Process for preparation of pegylated therapeutic proteins

Patent metadata

Field	Value
Publication number	US-12540158-B2
Application number	US-202017782161-A
Country	US
Kind code	B2
Filing date	Dec 3, 2020
Priority date	Dec 3, 2019
Publication date	Feb 3, 2026
Grant date	Feb 3, 2026

Abstract

The present invention provides a novel process of PEGylation and purification of the PEGylated therapeutic proteins. The developed PEGylation protocol provides high yield of PEGylated recombinant proteins (with % mono-PEG conversion of ≥70%) with a much faster reaction completion time indicating high productivity and fast kinetics. Thereafter a displacement mode cation exchange chromatography (CEX) is utilized which is able to remove all the multi-PEGylated impurities in the loading flow-through, simplifying the overall purification process. The novel PEGylation and purification process can be integrated to any existing manufacturing process to offer an end-to-end integrated assembly from inclusion bodies to purified PEGylated product with higher productivity and improved resin utilization.

First claim

What is claimed is:

1 . A process of preparation and purification of PEGylated therapeutic proteins comprising:
a. PEGylating a therapeutic protein by incubating the therapeutic protein with a functionalised PEG until % mono-PEG conversion reaches ≥70% to obtain a PEGylated protein; b. quenching the mixture obtained from step (a) by addition of a base; c. diluting the mixture of step (b) with a dilute acid to reduce the pH of the PEGylated protein in the mixture in the range 3-6 and conductivity to <3 mS/cm bmin in order to condition the mixture for binding on cation exchange chromatography (CEX); and d. subjecting the PEGylated protein obtained from step (c) to cation exchange chromatography (CEX) to obtain a PEGylated and purified therapeutic protein, wherein the CEX is performed in a displacement mode by loading the PEGylated protein onto a CEX column comprising a cation exchange resin.

2 . The process as claimed in claim 1 , wherein the process is operated in a batch, semi-continuous or fully continuous mode.

3 . The process as claimed in claim 1 , wherein said step (a) comprises:
a. reacting the therapeutic protein with the functionalised PEG in a ratio of 2:1 to 10:1 in presence of a catalyst; and b. incubating the therapeutic protein with the functionalised PEG with or without a reducing agent at temperature in the range of 10-37° C. for a time period in the range of 50-70 min or until % mono-PEG conversion reaches ≥70%, to obtain the PEGylated protein.

4 . The process as claimed in claim 1 , wherein the PEGylated protein is quenched by addition of the base in the concentration range of 0.05-2 M.

5 . The process as claimed in claim 1 , wherein the therapeutic protein is a cytokine selected from the group consisting of interferons, growth factors, tumour necrosis factor, interleukins, and colony stimulating factors.

6 . The process as claimed in claim 3 , wherein the functionalised PEG is selected from the group consisting of Methoxy PEG Propionaldehyde (mPEG-ALD), Methoxy PEG Succinimidyl propionate, Methoxy PEG N-hydroxy succinimide, Methoxy PEG Succinimidyl carbonate, and Methoxy PEG Maleimide.

7 . The process as claimed in claim 3 , wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride, and sodium borohydride, in the range of 1-100 mM.

8 . The process as claimed in claim 1 , wherein the dilute acid is selected from the group consisting of acetic acid, citric acid, hydrochloric acid, and any mixtures thereof so as to reduce pH in the range of 3-6 and achieve dilution in the range of 10-15 folds.

9 . The process as claimed in claim 1 , wherein the base is selected from the group consisting of Tris, NaOH, and Ammonium hydroxide.

10 . The process as claimed in claim 3 , wherein the catalyst is selected from the group consisting of sodium cyanoborohydride, and sodium borohydride.

11 . The process as claimed in claim 5 , wherein the therapeutic protein is granulocyte colony-stimulating factor (GCSF).

12 . The process as claimed in claim 1 , wherein the process is performed as a continuous mode in a Coiled flow inversion reactor (CFIR), the continuous mode comprising:
a. performing PEGylation in the CFIR with the therapeutic protein and the functionalized PEG pumped at equal flow rate at an entry of the CFIR; b. quenching output from step (a) by pumping the base at a flow rate of 5-10% v/v of the output flow rate; and c. conditioning output from step (b) by pumping the dilute acid to achieve dilution in the range of 10-15 fold, and wherein combined flow from step (a), step (b) and step (c) constitutes a loading flow rate for the cation exchange chromatography (CEX).

13 . The process as claimed in claim 1 , wherein the cation exchange chromatography (CEX) comprises:
subjecting the therapeutic protein, which has been PEGylated, quenched and conditioned, to the cation exchange chromatography (CEX) in the displacement mode by loading the PEGylated protein onto the CEX column to perform the cation exchange chromatography (CEX), and wherein the CEX is carried out by loading the cation exchange resin between 5-100% of breakthrough capacity or until a predetermined displacement of multi-PEGylated impurities is obtained.

14 . The process as claimed in claim 13 , wherein the cation exchange resin is a weak cation exchange resin selected from the group consisting of Fractogel COO − , CM Sepharose, Toyopearl CM 650 M, and Ceramic HyperD CM.

15 . The process as claimed in claim 13 , wherein the cation exchange resin is a strong cation exchange resin selected from the group consisting of Eshmuno CPX, Poros HS, Poros XS, Fractogel SO 3 , SP Sepharose, Capto S, Capto SP ImpRes, S Hypercel, and UNOsphere S.

16 . The process as claimed in claim 13 , wherein the cation exchange resin is a multimodal resin selected from the group consisting of Capto MMC, Capto Adhere, MEP Hypercel, HEA Hypercel, and PPA Hypercel.

17 . The process as claimed in claim 13 , wherein the cation exchange chromatography is performed in a batch or continuous chromatography mode.

18 . A system for carrying out the process as claimed in claim 1 , comprising:
a coiled flow inversion reactor for continuous PEGylation; and a system for continuous chromatography in the displacement mode, wherein unit operations of PEGylation and chromatography are in a continuous mode, and a continuous manufacturing train for the continuous mode comprises: a first surge vessel to receive the therapeutic protein from a plurality of inline concentrators; an inline dynamic mixer for mixing the functionalized PEG, a catalyst and the therapeutic protein; the coiled flow inversion reactor for continuous PEGylation; a depth filtration unit; and a second surge vessel with volume that is five times a cation exchange loading volume placed in between the PEGylation and chromatography step wherein the second surge vessel is for in-process analysis with offline, atline, or online sampling, and wherein the CEX is performed in a continuous chromatography setup.

Assignees

• Indian Institute Tech Delhi

Inventors

• Rathore Anurag S
• Kateja Nikhil
• Nitika

Classifications

• C07K14/535

  Granulocyte CSF; Granulocyte-macrophage CSF · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• C07K1/1077 Primary

  by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids · CPC title

• B01J19/243

  spirally, concentrically or zigzag wound · CPC title

• A61K47/60

  the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol · CPC title