GB2225017A - Immobilization of proteins on solid carriers - Google Patents

Description

9.

2225017- 1 54 620.505 is IMMOBILIZATION OF PROTEINS ON SOLID CARRIERS The present invention relates to an improved process for immobilizing proteins on solid carriers with a high degree of preservation of biological activity.

The immobilization of biologically significant matter on solid carriers has been suggested using a variety of methods (c.f. K. Buchholz "Characterization of Immobilized Biocatalysts" in Dechema Monographs Vol. 84, No. 1724-1731, Verlag Chemie 1979). Inorganic as well as organic materials have proved to be suitable solid carriers. Sometimes naturally occurring starting materials and sometimes synthetically produced materials are used.

The present invention is focussed on carriers bearing functional groups, in particular those groups which are capable of reacting with nucleophilic groups of biologically significant matter such as enzymes, or cells, and thus forming a covalent bond. Quite a few reactive functional (electrophilic) groups known from preparative organic chemistry, which react with the (nucleophilic) amino, thio or hydroxy functions of biologically significant matter under conditions which are biologically acceptable have been used more or less successfully for covalent binding. However it should be noted that the reactive groups on the carrier available for binding may become a limiting factor. Detailed information on this may be found in K. Buchholz loc.cit., also Ullmann's Encyclopadie der Technischen Chemie 4th edition vol. 10, pp.539-545, Verlag Chemie 1975 and Ullmann's Encyclopedia of Industrial Chemistry 5th Ed., Vol.A9, pg.382-383, Verlag Chemie 1988.

Methods of activation which are frequently used comprise for example: reacting carriers bearing OHgroups with cyanogen bromide to form the imides or reacting with halogen substituted S-triazines, diazotisation or reaction with glutardialdehyde with 2 is carriers bearing -NH2- groups and reaction to produce acid azide groups in the case of carriers with -COOHgroups, or the activation methods known from peptide synthesis, e.g. with carbodiimide. Carriers with anhydride or epoxy functions are suitable for an immediate reaction with nucleophiles present, in particular with the amino functions of proteins. Covalent immobilization of biologically significant material has quite a few advantages over other methods of binding. These include, among others: non-bleeding of the material, ready accessibility of active structures which are in general attached to the carrier surface and not infrequently increased thermal stability. on the other hand there are occasional-drawbacks to be contended with: e.g. a) impairment of biological activity on account of partial modification of active centres; b) undesirably strong interaction between carrier and immobilized matter, which reduces mobility and accessibility; C) difficulties in finding the optimum reaction conditions depending on the nature of the biological material concerned (c.f. Ullmann's Encyclopedia of Industrial Chemistry, 5th Ed. Vol.9A, loc.cit. pg.384).

Ulmann's Encyclopedia also mentions another drawback of covalent binding, namely that it is not applicable to entire cells, as well as the fact that the carriers cannot usually be regenerated. It is fair to say that in certain cases the consequences outlined under a) and b) will constitute a serious handicap for this method.

The following example serves to illustrate the point: for the purpose of immobilizing all kinds of protein, a carrier system consisting of crosslinked copolymers of k r J.

1 3 (meth)acrylamide with glycidyl (meth)- acrylate, preferably in bead form, has proved to be excellent. (c.f. the commercial product EUPERGIT C, cf. DE-C2722751, US-A-4190713, US-A-4511694). The main interaction between the carrier system and the protein occurs between the epoxy groups of the carrier and the free amino groups of the protein. This interaction is, however, not confined to only one or just a few sites of binding but may occur at a multitude of sites. In the final analysis the covalent binding is tantamount to inactivation of the fixed protein. The blocking of amino groups essential for biological activity-as an effect of binding to the carrier may also lead to inactivation of the immobilized protein.

Accordingly there still remains a need to find means of covalently binding biologically significant material using, if possible, proven carrier systems and at the same time to avoid the drawbacks mentioned above, in particular to avoid complete or partial biological inactivation. The present invention relates to a process which can be carried out in conjunction with established methods of covalent binding of biologically significant matter with protein structure according to the prior art, whilst maintaining the biological. activity thereof.

Thus, according to one aspect of the present invention there is provided a process for covalently binding biologically active proteins onto solid ca rrier materials having functional groups thereon, said process comprising the following steps:

a) modifying the most active amino groups of said proteins by means of a reagent which can be reversibly removed under controlled conditions; b) reacting said proteins with the functional groups of a solid carrier material; c) chemically blocking the remaining functional groups 4 of said carrier material; and d) splitting off the modifying groups formed from said reagent in a controlled reaction.

There is also provided a solid carrier material having biologically active proteins bound thereon when prepared by a process according to the invention.

Dimethyl maleic anhydride is particularly useful as a modifying reagent which can be removed reversibly under controlled conditions.

The process of the present invention in principle lends itself to any carrier material that is liable to pose the above-mentioned problems, i.e. whenever there is too strong an interaction between the carrier and the entity to be immobilized or whenever fixing occurs at the site of biologically significant matter, which is essential for biological activity and whose blocking means a reduction in or even loss of said biological activity. The danger of this happening may vary for individual types of reactive groups producing covalent binding and for individual carrier systems, but it is always present, in particular with a high density and high reactivity of the electrophilic functional groups under biologically acceptable conditions (something which is not undesirable per se) (c.f. K. Buchholz loc.cit. pp.49-63; Ullmann 4th Ed.Vol.16, loc. cit.pg.540).

Preferred functional groups of this description are for example: acid derivatives such as acid halides, azides, activated esters, imidoesters, anhydrides, in a broader sense also imidocarbonates, isothiocyanates and halogenated triazines as well as aziridine and in particular epoxide (=oxirane) functions.

Again a carrier system consisting of crosslinked copolymers of methacrylamide with glycidyl methacrylate (c.f. the product EUPERGIT mentioned above), which is suitable in other respects may serve as an example.

1 is Investigations into the structure and activity of this carrier system have implied a density of epoxy functions at the surface which is characterized by a 5A distance between individual functions in combination with a high degree of permeability of the system. It is immediately evident that under these circumstances, with certain materials to be immobilized, which usually means with certain proteins, there will be "Multi-Point Attachment", which will result in the undesirable effects outlined above.

it should, however, be stressed that in other carrier systems, too, there will be multi-point attachment with its negative consequences, given the prerequisites such as high density of functional groups and favourable steric conditions e.g. ready accessibility.

Keeping those considerations in mind neither established inorganic carriers such as surface modified, in particular silanized glass, silica gel, clays such as bentonite and hydroxylapatite, metals and metal derivatives such as nickel, nickel oxide, titanium oxide, zirconium oxide among others (for details c.f. E. Katchalski-Katzir, L. Goldstein Ed. Applied Biochemistry and Bioengineering Vol.1, pg.23ff; H.H. Weetall Ed. Immobilized Enzymes, Antigens, Antibodies and Peptides, Marcel Dekker, New York 1975, pg.lff) nor,naturally occurring polymers after modification or synthetically produced organic polymers are exempt.

As an example of modified naturally occurring polymers, polysaccharides in particular deserve mention. They can be converted into cyclic imides by reaction with cyanogen bromide or into carboxymethyl derivatives by reaction with chloroacetic acid or can be transformed into azides by the introduction of an amino group which can be diazotised or by introduction of a glycidyl (ether) group, in this way producing the desired functional groups. (c.f. K. Buchholz, Characterization of Immobilized Biocatalysts loc.cit. pg. 52-54).

6 As is immediately apparent, the highest degree of variability exists with synthetic organic polymers. on the one hand, polymers are used into which reactive groups are introduced by polymer-like reactions, on the other hand polymer carriers can also be produced by copolymerizing monomers containing reactive units. Apart from the functional groups being present, the carrier material is expected to provide sufficient mechanical resistance, as well as physical, chemical and biological stability and toxicological safety.

The significance of the present invention will now be illustrated taking the example of a carrier which contains epoxy groups: The prototype may be the abovementioned crosslinked copolymer of (meth)acrylamide and glycidyl (meth)acrylate, preferably in bead form. Such carrier polymers are described in German Patent. No. 2,722,751, and U.S. Patents. Nos. 4, 190,713 and 4,511,694. The beads generally have a diameter of 51000 microns, particularly 30-1000 microns, and have an inner hollow space, i. e., they are hollow beads. As a guideline for the content of glycidyl groups available for reaction in the carrier, about 0.8-2.5 gmol per mg dry carrier material is available. Additional characterizing parameters for a common commercially available matrix polymer (Eupergit C, of Roehm GmbH), typical of carriers of the type specified, may be obtained from the following Table:

Characteristics Dimension Mean particle size 140-180 micron Pore diameter 40 nm Exclusion limit (=MLIm) 2 x 10-5 Dalton Binding-active surface 180 m2/g (dry) Epoxide content 800-1000 mmol/g dry weight Water uptake 2.3 M1/g (dry) Specific gravity, d. 20 1.20 Bulk density 0.34 g/M1 i 7 Combining capacity (under ordinary conditions):

- Human albumin 48 mg/g (moist) - Human IgG 34 mg/g (moist) Swelling by water 1:1.4 (lml dry yields 1.4 ml moist) Solubility in water, buffers, and organic solvents Insoluble Pressure stability 300 bar is Under an electron microscope the macroporous structure of the beads can be observed, with channels and voids having a diameter of 0.1-2.5gm (1, 00025,OOOA), so that enzyme and substrate molecules with a size of 10100A can reach the entire interior of the macroporous carrier.

The materials of biological significance, whose immobilization at a carrier T is the object of the present invention, have been characterized above (c.f. State of the Art).

As a rule such materials possess nucleophilic groups, especially amino groups which will react with the functional groups FG of the carrier T under biologically acceptable conditions. The materials of biological significance are usually proteins. Mention should be made of biocatalysts like enzymes, as well as cells, organelles and immunologically active materials and structures, in particular antibodies.

Immobilized biocatalysts may be used for the production or the transformation of widely different substrates such as amino acids and peptides, sugars, organic acids, antibiotics, steroids, nucleosides and nucleotides, lipids, terpenoids and basic organic chemicals (c.f. Ullnann 5th Ed., Vol.9A, loc.cit. pg.389-390).

As immunologically active structures, microorganisms 8 such as gram-positive and gram-negative bacteria, spirochetes, mycoplasmas, mycobacteria, vibrionaceae, actinomyces, protozoa such as intestinal protozoa, amoebas, flagellates, spores, intestinal nematodes and tissue nematodes (worms), trematodes (schistosomes, leeches), cestodes, and toxoplasmas can be mentioned. Also, fungi such as sporotrichum, cryptococcus, blastomyces, histoplasma, coccidioides, and candida, viruses and rickettsia such as canine hepatitis, Shopepapilloma, influenza A and B, chicken pox, herpes simplex, adeno-viruses, polyomas, Rous-sarcoma, smallpox, polio virus, measles, canine distemper, leukemia, mumps, Newcastle-disease, sendai, echovirus, hoof-and-mouth disease, psittacosis, rabies, extromelia, and tree viruses. Further tissue antigens, enzymes such as pancreatic chymotrypsinogen, procarboxypeptidase, glucose-oxidase, lactate dehydrogenase, uricase, amino acid-oxidase, urease, asparaginase, and proteases. Further, blood cell antigens, blood group substances and other isoantigens such as blood platelets, leucocytes, plasma proteins, milk proteins, saliva proteins, urine proteins, and antibodies, including auto-antibodies, can be mentioned.

- Bacterial antigens: Tetanus toxoid; H. influenzae type b polysaccharide; Diphtheria toxin; Chlanydia trachomatis; M. leprae; Lipopolysaccharide endotoxin; Pneumococci; LPS of P. aeruginosa; Exotoxin of P._ aeruginosa; Streptococci group A Carbohydrate.

- Viral antigens: X31 influenza virus nucleoprotein; Measles virus; HSV glycoprotein D; Measles virus nucleocapsid; Cytomegaly virus; Influenza A viruses; Rubella virus antigen; HTLV I; Varicella zoster, HBsAg.

- Autoimmune antigens: Double strand DNA; islet cells (diabetes mellitus); Myasthenia gravis antiidiotypes; Thyrotropin (TSH) receptor; Rheuma factor; Acetylcholine receptor; Thyroid extract; Semen.

- Tumor antigens: Mammary carcinoma; Prostate carcinoma; Lung carcinoma; Gastric carcinoma; Melanoma; 1 9 BD2 (human melanoma); Glioma; Rectal carcinoma; Leukemia; Cervical carcinoma.

-Tissue and blood antigens: Rhesus D; Blood group antigen A; HL-A antigens A, B, C, or DR; Intermediary filaments.

- other antiqens: Malaria; Forssman antigen; Sheep erythrocytes; Nitrophenol; Dinitrophenol; Trinitrophenol; Keyhole limpet hemocyanin (KLH); Rheuma factor; Insulin.

is Using the process in a first reaction step the most active nucleophilic groups, which in practice are invariably the amino groups of the protein, are modified by means of a reagent R. This reagent is selected to react with the protein under controlled conditions and reversibly, i.e. if possible having no detrimental effect on the activity of the protein P.

As reagents, compounds with the following structure are preferred: 0 R A C/ -" I \ C 1 0 9 (wherein R represents a methyl group; R' represents a hydrogen atom or a methyl group; and A and A' each represents a methyl group or together form a double bond).

In particular dimethyl maleic anhydride and citraconic anhydride are preferred as reagents.

The use of the reagent 2,3-dimethyl maleic anhydride has already been reported for the covalent modification of the amino groups of certain antibodies (c.f. N.Endo, N.Umemoto, Y. Kato, Y. Takeda and T. Hara J. Immunolog. Methods 104, (1-2) 253-258, 1987).

The actual procedure is to block those amino groups which are responsible for the antigen binding capacity by means of the reagent, thereafter to modify the remaining amino groups and eventually to split off the dimethylmaleiyl- protecting group by hydrolysis. The method has been used for covalent conjugation of methotrexate (MTX) with two monoclonal antibodies via the MTX-N-succinimidylic ester.

In a similar manner monoclonal melanoma antibodies have been reacted first with dimethyl maleic anhydride and subsequently with N-acetyl homocysteine thiolactone for the introduction of reactive thiol groups capable of reacting further forming an S-S-linkage (c.f. PCT Patent Application No. W087/4171). Using this concept a reaction with thiopropyl- sepharose 4B was achieved.

The present invention aims primarily at the immobilization of biologically significant matter directly through its nucleophilic groups, particularly through amino groups, whilst surprisingly, in spite of a large excess of reagent, enough of these groups are still available with a steric accessibility sufficient for immobilisation. Modification, in particular activation of such amino groups which were left over after the blocking with the reagent in order to effect binding to the carrier is thus not necessary, which is a major advance on the prior art.

In general the process of the invention may be carried out as described in the following procedure: The biologically significant matter, usually a protein, in particular an antibody, is transferred into anaqueous medium preferably in an alkaline buffer solution (for example of pH 8.7 and with a content of NaCl, preferably 1 molar NaCl). This solution is mixed with the reagent preferably in excess (200-500 l/mol) dissolved in a suitable solvent which is preferably immiscible with water, e.g. with dimethyl formamide. The amount of the solvent used is usually kept below 5% by weight with respect to the entire solution. The solution is incubated advantageously with cooling preferably in an ice bath for 45 15 minutes, in this way effecting the desired modification. The liquid obtained 1 1 in this manner is then transferred into a suitable flask, e.g. a centrifuge beaker, containing the carrier and is incubated usually first at room temperature for several hours (end point about four hours) and afterwards for a considerably longer period of time, for example four times as long at a reduced temperature however usually above zero degrees C. e.g. at 4C. Thereafter blocking of the functional groups of the carrier by means of chemical reagents is performed. In the case of epoxy- functions the carrier is reacted with 2-mercapto- ethanol, e.g. in the form of a 0.2 molar solution and at ambient temperature. Generally for this step a reaction time of several hours e.g. about four hours will suffice. Thereafter the loaded carrier material is washed, for example with phosphate standard buffer solution (PBS). The cleavage of the protecting groups (which had been attached utilizing the reagent) is effected preferably by hydrolysis at a suitable pH, for example in the weakly acidic range, e.g. about pH 5.5, conveniently for a hydrolysis period of about 1 hour. Finally the loaded carrier material is washed again with PBS.

Determination of the Immunocapacitv of the Antibodies immobilized according to the invention:

The combining capacity of the inventively immobilized antibodies is determined by measuring antigens, which may be in particular enzymes such as anticarboxypeptidase (CPA), which combine with the antibodies. For this purpose, CPA for example, is added to test tubes in equal amounts, in PBS, the test tubes each containing 100 ing of the antibody loaded carrier material as well as differing amounts of immobilized monoclonal antibodies. The amount of CPA combined with the antibodies of the matrix polymer is determined as follows:

i) By determining the difference in protein content, using the Bradford test, and by the enzymatic activity 12 is difference between the starting and residual solutions; ii) by determining the enzymatic activity of the CPA enzyme immobilized on the carrier beads, e.g. with the aid of the ninhydrin method.

Enzyme activity should be determined whenever antibodies are present which do not affect the enzyme activity. It is apparent that the antibodies fixed to the carrier material in a manner according to the invention retain their entire immunological activity for bonding the enzyme, such as CPA. The ratio of monoclonal antibodies to-CPA usually is in the range of 2:1 to 1:1 mole/mole.

The advantageous effects of the process according to the present invention will be described with reference to a particular embodiment whereby antibodies are fixed to a carrier having epoxy groups as functional groups. By comparing antibodies immobilized according to the present invention on the one hand and antibodies immobilized according to the immobilizing procedures of the art on the same carrier system (i.e. EUPERGIT C) on the other hand, the results given in Table 1 below have been obtained (with an antibody load of 10 mg/g EUPERGIT C). The activity of all antibodies that were immobilized according to the present invention has increased. The increase in the activity of the different antibodies ranged from 3 to 10.7 fold apparently depending on the specific characteristics of individual antibodies. So far as the results obtained hitherto can allow one to comment, only the anti-horse radish peroxidase mAb, HRP2 did not show an increase in activity when immobilized by the method of the invention as compared with standard immobilization. The effect of the reagent was tested with different loads of antibody per gram of bead carrier. In order to allow a direct comparison between the two methods the same antibody (CPAB) was immobilized by both procedures in the same experiment. As shown in Fig. 1, the highest increase in X 1 13 activity of the immobilized antibody was obtained at low antibody loads.

Table 1: Comparison of the activities of anti-CPA,antibodies immobilized on Euvergit C by the standard and DMA Drocedures.

Monoclonal Antibody activity Improvement Antibodv Standard method DMA method factor CPA1 0.040 0.362 9.0 CPA8 0.060 0.216 3.6 CPA9 0.082 0.242 3.0 CPA14 0.102 0.300 3.0 CPA18 0.030 0.322 10.7 [Antibody activity is expressed by the activity of CPA immobilized by the antibody. CPA activity was measured by the ninhydrin method and the results shown in Table 1 are net absorbance at 550 nm values after blank substractionj.

The favourable results obtained when using the process of the invention suggest an interpretation of the efficacy of antibodies attached to R EUPERGIT C, which shall, however, in no way limit the scope of the present invention.

The success of the process relies primarily upon the blocking of free oxirane groups before reducing the concentration of the reagent. When the process is carried out in such a manner it is expected that two mechanisms may be in operation. Firstly amino groups that are essential for antibody activity and that are protected by the reagent from binding to the carrier may not be modified thus leading to enhanced antibody activity. Secondly the number of attachment points between the antibody and the carrier would be low compared with the number of attachment points i 14 anticipated upon the interaction of non-pre-treated antibodies with the carrier.

Also it has to be borne in mind that the major binding sites should originate from the interaction of the oxirane groups.

The present invention will now be further illustrated by reference to the following non-limiting examples.

1 EXAMPLES

1 Example 1 General method o.sing of carrier beads (EUPERGIT C of R6hm GmbH) are placed in Beckman centrifuge tubes with a capacity of 510Al. A solution of the antibodies (5-10 Mg per sample, 0.3 - 1.3 Mg/gl, 5-30 Al) in 25 mM sodium borate buffer at pH 8.7, containing 1 M NaCl, is mixed with an excess (x 400 mol/mol) dimethylmaleic anhydride in dimethylformamide (DMF), whilst the DMF concentration obtained should not exceed 5% by weight. After the solution has been incubated in an ice bath for 30-60 minutes, the modified antibodies are added to the carrier beads T and then kept for 16 hours at 4C.

Any oxirane (epoxide) groups still present are then chemically blocked by treating the carrier-antibody conjugate with 2-mercaptoethanol solution (0.2 M, 4 hours at ambient temperature). The beads are then washed with standard phosphate buffer (PBS). The protecting groups formed from dimethylmaleic anhydride are cleaved from the antibodies by incubation with 50 mM citrate buffer, pH 5.5 (1 hour at ambient temperature). Finally, the beads are thoroughly washed with standard phosphate buffer.

The following were used as antibodies, by way of example: 1) 4) Murine monoclonal antibodies specifically for carboxypeptidase A.

2) Murine monoclonal antibodies specifically for human choriogonadotropin. Murine monoclonal antibodies specifically for human insulin. A preparation of polyclonal goat antibodies specifically for the FG fragment of murine IgG.

Example 2 The activities of anti-carboxypeptidase-A (anti-CPA) 1 16 is antibodies, immobilised either according to the prior art or according to the process of the invention, were compared in an amount of iOmg of antibody per gram of EUPERGIT C. The activities of all the antibodies immobilized by the process according to the invention had increased (see the Table). The increase in activity ranged from a 3-fold to a 10.7-fold increase in the various antibodies, apparently depending on the specific characteristics of the individual antibodies.

According to the results obtained hitherto, only anti-horseradish peroxidase mAB, HRP2 showed no increase in activity after immobilisation according to the invention, compared with the immobilisation products obtained by the process according to the prior art. Example 3

In the preceding Examples, a 400-fold molar excess of dimethyl-maleic anhydride was used, based on the antibodies. The investigations into the variation in the ratio of antibody to dimethylmaleic anhydride seem to indicate that relatively small doses of dimethyl maleic anhydride are sufficient to obtain the full effect. A not particularly marked optimum was recorded in the region of a 200-500 molar excess.

If the excess is increased to 10,000 times the antibody concentration, there is a gradual drop in the activity of the immobilised antibodies.

17

Claims (17)

1. A process for covalently binding biologically active proteins onto solid carrier materials having functional groups thereon, said process comprising the following steps:

a) modifying the most active amino groups of said proteins by means of a reagent which can be reversibly removed under controlled conditions; b) reacting said proteins with the functional groups of a solid carrier material; c) chemically blocking the remaining functional groups of said carriers; and d) splitting off the modifying groups formed from said reagent in a controlled reaction.

2. A process as claimed in claim 1 wherein said reagent has the formula 0 1-1 R A c 10,11 1 \,\ c 1 i---, cl.., A C \0 0 (wherein R represents a methyl group, R' represents a hydrogen atom or a methyl group; and A and A' each represents a methyl group or together form a double bond).

3. A process as claimed in claim 1 or claim 2, wherein said reagent is dimethyl maleic anhydride or citraconic anhydride.

c 18

4. A process as claimed in any of the preceding claims wherein said reagent is used in a 200-500 fold molar excess over said proteins.

5. A process as claimed in any of the preceding claims wherein said proteins are antibodies.

6. A process as claimed in any of the preceding claims wherein the functional groups on said carrier material are oxirane groups.

7. A process as claimed in any of the preceding claims wherein said carrier material is in the form of hollow beads with oxirane groups thereon.

8. A process as claimed in any of the preceding claims wherein step (a) is carried out in a weakly alkaline buffer.

9. A process as claimed in any of the preceding claims wherein step (a) is carried out at pH 8.7.

10. A process as claimed in claim 8 or claim 9, wherein said buffer solution is also one molar in sodium chloride.

11. A process as claimed in any of the preceding claims wherein in step (b) the protein modified by said reagent is incubated with said carrier first for several hours at room temperature and subsequently for about four times as long at a temperature slightly above OC.

12. A process as claimed in any of the preceding claims wherein the conjugate of protein and carrier formed in step (b) is treated in step (c) with a 2-mercaptoethanol solution to block any oxirane groups still present.

A 19

13. A process as claimed in any of the preceding claims wherein the protective group formed from said reagent is split off by hydrolysis from the product of step (c).

14. A process for covalently binding biologically active proteins onto solid carrier materials having functional groups thereon substantially as herein described with reference to the Examples.

15. A solid carrier material having biologically active proteins bound thereon, when prepared by a process as claimed in any of claims 1 to 14.

16. A solid carrier material having biologically active proteins bound thereon substantially as herein described with reference to the Examples.

17. Antibodies when immobilized using a process as claimed in any of claims 1 to 14.

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