DE1792555C3 - Process for the preparation of a gamma globulin solution suitable for intravenous use - Google Patents

Description

The invention relates to a method of manufacture a gamma globulin solution suitable for intravenous use.

Gammagio produced by fractionation As is well known, bulin has the property. Fix complement. (Parandum, p. "The Gamma Globulin Therapy", S. Karger Verlag, Basel 1964, 32). It is for this reason for intravenous use not suitable, but only for intramuscular use. Because the antibody effectiveness but when administered intravenously it is more complete and, above all, occurs immediately, it has There have been no lack of attempts to produce intravenously tolerated gamma globulin preparations.

The biological function of the antibodies consists in the elimination of bacteria and viruses as well as in animal neutralization of toxins. The antigen-binding fragment of the antibody molecule takes care of this first for the detection and neutralization of pathogens. The resulting antigen-antibody complex activates the complement-binding fragment of the molecule. This will be set in motion a series of subsequent immunological reactions, the most essential of which is phagocytosis and the specific activation of the complement system. The specific activation of the complement system, which must be clearly distinguished from the unspecific (anti-complementary effect), causes bacteriolysis in bacterial infections. As a further consequence of complement activation Anaphylatoxins and chemotactic factors released "dosed". The anaphylatoxins take care of it Increase in tissue permeability for an increased antibody concentration at the site of infection, while the chemotactic factors support phagocytosis Defense against infection the complement-binding fragment (Fc part) of the immunoglobulin G molecule is an outstanding one Meaning / u. Of particular clinical importance is the in vivo half-life of that in intravenous Antibodies containing gamma globulin. The longer the in vivo half-life. all the longer the supplied antibodies circulate in the blood and the longer they stand the door of prophylactic effectiveness ZUf Disposal «This is critical Significance in diseases in the course of which the organism is not able to form antibodies. In such cases, one is forced to to continuously supply the body. The less often this is necessary, the simpler one in particular is to carry out outpatient treatment.

Processes are already known to convert from complement-fixing human standard gamma globulin to produce an intravenously tolerated preparation. These procedures are based on the fact that the by conventional fractionation methods such as Cohn alcohol fractionation and -2-ethoxy-6,9-diaminoacridine ammonium sulfate fractionation obtained complement-fixing human standard gamma globulin hydrolytically into the individual fragments split and so the complement fixation is eliminated. One of these two methods carries out the splitting with pepsin (Schulze, H. E. and Schwick, G., DMW 87, 1643 [1962]), the other method uses hydrolysis with hydrochloric acid at a pH of 4.0 (B a rand um, S., Kistler, P., Vox. Sang. 7, 157 [1962]). The preparations obtained ran in hers Half-life under the native Ga.nmaglobulin (G. Riva, Helvetica Medica Acta 415, 286 [1963]).

Surprisingly, it has now been found that a PJr is suitable for intravenous use Gamma globulin with an antibody half-life that is longer than that of known products obtained when standard human globulin is treated with critical amounts / f-propiolactone. While protein degradation or denaturation occur in the known processes, remains with that product manufactured according to the invention for the mobilization of the immunological auxiliary mechanisms mentioned got responsible fc-fore of the antibody molecule. Proof of the intactness of the Fc part is the fact that antibodies in addition to the neutralization and hemagglutination method also with the Kom:> element binding reaction (KBR) The half-life of the invention Gamma globulins corresponds to the natural half-life of immunoglobulins.

One already has ^ -propiolactone for treatment used by rabbit immune sera. The applied however, high concentrations caused a destruction of the antibody properties, so that the proteins denatured by the modification are only in shape for emc cosmetic use of creams and ointments come into question (J oos.

Chem. Abstracts. 55 25 74 [1961]).

The invention relates to a process for producing a gamma globulin solution suitable for intravenous use, which is characterized in that 0.043 ml of ί-propiolactone per g protein is added to a solution of standard gamma globulin at room temperature and a pH of 8.0 and left to stand for 1 hour while adjusting the pH to 8.0, then left to stand at 37 ° C. for about 3 ' ₂ hours while adjusting the pH to 8.0, dialyzed and sterile-filtered.

The beneficial properties of mit, i-propiolactone Gammaglobuhns modified according to the invention show the following studies. with a 5 "solution were carried out:

L. Clinical Tolerance

A 5% solution was injected. The compatibility of the überwiegönd immunoglobulin G-keeping preparation was good even with rapid injection. No incidents were observed in 46 Pföbänder with a total of 95 injections; The effectiveness corresponds to

so far it has been established that it is intramuscularly applicable Preparations whose disadvantages such as painfulness, hematoma formation in hemorrhagic diathesis, Infiltrates, losses through proteolysis and slow resorption, however, are absent.

2. Acute tolerance in the mouse 100 ml / kg are tolerated without reaction.

3. Chronic tolerance

The preparation was injected intravenously into rats every 2 days for 3 weeks at a dose of 20 ml / kg in total Each rat received 10 injections with a total of 200 ml kg. The starting weight of the rats was about 80 to 100 g (not more than 120 g). The weight was checked once a day weekly differential blood counts as well Blood cell sedimentation rate and white blood cell count determined. A kidney function test was carried out at the end of the trial (Phenol red excretion) and a macroscopic dissection finding was also made: it was found in comparison to the NaCl control no pathological changes.

4. Antigenicity

No precipitants were found by intramuscular and intravenous immunization of rabbits Antibodies to those caused by the / J-propiolactone treatment received groups.

5. Complement Fixation and Antibody Activity Table I.

Priiparai

'/ -Globulin produced by ^ -ethoxy-o ^ -diaminoacridine ammonium sulfate fractionation of human serum

j-globulin according to the invention

measles

1: 8192 1: 8192

Polio!

: 50: 50

Poüoü Pockcn-
vaccine 1:50 1.32 1:50 1:32

Knmplfmenihr 0.5 ml

0.40 ml

corresponds to a 0.9% strength

NaCI solution

6. Antibody Activity Table 2

Antibodies against

Measles virus

Herpes simplex virus ...

Influenza A ₂

Influenza B

Parainfluenza 1,2,3

Cytomegalovirus

Rubella Virus

Smallpox virus

Aü / SH antigen

Polio I

Proteus vulgaris

Pseudomonas aerogenes

E. Coli

Klebsiella

Salmonella

Antistreptolysin O

Γι tnik

KBR

KBR

KBR

KBR

KBR

KBR

HHT

HHT

HHT

NT

HT

HT

HT

HT

HT

HT

Reciprocal tilers

20th

40

40

40

40

20 512

16

16

64

80

80 160

80

80 450 I.U./ml antibody deficiency and by measuring specific Antibody activities determined.

Results

method

Specific antibody activity

Half-life
Time

(Days)

literature

HFI Γ Mamagglulination Inhibition Tesl.

M Γ HamagglutirKiltons-Tesl

N Γ neutralization-proof.

KBR K'implemcnlbindungsrcaktion.

7, half-life of immunoglobulin G

The half-life of the / ^ propiolactone modified ImmUnglobuHns G was determined by following the total immunoglobulin G content in patients with 17 B. Kornhuber, Klin. Wochenschrift, 51 (3), 135 (1973)

Total immunity 14 W. S tan gel and

globulin content H. Deicher, Bio

test bulletin 29, 59 (1972)

The half-life is thus comparable to the half-life found for normal human immunoglobulin from 14 to 28 days.

example

Standard gamma globulin solution (protein concentration 7 to 10%) is ^{adjusted to a pH of 8.0 at 20 °} C. and 0.043 ml / i-propiolactone per g protein is added. The mixture is left to stand at room temperature for 1 hour, during which the pH is readjusted to 8.0 every 20 minutes. The hydrolysis is then carried out at 37 ° C., the pH value being readjusted every 20 minutes (hydrolysis time approx.3V2 hours), then dialyzed against physiological saline solution for 20 hours, centrifuged at 5000 rpm for 10 minutes if necessary, the solution adjusted to 5, 0% protein and filtered sterile through membrane filters.

Claims (1)

Claim: A process for the preparation of a gamma globulin solution suitable for intravenous use, characterized in that 0.043 ml / i-propiolactone / g protein is added to a solution of standard gamma globulin at room temperature and a pH value of 8.0, and the mixture is adjusted for 1 hour The pH value is left to 8.0, then left to stand at 37 ° C. for about 3 ¹ I ₂ hours while the pH value is readjusted to 8.0, dialyzed and sterile-filtered.