WO2014155365A2 - Purification method - Google Patents

Purification method Download PDF

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Publication number
WO2014155365A2
WO2014155365A2 PCT/IB2014/060287 IB2014060287W WO2014155365A2 WO 2014155365 A2 WO2014155365 A2 WO 2014155365A2 IB 2014060287 W IB2014060287 W IB 2014060287W WO 2014155365 A2 WO2014155365 A2 WO 2014155365A2
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WO
WIPO (PCT)
Prior art keywords
gcsf
cation exchange
exchange chromatography
purification
pegylated
Prior art date
Application number
PCT/IB2014/060287
Other languages
French (fr)
Other versions
WO2014155365A3 (en
Inventor
Vivek ARTHANARI
Gopinath GOVINDARAJAN
Jaby Jacob
Bharata Ratnam PARAYITAM
Original Assignee
Dr.Reddy's Laboratories Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr.Reddy's Laboratories Limited filed Critical Dr.Reddy's Laboratories Limited
Publication of WO2014155365A2 publication Critical patent/WO2014155365A2/en
Publication of WO2014155365A3 publication Critical patent/WO2014155365A3/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF

Definitions

  • the present invention relates to a purification process for pegylated Granulocyte Colony Stimulating Factor (GCSF).
  • GCSF Granulocyte Colony Stimulating Factor
  • CSFs colony stimulating factors
  • GCSF Granulocyte Colony Stimulating Factor
  • GCSF is one of several proteins produced by recombinant DNA technology for therapeutic use.
  • lenograstim the glycosylated form of GCSF is expressed in mammalian cells
  • filgrastim the non- glycosylated form is expressed in Escherichia coli ⁇ E. coli).
  • Proteins expressed by recombinant DNA methods in bacteria such as E. coli are usually expressed as insoluble aggregates called inclusion bodies. These protein aggregates are separated, solubilized in the presence of protein denaturing agents, refolded and then purified by appropriate column chromatographic procedures.
  • WO2008096370 explains a two-step chromatography, cation exchange followed by size exclusion after a one step refolding of GCSF for its purification.
  • GCSF GCSF
  • the stability of GCSF can be improved and the immune response against these proteins reduced when these proteins are coupled to polymeric molecules
  • Veronese et al discusses the impact of PEGylation on biological therapies (BioDrugs. 2008; 22(5): 315-29).
  • WO94/28024 discloses that physiologically active proteins modified with PEG (Poly Ethylene Glycol) exhibit reduced immunogenicity and antigenicity and circulate in the bloodstream considerably longer than unconjugated proteins.
  • the principle object of the present invention is to provide an improved method for purification of pegylated GCSF with effective separation of high and low molecular weight impurities.
  • the present invention discloses a method for purification of pegylated GCSF that effectively separates low and high molecular weight impurities from desired mono- pegylated GCSF by using a cation exchange chromatography.
  • FIG 1 is an illustration of a chromatogram from the procedure as described in Example 2.
  • the line marked “Cond” represents the increase in conductivity in mS/cm.
  • Peak A represents the eluate (pegylated GCSF) obtained from cation exchange chromatography resin.
  • GCSF is a human endogenous secretory protein which selectively induces the development of granulocyte committed progenitors from multipotent hematopoietic cells.
  • the term "Peg-GCSF” or “pegylated GCSF” refers to a GCSF protein which is covalently linked with one or more polyethylene glycol moieties.
  • wash buffer refers to a buffer that is used to wash or re-equilibrate the ion exchange resin, or to elute one or more impurities from the ion exchange resin, prior to elution of the protein of interest.
  • low molecular weight impurities refers to forms of protein with lower molecular weight than the desired protein which includes but is not limited to non-pegylated forms of the protein, fragmented forms of the protein.
  • high molecular weight impurities refers to forms of protein with higher molecular weight than the desired protein which includes but is not limited to more than one pegylated form of the protein.
  • the invention provides a method of purification of pegylated GCSF by cation exchange chromatography wherein a combination of post load washes of varying conductivity are employed.
  • the invention provides a method of purification of pegylated GCSF by separating low molecular and high molecular weight impurities from a composition comprising Peg GCSF using cation exchange chromatography comprising a. Loading the said composition onto a cation exchange chromatography column b. Washing the cation exchange chromatography column with 1 st wash buffer
  • the invention provides a method of purification of pegylated GCSF by separating low molecular and high molecular weight impurities from a composition comprising Peg GCSF using cation exchange chromatography comprising a. Loading the said composition onto a cation exchange chromatography column b. Washing the cation exchange chromatography column with 1 st wash buffer
  • the second wash step may be followed by additional wash steps.
  • Cation exchange chromatographic step mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane, which could function as a weak or a strong cation exchanger.
  • Commercially available cation exchange support include a resin, but are not limited to, those having a sulfonate based group e.g., MonoS, MiniS, Source 1 5S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh.
  • a resin but are not limited to, those having
  • the support could be a monolithic column, disk or tubular, that performs the function of a cation exchanger.
  • a strong cation exchange resin such as SP-Sepharose® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked, 6 % agarose matrix attached to a sulfopropyl functional group.
  • SP-Sepharose® GE Healthcare Life Sciences
  • the pegylated GCSF obtained from example 1 was loaded onto a cation exchange resin, pre-equilibrated with sodium acetate (1 .6 mS/cm). This was followed by a first post load wash with 20 mM sodium acetate at a conductivity of 1 .6 mS/cm followed by a second post load wash with 20 mM sodium acetate, 175 mM NaCI at a conductivity of 18-21 mS/cm. The bound protein was eluted using a 20 mM sodium acetate buffer containing NaCI at a conductivity between 30 -32.5 mS/cm.

Abstract

The present invention is related to a method for purification of pegylated GCSF. In particular, the invention employs a cation exchange chromatography with varying conductivity of wash buffers to separate low and high molecular weight impurities from the desired pegylated GCSF.

Description

PURIFICATION METHOD
RELATED APPLICATION
This application is related to and takes priority from Indian Provisional Application 1430/CHE/2013 filed 29th March 2013 and is herein incorporated in its entirety. FIELD OF THE INVENTION
The present invention relates to a purification process for pegylated Granulocyte Colony Stimulating Factor (GCSF).
BACKGROUND OF THE INVENTION
Differentiation and proliferation of hematopoietic cells are regulated by glycoproteins referred to as colony stimulating factors (CSFs). Of the various CSFs, the Granulocyte Colony Stimulating Factor (GCSF) stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. When administered to mammals, GCSF promotes a dramatic increase in circulating granulocyte populations.
GCSF is one of several proteins produced by recombinant DNA technology for therapeutic use. Of the two types of GCSF clinically available, lenograstim, the glycosylated form of GCSF is expressed in mammalian cells, and filgrastim, the non- glycosylated form is expressed in Escherichia coli {E. coli). Proteins expressed by recombinant DNA methods in bacteria such as E. coli, are usually expressed as insoluble aggregates called inclusion bodies. These protein aggregates are separated, solubilized in the presence of protein denaturing agents, refolded and then purified by appropriate column chromatographic procedures.
To purify GCSF, many processes have been disclosed in patent and scientific literature that comprise ion exchange chromatography, chromatofocusing, reverse phase chromatography, hydrophobic interaction chromatography, and combinations of these and other methods. l WO05077973 teaches a method of using a reverse phase column for purification of human recombinant proteins and US2012093765 explains a two phase extraction system for the preparation of pure recombinant human GCSF.
US20080171 857 describes a method for purifying GCSF by using a cation exchange and a hydrophobic interaction chromatography step.
WO2008096370 explains a two-step chromatography, cation exchange followed by size exclusion after a one step refolding of GCSF for its purification.
US20050159589 teaches a method for purification of GCSF by using
Immobilised Metal Affinity Chromatography. EP1200471 describes the use of hydrophobic interaction chromatography and hydroxyapatite column for the purification of GCSF.
The stability of GCSF can be improved and the immune response against these proteins reduced when these proteins are coupled to polymeric molecules (Rajan, et al., Protein Sci. 2006 May; 15(5): 1 063-1075). Veronese et al, discusses the impact of PEGylation on biological therapies (BioDrugs. 2008; 22(5): 315-29). WO94/28024 discloses that physiologically active proteins modified with PEG (Poly Ethylene Glycol) exhibit reduced immunogenicity and antigenicity and circulate in the bloodstream considerably longer than unconjugated proteins.
Various methods of conjugating polypeptides like GCSF with polymeric moieties like PEG are well known and extensively described in the prior art. The preparation of glyco PEGylated GCSF, for example, is described in WO2005055946 and
WO2006074467 is directed to the preparation of conjugates between GCSF and PEG moieties.
The prior art discussed above describe various ways which can be employed to purify GCSF. However the modification of polypeptides with polymeric moieties like PEG, causes significant shift in the chemical and physical properties of those
polypeptides. Therefore, methods, which are useful for the purification of non-modified polypeptides may not necessarily be effective in purification of their modified versions. For example, a pegylation process may generate low and high molecular weight impurities which may require optimization of purification process for their removal.
Removal of such impurities poses a significant challenge in the development of methods for the purification of pegylated GCSF. The principle object of the present invention is to provide an improved method for purification of pegylated GCSF with effective separation of high and low molecular weight impurities.
SUMMARY OF THE INVENTION
The present invention discloses a method for purification of pegylated GCSF that effectively separates low and high molecular weight impurities from desired mono- pegylated GCSF by using a cation exchange chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1 :is an illustration of a chromatogram from the procedure as described in Example 2. The line marked "Cond" represents the increase in conductivity in mS/cm. Peak A, represents the eluate (pegylated GCSF) obtained from cation exchange chromatography resin.
DETAILED DESCRIPTION OF THE INVENTION
GCSF is a human endogenous secretory protein which selectively induces the development of granulocyte committed progenitors from multipotent hematopoietic cells. The term "Peg-GCSF" or "pegylated GCSF" refers to a GCSF protein which is covalently linked with one or more polyethylene glycol moieties.
The term "wash buffer" as used herein refers to a buffer that is used to wash or re-equilibrate the ion exchange resin, or to elute one or more impurities from the ion exchange resin, prior to elution of the protein of interest. The term "low molecular weight impurities" as used herein refers to forms of protein with lower molecular weight than the desired protein which includes but is not limited to non-pegylated forms of the protein, fragmented forms of the protein.
The term "high molecular weight impurities" as used herein refers to forms of protein with higher molecular weight than the desired protein which includes but is not limited to more than one pegylated form of the protein.
In current invention provides a method of purification of pegylated GCSF by cation exchange chromatography wherein a combination of post load washes of varying conductivity are employed. In an embodiment, the invention provides a method of purification of pegylated GCSF by separating low molecular and high molecular weight impurities from a composition comprising Peg GCSF using cation exchange chromatography comprising a. Loading the said composition onto a cation exchange chromatography column b. Washing the cation exchange chromatography column with 1 st wash buffer
solution with a buffer of low conductivity.
c. Washing the cation exchange chromatography column with 2nd wash buffer solution with a buffer of high conductivity.
d. Eluting the desired Peg GCSF molecule from the column
In an embodiment, the invention provides a method of purification of pegylated GCSF by separating low molecular and high molecular weight impurities from a composition comprising Peg GCSF using cation exchange chromatography comprising a. Loading the said composition onto a cation exchange chromatography column b. Washing the cation exchange chromatography column with 1 st wash buffer
solution with a buffer of low conductivity.
c. Washing the cation exchange chromatography column with 2nd wash buffer solution with a buffer of high conductivity,
d. Eluting the desired Peg GCSF molecule from the column wherein the said wash buffer solution of low conductivity has a value that is less than about 2 mS/cm and wherein the said wash buffer of high conductivity has a value that is about 18 mS/cm to about 21 mS/cm.
In another embodiment the second wash step may be followed by additional wash steps.
Cation exchange chromatographic step mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane, which could function as a weak or a strong cation exchanger. Commercially available cation exchange support include a resin, but are not limited to, those having a sulfonate based group e.g., MonoS, MiniS, Source 1 5S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh. Alternatively, the support could be a monolithic column, disk or tubular, that performs the function of a cation exchanger. In embodiments of the invention, a strong cation exchange resin, such as SP-Sepharose® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked, 6 % agarose matrix attached to a sulfopropyl functional group. The invention is more fully understood by reference to the following examples.
These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLE 1
Peqylation of GCSF Purified GCSF and methoxy-Poly Ethylene Glycol Propionaldehyde are mixed in a molar ratio of about 1 :5 at a pH pH of 5.0±0.2. The mixture is incubated for 7 to 9 hours and then quenched with 75 mM acetic acid. EXAMPLE 2
Cation exchange Chromatography
The pegylated GCSF obtained from example 1 was loaded onto a cation exchange resin, pre-equilibrated with sodium acetate (1 .6 mS/cm). This was followed by a first post load wash with 20 mM sodium acetate at a conductivity of 1 .6 mS/cm followed by a second post load wash with 20 mM sodium acetate, 175 mM NaCI at a conductivity of 18-21 mS/cm. The bound protein was eluted using a 20 mM sodium acetate buffer containing NaCI at a conductivity between 30 -32.5 mS/cm.

Claims

Claims
We claim:
1 - A method of purification of pegylated GCSF by separating low molecular and high molecular weight impurities from a composition comprising pegylated GCSF using cation exchange chromatography, comprising the steps of;
a. loading the said composition onto a cation exchange chromatography support
b. washing the cation exchange chromatography support with 1 st wash buffer solution with a buffer of low conductivity.
c. washing the cation exchange chromatography support with 2nd wash buffer solution with a buffer of high conductivity and
d. eluting the desired pegylated GCSF molecule from the support
2- A method according to claim 1 , wherein the said wash buffer solution of low
conductivity has a value less than 2 mS/cm.
3- A method according to claim 1 , wherein the said wash buffer of high conductivity has a value about 18 mS/cm to about 21 mS/cm.
4- A method according to claim 1 , wherein the second wash step may be followed by additional wash steps.
PCT/IB2014/060287 2013-03-29 2014-03-29 Purification method WO2014155365A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1430/CHE/2013 2013-03-29
IN1430CH2013 2013-03-29

Publications (2)

Publication Number Publication Date
WO2014155365A2 true WO2014155365A2 (en) 2014-10-02
WO2014155365A3 WO2014155365A3 (en) 2015-02-19

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Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1663962A (en) * 2004-03-01 2005-09-07 重庆富进生物医药有限公司 Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof
CN1313612C (en) * 2005-12-20 2007-05-02 山东泉港药业有限公司 Method for producing recombinant human granulocyte colony stimulating factor
MX2012010661A (en) * 2010-03-17 2013-03-25 Biogenerix Gmbh Method for obtaining biologically active recombinant human g-csf.
CN102234310B (en) * 2010-04-30 2017-02-08 杭州九源基因工程有限公司 Polyethylene glycol modified protein separating and purifying method
KR101831300B1 (en) * 2010-10-29 2018-02-23 한미사이언스 주식회사 Method of purifying human granulocyte-colony stimulating factor from recombinant e. coli
CN102485742A (en) * 2010-12-02 2012-06-06 山东新时代药业有限公司 Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor

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