JPH0772141B2 - Gamma and globulin preparations suitable for intravenous administration - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to gamma globulin. The present invention relates to gamma globulin and its intravenous preparation, which are particularly suitable for administration by intravenous injection.

[0002]

Gamma globulin, or immunoglobulin G (IgG), obtained from mixed plasma contains antibodies to many infectious pathogens. Immunoglobulins are effective in the clinical management of a wide variety of medical conditions. Immunoglobulins are used to prevent or treat infections in patients with various anti-deficiency states. For patients with normal immunoglobulin levels, immunoglobulins include, for example, hepatitis, measles, rubella, chickenpox, mumps, yellow fever, rabies, herpes, viral infections such as smallpox, diphtheria, whooping cough, It is also used in the prevention of bacterial infectious diseases such as tetanus, Rh incompatibility, and the like, and in the treatment of serious wound infection due to antibiotic resistance (septicemia due to staphylococcus and Escherichia coli, sepsis due to Pseudomonas aeruginosa). The full clinical potential of immunoglobulins has not yet been fully revealed.

Human immunoglobulins became first to be separated on a large scale during the Second World War. Over time, these formulations were observed to produce shock reactions in patients following intravenous administration, followed by IgG.
It was also demonstrated that the anti-complement activity of the formulation is the cause of the shock response. This anti-complement activity is based on the aggregation of IgG molecules generated during the fractionation process.

In view of these shock reactions associated with the intravenous administration of immunoglobulins, these therapeutically useful substances were administered intramuscularly instead of intravenously. However, intramuscular administration of immunoglobulin has many restrictions as described below. a It hurts, b There is a limit to the amount that can be administered, and C is that the proteolytic action at the injection site attenuates the amount of IgG that is administered. d It reaches the maximum concentration in blood unless 3 or 4 days after administration. Don't do it. This is a significant obstacle for cases requiring high levels of IgG immediately after administration.

On the contrary, the intravenous administration of immunoglobulin does not undergo decomposition at the injection site, and the entire dose immediately enters the bloodstream, and a considerably high blood concentration is obtained. It has a wider range of clinical applications. This idea has prompted the development of a method for producing IgG having low anti-complement activity suitable for intravenous administration. The methods developed to date are based on proteolytic or chemical treatments to reduce the anti-complementary action of aggregation molecules.

Examples of the preparations obtained by these production methods are as follows. 1 Pepsin-treated immunoglobulin In this formulation, the protein is the antibody fraction (5S, F (a
It has been greatly decomposed to b ') ₂ ). It disappears in a short time (from the blood) (unchanged IgG)
Is as short as 30 hours compared to 20 to 30 days) There is a limitation in the efficacy of this formulation for antagonizing bacterial infection. After binding to the antigen, the 5S scrap does not fix complement. This formulation is not applicable for prophylactic purposes. 2 Plasmin-treated immunoglobulin In this preparation, 60% or more of the globulin is Fab.
And Fc debris. The remaining 7S globulin retains a normal half-life (3-4 weeks), but has a limited spectrum of antagonists. 3 pH 4 treated globulin This formulation tends to become anti-complementary during storage. Therefore, its compatibility is limited and large doses cannot be administered. Half-life is slightly reduced (12-1
4 days), the antibacterial activity is attenuated to a measurable degree. 4 β-Propiolactone-treated globulin molecules can be significantly altered, giving rise to new antigenicity. The half-life is about 10 days. Lytic activity is reduced.

The four subclasses (subtypes) of IgG have different sensitivities to proteolytic action. Therefore, the pepsin, plasmin, and pH4 (coexisting with pepsin) -treated preparations are very different from untreated IgG in terms of the amount of IgG subclass distribution. As mentioned above, the undesired anti-complementary action underlying the shock response caused by the intravenous administration of IgG is due to the aggregation of molecules in IgG, which is produced during the fractionation process. The above-mentioned preparations are obtained by a method in which after the formation of agglomerates, they are decomposed mainly by chemical and enzymatic actions.
However, such a degrading treatment also causes the degrading of IgG with the inevitable loss of activity. As a result, the formulations as described above do not retain the expected activity. I, which blocks the formation of aggregates and has essentially no anti-complementary action
Little has been done to date on the development of gG formulations.

[0008] Most recently, in West German Published Patent No. 2357800 issued in 1974-6-6, gamma which is suitable for intravenous administration.
A method for producing a globulin preparation was announced. Similar to the other gamma-globulin preparation methods that have been published, this method is based on the fact that gamma-globulin fraction having a relatively high purity is used as a raw material. However, a very important point is that the gamma globulin obtained by the above-mentioned patent method still has an excessive anti-complementary effect for intravenous administration.

A method for obtaining a substance suitable for intravenous administration from Fraction 1 has also been proposed (US Pat. No. 3,763,1).
No. 35). However, the gamma globulin obtained by this method also contains many anti-complementary active substances, and the yield is low.

FD for gamma globulin for intramuscular injection
There is an A standard, but IV gamma globulin does not. Similar criteria are required to clearly distinguish between gamma globulin, which causes a shock-like response when administered intravenously to susceptible humans, and gamma globulin, which does not elicit such a response. Past 1
It has been recognized that over the course of five years, with sufficiently low levels of anti-complement activity gamma globulin, no clinical symptoms may be observed, even in highly sensitive patients. Standard Mayer 2
unit assay (Experimental
Immunochemistry, E.I. A. Kab
at, M .; M. Mayer Co-authored Second Edition, P133.
Thomas, Springfield, 1961
As a safe level in terms of (Year), the anti-complementary substance per mg of immunoglobulin G is 0.04 to 0.
02 units or less, but may be slightly higher than 0.04. However, at the level of 0.6 unit per mg, the reaction is always observed. Focusing on the absence of clinical side effects, it can be pointed out that gamma globulin preparations are suitable for intravenous infusion depending on whether or not their anti-complement activity is at specifically low levels. Furthermore, physiological antibody activity and specificity must be preserved in order to provide a clinically safe and effective preparation.

[0011]

The object of the present invention is to maintain the properties of a normal gamma globulin molecule, essentially no aggregation of the molecule and thus anti-complement activity, and therefore safe for intravenous infusion. And to obtain a formulation that is effective.

That is, the object of the present invention is: (1) supply of a gamma / globulin preparation suitable for intravenous injection, (2) in vitro, gamma for intravenous injection which has essentially no anti-complement activity.・ Providing globulin preparations,
(3) Supplying an intravenous gamma globulin preparation having a biological half-life of 3 to 4 weeks, (4) Having the ability to fix complement when bound to the corresponding antigen, Intravenous gamma globulin preparation having an antibody spectrum that is essentially unchanged from the type and level of antibodies present in standard gamma globulin obtained by the classical plasma ethanol fractionation method of mixed plasma and Cohn Is to supply.

The present inventor has for the first time provided an active gamma globulin suitable for intravenous administration and a stable formulation containing this gamma globulin. Therefore, in the present invention, the anti-complement activity is substantially reduced to the extent that a shock-like reaction does not occur when administered intravenously, and the sedimentation constant is 7S, which consists essentially of monomeric and dimeric forms. It does not contain degradation products and modified products such as F (ab), F (ab) ₂ and Fc, has an amount of IgA of about 1.0% or less, is colorless and transparent when dissolved in water, and its subclass distribution is Is within:

IgG1 50-80%; IgG2
15-50%; IgG3 1 = 7%; and I
gG4 0.5-3% (However, IgG1 50-72%
IgG2 24-50%; IgG3 1-
4.5%; and IgG4 1.5-3%) and the antibody spectrum is substantially the same as that of the source plasma, and relates to a gamma globulin preparation suitable for intravenous administration. is there.

Particularly preferred as the gamma globulin preparation of the present invention is the Kabat-Meyer method (Ka
m when measured by bat-Mayer method)
It will be understood for the purposes of the present invention that it may include, but is not limited to, those exhibiting from 0 to about 0.04 units per gram.

The gamma globulin suitable for intravenous administration, which is the object substance of the present invention, is described in, for example, J. Am. Chem. S
oc. 68 . It can be easily obtained by the fractionation method developed by the present inventors from the fraction II + III by the method of Kohn et al. Described on pages 459-475 (1946). When this fraction containing almost all immunoglobulins together with other contaminating proteins is treated with the fractionation technique developed by the present inventor, it prevents the aggregate formation of molecules that occurred during the fractionation treatment in the conventional technique. Thus producing active gamma globulin, which is essentially free of anti-complement activity and suitable for intravenous administration.

Another useful source of raw material is Fraction II, which is readily available as human serum globulin. It is economical, stable in lyophilized powder and free of hepatitis virus. This can be processed in the same way as Fractions II + III.

The gamma globulin which is the target substance of the present invention is obtained by the following steps.

[0019]

[Table 1] A preferred embodiment of the above method will be described below. A plasma protein fraction II or II + III paste was approximately 4.
P from 8 to 6.5, preferably from about 5.5 to 5.9
Extract with water at H. Protein content of about 25 to 3
About 25 to 45 liters, preferably 30 liters of pyrogen-free water are used per kg of 0% paste. The pH of non-toxic pharmaceutically acceptable organic or inorganic acids such as acetic acid, lactic acid, hydrochloric acid, sulfuric acid, etc. is used for adjustment. Water-insoluble substances were separated, and polyethylene glycol was used as the filtrate in order of 4 w / v% and 5 w.
/ V% and 12 w / v% at different concentrations. 1
The pH at the 2 w / v% concentration step is about 8.0. Impurities are removed by the first two precipitations, and the final precipitation gives gamma globulin, which is the target substance of the present invention, suitable for intravenous administration. The preferred polyethylene glycol molecular weight is about 4000 to 6000. Non-toxic pharmaceutically acceptable salts of organic or inorganic acids are used to adjust the pH to approximately 8.0. The operation may be carried out at temperatures of approximately 0 ° to 20 ° C, but low temperatures of 0 ° to 5 ° C are preferred.

The gamma globulin suitable for intravenous administration, which is the objective substance of the present invention, is one in which the anti-complement activity is substantially reduced to such an extent that no shock-like reaction occurs during intravenous administration. The preferred one has substantially zero anti-complementary activity. In addition, it has a sedimentation constant of 7S and is free of molecular aggregates and degradation products such as F (ab) ₁ F (ab) ₂ , Fc. The aqueous solution of this product is colorless and transparent and has no opal color or cloudiness like the aqueous solution of gamma globulin obtained by other methods. Gamma obtained by digestion method
Unlike globulin, the gamma globulin of the present invention is
It has an antibody spectrum that is no different from that of raw plasma. The subclass distribution (ie, relative amounts of IgG1,2,3,4) in the gamma globulin of the present invention is substantially unchanged from that of the source plasma.

The gamma globulin of the present invention can be immediately put into practical use as an intravenous preparation. In the formulation of the preparation, gamma globulin is dissolved in a buffer solution containing glycine, albumin and a nonionic surfactant and having a pH of 5.4 to 6.7. The DH of the preparation liquid has a pH of 5.4 to 6.
Adjust to the desired value between 7 and gamma globulin concentration is 5
Adjust to%. Suitable buffers include phosphoric acid and the sodium acetate-acetic acid system.

In the case of solutions, it is convenient to add a surfactant to the formulation composition to prevent or reduce denaturation of the formulation which may occur at the liquid-air or liquid-solid interface. A preferable surfactant is Pluronic 68 (poloxamer 18).
8) block copolymers of propylene and ethylene oxide such as 8), esters of sorbitol or C
TFA Cosmetic Ingredient D
motionary (Cosmetic, Toilee
try and Fragrance Associa
Tween 20, 40, 60, 80, 85 (Poly), which is a water-soluble substance described in ed.
Sorbate 20, 40, 60, 80, 95) and long-chain fatty acid polyoxyethylene oxides and Z
There are nonionic surfactants such as fluorosurfactants such as onyl FSA, FSB, FSC, FSN. These nonionic surfactants stabilize the protein against interfacial denaturation and do not contain any chemically reactive group in the molecular structure that affects or denatures the protein.

The details of the method for obtaining gamma globulin suitable for intravenous administration, which is the objective substance of the present invention, are described in Examples 1 and 2 for the pharmaceutical composition of the preparation containing the intravenous gamma globulin of the present invention. Described in Example 3.

[0024]

【Example】

Example 1 Plasma protein fraction II + II with a protein content of 25-30%
1 kg of I paste is suspended in 30 liters of pyrogen-free distilled water and stirred to make a uniform yellow suspension. The temperature is maintained at 5 ° C. Add 20 ml of 10% acetic acid per kg of Fraction II + III paste to lower the pH to 5.8. After stirring for a further 15 minutes, the precipitate is allowed to stand for 2-3 hours. After that, No. Clarify the supernatant using filter paper such as a 9 pad. 400 per mole
U.S. Pharmacopoeia polyethylene glycol (PEG) having an average molecular weight of 0 g is added in powder or flake form to a concentration of 4 g per 100 ml of solution. PE
After stirring until G was completely dissolved, the precipitate formed was allowed to stand for 1 to 2 hours. No. Collect the supernatant by filtration through a 9 pad or Millipore membrane. Then change the PEG concentration to 5
Increase to w / v%. The PEG is dissolved by stirring and the dissolution is left to stand for 1 to 2 hours. After that, the supernatant was added again to Filter with an asbestos pad of 9.

Next, 6% trishydroxyethylaminomethane (THAM) is added to adjust the pH to 8.
For gamma globulin, PEG is added continuously
Precipitate by increasing the concentration to 12%. When the white precipitate has settled, collect it by centrifugation.

The gamma globulin thus obtained is an immunologically active non-modified IgG, which is Kabat and M
aper, EXPerimental Immuno
Chemistry 2nd edition, Thomas P905f
f has an anti-complement activity of approximately 0 to 0.02 units per mg as determined by the method of Mayer et al.
It can be a formulation for immediate intravenous administration. The anti-complementary activity of 0 is due to the absence of molecular aggregation. No molecular aggregation occurs during the fractionation process in the method of the invention. The lack of agglomeration formation is attributed primarily to the use of polyethylene glycol and low ionic strength solvent in the fractionation operation. These serve to significantly suppress protein denaturation. Conductivity of solvent (3
A low ^{value of} 00 × 10 ⁻⁶ cm ⁻¹ ohm ⁻¹ ) indicates that the ionic strength thereof is low.

Gamma globulin and Folia Haematology obtained by the process of Example 1 above
ca, Leipzig 103 (1976) 6, p. 9
No. 38-945, the anti-complementary activity of the produced product was confirmed by Kabat-Mayer.
2 unit assay method and its purity was examined by gel filtration method.

As a result, the gamma-globulin of the present invention obtained by the process of Example 1 showed substantially zero anti-complement activity, while Folia Haematol.
ogica, Leipzig 103 (1976) 6,
p. At least 4 as described in 938-945
It has more than twice as high anti-complementary activity. Further, especially as a result of gel filtration and the like, the gamma-globulin of the present invention obtained by the process of Example 1 is substantially composed of its monomeric body and dimer body.
₂ and Fc, which did not include degradation products and modified products and IgG aggregates.
Degradation products and IgG according to matologica
Of aggregates.

Further, in the preparation of the present invention, the content of IgA is very low, about 1% or less, while in the case of Folia.
Haematological Leipzig 10
3 (1976) 6, p. Those described in 938-945 have a high IgA content, such as 3%. Clinically, patients with IgA deficiency or the like may have anti-IgA antibody, which causes a reaction such as hypersensitivity, which is a problem.

It has been reported that when the bentonite treatment is carried out, disappearance of subclass IgG3 is observed, but in the present invention, subclass IgG3 maintains a good value corresponding to the subclass distribution of normal plasma.

Furthermore, the ones shown in the Palson reference which disclose some similar methods are not sufficiently safe formulations. Although they are described as having no anti-complementary activity, they have not been measured in practice. The characteristics of the gamma globulin of the present invention, such as very low anti-complement activity, substantially no degradation products and modified products, and aggregates of IgG, characteristic subclass distribution, etc. are in accordance with Palson. It cannot be found among things.

Actually, (1) Japanese Patent Laid-Open No. 50-46814, (2) Vox Sang 23 (1972) p.
107-118 and (3) Biochem. Bioph
ys. Acta 82 (1964) p. 463-473
In the preparation obtained by the method described in 1., the IgG aggregate content is (1) 1-2%, (2) 10% and (3) 10%.
As described above, a large value is shown, and reduction of IgG aggregates cannot be achieved.

The above is supported by the following experimental data. 1 Removal of anti-complementary activity (ACA) from Cohn's fractions II + III

[Table 2] 2 shows the results of analysis of typical gamma globulin obtained in the process of Example 1. 2-1 The analysis result of the molecular composition of the product was as follows.

[Table 3] The relative distribution of 2-2 IgG subclasses is shown in the table below.

[Table 4] The anti-complement activity was measured according to the following method.

A dilution series of the primary composition is prepared.

[Table 5] Here, one unit means the amount of hemolysis of 2.25 × 10 ⁸ sheep red blood cells in 7 ml when 45% is hemolyzed with 0.0025 ml of guinea pig serum. VBSPA; Veronal buffer +
Saline + PEG-4000; (0.5)
%) + Human serum albumin (0.1%) 2 solution mixture is incubated at 37 ° C. for 30 minutes. 3 Then add 1 ml of freshly prepared sheep red blood cells (5 × 10 ⁸ cells / ml) sensitized to each tube. 4 Incubate the solution mixture again at 37 ° C. for 30 minutes. 5 After cooling, centrifuge and remove the supernatant. 6. Measure the absorbance at 541 nm to check the hemolytic property.

Example 2 2 g of Fraction II was suspended in 1 liter of a 4% PEG4000 aqueous solution at 3 ° C. The mixture is gently stirred and 0.05 mol acetic acid is added to adjust the pH to 5.1. After stirring for several hours, the solution is filtered to obtain a supernatant. Then the PEG concentration is increased to 5 w / v%. The PEG is dissolved by stirring, and the solution is allowed to stand for 1 hour or more. Then the supernatant is filtered again. Adjust pH to 8 by adding 6% THAM. Subsequently, PEG is added to 12% to precipitate gamma globulin. The precipitate is allowed to stand and is collected by centrifugation. The gamma globulin thus obtained is assayed for anti-complement activity as described in Example 1. The titer is 0.02 to 0.005 per mg.
It is a unit.

Furthermore, the results of comparison of the antibody titers of typical gamma globulin obtained according to Example 2 with those of the starting plasma are shown. The above results indicate that the antibody spectrum is substantially the same as that of the source plasma.

Example 3 The precipitate prepared in Example 1 was added to a solution (5 ° C.) containing albumin: 5 g / l Tween 80: 0.1% glycine: 0.15M sodium acetate: 0.025M acetic acid: 0.0125M. Dissolve as little as possible. The resulting pH is 5.4-5.5. If desired, 0.05M THAM is carefully added to adjust the pH to 6.4. IgG concentration was measured and 5 per 100 ml of solution
The dilution or precipitation of Example 1 is added to adjust to contain g of IgG. Instead of Tween 80, other species of Tween or Pluronic 68 may be used.
In order to obtain a liquid formulation, the solution is sterilized by filtration and filled in a container. To prepare a dry formulation, the liquid formulation is divided into vials and freeze-dried. Before use This product is dissolved in distilled water containing no pyrogens.

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 formulations currently on the market. Intravenous injection is desired without the undesirable side effects normally associated with intravenous gamma globulin administration, but under all or any conditions, the gamma globulin of the present invention is useful for intravenous administration. Proved to be.

[Table 6]

Claims (3)

[Claims] 1. The anti-complement activity is substantially reduced to such an extent that a shock-like reaction does not occur upon intravenous administration,
The sedimentation constant was 7S, which consisted essentially of monomeric and dimeric forms, but did not contain decomposition products and modified products such as F (ab), F (ab) ₂ and Fc, and IgA was about 1.0% or less. And is colorless and transparent when dissolved in water, and its subclass distribution is within the following range: IgG1 50
-80%; IgG2 15-50%; IgG3
1-7%; and IgG4 0.5-3% (however, IgG1 50-72%; IgG2 24-5
0%; IgG3 1-4.5%; and IgG4
A gamma globulin preparation suitable for intravenous administration characterized in that the antibody spectrum is substantially the same as that of the starting plasma (except for 1.5-3%). 2. The anti-complement activity is 0 to about 0.04 unit per mg and the sedimentation constant is 7 when measured by the Kabat-Mayer method.
S is substantially composed of a monomer body and a dimer body,
It does not contain degradation products and modified products such as F (ab), F (ab) ₂ and Fc, has an amount of IgA of about 1.0% or less, is colorless and transparent when dissolved in water, and its subclass distribution is Within the range: IgG1 50-80%
IgG2 15-50%; IgG3 1-
7%; and IgG4 0.5-3% (however, IgG4
1 50-72%; IgG2 24-50%; I
gG3 1-4.5%; and IgG4 1.5-
The preparation according to claim 1, characterized in that the antibody spectrum is substantially the same as that of the starting plasma (except for 3%). 3. A formulation according to claim 1 wherein the formulation is stabilized with other compounds such as albumin or nonionic detergents.