FIELD OF THE INVENTION

The present invention relates to a novel purification process for Granulocyte Colony
Stimulating Factor (G-CSF) using hydrophobic charge induction chromatography.

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 (G-CSF) stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. When administered to mammals, G-CSF promotes a dramatic increase in circulating granulocyte populations.

G-CSF is one of several proteins produced by recombinant DNA technology for therapeutic use. Of the two types of G-CSF clinically available, lenograstim, the glycosylated form of G-CSF is expressed In mammalian cells, and filgrastim, the non-glycosyiated form is expressed in Escherichia coli (E.coli). Herman et al (Herman, et al, Formulation, Characterization and Stability of Protein Drugs, R. Pearlman and Y. J. Wong, Eds., Plenum Press, New York, 1996.) provides a description of the structure and properties of G-CSF. Komrokji and Lyman (Komrokji and Lyman, Expert Opin. Biol. Ther. 4, 1897 - 1910, 2004) review the use of colony stimulating factors.

The processes for purification and/or isolation of G-CSF disclosed in patent and scientific literature comprise ion exchange chromatography, chromatofocusing, reverse phase chromatography, hydrophobic interaction chromatography, and combinations of these and other methods. These processes are described in EP 169566, EP 237545, EP 215126, EP 243153, US 5,055,555 and WO 0104154.

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, solublized in the presence of protein denaturing agents, refolded and then purified by appropriate column chromatographic procedures. A typical production process for G-CSF, as described in US 5,849,883, involves expression of recombinant G-CSF as inclusion bodies in E. coli. The fermentation broth is homogenized and inclusion bodies are separated by centrifugation. The inclusion bodies are then solubilized in a suitable denaturant and the protein is refolded under appropriate conditions. The refolded mixture is then applied to ion-exchange chromatography column(s) to achieve purification. The eluate from the chromatography column is then dialyzed and formulated. Most of the processes described in the prior art for the purification of G-CSF involve multiple chromatography steps where losses in the yield at the end of purification steps are significant. Thus new processes need to be developed to improve the yield of correctly folded and biologically active G-CSF.

SUMMARY OF INVENTION

The present invention discloses process for isolation and purification of recombinant G-CSF subjecting G-CSF containing protein mixture to hydrophobic charge induction chromatography (HCIC) column which purifies the G-CSF without exposing it to high pH or high salt concentration after binding on the column.

DETAILED DESCRIPTION OF THE INVENTION

G-CSF is a human endogenous secretory protein which selectively induces the development of granulocyte committed progenitors from multipotent hematopoietic cells. As used herein G-CSF refers to a native human G-CSF as disclosed in EP220520 which is incorporated herein by reference in its entirety, proteins having substantial amino acid identity (80% or more) to native human G-CSF. G-CSF as used herein also encompasses analogs of G-CSF as disclosed in US6931458 and US7029748 that are incorporated herein by reference. Thus the process of purification disclosed in the present invention applies to native as well as the analogs of G-CSF. The present invention discloses and enables a novel process for isolation and purification of recombinant G-CSF using HCIC. The process especially involves purifying the said recombinant protein without exposing it to high pH or high salt concentration after binding to the column.

Preferably G-CSF is produced by recombinant microbial expression systems such as bacteria. This requires that the DNA containing G-CSF gene, such as human G-CSF gene, be isolated and spliced into suitable expression vector, and the vector used to transform an appropriate host strain. The host strain is then grown in culture under conditions promoting expression of the DNA to provide the desired protein. Appropriate expression vectors, hosts, cloning, transformation and expression techniques are described in Maniatis et al. Molecular Cloning, 1982, and incorporated herein by reference. PCT applications WO 8801297 and WO 8701132 disclose suitable methods for expressing analogs of G-CSF. The following US Patents 4,810, 643; 4,999, 291; 5,055, 555; 5,849, 883; 5,582, 823; 5,580, 755; and 5,830, 705, describe various aspects of recombinant expression and purification of the G-CSF protein from various expression systems ranging from bacterial cells to yeast and mammalian cells. Expression of G-CSF as inclusion bodies in a bacterial system is described in US Patent 4,810, 643.

Proteins expressed by recombinant DNA methods in prokaryotic systems such as E. coli, are usually expressed as insoluble aggregates called inclusion bodies which require denaturation and renaturation (refolding) in order to recover the correctly folded biologically active form. The term "inclusion bodies" refers to the insoluble aggregates of proteins expressed by recombinant DNA methods in microbial expression systems. The method of isolation of inclusion bodies, their subsequent solubilization and refolding have been described in US Patents 4810643, 499291, 5849883 and have been included herein by reference in its entirety. Briefly the instant invention provides a process of isolating biologically active recombinant proteins, for example, G-CSF, involving the following steps:

a) solubilization of the insoluble and/or aggregated G-CSF by exposure to a denaturing agent;

b) refolding the solubilized G-CSF to obtain active refolded protein; and

c) purification of the correctly folded biologically active recombinant G-CSF by hydrophobic charge interaction chromatography.

The pellet of isolated inclusion bodies is solubilized in a solution comprising a denaturing agent such as urea at about 8 M concentration or guanidium hydrochloride at 6 M concentration and refolded by dilution of the solubilized inclusion body solution into a solution devoid of the denaturing agent.

In one embodiment, the refolded mixture is filtered through appropriately designed trains or composite filters that are readily recognized by a person skilled in the art, to remove aggregates that may otherwise bind irreversibly to the chromatography column. The refolded mixture is filtered through a composite filter containing cellulosic fibers and filter aid, as well as a final layer of membrane made of cellulosic ester polymer. In an alternative embodiment, refolded mixture is passed through filtration train containing filters made of ceilulosic fibers and filter aid, followed by polymeric membrane filters. By conducting the filtration after pH adjustment, the oxidized forms of G-CSF are minimized and the soluble aggregates are removed. Examples of such filters are commercially available Millistak AlHC filter from Millipore Corporation(USA), CUNO Zeta Plus I OS, 60S filters (Cuno Corporation, USA) and Milligard filters from Millipore Corporation.

The clarified refolded mixture is then applied to a hydrophobic charge induction chromatography (HCIC) media. HCIC or dual mode chromatography (DMC) is based on the pH-dependent behavior of the ionizable, dual-mode ligands. HCIC exploits the unique properties of chromatographic ligands that show either hydrophobic or charged interactions or both (Boschetti et al, Gen. Eng. News, 20, 2000; Ghose et al, Biotechnol. Prog., 21, 498 - 508, 2005). Adsorption is based on mild hydrophobic interaction while desorption is based on charge repulsion achieved by altering the mobile phase pH (US patent US5652348). Thus HCIC medium operates in two modes - at high pH, it behaves as a hydrophobic interaction chromatography medium and at low pH, it behaves as an anion exchange medium. Under mild acidic conditions (pH 4.0^.5), both the ligand and target molecule take on a net positive charge. Binding is thus disrupted and elution occurs. Examples of resins for HCIC or DMC include MEP-Hypercel manufactured by Pall Corporation (USA). This is a regenerated cellulose media with 4-mercaptoethyl pyridine (MEP) as the functional group. The ligand has a pKa of 4.8 and the nitrogen atom in pyridine ring acquires a positive charge at low pH. In the present invention, MEP hypercel is used as the chromatographic media for the purification of G-CSF. However any other alternative HCIC media for example HEA, PPA media from Pall Corporation and Capto MMC from GE Healthcare can also be used for the purification of G-CSF. The most significant advantage of using an HCIC column is that G-CSF is not exposed to high pH or high salt concentration after binding on the column.

The HCIC column is equilibrated with a similar buffer as that used in the step prior to HCIC. Thus in one embodiment, the starting buffer for HCIC is similar to the refolding buffer comprising about 25 mM Tris. The temperature during the loading can be maintained in the range of about 4 °C to about 25°C, preferably about 12 °C to about 18°C, more preferably about 15°C. Maintaining a lower temperature during loading improves yield, prevents the leakage of the non-native form(s) in the subsequent elution step and decreases the amount of non-native forms in a second subsequent elution step.

The column is loaded at about 1 mg to about 15 mg, preferably about 2 mg to about 12 mg, more preferably about 3 mg to about 10 mg, and most preferably about 4 mg of protein per ml of resin. The HCIC ligand for example MEP binds G-CSF via hydrophobic interactions. In the present invention G-CSF is bound to the MEP at a pH above the pi of G-CSF preferably above pH 5.4, more preferably above pH 6.5 most preferably about pH 7.2. After loading the refolded protein, the column wash and elution is carried out at room temperature.

The elution is carried out at pH below the pi of the protein. Thus the elution is carried out at pH below the pi of G-CSF, preferably at about 4.6 to about 5.0, more preferably at about 4.7 to about 4.9, and most preferably about 4.8. At pH less than 4.0, both native and the non-native forms of G-CSF elute. The eluate from the MEP column is diluted two-fold into a 10 mM acetic acid solution at about pH 3.5. The pH of the diluted MEP eluate is further adjusted to about 3.5. The diluted G-CSF containing eluate from MEP may be diafiltered against a 10 mM acetic acid solution of pH about 3.5 on a tangential flow filtration (TFF) system for 5 to 6 dia-volumes in a continuous dia-filtration mode and further concentrated until a desired concentration of the protein is obtained. The TFF retentate may be loaded on an anion exchange chromatography column for example Q-Sepharose column from GE health care under conditions where the impurities like endotoxins are bound to the column. G-CSF is collected in the flow through and filtered through a sterile grade 0,2 μim filter which serves as a bioburden removal step. Alternatively, anion exchange membrane chromatography devices, for example a Mustang Q device from Pall Corporation could be used to remove impurities.

The invention is more fully understood by reference to the following examples and figures. These examples should not, however, be construed as limiting the scope of the invention.

DESCRIPTION OF FIGURES

Figure 1 G-CSF elution profile of HCIC from MEP column showing pH based
elution of G-CSF at pH 4.8. Fractions containing the G-CSF peak are analyzed using RP-HPLC.

Figure 2 RP-HPLC profile of the G-CSF purified using HCIC.

Example 1
(Isolation of inclusion bodies)
Cells containing recombinant G-CSF in the form of inclusion bodies are resuspended in phosphate buffered saline (PBS buffer) (140 mM NaCl, 16.0 mM Na2HP04, 2.00 mM KH2PO4, 3.75 mM KCl, 10.0 mM LiCl, 1.00 mM EDTA-2Na, pH 7.4) in the ratio of 5 mL PBS buffer per gram of cell pellet. The cell suspension in PBS buffer is stirred on a magnetic stirrer for 20 min to make a homogenous solution. The cell suspension is centrifuged at a relative centrifugal force (RCF) of 13000 for 30 min at a temperature of 4 °C. After centrifugation, supernatant is discarded and the pellet is resuspended in lysis buffer (50 mM Tris and 10 mM EDTA) in the ratio of 10 ml lysis buffer per gram of pellet. The cell suspension in lysis buffer is stirred gently on a magnetic stirrer for 20 min. The cell suspension is passed through the homogenizer two times at a pressure of 900-1000 bar till a drop in ODaoo equivalent to 70% is achieved. The cell lysate is collected and centrifuged at 13000 RCF for 30 min at 4 °C. The pellet obtained is of the inclusion bodies.

Example 2
(Solubilization of inclusion bodies)
Inclusion bodies contained in the bottles are brought out of storage at - 20 °C and allowed to stand at room temperature for at least 30 minutes. Approximately 25 ml of water for injection (WFl) is added to each bottle and the inclusion bodies are dispersed into the WFI using a spatula/glass rod. The dispersed inclusion bodies are placed in a single glass beaker and approximately 40 mL of 8.0 M urea solution is added per gram of inclusion bodies and is mixed using a magnetic stirrer. The pH of this suspension is adjusted to 11 to 13 preferably 12.0 by adding small quantities of 1.0 M sodium hydroxide solution. The suspension is then stirred for 30 ± 5 minutes to solubilize the inclusion bodies. The solubilized inclusion bodies are centrifuged at 6000 rpm for 30 minutes. The supernatant is collected and optical density (OD) of the solution is measured at a wavelength of 280 nm. The solubilized inclusion bodies solution is then diluted 20-fold in a solution devoid of urea. The refolding mixture is gently mixed for ten minutes and incubated at 5 °C for 15 - 17 hours.

Example 3
(Filtration of refolded mixture)
The refolded protein solution of example 2 may be filtered through a series of filters into a vessel maintained at 15 ± 3 °C. The first filter is a depth filter made of cellulose fibers containing positively charged resin. This filter also contains a 0.1-μm (nominal) cellulose ester polymeric membrane final layer. This is followed by a 0.2-μm (absolute) polyvinylidene fluoride (PVDF) filter that serves as a bioburden reduction step.

Example 4
(Purification of refolded protein using HCIC)
The refolded protein solution from example 3 is applied to a hydrophobic charge induction chromatography (HCIC) column. The ligand of the chromatography media is Mercapto Ethyl Pyridine (MEP) that binds G-CSF via hydrophobic interactions at pH above 5.4 and becomes progressively more positively charged as the pH is reduced. The HCIC column is equilibrated with a loading buffer (containing 50 mM sodium acetate) at pH 6.5 and 15 °C for 15 column volumes. After equilibration filtered refolded protein solution is loaded on the MEP column and the column is washed using 50 mM sodium acetate at room temperature at a pH of 6.5. After washing the column for 5 column volumes, another wash is given for 10 column volumes at room temperature with 50 mM sodium acetate solution at a pH of 5.4. At pH 4.8, the native form of G-CSF is eluted. The eluate is collected as fractions of 20-20 mAU (OD280) into a 10 mM acetic acid solution at pH 3.5, such that approximately a two-fold dilution is achieved. The fractions are analyzed by reverse phase (RP-HPLC) and size exclusion (SE-HPLC) chromatography and pooled together. The pH of this solution is adjusted to 3.5 for further processing. The volume of the poo! is then made up to 40 L with 10 mM acetic acid, pH 3.5. A second elution is conducted at a pH of 4.0; the highly reduced form of G-CSF with some less reduced, native and oxidized forms is eluted at this lower pH.

Example 5
(Q-Sepharose chromatography)
The pooled MEP eluate (40 L) of example 4 can be concentrated to 7.8 ± 0.2L using three 0.5 m5 kDa UF cassettes. This concentrated protein solution is diafiltered against 10 mM acetic acid pH 3.5. It is further concentrated till the OD280 reaches 1.0. The cassettes are flushed with 10 mM acetic acid pH 3.5 to recover the remaining protein. This causes dilution of the protein solution and the final OD reaches about 0.8-0.9. The tangential flow filtration (TFF) retentate may be loaded on an anion exchange chromatography column, for example Q-Sepharose column where the protein does not bind but the impurities may bind to the column. G-CSF is collected in the flow through from 50-50 mAU OD28oand filtered through a sterile grade 0.2 ^m filter which serves as a bioburden reduction step.

We Claim

1. A process for isolating biologically active G-CSF comprising;

a) Loading a solution comprising G-CSF and a buffer substance to a
HCIC resin

b) Eluting G-CSF from the HCIC resin

2. A process according to claim 1, wherein the pH of the said buffer substance is
about 5.0 to about 8.0

3. A process according to claim 2, wherein the pH of the buffer substance Is
about 6.5.

4. A process according to claim 1, wherein the GCSF Is eluted at a pH of about
4,5 to about 5.0.

5. A process according to claim 4, wherein the GCSF is eluted at a pH of about
4.8

6. A process according to claim 1, wherein the amount of protein loaded on to the HCIC resin is about 3 mg to about 10 mg/ml of resin.

7. A process for isolating biologically active G-CSF comprising;

a) Loading a solution comprising G-CSF and a buffer substance to a
HCIC resin

b) Eluting G-CSF from the HCIC resin

c) And further purifying the GCSF by anion exchange chromatography.

8. A process according to claim 7, wherein the anion exchange chromatography
is column anion exchange chromatography.

9. A process according to claim 7, wherein the anion exchange chromatography
is a membrane anion exchange chromatography.