KR860002096B1 - Process for preparing immuno-globulin suitable for intravenous injection - Google Patents

Abstract

Ig for i.v. injection is prepd. by acid treatment of plasma or fractions obtained from plasma by Cohn's cold alc. fraction method and followed by treating the fractions with an alkylene oxide polymer or copolymer having a mol. wt. of 2000 - 20000. Thus, Cohn's fractions II and III were dissolved in aq. NaCl, the pH was adjusted to 3.8 with 1N HCl, and PEG was added. The pH was increased gradually to 5.0 and the ppt. centrifuged. The recovery of IgG in the supernatant was 85% and the purity was 97%. The supernatant was mixed with more PEG, the pH was adjusted to 8.5 and the ppt. was recovered by centrifugation. The ppt. was dissolved in 0.02 M acetate buffer (pH=6.6) contg. 1% human albumin. The ultimate yield of IgG was 84%.

Description

Method for preparing immunoglobulin suitable for intravenous injection

The present invention relates to a method of making an immunoglobulin suitable for intravenous injection.

Because immunoglobulins have antibody activity against various pathogens, they are administered to patients deficient in various antibodies for protection from therapeutic diseases or for therapeutic purposes. Immunoglobulin preparations have been developed in two forms, one for intravenous injection and the other for intramuscular injection, both of which are widely used clinically.

Known methods of preparing intravenous dosage forms include treating crude immunoglobulins with proteases such as pepsin and plasmin. Fractionation of Plasma or Cohn's Plasma Fractions Using Chemical Changes in Immunoglobulins by Acylation and Other Chemical Methods, Light Polyethyleneglycol, Pulnix (Polyoxyethylene-Polyoxypropylene Copolymer), etc. There is a method such as using.

In the intravenous immunoglobulin obtained by the pepsin treatment method, 7S IgG is modified to F (ab ') ₂ by pepsin digestion, which is disadvantageous because it is very short lived in vivo. In the plasmin treatment method, although 60 to 70% of the immunoglobulin remains in the form of normal globulin, the residual amount is converted to a low molecular substance which reduces the in vivo lifetime of the product as in the case of pepsin treatment. In the case of chemical modifications, safety issues arise when modified immunoglobulins must be administered repeatedly, so there is a possibility of developing new antigenicity depending on the type of modifying group and other conditions.

Fractionation by polyethylene glycol or pluronics is generally believed to be the most preferred method for recovering the forms present in vivo, i.e., unmodified and undigested immunoglobulins. The method is already described in detail in Paulson and Cobal (Japanese Patent Application “close to” (published) No. 46, 814/1975, 91, 321/1976 and 20, 415/1978). However, if polyethylene glycol or pluronics fractionation is carried out under known conditions, an immunoglobulin preparation containing no aggregated immunoglobulin and suitable for intravenous injection can be obtained, but the yield is always low. Therefore, there is a strong demand for improving the yield.

The present inventors conducted a study to determine the cause of the decrease in the yield, and the results showed that the non-aggregated immunoglobulin was also simultaneously removed in the step of adding polyethylene glycol or pluronic at low concentration to live immunoglobulin to remove the aggregated immunoglobulin. I found the facts. It has also been found that globurin materials containing relatively large amounts of aggregated immunoglobulins are fractionated into polyethyleneglycol or pluronic and the loss of non-aggregated immunoglobulins is large.

The present inventors also endeavored to solve the new technical problems associated with the above-mentioned difficulties inherent in the prior art, resulting in the acid treatment of live immunoglobulins to separate aggregated immunoglobulins, followed by By fractionation with glycol or polyoxyethylene-polyoxypropylene copolymers, it has been found that the yield of non-aggregated immunoglobulins is significantly improved. Based on this finding, the present invention has been accomplished.

An object of the present invention is to provide a high yield of immunoglobulin suitable for intravenous injection from fractions I + II + III, fractions II + III, fractions II or III of corns obtained by fractionating plasma or plasma with cold alcohol of corn. It is to provide a method of manufacturing.

Other objects and advantages of the present invention will become apparent from the following description.

According to the present invention, fractions I + II + III, fractions II + III, fractions II, or fractions III of the cones obtained by cold alcohol fractionation of the plasma alcohol cones, or plasma, at 4 ° to 15 ° C. for 30 to 180 minutes. treated with acid at pH 3. 2-5. 0, and the resulting material was prepared with an alkylene oxide polymer or copolymer having a molecular weight of 2000-200,000 at pH 4. 6-5. To 5% (W / V), to remove precipitated aggregated immunoglobulins and to recover precipitated non-aggregated immunoglobulins that actually contain non-aggregated immunoglobulins, pH 8 From 0 to 9. 0, there is provided a method for preparing an immunoglobulin suitable for intravenous injection, characterized in that the above-mentioned polymerization or the copolymer is added again at a concentration of 6 to 13% (W / V).

Plasma used as a starting material for the present invention is preferably derived from human blood in view of the problem of antigenicity. Plasma fractions I + II + III, II + III and III of cones are, in fact, α-, β-, and VIII-globulin (IhG, IgA, IgM). Plasma shock-globulin fractions are obtained by continuing to immerse with cold ethanol, as described in detail in Journal of Clinical Investigations, 23, 417 (1944) and American Chemistry, 68, 479 (1946).

Acid treatment is carried out by maintaining the starting material under acidic conditions. By this treatment, the aggregated immunoglobulin in the raw material is first separated from the non-aggregated type. The acid conditions are pH 3. 2 to 5. 0, preferably 3. 8 to 4. 2, and the ionic strength is 0.02 to 0.30, preferably 0.1 to 0.20. If the pH is 3.2 or less, protein denaturation occurs, and if it is 5. 0 or more, the above-described separation action of the aggregated immunoglobulin is insufficient, so other pH conditions are not preferable. The concentration of the protein is not limited, but 2 to 10% (W / V) is preferred because of its ease of manipulation. The temperature of the acid treatment is 4 ° to 15 ° C. If the temperature is 4 ° C. or lower, the above-described separation action is insufficient, and if the temperature is higher than 15 ° C., decomposition of the protein occurs, so that other temperatures are not preferable. The acid treatment time is 30 to 180 minutes. If the treatment time is shorter than 30 minutes, the above-mentioned separation is insufficient, and if the treatment time is too long, time is wasted, and may even lead to undesirable results. Acids used in this treatment include hydrochloric acid, inorganic acids such as phosphoric acid and organic acids such as acetic acid and citric acid.

After the acid treatment as described above, in order to prepare a high-purity, non-aggregated immunoglobulin in high yield, fractionation is performed using an alkylene oxide polymer or copolymer having a molecular weight of 2000 to 20, 000. The alkylene group of the alkylene oxide polymer used for fractionation is a group having 1 to 4 carbon atoms such as methylene, ethylene, propylene or butylene groups. Alkylene oxide copolymers include copolymers of two or more alkylene oxides, such as polyoxaethylene-polyoxypropylene copolymers.

The inventors belong to a copolymer of alkylene oxide having a molecular weight of 2000 to 20, 000.

In the fractionation using an alkylene oxide polymer or copolymer having a molecular weight of 2000 to 20, 000, the present inventors found that pH conditions are a very important factor, and the yield and purity of immunoglobulins are 4. 6 to 5. 4, It has been found that the remarkable improvement is preferably only in a very limited range of 4. 8 to 5. 2. This also dissolves the powder II + III paste in a 0.6% aqueous sodium chloride solution at a protein concentration of 5%, the resulting solution is treated with an acid for 60 minutes at pH 3. 5, and the solution is subjected to 4. 0 to 6. It is evident from the experimental results (Table 1) obtained by fractionation with polyethylene glycol (5% concentration) at varied pH in the 0 range and measuring the yield and purity of non-aggregated immunoglobulins.

TABLE 1

PH condition of polyethylene glycol fractionation

When polyoxyethylene-polyoxypropylene copolymers having an average molecular weight of 15, 000 are used instead of polyethylene glycol, higher yields and higher purity, non-aggregated immunoglobulins are obtained at the pH conditions described above than at other pH conditions.

The polymer or copolymer is added to the starting material at a concentration of 4.5 to 5. 5%, preferably 5% (W / V), and the precipitated impurities, i.e., the aggregated immunoglobulins, are centrifuged (1, 000 to 5,000 rpm). To the fractionated supernatant is added the above polymer or copolymer at a concentration of 6 to 13%. By adjusting the pH from 8. 0 to 9. 0, the desired non-aggregated immunoglobulin is precipitated and can be recovered by conventional methods such as centrifugation (1000-5000 rpm). The recovery of the target compound under the above conditions is 60% or more. The precipitate formed is re-dissolved, for example, in 0.02 M acetate buffer solution or physiological saline mixed with 0.6% sodium chloride, 2% mannite and 1% albumin, and the resulting solution is suitable for clinical use. To obtain an immunoglobulin solution, it is passed through a bacterial strainer. Immunoglobulin in this solution does not change its properties even when the solution is in a vial and lyophilized. Thus, if the product is to be stored for a long time, it can be made into a lyophilized formulation.

The immunoglobulins prepared according to the present invention contain substantially non-aggregated immunoglobulins, and when analyzed with 5% concentration solution, the anti-complement activity is 20 units or less, and the purity of IgG is 90% or more.

The present invention will be described in more detail with reference to the examples but is not intended to limit the invention.

In the examples, the recovery of immunoglobulin is measured by single immune diffusion and electrophoresis using cellulose acetate membrane. Anti-complement activity was determined by the Kovar and Mayer method [experimental immunochemistry, p225 (1961)] and the Nishi-oka and Odakan method [immunobiochemistry, p. 103 (1971), Kyoritsu Publishing Co., Ltd.].

Example 1

The fraction II + III paste (1 kg) of cones obtained by cold alcohol fractionation is dissolved in 10 L of 0.6% aqueous sodium chloride solution. The pH of the solution was adjusted to 3. 8 with 1N hydrochloric acid and stirred at 4 ° C. for 60 minutes to effect acid treatment. 500 g of polyethylene glycol (average molecular weight 4,000) is added to the solution. While dissolving polyethylene glycol, the pH of the solution is gradually increased using 1N aqueous sodium hydroxide solution. As soon as the pH reaches 5.0, the precipitate is removed by centrifugation (4,000 rpm) to give a clear supernatant. Based on fraction II + III, the recovery of IgG in the supernatant is 85% and the purity of IgG is 97%. To the supernatant, 500 g of polyethylene glycol (molecular weight 4,000) is added. While stirring gently, adjust the pH of the supernatant to 8. 5 with 1N sodium hydroxide solution. The immunoglobulin precipitated under the above conditions is recovered by centrifugation (4,000 rpm). The total recovered precipitate is dissolved in 0.02M acetate buffer containing 1% human albumin (pH 6. 6) and the concentration is adjusted to 5% using the same solvent. The solution is sterilized by passing through a Millipore filter (Millipore Inc.) and aseptically dispersed in a small vane. Half of the dispersion is lyophilized immediately to obtain a drying aid.

The final yield of IgG from the starting material was 84%, whereas the starting material was pH3. 8 is 63% excluding acid treatment. Except for adding more albumin, the purity is 95%. The anticomplement activity (protein concentration 5%) of the liquid and lyophilized preparations is 14 and 16, respectively.

The 5% solution is administered to 5 mice weighing about 20 g body weight in an amount of 1 ml / horse. No abnormalities were observed in weight loss and hair growth during the one week observation period. The lyophilizer was tested after 1 year storage at 4 ° C., but no change in solubility and anticomplement activity was observed compared to the initial preparation.

Example 2

Fraction II paste (500 g) of cones obtained by cold methanol fractionation is dissolved in 10 l of 0.1% sodium chloride solution. Immunoglobulins that do not contain aggregates are recovered in a similar manner as in Example 1. The yield of IgG is 80% compared to 62% when the acid treatment (pH 3. 8) is omitted. Purity is 97%.

In a manner similar to Example 1, the immunoglobulin obtained above was dissolved in a 0.5% sodium chloride solution at a protein concentration of 5%. After 1% mannite is added, the solution is lyophilized through a bacterial filter. The lyophilizer is dissolved in distilled water for injection at a protein concentration of 5%. The anticomplement activity of this concentration was found to be 13.

Example 3

The procedure of Example 1 is repeated except that an equivalent amount of polyoxyethylene-polyoxypropylene copolymer (average molecular weight 15,000) is used instead of polyethylene glycol (average molecular weight 4,000). Immunoglobulins are recovered with the same results as in Example 1.

Claims (6)

Plasma was obtained by fractionation of cold alcohol from corn, and fractions I + II + III, fractions II + III, fractions II or III of corn, or plasma, at pH 3. 2 to 5. 0 and 4 ° to 15 ° C and 30 ° C. To acid for 180 to 180 minutes, and the resulting material was subjected to an alkylene oxide polymer or copolymer having a molecular weight of 2,000 to 20, 000 at a pH of 4. 6 to 5. 4, from 4.5 to 5. 5% (W / V) to remove the precipitated aggregated immunoglobulin and recover the precipitation of non-aggregated immunoglobulin that does not actually contain aggregated immunoglobulin, at pH 8. 0 to 9. 0, Method for producing an immunoglobulin suitable for intravenous injection, characterized in that the polymer or copolymer is added again at a concentration of 6 to 13% (W / V). The method of claim 1 wherein the protein concentration of the acid treatment is between 2 and 10% (W / V). The method of claim 1 wherein the acid is hydrochloric acid, phosphoric acid, acetic acid, citric acid. The method of claim 1, wherein the plasma is derived from human blood. The method of claim 1 wherein the alkylene oxide polymer has methylene, ethylene, propylene or butylene groups. The method of claim 1 wherein the alkylene oxide copolymer is a polyoxyethylene-polyoxypropylene copolymer.