DD289204A5 - METHOD FOR REMOVING AND / OR OBTAINING IMMUNE LOBULIN M FROM WAESSRESS SOLUTIONS - Google Patents

Abstract

The invention relates to a process for the removal and / or recovery of class M immunoglobulins from aqueous solutions, in particular from blood plasma or blood serum. According to the invention, the process consists of selectively adsorbing specific plasma proteins, such as immunoglobulin M, onto polymeric adsorbents based on crosslinked acrylonitrile-vinyl acetate copolymers functionalized by reaction with polyalkylene polyamines. As a result, the removal of said substances from the initial aqueous solution is possible. The adsorbed special proteins can be recovered by desorption and optionally purified by standard techniques. The applications of the invention are in the fields of medicine, medical technology and protein preparation. {Adsorbent, selective; Plastic; proteins; Blood; Immunoglobulin M; Preparation; Medical}

Description

are prepared, adsorbed and enriched and then removed by means of an electrolyte solution and optionally purified in a second step by conventional methods.

Fields of application of the invention

The invention relates to a process for the removal and / or recovery of immunoglobulins of class M from aqueous solutions, in particular from blood plasma or blood serum and is applicable in the fields of medicine, medical technology and protein preparation.

Characterization of the known prior art

Class M (IgM) immunoglobulins are the antibodies of the primary humoral immunological reaction and play an important role in the defense against bacterial disease processes. IgM antibodies are able to agglutinate larger particles such as bacteria, large viruses and cells particularly well and to dissolve them with the help of the complement. Here they are the IgG antibodies superior (J.H.Humphrey and R.G.White: Short textbook of immunology, Thieme Verlag Stuttgart, 1971, p 110-112). These skills, which have been known for quite some time, have recently returned to the center of interest since investigations have shown that surgical procedures in septic (bacterial-purulent) diseases avoid or mitigate the particularly frequent complications of administering IgM-enriched preparations ( J. Seifert and D. Nietschke: "Immunoglobulin M", Deut.Med.Wschr.112, 1267-1271 (1987)).

The preparation of polyclonal IgM, if possible from mixed sera of many donors, thus regains interest. So far, this preparation required at least four steps (J. Brock: "Preparation of immunoglobulins" in: H. Friedrich [Hrsg.]: Immunological working methods, Fischer, Jena, 3rd ed. 1984, p 432 ff .; K. Heide and HG Schwick: "Salt fractionation of immunoglobulins" in: DM Weir (ed.): Handbook of Experimental Immunology, Blacwell Publication, Oxford, 3 ^rd edition, 1979, Chapter 7)

 In detail, the following steps are required to obtain pure IgM from blood plasma:

1. Separation of lipoproteins by precipitation with dextran sulfate.

2. Fractional precipitation of the remaining plasma proteins by salt addition or polyethylene glycol.

3. Chromatographic separation on DEAE-Sephadex and / or gel chromatography.

4. Electrophoretic separation.

The known methods for obtaining IgM are thus complicated, laborious and costly.

Certain antibodies, so directed against blood group specific Ervthrozyten structures (H, A, B) so-called. Isoagglutinine, also a large part of rheumatoid factors, also monoclonal antibodies in certain tumors of the bone marrow (Waldenström's disease) belong to the IgM class.

Your removal from the blood is u. U. offered as a therapeutic measure, z. As the removal of isoagglutinins before transplantations, if between donor and recipient ABO incompatibility (intolerance of blood groups) exists. For this purpose, immunoadsorbents have been developed, the production of which is relatively complicated and expensive and which are therefore expensive (Bensinger, WI, et al .: "Immunoadsorption for removal of A and B blood group antibodies" New Engl. J.Med.304, 160-162 Bannen, AD, et.al .: "Immunoadsorption and renal transplant in two patients with major ABO incompatibility." Transplantation 43, 909-911 [1987]; Osterwalder, β., Et al .: "Immunoadsorption for removal of anti-A and anti-B bloodgroup antibodies in ABO-incompatible bone marrow transplantation. "Blood 53, 379-390 [19861].

These immunoadsorbents consist of Siiikatpartikel to which the corresponding blood group antigens are covalently coupled. These, by their structure, oligosaccharide antigens, can be synthesized. To remove the isoagglutins from the plasma, the latter is passed through columns containing silicate granules loaded with the specific blood group antigen.

The "matching" IgM isoagglutinins (specific IgM antibodies) bind to it.

Object of the invention

The aim of the invention is to demonstrate a simple and inexpensive process for the removal and / or recovery of IgM from aqueous solutions, in particular blood plasma and serum.

Explanation of the essence of the invention

It is an object of the invention to develop, using polymeric selective adsorbents based on plastics, an enrichment method for class M immunoglobulins which simplifies further preparation. According to the invention, this object is achieved in that IgM to polymeric adsorbents by copolymerization of acrylonitrile, vinyl acetate and a crosslinker in the presence of a filling and / or swelling agent for the copolymer by the process of suspension polymerization in a particle size of 0.1-0, 3 mm and subsequent reaction with a polyalkylenepolyamine of the formula

, R * R-

f = 2-8

H. R * R- = H or alkyl group

/ Π χ,

R * R ^

ι ι

are prepared, adsorbed and enriched and then removed by means of an electrolyte solution and optionally purified in a second step by conventional methods.

In incubation and perfusion experiments, it has been found that these adsorbents selectively bind certain plasma proteins, including class I immunoglobulin.

Before use, the adsorbents should be adjusted to pH values of 5.5 to 7.5. To remove the proteins from the blood plasma or serum, the adsorbents may be directly suspended therein or applied as a packing in a chromatography column. In both cases, the adsorbents are first several times with distilled water. washed and the washing liquid, z. B. by centrifugation, largely removed. Thereafter, they are contacted with 0.1 M glycine-HCl buffer, pH 2.8 or 0.01 M HCl, after removal of the acidic solutions with phosphate-buffered 0.15 M NaCl solution (PBS), pH 7.4 washed several times until the adsorber is equilibrated in the desired pH range. Before use, the PBS is removed as completely as possible and the residual moist adsorber substances with the protein-containing aqueous liquid, eg. As serum, brought into contact. This can be done in a simple manner so that one suspends the adsorbents in the serum and with gentle shaking 10 to 120min z. B. incubated at room temperature or 37 ⁰ C. This method can be used to prepare IgM. For this purpose, the adsorbents are washed after contact with serum with 0.15 M NaCl solution and then the desorption with acidic solutions or higher concentrated saline solution, pH 4.5 to 6.5 made. The eluates contain, besides the high molecular weight IgM some plasma proteins having molecular weights below 200,000 daltons as the complement factors C3, C4 and C9, prealbumin, α, - and C ₂ -GIycoprotein and ceruloplasmin. You can in a Nachreinigungsschritt, z. B. by gel chromatography on Sephadex G 200, are separated. The described method for IgM preparation represents a substantial simplification of the hitherto conventional methods, since instead of the four process steps required according to the prior art, only two steps are required to obtain pure IgM. The crucial step here is the enrichment of the IgM on the adsorber used.

In contrast to the known adsorbers, such. B DEAE-Sephadex, only IgM and plasma proteins with a molecular weight below 200,000 daltons are adsorbed on the adsorber used according to the invention. Thus, pure IgM is already obtained after the process stages adsorption / elution and gel chromatography. In the following examples, the adsorbents of the invention and their use should be described more closely.

example 1

Binding pattern of adsorbents for serum proteins

The adsorbent material is first three times, each with about five times the amount (parts by weight of water-moist adsorbent to weight or volume of washing liquid) distilled water. washed, then treated two to three times with also five times the amount of glycine-HCl buffer, 0.1 M, pH 2.8 or 0.01 M HCl and finally with phosphate buffered 0.15 M NaCl solution (PBS), pH 7.4, washed several times and brought to a pH of 7.2 to 7.4.

By centrifugation (5 min, 1000 g) followed by decantation or suction through a frit or a filter, the PBS is removed and 1 g of the residual moist adsorbent mixed with 2ml mixed serum (obtained from a mixture of equal volumes serum of at least 10 blood donors) and 30min at After incubation in a shaking water bath or shaking at room temperature, the serum is separated from the adsorbents and analyzed.

The total protein was determined by the biuret method, that of the individual proteins by means of simple radial immunodiffusion according to Mancini and coworkers. Since mixed serum was used by many individual donors, d. H. If values of the normal ranges could be assumed approximately, it was sufficient to compare the values of the mixed serum before and after incubation and to indicate the latter as relative values of the former in%.

For the determination of a control area each of transferrin were a _r Macroglobülin, beta-lipoprotein, and immunoglobulin G influenced, of which a selective binding has never been observed. The decreases in their values after incubation are rather an expression of the dilution that the serum undergoes by residues of the suspending agent (PBS) and of a presumable, low-grade nonspecific adsorption on the inner and outer surfaces of the porous particles of the adsorber substances. The mean values of the four reference proteins plus or minus twice the standard deviation (x ± 2 s) form the control area. In general, it is 80 ± 10%. Selective binding is only assumed if the protein values after incubation (mean values of three determinations including the standard deviations) are below this control range.

The results of the determinations of serum proteins are shown in Table 1.

Example 2

Binding of Isoagglutinins of the Immunoglobulin Class M

Example 1 demonstrated the binding of polyclonal and polyspecific IgM of human serum. But also defined IgM specificities are bound, as the following experiment shows.

The naturally occurring antibodies directed against structures of human erythrocytes of blood group O, A

or B, the so-called isoagglutinins, belong to the IgM class. Up to serum dilutions of 1: 128 to 1: 256, red blood cells were agglutinated in 1% suspension of the respective blood groups; Α serum agglutinates 8-erythrocytes and vice versa, O-serum both A- and B-erythrocytes. Table 2 shows that after analogous preparation of the adsorbents and incubation with the serum samples, as stated in Example 1, the agglutinate monosters decrease by two to three stages. This corresponds to 75-87% isoagglutinin binding, i. H. Removal of specific IgM antibodies from the serum samples.

Example 3

Binding of Rheumatoid Factors (RF)

After analogous preparation of the adsorbent and incubation with serum samples from three patients suffering from Rheumatoidarthritiden, such as. in Example 1, a titer drop of the RF was again detectable by two to three steps with the tests of Podliachouk and Harboe, the latex fixation test and the acrylic fixation test. The total IgM also dropped to 12 to 21% of the starting concentration.

Example 4

Regeneration of adsorbents and repeated use

The first incubation with serum was carried out analogously to Example 1, after which the adsorber materials were washed once with 0.15 M NaCl solution, after removal of this solution with approximately five times the volume of glycine-HCl buffer 0.1 M, pH 2.8 , Incubated for 15 min at room temperature and shaken, then the buffer liquid removed and replaced with fresh solution. This treatment was done a total of three times. Thereafter, the plastics were treated similarly with PBS, pH 7.4, until the pH of the supernatant PBS was 7.2-7.4.

After removal of the PBS, serum could again be added for adsorption on the regenerated adsorbents. A total of ten adsorptions were made on the total of nine times regenerated Adsorberstoffen. Table 4 shows that the adsorbents were always regenerable. A drop in their binding capacities was not apparent.

Regeneration is also feasible by perfusion if the adsorbents are used as column packing

become. Instead of the glycine-HCl buffer, 0.5 M NaCl, dissolved in SORENSEN buffer, VisM, pH range 5 to 6, can be used with at least the same effectiveness. In the eluates can be detected 60-70% of the serum IgM.

Example 5

Preparation of immunoglobulin M

Prior to application of the resin, it is adjusted to a pH of 7.2 to 7.4 in the manner described in Example 1.

After reaching the desired pH, the adsorbent resin is separated from the buffer by filtration. The moist adsorber is stirred at room temperature in twice the amount of serum. Allow to stir for a little while, stirring occasionally, then separate the adsorber from the serum by filtration and wash twice with 0.15M NaCI solution.

The desorption of the bound proteins is carried out by repeated treatment with 0.5 M NaCl solution buffered with ¹ AsM phosphate buffer, pH 5.5. The combined eluates contain IgM as the main component, as well as proteins with molecular weights below 200,000 daltons. After concentration of the eluates, a fine purification by means of gel filtration on a Sephadex G-200 column in the usual manner.

With the described method it is possible to isolate pure IgM in a simple manner. The yield of pure IgM is 24-31%, based on the amount present in the starting material (serum).

Table 1

Protein amounts in the plasma after incubation with the adsorbers 1 and 2, based on the protein amounts before the adsorption of the plasma (= 100, stated in%)

protein Adsorber 1 Adsorber 2 prealbumin 0 0 albumin 73 ± 0 67 ± 6 α, glycoprotein 40 ± 6 - dj-glycoprotein 30 ± 6 - haptoglobin 67 ± 1 53 ± 4 hemopexin 79 ± 7 88 ± 3 ceruloplasmin 0 0 a ₂ -macroglobulin 78 ± 5 84 ± 4 transferrin 86 ± 5 81 ± 3 ß-Lipoproiein 85 ± 3 80 ± 1 IgG 79 ± 5 84 ± 3 IgA 70 ± 4 72 ± 4 IgM 17 ± 0 20 + 2 IgD 90110 90 + 2 IgE 79 + 3 85 ± 3 C, q 76 ± 4 76 ± 9 C4 0 - C3 32 + 6 5 ± 0 C9 0 _ C 1INA 26 ± 4 - C3-activator 85 ± 4 -

Adsorber 1 acrylonitrile-vinyl acetate-divinylbenzene copolymer, functionalized by reaction with 1,3-diaminopropane.

Adsorber 2 Acryinitrile-vinyl acetate-divinylbenzene copolymer, functionalized by reaction with 3-dimethylamino-i-propylamine.

Table 2

erythro agglutination after incubation with Adsorber 2 Serum · cytes before incubation Adsorber 1 1:32 B 1:32 1:16 A A 1: 128 1:16 1:64 B A 1: 128 1:64 1:32 O B 1: 256 1:32 O 1: 128

Table 3 a

RF titres in the sera from 3 patients before (upper word) and after incubation (lower value) at the adsorbent 1 determined in different tests

patient

Podhachouk- Latexfixa- Acrylfixa- titres Harboe test tion test tion test waste 1: 128 1: 256 1: 256 2,3,3 1:32 1:32 1:32 1: 512 1: 1024 1: 1024 2,2,2 1: 128 1: 256 1: 256 1: 256 1: 256 1: 256 2,3,3

1:64

1:32

1:32

Table 3 b

IgM concentrations (g / L) in the sera of the patients before and after incubation on the adsorber

patient

in front

IgM adsorption

to

Concentration of the IgM after adsorption (before = 100%)

K.G

 M.L.

 K.E.

1.46 2.55 1.36

0.21 0.54 0.16

14 21 12

Table 4

Relative IgM concentrations after repeated adsorption on regenerated adsorbents (in%, in each case in comparison to the serum samples before incubation = 100%)

adsorber Adsorption 1. Third 5th 7th 9th 10th 1 2 29 ± 5 24 ± 5 46 + 1 39 + 1 30 ± 3 16 + 3 29 ± 1 19 ± 2 33 ± 2 26 + 1 31 ± 2 2 <»± 3

Claims (1)

Process for the removal and / or recovery of class M immunoglobulins from aqueous solutions, characterized in that the immunoglobulin is based on polymer-based adsorbents obtained by copolymerization of acrylonitrile, vinyl acetate and a crosslinking agent in the presence of a filler and / or swelling agent for the copolymer according to the process of suspension polymerization in a particle size of 0.1 to 0.3 mm and subsequent reaction with an α / ω-polyalkylenepolyamine of the formula H ft, R ₁ β H or alkyl group ^{S is} N- (CH ₂ ) _n -N ^ η s 2 to 8