Field of the Invention
The invention is related to process for purification of colony stimulating factors using
at least one step of aqueous two phase extraction process. Particularly the invention is
related to the process for the purification of the recombinant human GCSF using
aqueous two phase extraction process. The invention is also related to purified
recombinant human GCSF produced by the processes of the invention resulting in
lesser oxidative forms, endotoxins and host cell proteins.
Background of the Invention
Colony-stimulating factors (CSFs) are secreted glycoproteins which bind to receptor
proteins on the surfaces of hemopoietic stem cells and thereby activate intracellular
signaling pathways which can cause the cells to proliferate and differentiate into a
specific kind of blood cell. Human granulocyte-colony stimulating factor (h-GCSF) and
human macrophage granulocyte-colony stimulating factor (h-GM-CSF) belongs to a
group of colony stimulating factors that play an important role in stimulating the
differentiation and proliferation of hematopoietic precursor cells and activation of
mature neutrophils. GCSF is capable of supporting neutrophil proliferation in vitro and
in vivo. GCSF protein has only one single O-glycosylafion site at threonine 133;
absence of glycosylation at this residue was not found to affect the stability of the
protein. For many protein therapeutics where glycosylation of the protein is known to
affect stability, it is necessary to undertake cloning and expression in yeast or
mammalian cells, using appropriate expression vectors. In the case of GCSF, the
recombinant protein expressed in E. coli was found to have the same specific activity as
the native protein (Oh-eda et. al. 1990 J. Biol. Chem. 256,11432-11435, Hill et. al.
1993 Proc. Nat. Acad. Sci. USA 90.5167-5171, and Arakawa et. al. 1993 J. Protein
Chem. 12, 525-531). Human GCSF in its naturally occurring form is a glycoprotein
having a molecular weight of about 20,000 Dalton and five cysteine residues. Four of
these residues form two intramolecular disulfide bridges which are of essential
importance for the activity of the protein. As GCSF is available only in small amounts
from its natural sources, recombinant forms of GCSF are mainly used for producing
pharmaceuticals, which can for example be obtained by means of expression in
mammalian cells like CHO (Chinese Hamster Ovary) cells or in prokaryotic cells like
E. coli. The recombinant proteins expressed in mammalian cells differ from naturally

occurring GCSF in that they have a different glycosylation pattern, while in the proteins
expressed in E. coli which can have an additional N-terminal methionine residue as a
result of bacterial expression, glycosylation is not present at all. The cloning and
expression of cDNA encoding human GCSF has been described by two groups
(Nagata, S. et. al., Nature 319, 415-418 (1986); Souza, L. M. et al., Science 232, 61-65
(1986)).
The recombinant production of GCSF has been described in patent literature for the
first time in 1987, in WO 87/01132 Al. The first commercially available GCSF is
produced and distributed by Amgen under the trade name Neupogen(R). While the
production of GCSF in prokaryotic cells is preferred as compared to the production in
mammalian cells, as the use of simpler expression systems and culture conditions is
possible. However a frequently occurring problem in the production of recombinant
proteins in prokaryotic cells is, the formation of hardly soluble intracellular aggregates
of denatured forms of the protein expressed called as inclusion bodies, which partially
have a secondary structure and can be found in the cytoplasm of the bacterial cells. The
formation of said inclusion bodies leads to the necessity of solubilizing and renaturing
the proteins subsequent to the isolation of the inclusion bodies by means of
centrifugation at moderate speed with the aid of suitable means in order to maintain
their active configuration. Herein, the competitive reaction between a transfer of the
denatured protein into the right folding intermediate and an aggregation of several
protein molecules is an essential factor limiting the yield of renatured protein.
Many earlier patents have described various aspects of recombinant expression and
purification of the GCSF protein from different expression systems ranging from
bacterial cells to yeast and mammalian cells. Some of the processes described are
multi-step processes where losses in yield at the end of the purification process can be
significant. The following U. S. Patents Nos. 4,810,643; 4,999,291; 5,582,823;
5,580,755; and 5,830,705, and PCT publications WO 87/03689, WO 87/02060, WO
86/04605 and WO 86/04506 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.

Various other methods have been reported in scientific literature for the purification of
GCSF expressed in E. coli, yeast or CHO cells. A method of purification of GCSF from
CHU-2 conditioned medium (human oral carcinoma cell line), which is known to
produce GCSF constitutively was developed by Nomura et. al. (EMBO J. vol 5,871,
1986). The process describes the use of a three-step chromatography procedure after
concentration and ultrafiltration of the conditioned medium. WO 87/01132 Al
describes the cation exchange chromatographic purification of GCSF.
Purification of GCSF in bacterial systems is disclosed in U.S. Patent Nos. 4,810,643
and 4,999,291. Several chromatographic based purification of GCSF has been
described in the prior art for example PCT publication Nos. WO 03/051922 Al, WO
01/04154 Al,
U.S. patent No. 5,055,555, describes a simplified process for purification of
recombinant hGCSF expressed from eukaryotic cells. After ion exchange
chromatography the protein is precipitated by salting our using sodium chloride. But
for recovery of GCSF from inclusion bodies expressed in bacteria, precipitation of the
protein by sodium chloride salt, increases the aggregation status resulting in loss of
yield and activity
The various purification protocols discussed in the above patents mention multiple
chromatography and other steps for the purification of GCSF. None of the above
literature disclosed a simple and viable processing method for the production of
pharmaceutical grade GCSF on industrial scale.
Purification technique known as aqueous two-phase extraction was introduced in 1956-
1958 with applications for both cell particles and proteins. Since then, it has been
applied to a host of different materials, such as plant and animal cells, microorganisms,
viruses, chloroplasts, mitochondria, membrane vesicles, proteins, and nucleic acids.
The basis for extraction by a two-phase system is selective distribution of substances
between the phases. For a soluble substance, distribution occurs mainly between the
two bulk phases, and the extraction is characterized by the partition coefficient, which
is defined as the concentration of partitioned substance in the top phase, divided by the
concentration of the partitioned substance in the bottom phase. Ideally, the partition

coefficient is independent of total concentration and the volume ratio of the phases. It is
mainly a function of the properties of the two phases, the partitioned substance, and the
temperature. The two-phase systems may be produced by mixing two phase-
incompatible polymer solutions, by mixing a polymer solution and a salt solution, or by
mixing a salt solution and a slightly apolar solvent. These types of systems, along with
aqueous two-phase extraction methods for separating macromolecules such as proteins
and nucleic acids, cell particles, and intact cells are described in the literature, for
example, in Albertsson, Partition of Cell Particles and Macromolecules, 3rd edition
(John Wiley & Sons: New York, 1986); Walter et al., Partitioning in Aqueous Two-
Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology, (Academic
Press: London, 1985).
Several low-cost two-phase systems are known that can handle protein separations on a
large scale. These systems use polyethylene glycol (PEG) as the upper phase-forming
polymer and crude dextran (e.g., Kroner et al., Biotechnology Bioengineering, 24:1015-
1045 [1982]), a concentrated salt solution (e.g., Kula et al., Adv. Biochem. Bioeng., 24:
73-118 [1982]), or hydroxypropyl starch (Tjerneld et al., Biotechnology
Bioengineering, 3.0:809-816[1987]) as the lower phase-forming polymer.
Two-phase aqueous polymer systems are extensively discussed in the literature. See,
e.g., Baskir et al., Macromolecules, 20: 1300-1311 (1987); Birkenmeier et al., J.
Chromatogr., 360:193-201 (1986); Birkenmeier and Kopperschlaeger, J. Biotechnol.,
21:93-108 (1991); Blomquist and Albertsson, J. Chromatogr., 73: 125-133 (1972);
Blomquist et al., Acta Chem. Scand., 29: 838-842 (1975); Erlanson-Albertsson,
Biochim. Biophys. Acta, 617: 371-382 (1980); Foster and Herr, Biol. Reprod., 46: 981-
990 (1992); Glossmann and Gips, Naunyn. Schmiedebergs Arch. Pharmacol., 282: 439-
444 (1974); Hattori and Iwasaki, J. Biochem. (Tokyo), 88: 725-736 (1980); Haynes et
al., AICHE Journal-American Institute of Chemical Engineers, 37: 1401-1409 (1991);
Johansson et al., J. Chromatogr., 331: 11-21 (1985); Johansson et al., J. Chromatogr.,
331: 11-21 (1985); Kessel and McElhinney, Mol. Pharmacol., 14: 1121-1129 (1978);
Kowalczyk and Bandurski, Biochemical Journal, 279: 509-514 (1991); Ku et al.,
Biotechnol. Bioeng., 33: 1081-1088 (1989); Kuboi et al., Kagaku Kogaku Ronbunshu,
16: 1053-1059 (1990); Kuboi et al., Kagaku Kogaku Ronbunshu, 16: 755-762 (1990);
Kuboi et al., Kagaku Kogaku Ronbunshu, 17: 67-74 (1991); Kuboi et al., Kagaku

Kogaku Ronbunshu, 16: 772-779 (1990); Lillehoj and Malik, Adv. Biochem. Eng.
Biotechnol., 40: 19-71 (1989); Mattiasson and Kaul, "Use of aqueous two-phase
systems for recovery and purification in biotechnology" (conference paper), 314, Separ.
Recovery Purif: Math. Model., 78-92 (1986); Ohlsson et al., Nucl. Acids Res., 5: 583-
590 (1978); Wang et al., J. Chem. Engineering of Japan, 25: 134-139 (1992); Zaslavskii
et al., J. Chrom., 439: 267-281 (1988); Zaslavskii et al., J. Chem. Soc, Faraday Trans.,
87:141-145 (1991); U.S. Pat. No. 4,879,234 issued Nov. 7, 1989 (equivalent to EP
210,532); DD (German) 298,424 published Feb. 20, 1992; WO 92/07868 published
May 14, 1992; and U.S. Pat. No. 5,093,254. See also Hejnaes et al., Protein
Engineering, 5: 797-806 (1992).
An aqueous two-phase extraction/isolation system is described by DE 288,837. In this
process for selective enrichment of recombinant proteins, a protein-containing
homogenate is suspended in an aqueous two-phase system consisting of PEG and
polyvinyl alcohol as phase-incompatible polymers. Purification of interferon has been
achieved by selective distribution of crude interferon solutions in aqueous PEG-dextran
systems or PEG-salt systems using various PEG derivatives as disclosed in German
Patent DE 2,943,016.
U.S. Patent No. 5,695,958 provides a method for isolating an exogenous polypeptide in
a non-native conformation from cells, such as an aqueous fermentation broth, in which
it is prepared comprising contacting the polypeptide with a chaotropic agent, preferably
a reducing agent and with phase-forming species to form multiple aqueous phases, with
one of the phases being enriched in the polypeptide which is depleted in the biomass
solids and nucleic acids originating from the cells.
U.S. Patent No. 6,437,101 describes the methods for the isolation of human growth
hormone, growth hormone antagonist, or a homologue of either, from a biological
source. The methods described in the '101 patent use multi-phase extraction process.
U.S. Patent No. 7,060,669 provides processes for extraction of proteins of interest in
aqueous two phase extraction by fusing said proteins to targeting proteins which have
the ability of carrying said protein into one of the phases.

The main benefits of the extraction technique are the method is efficient, easy to scale
up, rapid when used with continuous centrifugal separators, relatively low in cost, and
high in water content to maximize biocompatibility. Currently there are relatively few
industrial applications of aqueous two-phase system for purifying proteins.
Purification of GCSF protein in its native form and absence of denaturant using
aqueous two phase extraction has not been described so far.
Summary of the Invention
In one aspect the invention is related to a process for the purification of recombinant
human GCSF obtained in the form of inclusion bodies from microbial cells, which
comprises at least one step of aqueous two phase extraction.
In another aspect the invention is related to a process for the purification of
recombinant human GCSF obtained in the form of inclusion bodies from microbial
cells, the process comprises the steps:
a) solubilizing the inclusion bodies of GCSF;
b) refolding the said solubilized GCSF proteins;
c) purifying the refolded GCSF by using aqueous two phase extraction;
d) optionally further purifying the native GCSF obtained in step c; and
e) isolating pure GCSF.
In another aspect the invention is related to the aqueous two phase extraction process
for isolating native form of GCSF.
Another aspect of the invention is the purified GCSF obtained by the process of the
invention comprising at least one step of aqueous two phase extraction process.
In further aspect the invention is related to the aqueous two phase extraction process for
separating more than 95% of the host cell proteins, endotoxins and DNA from the
refolded protein GCSF in the lower phase wherein the refolded protein is a mammalian
polypeptide, (polypeptide that were originally derived from mammalian organism) that
are expressed in the form of inclusion bodies in prokaryotic cells. This process could

also be applied to GCSF purification from natural sources such as tissues and blood
samples.
In another aspect the invention also relates to pharmaceutical composition comprising
therapeutically effective amount of the biologically active GCSF obtained according to
the process of the present invention comprising at least one step of aqueous two phase
extraction process.
The details of one or more embodiments of the inventions are set forth in the
description below. Other features, objects and advantages of the inventions will be
apparent from the description and claims.
Brief Description of the Drawings
Fig. 1: Schematic Description of the aqueous two phase extraction process for
purification of GCSF.
Fig. 2: SDS-PAGE profile of purification
Detailed Description of the Invention
As used herein, "reducing agent" refers to a compound that, in a suitable concentration
in aqueous solution, maintains sulfhydryl groups so that the intra- or intermolecular
disulfide bonds are chemically disrupted. Representative examples of suitable reducing
agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol
(BME), cysteine, cysteamine, thioglycolate, glutathione, and sodium borohydride.
As used herein, "chaotropic agent" 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. The alterations may occur by changing,
e.g., the state of hydration, the solvent environment, or the solvent-surface interaction.
The concentration of chaotropic agent will directly affect its strength and effectiveness.
A strongly denaturing chaotropic solution contains a chaotropic agent in large
concentrations which, in solution, will effectively unfold a polypeptide present in the

solution. The unfolding will be relatively extensive, but reversible. A moderately
denaturing chaotropic solution contains a chaotropic agent which, in sufficient
concentrations in solution, permits partial folding of a polypeptide from whatever
contorted conformation the polypeptide has assumed through intermediates soluble in
the solution, into the spatial conformation in which it finds itself when operating in its
active form under endogenous or homologous physiological conditions. Examples of
chaotropic agents include guanidine hydrochloride, urea, and hydroxides such as
sodium or potassium hydroxide. Chaotropic agents include a combination of these
reagents, such as a mixture of base with urea or guanidine hydrochloride.
As used herein, the term "inclusion bodies" refers to dense intracellular masses of
aggregated polypeptide of interest, which constitute a significant portion of the total
cell protein, including all cell components. These aggregated polypeptides may be
incorrectly folded or partially correctly folded proteins. In some cases, but not all cases,
these aggregates of polypeptide may be recognized as bright spots visible within the
enclosure of the cells under a phase contrast microscope at magnifications down to
1000 fold.
The term "therapeutically effective amount" used herein refers to the amount of
biologically active G-CSF which has the therapeutic effect of biologically active G-
CSF.
The term "biologically active G-CSF" used herein refers to G-CSF which is capable of
promoting the differentiation and proliferation of hematopoietic precurser cells and the
activation of mature cells of the hematopoietic system.
In an embodiment the invention provides a process for large scale purification of
recombinant GCSF in native form obtained from microbial cells.
The process according to the present invention comprises the steps of:
a) solubilizing the inclusion bodies of GCSF;
b) refolding the said solubilized GCSF proteins;
c) purifying the refolded GCSF by using aqueous two phase extraction;
d) optionally further purifying the native GCSF obtained in step c; and

e) isolating pure GCSF.
According to one embodiment of the invention the inclusion bodies are dissolved in a
suitable solublizing buffer and a suitable chaotropic agent at a pH in the range of 7 to
12. The suitable buffer includes but not limited to Tris (chloride/maleate) buffer,
phosphate (sodium and potassium) buffer, glycine sodium hydroxide buffer, boric acid-
borax buffer, borax-sodium hydroxide buffer, carbonate-bicarbonate buffer etc
The suitable chaotropic agents include urea and salts of guanidine or thiocyanate,
preferably urea, guanidine hydrochloride, or sodium thiocyanate. The amount of
chaotropic agent necessary to be present in the buffer depends, for example, on the type
of chaotropic agent and polypeptide present. The amount of chaotropic agent required
should be sufficient to unfold a polypeptide present in the solution. The pH of the
solution will depend on the chaotropic agent, for urea the pH of the solution is
maintained in the range of 9 to 12, for guanidine hydrochloride the pH is in the range of
7 to 9. The OD of the solution is in the range of about 2 to about 12.
The surfactants and other agents that could be used for soulubilizing microbial
inclusion bodies include SDS, CTAB, CHAPS, Tween 20, Triton X100, Sarcosyl,
Octyl betaglucoside, Nonidet P-40, dodecyl maltoside, NDSB.
(From ref: Process Scale Bioseparations for the biopharmaceutical industry, Ed by
Abhinav A Shukla, Mark R Etzel and Shishir Gadam, Taylor and Francis, 2007 page
129, which is incorporated herein by reference in its entirety).
The solution containing solubilised inclusion bodies is treated with a reducing agent at
a temperature in the range of 10 to 30 °C. The reducing agent includes one or more of
the dithiothreitol (DTT), betamercaptoethanol (BME); cysteine, thioglycolate, and
sodium borohydride. The amount of reducing agent to be present in the buffer will
depend mainly on the type of reducing agent and chaotropic agent, the type and pH of
the buffer employed, and the type and concentration of the polypeptide in the buffer.
An effective amount of reducing agent is that which is sufficient to eliminate
intermolecular disulfide-mediated aggregation. The preferred reducing agent is DTT.

In an embodiment of the invention the protein GCSF is obtained in the native form by
refolding the solubilized GCSF in the refolding buffer. Typically a refolding buffer
may contain a suitable buffer, an amino acid such as arginine or proline, sucrose,
EDTA, sodium ascorbate, urea. 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 dxidised and reduced glutathione can also be used.
The refolding is carried out at a temperature in the range of 5 to 20 °C, preferably at
temperature of 6 to 10 °C. The time required for the refolding may take from about 6 to
24 hrs, preferably between 15 to 20 hrs.
After the refolding of the protein is complete diafiltration may be performed. For the
removal of the denaturant a buffer exchange may be carried out by using Tris buffer
having sucrose or sorbitol.
In an embodiment of the invention the protein GCSF is further isolated and purified by
using aqueous two phase extraction. To the diafiltered solution containing the refolded
GCSF protein a phase forming polymer-salt combinations is added. Examples of phase
forming agents, combinations of phase forming agents and parameters to consider in
selecting suitable phase forming agents are discussed in Diamond et al., 1992, supra,
and Abbott et al., 1990, Bioseparation 1:191-225, both of which are incorporated herein
by reference in their entirety. The polymer and the salt are used under such conditions
and at such concentrations so that a two-phase system is created.
Suitable polymers examples include but not limited to polyethylene glycol (PEG) or
derivatives thereof having molecular weight of about 2000 to 8000 for example PEG
2000, PEG 4000, PEG 6000 and PEG 8000.
A phase forming- salt includes inorganic or organic and preferably do not act to
precipitate the polypeptide. Anions are selected that have the potential for forming
aqueous multiple-phase systems. Examples include ammonium sulfate, sodium dibasic
phosphate, sodium sulfate, ammonium phosphate, potassium citrate, magnesium
phosphate, sodium phosphate, calcium phosphate, potassium phosphate, potassium
sulfate, magnesium sulfate, calcium sulfate, sodium citrate, ammonium citrate,

manganese sulfate, manganese phosphate, etc. Types of salts that are useful in forming
bi-phasic aqueous systems are evaluated more fully in Zaslavskii et al., J. Chrom., 439:
267-281 (1988), which is incorporated herein by reference in its entirety. Preferred salts
for the phase forming are sodium sulfate, potassium sulfate and ammonium sulfate.
In an embodiment of the invention the concentration of the phase forming agents may
be varied. The concentration of the phase forming polymer, expressed in
weight/volume is in the range of about 4% to about 18%, preferably from about 8% to
about 12%. In yet another embodiment the concentration of the phase forming salt
expressed in weight/volume is in the range of about 4% to about 18%, preferably from
about 6% to about 12%.
The resulting extraction mixture is processed to form distinct phases, one of which
contains an enrichment of the protein GCSF in the native form. Such processing can be
accomplished, for example, by centrifuging the extraction mixture or by letting the
mixture sit undisturbed for several hours (settle or coalesce at l.times.gravity). In a
further aspect, once distinct phases have been formed, the phase that contains an
enrichment of the protein GCSF, i e., typically the upper light phase, may be removed.
Optionally, after removal of the phase that contains the protein GCSF, the phase that
does not contain the protein GCSF may be reextracted ("two-stage extraction").
Reextraction can be performed by adding a solution containing a phase forming agent
capable of forming a second light phase so that it will form a phase in the reextraction
that is enriched in the protein GCSF. In another aspect, during two-stage extraction, the
extraction mixture is stirred to dissolve the phase forming agents and to thoroughly mix
the system. The resulting reextraction mixture is processed to form distinct phases of
which one contains the enriched protein GCSF.
Following extraction purification, the protein GCSF can be detected in the phase
removed from the extraction system. For example, the protein can be detected by a
variety of methods including, but not limited to, bio assays, HPLC, amino acid
determination or immunological assays, e.g., radioimmunoassay, ELISA, Western blot
using antibody binding, SDS-PAGE. Such antibodies include but are not limited to
polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric

antibodies, single chain antibodies, Fab fragments, fragments produced by a Fab
expression library, and epitope-binding fragments of any of the above. The amount of
the purified protein and their level of purity can be determined by methods well known
in the art.
The protein obtained using the method of the present invention can be further
processed, for example, in order to provide the protein or polypeptide having high
purity. Further purification may be necessary to remove related impurities. The
impurities may include oxidized forms, deamidated forms, aggregated GCSF and also
degraded forms such as biologically inactive monomeric forms, incorrectly folded
molecules of G-CSF, denaturated forms of G-CSF, host cell proteins, host cell
substances such as DNAs, (lipo)polysaccharides etc and additives which had been used
in the preparation and processing of G-CSF. Such higher purity may be required
depending on the use for which the protein or polypeptide is intended. For example,
therapeutic uses of the protein will typically require further purification following the
extraction methods of the invention. All protein purification methods known to the
skilled artisan may be used for further purification. Such techniques have been
extensively described in Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Volume 152, Academic Press, San Diego, Calif. (1987);
Molecular Cloning: A Laboratory Manual, 2d ed., Sambrook, J., Fritsch, E. F., and
Maniatis, T. (1989); Current Protocols in Molecular Biology, John Wiley & Sons, all
Viols., 1989, and periodic updates thereof); New Protein Techniques: Methods in
Molecular Biology, Walker, J. M., ed., Humana Press, Clifton, N.J., 1988; and Protein
Purification: Principles and Practice, 3rd. Ed., Scopes, R. K., Springer-Verlag, New
York, N.Y., 1987, the above are incorporated herein by references in its entirety. In
general, techniques including, but not limited to, ammonium sulfate precipitation,
centrifugation, ion exchange, reverse-phase chromatography, affinity chromatography,
hydrophobic interaction chromatography may be used to further purify the protein.
In a preferred embodiment the upper phase containing the GCSF protein is diluted to
adjust the conductivity in the range of 3 to 6 mS/cm, preferably in the range of 4 to 5
mS/cm. The pH is also adjusted in the range of 3-5.5 preferably in the range of 4 to 5.
The resultant solution containing GCSF along with related impurities can be further
purified to remove related impurities by using cation-exchange chromatography. In

another option the upper phase can also be subjected to hydrophobic interaction
chromatography by proper salt addition.
The yield of the pure protein GCSF obtained by the processes of the invention are in
the range of 40 to 50%.
According to one embodiment of the invention the aqueous two phase extraction is
useful for separating more than 95% of the host cell proteins, endotoxins and DNA
from the refolded protein GCSF. The protein GCSF obtained by the processes of the
invention has a purity 99 % or more. The GCSF obtained by the processes of the
invention have very low oxidative impurities. The presence of endotoxins in the pure
GCSF obtained by the processes of the invention is less than 2IU/ml. The content of
host cell protein in the pure GCSF is less than 20 ppm.
The purification of GCSF in native form comprising at least one step of aqueous two
phase extraction process according to the invention can be used for the native GCSF
obtained from any of the natural sources like mammalian tissues and blood. The
described process is particularly suitable for the industrial production of GCSF.
The process of obtaining pure GCSF as described herein further comprises of forming
the pure GCSF into a finished dosage form for clinical use.
The biologically active G-CSF obtained by the entire process for the purification and/or
isolation of the present invention is suitable for the preparation of pharmaceutical
composition, which comprises the therapeutically effective amount of biologically
active G-CSF and one or more pharmaceutical excipients and is suitable for clinical
use. The possibility of maintaining the active form of G-CSF in a short purification and
isolation process contributes not only to an improved yield, but also to an improved
purity and effectiveness of the biologically active G-CSF and the pharmaceutical
composition containing it.
Suitable pharmaceutically acceptable excipients include but not limited to suitable
diluents, adjuvants and/or carriers useful in G-CSF therapy.

In yet another embodiment the invention relates to pharmaceutical compositions
containing the GCSF obtained according to the present invention. The GCSF obtained
can either be stored in the form of a lyophilisate or in liquid form. It is administered
either subcutaneously or intravenously. Suitable adjuvants in the formulations of the
recombinantly expressed GCSF are, for example, stabilizers like sugar and sugar
alcohols, amino acids and tensides like for example polysorbate 20/80 as well as
suitable buffer substances. Examples for formulations are described in EP 0674525, EP
0373679 and EP 0306824 both of which are incorporated herein by reference in its
entirety.
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 : General Method for obtaining pure GCSF
Step A: Inclusion bodies of GCSF are solubilized in buffer containing 100mM Tris 6M
GuHCl pH 8.0. Solubilization takes around 45 min.The OD of the solubilized 1B is
adjusted with solubilization buffer to 8.0. (Generally 45ml solubilization buffer for lg
of IB is used). The solution is filtered through 0.45μm filter. DTT is added up to 5mM
to reduce the protein. Reduction is carried out for 30 min at room temperature (25 °C).
Step B: The solubilized GCSF is added to refolding buffer with stirring in a period of
30-45 minutes. Refolding buffer contains 75mM Tris pH 8.8, 0.1M L-Arginine, 10%
Sucrose, 2mM EDTA, 10mM Sodium ascorbate, 2M Urea. For lg of IB 1 liter of
refolding buffer is used. The temperature of the buffer is maintained at around 8.0 °C.
Refolding is carried out for 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 is over, buffer exchange of the refolded protein is carried out in 20mM
Tris pH 8.0, 5% Sucrose or 5% D + Sorbitol to remove the denaturant.
Step C: To the diafiltered solution containing the protein, PEG 4000 is added such that
its concentration in the final solution would be 10% w/w. After the PEG is dissolved

salt (Sodium sulfate) is added such that its concentration in the final solution would be
8% w/w. After the salt is dissolved the solution is left without stirring so that phase
formation will take place. Two phases are formed, namely, Salt Phase and PEG Phase.
The GCSF comes in the upper phase (PEG Phase). The lower phase is discarded, where
impurities get removed. The upper phase is checked for purity. The pH of the upper
phase, which contains GCSF protein, is adjusted to 4.5 and then either diafiltered or
diluted to bring the conductivity to around 4-6mS/cm.
Step D: This solution is then loaded on a cation exchanger (SP FF Sepahrose) at pH
4.5. The column is pre-equilibrated with 20mM sodium acetate buffer pH 4.5. After
loading is over the column is washed with 20mM sodium acetate pH 5.5 buffer. After
washing is over the bound protein is eluted with a linear gradient of NaCl in 20mM
sodium acetate pH 5.5 buffer.
Step E: The purified GCSF was then buffer exchanged into formulation buffer (10mM
sodium acetate, pH 4.0, 5% sorbitol, 0.004% Tween 80)
The purified GCSF protein is similar in-vitro bioactivity as the available commercial
GCSF product.
Example -2
2 g of inclusion bodies were solubilzed in 100mM Tris pH 8.0, 6M Guanadium
hydrochloride buffer. Solubilization was carried out at 25°C and for 45min. The
solubilized IBs solution was filtered through 0.45micron Polyether sulfone filter. The
OD at 280nm of the filtered solution was checked and adjusted to 8.0 by adding the
required amount of solubilization buffer. To 90 ml of solubilized IB solution DTT was
added such that the final concentration is 5mM. Reduction was carried out for 30 min.
After reduction the IB solution was slowly added to the 2000 ml refolding buffer with
following composition: 75mM Tris-Cl pH 8.8, 10% Sucrose, 2M Urea, 0.1M L-
Arginine, 2mM EDTA. The temperature was maintained at 8-10C. After the inclusion
body solution is added cystine and cysteine are added such that the final concentration
is 1mM and 4mM respectively. The refolding was carried out for 15 hrs at 10°C.
After the refolding was over the refolded protein was concentrated to 1 litre and
diafiltered against 3 diafiltration volume of 20mM Tris pH 8.0, 5% sorbitol using
Tangential Flow Filtration (TFF). To the diafiltered solution 122 g of PEG 4000 was

added. After the PEG was dissolved 97.6 g of sodium sulfate was added. The solution
was then left for gravity settling. The upper phase was then recovered and diluted with
20mM sodium acetate pH 4.5, 5% Sorbitol to adjust the pH to 4.5 and conductivity to
5.5mS/cm. This diluted solution was then loaded on SP Sepharose column equilibrated
with 20mM sodium acetate pH 4.5, 5% sorbitol. After loading and washing with 20mM
sodium acetate pH 4.5 5% sorbitol buffer a further wash of 20mM sodium acetate pH
5.5, 5% sorbitol buffer is provided. The bound protein was then eluted with a linear
gradient of 20mM sodium acetate pH 5.5, 5% sorbitol, 1M NaCl in 70CV. The eluted
fractions containing purity more than 99% by RP-HPLC were pooled. The pooled
fractions were then buffer exchanged against 10mM sodium acetate, pH 4.0, 5%
sorbitol, 0.004% Tween 80 using Sephadex G-25 medium gel filtration column.
200 mg of therapeutic grade Pure GCSF was obtained from the above process. Fig 2
provides the SDS-PAGE profile of the Purification. The upper phase shows purity of
more than 99% by SDS-PAGE. The final purified protein after ion-exchange shows
purity more than 99% by SDS-PAGE and more than 98.5% by RP-HPLC. The yield
obtained was 45%.
While the present invention has been described in terms of its specific embodiments,
certain modifications and equivalents will be apparent to those skilled in the art and are
intended to be included within the scope of the present invention.

We Claim:
1. A process for the preparation of pure recombinant human G-CSF obtained from
microbial cells, the process comprising the steps of:
a) solubilizing the inclusion bodies of GCSF;
b) refolding the said solubilized GCSF proteins;
c) purifying the refolded GCSF by using aqueous two phase extraction;
d) optionally further purifying native GCSF obtained in step c; and
e) isolating pure GCSF.

2. The process as claimed in claim 1, wherein the aqueous two phase extraction
system comprises a polyethylene glycol (PEG) and a salt phase.
3. The process as claimed in claim 2, wherein the PEG has a molecular weight of
about 2000 to about 8000.
4. The process as claimed in claim 2, wherein the salt phase comprises one or more of
sodium sulfate, potassium sulfate and ammonium sulfate, sodium citrate, potassium
citrate, ammonium citrate, sodium phosphate, ammonium phosphate and potassium
phosphate.
5. The process as claimed in claim 2, wherein the concentration of the phase forming
polymer is in the range of about 4 % to about 18% w/v.
6. The process as claimed in claim 2, wherein the concentration of the phase forming
salt is in the range of about 4 % to about 18% w/v.
7. The process as claimed in claim 1, wherein native G-CSF obtained in step c) is
further purified by additional chromatographic purification step comprising one or
more of ion exchange chromatography, reverse phase chromatography, affinity
chromatography, hydrophobic interaction chromatography.
8. The process as claimed in claim 1, further comprising forming the pure GCSF into
a finished dosage form.

9. Pure G-CSF having purity of 99% or more, having endotoxins less than 2IU/ml and
host cell protein less than 20 ppm.
10. Pure GCSF prepared by a process comprising at least one step of aqueous two
phase extraction.
11. A pharmaceutical composition comprising a therapeutically effective amount of
biologically active GCSF obtained by a process comprising at least one step of
aqueous two phase extraction and one or more pharmaceutically acceptable
excipients.

The present invention describes a novel process for large-scale purification of therapeutic grade quality of recombinant human GCSF from microbial cells, wherein the protein is expressed as inclusion bodies. The Inclusion bodies are solubilized and refolded under redox condition. The Redox condition is provided by using ascorbic acid, dehydroascorbic acid and reduced gluthathione. The process involves the novel use of aqueous two phase extraction step to purify refolded GCSF after removal of
denaturant. After this step GCSF is further purified using chromatography techniques
for removal of related impurities. The GCSF obtained has good purity and yields which
are essential for a production scale process. The host cell related contaminants like proteins, DNA and endotoxins are reduced using the purification processes of the invention.