FIELD OF THE INVENTION

The present invention relates to a method for purification of recombinant human granulocyte colony stimulating factor (rh-GCSF) from one or more process and product related impurities by using an anion exchange chromatography, operated in flow-through mode.

BACKGROUND OF THE INVENTION

G-CSF is a glycoprotein that stimulates differentiation of progenitor“stem cells” into granulocytes. In biotherapeutics, G-CSF has been used in treatment of neonatal infections, neutropenia, and to tackle severe infections and sepsis in acute myeloid leukaemia's. Recombinant human Granulocyte cell stimulating factor (rh-GCSF) has been therapeutically used and is indicated for treatment of chemotherapy induced neutropenia in cancer patients.

GCSF is one of several protein therapeutics which are produce by recombinant DNA technology. Recombinant GCSF produced in both bacteria (Filgrastim) and mammalian cells (Lenograstim) are clinically available. Recombinant Filgrastim, produced in bacteria, such as E.coli., are usually expressed in the form of inclusion bodies. Therefore, preparation of therapeutically effective Filgrastim involves processes, such as inclusion bodies isolation, solubilization in the presence of protein denaturing agents, followed by refolding the protein to its native state. The refolded rh-GCSF is then purified by various chromatographic approaches. Filgrastim produced in E.coli has a molecular weight of about 18Kda, with an isoelectric point of about 5.5.

U.S. patents 4810643, 4999291, 5055555, 5849883, 5582823, 5580755 and 5830705, describe various aspects of recombinant expression and purification of the h-GCSF protein from various expression systems ranging from bacterial cells to yeast and mammalian cells. Expression of rhG-CSF as inclusion bodies in a bacterial system is described in U.S. Patent 4810643. Recombinant protein production always leads to two broad classes of impurities - Process related and Product related. Process related impurities primarily constitute of impurities such as host cell DNA (HCDNA), host cell proteins (HCP), bacterial endotoxins (BET), while impurities, such as high molecular weight aggregates (HMW) or low molecular weight variants (LMW), constitute product related impurities. Various types of impurities in therapeutic proteins can significantly affect the biological property of the protein. Therefore, regulatory authorities pose strict specifications for content of various impurities within the pharmaceutical protein preparations.

Protein chromatography at commercial scale has been used to address the challenges associated with impurity removal from therapeutic protein preparations. Chromatography is primarily carried out in two modes i.e. bind-elute mode and flow-through mode.

In a bind-elute mode i.e. the protein of interest is first allowed to bind to the chromatography column under suitable conditions and then the conditions are so altered by means of suitable elution solvent or buffer system such that the bonding of the protein to the column could be reversed post. Washing the column with suitable wash solvents of buffer systems allow for impurities to be separated from the protein of interest. Contrary to a bind elute mode chromatography, flow-through chromatography relies on the property that the protein of interest does not bind or binds minimally to the column and purified protein is recovered in flow-through, while the impurities are allowed to bind to the column.

Despite significant advances in the understanding of various chromatography techniques and availability of various buffer systems, protein purification platforms, therapeutic proteins chromatography still remains significantly challenging and unpredictable.

Unpredictability in the art is primarily attributed to the diverse physiochemical property that the protein of interest as well as the process and product related impurities demonstrate. Since Chromatographic techniques primarily rely on fractionating the protein of interest and the related impurities based on these physiochemical properties, understanding physiochemical properties of both the impurity as well as the protein of interest is of utmost importance, and a tailored approach to purification has to be adopted. The purification process becomes significantly unpredictable, when the protein of interest and the impurities demonstrate similar or proximal physiochemical properties, for example when both, the impurity and the protein of interest, are acidic or are basic or share similar hydrophobicity etc.

Various approaches to purification of GCSF has been disclosed in several prior art references. US5849883 discloses purification of refolded bovine GCSF by using an ion exchange chromatography. Specifically, US ‘883 utilizes a bind elute mode chromatography using a CM Sepharose IEX column. US6489447 broadly describes protein purification using an ion exchange chromatography in bind-elute mode, and particularly attempts to address separation of deamidated variants. It also describes the importance of changing wash buffer conductivity and/or pH in protein purification. US9453045 discloses use of multimodal chromatography for purification of recombinant GCSF obtained from culture supernatant.

The state of art has described various approaches to purification of recombinant GCSF, however the state of art has been limited to a bind-elute mode chromatography. As could be understood a bind elute mode chromatography would require multiple buffer systems to fractionate the protein of interest with the impurities. More importantly, the prior arts as a whole is silent about how specifically impurities such as host cell proteins and certain process related impurities could be removed and the extent to removal that is possible. Thus, the main aim of instant invention is to develop a fast and cost-effective flow-through based process for purification of rh-GCSF, which is effective at removal of various process related impurities, such as HCP and HCD as well as product related impurities such as HMW species of refolded a rh-GCSF. As could be understood a flow-through based process could diminish the need of multiple buffer systems that may be needed in a bind elute mode chromatography, and hence provide significant operational and cost advantages at an industrial set up.

SUMMARY OF THE INVENTION

The present invention provides a method of purifying GCSF using anion exchange chromatography for impurities removal.

In one embodiment, the invention provides a method for purifying refolded rh-GCSF using an anion exchange chromatography in flow-through mode.

In another embodiment, the invention provides a method for purifying GCSF comprising the steps:

a) recovering refolded rh-GCSF from inclusion bodies, expressed in an E coli host cell

b) subjecting the refolded rh-GCSF to an anion exchange chromatography c) collecting purified GCSF as part of flow-through.

In a further embodiment, the refolded GCSF is loaded onto anion exchange resin with a buffer at conductivity not less than 4.50 mS/cm and pH about 7.1 -7.8.

In another embodiment, the refolded GCSF may be subjected to ultrafiltration/Diafiltration before loading onto anion exchange column.

In an embodiment, the anion exchange chromatographic step may be preceded by a tangential flow filtration step.

In another embodiment, the invention provides a method of purification of GCSF by anion exchange chromatography, wherein the anion exchange flow-through is collected as a whole or as a fraction from the anion exchange resin.

In another embodiment, the impurity removal using anion exchange chromatography is more that 70%. In an embodiment, the invention provides a method for purifying rh-GCSF comprising following steps: a) recovering the refolded GCSF from inclusion bodies produced in the recombinant expression

b) subjecting the biologically active GCSF to anion exchange chromatography c) collecting the purified GCSF in flow-through

wherein the anion exchange chromatography step removes about greater than 70% impurities.

In another embodiment, the anion exchange chromatography step may be followed by another chromatographic step.

In an embodiment, the invention provides a method for purifying GCSF comprising the following steps a) isolating the inclusion bodies of GCSF bacterial host cell;

b) solubilizing the inclusion bodies in a buffered solution comprising chaotropic agent and/or reducing agent;

c) refolding of solubilized GCSF in a refolding solution comprising a oxidizing and reducing agent under suitable conditions for at least about 2 hours;

d) recovering said refolded GCSF

e) subjecting the refolded GCCF to ultrafiltration/diafiltration

f) subjection the refolded GCSF to anion exchange chromatography

g) collecting the purified GCSF in flow-through from the anion exchange column in step (f) wherein the anion exchange chromatography step is capable of removing at least 90% of host cell proteins (HCP), host cell DNA (HCDNA), bacterial endotoxin (BET), and high molecular aggregates. The embodiments mentioned herein may further include one or more sterile filtration or nano filtration steps.

Brief description of Drawings

Fig-1 Anion Exchange Chromatography (Flow-through mode)

Detailed Description of the Invention

The invention provides a method for removal of process and product related impurities from a GCSF using anion exchange chromatography, operated in flow through mode.

In an embodiment, the present invention provides a method of purification of GCSF from one or more impurities using anion exchange chromatography.

In an embodiment, the invention provides a method of purification of an GCSF from a sample comprising one or more impurities, comprising a) loading the sample onto an anion exchange chromatographic resin b) collecting the flow through.

In another embodiment, the invention describes a method for purification of refolded GCSF using anion exchange chromatography, wherein HCP, HCDNA, BET and HMW are reduced by at least 90%.

The term“GCSF” or G-CSF or rh-GCSF has been used interchangeably herein, refers to recombinant human granulocyte colony stimulating factors or its analogues with isoelectric point is 5.5+ 1.

As used herein, "chaotropic agent(s)" refers to a compound that, in a suitable concentration in aqueous solution, is capable of changing the spatial configuration or conformation of polypeptides through alterations at the surface thereof so as to render the polypeptide soluble in the aqueous medium. A few non-limiting examples of chaotropic agents include guanidine hydrochloride, urea, and hydroxides such as sodium or potassium hydroxide. Chaotropic agents may also include a combination of these reagents, such as a mixture of a hydroxide with urea or guanidine hydrochloride.

As used herein, "reducing agent(s)" refer to a compound that, at a suitable concentration in aqueous solution, maintain the free sulfhydryl groups, such that intra- or intermolecular disulfide bonds are chemically disrupted. Representative non-limiting examples of suitable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), cysteine, cysteamine, thioglycolate, glutathione, and sodium borohydride.

As used herein, "buffered solution" refers to a solution which resists changes in pH by the action of its acid-base conjugate components.

As used herein, "solubilization buffer" refers to a solution used for solubilization of inclusion bodies.

As used herein, "refolding solution" refers to a solution used for recovering a desire refolded protein confirmation from solubilized inclusion bodies.

The term "Flow-through mode" as used herein refers to a chromatography process, wherein the protein on interest is allowed to flow though the column during the loading of the column as obtained as part of the unbound or "flow-through" fraction during loading or post load wash of the chromatography resin.

"Anion exchange resin" mentioned in the embodiments refers to a solid chromatographic support, which has a positively charged ligand such as a quaternary amino group attached thereto, capable of ionic interaction with a negatively charged protein or a functional group under suitable conditions. The anion exchange resin can be any weak or strong anion exchange resin or a membrane which could function as a weak or a strong anion exchanger. Various commercially available anion exchange resins are known in the art and include without any limitation DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation.

The terms "purification" or "purifying" or fractionation has been used interchangeably herein, refer to increasing the degree of purity of a protein of interest in particular rh-GCSF, from a sample or a preparation comprising the refolded rh- GCSF and one or more process or product related impurities.

The term "impurity" or“impurities” as used herein refers to any proteinaceous or non-proteinaceous molecular entity distinct from the than the protein of interest. The impurity includes, without limitation: host cell protein, host cell DNA, fragment, aggregate, another polypeptide, endotoxin, bacterial cell culture media component etc. The protein of interest may be described as the fraction of protein, substantially free of impurities, as described herein, and can be obtained as part of the flow through from the anion exchange chromatography.

The following examples are provided to further illustrate the present invention but are not provided to in any way limit the scope of the current invention.

Example 1:

Solubilization and refolding of GCSF inclusion bodies

Solubilization and refolding of GCSF has already been disclosed in WO2010/146599. In brief, inclusion bodies (IBs)of rh-GCSF were solubilized in lOOmM Tris, 6M GuHCl at pH 8.0 over a period of around 45 min. The OD of the solubilized IB was adjusted with solubilization buffer to 8.0. The solution containing solubilized inclusion bodies was filtered through 0.45pm filter. DTT was added up to 5mM to reduce the protein. Reduction was carried out for 30 min at room temperature (25 °C). The solubilized GCSF was added to a refolding buffer, containing 75mM Tris pH 8.8, 0.1M L- Arginine, 10% sucrose, 2mM EDTA, lOmM Sodium ascorbate, 2M Urea, and stirred over a period of 30-45 minutes. Refolding buffer temperature of the buffer was maintained at around 8.0°C, and refolding was continued for a period of 15- 20 hrs. When sodium ascorbate is used in refolding buffer dehydro ascorbate and reduced glutathione are also added in refolding buffer to provide redox condition while refolding. Alternately, oxido- shuffling agents such as Cysteine/Cystine or Oxidised and reduced glutathione can also be used.

After refolding, the solution was buffer exchanged to 20mM Tris pH 8.0, 5% Sucrose or 5% D + Sorbitol and the denaturant was removed.

Example 2: Anion exchange chromatography

5 CV of AEX equilibration buffer with 20mM Tris and 5 % Sorbitol, pH 7.45 ± 0.15, conductivity (4.90 - 5.40 mS/cm) was passed to equilibrate the column. After equilibration, the refolded GCSF obtained from Example 1 was loaded onto anion exchange resin. Flow-through fractions were collected from the anion exchange Q sepharose fast flow resin when absorbance reached to 50 mAU. After loading was over, AEX equilibration buffer was passed and peak collection was stopped after anion exchange flow-through absorbance reaches to 70 mAU (Peak Descending). Anion exchange chromatography was carried out for three different batches.

AEX chromatogram is shown in Fig- 1.

Example 3: Size exclusion HPLC (SE-HPLC) and Reverse Phase HPLC (RP- HPLC)

Anion exchange chromatography load and flow-through were analyzed using Size exclusion HPLC (SE-HPLC) for GCSF purity and HMW and Reverse Phase HPLC (RP-HPLC) for HCP, HCDNA and BET using techniques known in the prior arts. Analysis results of anion exchange chromatography load and flow-through are shown in Table 1, 2, 3 and 4. From the table 1, it was observed that HCP reduction takes place in AEX step. Percentage reduction from load to flow through was ~ 90 % and reduction was approximately 1.5 log in all the three batches.

From Table 2, it was observed that HCDNA reduction takes place in this step. Percentage reduction from load to flow through was ~ 99.80 % and log reduction was approximately 2.6 in all the three batches.

From Table 3, The reduction of high molecular weight impurities (HMW) was observed from around 9 -10.5 % to 0.5-1.34% in AEX Flow through which corresponds to greater than 90% reduction.

From Table 4 it was about lOOOfold reduction was observed in all three batches and the amount of BET in AEX flow-through was less than 10 EU/mg.

Table 1: HCP Content

Table 2: HCDNA Content

Table 3: HMW Reduction

Table 4: BET Reduction