Catalyst – solid sorbent – or support therefor: product or process – Solid sorbent – Organic
Reexamination Certificate
2001-05-22
2003-12-02
Lovering, Richard D. (Department: 1712)
Catalyst, solid sorbent, or support therefor: product or process
Solid sorbent
Organic
C252S179000, C424S179100, C435S178000, C435S181000, C435S810000, C502S404000, C516S100000, C516S101000
Reexamination Certificate
active
06656876
ABSTRACT:
This application is a 371 of PCT/SE99/01861 filed Oct. 14, 1999.
The present invention relates to storage stable, activated carrier in the form of matrices including surfaces. In particular it relates to such matrices suitable as carrier to which compounds containing an amine group may be covalently bound.
The invention also relates to a kit comprising such activated carrier, the kit being shippable to customers by virtue of the long term stability of said carrier.
In the context of this description of the invention the words carrier/carriers, matrix/matrices and medium/media are used interchangeable, if not otherwise said.
BACKGROUND OF THE INVENTION
The immobilization of biomolecules on various carriers finds great utility in research and industry, and many methods have been disclosed in the literature. These methods comprise an activation of a carrier, e.g. a matrix in the form of a gel, a surface of a polymer bead etc. The activation process generally comprises subjecting the carrier to a reactive agent that reacts with —OH groups or amine groups on the carrier, thereby, either directly or subsequent to further steps, forming intermediate reactive structures/sites. These structures may then be reacted with the compound in question, thereby immobilizing the compound.
Illustrative examples of such methods are the CNBr method, and epoxy activation. The activated intermediates obtained by these methods must be freeze dried in order to be shippable to customers. Freeze drying is fairly complicated and in addition require processing at the customer before the freeze dried intermediates can be used.
These and other activation and immobilization methods are described in “Immobilized affinity ligand techniques” by G. T. Hermanson, Academic Press, 1992.
In WO 95/15983 (Glüsenkamp et al) there is disclosed a process for immobilizing biomolecules and affinity ligands on carriers. This process comprises reacting an aminated polymer carrier with a bifunctional electrophilic squaric acid derivative as activating agents to form an activated intermediate (activated matrix or activated media or activated carrier), and coupling a biomolecule to the activated carrier.
For most of the above-mentioned activation processes it has been commonly known that the activated carriers are relatively unstable towards hydrolysis. Remembering that commercially available activated carrier media should have as long shelf time as possible, for instance from one up to several months, it is no surprise that commercial products comprising activated matrices typically have been sold in form of lyophilized compositions. For squarate activated carrier media we are not aware of any commercial products differing from what has been said above. When looking into the scientific literature it appears that most synthetic routes in which squarate activated material are obtained typically end up in crystallized and/or dried material. In the alternative the activated material is used more or less directly upon synthesis. See for instance Kamath et al., Glycoconjugate J. 13 (1996) 315-319; and Ola Blixt, Thesis: Enzymatic solid phase synthesis of carbohydrates, Swedish University of Agricultural Sciences, Uppsala, Sweden (1999) (see in particular page 50); DE 4341524 (Glüsenkamp et al); and DE 19624990 (Glësenkamp et al). An investigation of the hydrolysis of soluble squarate activated materials (monoamides) has been published (Glësenkamp et al., Z. Naturforsch. 46c (1991) 498-501). The storage of squarate activated aminated matrices moistured with methanol or in ethanol atmosphere has been described in the experimental part of WO. 9515983 (Glësenkamp et al). The reactivity is expected to be retained during these storing conditions because reaction with methanol and ethanol, respectively, will only mean a transesterification retaining the ester status of the activated group and thus also maintaining its reactivity.
It would be advantageous to provide a commercial product in form of an activated carrier comprising reactive squaryl groups said activated carrier being dispensed in aqueous media and having a shelf life of from 1-2 months up to several months without showing any significant loss in activity. This is in particular true in case the use is for immobilizing water-soluble compounds to the activated medium, for instance amine-containing biomolecules such as proteins. The aqueous media is typically an aqueous liquid.
It would also be advantageous in case such storage stable compositions would be resistant towards microbial growth.
Other publications connected to squarate activated materials are Tietze et al., Chem. Ber. 124(5) (1991) 1215-1221; Tietze et al., Bioconjugate Chem 2 (1991) 148-153; Hällgren et al., J. Carbohydrate Chem. 14(4&5) (1995) 453-46; Ausanneau et al., Bioorganic & Medicinal Chemistry 4 (1996) 2003-2010; and Pozsgay et al., J. Org. Chem. 62 (1997) 2832-2846.
SUMMARY OF THE INVENTION
The object of the present invention is therefore to provide a storage stable activated carrier comprising reactive squaryl groups, said carrier being dispersed in an aqueous medium for use in the immobilization of nucleophilic compounds, typically amine containing compounds. The activated medium should be stable during storage and/or resistent against bacterial growth over extended periods of time.
For the purpose of the invention the term “activated medium” or “activated carrier” or “activated matrix” mean a carrier to be used as a carrier for a nucleophilic compound. Said carrier has been provided with reactive squaryl groups enabling easy coupling of said compound to said carrier. The activated carrier should be regarded as an intermediate in the process of making immobilized forms of said compounds.
Nucleophilic compounds that are contemplated in the context of the invention typically contain a primary and/or a secondary amine group, for instance proteins or other compounds exhibiting peptide structure or other biomolecules and other organic compounds exhibiting this type of amine groups.
The inventor has now surprisingly discovered that a carrier as defined above comprising amine groups activated with an electrophilic squaric acid derivative may be stable in aqueous media. Unexpectedly it has been recognized that the rate of hydrolysis of reactive squarate groups bound; to carriers is sufficiently slow for enabling storage of the activated carriers in aqueous media.
Thus, in accordance with the present invention there is provided a storage stable composition comprising a carrier as defined above and usable for the immobilization of nucleophilic compounds. The carrier comprises primary or secondary amino groups that have been activated to carry reactive squaric acid derived groups linked to said primary or secondary amine sites on said carrier, said carrier being in contact with an aqueous medium, e.g. an aqueous liquid.
In a second aspect of the invention there is provided a method of preparing such a composition. This method is defined in claim
11
.
Furthermore, in accordance with the invention there is provided a kit for storing the composition over extended periods of time, this being defined in claim
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.
The composition according to the present invention is stable for an extended period of time as defined above, for instance for a week, a month or even longer periods of time, such as two, three or more months.
Preferred embodiments are defined in the dependent claims.
DETAILED DESCRIPTION OF THE INVENTION
The squaric acid derivatives useful for making the activated carriers may be selected among bifunctional electrophilic squaric acid derivatives, such as dialkoxyesters (e.g. dimethylester, diethylester, dibutylester and also corresponding mixed diester (squarates)), dihalides and diimidazolides. Other reactive derivatives may also be used. The activation reaction is normally run in inert media. See Glüsenkamp WO9515983. In case the reactive group formed on the carrier is too reactive to be stored in the contemplated aqueous media, it may be easily transformed to a group of lower reactivity. For instance, in
Amersham Biosciences AB
Lovering Richard D.
Ronning, Jr. Royal N.
Ryan Stephen G.
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