# US 10,864,512 B2 · Chromatography matrix

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- Number: US 10,864,512 B2
- Document ID: US10864512B2
- Status: Granted
- Kind: B2
- Application number: 16/090,609
- Filed: 2017-03-30
- Published: 2019-04-18
- Granted: 2020-12-15
- Earliest claimed: 2016-04-06
- Assignees: Cytiva BioProcess R&D AB
- Applicants: Ge Healthcare Bioprocess R&D AB
- Inventors: Jean-Luc Maloisel; Tobias Söderman; Ola Lind; Ida Eklind
- Web page: https://patentgrep.com/patent/US10864512B2

## CPC

- B01J 47/02: Column or bed processes
- B01D 15/327: {with hydrophobic interaction}
- B01D 15/362: {Cation-exchange}
- B01D 15/363: {Anion-exchange}
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- B01D 15/3847: {Multimodal interactions}
- B01J 20/267: {Cross-linked polymers}
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/289: bonded via a spacer
- B01J 20/3208: {Polymeric carriers, supports or substrates}
- B01J 20/3217: {Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond}
- B01J 20/3219: {involving a particular spacer or linking group, e.g. for attaching an active group}
- B01J 20/3278: {Polymers being grafted on the carrier}
- B01J 20/3285: {Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic}
- B01J 20/3293: {Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers}
- B01J 39/07: in the weakly acidic form
- B01J 39/19: obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
- B01J 39/26: Cation exchangers for chromatographic processes
- B01J 41/07: in the weakly basic form
- B01J 41/13: obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
- B01J 41/20: Anion exchangers for chromatographic processes
- C07K 1/18: Ion-exchange chromatography
- B01D 15/361: {Ion-exchange}
- B01D 15/3804: {Affinity chromatography}
- B01J 2220/54: Sorbents specially adapted for analytical or investigative chromatography
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention discloses a separation matrix for purification of biomacromolecules, comprising a plurality of particles (1) having a core region (2) and a shell region (3), wherein: a) said shell region is accessible to a target biomacromolecule; b) said core region is less accessible to the target biomacromolecule than the shell region; and c) the core region comprises a grafted polymer comprising residues of at least one polymerizable monomer.

## Claims

### Claim 1

A separation matrix for purification of biomacromolecules, comprising a plurality of particles having a core region and a shell region, wherein:
a) said shell region is accessible to a target biomacromolecule, wherein said shell region does not comprise polymers grafted to a porous support material and does not comprise polymers grafted to said shell;
b) said core region is less accessible to said target biomacromolecule than said shell region; and
c) said core region comprises at least 50 mg/ml of a vinyl polymer grafted to the porous support material and comprising residues of at least one polymerizable monomer, wherein said vinyl polymer comprises vinyl amide monomer residues selected from the group consisting of N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl formamide and N-vinyl acetamide.

### Claim 2 (depends on 1)

The separation matrix of claim 1, wherein said core region is substantially inaccessible to said target biomacromolecule.

### Claim 3 (depends on 1)

The separation matrix of claim 1, wherein a diffusion rate for said target biomacromolecule in the core region is less than 10% of the diffusion rate for said target biomacromolecule in the shell region.

### Claim 4 (depends on 1)

The separation matrix of claim 1, wherein said target biomacromolecule is a protein with a molecular weight of over 5 kDa.

### Claim 5 (depends on 1)

The separation matrix of claim 1, wherein said target biomacromolecule is an immunoglobulin.

### Claim 6 (depends on 1)

The separation matrix of claim 1, wherein said shell region has an average thickness (d) of 1-15 micrometers.

### Claim 7 (depends on 1)

The separation matrix of claim 1, wherein said shell region has an average thickness of 0.5 to 6% of the diameter or the sphere-equivalent diameter of said particles.

### Claim 8 (depends on 1)

The separation matrix of claim 1, wherein said core region has a molecular weight cutoff for globular proteins of 1 to 500 kDa.

### Claim 9 (depends on 1)

The separation matrix of claim 1, wherein said particles are substantially spherical.

### Claim 10 (depends on 1)

The separation matrix of claim 1, wherein said particles have a volume-weighted median diameter of 10-400 micrometers.

### Claim 11 (depends on 1)

The separation matrix of claim 1, wherein said shell region and said core region both comprise said porous support material.

### Claim 12 (depends on 11)

The separation matrix of claim 11, wherein said porous support material comprises a polysaccharide.

### Claim 13 (depends on 12)

The separation matrix of claim 12, wherein said polysaccharide comprises agarose or agar.

### Claim 14 (depends on 12)

The separation matrix of claim 12, wherein said polysaccharide is crosslinked.

### Claim 15 (depends on 1)

The separation matrix of claim 1, wherein said vinyl polymer is grafted to said support material via bonds or linkers.

### Claim 16 (depends on 1)

The separation matrix of claim 1, wherein said vinyl polymer comprises N-vinyl pyrrolidone monomer residues.

### Claim 17 (depends on 1)

The separation matrix of claim 1, wherein said shell region comprises ligands capable of binding said target biomacromolecule.

### Claim 18 (depends on 17)

The separation matrix of claim 17, wherein said ligands comprise positively or negatively charged groups.

### Claim 19 (depends on 17)

The separation matrix of claim 17, wherein said ligands comprise hydrophobic groups.

### Claim 20 (depends on 17)

The separation matrix of claim 17, comprising multimodal ligands.

### Claim 21 (depends on 17)

The separation matrix of claim 17, wherein said ligands comprise affinity groups.

### Claim 22 (depends on 17)

The separation matrix of claim 17, wherein said shell region comprises 10-20 000 micromol/ml ligands.

### Claim 23 (depends on 4)

The separation matrix of claim 4, wherein said protein has a molecular weight of over 20 kDa.

### Claim 24 (depends on 5)

The separation matrix of claim 5, wherein said target biomacromolecule is IgG.

### Claim 25 (depends on 15)

The separation matrix of claim 15, wherein said linkers have a structure selected from the group consisting of structures I, II, III, IV or V:
Sup-O-CH2-CH2-CH2-Pol (I)
Sup-O-CH2-CH(CH3)-Pol (II)
Sup-O-CH2-CHOH-CH2-O-CH2-CH2-CH2-Pol (III)
Sup-O-CH2-CHOH-CH2-O-CH2-CH(CH3)-Pol (IV)
Sup-O-Pol (V),
wherein Sup denotes the support material and Pol the vinyl polymer.

### Claim 26 (depends on 20)

The separation matrix of claim 20, wherein said multimodal ligands are selected from the group consisting of N-benzyl-N-methyl ethanolamine, N-benzoyl homocysteine, 4-mercaptoethyl pyridine, p-aminobenzamidoacetic acid, tryptophan and phenylalanine.

### Claim 27 (depends on 1)

A method of manufacturing a separation matrix of claim 1, comprising the steps of:
a) providing a plurality of particles comprising a porous support material with grafting sites;
b) inactivating said grafting sites in a shell region;
c) incubating said particles in a composition comprising a polymerizable monomer wherein said polymerizable monomer is a vinyl amide; and
d) initiating polymerization of said polymerizable monomer.

### Claim 28 (depends on 27)

The method of claim 27, further comprising, before step a), a step a′) of introducing grafting sites on a porous support material.

### Claim 29 (depends on 27)

The method of claim 27, further comprising a step f) of reacting said particles with a ligand precursor reagent to couple ligands to said shell region.

### Claim 30 (depends on 27)

The method of claim 27, wherein said porous support material comprises carbon-bound hydroxyl groups and wherein said method comprises:
in step b) reacting said particles with an allylation reagent to obtain at least 50 micromole/ml allyl groups covalently attached to the particles; and
in step c) reacting said particles with a halogen for a period of up to 30 minutes and then with an alkaline aqueous solution.

### Claim 31 (depends on 27)

The method of claim 27, wherein said vinyl amide is N-vinyl pyrrolidone.

### Claim 32 (depends on 1)

A method of separating a target biomacromolecule, comprising the steps of:
a) providing a sample comprising said target biomacromolecule;
b) conveying said sample through a column packed with the separation matrix of claim 1;
c) optionally conveying a wash liquid through said column; and
d) conveying an eluent through said column and recovering said target biomacromolecule in an eluate.

### Claim 33 (depends on 32)

The method of claim 32, wherein a flow velocity through the column in step b) and/or d) is at least 70 cm/h.