FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. -
PROCESS FOR PURIFICATION OF RECOMBINANT
HUMAN IL-11
2. Applicant(s)
(a) NAME : LUPIN LIMITED
(b) NATIONALITY : An Indian Company
(c) ADDRESS : 159 CST Road, Kalina, Santacruz (East)
Mumbai - 400 098, State of Maharashtra
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed :

PROCESS FOR THE PURIFICATION OF RECOMBINANT HUMAN IL-11
Field of the invention
The invention relates to process for purification of recombinant human IL-1 1 from a
soluble IL-1 1 fusion protein expressed in bacterial system. The method involves
purification of the soluble fusion protein, digestion to separate the IL-1 1 from the fusion
tag followed by hydrophobic interaction chromatography and ion exchange
chromatography.
Background of the invention
The development of techniques and methods for protein purification has been an essential
prerequisite for many of the advancements made in biotechnology. Protein purification
varies from simple one step precipitation procedures to large scale validated production
processes. The key to successful and efficient protein purification is to select the most
appropriate techniques, optimize their performance to suit the requirements and combine
them in a logical way to maximize yield and minimize the number of steps required.
Most commonly used purification methods include use of affinity tags, fusion tags, metal
binding, immunoaffinity chromatography, ion exchange chromatography, size exclusion
chromatography, hydrophobic interaction chromatography and HPLC.
Recombinant DNA developments over the past decade have revolutionized the
production of proteins in large quantities. While production of heterologous proteins in
bacterial hosts has been implemented successfully in the biotechnology industry, there are
numerous instances where bacterial expression systems have given less than satisfactory
results. Often, over expression leads to the production of inclusion bodies, that are
insoluble aggregates of misfolded proteins. For example, when high-expression levels are
achieved, recombinant proteins are frequently expressed in Escherichia coli as insoluble
protein aggregates termed "inclusion bodies" that have been the subject of many protein
folding studies. The conditions for refolding the denatured protein must be optimized for
each specific protein, and the renaturation yield may be low even in an optimized system.
Thus it is often desirable to maximize the expression of the protein in a completely
soluble form.
US 5215895 relates to a novel cytokine that stimulates the function of cells of the immune
and hematopoietic systems, and to processes for obtaining the factor and producing it by
recombinant genetic engineering techniques. It describes expression of IL-l l in bacterial
cells where the purification involves four ion exchange steps and cleavage of the fusion
protein by hydroxylamine.
WO9516044A2 relates to proteins and peptide(s) fused to thioredoxin or thioredoxin-like
molecules - useful for production of large amounts of heterologous proteins. The fusion
molecule is modified to introduce one or more metal-binding/chelating amino-acid
residues to aid in purification. Expression of this fusion molecule under the control of a
regulatory sequence capable of directing its expression in a desired host cell produces
high levels of stable and soluble fusion protein.
EP 1598364A1 discloses novel polynucleotide encoding fusion interleukin (IL)-l l
receptor and IL-1 1 polypeptide.
US 20090036652A1, WO 06126102A2 and US 5646016 disclose method for purification
of fusion using different purification protocols.
US 20070 141 662A1 discloses a method for recombinantly producing a peptide
comprising expressing the peptide as a fusion protein and applying the protease to cleave
the fusion protein.
WO 9521 197A1, US 20070 14 1662A1, EP 888384B1, US 7585943, and US 7442371
relates to fusion products prepared by recombinant DNA procedures.
Summary of the invention
In one general aspect the invention is related to a method for purification of recombinant
human Interleukin- 11 from bacterial cells, the process comprising the steps of:
(a) purifying the fusion protein using hydrophobic interaction chromatography,
(b) cleaving the fusion protein with enterokinase, and
(c) purifying IL-1 1 by ion exchange chromatography,
wherein the purification steps may be in any order.
In another aspect of the invention the hydrophobic interaction chromatography is
performed using resins selected from butyl sepharose, phenyl sepharose or octyl
sepharose. In another aspect of the invention the Ion exchange chromatography is selected
from cation exchange or anion exchange or both in either order.
In yet another aspect of the invention the method for purification of recombinant human
Interleukin 11 comprises use of one or more fusion tags selected from SD tag, GM tag,
T7 tag, GST tag, His tag, Trx Tag or MBP tag. The SD tag used in the invention having
sequence Id 1, comprises of 49 times repeated, Serine Aspartate residues. The fusion tag
(having a pi of 4.0) has a total of 107 amino acids.
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 accompanying drawings:
Figure 1: SDS PAGE separation of Interleukin 11.
DESCRIPTION OF SEQUENCE ID
SEQ ID 1: Amino acid sequence of SD tag
MSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD
SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSGSDDDDK.
SEQ ID 2: Nucleotide sequence of SD tag
ATGAGCGATTCCGATTCAGACTCGGACTCGGATTCCGATTCCGACAGTGATTCAGATTCTGACT
CAGATTCCGATTCTGATTCTGATTCGGATTCCGACTCCGATAGCGACTCAGATAGTGACTCTGA
CTCGGACAGCGATTCTGATAGCGACTCTGATTCCGATAGCGATAGCGATTCAGATAGCGATTC
TGACTCGGATTCTGATTCCGATTCTGACTCTGACAGCGATTCCGATAGCGACAGCGACTCTGAT
AGTGATTCAGACTCTGATTCTGATAGTGATAGCGATTCGGATAGTGGATCCGATGATGATGAT
AAA.
SEQ ID 3: Amino acid sequence of IL 11
GPPPGPPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVL
TRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPP
LAPPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL.
SEQ ID 4: Nucleotide sequence of IL 11
GGTCCACCACCTGGACCACCTCGGGCCGAGCTGGACAGCACCGTGCTCCTGACCCGCTCTCTC
CTGGCGGACACGCGGCAGCTGGCTGCACAGCTGAGGGACAAATTCCCAGCTGACGGGGACCA
CAACCTGGATTCCCTGCCCACCCTGGCCATGAGTGCGGGGGCACTGGGAGCTCTACAGCTCCC
AGGTGTGCTGACAAGGCTGCGAGCGGACCTACTGTCCTACCTGCGGCACGTGCAGTGGCTGCG
CCGGGCAGGTGGCTCTTCCCTGAAGACCCTGGAGCCCGAGCTGGGCACCCTGCAGGCCCGACT
GGACCGGCTGCTGCGCCGGCTGCAGCTCCTGATGTCCCGCCTGGCCCTGCCCCAGCCACCCCC
GGACCCGCCGGCGCCCCCGCTGGCGCCCCCCTCCTCAGCCTGGGGGGGCATCAGGGCCGCCCA
CGCCATCCTGGGGGGGCTGCACCTGACACTTGACTGGGCCGTGAGGGGACTGCTGCTGCTGAA
GACTCGGCTGTGA.
Detailed description of the invention:
IL-1 1 is a 19 kDa polypeptide consisting of 178 amino acids, which does not contain
potential glycosylation residues, disulphide bonds or other post-translational
modifications and has a close similarity to IL-6. It binds to a multimeric receptor complex
which contains an IL- 11 specific a-receptor subunit and a promiscuous subunit (gpl30).
IL-1 1 has been demonstrated to improve platelet recovery after chemotherapy-induced
thrombocytopenia, induces acute phase proteins, modulates antigen-antibody responses,
participates in the regulation of bone cell proliferation and differentiation and could be
use as a therapeutic for osteoporosis. Besides from lymphopoietic/hematopoietic and
osteotrophic properties, it has functions in many tissues such as brain, gut and testis. IL-
1 stimulates the growth of certain lymphocytes and, in the murine model, stimulates an
increase in the cortical thickness and strength of long bones.
SD is an acidic protein having low pi of 4.0 present at the N terminus portion of the target
protein fused (IL-1 1) having a pi of 11.16. This information can be used for the
purification of protein of interest from the tag by using cation exchange chromatography
at pH 8.0 wherein the fusion tag, due to its low pi does not bind to the resin whereas
protein of interest will bind to the cation exchange resin. In this way, SD can be used as
an effective tool to remove the fusion tag after cleavage by simple cation exchange
chromatography. This tag can be fused to any other protein of therapeutic value having a
high pi point and similar strategy can be applied for their purification as well for example
Interferon Beta pi, 9.69, Nesiritide pi 10.9 etc.
In an embodiment of the invention the protein obtained from the fermentation is further
purified by subjecting the fusion protein to hydrophobic interaction chromatography.
Hydrophobic interaction chromatography (HIC) is used as the first step for the
purification. There are several resins which can be used for the particular application like
butyl sepharose, phenyl sepharose; octyl sepharose etc. The purified fusion protein may
further be treated with enterokinase to cleave the fusion protein to obtain the protein of
interest. The above digested protein is purified by weak or strong cation exchange
chromatography, preferably weak cation exchange chromatography. Several ion exchange
resins can be used such as CM sepharose, SP sepharose etc.
In an embodiment of the invention enterokinase from any sources such as bovine
enterokinase, human enterokinase, porcine enterokinase or recombinant enterokinase may
be used. Recombinant enterokinase may be obtained from bacterial such E.coli or yeast
such as Pichia pastoris may be used. Enterokinase can then be removed from the IL 11 by
the same step of cation exchange chromatography as enterokinase with pi of 5.5 will not
bind to cation exchange resin and will come in the flow through along with other protein
impurities. On the other hand, target protein binds to the column which can be eluted by
giving salt gradient.
In another embodiment, enterokinase used in the process is recovered from the flowthrough
of cation exchange chromatography by loading it in an affinity chromatography
resin (eg. soyabean trypsin inhibitor agarose) Sepharose resin. This way, the IL- 11
downstream operations can be made cost effective by recycling enterokinase.
In an another embodiment of the invention the fusion protein may be subjected to ion
exchange chromatography followed by HIC followed by enterokinase digestion and
further purifying with ion exchange chromatography.
In the purification process carried out by this method IL-1 1 fusion protein is active before
and after cleavage with enterokinase and since it is active before cleavage also, this can
be exploited to check whether the fusion protein is active or not through out the process.
The advantage of the present invention is that the protein of interest is having a novel
fusion tag which imparts solubility to the target protein. Also the protein does not have to
go through the complex process of refolding in order to have the biologically active form.
In addition to it, the present invention provides a very simple and cost effective process to
achieve the purity level of more than 99%.
Definitions
The term fusion tag used herein refers to a fusion tag comprising Serine Aspartate residue
sequence, repeated 49 times, having a total of 107 amino acids (having pi of 4.0) for
separation of fusion tag from the protein of interest after cleavage using simple and cost
effective purification steps. The fusion tag as described provides solubility to the protein
and also simplifies the purification of the target protein.
As used herein the term Hydrophobic Interaction Chromatography refers to a separation
technique that uses the properties of hydrophobicity to separate proteins from one
another. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or
butyl, are attached to the stationary column. Proteins that pass through the column that
have hydrophobic amino acid side chains on their surfaces are able to interact with and
bind to the hydrophobic groups on the column. In this separation, a buffer with a high
ionic strength, usually ammonium sulfate is initially applied to the column. The salt in the
buffer reduces the solvation of sample solutes thus as solvation decreases, hydrophobic
regions that become exposed are adsorbed by the medium. To elute the proteins, the salt
concentration is gradually decreased in order of increasing hydrophobicity. Additionally,
elution can also be achieved through the use of mild organic modifiers or detergent.
In an embodiment the hydrophobic interaction chromatography is carried out using a salt
concentration of more than 0.5 M.
As used herein the term Enterokinase is a Serine Protease enzyme which converts inactive
trypsinogen into active trypsin by cleavage at the C-terminal end of the sequence. It
consists of a disulfide-linked 82-140 kDa heavy chain which anchors enterokinase in the
intestinal brush border membrane and a 35-62 kDa light chain which is the catalytic
subunit. Enterokinase cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys. It
will sometimes cleave at other basic residues, depending on the conformation of the
protein substrate. Enterokinase will not cleave at site followed by proline.
As used herein the term Ion Exchange Chromatography relies on charge-charge
interactions between the proteins and the charges immobilized on the resin. Ion exchange
chromatography can be subdivided into cation exchange chromatography, in which
positively charged ions bind to a negatively charged resin; and anion exchange
chromatography, in which the binding ions are negative, and the immobilized functional
group is positive. Once the solutes are bound, the column is washed to equilibrate it in
your starting buffer, which should be of low ionic strength, then the bound molecules are
eluted off using a gradient of a second buffer which steadily increases the ionic strength
of the eluent solution. Alternatively, the pH of the eluent buffer can be modified as to
give the protein or the matrix a charge at which they will not interact and the molecule of
interest elutes from the resin.
The invention is further illustrated by the following examples which are provided merely
to be exemplary of the invention and do not limit the scope of the invention. Certain
modifications and equivalents will be apparent to those skilled in the art and are intended
to be included within the scope of the invention.
Example 1 : In vitro Biological Assay
Cells were cultured in RPMI medium containing 10% fetal calf serum, penicillin (100
units ml 1), streptomycin ( 1 mg ml 1) and human IL-3 ( 1 ng ml ) (Medium A). For cell
proliferation assay, cells were pre-incubated in medium A devoid of IL-3 for 3 hours.
Cells (5xl0 /well/100 ΐ in Medium A devoid of IL-3) were then incubated with purified
IL-1 1 (200 ng ml 1) in 96 well tissue culture plates for 48 hours at 37°C, 5% C0 2. MTS
(20 ΐ, Cell Titer 96 Aqueous One Solution, Promega) was added to each well and A490
was monitored after 4 hours of incubation at room temperature. A490 of control wells
(without IL- 11) were subtracted from IL-1 1 treated wells to get specific absorbance of
treated wells.
IL-1 1 obtained in this manner was seen to be 95 % active when compared with the
marketed formulation.
Example 2: Processing the Cell Lysate
Cell lysate of the fusion protein is centrifuged at 15000 rpm for 15 minutes at room
temperature. Supernatant is collected and to the supernatant, NaCl is added to the final
concentration of 500-2000 mM preferably 1000 mM. When NaCl is completely
dissolved, Polyethylene Imine is added with constant stirring from 0.05 % to 5 %
concentration, preferably 0.05% to precipitate DNA (both plasmid and host chromosomal
DNA) from the cell lysate.
After DNA precipitation, cell lysate is centrifuged at 13 to 15,000 rpm for 15 minutes
between 4-25 °C. Supernatant obtained is used as starting material for purification.
Example 3 : Purification Using Hydrophobic Interaction Chromatography
The column is pre-equilibrated with a suitable buffer which may include 10-50 mM Tris-
Cl pH 8.0, 20-50mM sodium acetate buffer, 50-100 mM sodium phosphate buffer etc. In
HIC, protein should bind to the resin at a reasonably high concentration of salt. The salt
concentration should be tried depending upon the hydrophobicity of the target protein
i.e. 1-2 M NaCl or 0.5-2 M ammonium sulphate can be used. After loading, the column is
washed with 5 column volumes equilibration buffer and the bound protein is eluted with
20-50 mM sodium acetate buffer, preferably 50-100 mM sodium phosphate buffer and
most preferably with 10-50 mM Tris-Cl, pH 8.0.
Example 4: Enterokinase Cleavage
HIC eluted protein is dialyzed against 10-200 mM, preferably 20 mM Tris pH 8.0 for 16
hours in cold (4-10 °C). To the dialyzed protein, CaCl2 is added to the concentration of 1-
10 mM, preferably 2-5mM. Enterokinase is added for the cleavage of fusion protein in the
range of 1-5 units of Enterokinase for 400 microgram of the GMSD-IL-1 fusion protein
at 10-30 °C, for 16 hours after which, there is almost 90 percent digestion of the fusion
protein. Higher concentrations of E could also be added for 100% fusion cleavage or by
adding the same amount of EK but carrying out the EJK digestion at a higher temperature
(RT-40 C).
Example 5: Purification using Ion Exchange Chromatography
The column is pre-equilibrated with sodium phosphate buffer 20-100mM, pH 7-8, 10-
lOOmM Tris, pH 8.0, preferably with 20-50mM Tris, pH 8.0 .After loading, the column is
washed with 5 column volumes of equilibration buffer to remove loosely bound
impurities. The bound proteins can then be eluted with 20-100mM sodium phosphate
buffer , pH 7-8, 20-50 mM Tris pH 7-8.0 containing 100-500mM NaCl.
The CM elution is loaded onto Q sepharose at the conductivity of 20-25 mS/cm , protein
of interest comes in the unbound fraction whereas the impurities are bound to the resin
and is eluted in 1M NaCl salt, the unbound protein at this step is >99% pure.
Example 6: Purification using High Performance Liquid Chromatography (HPLC)
Reverse phase HPLC runs were carried out in Shimadzu Model 20 0 CHT. The analysis
was done on a Vydac C4 4.6*50 50mm, 5column and a 2ml/min flow rate. Gradient
chromatography used 0.1% trifluoroacetic acid as buffer A and 0.1% trifluroacetic acid in
90% aqueous acetonitrile as buffer B. A linear gradient of 10 min from 30 to 70% B was
used .Samples for analysis were injected. RP-HPLC Profile of in house and Innovator IL-
11 was performed. HPLC runs used Shimadzu Model 2010 CHT. The analysis used a
Vydac C4 4.6*50 50mm, 5um column and a 2ml/min flow rate. Gradient chromatography
used 0.1% trifluoroacetic acid as buffer A and 0.1% trifluroacetic acid in 90% aqueous
acetonitrile as buffer B. A linear gradient of 10 min from 30 to 70% B was used. Samples
for analysis were injected.
CLAIMS
. A method for purification of human Interleukin 1 from bacterial cells comprising
steps of :
(a) purifying the fusion protein using hydrophobic interaction chromatography,
(b) cleaving the fusion protein with enterokinase, and
(c) purifying IL-1 1 by ion exchange chromatography,
such that the purification steps may be in any order.
2. The method as claimed in claim 1, wherein hydrophobic interaction chromatography is
performed using resins selected from butyl sepharose, phenyl sepharose or octyl
sepharose.
3. The method as claimed in claim 2, wherein hydrophobic interaction chromatography
uses a salt concentration of more than 0.5 M.
4. The method as claimed in claim 1, wherein Ion exchange chromatography is selected
from cation exchange and anion exchange in any order.
5. The method as claimed in claim 1, further comprises use one or more fusion tags
selected from the group consisting of SD tag, GM tag, T7 tag, GST tag, His tag, Trx Tag
or MBP tag.
6. The method as claimed in claim 5, wherein the fusion tag is SD tag comprising
nucleotide sequence
ATGAGCGATTCCGATTCAGACTCGGACTCGGATTCCGATTCCGACAGTGATTCAGATTCTGACT
CAGATTCCGATTCTGATTCTGATTCGGATTCCGACTCCGATAGCGACTCAGATAGTGACTCTGA
CTCGGACAGCGATTCTGATAGCGACTCTGATTCCGATAGCGATAGCGATTCAGATAGCGATTC
TGACTCGGATTCTGATTCCGATTCTGACTCTGACAGCGATTCCGATAGCGACAGCGACTCTGAT
AGTGATTCAGACTCTGATTCTGATAGTGATAGCGATTCGGATAGTGGATCCGATGATGATGAT
AAA.
7. The method as claimed in claim 5, wherein the fusion tag is SD tag comprising amino
acid sequence
MSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSD
SDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSGSDDDDK.
8. The method as claimed in claim 4, wherein ion exchange chromatography is cation
exchange chromatography.
9. The method as claimed in claim 8, wherein ion exchange chromatography is performed
using resins selected from CM sepharose or SP sepharose.