JPS6016406B2 - Method for producing intravenously administrable gamma globulin and gamma globulin prepared thereby - Google Patents

Description

[Detailed description of the invention] The present invention relates to gamma globulins.

The present invention relates to gamma globulin preparations and methods of making the same, particularly for administration by intravenous injection. Gamma globulin or immunoglobulin G (1gG) obtained from mixed blood counts contains antibodies against many infectious agents.

Immunoglobulins have efficacy in the clinical management of a wide variety of disease states. Immune globulins are used to prevent or treat infections in patients with various antibody deficiencies. For patients with normal immunoglobulin levels, immunoglobulins are used to treat viral infections such as hepatitis, measles, rubella, chickenpox, mumps, yellow fever, rabies, herpes, smallpox, diphtheria, and pertussis. It is also used to prevent bacterial infections such as tetanus and Rh incompatibility, and to treat severe infections caused by antibiotic-resistant bacteria (sepsis caused by Staphylococcus and Escherichia coli, sepsis caused by Pseudomonas aeruginosa).
The full clinical potential of immunoglobulins is
It has not yet been completely clarified. Human immunoglobulins first became isolated on a large scale during World War II.

It was soon observed that these preparations produced shock reactions in patients when administered intravenously, and it was subsequently established that the anticomplement activity of 1 and the preparations was responsible for the shock reactions. . This anti-complement activity is based on the aggregation of 1gG molecules that occurs during the fractionation process. In view of these shock reactions associated with intravenous administration of immunoglobulins, these therapeutically useful substances were administered intramuscularly instead of intravenously.

However, intramuscular administration of immunoglobulin has many limitations as described below. a) it is painful, b) there is a limit to the amount that can be administered, c) the proteolytic effects that occur at the injection site attenuate the amount of 1 gG administered, and d) the maximum concentration in the blood is not reached until 3 or 4 days after administration. What not to do.

This presents a significant obstacle for cases requiring high levels of 1 or concentration immediately after administration. In contrast, intravenous administration of immunoglobulin is more widespread because it is not subject to degradation at the injection site, the entire dose immediately enters the bloodstream, and significantly higher blood concentrations are obtained. It has clinical applications.

This idea has prompted the development of a method for producing 1gG with low anti-complement activity suitable for intravenous administration. Methods developed to date are based on proteolytic or chemical treatments to reduce the anti-complement effect of aggregated molecules. Examples of preparations obtained by these manufacturing methods are as follows. 1. Pepsin-treated immunoglobulin In this preparation, proteins are degraded into fragments of antibodies ($, F(ab')2).

Because it is cleared (from the blood) over a short period of time (3q hours compared to 20-30 days for unchanged 1gG), the efficacy of this preparation in combating bacterial infections is limited. . After binding to the antigen, the $ fraction does not immobilize the barrel.
This preparation is not applicable for prophylactic purposes. 2 Plasmin-treated immunization.

Purines In this formulation, more than 60% of the glopurins are degraded to Fab and Fc fragments.

Remaining globulin has a normal half-life (3-4 weeks)
However, the antibody spectrum is limited. 3. pH4-treated globulin This formulation tends to develop anti-complementary effects during storage.

Its suitability is therefore limited and it cannot be administered in large quantities. The half-life is slightly decreased (12-14 days),
Antibacterial activity was weakened to the extent that it could not be measured. 4
8-Propiolactone-treated globulin molecules may undergo major changes, giving rise to new antigenic properties.

Half-life is approximately 10 days. Bacteriolytic activity is reduced. 1
The four subtypes of gC have different susceptibilities to proteolytic effects.

Therefore, the pepsin, plasmin and pH 4 (with pepsin) treated formulations are very different from the untreated 1gG with respect to the amount of distribution of each 1gG subtype. As mentioned above, the undesirable anti-complement effect responsible for the shock response caused by intravenous administration of 1gG is due to the aggregation of molecules in 1gG, which are generated during the fractionation process.

The above-mentioned preparations are obtained using a method in which the aggregates are decomposed mainly by chemical and enzymatic action. However, this kind of decomposition process
This will inevitably lead to decomposition of 1 and 2 with a loss of activity. As a result, formulations such as those described above do not retain the expected activity. To date, little has been done to develop methods to prevent the formation of aggregates and create 1g0 formulations that are essentially anti-complementary. Very recently 1974-6-6
A method for preparing gamma globulin preparations suitable for intravenous administration was published in West German Published Patent No. 2,357,80. Like other published gamma globulin preparation methods, this method relies on the fact that it uses a relatively pure gamma globulin fraction as a raw material. However, a very important point is that the gamma globulin obtained by the patented method still has too much anti-complementary action for intravenous administration. A method for obtaining a substance suitable for intravenous administration from fraction 1 has also been proposed (US Pat. No. 3,763,135). However, the method according to the invention yields much higher yields containing much less anticomplementary active substance. Gamma for intramuscular injection
There are FDA standards for globulin, but there are no such standards for gamma globulin for static methods.

Similar criteria are needed to clearly distinguish between gamma globulins that cause a shock-like response when administered intravenously to susceptible humans and those that do not. In the past 1g main period,
It has been established that at sufficiently low levels of gamma globulin anti-complement activity, no clinical symptoms are observed, even in highly susceptible patients.

StandardNbyer 2 unit as
say ( ExperimentallmmumXh
emjsthi, E. A. Kabi t, M. M. Mayer
Co-authored 2nd edition, P133 Thomas, Springie
ld company, 1961), the anti-complement active substance per unit of immunoglobulin G is 0 as a safe level.
．． 04 to 0.02 units or less, but 0.0
It may be slightly higher than 4. However, at a level of 0.8 units per female, the reaction is always observed. Emphasizing the absence of clinical side effects, the suitability of gamma globulin preparations for topical use depends on whether anti-complement activity is at a specifically low level. Furthermore, in order to provide clinically safe and effective preparations, it is necessary to maintain physiological antibody activity and specificity. The patented method maintains the properties of the normal gamma globulin molecule and yields a formulation that is essentially free of molecular aggregation and consequent anti-complement activity, thus making it safe and effective for topical use. I've been thinking about it.

Accordingly, the objects of the present invention are: '1' To provide a gamma globulin preparation for intravenous injection; and '2) An intravenous gamma globulin preparation that essentially has no anti-complement activity in vitro. 4) Incorporating a gamma globulin preparation with a biological half-life of 3 to 4 weeks, which has the ability to immobilize the cartilage body when combined with the corresponding antigen. In comparing the types of antibodies present in the starting material mixed dish bran and the standard gamma globulin obtained by Cohn's classical blood bran ethanol fractionation method, a static antibody with an essentially unchanged antibody spectrum was found. To provide injectable gamma globulin preparations, ■ To specify methods for preparing intravenous gamma globulin preparations, and ■ To specify methods for preparing intravenous gamma globulin preparations from readily available dish protein fractions. [71] To define a method for preparing intravenous gamma globulin preparations in such a way that essentially no aggregation of the molecules occurs.

The present invention provides active gamma globulin suitable for intravenous administration,
A method for producing this gamma globulin and a stable formulation containing this gamma globulin are provided.

According to one of the production methods of the present invention, this gamma globulin is produced by J. Am. Chem. Soc. Spots. 459-475, (
It can be easily obtained from the fraction 00m by the method of Cohn et al. (1946).

This fraction, which contains almost all the immunoglobulins along with other hybrid proteins, can be treated with the fractionation technique of the present invention to prevent the formation of molecular aggregates that would occur during the fractionation process using conventional techniques. to produce active gamma globulin suitable for intravenous administration with essentially no anti-complement activity. Another useful raw material source is Fraction D, which is readily available as human serum globulin.

This product is economical, stable as a freeze-dried powder, and does not contain hepatitis viruses. This can be processed in the same way as the fraction 00m. The manufacturing method of the present invention consists of the following steps.

Dish protein fractionation day + m Preferred embodiments of the invention will be described below.

Blood bran protein fraction 0 or D+m Beist is approximately 4.8
PH of about 6.5, preferably about 5.5 to 5.9
Extract with water. Protein amount is about 25%, 3%
About 25 to 45, preferably 30,000 pyrogen-free water is used per 0% Beist k9. Non-toxic, pharmaceutically acceptable organic or inorganic acids such as harsh acids, lactic acid, hydrochloric acid, sulfuric acid, etc. are conveniently used to adjust the pH. Substances that do not fall into water are separated, and the filtrate is made of polyethylene glycol, sequentially 4W/V%, 5W/V% and 1
It is fractionally precipitated at a concentration of 2W/V%. The pH at the 12 W/V% concentration step is approximately 8.0.

The first two fractional precipitations remove impurities, and the final precipitation provides the gamma globulin sought in the present invention. The preferred polyethylene glycol molecular weight is about 4,000 to 6,000. A non-toxic, pharmaceutically acceptable salt of an organic or inorganic acid that lowers the pH to approximately 80
Used to adjust. The operation is approximately oo~2
It may be carried out at a temperature of 0°C, but lower temperatures of 00 to 5°C are preferred. Details of the operation are described in Examples 1 and 2.

These examples are intended to be illustrative and not limiting. Example 1 30 m of dish bran protein fraction LK9 with a protein content of 25-30% is suspended in pyrogen-free distilled water and stirred until a homogeneous yellow suspension is obtained. .

The temperature is maintained at 5°C. Add 20' of 10% harsh acid per 90m Beist lk9 to lower the pH to 5.8. After praying for an additional 15 minutes, the precipitate is allowed to settle for 2 to 3 hours. After that, the gunwale. The supernatant is clarified through a clarifier using paper such as a 9 pad. USP polyethylene glycol (PEG) having an average molecular weight of 4,000 moles per mole is added in powder or flake form to a concentration of 4 moles per 100 moles of solution.

After coagulating until PEG is completely dissolved, the precipitate formed is
Wait for 2 hours. The gunwale. Collect the results by filtration using a 9-pad or Millipore membrane. Subsequently, the PEG concentration is increased to 5 W/V%.

The PEG is dissolved by stirring and the solution is allowed to stand for 1 to 2 hours. Thereafter, the supernatant was redistributed to M. 9. Filter with asbestos pad. The pH is then adjusted to 8 by adding 6% tris hydroxyethylaminomethane (THAM).

Gamma globulin is further added with PEG to make PEG.
Precipitate by increasing the concentration to 12%. A white precipitate settles out and is collected by centrifugation.
The gamma globulin thus obtained is immunologically active, unmodified 1&, Kabat and Mayer, E.
experimentallmmumXhemistry
May described in 2nd edition, Momas P908f
When measured by the method of Er et al., it is approximately 0 to 0.
It has an anti-complement activity of up to 0.02 units and can be made into a ready-to-use formulation for intravenous administration.

Zero anti-complement activity is due to the absence of molecular aggregation. No aggregation of molecules occurs during the fractionation step in the method of the invention. The lack of formation of aggregates is primarily attributable to the use of polyethylene glycol and low ionic strength solvents in the fractionation procedure. These serve to considerably suppress protein denaturation. The low conductivity of the solvent (-lohm-1 of 300 x 10-8) indicates its low ionic strength. In addition to having virtually no anti-complement activity, the gamma globulin prepared by the method described in Example 1 has a modest sedimentation constant and is free from aggregates and F(ab). F(ab)2, F
It does not contain decomposition products such as c.

The aqueous solution of this product is clear and colorless, and does not have the opalescent color or turbidity that aqueous gamma globulin solutions obtained by other methods have.
Unlike the gamma globulin obtained by the digestion method, the gamma globulin of the present invention has an antibody spectrum that is completely the same as that of the raw material. Distribution of subclasses in the gamma globulin of the present invention (i.e., 1gG.1,
Relative amounts of 2, 3, and 4) remain unchanged from those of raw blood acid.
Example 2 Two fractions are suspended in a 4% PEG4000k solution at 3°C.

Slowly stir and add 0.05M harsh acid to adjust the pH to 5.
Set to 1. After soaking for several hours, pass through the oven to obtain the liquid. Then P
Increase the EG concentration to 5W/V%. PEG is dissolved with a rope and the solution is allowed to settle for at least 1 hour. Afterwards, he expressed his feelings again. Adjust pH to 8 by adding 6% THAM.

Subsequently, PEG is added to 12% to precipitate gamma globulin. The precipitate is collected by static pupil centrifugation. The gamma globulin thus obtained is assayed for anti-complement activity as described in Example 1. The titer is
It is 0.02 to 0.009 units per land. The gamma globulin of the present invention can be immediately put to practical use as an intravenous preparation. In the formulation, gamma globulin is dissolved in a pH 54-67 buffer containing glycine, albumin, and a nonionic surfactant. The pH of the formulation is adjusted to the desired value between pH 5.4 and 6.7, and the gamma globulin concentration is adjusted to 5%. Suitable buffers include phosphoric acid and sodium chloride systems.
In the case of solutions, it is advantageous to add surfactants to the formulation composition in order to prevent or reduce denaturation of the formulation that occurs at liquid-air or liquid-mass interfaces.

Preferred surfactants include block copolymers of propylene and ethylene oxide such as Pluronic (poloxamer 1) to esters of sorbitol or CTFA Cosmeticln.
ent DictionaryMry(Cosmetic,To
ilet hiaMFragame sMation line,
Tween, a water-soluble substance described in
20, 40, 60, 80, 85 (Pol $ or port te2
Polyoxyethylene oxides of long chain fatty acids such as 0,40,60,80,95) and nyIFSA, FS
There are nonionic surfactants such as fluorine surfactants such as B, FSC, and FSN. These nonionic surfactants stabilize proteins against interfacial denaturation and do not contain any chemically reactive groups in their molecular structures that would affect or denature proteins. Example m describes the pharmaceutical composition of a formulation containing intravenous gamma globulin according to the invention.

Example m The precipitate prepared in Example 1 was added to a solution (at 5°C) containing Alpmin: 5 ml/So Tween 80: 0.1%, Glycine: 0.18 M, Sodium chloride: 0.029 M, and 0.012 M chloride. Dissolve as much as possible without creating bubbles.

The resulting pH is 5.4-5.5. 0 if desired.
Carefully add 09MTHAM and adjust pH to 6.4. The concentration of 1g0 was measured and the concentration was 1g0 per 100w' of solution. The mixture is adjusted to contain 1 g of 500 g by further dilution or addition of the precipitate of Example 1.

Other types of Tween or Pluronic Satoshi may be used instead of Tw■n80. To make a liquid preparation, the solution is placed in a sterilized oven and filled into containers.

To prepare a dry formulation, the liquid formulation is aliquoted into vials and lyophilized.

Before use, dissolve the product in pyrogen-free distilled water.
When stored under the pharmaceutical composition according to the invention, the gamma globulin of the invention has a longer half-life than other gamma globulin preparations currently on the market.

The gamma globulin of the present invention is useful for intravenous administration in all cases or under any conditions in which administration of gamma globulin is desirable. It has been proven that there is.

Claims (1)

[Claims] 1. Plasma protein fraction II+I with a protein content of about 25-30%
In a method for producing gamma globulin essentially free of anti-complement activity and suitable for intravenous administration from a raw material selected from Paste II and Fraction II Paste, a. Approximately 300× at 8-6.5
Make a low ionic strength solution with a conductivity of 10^-^6cm^-^1ohm^-^1, obtain a precipitate and a filtrate, b
Impurities are separated and precipitated from the filtrate in step a by adding polyethylene glycol having a molecular weight of about 4,000 to 6,000 to a concentration of about 4 W/V% to obtain a filtrate, and c
Subsequently, by adding polyethylene glycol having a molecular weight of approximately 4,000 to 6,000 to a concentration of approximately 5 W/V%, impurities are separated and precipitated from the filtrate in step b to obtain a filtrate, and d the pH is approximately 8. At 0, the molecular weight is about 4,
Approximately 1 000 to 6,000 polyethylene glycol
Precipitating gamma globulin from the filtrate of step c by adding 2 W/V%,
The method for producing gamma globulin as described above, characterized in that all of the above operations are carried out at a temperature of about 0 to 20°C. 2. The method according to claim 1, wherein the pH at which the paste is extracted is about 5.7 to 5.9. 3 At pH 5.8, 1 kg of fraction II+III paste
The method according to claim 2, wherein the extract is extracted with 30 liters of pyrogen-free distilled water. 4. The method of claim 1, wherein the operating temperature is about 0-5°C. 5 Anti-complement activity from 0 to about 0.02 units per mg, sedimentation constant 7S, molecular aggregates and degradation products F(ab)_
1F(ab)_2, a gamma globulin that does not contain Fc, is colorless and clear when dissolved in water, and has an antibody spectrum and subclass distribution that is not different from that of the raw plasma, and is suitable for intravenous administration. 6. The gamma globulin of claim 5 prepared by the method of claim 1 and suitable for intravenous administration. 7 Claim 6 consisting of gamma globulin dissolved in an aqueous solution containing albumin, a nonionic surfactant, and a glycine acetic acid-acetate buffer (pH 5.4 to 6.7).
intravenous gamma globulin solution. 8. The gamma globulin solution according to claim 7, wherein the nonionic surfactant is Tween 80 or Pluronic 68.