FIELD OF THE INVENTION

The present invention relates to a novel refolding process for Granulocyte Colony Stimulating Factor (G-CSF) from inclusion bodies.

BACKGROUND OF THE INVENTION

Differentiation and proliferation of hemtopoietic 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-glycosylated 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.

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.

Unpurified G-CSF, obtained by solubilization and refolding of inclusion bodies, contains the native, oxidized and the reduced forms of G-CSF. The reduced forms of G-CSF are reported to form aggregates, whereas the oxidized form of G-CSF has reduced bioactivity (Lu, et al, J. Biol. Chem. 267, 8770-8777, 1992; Reubsaet, et al, J. Pharm. Biomed. Anal. 17, 283-289, 1998). 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, lysing the cells and separating G-CSF from soluble proteinaceous material; extracting the material with deoxycholate (optionally); solubilizing and oxidizing the G-CSF in the presence of a denaturant solubilizing agent and oxidizing agent; removing the denaturant solubilizing agent from the G-CSF to allow its refolding. Refolded protein is further purified using two ion exchange chromatography steps. Refolding of G-CSF by conventional methods, consisting of inclusion body isolation, solubilization and refolding, results in suboptimal yields of the correctly folded biologically active protein. The present invention discloses a novel process to improve the yield of correctly folded and biologically active G-CSF.

SUMMARY OF INVENTION
The present invention discloses a process for refolding of recombinant G-CSF that minimizes product related impurities by optimizing the refolding of inclusion bodies containing recombinant G-CSF. The invention discloses a method of the pH adjustment to improve the yield of correctly folded form of G-CSF after refolding of solubilized inclusion bodies in the presence of redox agents cystine and cysteine. In an embodiment of present invention, the refolding is carried out at a pH higher than the pi of G-CSF. Using a two step procedure, the pH of the refolded mixture is adjusted after refolding to a pH lower than the pH at which refolding is carried out, but higher than the pl of G-CSF. The method of pH adjustment post refolding results in a decrease in the proportion of oxidized form of G-CSF which has reduced biological activity compared to the native form.

DETAILED DESCRIPTION OF THE INVENTION

As used herein G-CSF refers to a protein having substantial amino acid identity with native human G-CSF, the sequence of which is disclosed in European Patent Application No. 220520 and incorporated herein by reference. G-CSF is a human endogenous secretory protein which selectively induces the development of granulocyte committed progenitors from multipotent hematopoietic cells. Analogs of G-CSF are disclosed in US application serial number 6931458 and 7029748. The process disclosed in the present invention applies to native as well as analogs of G-CSF.

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 application numbers 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 refer to the insoluble aggregates of proteins expressed by recombinant DNA methods in microbial expression systems. The formation of inclusion bodies presents complications in recovery of the desired product from the cells in which it is produced. Therefore, effective ways are desired to isolate the proteins expressed as inclusion bodies.

The invention provides methods of increasing the recovery of active polypeptides. In particular, the invention involves promoting the formation of the native conformation in preparations of recombinant G-CSF in bacterial systems. A native conformation for a polypeptide, herein a recombinant G-CSF, is the conformation of a protein that most closely resembles, and duplicates the function of, the naturally occurring protein. In an embodiment of the present invention, the inclusion bodies pellet is solubilized in a solution comprising a denaturing agent such as urea at about 8 M concentration, wherein the ratio of inclusion bodies (g) to urea solution (mL) is about 1:20 to about 1: 60, preferably a ratio of about 1:40, and adding small amounts of 1 M sodium hydroxide solution at a pH of about 11 to about 13, preferably about 12. The solubilized inclusion body is then refolded by addition of the solubilized inclusion body solution to a refolding buffer containing about 10 to about 100 mM Tris, preferably about 10 to about 50 mM Tris, most preferably about 26.3 mM Tris; about 0.4 to about 0.8 M, preferably about 0.63 M Arginine; about 1 % to about 10 %, preferably about 5.26 % sorbitol; and about 0 to about 4 mM, preferably about 1.05 mM EDTA.

The solubilized inclusion body is diluted by about 10 to about 40 fold, preferably about 15 to about 25 fold, more preferably about 20 fold in the refolding buffer. This dilution step takes about 15 to about 45 minutes, preferably about 20 to about 40 minutes, and most preferably about 30 minutes. It is expected that the final concentrations of the various components of the refolding mixture after the addition of inclusion bodies is about 25 mM Tris, about 0.6 M Arginine, about 5 % sorbitol and about 1.0 mM EDTA.

Refolding of proteins is markedly influenced by pH. The iso-electric point or pi of G-CSF is 5.8 to 6.1. Thus, refolding at a pH less than 6.5 may result in aggregation losses of the protein. In a preferred embodiment of this invention the refolding is carried out at a pH above pi, preferably at about pH 8 to about pH 10, more preferably at about pH 8.5 to about pH 9.5, most preferably at about pH 9. The pH is maintained at the desired setting by the addition of small amounts of glacial acetic acid. The temperature during the refolding of G-CSF is maintained at a temperature of about 4 to about 15 °C, preferably at about 4 to about 8 °C, more preferably at about 5 "C. Correctly folded biologically active GCSF can be obtained by the addition of redox shuffling agents. Examples of such redox shuffling agents include cysteine and cystine. To achieve maximum yield of correctly folded active G-CSF, the refolding can be carried out in the presence of cysteine-cystine. The cysteine-cystine may be added sequentially or simultaneously. Thus the refolding can be carried out by a first addition of an amount of cystine, followed by the addition of cysteine, and then followed by a second addition of an amount of cystine. The final concentration of the cysteine in the refolding mixture could be about 0.6 to about 2.4 mM, preferably about 1.2 to about 2.4 mM, more preferably about 1.5 to about 2.4 mM, and most preferably about 1.8 mM. The final concentration of the cystine in the refolding mixture could be about 0.2 to about 2.4 mM, preferably about 0.2 to about 1.2 mM, more preferably about 0.3 to about 0.5 mM, and most preferably about 0.4 mM.

Thus the cysteine-cystine additions may be conducted in the following manner: From a freshly prepared stock solution of cystine, about 10 to about 90%, preferably about 50% of the required amount is added to the refolding mixture and mixed for about 5 to about 15 minutes, preferably about 10 minutes. The desired amount of cysteine is then added into the refolding mixture from a freshly prepared stock solution. The refolding mixture is again mixed for about 5 to about 15 minutes, preferably about 10 minutes. The remaining about 90 to about 10 %, preferably about 50 % of the cystine solution is then added to the refolding mixture and mixed for about 5 to about 15 minutes, preferably about 10 minutes. In another example 100% of cystine is added to the refolding mixture followed by addition of required amount of cysteine. In yet another embodiment, cysteine and cystine are not added to the refolded mixture.

The refolding mixture is incubated at about 2 to about 8 °C most preferably at about 5 °C for about 2 to about 24 hours, preferably about 2 to about 8 hours, more preferably about 2 to about 4 hours, and most preferably about 3 hours. In a preferred embodiment of this invention, the temperature of refolded protein solution is increased to about 15 °C and the pH is reduced to a value below the pH at which refolding is carried out but above the pl of G-CSF, preferably at pH of about 6.5 to about 8.5, more preferably about 7.0 to about 8.0, most preferably about 7.2 using acetic acid solutions of different strengths. The pH adjustment results in a decrease in the oxidized form(s) to less than about 0.5% as well as a substantial increase in the yield of the native forms of the protein. Whereas, In the absence of pH adjustment after refolding the oxidized form(s) comprise about 1 to about 3 % of the total protein. The various forms are analyzed by reverse phase (RP-HPLC) or size exclusion (SE-HPLC) chromatography. The pH is adjusted slowly in two steps to prevent the formation of aggregates and the loss of protein in the subsequent filtration step.

Soluble aggregates present in the refolded mixture in small quantities may bind irreversibly to the chromatography column(s) used subsequently for the purification of protein, thereby decreasing yield as well as resin lifetime. Applying the refolded mixture directly to polymeric membrane filters results in clogging of the filters and loss of product. Filtration through appropriately designed trains or composite filters removes such aggregates and extends the life of chromatography media. In accordance with another aspect of the present invention 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. Alternatively, refolded mixture can also be passed through filtration train containing filters made of cellulosic 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 A1HC filter from Millipore Corporation (USA), Cuno 10S and 60S Zeta Plus depth filters from Cuno Corporation (USA), and Milligard polymeric membrane filters from Millipore Corporation.

The clarified refolded mixture is then applied to any suitable chromatography media for further purification. Various chromatography methods for the purification of E. colt expressed G-CSF have been described in the US patent no 5849883, 4810643 and 4999291 and have been included herein as reference in its entirety.
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 shows the RP-HPLC profile of partially refolded refolded G-CSF without the Ph adjustment showing native, reduced and oxidized forms of G-CSF.

Figure 2 shows the RP-HPLC profile of refolded G-CSF after pH adjustment carried out in accordance to the disclosed procedure, showing only native and reduced forms of G-CSF.

Example 1

(Isolation of inclusion bodies)

Cells containing rG-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 KH2P04, 3.75 raM KCl, 10.0 mM LiCl, 1,00 mM EDTA-2Na, pH 7.4) in the ratio of 5 mL PBS buffer per g 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 g 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 OD GOO 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)
Bottles containing inclusion bodies 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 (WFI) 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 20 to 60 mL, preferably 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.

Example 3

(Refolding of protein from inclusion bodies)

The solubilized inclusion bodies solution is diluted 10 to 40 fold preferably 20-fold into a refolding buffer solution comprising stabilizing chemicals at a controlled flow rate over a period of 20 min and held at 2 to 8 preferably 5 "C. The refolding buffer is prepared to contain 0.6 M Arginine, 5.26% D-Sorbitol, 26.3 mM Tris and 1.05 raM EDTA at pH 9, after dilution with the solubilized inclusion bodies. The dilution is conducted at a low flow rate to minimize temperature and pH fluctuations. The refolding mixture is gently mixed for ten minutes and incubated at 5 °C for 15 - 17 hours. Optionally refolded mixture can be mixed with redox agents like cystiene and cystine as described in examples 4 or can be used directly for pH adjustment as described in example 5.

Example 4

(Refolding using redox agents)

To the refolding mixture of example 3, 50 % of the amount required to attain 0.4 mM concentration of L-Cystine, can be added from a freshly prepared 95.7 mM stock solution of L-Cystine. The diluted inclusion bodies solution is gently mixed for 10 min before the addition of the required amount for attaining !.8 mM L-Cysteine from a freshly prepared 6.37 mM L-Cysteine stock solution. The refolding mixture is gently mixed for another 10 min and the remaining 50% of the amount required to attain 0.4 mM concentration of L-Cystine is added from the 95.7 mM stock solution of L-Cystine. Alternatively, cystiene and cystine can also be added simultaneously to the refolding mixture to attain a final concentration of 0.4 mM L-Cystine and 1.8 mM L-Cysteine in the refolding mixture. The refolding mixture is gently mixed for another ten minutes and incubated at 5 °C for 15 - 17 hours.

Example 5

(pH adjustment of refolded mixture)

The temperature of the refolded protein solution obtained from example 3 or 4 is increased to 15 °C. The pH is then adjusted to 7.5 by the addition of 2.0 M acetic acid at a controlled flow rate over a period of 40 min. The pH is further reduced to 7.2 by adding 0.5 M acetic acid at the same flow rate to minimize pH fluctuations. The temperature is maintained at 15 ± 3 °C throughout the pH adjustment step.

Example 6

(Filtration of refolded mixture)

The pH adjusted refolded protein solution is 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-um (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 7

(Analysis of refolded mixture)

Yield and purity of the refolded G-CSF can be analyzed by reverse phase high performance liquid chromatography (RP-HPLC) using a Q (15 cm x 4.6 mm) column (Zodiac corp., Germany) under an isocratic gradient of 0.1 % Trifluoroacetic acid (TFA) and 0.1 % TFA in 90% acetonitrile as the mobile phase. Flow rate of mobile phase is maintained at 1 mL/min and the column temperature is maintained at 60 °C. Detector (UV) wavelength is set at 215 nm.

We Claim;

1. A method of preparing correctly folded biologically active G-CSF from insoluble or
aggregated G-CSF comprising:

a. Solubilization of the insoluble or aggregated G-CSF by exposure to a denaturing
agent,

b. Refolding the solubilized G-CSF at a pH higher than the pI of G-CSF,

c. Reducing the pH of the above refolding mixture to a value lower than the pH of
step b but higher than the pI of G-CSF.
2. A method according to claim 1, comprising further addition of redox reagents to the refolding mixture.

3. A method according to claim 2, wherein the said redox reagents are cysteine and cystine.

4. A method according to claim 1, wherein the said denaturing agent is urea.

5. A method according to claim 1, wherein the said denaturing agent is guanidine hydrochloride.

6. A method according to claim 1, wherein the said refolding is done at a pH of about 8-10.

7. A method according to claim 1, wherein after the said refolding, the pH is reduced to about 6.5 -8.5.

8. A method of preparing correctly folded biologically active G-CSF from insoluble or aggregated G-CSF comprising:

a. Solubilization of the insoluble or aggregated G-CSF by exposure to a denaturing
agent,

b. Refolding the solubilized G-CSF at a pH higher than the pi of G-CSF,

c. Adding a redox reagent to the above refolding mixture.
d. Reducing the pH of the above refolding mixture to a value lower than the pH of
step b but higher than the pI of G-CSF.
e. Filtering the above refolded mixture of by a series of filters.