HU200345B - Process for producing immunoglobin and albumin by combination of 2-ethoxy-6,9-diaminoacridine and chromatography - Google Patents

Abstract

The present invention relates to a process for the production of immunoglobulin and albumin by a combination of 2-ethoxy-6,9-dinynoacrid and chromatography. A suspension of 2-ethoxy-6,9-diaminoacridine at pH 7.2-7.8, preferably pH, is added to the plasma in desalted pH 4.7 with stirring. After reaching at least 7.5, after stirring for at least 15 minutes, the supernatant (immunoglobulin fraction) was drained from the precipitate (albumin fraction) and the pH of the precipitate or supernatant diluted with water equal to the initial plasma volume was 4.0-4.2. pH is adjusted, preferably to pH 4.1, and then 22 to 2.8, preferably 2.4% (w / v) of activated carbon is added to the initial plasma volume, and the carbon is removed from the mixture by a membrane filter known per se. The resulting 2-ethoxy-6,9-diaminoacridine-free albumin or immunoglobulin solution is purified by a known chromatographic purification method known per se. The process of the present invention significantly increases the amount of immunoglobulin that can be recovered from plasma. 200345A Scope of the description: 4 pages, Figure 1

Description

BACKGROUND OF THE INVENTION The present invention relates to a new process pharmacopoeia. Anti-D immunoglobulin, other specific immunoglobulin fractions, and albumin by the combination of 2-ethoxy-6,9-diaminoacridine and chromatography.

The industrial production of plasma proteins is still mostly carried out by the so-called Cohn et al .; Plasma Protein Fractionation, J. Am. Chem. Soc., 459 (1946). However, in addition to the technological advantages of this process, there are several disadvantages. Among other things, the risk of protein denaturation and high cooling and, in particular, that the hepatitis virus is detectable in all alcoholic fractions (Duana D. Schoeder, Milton MMozen: Australia Antigen: Distribution during Cohn's Ethanol Fractionation of Human Plasma, 1964, 168, 1462).

The discovery and production of chromatographic materials (Prath, J. and Flodín, P. Natúré 183,1959) has allowed their wider application, and in recent years several plasma fractionation methods have been developed [Curling, J. M., Berglöf, J. H., Lindquist, LO. , Erikson, S., Vox Sang. 33: 97-107 (1977); AD. Friesen, J.M.Bowman, W.C.H. Bees, 19th Congress of the International Society of Hematology and 17th Congress of the International Society for Transfusion, Budapest, 08-07-1982, Congressional Journal, pp. 117-1266], with the combined use of gel filters and ion exchangers. However, these methods could not solve the hepatitis virus clearance of the obtained plasma fractions.

In recent years, a new, so-called, antiseptic for plasma fractions has been introduced. a rivanole method has been developed [H.Geiger, Th.Kranz and Haupt, The Fate of Australia during the 13th International Congress of Plasma Fractionation of LABS, Budapest: Part A: Purification of Proteins; Develop.bioLStandard, 27. 158-165, 166-171 (Karger, Basel 1974); N. Chariatte, Hepatitis B: Au / Ha-Antigen during Production of Human Albumin and Gammaglobulin, 13th International Congress of LABS, Budapest 1973; Purification of Proteins] In these methods, albumin is separated from desalted plasma by rivanola (2-ethoxy-6,9-diaminoacridine lactate) while adjusting the pH to 7.5-8.0 with sodium hydroxide solution. . However, prior to further purification procedures, rivanol should be removed in both the albumin fraction and the gamma globulin fraction. This operation is reported in the literature to be carried out by adsorption on activated carbon above pH 6.0 (AR. Neurath, Z. Malik and C. Altner: Immune Chemistry Studio Modified by Verfabrens). 121, 240, 1961. However, this process for the removal of rivanol has a high loss of immunoglobulin, with a yield of 10-20% relative to the starting plasma. Removal of most of the rivanol can be accomplished by sodium chloride salting, but also by activated charcoal purification. , and finally, due to the high concentration of sodium chloride (3-10%), even desalination is required.

Chromatographic methods are also recommended for rivanol removal, but this method is not feasible on a commercial scale since gel-bound rivanol must be washed with formic acid Frantisek Franek: Purification of IgG Monoclonal Fluid Based on Rivanol precipitation [Matods in Enyzm, 1986]. ].

SUMMARY OF THE INVENTION The object of the present invention is to overcome the drawbacks of known methods for the decontamination of castle plasma by a process which can be carried out on a simple large scale that provides significantly higher production of recoverable protein fractions than before.

The present invention is based on the discovery that an aqueous suspension of 2-ethoxy-6,9-diaminoacridine (prepared as described in Example 1) is added to lactic acid 4.6-4.8 instead of rivanol to precipitate the plasma albumin fraction of the blood and thereby decontaminate the plasma. For pH adjusted plasma, the pH rises continuously, reaching pH 7.5-8.0, albumin precipitates The prior art methods described above for precipitation of albumin employ an aqueous solution of rivanol and adjust the pH with an aqueous solution of sodium hydroxide 7, 5 to 8.0, so that the amount of ions introduced will be significantly higher and the ionic strength will be significantly higher than in our process, which will influence the subsequent processing and probably the low production levels found in the literature. Although it is known in the literature that precipitation of albumin results in the formation of a 2-ethoxy-6,9-diaminoacridine-albumin complex [S.E. Tukachinsky and V.P.Moyseyeva: Svyazivanyi rovanola v civorotocsnimi belkami, Biochemistry 26.1 (1961); Mitterhauzerova, K. Králová and L. Krasnec, Interaction of human serum albumin with acridine and phenazine, Zvesti, 32, 124, (1978)], however, rivanol is always used to precipitate the albumin. The pH of the precipitation is also adjusted here with an aqueous solution of sodium hydroxide.

According to the literature, rivanol is removed from the precipitated albumin fraction and the fraction containing the immunoglobulins obtained after precipitation by carbon adsorption at pH 6.0 or higher. In our experiments, it was surprisingly observed that by adsorption of activated carbon at pH 4.1, the production of immunoglobulin is significantly increased compared to the starting plasma, because at this pH the 2-ethoxy-6,9-diaminoacridine used immunoglobulins and thus do not bind to activated carbon. Our results are shown in Figure 1, where, after adsorption with activated charcoal, they were centrifuged and the concentration of immunoglobulin in the supernatant was measured as a function of the pH used for the sensitization. Such a large deviation from physiological pH does not pose a risk of denaturation, since immunoglobulin formulations for intravenous use are now being marketed at this pH to avoid aggregation (R.Toldold et al., Properties and Characteristics of a New Immunoglobulin-G intravenous preparation.

-2HU 200345 A infectious Dis., Fi. Supplement 4., July-August 1986).

The present invention relates to a process for the preparation of immunoglobulin and albumin by combining 2-ethoxy-6,9-diaminoacridine with chromatography, wherein the anti-D immunoglobulin-containing plasma, normal blood plasma, fibrinogen-depleted plasma, melt-depleted plasma is deoxygenated. Sang 32.97-107 (1977), the pH of the eluate is adjusted to 4.6-4.8, preferably 4.7 with aqueous lactic acid, and a sterile distilled aqueous suspension of 2-ethoxy-6,9-diaminoacridine As described in Example 1) is added until the pH reaches 7.2-7.8, preferably 7.5, then the reaction mixture is stirred for at least another 15 minutes, preferably 30 minutes, and preferably after 20 minutes of precipitation. (Fraction L: albumin fraction), the supernatant is aspirated (II / fraction: immunoglobulin fraction) with an aqueous lactic acid solution of 4.0-4.2, preferably 4.1 The activated carbon is adjusted to 2.2-2.9%, preferably 2.4% by weight of the starting plasma, the carbon is removed on a Seitz filter or a supercentrifuge, and then sterilized on a membrane filter, preferably a 0.2 mm Millipore filter. The filtrate is further purified by any of the ion exchange purification methods known in the art (Vox Sang 33.97, 077 (1977)) (e.g., 5.0%, above 99% purity as in Example 5) and lyophilized. The removal of 2-ethoxy-6,9-diaminoacridine from fraction 1b-1bumin is the same as that of Π. The filtrate obtained after active carbonation according to the method described in Fraction II is further processed by any of the ion exchange purification methods described in the literature to a purity of above 20.0% and greater than 99% (e.g. as in Example 4).

The main advantages of the process according to the invention are the following:

the. ), the production of the virus-free plasma fraction increases from 10-20% in the literature to 30-40%;

b. ) the process is feasible on a large scale;

c. ) the quality of the plasma fractions obtained after purification is in accordance with the requirements of the pharmacopoeia.

The following examples illustrate the process according to the invention

Example 1

Preparation of 2-eiPXi-6.9-thiamine-Q-acridia l-Dilylation * In sterile pyrogen-free distilled water at 70 ° C, 70 g of rivanole (2-ethoxy-6,9-diamino-acridine-lactate) is dissolved in 1000 ml of sterile pyrogen-free distilled water. After cooling, 170 ml of 1 M sodium hydroxide are added under constant stirring, and the reaction mixture is stirred for 30 minutes. The precipitate is filtered off and washed with sterile distilled water to make it alkaline. The precipitated 2-ethoxy-6,9-diamino acrylide precipitate was suspended in sterile pyrogen-free distilled water equal to its wet weight at room temperature.

Example 2

Saline depletion of plasma and precipitation of fraction I (albumin) was determined by the addition of Sephadex G-25 Coarse, pH 1.0.0, Siemens Conductivity 0.02 M Sodium Lactate, pH 4.0 4.0 [Pharmazia Cat. No. 17-0034-01 ( 02)] desalted on a Sephamatic CF-6 (Pharmacia Cat. No. KS 370) packed with a gel filter. Desalting is performed in 12-13 liter cycles. Set to 7 and, while stirring, add a distilled aqueous suspension of 2-ethoxy-6,9-diaminoacridine (prepared as in Example 1) to pH 7.5 (1300 mL). After reaching pH 15, stirring was continued for another 30 minutes and the mixture was allowed to stand for 20 minutes and the supernatant (fraction II immunoglobulin fraction) was aspirated from the precipitate (fraction I albumin fraction).

Example 3

I. and g obtained according to Example 2. Fraction 2-ethoxy-6,9-diaminoacridine

The pH of the precipitate diluted with sterile pyrogen-free distilled water (fraction I albumin fraction) equal to the volume of the starting plasma (25 liters) and the supernatant obtained according to example 2 (fraction imm immunoglobulin fraction) were obtained according to Example 2 with constant stirring. With 1 mol of lactic acid pH

Adjust to 4.1 and continue stirring, adding 600 g of activated carbon, which adsorbs the 2-ethoxy-6,9-diaminoacridine on a Seitz filter, or centrifuges the supernatant through a supercentrifuge and passes the supernatant through a 0.2 nm Millipore filter, the resulting 2-ethoxy-6,9-diaminoacridine-free albumin or immunoglobulin fraction is then worked up in known manner, preferably by chromatographic purification, into the appropriate albumin, or immunoglobulin formulations (see Examples 4 and 5).

Example 4

Chromatographic purification of the 2-ethoxy-6.9-diaminoacrylidinium-free albumin fraction (fraction I) obtained in Example 3 (Seq. 30/87, entitled "Procedure for the preparation of a yellow stable human albumin solution from human plasma"). )

To the 2-ethoxy-6,9-diaminoacridine-free albumin solution (fraction I) obtained in Example 3 was added 1 mol of sodium caprate with constant stirring.

5.2 Up to pH value. After stirring for 30 minutes, the supernatant was centrifuged, the supernatant was filtered through a 0.2 nm Millipore filter, and the conductivity of the filtrate was adjusted to 1.6 ml Siemens, pH 5.2 with 1 M sodium hydroxide and pH 5.2 by addition of distilled water. onto a 1.6 mL Siemens conductive 0.02 M sodium acetate buffer equilibrated with DEAR Sepharose FF (Pharmacia formulation) anion exchange resin column (16 L). Elution is carried out in three steps with the following buffer vessels; 1) 0.02

13GB 200345 Molar Siemens Conductivity pH 5.2 Molar Sodium Acetate: 20 liters, 2) 200 L 0.025 M pH 2.0 Million Siemens Conductivity Sodium Acetate, 3) 150 L 0.125 M pH 4.8 -8,8 million Siemens conductive sodium acetate. Elution with buffer 2 yields an albumin fraction. The albumin fraction was then loaded on a 16 liter CMSepharose FF (Pharmacia formulation) cation exchange resin column equilibrated with 1.8 ml Siemens conductive sodium acetate buffer, pH 4.5, 0.025 M, eluted in 3 steps with 1/172 liter 0.025 1.8 M Siemens Conductive Sodium Acetate, pH 4.5, 2/175 liters 0.11 M pH 6.0 Million Siemens Conductive Acetate, 3) 75 L 0.4 M pH 8.2 - 23 million Siemens conductivity and sodium acetate. The eluate obtained with buffer 2 contains the albumin fraction. Its pH was adjusted to pH 6.5 with a 1 M aqueous solution of sodium hydroxide and then transferred to a gel filter Sephacryl S-200 (Pharmacia formulation) equilibrated with 0.05 M sodium chloride with 0.3 M pH 4.0 Condensate Acetate Buffer. elute, and concentrate the albumin fraction by ultrafiltration to a 20% Protein content, then adjust the sodium ion content to 130 mmol / l by adding 1 M hydrochloric acid or 1 M aqueous sodium hydroxide and adjusting to 7.0. sodium caprate solution was added to a final concentration of 0.04 M sodium caprate, finally the solution was sterile filtered and bottled, and the bottled albumin solution was pasteurized for 10 hours at 60 ° C. A pale yellow albumen composition with a viral content of 20% and a purity of greater than 99% is obtained. The product is stable and no precipitation occurs after standing.

Example 5

Lyophilization of the 2-ethoxy-6,9-diaminoacridine-free immunoglobulin fraction (fraction Π) obtained in Example 3 To the 2-ethoxy-6,9-diaminoacridine-free immunoglobulin solution obtained in Example 3 (fraction Π) is of type KS 370. Anion exchanger DEARG (Pharmacia formulation, composition: DEAE Sepharose Fás Flow: 60% Arginine Sepharose 4B: 40%) in 0.02 M (Pharmacia) equilibrated with 0.01 M pH 5.6 Sodium Conductive Buffer resin (16 L) and flow the solution into a cation exchange resin (KL 370) equilibrated in CM-Sepharose FF (Pharmacia) 0.02 M pH 5.6, 1.4 M Siemens Conductive Acetate Buffer equilibrated with KS 370 let's put it on. The column was washed with 20 L of 0.02 M sodium acetate followed by 30 L of 0.01 M glycine and the immunoglobulin was eluted with pH 9.0 buffer containing 0.15 M glycine and 0.1 M sodium hydroxide. The eluted immunoglobulin fraction was adjusted to pH 7.0 with 1 M aqueous hydrochloric acid and then concentrated by ultrafiltration to a protein content of 5.0% and then sterilized by filtration. The resulting immunoglobulin solution was stored at -25 ° C until ampouling. The immunoglobulin composition is lyophilized according to Ph.Hg. VII.

Claims (1)

Process for the preparation of immunoglobulin and albumin by combining 2-ethoxy-6,9-diaminoacridine and chromatography from plasma by desalting the plasma by gel filtration according to a known method, characterized in that the pH of the desalted plasma is constantly adjusted to pH 4.6-4.8 with aqueous lactic acid solution. is preferably adjusted to pH 4.7 and a suspension of 2-ethoxy, 9-diaminoacridine is added with stirring to pH 7.2-7.8, preferably pH 7.5, preferably for at least 15 minutes. After 20 minutes, the supernatant is aspirated off and the pH of the supernatant diluted with water equal to the initial plasma volume and the supernatant is adjusted to pH 4.0-4.2, preferably pH 4.1 lactic acid solution, followed by stirring at 2.2-2.8, preferably 2.4% w / v of the starting plasma volume. RFoG% active carbon was added thereto, and the carbon removed from the mixture and the resulting solution is filtered sterile membrane filter known per se manner, to give 2-ethoxy-6,9-diaminoakridinmentes albumin or immunoglobulin solution is purified to conventional chromatographic purification procedure.