Chromatography resin, production and use thereof

Patent metadata

Field	Value
Publication number	US-11124539-B2
Application number	US-201716348534-A
Country	US
Kind code	B2
Filing date	Nov 14, 2017
Priority date	Nov 16, 2016
Publication date	Sep 21, 2021
Grant date	Sep 21, 2021

Abstract

The present invention relates to the field of chromatography and more specifically to producing protein affinity chromatography resins comprising affinity ligands based on a N-terminal fragment of a split intein, such as DnaE from Nostoc punctiforme, as well as methods for using the same. The N-terminal fragments are produced in inclusion bodies in bacterial cells.

First claim

The invention claimed is:

1. A method for production of an affinity chromatography resin comprising an N-terminal split intein fragment as an affinity ligand, comprising the following steps:
a) expression of an N-terminal split intein fragment, wherein the N-terminal split intein fragment comprises a sequence selected from SEQ ID NO:s 1-5 or a sequence having at least 95% homology therewith and an Alanine at the fourth position and a Serine at the thirty-first position, as insoluble protein in inclusion bodies in bacterial cells,
b) harvesting said inclusion bodies;
c) solubilizing said inclusion bodies and releasing expressed protein;
d) binding said protein on a solid support;
e) refolding said protein;
f) releasing said protein from the solid support; and
g) immobilizing said protein as ligands on a chromatography resin to form an affinity chromatography resin—wherein the ligand density on said chromatography resin is 5-10 mg ligand/ml resin.

2. The method according to claim 1 , wherein the N-terminal split intein fragment comprises a sequence selected from SEQ ID NO:s 1-5.

3. The method according to claim 1 , wherein said chromatography resin is selected from the group consisting of agarose, polystyrene, methacrylate and cellulose beads.

4. The method according to claim 1 , wherein the immobilization in step g) is by covalent attachment.

5. The method according to claim 1 , wherein said immobilization is preceded by concentration to a protein concentration between 5-30 mg/ml.

6. The method according to claim 1 , wherein the chromatography resin is packed in a column, fluidized bed, porous monolith or capillary bed.

7. The method according to claim 1 , comprising binding of the N-terminal fragment on an ion exchange column in step d).

8. The method according to claim 1 , wherein the N-terminal fragment is tagged, and the solid support in step d) is an IMAC column.

9. The method according to claim 1 , wherein said chromatography resin is agarose beads.

10. The method according to claim 1 , wherein the N-terminal fragment is poly-histidine tagged, and the solid support in step d) is an IMAC column.

11. The method according to claim 1 , wherein the N-terminal split intein fragment a sequence having at least 95% homology to one of SEQ ID NO:s 1-5, an Alanine at the fourth position, a Serine at the thirty-first position, and a Serine at the sixty-second position.

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Ohman Johan William
• Ander Mats
• Graner Therese
• Jorsback Anneli
• Zou Jinyu

Classifications

• C07K2319/95

  containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence) · CPC title

• B01J20/3217

  Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond · CPC title

• B01J20/3212

  consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds · CPC title

• B01J20/321

  consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds · CPC title

• C07K2319/92

  containing an intein ("protein splicing")domain · CPC title