Process for chromatographic separation of peptides and nucleic a

Details Process for chromatographic separation of peptides and nucleic a Process for chromatographic separation of peptides and nucleic a

1998-12-29

2000-07-18

Therkorn, Ernest G.

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

210656, 2101982, 2105021, 530413, 530416, 521 30, 536 254, B01D 1508

Patent

active

060902884

DESCRIPTION:

BRIEF SUMMARY
REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/SE97/00237 filed Feb. 14, 1997.


FIELD OF THE INVENTION AND DEFINITIONS

The present invention relates to weak anion exchangers, i.e. ion exchangers exhibiting protonated forms of primary, secondary or tertiary amino as anion exchanging ligand. The invention is based on our discovery that this type of anion exchangers, when having certain hydrogen binding atoms at a distance of 2 or 3 atoms away from the nitrogen in the weak anion exchanging group of the ligand, can exhibit enhanced binding properties to peptides or nucleic acids. Because the binding is broken by guanidine or urea, this has been construed as a presence of hydrogen bonds between peptide
ucleic acid and anion exchanging ligand.
Our discovery has led to both a new process for separating nucleic acid or peptide, and new ion exchange adsorbents (ion exchanging matrices) which can be used in the method.
By amino group (structures and expression) is meant, if not otherwise stated, a protonated and non-protonated primary, secondary, tertiary amino group as well as a quaternary ammonium group.
By the expression "to separate" is meant removing or separating off one or more nucleic acids/peptides from a more or less complex mixture of substances. Separation can be used in connection with isolation, purification, and analysis of nucleic acid/peptide. Separation is effected by way of substances interacting with the ligands of the anion exchanger. The interaction is different for different substances which leads to separation.
By nucleic acid is meant both RNA and DNA, including single and double stranded or modified forms of these, such as phosphothionate forms which exhibit protecting groups and markers (isotope, biotin, hapten etc.). Nucleic acid can be an oligo- or poly-nucleotide. The number of bases and basepairs, respectively in oligonucleotides is typically.ltoreq.200. The invention is preferably applied to isolation and purification of synthetically produced oligonucleotides.
By peptide is meant compounds exhibiting polypeptide structure, i.e. proteins, such as lipoproteins, glycoproteins, proteoglycans etc., and oligopeptides (i.e. peptides having 2-10 amino acids).


PRIOR ART

EP 276,138 and DE 3,935,098 describe anion exchangers exhibiting weak anion exchanging groups having a hydroxyl group at a distance of two carbon atoms away from a nitrogen in the ion exchanging group. The ion exchanging group has been introduced using a bifunctional reagent which, through a silane group couples to a glass matrix and through an epoxide to a primary or secondary amino --NH--(CH.sub.2).sub.2 --OH available pH values. EP 276,138 suggests that the matrix also can be an organic polymer. However, concerning organic matrices, it is uncertain if enough stable products can be obtained in the suggested way. Both publications are related to separation of polynucleotides. DE 3,935,098 does also use an eluent which contains the denaturating agent urea. A more detailed explanation is not given.
EP 167,488 also describes ion exchangers exhibiting weak ion exchanging groups having a hydroxyl group at a distance of two carbon atoms away from a nitrogen atom. The ion exchanging group binds through an amide to the matrix. Nothing is mentioned regarding separation of peptides or nucleic acid.
The inventors of EP 276,138 turn down our types of ligands since they find their binding ability too strong. The explanation is wrong which probably also is the reason for them not to understand to utilize the general feature on which the strong binding ability of our novel ligands is based (hydrogen-bonding). Probably, they have not even realized the need for binding at high ionic strength.
There is also a relatively wide range of literature regarding ECTEOLA cellulose for purification of nucleic acid. ECTEOLA cellulose has been produced by coupling triethanolamine, N(CH.sub.2 CH.sub.2 OH).sub.3 to cellulose using epoxide. It has been considered that the resulting groups are --CH.sub.2 CHOHCH.sub.2 --N.sup.+ (CH.s

REFERENCES:
patent: 4767670 (1988-08-01), Cox
patent: 5087359 (1992-02-01), Kakodkar
patent: 5147536 (1992-09-01), Engstrom
patent: 5466368 (1995-11-01), Arvidsson

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