FI61191B - FOERFARANDE FOER UTVINNING AV IMMUNOGLOBULIN FOER INTRAVENOES ADMINISTRATION - Google Patents

Description

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C Patent myonne-Lty ID 06 1902 ^ Patent meddelat (51) kv.ik.Vcl3 c 07 G 7/00, A 61 K 39/395 FINLAND — FINLAND (21) Patent application - PateotaneBknlnf 771071 (22) HtkumtopWvl— Anettknlnfedag 05.0¾ . 77 '' (23) AlkupUvt — GlkighMsdtg 05.0k.J7 (41) Tullut (outdoor - BlWIt oBentlig 07.10.77

Patent and Registration Office .... , ,,,, " . (44) Date of issue of the contract. -

Patent and registration authorities Antokin utiigd och uti.akriften pubUcend 26.02.82 (32) (33) (31) Pyydutty »tuolkuu * —Begird priori! * Q6.0U. ^ 6

Denmark-Danmark (DK) 1628/76 (71) Nordisk Insulinlabatorium, Ved Stadion 2, DK-2820 Gentofte, Denmark-Danmark (DK) (72) Jurgen Frederik Hansen, R ^ Sdovre, Denmark-Danmark (DK) (7M Berggren The present invention relates to a method for the recovery of an intravenous immunoglobulin having very little or no anti-complementary activity. in steps, blood plasma, serum or an immunoglobulin-containing fraction thereof with a precipitant consisting of polyethylene glycol having a molecular weight between 2000 and 6000.

Immunoglobulins, which also comprise gamma globulins, have therapeutic significance because they can be used to prepare immunizing preparations.

Immunoglobulin is present in the blood plasma of animal origin, from which it can be recovered by various precipitation and purification processes. Thus, gamma globulin can be obtained as concentrated solutions by the method developed by Cohn et al. (J. Clin. Invest. Chem. Soc. 68, 479-75 (1946)). According to Cohn, fraction II, 61191, which is a 16 ', 5% concentrate and can be injected intramuscularly can be obtained.

In said known method, gamma globulin molecular aggregates are formed, which makes the preparations unsuitable for intravenous administration. Cohn's method involves precipitation with alcohol, which appears to displace water and can cause irreversible denaturation, making the globulin anticomplementary. Attempts have been made to avoid this by using special separation processes and / or by chemical modification of gamma globulin.

Thus, an improved fractional precipitation process is known in which gamma globulin is precipitated from plasma using polyethylene glycol (PEG) as a precipitant (U.S. Patent 3,415,804). This avoids harmful denaturation, but the purity of the precipitated product is not satisfactory.

According to Danish Patent Publication 135,270, this precipitation process can be improved by using certain types of block polymers of ethylene oxide and polyoxypropylene polymer instead of polyethylene glycol and by maintaining certain precipitation conditions.

In this way, it is possible to improve the yield in gamma globulin recovery. However, in all cases the doped products do not have a satisfactory purity and a sufficiently low anticomplementary activity.

The present invention is based on the finding that the precipitation of immunoglobulins, also called gamma globulins, in successive steps with a precipitant consisting of polyethylene glycol having a molecular weight between 2000 and 6000 can be made more specific if during at least one additional polyethylene glycol precipitation Alkanoic acid containing -9 carbon atoms while removing fibrinogen and lipoproteins.

3,61191

The method according to the invention provides a surprisingly pure immunoglobulin in a simple manner, with a high yield. The yield is as high as possible by the best methods mentioned above.

The process shown uses polyethylene glycol / having a molecular weight ranging from 2000 to 6000, preferably from 4000 to 6000.

Of the alkanoic acids having 6 to 9 carbon atoms, caprylic acid is most preferred. Examples of other suitable alkanoic acids are caproic acid and nonanoic acid. Branched alkanoic acids can also be used.

The immunoglobulin-containing solution is preferably mixed with 1 to 8% by weight of polyethylene glycol and 0.1 to 5% by weight of caprylic acid or another alkanoic acid having 6 to 9 carbon atoms. The precipitation step, in which both polyethylene glycol and alkanoic acid are used as precipitant, is carried out according to the invention at a pH of between 3 and 7, preferably between 4.5 and 5.5.

The method according to the invention is described in more detail below by means of some examples.

Example 1 415 ml of human blood plasma containing 10 g of immunoglobulin per liter of plasma is adjusted to pH 5.0. Compound PEG 3000 is added to a concentration of 6% and the solution is allowed to stand for 45 minutes. The precipitate, which consists mainly of fibrinogen, is separated by centrifugation and the pH of the liquid phase is adjusted to 7.0. After additional PEG 3000 has been added to a concentration of up to 12%, the mixture is allowed to stand for 45 minutes. The mixture is centrifuged and the liquid phase containing albumin and α- and β-globulin is removed. The precipitate formed by the crude immunoglobulin is dissolved in 0.9% sodium chloride solution to achieve a protein content of about 4%. The solution is filtered clear with a Seitz EK filter. To the resulting solution 4,61191 are added 5 g of caprylic acid and 30 g of PEG per 3000 liters, and the pH of the solution is adjusted to 4.9. After standing for 2 hours at 20 ° C, the mixture is centrifuged and the liquid phase is filtered to record the solution. The pH of the solution is adjusted to 7.0 and PEG 3000 is added until the PEG concentration is 12%. This precipitates pure immunoglobulin, which is recovered by centrifugation and drying. The yield is 60%. In the purity assay performed by DISC PAGE (using 5% polyacrylamide), only two bands corresponding to the immunoglobulins IgG and IgM are present. The analysis was performed on a 500 microgram product batch.

In the Grabar & Williams immunoelectrophoresis experiment using a rabbit antibody against total human serum protein, there were essentially only three peaks corresponding to IgG, IgM, and IgA.

If desired, the purified product can be converted to an intravenous injectable by dissolving it in 0.9% NaCl solution until the protein content of the solution is about 5%. The preparation is characterized by very low anticomplementary activity and is therefore very suitable for intravenous administration.

Example 2 100 l of serum are mixed with 100 liters of a solution containing 600 g / l of PEG 3000. The pH of the mixture is adjusted to 6.5. The precipitate is filtered off and dissolved in 100 liters of distilled water to which 0.015 M NaCl has been added. To the solution are added 40 g of PEG per 3000 liters and 5 g of caprylic acid per liter. The pH of the solution is adjusted to 5.0 and allowed to stand for 45 minutes, after which the precipitate is separated by centrifugation. To the solution remaining in the centrifugation, a further 40 g of PEG per 3000 liters are added and the pH is adjusted to 7.5. The precipitate is separated by centrifugation and dissolved in distilled water containing 0.002 M NaCl. The solution is filtered clear (for example with a Seitz® EK filter).

5,61191

The protein content must be between 50 and 100 g / l. To purify the solution, 20 g of DEAE-Sephadex-A25 per 100 g of protein are added. After standing for 1 hour under agitation, the DEAE-Sephadex is separated by filtration. The cleaning treatment with DEAE-Sephadex is repeated. After sterile filtration, an immunoglobulin G preparation is obtained which has no anticomplementary activity and is suitable for use in intravenous administration.

Comparative Test

In the comparative experiment, an immunoglobulin preparation prepared according to Example 2 and an immunoglobulin preparation prepared by the method described in Example 2 were used, with the difference that caprylic acid was omitted. This latter method corresponds to the method disclosed in U.S. Patent No. 3,763,135.

The anticomplementary content of the two preparations was determined by the method described in J. Immunology 89, 336 (1962), according to which the preparations were diluted until anticomplementary activity could just be detected. The anticomplementary content of the preparation prepared according to Example 2 was so small that it could not be detected in the undiluted preparation. The anticomplementary content of the preparation prepared without caprylic acid could just be detected at a dilution ratio of 1:10. The content of the immunoglobulin preparations precipitated by the Cohn method was so high that it corresponded to a dilution ratio of about 1: 100.