the evolution of which is always fatal and whose causative agents are not always identified.

These transmissible spongiform subacute encephalopathies concern both humans and animals: o in humans: Kuru, Creutzfelt-Jakob disease, Gerstmann syndrome
Straussler-Scheinker, fatal familial insomnia and, for some, Alpers disease.

 o in animals: scrapie in sheep, transmissible mink encephalopathy, chronic wasting syndrome in wild ruminants in captivity and in freedom, and bovine spongiform encephalopathy.

 When albumin is extracted from biological material, whether human or animal, and this albumin is intended for therapeutic use, it is essential to treat it in order to eliminate the non-conventional transmissible agents which would eventually be there.

We know from the article "Viral Validation of the manufacturing process of high purity albumin from placentas" published in Brown F (ed): Virological Safety Aspects of
Plasma Derivatives Dev. Bio Stand. Basel, Karger, 1993, vol 81, pp. 237-244, a process for the purification of albumin from human placentas, presenting all the guarantees of safety vis-à-vis possible viral contaminations. Likewise, as mentioned in the article "Safety ofplacental blood derivatives" published in Lancet, Vol 343. January 15, 1994, this process was considered particularly suitable for the elimination of prions. Indeed, the purification steps carried out in this process include in particular three steps FC 1, FC2 and Hcol of alcoholic precipitation in acid medium associated with filtrations. Since prions tend to coprecipitate with denatured proteins, these three stages, which are characterized by
Elimination of large quantities of protein precipitates are particularly suitable for the elimination of prions.

 However, this method, if it is well suited for the purification of placental albumin, is not at all suitable for the purification of albumin of plasma origin.

The precipitation step FC i, for example, is a step whose conditions of production are such that the hemoglobin itself is denatured and precipitated in a specific manner; in the case of plasma, the amount of hemoglobin is very low, this step would therefore not have the same characteristics and in particular the albumin itself would be exposed to denaturation by the chemical agent. It is therefore desirable to be able to have a method for removing unconventional transmissible agents from an albumin solution, even without carrying out these precipitation steps.

 To this end, the invention provides a method for removing the ATNC from an albumin solution, characterized in that it consists in treating the solution by hydrophobic and electrostatic adsorption chromatography in 2 stages, one stage being carried out Acid pH, The other step being carried out at substantially neutral pH.

 According to a particular characteristic, the step at acid pH is carried out before the step at substantially neutral pH.

 The acid pH stage in fact performs most of the purification, the neutral pH stage completing it.

 According to a particular embodiment of the invention, the albumin solution is treated beforehand by anion exchange chromatography.

 Thus, the albumin solution has a very high degree of purity.

 According to another characteristic, the method according to the invention also consists in first filtering the albumin solution, by means of a filter whose cut-off threshold is substantially equal to 0.2 μm.

 Thus, the removal efficiency of the NCTAs is further increased.

 The present invention will be better understood on reading the detailed description which follows.

 The albumin solution for which it is necessary to eliminate the ATNCs which may be present can have various origins. It is however preferable, in order to submit it to the process according to the invention, that its degree of purity, expressed as a percentage of albumin relative to all of the proteins, that is to say approximately 98% or more.

 It may in particular be a solution of albumin of plasma origin obtained by any suitable method, and in particular by a method including the COHN fractionation technique.

 According to the invention, the albumin solution is passed successively through 2 columns of adsorption chromatography at 2 different pH: the passage in one of the columns being carried out at an acidic pH and the passage in the other column to a Substantially neutral pH. The gel used can be the same for the 2 columns. It has functional groups, such as reticulates of diethylaminopolystyrene, of diethylaminopolyphenyl or any other association of radicals carrying a positive charge and hydrophobic molecules, linked to an inert matrix (silica, cellulose, sepharose, acrylamine ...). We use for example for each of the 2 columns of the DEA Spherodex / LS supplied by BIOSEPRA (US). In this case, the impurities are retained by hydrophobic and electrostatic adsorption on the columns, while the albumin crosses the column without being fixed.

 According to their isoelectric point, the ATNCs are retained either during the chromatography at acid pH, or during the chromatography at substantially neutral pH.

 Among the other fixed impurities, there may be mentioned pigments, degraded hemoglobin, fatty acids.

 Surprisingly, although albumin has, like prions, a tendency to adsorb all that is hydrophobic, it has been observed that by carrying out these 2 steps of hydrophobic and electrostatic adsorption chromatography, we were able to separate albumin ATNC.

 According to a particular mode, the chromatography at acidic pH is carried out prior to the chromatography at neutral pH.

 For safety, an additional step of non-specific adsorption chromatography is also carried out using for example a silica column. It is thus possible to eliminate the elements which may have been released during the preceding steps.

 Advantageously, it is possible to use the same columns several times, which makes it possible to implement this process on an industrial level.

 The albumin solution obtained at the end of this process is not only more than 99% pure as regards its albumin content, but also completely devoid of contaminants such as prions or ATNC, as could be be demonstrated in animal tests.

 It can be directly concentrated to the desired commercial concentration.

 According to a particular embodiment of the invention, the albumin solution is prepurified by anion exchange chromatography, in particular by passage through a column loaded with diethylaminoethyl groups grafted onto an inert matrix (dextran-silica, cellulose, Sepharose, acrylamide, etc.). .); for example, a column of DEAE Spherodex / LSB supplied by BIOSEPRA is used which is balanced beforehand by a buffer so that the electrical charge on the surface is positive. For example, a sodium phosphate buffer of pH 6.8 is used.

 It is thus possible to start from an albumin solution constituted by fraction V obtained by the alcoholic fractionation of the COFIN method or of derived methods and the degree of purity of which is approximately 95%.

 When the albumin solution is injected through the column, Albumin is fixed, while certain impurities and alcohol possibly present in the solution (especially when it is obtained by fractionation of COHN) are not fixed. After rinsing with an alcoholic solution and then with water, the albumin is eluted by increasing the ionic strength, for example during the passage through the column of an aqueous solution of sodium chloride. The albumin solution obtained is then approximately 98% pure or more.

 Before even passing through the chromatography column, the albumin solution is preferably filtered using a filter whose cutoff threshold is 0.2 μm.

When this filter is located at the upper entry of the chromatography column, the conditions are such that the aggregation of hydrophobic particles already makes it possible to eliminate a certain number of the ATNCs possibly present in the solution.

Example 1
We have 150 ml of a solution of albumin of placental origin partially purified according to the 4 precipitation steps and the dilution step described on p: 239 of the article "Viral Validation of the Manufacturing Process of High Purity Albumin From
Placentas "mentioned above.

 This solution is a hydroethanolic albumin solution containing 50 g / l of albumin.

This solution is deliberately contaminated with 2% (v / v) of an inoculum containing NCTAs. This inoculum is obtained by grinding the brains of animals (C 57BL / 6 male mice) infected with the scrapie agent of the mouse (strain C 506 / M3 in the 7th passage provided by Drs. DC Gajdusek and P. Broun,
NIH Bethesda USA, which has an infectious titer of around 108 LD50 per gram of brain) and has reached the terminal stage of the disease. The ground material is made in a sterile 5% glucose solution, at a weight / volume ratio of 10%. The inoculum is sonicated, then clarified by centrifugation and filtered on a 0.45 μm filter before use.

 The contaminated albumin solution is filtered through a 0.2 ptm filter and then treated successively by DEAE-Spherodex / LS chromatography, then DEA-Spherosil / LSB at pH 4.8 and finally DEA-Spherosil / LS (3) at pH 6, 8.

 The 1st column or DEAE-Spherodex / LS (g) containing 30 g of gel is first of all equilibrated with monosodium phosphate buffer at pH 6.8.

 The albumin solution is then injected into the column. Albumin, negatively charged, is gradually fixed on the support by ionic interaction. Alcohol and protein impurities which are electrically neutral or which are positively charged are directly eliminated.

 The column is then rinsed with a 12.5% alcoholic solution and then with purified water.

The albumin is then eluted from the column by passing a 10 g / l NaCl solution. The column is then regenerated using a 60 g / l solution of
NaCI then sterilized by an acid regeneration phase by washing with HCl 0.1 N; the column is then rinsed in a 70% alcoholic solution and stored in this solution.

 The albumin saline solution obtained is then subjected to chromatography on a DEA Spherosil / LSs column containing 15 g of gel at pH 4.8. This chromatography combines electrostatic adsorption thanks to DEA groups and hydrophobic adsorption thanks to polystyrene reticulates. The support of the column is beforehand balanced with a NaCl solution at 0.7 g / l. When the albumin solution passes through this column, certain hydrophobic impurities are fixed while the albumin remains in solution.

 The column is then rinsed by passing a NaCl solution at 4 g / l.

 The column is then regenerated by washing with 0.1 N HCl, then washed with a 70% alcoholic solution and stored in alcohol.

 The albumin solution obtained at the outlet of this column is then subjected to a new chromatography step on an identical support, but this time at pH 6.8. The column here contains 3 g of gel and is associated with a column containing 3 g of silica. This step makes it possible to adsorb hydrophobic impurities, which are not fixed at the pH at which the previous step is carried out; it also makes it possible to eliminate possible molecules of DEAE - dextran originating from the 1st chromatography.

The 2 columns are then subjected to a first rinsing with a solution of
NaCl at 6 g / l in order to recover the albumin which may be retained.

 Then the columns are subjected to a second rinsing with a concentrated NaCl solution at 60 g / l in order to remove any traces of denatured albumin.

 The columns are then regenerated by washing with 0.1 N HCl, then they are sterilized by washing with a 70% alcoholic solution and they are stored in alcohol.

 A solution of pure albumin, then concentrated to 7.6 ml, is obtained at the outlet of the silica column.

 In order to verify the presence of ATNC, a test is carried out on approximately 200 healthy mice C 57BL / 6 males of 18-20 grams, each of which is inoculated with 20 μl of albumin solution by intracerebral route in the right hemisphere. . The first 24 mice receive concentrated albumin solution. Then, the albumin solution is diluted 10 to 10 before being injected, each dilution being injected into 12 mice.

 The animals are kept under observation in order to verify the signs of the appearance of the experimental scrapie of the mouse. A mouse is considered positive after the second positive evaluation of a clinical sign. When a mouse dies, its brain is frozen at -80 "C and a histopathological examination is carried out to confirm the cause of death.

 The results obtained show that, thanks to the method according to the invention, the reduction in the infectious titre of the starting albumin solution expressed in LD50 (which is determined by the method of Reed and Muench for dilutions carried out from 10 to 10 of the sample tested), is 10 5ç5, a difference in titer of 5.5 log.

Example 2
After carrying out Example 1, 150 ml of an albumin solution at 50 g / l identical to that of Example 1 are available, except for the voluntary contamination with an inoculum containing ATNC; this albumin solution is passed through the same chromatography steps as those described in Example 1, in order to verify that the
ATNC adsorbed by the chromatography columns during Example 1, are not released into the new albumin solution. This time, 6.6 ml of punctured solution is obtained.

 The same test is therefore carried out as in Example 1, that is to say intracerebral inoculation, to each animal, of 20 μl of the albumin solution thus obtained, then of the albumin solution diluted by 10 in 10. About 100 mice are thus inoculated.

 The results obtained show that the mice are not contaminated; the albumin solution is therefore free from any ATNC.

 It is therefore possible to use the chromatography columns several times.

Example 3
The same experiment is carried out as in Example 1, this time starting with an albumin solution constituted by fraction V of the COHN technique and filtered on a 0.45 μm filter.

 The albumin solution then obtained has the same characteristics as that obtained in Example 1.

Claims (6)

1. Method for eliminating non-conventional transmissible agents (ATNC) from a  albumin solution, characterized in that it consists in treating the solution with  2-step hydrophobic and electrostatic adsorption chromatography, one step  being carried out at acidic pH, the other step being carried out at substantially neutral pH. 2. Method according to claim 1, characterized in that the step at acidic pH is carried out  before the step at substantially neutral pH. 3. Method according to one of the preceding claims, characterized in that the  chromatography is carried out using a support having diethylaminopolystyrene groups. 4. Method according to one of the preceding claims, characterized in that it consists of  in addition to pre-treating the albumin solution by exchange chromatography  anions. 5. Method according to claim 4, characterized in that the chromatography  ion exchange is carried out by means of a support having diethylaminoethyl groups. 6. Method according to one of the preceding claims, characterized in that it consists of  in addition to filtering the albumin solution beforehand, by means of a filter whose threshold  cutoff is substantially equal to 0.2 pm.