Affinity supports containing ligands bound to oxirane groups

Details Affinity supports containing ligands bound to oxirane groups Affinity supports containing ligands bound to oxirane groups

1996-02-27

2001-09-18

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Carrier-bound or immobilized enzyme or microbial cell;...

Enzyme or microbial cell is immobilized on or in an organic...

C435S177000, C435S179000, C435S180000, C435S815000, C502S401000, C502S402000, C530S413000, C530S812000, C530S813000, C530S814000, C530S815000

Reexamination Certificate

active

06291216

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to activated support materials, and to affinity supports and immobilized enzymes which can be prepared from these activated support materials.
In affinity chromatography processes, specific interactions between the material to be analyzed and so-called affinity ligands are utilized to remove concomitant substances. The materials to be analyzed are bonded reversibly to the affinity ligands bonded to a support. Concomitant substances are not bonded and can therefore easily be washed out. The material to be analyzed is then liberated, utilizing the reversibility of the bonding. Liberation is effected, for example, by changes in pH, a change in ionic strength or by addition of dissolved affinity ligands to the eluting agent. Details of these processes and process variants are known to the expert.
The processes of affinity chromatography include purification of antibodies directed against a certain protein. The protein is bonded to a chromatographic support material. The solution which contains the antibodies to be purified is applied to the support material. The non-bonding portions of the antibody preparation are washed out and the purified antibodies are then eluted. In a reversal of this process, certain proteins can also be purified by means of affinity chromatography, an antibody directed against this protein being bonded to the chromatographic support material. In other applications of affinity chromatography, for example, coenzymes are bonded to the chromatographic support and serve to purify enzymes which bond this coenzyme. Instead of the coenzymes, dyestuffs can also be employed with similar success. A number of affinity ligands are summarized by way of example in Table 1. Other affinity ligands and materials to be analyzed as well as variants of processes are to be found in handbooks, for example in Kirk-Othmer Encyclopedia of Chemical Technology (page 35-40, 3rd edition, 1978, John Wiley and Sons) and in Protein Purification, Janson, J.-C. and Ryden, L. (editors) (page 275-325, 1989, VCH Publishers), or else in Vijayalakshimi, M. A. (1989; TIBTECH 7, page 71-76.
TABLE 1
Affinity ligand
Material to be analyzed (Example)
Protein A
Immunoglobulins
Concanavalin A
Glycoproteins
Biotin
Avidin/streptavidin
Avidin
Biotin
Streptavidin
Biotin
5′-adenosine monophosphate
AND-dependent oxidoreductases
2′,5′-adenosine diphosphate
NADP-dependent oxidoreductases
Aminoacridine
RNA or DNA
Boronic acid
Catecholamines
Boronic acid
Glycosylated hemoglobin
Iminodiacetic acid
Metalloproteins
“Thiophilic” ligands
Immunoglobulins
Cibachromium [sic] Blue
Monoclonal antibodies
Different ligands are often required for affinity chromatography. It has therefore become established to provide activated support materials onto which the particular ligand can be bonded by simplified processes. One of the first products was BrCN-agarose: crosslinked agarose activated with cyanogen bromide. This reacts with primary amino groups of the ligand and thus bonds this to the support material. Improvements in respect of the stability of the support materials under pressure were obtained, for example, by introduction of crosslinked poly-(meth)acrylic acid derivatives or by the use of silica gel as the base material.
According to EP 064 833, silica gel is reacted with gamma-glycidoxypropyltrimethoxysilane, an activated support material being formed, onto the oxirane group of which affinity ligands can be bonded. According to DE 40 02 044, for example, “thiophilic” ligands are bonded to oxirane groups; these affinity supports are particularly suitable for the purification of immuno-globulins, for example of monoclonal antibodies. U.S. Pat. No. 4,737,560 describes activated support materials based on crosslinked polymers which comprise azlactone compounds as the active grouping. However, the bonding properties of these materials are unsatisfactory.
EP 0 172 579 describes support materials which have a core of silica gel, onto which a crosslinked polymer is applied. These polymers can contain the oxirane group, for example, as the activated group. The polymer is bonded to the support by reaction of some of the oxirane groups. This results in Si-O
9
bonds, which are sensitive to hydrolysis. Since preshaped polymers are bonded to the silica gel, the charging density is limited. Crosslinking of the polymer moreover impedes access of high molecular compounds, such as proteins or nucleic acids.
Since the material to be analyzed in affinity chromatography is often a compound of high molecular weight (>10
3
), the rate of mass transfer and therefore the productivity of the separation process is restricted by diffusion processes. U.S. Pat. No. 5,019,270 discloses chromatographic supports with which an accelerated mass transfer can be achieved, on the basis of a specific geometry of the support particles and the resulting liquid dynamics.
EP 0 295 073 discloses another method for accelerated mass transfer: the affinity ligands are rendered more accessible by using polyethylene glycol as a spacer. However, the bonding capacity of these materials is limited in a manner similar to that of materials according to EP 0 064 833.
Activated support materials such as are used for the preparation of affinity supports furthermore are in principle suitable for immobilizing enzymes. Such immobilized enzymes often have an improved stability. Moreover, they can easily be removed from the reaction batch and can also be re-used. Another use of immobilized enzymes is the provision of enzyme reactors with which a continuous reaction procedure is possible under flow-through conditions. However, it has been found that the the enzymes are often inactivated during the immobilization, and the yield of bonded enzyme after the bonding reaction is extremely low. In particular, the reactivity toward high molecular weight substrates (molecular weight >10
3
) thereby drops. Benzonase, a nucleic acid hydrolase which hydrolyzes RNA and single- and double-stranded DNA into small, biologically inactive oligonucleotides, and proteinase K may be mentioned as examples of such enzymes which preferentially convert high molecular weight substrates and cannot be immobilized by methods known to date. Benzonase is capable of breaking down any nucleic acids still present in biologically obtained pharmaceutical active compounds, and thus of excluding undesirable transfer of genetic material. Such a process would preferably be carried out in a flow-through reactor. A precondition of this is immobilization of the benzonase on a suitable activated support while retaining the reactivity toward high molecular weight nucleic acids.
Other enzymes which present similar problems in their immobilization are known to the expert from the literature.
SUMMARY OF THE INVENTION
There is thus the object of providing an activated support material onto which affinity ligands or enzymes can be bonded in a simple manner. The resulting affinity supports should have a high bonding capacity and allow high mass transfer, and during immobilization of enzymes, the reactivity thereof, especially towards high molecular weight substrates, should be retained.
Surprisingly, it has been found that (meth)acrylic acid derivatives which contain an oxirane or an azlactone group, or the precursor of an azlactone group, can be grafted onto base supports which contain aliphatic hydroxyl groups on their surface. According to the invention, the polymerization is started here with cerium(IV) ions: G. Mino and S. Kaizerman (1958) J. Polymer Science 31, 242-243; G. Mino et al. (1959) J. Polymer Science 38, 393-401. The support materials obtainable by this method can be converted into affinity supports in a simple manner and have a high bonding capacity and a high mass transfer. Activated support materials with an azlactone grouping, the azlactone group of which is bonded to the support via a thioether bond, also show outstanding properties. These activated support materials also show particularly advantageous properties if the thio

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