JPH0375533B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to gamma globulins. The present invention relates to gamma globulin and intravenous formulations thereof particularly suitable for administration by intravenous injection. Gamma globulin or immunoglobulin G (IgG) obtained from mixed plasma contains antibodies against many infectious agents. Immunoglobulins have efficacy in the clinical management of a wide variety of disease states. Immunoglobulins are used to prevent or treat infections in patients with various antibody deficiencies.
For patients with normal immunoglobulin levels,
Immune globulin is effective against viral infections such as hepatitis, measles, rubella, chickenpox, mumps, yellow fever, rabies, herpes, smallpox, bacterial infections such as diphtheria, pertussis, and tetanus, and Rh incompatibility. It is also used for prophylaxis and treatment of severe infections due to antibiotic resistance (staphylococcal and E. coli sepsis, aeruginosa sepsis). The full clinical potential of immunoglobulins has not yet been fully realized. Human immunoglobulins first became isolated on a large scale during World War II. It was soon observed that these preparations produced a shock response in patients upon intravenous administration, and it was subsequently established that the anti-complement activity of IgG preparations was responsible for the shock response. Ta. This anti-complement activity is based on the aggregation of IgG molecules that occurs during the fractionation process. In view of these shock reactions associated with intravenous administration of immunoglobulin, 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, and c) The proteolytic effect that occurs at the injection site
Attenuate the amount of IgG; d) Maximum concentration in the blood should not be reached until 3 or 4 days after administration. This poses a significant obstacle for cases requiring high levels of IgG concentration immediately after administration. In contrast, intravenous administration of immunoglobulin
It has broader clinical applications 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. This idea has prompted the development of a method for producing IgG 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, the protein is antibody fragments (5S,
F(ab′) ₂ ) is decomposed into large widths. Because it is cleared (from the blood) over a short period of time (30 hours, compared to 20-30 days for unchanged IgG), the efficacy of this preparation in combating bacterial infections is limited. After binding to antigen, 5S fragments do not fix complement. This preparation is not applicable for prophylactic purposes. 2 Plasmin-treated immunoglobulin In this preparation, more than 60% of the globulin is
Decomposed into Fab and Fc debris. remain
7S globulin has a normal half-life (3-4 weeks)
However, the spectrum of antibodies is limited. 3 PH4-treated globulin This preparation tends to become anti-complementary during storage. Its suitability is therefore limited,
Large doses cannot be administered. The half-life is slightly decreased (12-14 days), and the antibacterial activity is attenuated to the extent that it cannot be measured. 4 β-propiolactone-treated globulin The molecule undergoes extensive changes, potentially giving rise to new antigenic properties. Half-life is approximately 10 days. Bacteriolytic activity is reduced. The four subtypes of IgG have different susceptibilities to proteolytic effects. Therefore, pepsin, plasmin and PH4 (with pepsin) treated preparations are very different from untreated IgG with respect to the amount of distribution of each IgG subtype. As mentioned above, the undesirable anti-complement effect responsible for the shock reaction caused by intravenous administration of IgG is due to the aggregation of molecules in IgG, 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, such a decomposition process inevitably involves a loss of activity.
This will also cause the degradation of IgG. As a result, formulations such as those described above do not retain the expected activity. To date, little progress has been made to develop IgG preparations that prevent the formation of aggregates and are essentially free of anti-complement effects. Most recently, West German published patent No. 1974-6-6 issued
No. 2357800 published a method for producing gamma globulin preparations suitable for intravenous administration. 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 above-mentioned patented method still has excessive anti-complementary activity for intravenous administration. A method for obtaining substances suitable for intravenous administration from fraction 1 has also been proposed (US Pat. No. 3,763,135).
issue). However, the gamma globulin obtained by this method also contains a large amount of anti-complement active substances, and the yield is also small. There are FDA standards for intramuscular gamma globulin, but there are no such standards for intravenous gamma globulin. Similar criteria are needed to clearly distinguish between gamma globulins that elicit a shot-like response when administered intravenously to susceptible humans and those that do not. Over the past 15 years, it has been recognized that at sufficiently low levels of gamma globulin anti-complement activity, no clinical symptoms will be observed, even in highly susceptible patients.
Standard Mayer 2 unit assay
(Experimental Immunochemistry, EA
Kabat, MMMayer, 2nd edition, P133.
Thomas, Springfield, 1961), the safe level of immunoglobulin G is 1.
The anti-complement agent per mg is 0.04 to 0.02 units or less, but may be slightly higher than 0.04. However, at a level of 0.6 units per mg, a reaction is always observed. With emphasis on the absence of clinical side effects, the suitability of gamma globulin preparations for intravenous administration depends on specifically low levels of anticomplement activity. Furthermore, physiological antibody activity and specificity must be preserved in order to provide clinically safe and effective preparations. The object of the present invention is to obtain a formulation which maintains the normal properties of the gamma globulin molecule and is essentially free from aggregation of the molecule and its anti-complementary activity, thus rendering it safe and effective for intravenous administration. That's true. That is, the objects of the present invention are; (1) to provide a gamma globulin preparation suitable for intravenous injection; (2) to provide an intravenous gamma globulin preparation that essentially has no anti-complement activity in vitro. (3) provide an intravenous gamma globulin preparation with a biological half-life of 3 to 4 weeks; (4) possess the ability to fix complement when bound to the corresponding antigen; and has an antibody spectrum essentially unchanged when compared to the types and levels of antibodies present in the starting mixed plasma and standard gamma globulin obtained by Cohn's classical plasma ethanol fractionation method. The aim is to supply intravenous gamma globulin preparations. The present inventors have provided for the first time an active gamma globulin suitable for intravenous administration and a stable preparation containing this gamma globulin. The target substance of the present invention, gamma globulin, suitable for intravenous administration is, for example, J.Am.Chem.Soc. 68 .
It can be easily obtained by the fractionation method developed by the present inventor from the fractionation + by the method of Cohn et al., described on pages 459-475 (1946). This fraction contains almost all immunoglobulins along with other contaminant proteins.
If treated with the fractionation technology developed by the present inventor, it will prevent the formation of molecular aggregates that occur during fractionation using conventional techniques, making it suitable for intravenous administration with essentially no anti-complement activity. It produces activated gamma globulin. Another useful raw material source is the readily available fraction of 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 Fraction+. Gamma globulin, which is the target substance of the present invention, can be obtained by the following process. [Table] Preferred embodiments of the above method are described below. Plasma protein fraction or paste with a pH of approximately 4.8 to 6.5, preferably approximately 5.5 to 5.9
Extract with water. Protein content is about 25 to
30% paste about 25 to 45 per Kg preferably
Use 30 pyrogen-free water. non-toxic pharmaceutically acceptable acetic acid, lactic acid, hydrochloric acid,
The pH of an organic or inorganic acid such as sulfuric acid is used for adjustment. Substances that are insoluble in water are separated, and the filtrate is prepared using polyethylene glycol.
It is fractionally precipitated at concentrations of 4w/v%, 5w/v% and 12w/v%. The pH at the 12 w/v% concentration step is approximately 8.0. The first two precipitations remove impurities, and the final precipitation yields gamma globulin, the target substance of the present invention, suitable for intravenous administration. The preferred polyethylene glycol molecular weight is about 4,000 to 6,000. Non-toxic pharmaceutically acceptable organic or inorganic acid salts 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 lower temperatures of 0° to 5°C are preferred. Gamma globulin, which is the target substance of the present invention and is suitable for intravenous administration, has a sedimentation constant of 7S in addition to having substantially no anti-complement activity, and is free from molecular aggregates and F It does not contain decomposition products such as (ab) ₁ F (ab) ₂ and Fc. The aqueous solution of this product is colorless and transparent, and does not have an opalescent color or cloudiness like gamma globulin aqueous solutions obtained by other methods. Unlike gamma globulin obtained by digestion methods, the gamma globulin of the present invention has an antibody spectrum that is exactly the same as that of the source plasma. Subclass distribution in the gamma globulins of the present invention (i.e.
The relative amounts of IgG.1, 2, 3, and 4) were unchanged from those of the raw plasma. The gamma globulin of the present invention can be immediately put to practical use as an intravenous preparation. In the formulation of the drug, gamma globulin has a pH of 5.4~ containing glycine, albumin and non-ionic surfactants.
Dissolve in the buffer solution of 6.7. The PH of the mixture is PH5.4
Adjust to desired value between ~6.7 and gamma globulin concentration to 5%. Suitable buffers include phosphoric acid and sodium acetate-acetic acid 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-solid interfaces. Preferred surfactants include block copolymers of propylene and ethylene oxide such as Pluronic 68 (poloxamer 188), esters of sorbitol or CTFA Cosmetic.
Ingredient Dictionary (Cosmetic, Toiletry
Tween 20, 40, 60, which is a water-soluble substance described in
There are nonionic surfactants such as polyoxyethylene oxide of long chain fatty acids such as 80, 85 (Polysorbate 20, 40.60, 80, 95) and fluorine surfactants such as Zonyl FSA, FSB, FSC, 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. Details of the method for obtaining gamma globulin suitable for intravenous administration, which is the target substance of the present invention, are described in Examples 1 and 2. Example 3 describes the pharmaceutical composition of a preparation containing the gamma globulin for intravenous administration of the present invention. Describe it. Example 1 Plasma protein fraction with protein content of 25 to 30% +
1 Kg of paste is suspended in 30 pyrogen-free distilled water and stirred until a homogeneous yellow suspension is obtained. The temperature is maintained at 5°C. Add 20ml of 10% acetic acid per 1Kg of fraction + paste and adjust the pH to 5.8
lower to After stirring for another 15 minutes, 2 to 3
Let the precipitate stand for an hour. Thereafter, the supernatant is clarified and filtered using a filter paper such as No. 9 Pad. USP specification polyethylene glycol (PEG) with an average molecular weight of 4000 grams per mole
Add in powder or flake form to a concentration of 4 g per 100 ml of solution. After stirring until PEG is completely dissolved, the resulting precipitate is allowed to stand for 1 to 2 hours. Collect the supernatant by filtration through a No. 9 pad or Millipore membrane. Subsequently, the PEG concentration is increased to 5% w/v.
PEG is dissolved by stirring, and the dissolution time is 1 to 2.
Let stand for a while. The supernatant is then filtered again through No. 9 asbestos pad. The pH is then adjusted to 8 by adding 6% tris hydroxyethylaminomethane (THAM).
Gamma globulin continues to add PEG,
Precipitate by increasing the PEG concentration to 12%. When the white precipitate settles, collect it by centrifugation. The gamma globulin thus obtained is an immunologically active unmodified IgG, as described by Kabat and Mayer.
Author, Experimental Immunochemistry 2nd edition,
It has an anti-complement activity of approximately 0 to 0.02 units per mg, as measured by the method of Mayer et al., described in Thomas Company P905ff, and can be made into a ready-to-use intravenous preparation. 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 are,
It helps to considerably suppress protein denaturation. The low conductivity of the solvent (300×10 ^-6 cm ^-1 ohm ^-1 ) indicates its low ionic strength. Gamma globulin and Folia Haematologica, Leipzig obtained by the process of Example 1 above
103 (1976) 6, p. 938-945 was prepared, and its anti-complement activity was tested by Kabat-Mayer's two unit assay method, and its purity was examined by gel filtration method. Ta. As a result, the gamma globulin of the present invention obtained by the process of Example 1 above showed substantially 0 anti-complement activity, whereas Folia
Haematologica, Leipzig 103 (1976) 6,
Those according to the description on pages 938-945 have at least 4 times higher anti-complement activity. Furthermore, especially as a result of gel filtration, the gamma globulin of the present invention obtained by the process of Example 1 consists essentially only of its monomer and dimer forms,
It did not contain degradation products and modified products such as F(ab), F(ab) ₂ and Fc, nor IgG aggregates, but
Those according to the above Folia Haematologica contained degraded products and aggregates of IgG. Furthermore, we disclose some similar methods
What is shown in Palson's literature is not a sufficiently safe formulation. Although they are described as having no anti-complement activity, this has not actually been measured. Characteristics of the gamma globulins of the invention, such as very low anti-complement activity;
Substantially no degradation products, modified products, or IgG aggregates, characteristic subclass distribution, etc.
It cannot be found in those who follow Palson. The above is also supported by the experimental data shown below. 1 Extraction step for removing anti-complement activity (ACA) from Cohn's fraction+ ACA units/mg IgG H ₂ O treatment 0.28 PEG4% extraction treatment 0.046 PEG5% extraction treatment 0.00 2 In the process of Example 1 The representative gamma obtained
The results of an analysis of globulin are shown. 2-1 The analysis results of the molecular composition of the product were as follows. [Table] 2-2 The relative distribution of IgG subclasses is shown in the table below. [Table] Note that the anti-complement activity was measured according to the following method. 1. Create a dilution series of the first order composition. [Table] * Complement level: 2 units per ml. Here, 1 unit means that when 2.25 x 10 ⁸ sheep red blood cells are hemolyzed with 0.0025 ml of guinea pig serum, 45% of them are hemolyzed in 7 ml. indicates the amount. VBSPA; Veronal Batsuhua +
Saline + PEG-4000; (0.5%) + Human Serum Albumin (0.1%) 2 Incubate the solution mixture at 37°C for 30 minutes. 3. Then add 1 ml of freshly prepared sensitized sheep red blood cells (5 x 10 ⁸ cells/ml) to each tube. 4. Incubate the solution mixture again for 30 minutes at 37°C. 5 After cooling, centrifuge and remove the supernatant. 6 Measure the absorbance at 541 nm to check hemolysis. Example 2 2 g of fraction was added to 4% PEG4000 aqueous solution 1 at 3°C.
Suspend in Stir gently and add 0.05 mol acetic acid to adjust the pH to 5.1. After stirring for several hours, the mixture is filtered to obtain a supernatant. Then increase the PEG concentration to 5% w/v.
The PEG is dissolved by stirring, and the solution is allowed to stand for at least 1 hour. The supernatant is then filtered again. 6%
Add THAM to adjust pH to 8. continue
Add PEG 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. Its titer is 0.02-0.005 units per mg. Furthermore, the results of comparing the typical gamma globulin antibody titer obtained according to Example 2 with that of raw plasma are shown. [Table] Globulin

* Indicates dilution level.
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 dissolved in a solution (5°C) containing albumin: 5g/Tween80: 0.1%, glycine: 0.15M, sodium acetate: 0.025M, and acetic acid: 0.0125M, avoiding foaming as much as possible. The resulting PH is 5.4-5.5. If desired, carefully add 0.05M THAM and adjust the PH to 6.4. Measure the IgG concentration, 5 per 100 ml of solution.
The dilution or precipitate of Example 1 is adjusted to contain g of IgG. Other types of Tween or Pluronic 68 may be used in place of Tween 80. To prepare a liquid preparation, the solution is sterilized and filtered and filled into containers. To obtain a dry formulation, the liquid formulation is divided 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 present 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 intravenous administration is desired without the undesirable side effects normally associated with intravenous administration of gamma globulin. It has been proven that there is.

Claims (1)

[Claims] 1. The anti-complement activity is 0 to about 0.02 units per mg when measured by the Kabat-Mayer method, the sedimentation constant is 7S, and the anti-complement activity is 0 to about 0.02 units per mg as measured by the Kabat-Mayer method. Consisting of F
It does not contain degraded or modified products such as (ab), F(ab) ₂ , and Fc, nor does it contain IgG aggregates, and has an IgA content of approximately 1.0% or less, and is colorless and transparent when dissolved in water. and its subclass distribution is in the following range: IgG1 50-72%; IgG2 24-50%; IgG3 1-4.5%; and IgG4 1.5-3%, and the antibody spectrum is not different from that of the source plasma. A gamma globulin preparation suitable for intravenous administration. 2. A formulation according to claim 1, wherein the formulation is stabilized with albumin or other compounds such as non-ionic detergents.