Functionalised Chromatography Medium Comprising Polymer Nanofibres and Process of Preparation Thereof

• US 20190308169A1
• Filed: 07/14/2017
• Published: 10/10/2019
• Est. Priority Date: 07/14/2016
• Status: Active Grant

• Current Assignee: Cytiva Bioprocess R&D AB (Danaher Corporation)
• Original Assignee: Puridify Ltd. (Danaher Corporation)
• Inventors: Hummersone, Marc, Wallis, Benjamin, Leroux, Frederic, Law, William
• Granted Patent: US 11,097,252 B2
• Time in Patent Office: Days
• CPC Class Codes: B01D 15/20 relating to the conditioning of the sorbent material
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/362 Cation-exchange
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01D 15/3847 Multimodal interactions
• CPC Class Codes: B01J 20/265 modified or post-treated polymers polymer carriers or substrates subjected to further impregnating or coating B01J20/3208
• CPC Class Codes: B01J 20/28023 Fibres or filaments fibres or filaments in the form of membranes B01J20/28038; B01J20/28007 takes precedence
• CPC Class Codes: B01J 20/28038 Membranes or mats made from fibers or filaments
• CPC Class Codes: B01J 20/28052 Several layers of identical or different sorbents stacked in a housing, e.g. in a column
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/289 bonded via a spacer
• CPC Class Codes: B01J 20/3085 Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
• CPC Class Codes: B01J 20/3092 Packing of a container, e.g. packing a cartridge or column of chromatography columns B01D15/206
• CPC Class Codes: B01J 20/321 consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3212 consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• CPC Class Codes: 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
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3227 by end-capping, i.e. with or after the introduction of functional or ligand groups
• CPC Class Codes: B01J 20/3248 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 Class Codes: B01J 20/3272 : Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/328 : Polymers on the carrier being further modified
• CPC Class Codes: B01J 20/3293 : Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
• CPC Class Codes: B01J 2220/62 : In a cartridge
• CPC Class Codes: C07K 1/16 : by chromatography
• CPC Class Codes: C07K 1/18 : Ion-exchange chromatography
• CPC Class Codes: C07K 1/22 : Affinity chromatography or related techniques based upon selective absorption processes

Abstract

The present invention provides a functionalised polymeric chromatography medium, prepared by a process which comprises (i) providing a substrate formed of one or more polymer nanofibres, (ii) grafting one or more neutral polymer chains from the substrate, and (iii) contacting the grafted product with a reagent which functionalises the product of step (ii) as a chromatography medium, wherein step (ii) comprises reacting a plurality of compounds of formula and/or its enantiomers, and/or its derivatives of formula (I) and/or enantiomers and/or diastereomers thereof: with one or more functional groups present on the nanofibre substrate, wherein R₁, R₂, R₃, R₄ and R₅ may be the same or different, and are chosen from H, halogen, C₁-C₄ alkyl, or C₁-C₄ alkoxy provided that at least one of R₁, R₂, R₃, R₄ or R₅ is not hydrogen.

Claims

1. A process for preparing a functionalised polymeric chromatography medium, which process comprises

(i) providing a substrate formed of one or more polymer nanofibres,

(ii) grafting one or more neutral polymer chains from the substrate, and

(iii) contacting the grafted product with a reagent which functionalises the product of step (ii) as a chromatography medium,

wherein step (ii) comprises reacting a plurality of compounds of formula

2. The process according to claim 1, wherein step (ii) comprises reacting a plurality of compounds of formula

3. The process according to claim 1, wherein the polymer nanofibres comprise a polymer selected from the group consisting of cellulose, cellulose acetate, polysulfones, polyamides, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide, and mixtures thereof, preferably wherein the polymer is selected from the group consisting of cellulose, cellulose acetate, and a mixture thereof.

4. The process according to claim 1, wherein the amount of polymer introduced in step (ii) is between 500-60,000 μ

mol/g of grafted product.

5. The process according to claim 1, wherein the step of contacting the grafted product with a reagent introduces one or more ligand groups into the grafted product which renders the functionalised product comprising the one or more ligand groups suitable for use as a chromatography medium.

6. The process according to claim 5, wherein the density of said ligand groups in the product of step (iii) is between 100-2,500 μ

mol/g of chromatography medium.

7. The process according to claim 1, wherein the pressure drop over the functionalised polymeric chromatography medium is less than 2 MPa when a liquid phase is passed through a thickness of 0.05 to 10 mm of the medium at a flow rate of between 1 to 640 membrane volumes per minute.

8. The process according to claim 1, wherein the productivity of the functionalised polymeric chromatography medium is 50 mg/mL/min to 75,000 mg/mL/min.

9. The process according to claim 1, wherein the productivity of the functionalised polymeric chromatography medium is greater than 200 mg/ml/min.

10. The process according to claim 1, wherein the grafting step (ii) has the effect of increasing the dynamic binding capacity (DBC) of the functionalised polymeric chromatography medium.

11. The process according to claim 1, wherein the substrate is treated between steps (i) and (ii) to introduce the one or more functional groups, or the substrate is treated between steps (i) and (ii) to deprotect or activate any functional groups on the substrate, or the substrate is treated between steps (i) and (ii) to increase the number/density of functional groups on the substrate.

12. The process according to claim 1, wherein the grafting step (ii) is carried out under conditions which additionally, in the same step, introduce the one or more functional groups, or deprotect or activate any functional groups on the substrate, or increase the number/density of functional groups on the substrate.

13. The process according to claim 12, wherein the grafting step (ii) is effected in the presence of aqueous alkali, preferably NaOH or KOH, in water or water:

ethanol, preferably in water.

14. The process according to claim 1, wherein the one or more functional groups present on the nanofibre substrate comprise hydroxyl groups.

15. The process according to claim 1, wherein in the functionalisation step (iii), the functionalising reagent is selected from the group consisting of glycidyltrimethylammonium chloride (GMAC), 1,4-butanesulfone, sodium chloroacetate, NaIO₄ followed by Protein A, divinylsulfone followed by Protein A, allyl glycidyl ether followed firstly by a halohydrin-forming reagent, preferably N-bromosuccinamide, and subsequently by Protein A, allyl glycidyl ether followed firstly by an epoxide-forming reagent and subsequently by Protein A, and combinations thereof.

16. The process according to claim 10, wherein one or more of the polymer chains is branched.

17. The process according to claim 1, wherein in step (iii) the reagent functionalises the grafted product so that the resultant functionalised chromatography medium is suitable for use in a chromatography method chosen from the group consisting of ion exchange, affinity capture, hydrophobic interaction and mixed mode methods.

18. The process according to claim 17, wherein

the chromatography method is a cationic exchange method, and the reagent functionalises the chromatography medium with one or more charged groups comprising one or more carboxylate, sulphonate or phosphonate moieties;

the chromatography method is an anionic exchange method, and the reagent functionalises the chromatography medium with one or more charged groups comprising one or more quaternary amino or diethylamine moieties;

the chromatography method is an affinity capture chromatography method, and the reagent functionalises the chromatography medium with one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine groups, or groups containing a metal cation;

the chromatography method is a hydrophobic interaction chromatography method, and the reagent functionalises the chromatography medium with one or more propyl, butyl, phenyl, or octyl groups;

or

the chromatography method is a mixed mode chromatography method, and the reagent functionalises the chromatography medium with one or more MEP, octylamine, N-benzyl methyl ethanolamine or N-benzoyl-homocysteine groups.

19. The process according to claim 1, wherein in step (iii) the reagent functionalises the grafted product with one or more Protein A molecules.

20. The process according to claim 1, wherein step (iii) involves multiple steps that together functionalise the product of step (ii) as a chromatography medium.

21. The process according to claim 20, wherein in step (iii):

(a) the grafted product is contacted with a reagent selected from the group selected from divinyl sulfone, allyl glycidyl ether, and combinations thereof;

(b) the product of step (a) is optionally treated with a halohydrin-forming reagent; and

(c) the product of step (b) is contacted with Protein A.

22. The process according to claim 1, wherein the substrate is in the form of a membrane.

23. The process according to claim 1, wherein the nanofibres have mean diameters from 10 nm to 1000 nm.

24. The process according to claim 1, which process comprises

(i) providing a substrate formed of one or more cellulose nanofibres,

(ii) grafting one or more polyglycerol polymer chains from the substrate by reacting a plurality of compounds of formula

25. The process according to claim 24, wherein step (ii) comprises grafting one or more polyglycerol polymer chains from the substrate by reacting a plurality of compounds of formula

26. A functionalised chromatography medium obtainable by the process according to claim 1.

27. A process for preparing a chromatography cartridge, which process comprises carrying out the process of claim 1 and incorporating the thus-obtained product into a cartridge.

28. A chromatography cartridge which (a) is obtainable by the process of claim 27, or (b) which comprises one or more functionalised chromatography media.

29. Use of a functionalised chromatography medium according to claim 26 or a chromatography cartridge in chromatography.

30. A process for isolating one or more biological molecules from a mobile phase, which process comprises contacting one or more biological molecules in a mobile phase with a functionalised chromatography medium according to claim 26 or a chromatography cartridge.

31. The process according to claim 30 wherein the one or more biological molecules in a mobile phase is contacted with the functionalised chromatography medium for a period of time of one minute or less.

32. The process according to claim 30, wherein the one or more biological molecules are one or more monoclonal antibodies, or proteins engineered to exhibit a site with an affinity for Protein A binding, and the functionalised chromatography medium carries at least one Protein A ligand group.