Separation matrices for purification of biological particles
The invention discloses a separation matrix for purification of biological particles, comprising a plurality of particles having a porous core entity and a porous shell entity covering the core entity, wherein the core entity comprises at least 50 micromole/ml primary amines present on covalently attached ligands displaying at least two primary amines per ligand and the shell entity comprises less than 20 micromole/ml primary amines The invention further discloses a method of purifying biological particles and a method of manufacturing a separation matrix.
1. A separation matrix for purification of biological particles, comprising a plurality of particles having covalently attached ligands displaying at least two primary amines per ligand, the particle having a shell and an inner radial portion, surrounded by the shell, wherein the combined content of nitrogen and oxygen atoms in said ligands is at least 20 wt % and wherein;
a) the inner radial portion of the particle comprises substantially evenly distributed at least 50 micromole/ml primary amines from the covalently attached ligands and
b) the shell of the particle having an average thickness of 0.5 to 6% of the volume-weighted average diameter or the sphere-equivalent diameter of the plurality of particles, comprises substantially evenly distributed less than 20 micromole/ml primary amines from the covalently attached ligands,
wherein the shell is formed by;
contacting allyl modified polysaccharide particles with an amount of halogen for a period of up to 30 minutes, wherein the amount of halogen is equivalent to the amount of allyls in a shell of the particles, to form polysaccharide particles with a halogenated shell;
washing the polysaccharide particles with a halogenated shell with water; and
adding an aqueous alkaline solution to the washed polysaccharide particles with a halogenated shell to at least partially inactivate the halogenated shell to form intermediate polysaccharide particles comprising remaining allyl groups, wherein the thickness of the shell is also controlled by the reaction time with the halogen.
2. The separation matrix of claim 1 , wherein said ligands are attached to the particle via secondary or tertiary amine links.
3. The separation matrix of claim 1 , wherein the content of primary amine nitrogen in said ligands is at least 18 wt %.
4. The separation matrix of claim 1 , wherein the ratio of displayed primary amines to the total amount of nitrogen atoms in said ligands is at least 0.5.
5. The separation matrix of claim 1 , wherein said ligands are selected from the group consisting of tris(2-aminoethyl)amine and polyallylamine, attached via an amine.
6. The separation matrix of claim 1 , wherein said ligands comprise polyallylamine of molecular weight at least 1 kDa.
7. The separation matrix of claim 6 , wherein the polyallylamine is non-crosslinked.
8. The separation matrix of claim 1 , wherein said particles are substantially spherical.
9. The separation matrix of claim 1 , wherein the shell of the particle has a molecular weight cutoff for globular proteins of 60 to 1000 kDa.
10. The separation matrix of claim 1 , wherein the shell of the particle has a molecular weight cutoff for globular proteins of 400-800 kDa.
11. The separation matrix of claim 1 , wherein the halogen is bromine.