Note: Descriptions are shown in the official language in which they were submitted.
CA 02402562 2002-09-11
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A PROCESS FOR THE PRODUCTION OF HUMAN INTERFERON ALPHA FROM
GENETICALLY ENGINEERED YEAST
CLAIM OF PRIORITY
This application claims priority to Indian Patent Application No. 826/MAS/98,
filed
March 19, 1999.
REFERENCE TO CTTATIONS
Complete citations to the cited references can be found in the Bibliography
preceding the
claims.
FIELD OF THE INVENTION
The invention relates to a process for the production of human interferon
alpha from
genetically engineered yeast. More particularly the invention relates to the
cloning and
expression of human interferon alpha gene in the methylotropic yeast, Pichia
pastoris and a
process for purification of the said protein.
DESCRIPTION OF THE RELATED ART
Interferon, the body's most rapidly produced defense against viruses, is a
protein secreted
by the body cells when they are exposed to viruses, bacteria, and different
types of
macromolecules. The secreted interferon then stimulates surrounding cells to
produce other
proteins, which in turn may regulate viral multiplication, the immune
response, cell growth, and
other cell functions. There are three classes of human interferons:
(i) interferon alpha, which is secreted by leukocytes,
(ii) interferon beta, which is secreted by fibroblasts,
(iii) interferon gamma, which is secreted by lymphocytes.
Interferon a and (3 have been referred to as type I interferon and interferon
y has been
referred to as type II interferon. Human interferon a proteins generally
contain 165 or 166 amino
acids and have molecular weights ranging from 17,000 - 20,000 daltons, as
determined by SDS-
PAGE.
Interferon a has been used for the treatment of various viral and cancer
related diseases,
for example to treat hepatitis B, C and D viral infections and cancer diseases
like hairy cell
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leukemia, AmS-related Kaposi's sarcoma, chronic myelogenous leukemia, and
renal cell
carcinoma.
The human leukocyte interferon alpha is produced either from human cell lines
grown in
tissue culture or through human leukocytes collected from blood donors.
Horawitz, et al. 1982,
S US patents 4680261, 5503828, 5391713, 4732683, 4696899, 5789551 and European
patent
EP0945463 . These processes are laborious, tedious and time consuming. The
medium employed
is costly and the yields of purified material obtained are low. There is a
risk from contamination
of the blood used for the preparation of leukocytes by an unidentified
infectious agent.
With the advent of recombinant DNA technology, it has been possible to clone
the human
interferon alpha gene in microorganisms and produce sufficient quantities of
human interferon
alpha from these microorganisms. Stahelin, et al. 1981, Ho, et al., 1989,
Yang, et al. 1992,
Tarnowski, et al. 1986, Thatcher, et al. 1986, Tiute, et al. 1982, US patents
5710027, 5661009,
4765903, 5196323, 4315852, 4845032, 4530901 and European patents EP 0032134
and EP
0679718 describe the process for production of human interferon alpha from
recombinant E. coli
and Saccharomyces cerevisiae. Although expression and purification of human
interferon alpha
in E. coli overcame the problems and potential risks associated with the
production from natural
sources, it has its own drawbacks. The expressed protein in some cases is not
correctly
processed. The purified protein should be free of bacterial endotoxins.
Additional purification
steps are required to remove the endotoxins. Accordingly, the process employed
comprises
multiple chromatographic steps and thus is time consuming. These processes are
difficult to scale
up, a prerequisite for large scale production. The yields of recombinant human
interferon alpha
expressed in Saccharomyces is low. Thus there exists a need for expressing
human interferon
alpha in a suitable host and a purification process which is simple, efficient
and easily scalable.
Methylotropic yeast Pichia pastoris is increasingly becoming popular as a
protein
expression system. Pichia has the following advantages: first, extremely high
yields of intra
cellular proteins; second, ease of fermentation to high cell density; third,
genetic stability and
scale-up without loss of yield; and fourth, no endotoxin contamination.
Thus, the drawbacks associated with E. coli expression and purification of
recombinant
interferon can be overcome by cloning and expressing the human interferon
alpha in the
methylotrophic yeast Pichia pastoris. Accordingly, the aim of the invention is
to clone and
express the human interferon alpha gene in Pichia pastoris. Another object of
the present
invention is to develop an efficient purification process which can easily be
scaled up for the
recombinant human interferon alpha expressed in Pichia pastoris.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration diagramming the interferon clone development.
FIG. 2 is a flowchart outlining a first preferred embodiment of the downstream
processing and purification of interferon alpha.
FIG. 3 is a flowchart outlining a second preferred embodiment of the
downstream
processing and purification of interferon alpha.
DETAILED DESCRIPTION
This invention provides a process for the production of physiologically-active
human
interferon alpha from genetically engineered yeast. The process has the
following steps. A
plasmid having a promoter and operationally linked to a human interferon alpha
gene in the
absence of a fi.~sion region is digested with an enzyme (preferably Note to
produce a linearized
plasmid. Pichia pastoris cells are transformed with the linearized plasmid by
homologous
recombination to form Pichia pastoris clones. The Pichia pastoris clones are
screened for high
interferon alpha expression to find a high interferon-yielding Pichia pastoris
clone. The high
interferon-yieldingPichiapastoris clone is grown. Physiologically-active human
interferon alpha
protein is purified from the high interferon-yielding Pichia pastoris clones.
Preferably, the plasmid is constructed by cloning human interferon alpha gene
into a
plasmid pHIL-D2 containing an AOX1 promoter. Other promoters, such as GAP,
MOX, FMD,
ADH, LAC4, XPR2, LEU2, GAM1, PGK1, GAL7, GADPH, CYC1, and CUP1, are known and
will work with similar success. E. coli is transformed with the plasmid pHII,-
D2 containing the
cloned human interferon alpha gene. The transformed E. coli is then screened
for a recombinant
clone carrying the interferon alpha gene in proper orientation with respect to
the AOXl promoter
in plasmid pHIL-D2. The pHIi,-D2 plasmid is available commercially from
Invitrogen
Corporation (Carlsbad, California, US) and has been described in their
catalog. The transformed
Pichia pastoris clone that expresses human interferon alpha was deposited at
the American Type
Culture Collection, 10801 University Blvd., Manassas, Virginia 20110-2209, US,
on February
3, 2000, and is available under accession number PTA-1276.
Preferably, the production of human interferon alpha from genetically
engineered yeast
uses the following steps:
1. cloning human interferon alpha gene into plasmid pHIL-D2;
2. transforming E. coli with the plasmid pHIL-D2 containing the cloned human
interferon gene;
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3. screening the transformed E. coli for a recombinant clone carrying the
interferon alpha gene in proper orientation with respect to AOX1 promoter in
the plasmid pHIL-
D2;
4. digesting the plasmid pHIL-D2 in E. coli and Notl enzyme to get a Notl
fragment of pHIL-D2.
5. transforming the Pichia pastoris cells with the Notl fragment of pHIL-D2
harboring interferon alpha gene by homologous recombination;
6. screening the Pichia pastoris clones for interferon alpha expression and
confirmation of nucleotide sequence of interferon alpha gene from a high
interferon-yielding
clone;
7. growing the high yielding Pichia pastoris clone in a fermentor under
optimal
conditions as herein described;
8. washing the Pichia pastoris cells obtained from the fermentor with a
buffer, as
herein described;
9. breaking the Pichia pastoris cells with glass beads in a bead mill in the
presence
of protease inhibitor, as herein described;
10. adding protein solubilizing agent, as herein described to final
concentration
of 4-8 M and stirnng for 2-10 hours at 200 - 300 rpm and 4-7°C with or
without centrifugation;
11. diluting the extract of the above step 10-30 fold with buffer, as herein
described followed by clarification either by centrifugation or filtration and
with or without
concentration of the extract;
12. adjusting the pH of the above extract with a buffer, as herein described
to pH
3-5 followed by centrifizgation or filtration;
13. adsorbing the above extract on cation exchange column of "SP-
SEPHAROSE" (Pharmacia Fine Chemicals, New Market, New Jersey, US) and eluting
the
protein containing human interferon alpha with alkali chloride;
14. adjusting the pH ofthe above eluted sample containing human interferon
alpha
to neutral pH and adsorbing it on immuno-affinity column containing monoclonal
antibodies
against human interferon alpha coupled to a matrix, as herein described and
eluting interferon
alpha at pH below 4.0; and
15. diafiltering followed by sterile filtering the eluted interferon alpha.
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The preferred conditions for growing said high yielding Pichia pastoris clone
in
a fermentor are pH 5.0, 28-30°C, and 500-1500 rpm for 2 days and
inducing it with methanol
for 48 hours.
The preferred buffer used to wash the Pichia pastoris cells obtained from the
fermentor is sodium phosphate buffer of molarity 25-100 mM and pH 6.5-8, 1-5
mM
ethylenediaminetetraacetic acid (EDTA).
The preferred protease inhibitor used during breaking in the bead mill is 0.5 -
2.0
mM phenylmethylsulfonyl fluoride (PMSF).
The preferred protein solubilizing agents used are guanidine chloride or urea
at
a concentration of 4-8 M.
The preferred bufferused forthe dilution ofthe extract 10-30 fold is Tris-HCl
25-
100 mM, urea 0-1 M, pH 6.5-8Ø
The clarification is preferably carried out by centrifugation or filtration
and
concentration is preferably by ultra-filtration.
The concentrated sample is diluted with citrate buffer (25-100 mM, pH 4-5)
followed by centrifugation and filtration.
The clarification is either by centrifugation or filtration and without
concentration
and the pH of the extract is adjusted with a citrate buffer (1-2 M, pH 2-5)
The buffer used in adjusting the pH of the extract to pH 3-5 preferably is a
citrate,
either 25-100 mM or 1-2 M, pH 2-5 depending upon the volume of the extract.
The alkali chloride for eluting the protein containing human interferon alpha
preferably is sodium chloride.
The preferred matrix used is one that is an affinity support for coupling of
ligands
via primary amines. A most preferred matrix is "AFFI-GEL-10" available from
BIO-RAD,
Hercules, California, US. Interferon alpha is preferably eluted at pH 2-4.
The invention will now be described with reference to the following flow
diagrams
and the examples:
EXAMPLE 1
Referring to FIG. 1, which outlines the steps for the cloning of human
interferon alpha
gene in Pichia pastoris, the human interferon alpha gene is amplified
(preferably by PCR) and
digested with EcoRI. pHIL-D2 plasmid carrying the AOX1 promoter is linearized
by digesting
with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-D2. E.
coli cells are
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transformed with pHIL-D2-IFN plasmid. The E. coli transformants are screened
for a
recombinant in which IFN alpha gene is in the correct orientation with respect
to the AOX1
promoter present in pHIL-D2 plasmid. Pichia pastoris is transformed with the
Notl digested
pHIi,- D2-IFN plasmid. This results in the integration of IFN gene into yeast
genome by
homologous recombination. Recombinants are selected by their ability to grow
on minimal
medium. Recombinants are screened for intracellular expression of human alpha
interferon.
Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a
minimal medium,
pH 5.0, 28-30°C, 500 - 1200 rpm for Z days and induced with methanol
for 48 hours.
Referring to FIG. 2, which outlines the steps for a first preferred method of
downstream processing and purification of human interferon alpha from
fermentation onwards,
the fermentor culture is harvested and the cells are washed with lysis buffer,
25 mM sodium
phosphate buffer pH 8.0, 2 mM EDTA. The washed cells are broken with glass
beads in a bead
mill in the presence of 1 mM PMSF.-To the broken cell extract, solid guanidine
chloride is added
to a final concentration of 7 M and stirred at 200 - 300 rpm for 4-6 hours
with or without
centrifugation. The extract of the above step is diluted twenty times with a
buffer, 25 mM Tris-
HCl pH 7.5 containing 1-10 p.M PMSF and clarified by centrifizgation or
filtration. The clarified
extract is concentrated 10 fold by ultra filtration. The 10-fold concentrated
extract is diluted
again 10 times with 50 mM citrate buffer pH 4.0 containing 1 p,M PMSF. The
citrate diluted
extract is clarified by centrifugation or filtration and concentrated 10 fold
by ultra-filtration. The
above concentrated extract is subjected to cation exchange chromatography on
SP-sepharose and
eluted with a gradient of NaCI.
The pH of the above eluted IFN fraction is adjusted to 7.0, and the fraction
is
loaded onto an immuno-affinity column containing monoclonal antibodies coupled
to an AFFI
GEL-10 matrix (BIO-RAD, Hercules, California, US). Pure interferon is eluted
with 0.2 M acetic
acid and 0.15 M NaCI. The eluted interferon is diafiltered and sterile
filtered.
EXAMPLE 2
The human interferon alpha gene is amplified and cloned in the same manner as
described
for Example 1.
Referring to FIG. 3, which outlines the steps for a second preferred method of
downstream processing and purification of human interferon alpha from
fermentation onwards,
the fermentor culture is harvested and the cells are washed with lysis buffer;
25 mM sodium
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phosphate buffer; and 2 mM EDTA, pH 8Ø The washed cells are broken with
glass beads in a
bead mill in the presence of 1 mM PMSF. To the broken cell extract solid
guanidine chloride is
added to a final concentration of 7 M and stirred at 200-300 rpm for 4-6 hours
with or without
centrifi~gation. The extract of the above step is diluted twenty times with a
buffer, 25 mM Tris-
HCl pH 7.5 containing 1- 10 pM PMSF, and clarified by centrifugation or
filtration. The pH of
the above clarified extract is brought down to 4 with 1-2 M citrate, pH 2-4.
The above pH-adjusted extract is subjected to cation exchange chromatography
on SP-
sepharose and eluted with a gradient of NaCI. The pH of the above-eluted
fraction containing
IFN is adjusted to 7.0 and loaded onto an immuno-affinity column containing
monoclonal
antibodies coupled to an AFFI-GEL-10 matrix. Pure interferon is eluted with
0.2 M acetic acid
0.15 M NaCI. The eluted interferon is diafiltered and sterile filtered.
Table 1 details the specific activity and the yield of purified recombinant
Interferon alpha.
Biological activity of interferon alpha was determined by viral cytopathic
effect reduction assay.
Madin Darby Bovine Kidney (MDBK) cells and vesicular stomatitis virus (VSV)
were used in
the assay. The assay was calibrated with an international reference standard
obtained from
National Institute for Biological Standards and Control, U.K. Data is
presented for 3 batches of
purified Interferon alpha.
TABLE 1
SPECIFIC
ACTIVITY
AND
YIELD
OF
PURIFIED
INTERFERON
ALPHA
S. Batch No. Avera a s ecific Total units/liter
No. activi
1 0399 4.4 x 10g IU/m 600 x 10g IU
2 0499 4.66 x 10g IU/m 620 x l Og
IU
3 0599 5.2 x 10g IU/mg 560 x 10g ICT
The above process for the production of interferon alpha from genetically
engineered
yeast has several advantages over earlier processes, which also used
recombinant DNA
technology. First, Pichia pastoris can be grown to very high cell densities,
and the interferon
gene can be expressed using a strong alcohol-oxidase promoter so that high
yields of the
recombinant human interferon alpha can be obtained. Furthermore, methanol is
an inexpensive
inducer. Second, because the interferon gene is stably integrated into the
yeast genome by
homologous recombination, there is no need to use an antibiotic to maintain
the plasmid. Third,
the purification process employed is simple, efficient, and results in high
recovery of the
expressed protein. Fourth, the process can be scaled up easily for large-scale
purification of
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human interferon alpha. Finally, because yeast is a eukaryote, it can provide
a more suitable
environment for the folding of the eukaryotic interferon protein. Perhaps, it
is for this reason that
the interferon produced by the above process was found to give much higher
specific activity than
those reported earlier for interferon purified from E. coli.
Thus the subject process for the production of recombinant human interferon
alpha from
the genetically engineered yeast Pichia pastoris is simple, efficient and
easily scalable for large
scale production. The yield and specific activity of purified interferon alpha
is higher than those
reported from other systems.
It is understood that the invention is not confined to the particular
construction and
arrangement of parts herein illustrated and described, but embraces such
modified forms thereof
as come within the scope of the following claims.
BIBLIOGRAPHY
PATENTS CITED
EP 4530901 July, 1985 Weissmann
EP 0043980 January, 1982 Goeddel, et al.
US 4680261 July, 1987 Nobuhara, et
al.
US 5503828 April, 1996 Testa, et al.
US 5391713 February, 1995 Borg, et al.
US 4732683 March, 1988 Georgiades, et
al.
US 4696899 September, 1987 Toth, et al.
US 5789551 August, 1998 Pestka
EP 0945463 September, 1999 Attalla , et
al.
US 5710027 January, 1998 Hauptmann, et
al.
US 5661009 August, 1997 Stabinsky
EP 0032134 July, 1981 Weissmann
US 105629 August, 1988 D'Andrea, et
al.
US 5196323 March, 1993 Bodo Gerhard,
et al.
US 4315852 February, 1982 Leibowitz, et
al.
EP 0679718 November, 1995 Ettlin, et al.
US 4845032 July, 1989 Obermeier
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OTHER REFERENCES
1. Staehelin, T., et al., 1981, Purification of recombinant human leukocyte
interferon with
monoclonal antibodies, Methods in Enrymology, Vol. 78, SOS-512.
S 2. Ho, L. J., et al., 1989, Production of human leukocyte interferon in E
coli by control of
growth rate in fed-batch fermentation, Biotechnology Letters, Vol. 11, 695-
698.
3. Yang, X. M., et al., 1992, Production of recombinant human interferon alpha
by E. coli
using a computer controlled cultivation process, Journal ofBiotechnology, Vol.
23, 291-
301.
4. Gwynne, D. L, et al. 1987, Genetically engineered secretion of active human
interferon
and a bacterial endoglucanase from Aspergillus nidulans, Biotechnology, Vol.
5, 713-
719.
5. Tiute, M. F., et al., 1982, Regulated high efficiency expression of human
interferon alpha
in Saccharomyces cerevisiae, The EMBO Journal, Vol. 1, 603-608.
6. Horowitz, B., 1986, Large scale production and recovery of human leukocyte
interferon
from peripheral blood leukocytes, Methods in Enzymology, Vol. 119, 39-47.
7. Tarnowski, J. S., et al., 1986, Large scale purification of recombinant
human leukocyte
interferon, Methods in Enzymology, Vol. 199, 153-165.
8. Thatcher, D. R., et al., 1986, Purification of recombinant human IFN-a2,
Methods in
Enzymology, Vol. 119, 166-177.
9. Sudbert, P. E., 1996, The expression of recombinant proteins in yeasts,
Current Opinion
in Biotechnology, Vol. 7, 517-524
10. Romanos, M., 1998, Advances in the use of Pichia pastoris for high level
gene
expression, Current Opinion in Biotechnology, Vol. 6, 527-533.
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