JPS6221359B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immunoglobulin suitable for intravenous injection.

Immunoglobulins have antibodies against many pathogens, and are therefore used to prevent or treat infections in patients with various antibody deficiencies.

Intravenous and intramuscular preparations of immunoglobulin have been developed and are already widely used clinically.

Methods for producing intravenous preparations include treating crude immunoglobulin with proteolytic enzymes such as pepsin and plasmin, chemically modifying immunoglobulin by acylation, and using polyethylene glycols such as plasma and Cohn plasma fractions. Fractionation methods such as Pluronik are known.

Intravenous immunoglobulin obtained by the pepsin treatment method can be obtained by pepsin digestion.
7SIgG changes to F(ab)' ₂ , which has a disadvantage in that it has an extremely short half-life in the body compared to normal immunoglobulin.

In the plasmin treatment method, 60-70% of the immunoglobulin is left in the state of normal immunoglobulin, but the rest is digested by plasmin and reduced to a low molecular weight, and like the pepsin mentioned above, this immunoglobulin is not absorbed in the body. has a short half-life.

In the chemical modification method, new antigenicity may appear depending on the modifying group and other conditions, which poses a safety problem when repeatedly administered.

Fractionation using polyethylene glycol or Pluronic is considered to be the most preferable method for recovering immunoglobulins in the same state as they exist in vivo, that is, unmodified and undegraded immunoglobulins.

Detailed reports on the fractionation method have already been made by Bolson et al.
46814, 51-91321 and 53-20415). However, when performing polyethylene glycol fractionation or Pluronic fractionation under the conditions of these known methods,
Although it is possible to obtain immunoglobulin that does not contain polymerized immunoglobulin, that is, immunoglobulin that is suitable for intravenous injection, the yield is low, and therefore, improvement in this point has been desired.

The inventors investigated the cause of this low yield and found that non-polymerized immunoglobulin was also removed at the same time as polymerized immunoglobulin was removed when polyethylene glycol was added at a low concentration. I found it.

Furthermore, we have found that when similar polyethylene glycol fractionation is performed on an immunoglobulin containing a relatively large amount of polymerized immunoglobulin, the loss of non-polymerized immunoglobulin also increases.

Therefore, the present inventors have carried out research in order to solve the above-mentioned shortcomings inherent in the conventional technology, that is, new technical problems, and to improve the yield of non-polymerized immunoglobulin. As a result, we found for the first time that by dissociating polymerized immunoglobulin into non-polymerized immunoglobulin by acid treatment and then performing the above-mentioned fractionation, the recovery rate of non-polymerized immunoglobulin was significantly improved. Heading: The present invention has been completed. In other words, the present invention was completed as a result of identifying the cause of the low yield in the prior art and conducting various further studies based on this finding, and is novel not only in its structure but also in its technical problem. be.

The present invention relates to a fraction obtained by subjecting plasma or plasma to Cohn's cold alcohol fractionation method.
, fraction +, fraction or fraction (hereinafter also collectively referred to as starting materials) is treated with acid at pH 3.2 to 4.2, and then subjected to fractionation with polyethylene glycol having a molecular weight of 2,000 to 20,000 for intravenous injection. This is a method for producing immunoglobulin that can be used for.

The plasma used as a starting material in the present invention is preferably derived from humans due to antigenicity issues. In addition, Cohn plasma fraction ++, +
, and are essentially alpha, beta and gamma globulins (IgG, IgA,
IgM). The gamma globulin fraction of plasma was obtained by serial precipitation with cold ethanol and was published in Journal of Clinical Investigation, 23 , 417, 1944 and Journal of Clinical Investigation, 23, 417, 1944;
The American Chemical Society, 68 ,
479, detailed in 1946.

Acid treatment is carried out by keeping the starting material under acidic conditions. The PH conditions when performing acid treatment are PH
The pH is 3.2-4.2, preferably 3.8-4.2, and the ionic strength is 0.002-0.30, preferably 0.10-0.20. Further, the protein concentration at this time does not need to be particularly regulated, but is preferably 2 to 10 w/v% from the viewpoint of ease of operation. The concentration in acid treatment is 3° to 20°C, preferably 4° to 15°C, and the time for acid treatment is
The duration is 30 to 180 minutes, and anything longer than this is uneconomical in terms of time and may even have the opposite effect.

Examples of acids used in the acid treatment include inorganic acids such as hydrochloric acid and organic acids such as acetic acid.

After such acid treatment, the molecular weight is 2000-20000.
By performing fractionation using polyethylene glycol, non-polymerized immunoglobulin can be obtained in high yield and purity.

By the way, the present inventors found that in the fractionation using polyethylene glycol with a molecular weight of 2000 to 20000,
Setting the PH condition is extremely important, and PH4.6~
Very limited PH of 5.4, preferably PH4.8-5.2
We found that only under these conditions did immunoglobulin recovery and purity become significantly higher. For example, using a solution in which fraction + paste is dissolved in a 0.6% sodium chloride solution to a protein concentration of 5% and acid-treated at pH 3.5 and temperature 110°C for 60 minutes, This is clear from the experimental results (Table 1) in which the yield and purity of non-polymerized immunoglobulin were investigated when polyethylene glycol fractionation was carried out at a pH between 4.0 and 7.0.

Add polyethylene glycol to 10-15% of the supernatant thus fractionated and adjust the pH to 7-8 to precipitate the desired non-polymerized immunoglobulin. Therefore, it can be recovered, for example, by centrifugation.

The precipitate obtained can be dissolved, for example, in physiological saline solution or
0.6% sodium chloride in 0.02M acetate buffer, 2
By redissolving the product in a solution containing 1% mannitol and 1% albumin, and performing sterilization filtration, it can be made into an intravenous immunoglobulin that can be used clinically.
Furthermore, even if this solution is dispensed in small quantities and freeze-dried, no change in properties is observed before and after freeze-drying, so it can also be made into a freeze-dried preparation if long-term storage is desired.

The immunoglobulin produced by the method of the present invention does not substantially contain polymerized immunoglobulin, has an anti-complement activity of 20 units or less when measured with a 5% protein concentration solution, and has a purity of 90% or more as IgG. show.

Table 1 PH conditions during polyethylene glycol fractionation PH IgG recovery rate IgG purity 4.1 60% 35% 4.3 58% 60% 5.0 70% 98% 5.5 45% 95% 6.0 10% 93% Making the features of the present invention easier to understand For this purpose, the present invention will be explained using examples, but the present invention is not limited thereto.

The recovery rate of immunoglobulin in the present invention was determined by one-way immunodiffusion method, and the purity thereof was determined by cellulose acetate membrane electrophoresis method.

Anti-complement titers were determined by the method of Kabat and Mayer [Experimental, Immunochemistry, 225,
(1961)] and the method of Nishioka and Okada [Biochemistry of Immunology, 103, 1972 (Kyoritsu Shuppan)].

Example 1 1 kg of fraction + paste obtained by Cohn's cold alcohol fractionation method was added to 0.6% sodium chloride 10
Dissolve in water, adjust pH to 3.8 with 1N-HCl, and cool at 4°C.
Acid treatment was performed by stirring for 60 minutes.

Add 500g of polyethylene glycol (average molecular weight 4000) to this solution and dissolve it while stirring 1N-
NaOH gradually increases the PH, and eventually the PH
Adjusted to 5.0. Immediately after reaching pH 5.0, the precipitate was removed by centrifugation to obtain a clear supernatant.

The amount of IgG recovered in this supernatant is fraction + 100.
The purity was 97%.
Furthermore, 700 g of polyethylene glycol (average molecular weight 4000) was added to this supernatant, and the pH was adjusted to 8.0 with 1N-NaOH while stirring gently.

Immunoglobulin precipitated under these conditions was collected by centrifugation.

The total amount of the collected precipitate was added to 0.6% sodium chloride,
Contains 2% mannitrate, 1% human albumin
It was dissolved in 0.02M acetate buffer (PH6.6), and the protein concentration was adjusted to 5% with the same solvent.

After sterile filtration using a Millipore filter (Millipore), the mixture was aseptically dispensed into small containers. Half of the dispensed amount was immediately freeze-dried to obtain a dry preparation.

Recovery of IgG from final starting material was 84%
(63% when the starting material was not acid-treated (PH3.8) as a control), and the purity was 95% excluding added albumin. In addition, for liquid preparations and lyophilized preparations, protein concentration 5
When anti-complement titers were measured in %, they were 14 and 14, respectively.
It was 16.

After administering 1 ml of the 5% solution to five mice (approximately 20 g) through the tail vein, the animals were observed for one week, and no abnormalities such as weight loss or piloerection were observed.

The lyophilized preparation was re-examined after being stored at 4°C for 1 year, but no changes were observed in solubility, anti-complement value, etc. compared to the time of manufacture.

Example 2 500 g of fractionated paste obtained by Cohn's cold ethanol fractionation method was dissolved in 0.1% sodium chloride 10, and immunoglobulin free of polymerized immunoglobulin was recovered in the same manner as in Example 1.

The recovery rate as IgG was 80% (62% when the starting material was not acid-treated (PH3.8) as a control)
The purity was 97%.

In the same manner as in Example 1, the protein was dissolved in a 0.5% sodium chloride solution to a protein concentration of 5%, and 1% mannitol was added thereto, followed by sterilization filtration and freeze-drying.

The lyophilized product was dissolved in distilled water for injection to a protein concentration of 5%, and the anti-complement titer at this concentration was determined to be 13.

Claims (1)

[Claims] 1 Plasma or a fraction ++, fraction +, fraction or fraction obtained by subjecting plasma to Cohn's cold alcohol fractionation method, after acid treatment at a pH of 3.2 to 4.2. , a method for producing an immunoglobulin usable for intravenous injection, which comprises fractionating with polyethylene glycol having a molecular weight of 2,000 to 20,000. 2. The method for producing immunoglobulin according to claim 1, wherein the pH conditions in the acid treatment are PH3.2 to 4.2, the temperature conditions are 4°C to 15°C, and the treatment time is 30 to 180 minutes. 3 Fractionation using polyethylene glycol with a molecular weight of 2000 to 20000 is carried out at pH 4.6 to 5.4, and the polymer is
After adding the polymer at a concentration of ~5 w/v% and removing the polymerized immunoglobulin, the polymer was further added at pH 7 to 8 to a concentration of 10 to 15 w/v% to form a polymerized immunoglobulin. The method for producing immunoglobulin according to claim 1, which is carried out by recovering immunoglobulin substantially free of immunoglobulin. 4 Fraction ++, fraction +, fraction or fraction obtained by subjecting plasma or plasma to Cohn's cold alcohol fractionation method, after acid treatment at a pH of 3.2 to 4.2, to a pH of 4.6 to 5.4 Add polyethylene glycol with a molecular weight of 2,000 to 20,000 to a concentration of 3 to 5 w/v%, collect the supernatant, and then add the polymer at a pH of 7 to 8 to a concentration of 10 to 15 w/v%. 2. The method for producing immunoglobulin according to claim 1, which comprises collecting a precipitate.