Note: Descriptions are shown in the official language in which they were submitted.
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1
EXPRESSION SYSTEM FOR RECOMBINANT PROTEINS
BACKGROUND OF THE INVENTION
Pichia Pastoris Expression S, std
P. pasto~is was recognized in the seventies as a potential source for
production of single-
cell proteins for feed supplements due to its rather unique ability to
anabolize methanol to very
high cell mass. Expression of recombinant proteins in P. pasto~is has been in
development since
the late 1980's and the number of recombinant proteins produced in P. pastoris
have increased
significantly in the past several years (Cregg, et al., 1993; Sberna, et al.,
1996). P. pastof°is is a
desirable expression system because it grows to extremely high cell densities
in very simple and
defined media free of animal-derived contaminants. The defined growth medium
used for the
cultivation of P. pastonis is inexpensive and free of toxins or pyrogens.
Furthermore, the yeast
itself does not present problems in terms of endotoxin production or viral
contamination.
Additionally Piclaia can secrete expressed proteins at very high levels (>lg/L
and up to
80% of total cellular protein for some proteins) (Sberna, et al 1996). Unlike
bacteria, it is
capable of producing complex proteins with post-translational modifications,
e.g., correct
folding, glycosylation, and proteolytic maturation (White, et al. 1994;
Sberna, et al. 1996).
Pichia are different than Saccha~ofnyces in that they do not tend to
hyperglycosylate proteins
(oligosaccharide chains of 8-14 mannose) (Grinna ~Z Tschopp 1989) and the
highly
immunogenic a1,3-mannose structure is not found (Cregg et al., 1993). Pichia
generally
secretes the expressed proteins into the medium in a fairly pure form (30-80%
of total secreted
proteins) (Sberna, et al.) thus allowing for easy purification. It is also
capable of growing in a
very wide pH range, from 3 to 7.
Traditionally, P. pastoris fermentations are performed in batch/fed-batch
modes using a
methanol inducible system, Chen et al. (1). Some researchers have adapted this
system to
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continuous or continuous perfusion fermentation with limited success (Brierley
et al.; Chen et al.
(2); Cregg; Digan et al., 1989). Recently, constitutive promoters (e.g.,
Glyceraldehyde-3-
phosphate Dehydrogenase, GAP) have been developed for the P. pastor°is
expression system
(Waterham et al., 1997). These vectors allow for continuous production of the
desired
recombinant protein without methanol induction and are now readily available
commercially
(Invitrogen, San Diego, CA). This system is more desirable for large scale
productions because
the hazard and cost associated with large volumes of methanol are eliminated.
Using constitutive
constructs, glucose can be chosen as an inexpensive and efficient carbon
source. P. pastof~is high
yield expression systems have been successfully utilized to produce large
quantities of
biologically active, highly disulfide-bonded recombinant proteins of
commercial interest e.g.
IGF-1, HSA, TNF, Human Interleukin-2. (Buckholz et al (1991)., Cregg et al.
(1993); Ohtani et
al. (1998); White et a1.(1994)).
EntreMed, Inc. was recently reported to have successfully used the P. pastoris
expression
system for the production of the proteins AngiostatinOO and EndostatinTM
(Wells (1998)).
Proteins produced by P. pastof°is are usually folded correctly and
secreted into the medium,
facilitating the subsequent downstream processing. P. pastoris has further
been proven to be
oapable of N- and O-linked glycosylation and other post-translational protein
modifications
similar to that found in mammalian cells (Buckholtz et al; Cregg et al (1995);
Cregg et al.
(1993)).
2 0 The continuous production mode offers, in comparison to fed-batch
fermentation,
advantages in teens of higher volumetric productivity, product quality, and
product uniformity as
the exposure of the product to proteolytic enzymes, the possibility of protein
aggregation,
oxidation or inactivation is significantly reduced. A continuous production
process for rh-
Chitinase using a constitutive P. pastor~is expression system was recently
developed by the
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inventors and cor~ipared very favorably in terms of cost effectiveness,
development time, and
effort to expression of rh-Chitinase in mouse C127 cells.
One major drawback of the P. pastoris system is the degradation of the
secreted protein
by its own proteases (Boehm 1999). Degradation is increased when high-density
fermentation is
employed since the concentration of proteases in the fermentation broth also
increases. Several
strategies have been tried including the addition of an amino acid-rich
supplement, changing of
growth pH (3-7), and use of a protease-deficient host, but they have only
worked with limited
success. Another potential disadvantage of P. pastoris compared to mammalian
cell expression
systems is hyperglycosylation, which may cause differences in immunogenicity,
specific activity,
and serum half life of the recombinant protein.
Lysosomal Storage Diseases
Several of the over thirty known lysosomal storage diseases (LSDs) are
characterized by
a similar pathogenesis, namely, a compromised lysosomal hydrolase. Generally,
the activity of
a single lysosomal hydrolytic enzyme is reduced or lacking altogether, usually
due to inheritance
of an autosomal recessive mutation. As a consequence, the substrate of the
compromised
enzyme accumulates undigested in lysosomes, producing severe disruption of
cellular
architecture and various disease manifestations.
Gaucher's disease is the oldest and most common lysosomal storage disease
known.
Type 1 is the most cormnon among three recognized clinical types and follows a
chronic course
2 0 which does not involve the central nervous system ("CNS"). Types 2 and 3
both have a CNS
component, the former being an acute infantile form with death by age two and
the latter a
subacute juvenile form. The incidence of Type 1 Gaucher's disease is about one
in 50,000 live
births generally and about one in 400 live births among Ashlcenazim (see
gehe~°ally Kolodny et
al., 1998, "Storage Diseases of the Reticuloendothelial System", In: Nathan
and Oski's
2 5 Hematology of Infancy and Childhood, 5th ed., vol. 2, David G. Nathan and
Stuart H. Orkin,
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Eds., W.B. Saunders Co., pages 1461-1507). Also known as glucosylceramide
lipidosis,
Gaucher's disease is caused by inactivation of the enzyme glucocerebrosidase
and accumulation
of glucocerebroside. Glucocerebrosidase normally catalyzes the hydrolysis of
glucocerebroside
to glucose and ceramide. In Gaucher's disease, glucocerebroside accumulates in
tissue
macrophages which become engorged and are typically found in liver, spleen and
bone marrow
and occasionally in lung, kidney and intestine. Secondary hematologic sequelae
include severe
anemia and thrombocytopenia in addition to the characteristic progressive
hepatosplenomegaly
and skeletal complications, including osteonecrosis and osteopenia with
secondary pathological
fractures.
Niemann-Pick disease, also known as sphingomyelin lipidosis, comprises a group
of
disorders characterized by foam cell infiltration of the reticuloendothelial
system. Foam cells in
Niemann-Pick become engorged with sphingomyelin and, to a lesser extent, other
membrane
lipids including cholesterol. Niemann-Pick is caused by inactivation of the
enzyme
sphingomyelinase in Types A and B disease, with 27-fold more residual enzyme
activity in Type
B (see Kolodny et al., 1998, Id.). The pathophysiology of major organ systems
in Niemann-Pick
can be briefly summarized as follows. The spleen is the most extensively
involved organ of
Type A and B patients. The lungs are involved to a variable extent, and lung
pathology in Type
B patients is the major cause of mortality due to chronic bronchopneumonia.
Liver involvement
is variable, but severely affected patients may have life-threatening
cirrhosis, portal hypertension,
2 0 and ascites. The involvement of the lymph nodes is variable depending on
the severity of
disease. CNS involvement differentiates the major types of Niemann-Pick. While
most Type B
patients do not experience CNS involvement, it is characteristic in Type A
patients. The kidneys
are only moderately involved in Niemann Pick disease.
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SUMMARY OF THE INVENTION
Accordingly, the present invention provides methods for the production of
recombinant
proteins, such as glucocerebrosidase, sphingomyelinase and others, with high-
mannose
carbohydrate structure. The methods comprise culturing cells ofPichia pastoris
which cells
5 have been recombinantly engineered to comprise a DNA molecule which encodes
the protein of
interest, such as glucocerebrosidase or sphingomyelinase, under conditions
suitable for the
expression of said DNA molecule. The methods of the present invention are
particularly
applicable for production of proteins intended to be targeted to macrophages,
including Kupffer
cells. The methods of the invention may also be useful for targeting other
cells which contain
surface mannose receptors.
The methods are preferably performed under conditions suitable for continuous
fermentation of Pichia pastonis. The DNA molecules for use in the present
invention preferably
comprise a constitutive promoter operatively linked to the coding sequence of
interest. One
particularly well-suited constitutive promoter is the GAPDH promoter from
yeast.
In other embodiments, the present invention also provides methods for the
purification of
recombinant proteins, such as recombinmt human glucocerebrosidase or
recombinant human
sphingomyelinase, with high-mannose carbohydrate structure. The method
preferably comprises
culturing cells of Pichia pastoris, which cells comprise a DNA molecule which
encodes the
protein of interest, such as glucocerebrosidase or sphingomyelinase, under
conditions suitable for
2 0 the expression of said DNA molecule to produce recombinant protein in a
cell culture, and
purifying said produce purified recombinant human protein from the cell
culture. The
purification can be accomplished~by any suitable conventional means for
isolating protein from
other components of cell cultures, including HPLC, affinity columns, column
chromatography,
gel chromatography.
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One important advantage of the present methods is that, because Pichia
produces proteins
with high-mannose glycosylation, fewer modification steps will be needed in
order to remove
other complex carbohydrates from the recombinant protein in order to expose
high-mannose
moieties. This will simplify the production of recombinant protein, if a high-
marmose
glycosylation product is desired. Such a product may be desirable, for
example, if targeting of
the recombinantly produced protein to macrophages is desired, such as with
certain of the
lysosomal storage enzymes, including glucocerebrosidase and sphingomyelinase.
The present invention provides methods for continuous high cell density
fermentation
system for the production of recombinant human proteins, including Chitinase,
glucocerebrosidase, sphingomyelinase and others, preferably using constitutive
promoters, such
as the GAPDH promoter, in which proteolytic degradation of the product was
reduced or even
undetectable. Among other advantages, the proteins that are produced using the
present system
result in a high mannose carbohydrate moiety. While often a disadvantage, this
glycosylation
pattern is useful for the targeting of certain proteins to macrophages. In
preferred embodiments
of the invention, a continuous fermentation process is employed to produce
recombinant humor
glucocerebrosidase with high mannose content.
Other lysosomal storage disorders, whose associated lysosomal enzymes which
may be
suitable for expression in Piclaia include Pompe's (alpha-glucosidase),
Hurler's (alpha-L
iduronidase), Fabry's (alpha-galactosidase), Hunters (MPS II) (iduronate
sulfatase), Morquio
2 0 Syndrome (MPS IVA)(galactosamine-6-sulfatase), and Maroteux-Lamy (MPS VI)
(arylsulfatase
B). Additional proteins that may be produced in accordance with the present
invention include
lysosomal acid lipase. In addition, any protein for which targeting to the
macrophages is desired
may be a suitable candidate for recombinant expression in Piclaia, for
example, by the
continuous fermentation processes provided by the present invention.
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Thus, in certain embodiments, the present invention comprises methods for the
production of recombinant proteins with high-mannose glycosylation by
expression in a Pichia
cell expression system. The production process is preferably a continuous
fermentation process.
In preferred embodiments, the process utilizes expression vectors comprising a
constitutive
promoter, such as the GAPDH promoter, operably linked to a coding DNA
sequence. The
preferred coding DNA sequences include any therapeutic protein for which
activity and targeting
are not adversely impacted by high-mannose glycosylation. In preferred
embodiments, the
coding DNA sequences comprise a sequence encoding a protein which is desired
to be targeted
to macrophages. In particular, preferred coding DNA sequences include those
sequences
encoding, glucocerebrosidase and acid sphingomyelinase, for the treatment of
patients with
Gaucher's Disease and Niemami-Pick Disease, respectively. Other preferred
coding DNA
sequences include those encoding alpha-glucosidase (Pompe's Disease), alpha-L
iduronidase
(Hurler's Disease), alpha-galactosidase (Fabry Disease), iduronate sulfatase
(Hunters Disease
(MPS II), galactosamine-6-sulfatase (MPS IVA), beta galactosidase (MPS IVB)
and arylsulfatase
B (MPS VI).
BRIEF DESCRIPTION OF THE FIGURES
Figure 1A. rh-Chitinase expression in methanol induced fed-batch culture with
P.
pasto~is (host SMD 1168, His-, vector pPICZoc). A 50% glycerol solution was
fed during
day one (0.3 ml/min). Subsequently, induction with methanol (0.12 mlhnin) was
initiated.
2 0 Figure 1B. SDS-PAGE. La~~e 2 af2d 3: rh-Chitinase standard containing full
length and
cleaved 37 kDa protein (both forms are active). Latae 4-7: supernatant from
fed-batch
culture, days 2-5.
Figure 2A. Constitutive rh-Chitinase expression in fed-batch culture with P.
pasto~is
(host SMD 1168, His , vector pGAPZa). A 50% glycerol solution was fed (0.16
ml/min).
2 5 Figure 2B. SDS-PAGE. Layae 2-5: supernatant from fed-batch culture, days 4-
7.
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Figure 3. Constitutive rh-Chitinase expression in fed-batch culture with P.
pasto~~is (host
SMD 1168, His , vector pGAPZa). A SO% glycerol solution was fed (0.16 ml/min)
containing casamino acids (graph not shown). SDS-PAGE. Lahe 2-S: supernatant
from
fed-batch culture, days 4-7.
Figure 4A. Constitutive rh-Chitinase expression in continuous culture with P,
pasto~is
(host SMD 1168, His-, vector pGAPZa). A 50% glycerol solution was fed (1.0
ml/min;
1.0 VVD).
Figure 4B. SDS-PAGE. Lane 2-8: supernatant from continuous culture, days 2-8.
Figure SA. Constitutive rh-Chitinase expression in continuous culture with P.
pastoris
(host X33, vector pGAPZa). A 30% glucose solution was fed (1.2 ml/min; 1.2
WD).
Culture was run successfully for 30 days.
Figure 5B. SDS-PAGE. Lah.e 2-5: supernatant from continuous culture, day 10-
30.
Figure 6. Glucose limited 1 S L continuous culture of P. pastoris for rh-
Chitinase production (D
= 0.04 h-'; one volume exchange per day, sparged with conventional air).
Figure 7. Comparison of 1.S L continuous culture of P. pastof~is (1.2 volume
exchanges per
day, sparged with molecular oxygen) with a cultivation at 1 S L scale (one
volume exchange per
day, sparged with conventional air).
Figure 8. kLa, OTR, and impeller speed (N) vs. PIVL in a 1,500 L STR (kLa and
OTR measured
with the steady-state method, with: F/VL = 0.6 1/1 min, p = 1.01 bar, pOz = 0
%, T = 30°C). In
2 0 comparison, the operational set point of the 21 L CSTR in terms of kLa and
OTR (steady-state
method) with: F/VL = 1.21/1 min, p = 1.61 bar, pOz = 3S %, T = 30°C.
Figure 9. Model prediction for rh-Chitinase productivity (QP) and oxygen
demand in terms of
OUR and kLa for further increased dilution rates (OUR based on p = 1.61 bar
and pOz = 3S %)
with: YDCwioz = 0.91, YP~cw = 1.4 x 10-3, and YDCwiol"~ose = 0.37.
Figure 10. 1.SL continuous Pichia pastof~is X33 culture for the expression of
rh-GCR
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Figure 11. 15L continuous Pichia pastoris SMD 1168 culture for the expression
of rh-LAL
DETAILED DESCRIPTION OF THE INVENTION
The continuous production processes of the present invention offer, in
comparison to
conventional fed-batch fermentation, advantages in terms of higher volumetric
productivity,
product quality, and product uniformity as the exposure of the product to
proteolytic enzymes,
oxidation or inactivation is significantly reduced. A continuous production
process for rh-
Chitinase using a constitutive P. pasto~is expression system was recently
developed by the
inventors and compared very favorably in terms of cost effectiveness,
development time, and
effort to expression ofrh-Chitinase in mouse C127 cells.
The P. pastof°is production process has an extremely high oxygen demand
due to the high
cell densities obtained in the reactor. The oxygen demand is usually met by
sparging with
molecular oxygen (Chen (1); Chen (2); Siegel et al.) which presents a major
economic and safety
concern, especially at large-scale. Aerobic microbial high cell density
cultures are usually run in
stirred tank reactors (STR) and require the creation of a large airlwater
interface. The formation
of the latter depends mainly on the realizable volume related power input into
the reactor which
is scale-dependent. The present invention provides methods for which air
provides sufficient
oxygen, and molecular oxygen is not needed. These methods have been scaled up
to 15 L, and
can potentially be further augmented for significantly larger scale processes,
of up to 1000 L or
2 0 more.
The present invention further provides processes for large-scale recombinant
protein
production using the constitutive P. pasto~is expression system.
It is known that glycosylation of proteins expressed in Pichia is closer to
that of
mammalian cells compared to other yeasts and microorganisms. However, there
are subtle
differences. If glycosylation is critical to the function of the protein,
e.g., activity and targeting,
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Pichia may not be suitable. However, many of the lysosomal enzymes, and in
particular,
glucocerebrosidase (Gaucher's Disease) and acid splungomyelinase (Niemann-Pick
Disease A &
B), are particularly good candidates for treatment with recombinant protein
produced in PicIZia.
This is because the majority of lysosomal storage enzymes naturally contain
high-mannose
5 oligosaccharides similar to Pichia derived proteins, and they have acidic
optimal pH ranges
which are found in lysosomes. For proteins that are targeted to macrophages by
terminal
mannoses, e.g., glucocerebrosidase and acid sphingomyelinase, the presence of
mannose-6-
phosphate may not be necessary. Pichia is an ideal expression system for
expression of these
proteins, because processing steps which may be necessary for trimming the
carbohydrate chains
10 produced by other expression systems, such as CHO, will not be required to
expose mannose
moieties.
Other lysosomal storage disorders, whose associated lysosomal enzymes which
may be
suitable for expression in Pichia include Pompe's (alpha-glucosidase),
Hurler's (alpha-L
iduronidase), Fabry's (alpha-galactosidase), Hunters (MPS II) (iduronate
sulfatase), Morquio
Syndrome (MPS IVA)(galactosamine-6-sulfatase), MPS IVB (beta-D-galactosidase),
and
Maroteux-Lamy (MPS VI)(arylsulfatase B). Other proteins that may be produced
in accordance
with the present invention include lysosomal acid lipase. There is evidence of
other
independent pathways, in addition to the mannose-6-phosphate pathway, that
function in the
transport of lysomal enzymes inside cells and of alternate mechanisms for the
internalization of
2 0 lysosomal enzymes by cell-surface receptors in addition to mannose-6-
phosphate receptors
(Scriver et al. 1995). In addition, any protein for which targeting to the
macrophages is desired
may be a suitable candidate for recombinant expression in Pichia, for example,
by the
continuous fermentation processes provided by the present invention.
T Thus, in certain embodiments, the present invention comprises methods for
the
2 5 production of recombinant proteins with high-mannose glycosylation by
expression in a Piclaia
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11
cell expression system. The production process is preferably a continuous
fermentation process.
In preferred embodiments, the process utilizes expression vectors comprising a
constitutive
promoter, such as the GAPDH promoter, operably linked to a coding DNA
sequence. Other
promoters of potential use in the present invention include constitutive
promoters, such as the
CMV promoter, the adenoviral major late promoter, and ubiquitin promoters, as
well as
inducible promoters, such as the alcohol oxidase promoter (Elks et al., Mol.
Cell. Biol. 9:1316-
1323 (1985)); and the tetracycline inducible promoter system. The preferred
coding DNA
sequences include any therapeutic protein for which activity and targeting are
not adversely
impacted by high-mannose glycosylation. In preferred embodiments, the coding
DNA sequences
comprise a sequence encoding a protein which is desired to be targeted to
macrophages. In
particular, preferred coding DNA sequences include those sequences encoding,
glucocerebrosidase and acid sphingomyelinase, for the treatment of patients
with Gaucher's
Disease and Niemann-Pick Disease, respectively. Other preferred coding DNA
sequences
include those encoding alpha-glucosidase (Pompe's Disease), alpha-L
iduronidase (Hurler's
Disease), alpha-galactosidase (Fabry's Disease), and iduronate sulfatase
(Hunters Disease (MPS
II), galactosamine-6-sulfatase (MPS IVA); beta-D-galactosidase (MPS IVB); and
arylsulfatase
B (MPS VI). In addition, a cDNA for any protein for which targeting to the
macrophages is
desired may be a suitable candidate for recombinant expression in Pichia, for
example, by the
continuous fermentation processes provided by the present invention.
2 0 Methods for the purification of recombinant human proteins are well-known,
including
methods for the production of recombinant human glucocerebrosidase (for
Gaucher's Disease);
sphingomyelinase (for Niemann-Pick Disease), alpha-galactosidase (for Fabry
Disease); alpha-
glucosidase (for Pompe's Disease); alpha-L iduronidase (for Hurler's
Syndrome); iduronate
sulfatase (for Hunter's Syndrome); galactosamine-6-sulfatase (for MPS IVA);
beta-D-
galactosidase (for MPS IVB); and arylsulfatase B (for MPS VI). See, for
example, Scriver et al.,
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eds., The Metabolic and Molecular Bases of Inherited Diseases, Vol. IL, 7"'
ed. (McGraw-Hill,
NY; 1995), the disclosure of which is hereby incorporated herein by reference.
While the invention is exemplified with respect to the production of specific
proteins,
these examples are not to be interpreted as limiting the invention in any
manner. As described
above, and as will be clear to those skilled in the art from reading the
specification, the methods
of the present invention are useful for production of numerous other
recombinant proteins,
including the lysosomal enzymes described above. Many modifications and
variations of the
methods and materials used in the present description will also be apparent to
those skilled in the
art. Such modifications and variations fall within the scope of the invention.
1 o The entire disclosures of all of the publications and references cited in
this specification
are hereby incorporated herein by reference.
EXAMPLES
Example 1. Cloning and Selection of the human Chitinase (hChitinase) Gene in
P. pasto~~is
a. Vector Construction
The hChitinase cDNA was received from Johannes Aerts, University of Amsterdam,
NL
(WO 9640940) and used as a template for all PCR reactions. The coding region
of hChitinase
without the secretion signal peptide and containing Eco RI sites at the 5' and
3' ends was
generated by PCR and inserted into Eco RI linearized pPICZoc and pGAPZoc,
which contain the
S. cef°evisiae ec-factor secretion signal. The coding region of
hChitinase with it's secretion signal
2 0 peptide and Eco RI sites at the 5' and 3' ends was generated by PCR and
inserted into Eco RI
linearized pGAPZcc. All vectors were obtained from Invitrogen(San Diego, CA).
h. Transformation
P. pastoris cells were made competent and transformed by electroporation as
previously
described (Becker et al., 1991) with slight modifications. P. pastof°is
strains X33 and SMD1168
2 5 (Invitrogen) were grown to OD6oo of 0.5-0. 8. in a 50 ml culture, pelleted
and resuspended in 10
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13
ml ice-cold 100 mM Tris, 10 mM EDTA buffer with 200 mM DTT (Sigma), and
incubated for
15 minutes at 30°C with shaking at 100 rpm. Cells were then washed 2x
with ice-cold sterile
water and lx with 1 M sorbitol (Invitrogen) and resuspended in 100 p.1 1 M
sorbitol to a final
volume of X200 ~.1. 80 ~l competent cells were electroporated with 2-6 p,g DNA
in 0.2 cm
cuvettes at 1500 V, 25 ~F and 200 S2 using a BioRad Gene Pulser with pulse
controller.
Immediately after pulsing, 1 ml of ice-cold sorbitol was added to the cuvette.
Cells were allowed
to recover overnight at room temperature, then plated (20-100 ~,l cells per
plate) directly on YPD
(Yeast Extract, Potato, Dextrose medium, Invitrogen) agar containing differing
amounts of
zeocin (Invitrogen) for selection. Plates were incubated at 30°C.
Resistant colonies appeared
after 2 days on 0.1-0.5 mg/ml zeocin and after 3-4 days on 1-2 mg/ml zeocin.
c. Selection of high producers
Several hundred clones that survived higher titers (0.5-2 mg/ml) of zeocin
were screened
in test tubes as follows. A single colony was inoculated into 5 ml of YPD in
50 ml conical
centrifuge tubes and incubated for 24 hours at 30°C with shaking at 250
rpm. Cell density was
measured by OD6oo and a fresh 5 ml YPD was inoculated with 2.5 x 106 cells and
incubated as
above. This process was repeated as necessary until cells from each clone
being analyzed were
synchronized in growth. Typically two or three passages were sufficient. Once
synchronized,
cells were grown for 60 hours as above. Aliquots of culture (50 ~I) were
aseptically removed at
24, 48 and 60 hours and conditioned medium was harvested and analyzed by the
pNP(Sigma)
2 0 activity assay as described below to identify top producers.
d. 1.5-L Fed-batch Fermentation
A shake flask containing 100 ml of YPD medium was inoculated with one vial (~1
ml,
OD6oo-25) containing a recombinant P. pastor°is cell line. The flask
was incubated at 30°C and
220 miri' for 16-24 hours, until the cell density reached OD6oo >15. YPD
medium (pH = 6.0)
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14
used in shake flask cultivation consisted of (per liter deionized water): D-
glucose 20 g, soy
peptone 20 g, yeast extract 10 g, yeast nitrogen base (w/o amino acids) 13.4
g, KH2P04 11.8 g,
K2HP04 2.3 g, D-biotin 0.4 mg.
The cells from this flask were used to inoculate a 3.0-L fermenter (Applikon,
Foster City,
CA) with a 1.5-L working volume at a density of 1.0 to 2.0 OD6oo units. The
fermenter contained
Basal Salts Medium plus 2 g/L Histidine for His strains. Basal Salts Medium
used for fermenter
batch cultivation contained (per Iiter deionized water): Glucose 40 g, H3P04
(85%) 26.7 ml,
K2S04 18.2 g, MgS04 ~ 7H20 14.9 g, KOH 4.13 g, CaS04 ~ 2H20 0.93 g, D-biotin
0.87 mg,
trace salts solution 4.35 ml; (trace salts solution(per liter deionized
water): Fe2(S04) ~ 7H20 65
g, ZnS04 42.19 g, CuS04 ~ SH20 6 g, MnS04 ~ H20 3 g, CoCl2 ~ 6H2O 0.5 g,
Na2Mo04
2H20 0:2 g, NaI 0.08 g, H3B03 0.02 g).
The cells were grown batchwise until the initial glucose was depleted (~24
hours) and the
wet cell weight (WCW) was ~80-100 g/L. When the initial glucose was depleted
as indicated by
a dissolved oxygen (pO2) spike, fed-batch fermentation was initiated by
starting the fed-batch
medium at a rate of 0.13-0.20 mL/L initial medium volume. The fed-batch medium
consisted of
(per liter deionized wafer): D-glucose 500 g, D-biotin 2.4 mg, trace salts
solution 12 mL, and
casamino acids 10 g (in circumstances when such use is mentioned in present
description of
production of specified proteins).
Fed-batch fermentation was continued until activity had plateaued (~5-7 days).
Samples
2 0 were taken daily for WCW and cell density by OD6oo. Supernatant was
obtained by
centrifugation at 4-6,000 g for 25 min. at 4°C and stored at -
20°C until assayed.
e. 1.5-L Continuous Fermentation
After the fed-batch fermentation had been established (see above), and allowed
to
continue for approximately 24 hours (WCW 200-220 g/L), continuous fermentation
was
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initiated at a rate of 0.7-0.8 Volumes/Working Volume/Day (VVD). The
continuous feed
medium (pH = 1.3) contained (per liter deionized water): D-glucose 300 g,
H3P04 (85%) 13.35
ml, K2S04 9.1 g, MgS04 ~ 7H20 7.45 g, KOH 2.07 g, CaS04 ~ 2H20 0.47 g, D-
biotin 0.87 mg,
and trace salts solution 4.35 ml.
5 After ~24 hours of continuous culture, the continuous flow rate was
increased to ~1.0-1.2
VVD, or ~0.7-0.85 ml/min/L working volume. Flow rate was maintained in this
range for the
duration of the run. Samples were taken daily for WCW and cell density by
OD6oo. Supernatant
was obtained and stored at -20°C for recombinant protein concentration
measurements.
The continuous outflow of culture was harvested daily and supernatant was
obtained by
10 centrifugation at 4-6,000 g for 25 min. at 4°C and stored at -
20°C until assayed.
f. rhChitinase Activity Assay
Crude supernatant (1:100 to 1:1000) or pNP standard (Sigma) (0-20 nM/well)
were
diluted in assay buffer pH 5.2. 100 p1 of standards and diluted crude
supernatant were placed
into duplicate wells in a 96 well microtiter plate. One hundred p1 of
substrate, 0.25 mg/ml pNP-
15 ~3-N,N'-diacetylchitobiose (Sigma), was then added to each well and the
plate incubated at 37°C
with shaking at 50 rpm. After 2 hours, 50 p1 of 1.0 N NaOH was added to each
well and the
absorbance at 405 nm against 650 nm(reference) was read using a microtiter
plate reader.
Activity was determined using a pNP standard curve. A specific
activity(determined using
purified material at Genzyme) of 1.67 U/mg was used to convert activity units
[LT/ml] to protein
units [mg/ml].
g. SDS Page and Gel Staining
About 10 ~1 supernatant of each sample (2-4 ~g protein) was mixed with 20 ~.l
Sx SDS
non-reducing sample loading buffer (BioRad, CA) and 30 ~,l was subjected to
electrophoresis on
4-20% Tris Glycine acrylamide mini-gels (Ready Gel, BioRad, CA) in tris-
glycine-SDS running
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Z6
buffer (BioRad, CA). Gels were stained with Coomassie-blue staining reagent
(BioRad, CA) for
about one hour, then destained with 40% methanol / I O% acetic acid for one
hour.
h. Results and Discussion
i. Fed Batch Production of rh-Chitinase by a Methanol-Inducible clone (pPICZa-
SMD 1168) Cell yield measured by WCW of the culture plateaued after 2 days at
200 g/L while
activity increased slowly through day 5. Final rh-Chitinase concentration in
the culture broth
reached a moderate level of 300 mglL (Fig. 1 a). However, degradation of the
rh-Chitinase was
evident on day 4 when time samples were analyzed on SDS page gel (Fig. 1b).
Two distinct
bands can be seen in samples collected on the 5'" day. These data may explain
why the rh
Chitinase activity only increased slowly with time under fed-batch mode.
ii. Fed Batch Production of rh-Chitinase by a Constitutive Clone (pGAPZa-SMD
1168) When pGAPZa-SMD 1168 was grown under fed-batch conditions, cell yield
reached 330
g/L WCW and a rh-Chitinase concentration of 450 mg/L was attained (Fig. 2a). A
similar
degradation pattern was seen with the recombinant protein. A second lower MW
band began to
appear after 6 days and the band became more prominent on day 7, suggesting
proteolytic
degradation (Fig. 2b).
iii. Protection of Enzyme from Proteolytic degradation by Casamino Acids
Supplementation
Casamino acids have been shown to protect proteins from proteolytic
degradation when
2 0 added to cultures. They were included in the fed-batch feed medium and
samples (4,5,6 &
7days) were collected and analyzed by SDS PAGE. A tight band at around 50 kDa
in each one
of the samples analyzed suggests intact rh-Chitinase(Fig. 3). This can be
compared to samples
from a fed batch fermentation without casimino acids which showed a low MW
band on day
6(Fig. 2b). These data suggests that rh-Chitinase was most likely degraded by
proteolytic
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17
enzymes under fed batch conditions and that rh-Chitinase can be stabilized by
addition of
casimino acids.
Athough casimino acids appeared to be effective in preventing proteolytic
degradation of
rh-Chitinase in the fermentation broth, this method may not be ideal for
production because of
the animal origin of such casimino acids.
iv. Stabilization of rh-Chitinase by Continuous Fermentation
Figure 4a shows rh-Chitinase and growth data of the constitutive clone (pGAPZa-
SMD
1168) in a continuous mode. Medium was exchanged at a rate of 1.0 VVD. The
culture reached
steady-state on day 2 of continuous mode and rh-Chitinase was produced at a
volumetric
productivity of 180 mg/L/d. The fermentation was continued for 26 days and
samples from day
2 through 8 were analyzed on SDS PAGE. The gel shows a single rh-Chitinase
band (~50 kDa)
in all samples (Fig. 4b) indicating that continuous fermentation can prevent
degradation of rh-
Chitinase for at least up to 8 days. It appears that little or no proteolytic
enzymes) is produced
and released by the culture into the medium under continuous cultivation. It
is also possible that
when the protein is harvested continuously, it is exposed to less concentrated
proteolytic
enzymes for a much shorter time period compared to rh-Chitinase production
under fed batch
conditions. SDS PAGE of samples after day 8 were not performed because the
onset of protease
typically occurred much before day 8.
v. pGAPZa-X33 Clone
2 0 The highest producing clone was created when the X33 host was used for the
transformation. This clone was grown in the continuous mode with an initial
dilution rate of 0.8
VVD. The feeding rate was ramped up slowly to 1.2 VVD on day 6 (fig. 5a). rh-
Chitinase
concentration increased steadily from 50 mg/L to 300 mg/L within a period of 8
days. Cell yield
plateaued on day 5 0400 g/L WCW) and rh-Chitinase concentration plateaued on
day 9 0300
mg/L). The culture was continuously fed with 30% glucose feed medium, as
discussed in the
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18
present description of the invention, at a rate of 1.2 WD for an additional 24
days. The cell
yield and rh-Chitinase volumetric productivity remained steady at 400g/L WCW
and 360 mg/Ld,
respectively. As far as we know, this is the first report describing a P.
pasto~is high cell density
fermentation continuing for 30 days. The culture showed no signs of decline,
both in cell and
product yields at run termination. SDS PAGE analysis of samples indicated that
the product was
not degraded even on the 30"' day of the fermentation (Fig. 5b). We have since
cloned two other
therapeutic proteins (one antiangiogenesis protein & one lysosomal enzyme)
with the GAP
promoter and produced them using continuous conditions. Both recombinant
proteins, which
normally were digested by proteases under fed-batch conditions, were not
degraded.
Conclusions
A process for the cultivation of P. pasto~is in continuous fermentation using
the
constitutive GAP promoter for the production of recombinant proteins has been
developed. To
our knowledge this is the first use of a continuous high cell density
fermentation process
employing the constitutive expression vector (pGAPZa) in P. pasto~is. Also,
the constitutive
expression system allows for the safe handling of the P. pasto~~is production
system, especially in
large scale, avoiding the use of methanol, which is flammable. This would
greatly reduce the
hazard and costs involved with large scale production of recombinant
therapeutic proteins in P.
pastoris by alleviating the need fox explosion proof GMP facilities.
This continuous system provides not only for greatly enhanced production of
2 0 recombinant proteins and reduction of down-time associated with fermentor
turn around
(approximately 6 fold higher productivity than fed-batch fermentation) but
also for the
production of intact proteins that are usually degraded in a fed-batch mode.
This may be due to
the continual separation of sensitive proteins from the culture broth. It is
believed that this
continuous Pichia expression system, employing the GAP promoter, is applicable
to a wide range
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19
of proteins which previously could not be produced in methylotrophic Pichia
due to proteolytic
degradation and/or economic reasons.
EXAMPLE 2. Scale-up of a High Cell Density Continuous Culture
with Pichia pastoris X-33 for the Constitutive Expression of rh-Chitinase
List of
symbols
cDCw ' g/1 dry cell weight concentration
cP g/1 product concentration in the supernatant
coz,r. g/1 dissolved oxygen concentration
c*oz,L g/1 oxygen solubility
CER g/1 h carbon dioxide evolution rate
d m impeller diameter
D h-' dilution rate
FN 1/min air flow rate (under standard conditions)
F/VL Ul min volume related aeration rate
He g/1 bar Henry constant
kLa h-I volume related oxygen transfer coefficient
MW g/mol molecular weight
N miri' impeller speed
2 OTR g/1 h oxygen transfer rate
0
OUR g/1 h oxygen uptake rate
p bar pressure
pN bar pressure (under standard conditions)
pOz % oxygen partial pressure in the liquid
phase
2 P/VL kW/m3 volume related power input
5
qOz h-1 specific oxygen uptake rate (g Oz /
g DCW h)
QP _ g/1 d volumetric productivity
r % percentage of solids in fluid
R bar 1/K gas constant
mol
3 RQ 1 respiratory quotient
0
TN K temperature (under standard conditions)
U ~M/min enzyme activity
VL 1 reactor liquid volume
xoz,~~ 1 mol fraction of Oz in inlet gas
3 xcoz,~a 1 mol fraction of COz in inlet gas
5
xoz,ouc 1 mol fraction of Oz in exhaust gas
xcoz,ou~ 1 mol fraction of COz in exhaust gas
Yocwioz yield coefficient (biomass formed / oxygen
1 consumed)
YP,~cw 1 yield coefficient (rh-Chitinase produced
/ biomass formed)
40
Greek
symbols
r~ Pas shear viscosity
h-' specific growth rate
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Introduction:
The feasibility of large-scale production of recombinant human chitinase using
a
constitutive Pichia pastor~is expression system was demonstrated in a 21 L
continuous stirred
tank reactor (CSTR). A steady-state recombinant protein concentration in the
supernatant of 250
5 mg/1 was sustained for one month at a dilution rate of D = 0.04 h-'
(equivalent to one volume
exchange per day), enabling a volumetric productivity of 144 rng/1 d (240 U/1
d). The steady-
state dry cell weight concentration in this high cell density culture reached
110 g/1. Considering
safety and economical aspects, all large-scale cultivations were conducted
without molecular
oxygen supplementation. Conventional air sparging was used instead. The oxygen
demand of the
10 process was determined by off gas analysis (OUR = 4.8 g Oz 1-' h-' with kLa
= 846. h-1) and
evaluated with regard to further reactor scale-up.
A. Microorganism and conditions of cultivation
Cultivations were carried out with the yeast Pichia pastor°is X-33,
wild type strain His+
(Invitrogen, San Diego, CA) using a pGapZa vector for constitutive expression
of rh-Chitinase.
15 Vials with 1 ml of frozen working stock of recombinant P. pastor°is
were stored at -80 °C and
used as inoculum for 2 L shake flask cultivations with 500 ml YPD medium. Two
2 L shake
flasks with 500 ml YPD medium were inoculated and incubated for ~24 h at
30°C on a orbital
shaker at 220 mini' until cell density reached OD6oo >50. YPD medium (pH =
6.0) used in shake
flask cultivations consisted of (per liter deionized water): D-glucose 20 g,
soy peptone (Type IV,
2 0 Sigma, MO) 20 g, yeast extract (HyYest 444, Quest, IL) 10 g, yeast
nitrogen base (w/o amino
acids) (Difco, MI) 13.4 g, KH2PO4 11.8 g, K2HP04 2.3 g, D-biotin 0.4 mg. Each
bioreactor
cultivation was seeded with the contents of two 2L shake flask cultures,
equivalent to 6.6 % v/v
culture suspension.
Bioreactor cultivations were performed in a 21 L stirred tank reactor (STR)
with 15 L
2 5 working volume (CF 3000, Chemap AG, Switzerland) and a height/diameter
ratio of 2Ø The
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21
reactor had four baffles and three Rushton impellers (d = 0.075 m) installed
on the shaft at 1/4,
1/2, and 3/4 of the liquid level. Cultivation parameters were set to T =
30°C, pH = 5.0 [adjusted
with NH40H (30 %) and H3P04 (40 %)], N = 600 - 1,000 miri', F/V = 1.0 - 2.0
1/1 min, p =
1.01 - 1.61 bar, and p02 >_ 20 %. The medium used for bioreactor batch
cultivations contained
(per liter deionized water): D-glucose 40 g; H3P04 (85%) 26.7 ml; K2S04 18.2
g;
MgS04~7H20 14.9 g; KOH 4.13 g; CaS04~2H20 0.93 g; D-biotin 0.87 mg; and trace
salts
solution 4.35 ml. Trace salts solution consisted of (per liter deionized
water): Fe2(S04)~7H20
65 g; ZnS04 42.19 g; CuSOq.~5H20 6 g; MnS04~H20 3 g; C~C12~6H20 0.5 g;
Na2Mo04~2H20 0.2 g; NaI 0.08 g; and H3B03 0.02 g. After ~24 h cultivation
time, the OD6oo
reached ~150 and the initial glucose was depleted. 4.5 L fed-batch medium in a
5 L bottle were
fed into the reactor at a feed rate of 3 ml/min. The fed-batch medium (pH 7.0)
consisted of (per
liter deionized water): D-glucose 500 g and D-biotin 2.4 mg. After ~24 h of
fed-batch mode, an
ODboo of ~450 was attained and the reactor was switched to continuous mode at
a medium feed
rate of 7 mllmin, which was increased to <_ 11 ml/min after 24 h. The feed
medium (pH = 1.3)
used for continuous cultivation contained (per liter deionized water): D-
glucose 300 g, H3P04
(85%) 13.35 ml, K2S04 9.1 g, MgS04~7H20 7.45 g, KOH 2.07 g, CaS04~2H20 0.47 g,
D-
biotin 0.87 mg, and trace salts solution 4.35 ml. Glucose limitation of the
culture was
maintained and monitored during fed-batch and continuous mode of operation.
Steady-state
condition was usually reached after 3-5 volume exchanges. The media used in 2
L shake flasks, 5
L bottles, and a 21 L bioreactor was sterilized for 30 min at 121°C.
The feed medium used for
continuous production was filter-sterilized into 200 L plastic bags. The
harvest was continuously
pumped into a sterile 130 L plastic bag which was placed in a 150 L chilled
vessel (4°C). Twice
a week, the harvest was filled into 1 L plastic bottles and centrifuged
(Sorvall RC3C, DuPont
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22
Corp.) at 4,500 miri' (5,900 g) for 45 min at 4°C. The supernatant was
concentrated by cross
flow filtration (Pellicon, Millipore; membrane cut-off: 10 kDa) and frozen at -
80°C for further
protein purification.
B. Analysis
OD was measured at 660 nm (spectrophotometer model 8452A, Hewlett Packard).
Wet
cell weight (WCW) was determined gravimetrically after centrifugation (Allegra
21R, Beckman)
of 50 ml cell suspension at 6,500 miri' (4,400 g) for 25 min at 4°C.
Dry cell weight (DCW)
estimation included washing of the pellet and drying of the sample at
40°C for 72 h. The D-
glucose concentration was measured with an Accu-Chek glucose analyzer
(Boehringer
Mannheim, Germany) for confirmation of glucose limitation in the CSTR. Exhaust
analysis of
carbon dioxide, oxygen, nitrogen, and water was measured by mass spectrometer
(MGA 1600,
Perkin-Elmer, USA). Carbon dioxide evolution rate (CER), oxygen uptake rate
(OUR), and
respiratory quotient (RQ) were evaluated from the gas phase material balance.
Bioreactor and
cultivation parameters such as N, T, pH, and p02 were documented via chart
recorder
(Yokogawa). Power input was determined by measurement of electrical voltage
and current at
the armature of the motor. Friction losses were subtracted.
C. rh-Chitinase activity assay
rh-Chitinase activity in the supernatant was determined via enzymatic essay.
Crude
supernatant or pNP (p-Nitrophenol) standard were diluted in assay buffer (0.02
% NaAzide, pH
2 0 5.2). 100 ~.1 of standards and diluted crude supernatant were placed into
duplicate wells in a 96
well microtiter plate. 100 ~1 of substrate (0.25 mg/ml pNP-[3-N,N'-
diacetylchitobiose) was then
added to each well and the plate was incubated for two hours in the dark at
37°C with shaking at
50 rpm. After two hours, 50 ~,l of 1.0 N NaOH was added to each well and the
absorbance at 405
nm to 650 nm reference was measured using a microtiter plate reader (340 ATTC,
SLT,
2 5 Salzburg). Activity was determined via pNP standard curve. A specific
activity of 1.67 U/mg
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23
was used to convert activity units [Ulml] to protein units [mg/ml] (determined
using purified
material at Genzyme).
D. Calculation of rh-Chitinase productivity
A change in the biomass concentration in the CSTR can be described by:
dcDCw / dt = ~ cDCw - D cDCw (1)
Steady-state condition of the continuous culture was reached when dcDCw / dt -
0 and
consequently ~ = D. With cP as the steady-state rh-Chitinase concentration in
the supernatant
and r as the percentage of solids in the reactor fluid, the rh-Chitinase
productivity can be
calculated as:
QP = cP D [1 - (r / 100)] (2)
The oxygen consumption utilized for biomass formation can be described as:
dcDCw / dt = OUR YDCwioz (3)
rh-Chitinase production is assumed to be associated with growth:
dcP / dt = YP~cw * dc~cw / dt (4)
E. Calculation of oxygen demand of process
The oxygen demand of the process in terms of kLa and oxygen transfer rate
(OTR) can be
estimated as follows. A change in the dissolved oxygen concentration in the
reactor can be
expressed as:
dcoz,L / dt = OTR - OUR = kLa (c*oz,L - coz,L) - O~ (5)
2 0 In a small time interval, dcoz,L / dt = 0 and consequently
OTR = kLa (c*oz,L - coz,L) = OUR (6)
The oxygen uptake rate (OUR) can be determined via oxygen mass balance derived
from exhaust
analysis:
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24
N N ~
F p xOZ,in MW02 x02,out (1 - x02,in - xC02,inJ
OUR = [1 - ] (7)
N N
VL R T x02,in (1 - x02,out - xC02,out)
Assuming an ideally mixed gas phase in the reactor, with
*
c oz,l. = xoz,oat p / He (8)
and coZ,L = c*oz,L pOz / 100 (9)
the kLa can be calculated (steady-state method).
A correlation for kLa using the parameters power input per volume (P/VL),
volume related
aeration rate (F/VL), and viscosity (r~) can be described as:
kLa = C (P/VL)° (F/VL)b (,0)d (10)
P/VL = idem. is .a common scale-up strategy to secure an equal oxygen supply
in the larger
reactor (to attain kLa = idem. and OTR = idem.). Assuming geometrical
similarity of the larger
reactor, turbulent flow, F/VL = idem., and r~ = idem.:
P ~ N3 d5 and P/VL ~ N3 d2 (11)
With P/VL = idem.: Nlarse / Nsmau _ (dsman / dlar~e) Z/3 (12)
The necessary impeller speed to secure an equal oxygen supply in the larger
reactor can be
calculated as shown in Eq. (13):
_ ~/3
Nlarge Nsmall (dsmall / dlarge) ~ (13)
Example 3: rh-LAL (lysosomal acid lipase
2 0 rh-LAL was expressed in a 15L continuous culture with Pichia pasto~is SMD
1168
(auxotrophic: His ) using the constitutive GAPDH promoter. Under steady-state
conditions and a
volumetric turnover rate of 1.0 VVD, an average LAL activity in the
supernatant of 25,000
[nM/ml h] was attained with an average WCW of 350 [g/1]. All culture
conditions were identical
compared to 15L rh-Chitinase production (e.g. pH at 5.0, DOT controlled at 30%
by air
2 5 sparging).
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It was found that a 75% reduction in TMS (compared to 'Pichia Fermentation
Guidelines'
by Invitrogen, CA) combined with a lower VVD of 1.0 could suppress protease
activity in the
medium and protect the LAL-product.
LAL activity was measured via fluorometric assay (similar assay published by:
5 Grabowski, J Biol Chem, 270, 27766 (1995)).
Example 4: Pichia Expression of rh-GCR I~GlucocerebrosidaseZ
rh-GCR was expressed in a 1.5L continuous culture with PicIZia pastof~is X33
(prototrophic strain) using the constitutive GAP promoter. Under steady-state
conditions, a
maximum volumetric productivity (VPR) of 466 [U/L day] Was attained at a
volumetric turnover
10 rate of 1.2 [volume/volume day] (WD). GCR activity in the supernatant was
388 [U/L] and the
wet cell weight (WCV~ was 388 [g/1]. All culture conditions were identical
compared to 1.5L
rh-Chitinase production (e.g. pH at 5.0, dissolved oxygen tension (DOT)
controlled at 30% by
oxygen sparging).
A 23% increase in VPR (from 380 [U/L day] to 466 [U/L day]) was achieved when
the
15 trace metal solution (TMS) in the medium was reduced by 50% on day 28
(compared to rh-
Chitinase process which was based on the 'Pichia Fermentation Guidelines' by
Invitrogen, CA).
It was assumed that trace metals may catalyze GCR degradation. The GCR-CHO
process adds
DTT to the medium to protect the GCR from oxidation. The GCR-Pichia process
did not need
the addition of DTT, as GCR is not being oxidized at pH 5Ø
2 0 GCR activity can be tested according to conventional assays, such as the
PNP-Beta-D-
Glucopyranoside activity assay.
