The method of obtaining intravenous immunoglobulin

 

(57) Abstract:

The invention relates to biotechnology. The method includes the ultrafiltration or dialysis products immunoglobulin with subsequent thermal treatment at 30-58°C for at least 2 hours and not more than 45 days and maintain at all stages of the process, the electrical resistance of the immunoglobulin solution is not less than 0.1 Moms. The method allows to obtain the product BB IG with AKA more than 10 mg/SN that do not contain aggregates. 3 table.

The invention relates to biotechnology, in particular to the production of medical biological preparations, and can be used to obtain immunoglobulins (IG), intended for intravenous (IV).

The main quality indicators CENTURIES IG is low anticomplementary activity (AKA), the absence of aggregates, the higher content of monomers and stability of these indicators during storage.

There are many methods of obtaining intravenous immunoglobulins.

The highest clinical tolerability and low anticomplementary activity have IG obtained by enzymatic treatment with pepsin (1), trypsin, plasmin (2). But the splitting of the functions.

Chemical modification of IG toxic b-propiolactone (3), sulfanilamide agents leads to antigenic effect of IG (4).

The use of surface-active substances remaining in the finished product, nefiziologichnoe (5).

CC IG obtained by ion-exchange chromatography (6), by treatment with polyethylene glycol (7), are characterized by low output and disturbed spectrum distribution of subclasses of antibodies in comparison with the original plasma (8).

Remove aggregates and reduced anticomplementary by ultrafiltration (9) through the membrane of about 1 MDA has not yet found industrial application because of ethnological and instability during storage.

Above the IG have a neutral pH of the final preparation that contributes to the appearance of aggregates during storage.

Currently the first in the world in terms of sales CENTURIES IG (10) are firm Cutter (USA), which produces immunoglobulin with a pH of 3.9 and 4.4, and the company Green Cross/alpha (Japan), offering to sell IG with a pH of 5.5. The pH from 3.9 to 5.5 is far from the isoelectric point of the IG, 5.8-7,3, so the molecules have a high charge and the product is stable.

A method of producing a drug with a pH of 5.5 (11) predusmotren and subsequent ion-exchange chromatography. The method is quite time-consuming, has low output. The only proposed stabilizer - sorbitol is a natural metabolite of the human body, which can lead to the disruption of the acid - alkaline balance.

The closest technical solution to the proposed method of obtaining CENTURIES IG is the method (12), comprising the following process stages. IG diluted or concentrated to a protein concentration of 0.5 to 20.0% at the temperature of 0 - 20oC. Adjusted the pH to 3.5 to 5.0 with hydrochloric acid. Reduce the ionic strength of the solution is less than 0.001 M by dialysis or diafiltration against distilled water, which corresponds to the reference and the calculated data to the electrical resistance of the solution BB IG equal 0,008-0,05 MOsm (13). Enter stabilizers, pH adjusted to 3.5-5.0 and the final concentration of the protein to 5-15%. In the resulting drug content of the monomers is more than 90%, and AKA more than 4 mg/2CH5O (examples 6 mg/2CH5O).

This method allows to get the native IG containing antibodies, consistent with their original contents in plasma, is characterized by low cost, lack of use of technology for hazardous and toxic substances, high yield. Positive moment australiasia finished product is accompanied by the appearance of the aggregate even in freshly prepared drug (14).

However anticomplementary activity CC IG obtained by a known method, exceeded the required FS 42-3159-95 (15) more than 2 times, and during storage was observed the appearance of aggregates. When testing of the drug in the test for accelerated aging by heating IG, with 57oC within 24 hours appeared more than 5-10% of the aggregated molecules (11), which is unacceptable for CENTURIES IG. According to the authors (12), the drug was stable for 6 months under normal storage life of such drugs from 2 to 3 years.

The problem to which the invention is directed, is to develop a method of obtaining drug CC IG, low anticomplementary activity and storage stability.

The technical result consists in obtaining medication CENTURIES IG with AKA more than 10 mg/2CH5O that do not contain aggregates.

The essence of the method is that the method includes the ultrafiltration or dialysis products immunoglobulin with subsequent thermal treatment at 30-58oC for at least 2 hours and not more than 45 days and maintain at all stages of the process, the electrical resistance of the solution u is not less than 0.1 Moms.

Unlike the prototype, claimed the processing. In addition, in this way at all stages of the process to maintain the electrical resistance of the solution u at the level not less than 0.1 Moms.

The set of distinctive features of the proposed method allowed us to obtain the following advantages. Heat treatment at acidic pH was significantly reduced AKA to allow clinical use - more than 10 mg/2CH5O, because when the temperature is stronger hydration of the molecules of the IG (PL. 1). In addition, temperature effects in the absence of ions in solution (high resistance solution) leads to inactivation or inhibition of the proteases of the IG, which prevents fragmentation of the drug during storage (table. 2). In turn, maintaining a high electrical resistance of the solution of the IG ensures its stability (table. 2, 3).

Preparations of intravenous immunoglobulin was evaluated by the following methods.

Molecular parameters were determined on Sephadex G200 (16,17), anticomplementary activity - in vitro method (18). Test thermal stability was carried out by heating the IG, with 57oC for 24 h (19). This test is considered as an analogue ur>C at 80 horizontal oscillations per minute. This bottle made of borosilicate glass type 1 with a capacity of 50 ml was aseptically filled with 20 ml of a sterile solution of IG with a concentration of 5%. This test can be considered as similar transportation conditions to the consumer.

The results are shown in table. 1-3. As can be seen from the presented data (table. 1), using the proposed method allows to obtain a product with a lower AKA, lack of units, the minimum content of dimers is less than 5%, a high content of monomers. The stability of these indicators are presented in the test for thermal stability (table. 2) test and mixing (PL. 3).

The method is as follows.

The raw material used immunoglobulin with electrophoretic purity not less than 99%, obtained by any known method. Bring the pH from 3.9 to 4.4 hydrochloric acid. Dialysis or diafiltration against water with a resistance of 10.0-18.2 Mω/cm, and concentrate the solution to a protein content of 0.5 to 20.0%. Then carry out a thermal treatment of the solution of the IG, maintaining at the temperature 30-58oC for at least 2 hours and not more than 45 days. The resulting solution of immunoglobulin which has been sterilizing filtration. At all stages of the process support the electrical resistance of the solution u is not less than 0.1 Moms.

Examples

Example 1. Raw filtrate III, obtained by the method of Cohn 6. 100 l of filtrate III, a protein content of 0.3% was adjusted to pH of 4.0 with 0.1 N HCl solution (20 l). The resulting solution was diluted to 200 l of water for injection with resistance 17 Moms and spend concentration to 3% protein and diafiltration against 8 volumes above water at a temperature of 4oC on the cartridge with a membrane YM (100000 Da) cellulose (firm Amicon). Then the concentration up to 7% protein. Enter maltose up to 30% and perform thermal processing in a water bath at a temperature of 52oC for 4 h Then perform dialysis against the above-mentioned water. In the resulting solution is injected glucose to 5%, glycine to 0.75%, set pH to 4.0,% protein, 5%, and subjected to sterilizing filtration. Get 5 liters of 5% solution for intravenous immunoglobulin with an electric resistance of 1.2 Moms, AKA 13 mg/2CH5O and the following molecular parameters units 0, dimers - 2,3, monomers - 96,4, fragments of 1.3%.

Example 2. Raw sludge II obtained by the method of Cohn 6. Precipitate II in the amount of 1 kg was dissolved in 1.7 l of 1% solution of glycine containing 0.45% of Natalino tube from acetylcellulose against 200 volumes of water resistance 18 MOsm at a temperature of 8oC. in a day otvetsvennyy solution with a pH of 4.1 sterile filtered and kept in a thermostat at a temperature of 37oC for 14 days. In the resulting solution is injected maltose up to 10%, set pH to 4.0,% protein -5% and subjected to sterilizing filtration. Get solution intravenous immunoglobulin volume of 5.0 l with an electric resistance of 2 Mω/cm, AKA 12 mg/2CH5O and the following molecular parameters - units Of the dimers is 2.1, the monomers 95,9, fragments -2,0%.

Example 3. Raw materials - the eluate obtained by ion-exchange chromatography on DEAE-Sephadex And 50 from the precipitate II at Stake (21), electrophoretic purity of 100% and a protein concentration of 0.6% in the volume of 10 L. the immunoglobulin Solution down to pH of 4.0 1N hydrochloric acid, concentrated by ultrafiltration to 7% protein and subjected to diafiltration against 7 volumes of water resistance 18 MOsm. Thermal processing and further operations are performed according to example 1. Get 5% solution of intravenous immunoglobulin in volume 1.2 l with electric resistance 3 Moms, AKA 15 mg/2CH5O and the following molecular parameters units 0, dimers of 2.5, the monomer - 97,0, fragments -0,5%.

Sources of information

1) Patent of Russian Federation N 2068695. The method of obtaining the enriched intravenous immunoglobulin. C 07 G 7/00. Publ. 21.01.1981.

3) U.S. Patent N 3916026. Method of obtaining intravenous immunoglobulin. A 61 K 37/00. Publ. 28. 10. 1975.

4) Stephan W. Einfurhung immunspezifischer Determinanten in Serumalbumin durch Imidoacylierung. Zeitschrift fur Immunitatsforschung 1967, 133, 153-157.

5) Patents PCT (WO) N 84/00890. The allocation of intravenous immunoglobulin. A 61 K 35/14. Publ. 15.03.1984.

6) Patent PCT/US83/01016. Method of obtaining intravenous immunoglobulin. A 61 K 39/395. Publ. 07.09.1982.

7) German Patent N 2751717. Intravenous immunoglobulin. A 61 K 39/395. Publ. 20. 10. 1980.

8) Beck O. E., Kaiser, P. E. Distribution of human lgG Subclasses in Commercial Intravenous Immunoglobulin Preparations. Vox Sang. 1981, 41, 79-84.

9) U.S. Patent N 5219999. Immunoglobulin G and the process of its products. C 07 K 3/12. Publ. 15.06.1993.

10) Faure A. Human blood Fractionation. 8th International Biotechnology Symposium, Paris 1988, 669-678.

11) Patent EP N 0278422 A2. Injections of immunoglobulin with a pH of 5.5. A 61 K 39/395. Publ. 17.08.1988.

12) U.S. Patent N 4499073. Vnutrepenialnymi serum immunoglobulin. A 23 J 1/06. Publ. 12.02.1985. Or similar Patent EPB N 0073371 B1. A 61 K 39/395. Publ. 09.03.1983.

13) Rabinovich C. A., Havin 3. I. Brief chemical reference: Ref, ed. Ed. by A. A. Potekhin, A. I. Efimov, 3rd ed. L.: Chemistry, 1991, S. 338.

14) Mateytschuk P., More J. Factors affecting the production of intra who nutrivene introduction.

16) guidelines define aggregates and fragments immunoglobulin products by gel-filtration. THE USSR MINISTRY OF HEALTH, 1986, 1-11.

17) FS 42-WS-90 Physico-chemical, chemical, physical and immunochemical methods for the control of medical immunobiological preparations.

18) guidelines. Definition anticomplementary activity of preparations of immunoglobulins. RSFSR MINISTRY OF HEALTH. Bitter 1988,1-11.

19) Patent JP N 2-32261. The stabilizer of serum immunoglobulin. A 61 K 47/26. Publ. 19.07.1990.

20) the Patent EP-A N 448075. Intravenous immunoglobulin. A 61 K 39/395. Publ. 06.03.1991.

21) Fractionation of sediment fraction II+III using an ion exchanger. In kN. C. M. Rusanov, A. I. Skobelev. Fractionation of plasma proteins in the manufacture of blood products. Moscow. "Medicine", 1983, 100-101.

The method of obtaining intravenous immunoglobulin, including ultrafiltration or dialysis products immunoglobulin at a pH of 3.9 and 4.4, the introduction of stabilizers and final sterilizing filtration, characterized in that after stage ultrafiltration or dialysis carry out thermal treatment at 30 - 58oWith not less than 2 hours and not more than 45 days, at all stages of the technology is

 

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