Ligand functional substrates
View Patent ↗A substrate comprising a crosslinked polymer primer layer, and grafted thereto a ligand-functionalized polymer is provided. The grafted polymer has the requisite affinity for binding neutral or negatively charged biomaterials, such as cells, cell debris, bacteria, spores, viruses, nucleic acids, and proteins, at pH's near or below the pI's of the biomaterials.
1. A method of separating a target biological species from a biological fluid comprising contacting the fluid with a ligand-functionalized substrate comprising:
a) a substrate
b) a primer layer disposed on the substrate comprising:
a crosslinked polyamine polymer having ethylenically unsaturated polymerizable groups; and
c) a ligand-functional alkenyl (co)polymer layer grafted to the ethylenically unsaturated polymerizable group of the primer layer;
whereby a complex comprising the functionalized substrate and the target biological species is formed, and separating the complex; wherein said target biological species is selected from biomacromolecules and microbiological species.
2. The method of claim 1 wherein said biomacromolecules are selected from proteins, enzymes, nucleic acids, and endotoxins.
3. The method of claim 1 wherein said microbiological species is selected from bacteria, viruses, cells, cell debris, and spores.
4. The method of claim 3 wherein the cells are selected from archaea, bacteria, and eucaryota.
5. The method of claim 1 wherein the biological fluid is derived from a cell culture or fermentation process.
6. The method of claim 1 wherein the biological fluid comprises a solution of a purified protein or enzyme after separating the target species.
7. The method of claim 6 wherein the separated target species comprises a purified protein or enzyme.
8. The method of claim 1 wherein the fluid has a salt content of at least 50 millimolar.
9. The method of claim 1 , wherein the crosslinked polyamine polymer having ethylenically unsaturated polymerizable groups is the reaction product of:
1) a polyamine polymer
2) a polyfunctional crosslinking agent for the polyamine polymer; and
3) a monomer having an amine-reactive functional group and an ethylenically unsaturated polymerizable group.
10. The method of claim 9 wherein the crosslinking agent of the crosslinked polyamine polymer is a polyfunctional crosslinking agent of the formula:
R 8 —(Z) y ,
where R 8 is a (hetero)hydrocarbyl group, Z is an amine-reactive group, and y is ≥2.
11. The method of claim 1 , wherein the ligand-functional alkenyl (co)polymer comprises polymerized monomer units of the formula:
where
R 1 is H or C 1 -C 4 alkyl;
R 2 is a (hetero)hydrocarbyl group;
X 1 is —O— or —NR 3 —, where R 3 is independently H or C 1 -C 4 alkyl;
R lig is a ligand-containing group,
p is 0 or 1; and
m is 0 or 1.
12. The method of claim 1 , wherein the ligand-functional alkenyl (co)polymer comprises polymerized monomer units of the formula:
wherein
R 1 is H or C 1 -C 4 alkyl;
R 2 is a (hetero)hydrocarbyl group;
each R 3 is independently H or hydrocarbyl;
R 4 is H, C 1 -C 4 alkyl or —N(R 3 ) 2 ;
R 5 is H or hydrocarbyl;
X 1 is —O— or —NR 3 —,
o is 0 or 1, and
n is 1 or 2.
13. The method of claim 1 , wherein the ligand-functional alkenyl (co)polymer further comprises polymerized polar monomer units.
14. The method of claim 13 , wherein the ligand-functional alkenyl (co)polymer further comprises polymerized cationic or anionic monomer units.
15. The method of claim 13 , wherein the polar monomer units are poly(oxyalkylene) (meth)acrylate monomer units.
16. The method of claim 15 , wherein the poly(oxyalkylene) (meth)acrylate monomer units are of the formula: CH 2 ═CR 1 —C(O)—X 1 —(CH(R 1 )—CH 2 —O) n —R 1 , wherein each R 1 is independently H or C 1 -C 4 alkyl, X 1 is —O— or —NR 3 —, where R 3 is H or C 1 -C 4 alkyl and n is 2 to 100.
17. The method of claim 1 wherein the polyamine polymer is selected from the group consisting of polyethylenimine, polylysine, polyaminoamides, polydimethylamine-epichlorohydrin-ethylenediamine, polyaminosiloxanes and dendrimers formed from polyamidoamine (PAMAM) and polypropylenimine.
18. The method of claim 1 wherein the ligand-functional alkenyl (co)polymer layer is of the formula:
-(M Lig ) y -(M Hydrophil ) x -(M crosslink ) z -, 10
where
(M Hydrophil ) x are hydrophilic monomer units having “x” polymerized monomer units,
(M Lig ) y are ligand functional monomer units having “y” polymerized monomer units,
(M crosslink ) are multifunctional (meth)acryloyl monomer units having “z” polymerized monomer units,
y is 10 to 100 wt. % of the monomer units;
x is 0 to 90 wt. % of the monomer units;
z is 0 to 5% wt. % of the monomer units,
based on 100 wt. % total monomers.