Polyvalent hyperimmunoglobulin preparation.

The invention relates to a new polyvalent hyperimmunoglobulin preparation and a method for obtaining this polyvalent hyperimmunoglobulin preparation.

The serological differences between bacteria are determined by surface-bound, species-specific antigens. As a result, specific antibodies, for example against Salmonella, do not or only very weakly to antigens of other germs, e.g. B. Pseudomonas bind. The consequence of this is that a special hyperimmunoglobulin or hyperimmune serum is required for the therapy and prophylaxis of bacterial infections for each particular germ. This significantly restricts the use of hyperimmunoglobulins.

On the other hand, it is known that the group of gram-negative germs (Salmonella, E. coli, Serratia, Pseudomonas and others have a common antigen. Von Braude and Ziegler ^* immune serum directed against this antigen was obtained and successfully used in patients with gram-negative infections. This hyperimmune serum was obtained by immunizing volunteer donors with a vaccine of the E. coli mutant J5. It is therefore known that by vaccinating donors with a vaccine of the E. coli mutant J5, an immune serum effective against the antigen common to the gram-negative germs can be obtained.

An object of the present invention is to obtain an immunoglobulin preparation with activities against gram-negative germs, which can be obtained without having to previously immunize voluntary donors with E. coli J5.

Another object of this invention is an immunoglobulin preparation which also has activities against gram-positive germs.

These goals are achieved with the present invention.

If the antibodies against the E. coli J5 antigen are determined in non-immunized plasmapheresis donors, it is found that even normal populations of non-vaccinated persons have activities against the antigen common to the group of gram-negative germs, ie that antibodies against the E coli J5 antigen occur naturally, so that a donor strain with high titers against E. coli J5 antigen can be determined. However, if one determines the entire spectrum of antibodies

^* Ziegler er al., The New England Joumal of Medicine 307. 1982, pp. 1225 to 1230

Plasmapheresis donors with high activities against E. coli J5 surprisingly show that the antibody activities against other gram-negative germs are also significantly increased. This is not the case in a donor population with low titers against E. coli J5; here (Fig. 1) only one donor shows less than 10 titers against other germs.

In addition to the titers against gram-negative germs, however, surprisingly, the titers against gram-positive germs are also significantly increased. This is all the more surprising since gram-positive germs differ from gram-negative germs by a fundamentally different wall structure.

While high titers against E. coli J5 of a plasma with high titers correlate against other germs, on the other hand a high titer (1: 320) against Pseudomonas with low titer against E. coli J5 (1:20) or against Staphylococcus (<10) be connected. It was also found that plasma can have a titer of 1: 160 against E. coli and at the same time <10 against staphylococci and streptococci. From this it follows that above all screening of plasma for high titers against E. coli J5, but not against other antigens, leads to a selection of plasmas which have high antibody titers against both gram-negative and gram-positive germs.

Screening among plasmapheresis donors with increased titers against E. coli J5 can thus also be used to determine those donors with increased titers against other gram-negative germs and also against gram-positive germs.

In addition to the E.coli J5 antigen, there is another common antigen in the group of gram-negative bacteria, Lipid A, which is also part of the antigen structure of these bacteria. Lipid A is also responsible for toxic reactions (pyrogenicity). By screening plasmapheresis donors with increased titers against lipid A, donors with increased titers against other gram-negative and gram-positive germs can thus be identified in the same way as described above in connection with the E. coli J5 antigen .

Provides from the plasma of such donors, for. B. via the Cohn fraction 11/111 or 111 according to known methods, an immunoglobulin preparation results in a preparation which is distinguished by high titers against gram-negative and gram-positive germs. Table 1 shows these titers in comparison with a conventional immunoglobulin preparation.

If the immunoglobulin preparation obtained in this way is examined, it is found that, in addition to IgG as the main constituent, it also contains substantial amounts of antibodies of the classes IgA and IgM. IgG and IgA are generally 7s molecules; IgM is generally a 19s molecule - (see Lexikon Biochemie, Leipzig 1976, p. 509). The fragments of the above-mentioned molecules obtained in a known manner can also be contained in preparations according to the invention.

In this way, an immunoglobulin preparation is obtained, which can be used against a broad spectrum of bacterial infections, including mixed infections, and at the same time has activities which correspond to special hyperimmunoglobulins against different germs, and which is therefore a polyvalent hyperimmunoglobulin preparation.

The invention is explained in more detail using the exemplary embodiments below.

Example 1:

Plasmas were screened for high titers against E. coli J5 using the passive hemagglutination test. The supernatant of an autoclaved overnight culture of E. coli J5 (ATCC 39041) was used as the antigen.

Plasmas with reciprocal titers from 1: 320 were selected and combined.

4240 ml of this plasma pool were fractionated according to Cohn, fraction II / III being worked up using octanoic acid and calcium phosphate. 207 ml of a 5% immunoglobulin solution were obtained, which was modified by means of β-propiolactone for intravenous administration. After intensive diafiltration and subsequent sterile filtration, approximately 180 ml of the final product were obtained.

The preparation contains 4160 mg / 100 ml IgG, measured using Kallestad Quantiplate, 480 mg / 100 ml IgA and 960 mg / 100 ml IgM. The determination of the antibody titers in the passive hemagglutination gave the values ​​shown in Table 1.

Example 2:

In accordance with the processing of Example 1, 200 ml of IgM-enriched immunoglobulin solution for intravenous administration were obtained from 4290 ml of screened plasma.

Example 3:

From 4.35 liters of screened plasma, 50 ml of an IgM-enriched immunoglobulin solution (5%) were obtained according to Example 1, but via the Cohn fraction 111. The antibodies against lipid A in this preparation were about 20 to 38 times higher than in a serum pool of unsanitary blood donors.

Example 4:

2.4 liters of screened plasma turned into 1.4 liters of a stabilized serum preserve by treatment with β-propiolactone, UV and Aerosil^R produced according to the process described in EU-A-14 333. Again, the antibody titers against lipid A were about 20 to 40 times higher than in a plasma from unsanitary blood donors. The antibody titers of this preparation against various other antigens are shown in Table 2.

Animal experimentation

To determine the effectiveness of the polyvalent hyperimmunoglobulin produced from screened plasma, 0.5 ml of a 5% strength solution was administered intravenously to mice, which were now intraperitoneally infected with 10 'CFU (colony-forming units) of Pseudomonas aeruginosa per animal. A group of animals which received immunoglobulin from unscreened plasma served as a comparison. In the control group, the infected animals remained untreated. Each group comprised 21 animals. 25th Hours after infection, only 14.3% of the animals survived in the untreated control group. Immunoglobulin from unscreened plasma protected 47.6% of the animals (= 10 out of 21); in the group of animals treated with immunoglobulin from plasma screened, 71.4% (= 15 of 21 animals) were protected - (Table 3). Thus, in the group treated with immunoglobulin from unscreened plasma, nearly twice as many animals died as a result of infection than in the group treated with immunoglobulin from plasma screened. The preparation is therefore much more effective.<tables id="tabl0001" num="0001"><img file="EP0201004A2_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0201004A2_D0002.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0201004A2_D0003.tif" /></tables><img file="EP0201004A2_D0004.tif" />