JPS6399022A - Gamma globlin for intravenous administration and medicine - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to gamma globulins. The present invention relates to gamma globulin and intravenous formulations thereof, which are 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, immunoglobulins are used to treat viral infections such as hepatitis, measles, rubella, chickenpox, mumps, yellow fever, rabies, herpes, smallpox, diphtheria, and pertussis. , bacterial infections such as tetanus, and I? Prevention against nonconformity, etc.
It is also used in the 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 immunoglobulin was first isolated on a large scale during the Second Ascension War. It was soon observed that these preparations produced shock reactions in patients upon intravenous administration, and it was subsequently established that the anti-complement activity of IgG preparations was also responsible for the shock reactions. This anti-complement activity is based on the aggregation of 1g0 molecules generated during the fractionation step.

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 effect that occurs at the injection site makes the amount of IgG administered questionable. (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 +3c>a degrees 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 a higher blood concentration is obtained. It has many clinical applications. 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.

■, Pepsin-treated immunoglobulin In this preparation, proteins are degraded by antibody degradation (5S, P(a)
b') 2) It is decomposed into large 111 parts.

Due to its rapid elimination (30 hours compared to 20-30 days for intact IgG), the efficacy of this preparation in combating bacterial infections is limited. After binding to antigen, it does not fix 58 minutes of degraded complement. This preparation is not applicable for prophylactic purposes.

2. Plasmin-treated immunoglobulin In this preparation, more than 60% of the globulin is Pab
and Pc decomposition. The remaining 7s globulin retains a normal half-life (3-4 weeks) but has a limited spectrum of antibodies.

3. p114-processed globulin This formulation tends to become anti-complementary during storage. Its suitability is therefore limited and it cannot be administered in large quantities. The half-life has decreased slightly (12-1
4th), the antibacterial activity was tested to the extent that it could not be measured.

4. β-propiolactone-treated globulin molecules may undergo extensive changes, resulting in new antigenicity. 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 pt14 (with pepsin) treated formulations are very different from untreated IgG with respect to the amount of IgG subtype separation, respectively.

As mentioned above, the undesired anti-complement effect responsible for the shock response caused by intravenous administration of IgG is due to the aggregation of molecules within the IgG, which is 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 also inevitably results in the decomposition of IgG with loss of activity. As a result, formulations such as those described above do not retain the expected activity.

IgG that prevents the formation of aggregates and is essentially anti-complementary
There has been little development of formulations to date.

Most recently, West German Published Patent No. 2, issued 1974-8-fi.
No. 857.800 published a method for preparing 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 patented method still has too much anti-complementary action for intravenous administration.

A method for obtaining substances suitable for intravenous administration from fraction 1 has also been proposed (US Pat. No. 3.7H, 135).

However, the gamma globulin obtained by this method also contains a large amount of anti-complement active substances, and the yield is also small.

Although there are FDA standards for gamma globulin for intramuscular injection, there are no standards for gamma globulin for intravenous injection. 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.

Over the past 15 years, it has been established that sufficiently low levels of gamma ψ globulin anti-complement activity do not cause any clinical symptoms, even in highly susceptible patients. 5 standard Mayer2 unl
t assay (Hxperlmental Ism
unochewlstry.

Co-authored by E. A. Kabat and M. M. Mayer, 2nd edition, P2S5. Thomas.

1 mg of immunoglobulin G is considered a safe level according to Springf'1eld, 1901).
0.04 to 0.02 units or less of anti-complement agent per unit, but may be slightly higher than 0.04. However, at a level of 0.6 units per a+g, the reaction is constantly 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, in order to provide clinically safe and efficacious preparations, it is necessary to preserve physiological antibody activity and specificity.

The object of the present invention is to create a formulation which maintains the normal properties of the gamma globulin molecule and which is essentially free from aggregation of the molecule and its anti-complementary activity, thus making it safe and effective for intravenous administration. It's about getting.

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) provide the ability to fix complement when bound to the corresponding antigen; intravenous infusion with an antibody spectrum essentially unchanged in comparison 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 gamma globulin preparations for

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.

Gamma globulin, which is the target substance of the present invention and is suitable for intravenous administration, can be obtained, for example, by fractionation by the method of Cohn et al. can be easily obtained by the fractionation method developed by the present inventor. This fraction, which contains almost all the immunoglobulins along with other contaminant proteins, can be processed using the fractionation technique developed by the present inventors to prevent the formation of aggregates of molecules that occur during fractionation 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 II, which is readily available as human serum globulin. This thing is economical and
The lyophilized powder is stable and does not contain hepatitis virus.

This can be processed in the same way as fractions ①+②.

Gamma globulin, which is the target substance of the present invention, can be obtained by the following process.

Plasma protein -n+m Preferred embodiments of the above method are described below.

The plasma protein fraction ■ or ■ + ■ is approximately 4.8
pH from about 6.5, preferably from about 5.5 to 5.9
Extract with water. Protein content is about 25 to 30
% of paste per kg of about 25 to 454 preferably 3
Use 01 pyrogen-free water.

Non-toxic, pharmaceutically acceptable organic or inorganic acids such as acetic acid, lactic acid, hydrochloric acid, sulfuric acid, etc. are used to adjust pl+. Substances insoluble in water are separated, and the filtrate is prepared using polyethylene glycol, sequentially at 4v/v% and 5v/v%.
and fractional precipitation at a concentration of 12 W/V%. 12W/
The pH at the V% concentration step is about 8.0. The first two fractional precipitations remove impurities, and the final precipitation yields gamma globulin, the object substance of the present invention, suitable for intravenous administration. Desirable polyethylene glycol molecular weights are about 4.000 to e,ooo. 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 a temperature of approximately 0 to 20°C, but θ.

A low temperature of 5°C to 5°C is preferred.

Gamma globulin, which is the target substance of the present invention and is suitable for intravenous administration, has an anti-complement activity of substantially O, and also has a sedimentation constant of 7S, and is free from molecular aggregates and F.
(ab)+F (ab)2, does not contain decomposition products such as Pc. The aqueous solution of quartz is colorless and transparent and does not have the opalescent color or cloudiness 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 plasma. Subclass distribution in the gamma globulins of the present invention (i.e., I
The relative amounts of gG, I, 2, 3.4) 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 formulating the drug, gamma◆globulin is dissolved in a pH 5.4-6.7 buffer containing glycine, albumin, and a nonionic surfactant. The pH of the preparation is p.

Adjust to the desired value between 5.4 and 6.7, and the gamma globulin concentration is adjusted 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 (boroxamer 168), esters of sorbitol or CTFA Co
smetic Ingredient Dictionary
ry (CosIIetic, Tollatory a
nd Fragrance Association,
Tween, a water-soluble substance described in the 1973 edition)
20.40.80.80.85 (Polysorba
te20, 40.60.80.95”) and Zonyl
FSA.

There are nonionic surfactants such as fluorosurfactants such as I'SI3, FSC, and PSN. These nonionic surfactants stabilize proteins against interfacial denaturation and do not contain any chemically reactive groups in their molecular structure 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 1 kg of plasma protein fraction Baste with a protein content of 25-30% is suspended in 304 pyrogen-free distilled water and stirred until a homogeneous yellow suspension is obtained.

The temperature is maintained at 5°C. Fraction ■ Add 2011 10% acetic acid per kg of 10m paste to lower pi to 5.8. After stirring for an additional 15 minutes, the precipitate is allowed to stand for 2 to 3 hours. Thereafter, the supernatant is clarified and filtered using a filter paper such as No. 9 Pad.

USP polyethylene glycol (PEG) having an average molecular weight of 4.000 g per mole is added 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 millibore fist membrane.

Subsequently, the PEG concentration is increased to 5 W/V%. PEG is dissolved by stirring, and the dissolution is allowed to stand for 1 to 2 hours. The supernatant is then filtered again through a No. 9 asbestos pad.

Next, 6% Tris hydroxyethylaminomethane (T
HAN) to adjust the pH to 8. For gamma globulin, continue to add PEG to increase the PEG concentration to 12%.
Precipitate by raising the temperature to . When the white precipitate settles, collect it by centrifugation.

The gamma globulin thus obtained is immunologically active unmodified 1gG, as described by Kabat and Mayer,
Experio+ental Imwunocheml
have an anti-complement activity of approximately 0 to 0.02 units per 11 g when measured by the method of Mayer et al. described in 2nd edition of Str. It is. Anti-complement activity is 0
This 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. Solvent conductivity (300
x 10-6 cm-'otv-') indicates that the ionic strength is low.

Example 2 Fractionation [2 g of 4% PT at 3°C! 04000 aqueous solution 14. Stir gently and add 0.05 mol acetic acid to adjust pH to 5.1. After stirring for several hours, the mixture is filtered to obtain a supernatant. Next, increase the PEG concentration to 5w/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. Add 6% THAN and p
Adjust H to 8. 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.

Its tlter is 0.02-0.005 units per Ig.

Example 3 The precipitate prepared in Example 1 was mixed with albumin: 5 g/Il Twccn80: 0.1% glycine
: O, 15M Sodium acetate : 0.025M Acetic acid : Q, Dissolve in a solution containing 0125M (5°C) while avoiding foaming as much as possible. The resulting pH is 5.4-5.5. 0.05 if you want
Carefully add M THAN and adjust 9H to 6.4. Measure the IgG concentration and add 5g IgG per 100* of solution.
The precipitate of Example 1 is diluted or further added to contain . Other species of Tween instead of Tween80
en or Pluronic 68 may also be used.

To make 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, the crystals are dissolved 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 intravenous administration is desired without the undesirable side effects normally associated with intravenous administration of gamma globulin. It has been proven that there is.

procedural amendment September 9, 1986

Claims (1)

[Claims] 1. Anti-complement activity is 0 to about 0.02 units per mg, sedimentation constant is 7S, molecular aggregates and degradation products F(ab)_
1F(ab)_2, a gamma globulin that does not contain Fc, is colorless and transparent when dissolved in water, and has an antibody spectrum and subclass distribution similar to that of the raw plasma, suitable for intravenous administration. Claim 1 consisting of gamma and globulin dissolved in an aqueous solution containing 2. albumin, a nonionic surfactant, and a glycine acetate-acetate buffer (pH 5.4 to 6.7).
intravenous gamma globulin solution. 3. The gamma globulin solution according to claim 2, wherein the nonionic surfactant is Tween 80 or Pronic 68.