Separation matrices for purification of biological particles

Patent metadata

Field	Value
Publication number	US-2016367966-A1
Application number	US-201515120845-A
Country	US
Kind code	A1
Filing date	Feb 18, 2015
Priority date	Mar 14, 2014
Publication date	Dec 22, 2016
Grant date	—

Abstract

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.

First claim

1 . A separation matrix for purification of biological particles, comprising a plurality of particles having a porous core entity and a porous shell entity covering said core entity, wherein;
a) said core entity comprises at least 50 micromole/ml primary amines present on covalently attached ligands displaying at least two primary amines per ligand and b) said shell entity comprises less than 20 micromole/ml primary amines.

2 . The separation matrix of claim 1 , wherein said matrix is capable of binding at least 20 mg ovalbumin per ml matrix, such as at least 40 mg ovalbumin, in an aqueous buffer having a conductivity of at least 10 mS/cm, such as at least 20 mS/cm.

3 . The separation matrix of claim 1 , wherein said ligands display at least two, such as at least three or at least four primary amines per ligand.

4 . The separation matrix of claim 1 , wherein said ligands are attached to said core entity via secondary or tertiary amine links.

5 . The separation matrix of claim 1 , wherein the content of nitrogen and oxygen atoms in said ligands is at least 20 wt %.

6 . The separation matrix of claim 1 , wherein the content of primary amine nitrogen is at least 18 wt %.

7 . The separation matrix of claim 1 , wherein the ratio of displayed primary amines to the total amount of nitrogen atoms is at least 0.5, such as at least 0.7 or at least 0.9.

8 . 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.

9 . The separation matrix of claim 1 , wherein said ligands comprise polyallylamine of molecular weight at least 1 kDa, such as at least 10 kDa.

10 . The separation matrix of claim 9 , wherein the polyallylamine is non-crosslinked.

11 . The separation matrix of claim 1 , wherein said shell entity has an average thickness of 1 to 10 micrometers.

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

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

14 . The separation matrix of claim 1 , wherein said shell entity has a molecular weight cutoff for globular proteins of 60 to 1000 kDa, such as 100-1000 or 400-800 kDa.

15 . A method for purification of biological particles, comprising the steps of:
a) providing a separation matrix according to claim 1 ; b) contacting said matrix with a liquid comprising biological particles and at least one contaminant protein, such that said contaminant protein binds to said matrix; c) separating said liquid from said matrix and recovering said liquid with purified biological particles.

16 . The method of claim 15 , wherein said liquid has a conductivity of at least 10 mS/cm.

17 . The method of claim 16 , further comprising the steps of:
d) regenerating said matrix by contacting it with a regeneration solution comprising less than 15% organic solvents, such as less than 5%, and; e) repeating steps a)-c) at least once, such as at least five, at least 10 or at least 50 times.

18 . The method of claim 17 , wherein the regeneration solution comprises alkali.

19 . The method of claim 18 , wherein the residual protein content in the matrix after step d) is less than 100 micrograms/ml.

20 . The method of claim 19 , wherein said biological particles are selected from the group consisting of viruses, virus-like particles, cells, organelles and plasmids.

21 . The method of claim 20 , wherein at least 40 mg/ml, such as at least 60 mg/ml, of said contaminant protein binds to the matrix.

22 . The method of claim 21 , wherein said liquid comprises a buffering substance.

23 . The method of claim 22 , wherein said buffering substance comprises buffering cations and/or monovalent buffering anions.

24 . The method of claim 23 , wherein the pH of said liquid is 6.0-8.5, such as 6.5-8.5 or 6.5-8.0.

25 . The method of claim 24 , wherein the contaminant protein is an albumin, such as ovalbumin.

26 . The method of claim 25 , wherein the biological particles comprise an influenza virus and said liquid comprises allantoic fluid derived from fertilized eggs.

27 . The method of claim 26 , further comprising, before step b), a step a′) of conditioning said liquid by crossflow filtration, such as ultrafiltration and/or microfiltration.

28 . The method of claim 27 , further comprising, after step c), a step c′) of ion exchange chromatography, such as anion exchange chromatography.

29 . Use of a separation matrix according to claim 1 for purification of biological particles.

30 . The use according to claim 29 , wherein said biological particles are selected from the group consisting of viruses, virus-like particles, cells, organelles and plasmids.

31 . The use of the separation matrix in the method of claim 15 .

32 . A method for manufacturing a separation matrix according to claim 1 , comprising the steps of:
a) providing a plurality of porous polysaccharide particles, such as crosslinked agarose gel particles; b) reacting said particles with an allylation reagent to obtain at least 50 micromole/ml allyl groups covalently attached to the particles; c) reacting said particles with a halogen for a period of up to 30 minutes and then with an alkaline aqueous solution; d) coupling a ligand precursor comprising a plurality of primary amines to the remaining allyl groups.

33 . The method of claim 32 , wherein the particles are substantially spherical, with a volume average diameter of 15-400 micrometers, such as 30-100 micrometers.

34 . The method of claim 33 , wherein the ligand precursor has at least three primary amines per molecule and is coupled to the allyl groups by first reacting the allyl groups with an aqueous halogen and then under alkaline conditions with the ligand precursor.

35 . The method of claim 34 , wherein the ligand precursor is selected from the group consisting of tris(2-aminoethyl)amine and polyallylamine.

36 . The method of claim 35 , wherein the allylation reagent is selected from the group consisting of allyl halides and allyl glycidyl ether.

Assignees

• Ge Healthcare Bioprocess R&D Ab

Inventors

• Maloisel Jean-Luc
• Busson Karolina
• Lacki Karol
• Noren Bjorn
• Skoglar Helena

Classifications

• B01D15/305

  Hydrophilic interaction chromatography [HILIC] · CPC title

• B01J20/3248

  the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such · CPC title

• B01J20/28019

  Spherical, ellipsoidal or cylindrical · CPC title

• B01D15/363

  Anion-exchange · CPC title

• B01J20/3219

  involving a particular spacer or linking group, e.g. for attaching an active group · CPC title