DD280174A1 - PROCESS FOR REMOVING IMMOBILIZATION LOBULIN AGGREGATES IN IMMUNE LOBULIN PREFERENCES - Google Patents

Abstract

The invention discloses a method for the removal of immunoglobulin aggregates from immunoglobulin preparations by biospecific binding thereof to the protein C1q, which is immobilized on siliceous solid phases by means of silane coupling agents and homo- or hetero-bifunctional reagents, whereby the bound immunoglobulin aggregates are separated by separation of the solid phase and the solid phase is regenerated by treatment with non-denaturing media. The invention finds application in the pharmaceutical industry.

Description

Field of application of the invention

The invention relates to a method of selective elimination of immunoglobulin aggregates from immunoglobulin preparations (gamma globulin) with repeated and reproducible use of a coated carrier. The method is applicable in the pharmaceutical industry as well as medicine.

Characteristic of the known state of the art

A number of different immunoglobulin preparations have been developed, which are used both for the prophylaxis of certain viral and toxic diseases (poliomyelitis, measles, rubella, hepatitis, tetanus) and for the substitution of antibody deficiency states as well as for treatment of serious bacterial infections with or without disturbing the humoral immune system become. Considering the given clinical situation, the use of these preparations should be critically weighed, as all these preparations - due to the manufacturing process - have advantages and disadvantages

By far the most common is still the standard Gammaglobulin obtained by the classical Cohnschen Alkoholfällungsverfahren. This preparation is characterized by the native structure and function of the IgG immunoglobulins, which is expressed in the normal biological half-life, a retained ability of complement activation and opsonization. However, these preparations also contain polymeric immunoglobulin aggregates which spontaneously activate the complement system even in the absence of antigens, so that life-threatening anaphylactoid reactions can occur, especially in immunodeficient patients. Therefore, such preparations should not be administered intravenously, so they z.T. can not be used in therapeutically adequate dosage and are absorbed only slowly from the muscle depot.

That's why you're on i. v. instructed applicable immunoglobulin preparations, which can trigger almost no complement activation. For this purpose, various methods and procedures have been developed, all of which, however, have disadvantages. Essentially, the following ways were labeled:

enzymatically split off of the Fc part of the IgG molecule by means of pepsin or plasmin,

chemical modification of the IgG molecule so that the IgG aggregates lose the ability to spontaneously activate complement,

selective removal of the aggregates formed by precipitation with polyethylene glycol, treatment with adsorbents or gentle acid treatment,

- Prevention or limitation of the formation of IgG aggregates during storage by the addition of albumin. All these methods lead to a more or less strong influence or change in the structure and function of immunoglobulins, so that the effectiveness is reduced. Thus, while the proteolytically cleaved fragments are well tolerated, their immunological effector functions, such as activation of the classical pathway of complement system, stimulation of phagocytosis and their biological half-life are greatly reduced due to the lack of Fc portion. Analogously, various biological effector functions are modified or even completely suppressed by chemical modification of the IgG (β-propiolactone, alkylation). On the other hand, unmodified, native immunoglobulins always present the risk of spontaneous aggregation during the production process and storage, especially since the agents used hitherto did not lead to quantitative or selective precipitation of the immunoglobulin aggregates.

French Patent 2279419 describes a method of removing immunoglobulin agregates from gamma globulin preparations using biospecific binding to rheumatoid factors and the liquid and solid phase protein C 1q.

In this case, the immunoglobulin aggregates to be removed are either precipitated by the addition of rheumatoid factors or C1 q or, after prior immobilization thereof, bound to sepharose. In the former case, the precipitated immunoglobulin aggregates are removed by centrifugation. In addition to this cumbersome for technological process step is a major disadvantage, the risk of contamination of the immunoglobulin preparations to be administered by rheumatoid factors or C 1q, which would lead to significant side effects of such preparations. While binding rheumatoid factors or C 1q to sepharose, these disadvantages are largely circumvented, however, the carrier used is in turn associated with a number of disadvantages. So polysaccharides such as Sepharose are easily attacked and degraded microbially. Furthermore, they are stable only up to 40 ° C and can therefore be difficult to sterilize.

The chemical stability of proteins bound to Sepharose® is in many cases not sufficient, so that upon elution, in particular using chutrophic ions such as ammonium thiocyanate or pH values below 4 or above 9, contamination of the immunoglobulin preparation with the bound protein may occur. which can give rise to the already mentioned side reactions. All of these disadvantages include as well as the described use of ammonium thiocyanine. to cleave the aggregates as a denaturing agent from the reusability of the matrix, so that using this method would incur significant costs.

Object of the invention

The object of the invention is to remove immunoglobulin aggregates selectively, simply and gently from immunoglobulin preparations. The carriers can be easily, quickly and reproducibly regenerated for reuse.

Explanation of the essence of the invention

The object of the invention is to biospecifically bind immunoglobulin aggregates of immunoglobulin preparations to a solid phase (support), in which case the solid phase should be reusable.

According to the invention the object is achieved in that the protein C 1q - a subunit of the first complement - to silicon dioxide-containing support, preferably porous glass or sheet-like glass fiber filter is covalently bonded via silane coupling agent and this carrier-bound C 1q is used for binding and removal of the immunoglobulin aggregates, wherein the immunoglobulin aggregates bound to the C 1q are separated from the immunoglobulin preparations by separation of the solid phase and the fosted phase is functionally regenerated by treatment with non-denaturing media.

The inventive method is characterized in that the protein occurring in human and animal blood and responsible for the binding of immune complexes and their physiological elimination protein C 1q to silica-containing support with sufficiently high surface area and porosity known per se prior chemical surface modification of the support with SilanhaK. nitteln and hetero or homobifunctional reagents is bound and this carrier-bound C1q is used for the in-vitro removal of immunoglobulin aggregates from immunoglobulin preparations and then the aggregates thus bound are cleaved again by non-denaturing media, so that the loaded with C 1q matrix used again can be.

The carrier has a different geometric shape, wherein spherical or fibrous carrier with or without additives, which can be arranged by packing, sintering or use of a binder to higher-level structures such as filters, frits and the like. With defined permeability, are particularly suitable. The silicon dioxide-containing materials are essentially porous glasses (see this: F. Janowski, W. Heyer: Porous glasses, Dt., Verlag für Grundstoffindustrie, Leipzig 1982), which can be known sterilized and kept sterile, with their particle size, structure and Porosity is precisely determined. Particularly advantageous are ideal spherical particles (DD-WP C03B2595548, DD-WP BO U2694382, DD-WP BOIJ 2694390) with particle sizes between 1-1000mm and pore widths of 0.05-100μπι, where the pore size and the available area, which crucially affects the capacity of the carrier, reciprocally proportional behave. The ideal spherical shape of the matrix has a decisive influence on the gentle conditions of the process, since the shearing forces acting on the proteins are drastically reduced. Furthermore, the ideal spherical and abrasion-resistant glass particles are distinguished by their erosion resistance, their favorable hydrodynamic properties, their variation in texture and thus also in the flow properties and by the absence of toxic constituents.

In particular from the aspect of technological processes, surface-shaped carriers or cartridges with raw material or membranes of porous glass having superordinate structures or planar carriers which are used as filters in suitable filtration systems should be emphasized. Drawing here are especially glass fiber-containing filter materials due to their mechanical strength. The non-denaturing media for the cleavage of the bound immunoglobulin aggregates are composed of buffered solutions of high ionic strength, in particular 0.5-3 m NaCl solutions are suitable. The durability, handling and reuse of the C1 q-loaded carrier is particularly advantageous in that silica-containing carriers show no swelling or shrinkage phenomena upon change of the ionic milieu.

Surface modification of the silica-containing supports for the purpose of creating conditions for covalent bonding with the protein C1 q is accomplished by so-called silane coupling agents such as

OR

x - (CH) -Si-OR), "^ OR

wherein R are alkyl radicals and χ amino, epoxy, diazo, carbonyl, carboxy, isocyano, isothiocyanato, sulfhydryl or nitroso groups, and by homo. By reacting the remaining functional group in a known manner or heterofunctional reagents or direct reaction with the reactive groups of the protein.

The matrix thus loaded with the protein C1 q is then used for binding and removal of the immunoglobulin aggregates, the matrix being able to be used both in the batch process and in the flow-through process. After completion of the reaction and removal of the matrix from the immunoglobulin preparations to be treated, the bound immunoglobulin aggregates are cleaved off by suitable non-denaturing media, such as 1 M NaCl solution, so that the covalently bonded C1 q is available for reuse.

The inventive method uses the physiological function of the protein C1 q for selective and gentle binding and thus removal of immunoglobulin aggregates of immunoglobulin preparations, so that the efficacy of these preparations is not affected, but the compatibility is significantly improved.

embodiments

In the following the invention is explained by some examples.

Example 1:

Porous glass beads having a pore size of 1350 Å are, after boiling with 30% nitric acid and washed neutral with a 2% solution of the silane coupling agent NB1114 (aminopropyltriethoxysilane, VEB Chemiewerk Nünchritz) in ethanol / water (1: 1), pH 3.5 4h at 6O ⁰ C and 6h at 12O ⁰ C activated. After washing with ethanol / water and washing several times with PBS buffer, pH 7.4, the thus-functionalized glass is incubated with 2% glutaric dialdehyde in 0.1 M phosphate buffer, pH 6.8, for 2 h at 37 ^° C. , After washing again with PBS buffer, the activated glass is mixed with C1 q (500 μg ZmI) in PBS buffer, pH 7.4, containing 10 mM EDTA and incubated at 37 ° ^{C. for} 4 h. After further washes, the remaining free aldehyde groups are blocked with 1 M ethanolamine solution. After repeated washing with 0.01 M phosphate buffer, pH 7.4,0.05 m NaCl, this C1 q-loaded matrix is used to bind the immunoglobulin aggregates. For this purpose, the thus loaded with C1 q porous glass with the corresponding immunoglobulin preparation in a batch process and incubated for 4 h at room temperature or 37 ^C C. After removal of the support by decanting or centrifuging, it is subsequently treated with 0.01 M phosphate buffer,

pH 7.4, 0.05m NaCl, and then regenerated with 2m NaCl in PBS buffer, pH 7.4.

The determination of the immunoglobulin aggregates was carried out with a C1 q-solid phase enzyme immunoassay, the determination of the protein concentration by measuring the absorbents at 280nm

 Table 1 shows some characteristic results of such experiments.

Table 1:

Aggregated. IgGlpg / ml)

Immunglobulinpräpsrat

before treatment 480 100

after treatment 148 31

Example 2:

As described in Example 1, C1 q is bound to porous glass. This matrix is used several times to remove immunoglobulin aggregates. After binding and removal of the immunoglobulin aggregates according to Example 1, the matrix is regenerated with 2 mM NaCl solution in PBS buffer, pH 7.0, and brought into contact again with the already treated immunoglobulin preparation, so that the remaining immunoglobulin aggregates are removed. Table 2 shows the results of two characteristic experiments.

Table 2: Aggregated. IgG (pg / ml) (° / Ό) 860 250 120 860 320 150 100 29 14 100 37 17 Immunoglobulin preparation before treatment after I. Treatment after 2nd treatment

Example 3:

Glass fiber filters are functionalized as described in Example 1, activated and loaded with C1 q. These filters are fitted in suitable filtration systems (Sartorius GmbH, Göttingen, FRG), so that the immunoglobulin preparations to be treated come into contact with the C1 q matrix during the filtration process (eg sterile filtration) and the immunoglobulin aggregates are removed. The regeneration of the loaded with C1 q glass fiber filter is then carried out analogously to Example 1 and 2 with

2m NaCl solution in PBS buffer, pH 7.0.

Table 3 shows a characteristic experiment using the C1 q matrix twice.

Table 3:

Aggregated. IgG (pg / ml)

immunoglobulin preparation

before treatment 520 100

after I. treatment 370 71

after 2nd treatment 200 38

Claims (12)

1. A method for the removal of immunoglobulin aggregates from immunoglobulin preparations on the basis of biospecific interactions on solid phases, characterized in that are used as the solid phase siliceous materials, after previous, known chemical surface modification with silane coupling agents and hetero or homobifunctional reagents or are loaded directly with the protein C1q and that after binding of the immunoglobulin aggregates to the Ciq of the solid Phi. By incubation with the immunoglobulin preparation and separation of the solid phase, regeneration of the solid phase occurs by treatment with non-denaturing media and reuse of the solid phase. 2. A process for the removal of immunoglobulin aggregates from immunoglobulin preparations according to item 1, characterized in that the silicon dioxide-containing materials are molded bodies of glass or glass-like materials. 3. Expired after item 2, characterized in that the shaped bodies are present as balls. 4. The method according to item 2, characterized in that the shaped bodies are present as fibers. 5. The method according to item 3, characterized in that the balls are porous and have a pore size of 0.05-100pm. 6. The method according to item 3, characterized in that the balls have a particle size of 1 to 1000 microns. 7. The method according to item 2-6, characterized in that the shaped bodies are formed by packing, sintering or by using a binder to higher-level structures with defined permeability. 8. The method according to item 4 and 7, characterized in that the parent structures have a predominantly planar expansion such as filters, frits, hollow cylinders. 9. The method according to item 4 and 7, characterized in that the fibers are present as nonwovens or tissue. 10. The method according to item 7, characterized in that the parent structures are mainly column packs. 11. The method according to item 1-10, characterized in that the surface modification of the glass is carried out by silane coupling agent and homo- or heterobifunctional reagents. 12. The method according to item 1-11, characterized in that the non-denaturing media are buffered solutions of high quality.