EP1409695A2 - Systeme d'expression de proteines recombinees - Google Patents

Systeme d'expression de proteines recombinees

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Publication number
EP1409695A2
EP1409695A2 EP01992338A EP01992338A EP1409695A2 EP 1409695 A2 EP1409695 A2 EP 1409695A2 EP 01992338 A EP01992338 A EP 01992338A EP 01992338 A EP01992338 A EP 01992338A EP 1409695 A2 EP1409695 A2 EP 1409695A2
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EP
European Patent Office
Prior art keywords
dna molecule
cells
glucocerebrosidase
expression
sphingomyelinase
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German (de)
English (en)
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Nick Wan
Henry Hoppe, Iv
Jason C. Goodrick
Bernhard M. Schilling
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Genzyme Corp
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Genzyme Corp
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Publication of EP1409695A2 publication Critical patent/EP1409695A2/fr
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • Pichia pastoris 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. pastoris 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. pastoris 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. pastoris is inexpensive and free of toxins or pyrogens. Furthermore, the yeast itself does not present problems in terms of endotoxin production or viral contamination.
  • Pichia 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 Saccharomyces in that they do not tend to hyperglycosylate proteins (oligosaccharide chains of 8-14 mannose) (Grinna & Tschopp 1989) and the highly immunogenic ⁇ l,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.
  • 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 continuous or continuous perfusion fermentation with limited success (Brierley et al.; Chen et al. (2); Cregg; Digan et al., 1989).
  • constitutive promoters e.g., Glyceraldehyde-3- phosphate Dehydrogenase, GAP
  • GAP Glyceraldehyde-3- phosphate Dehydrogenase
  • glucose can be chosen as an inexpensive and efficient carbon source.
  • P. pastoris 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 al.(1994)).
  • P. pastoris was recently reported to have successfully used the P. pastoris expression system for the production of the proteins Angiostatin® and EndostatinTM (Wells (1998)). Proteins produced by P. pastoris are usually folded correctly and secreted into the medium, facilitating the subsequent downstream processing. P. pastoris has further been proven to be capable 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)).
  • the continuous production mode offers, in comparison to fed-batch fermentation, advantages in terms 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. pastoris expression system was recently developed by the inventors and compared very favorably in terms of cost effectiveness, development time, and effort to expression of rh-Chitinase in mouse C127 cells.
  • P. pastoris 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. Lvsosomal Storage Diseases
  • LSDs lysosomal storage diseases
  • a compromised lysosomal hydrolase a lysosomal hydrolase
  • the activity of a single lysosomal hydrolytic enzyme is reduced or lacking altogether, usually due to inheritance of an autosomal recessive mutation.
  • 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 common among three recognized clinical types and follows a chronic course 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 Ashkenazim (see generally Kolodny et al., 1998, "Storage Diseases of the Reticuloendothelial System", In: Nathan and Oski's Hematology of Infancy and Childhood, 5th ed., vol. 2, David G.
  • 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.
  • 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.
  • 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, 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.
  • 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 of Pichia pastoris which cells 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 pastoris.
  • the DNA molecules for use in the present invention preferably comprise a constitutive promoter operatively linked to the coding sequence of interest.
  • a constitutive promoter is the GAPDH promoter from yeast.
  • the present invention also provides methods for the purification of recombinant proteins, such as recombinant 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 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.
  • 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-mannose glycosylation product is desired.
  • 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.
  • constitutive promoters such as the GAPDH promoter
  • 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 INA)(galactosamine-6-sulfatase), and Maroteux-Lamy (MPS NI) (arylsulfatase B). Additional proteins that may be produced in accordance with the present invention include lysosomal acid lipase.
  • 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.
  • 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.
  • 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.
  • the coding DNA sequences comprise a sequence encoding a protein which is desired to be targeted to macrophages.
  • 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.
  • 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 INA), beta galactosidase (MPS INB) and arylsulfatase B (MPS NI).
  • FIG 1A rh-Chitinase expression in methanol induced fed-batch culture with P. pastoris (host SMD 1168, His " , vector pPICZ ⁇ ). A 50% 0 glycerol solution was fed during day one (0.3 ml/min). Subsequently, induction with methanol (0J2 ml/min) was initiated.
  • Figure IB SDS-PAGE. Lane 2 and 3: rh-Chitinase standard containing full length and cleaved 37 kDa protein (both forms are active). Lane 4-7: supernatant from fed-batch culture, days 2-5.
  • FIG. 2A Constitutive rh-Chitinase expression in fed-batch culture with P. pastoris (host SMD 1168, His " , vector pGAPZ ). A 50% glycerol solution was fed (0J6 ml/min).
  • Figure 2B SDS-PAGE. Lane 2-5: supernatant from fed-batch culture, days 4-7.
  • Figure 3. Constitutive rh-Chitinase expression in fed-batch culture with P. pastoris (host
  • FIG. 4B SDS-PAGE. Lane 2-8: supernatant from continuous culture, days 2-8.
  • FIG. 5A Constitutive rh-Chitinase expression in continuous culture with P. pastoris (host X33, vector pGAPZ ⁇ ). A 30% glucose solution was fed (1.2 ml/min; 1.2 WD).
  • FIG. 5B SDS-PAGE. Lane 2-5: supernatant from continuous culture, day 10-30.
  • 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. pastoris expression system was recently developed by the inventors and compared very favorably in terms of cost effectiveness, development time, and effort to expression of rh-Chitinase in mouse C127 cells.
  • the P. pastoris 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 air/water 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 more.
  • the present invention further provides processes for large-scale recombinant protein production using the constitutive P. pastoris expression system.
  • 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, Pichia may not be suitable. However, many of the lysosomal enzymes, and in particular, glucocerebrosidase (Gaucher's Disease) and acid spliingomyelinase (Niemann-Pick Disease A & B), are particularly good candidates for treatment with recombinant protein produced in Pichia.
  • lysosomal storage enzymes naturally contain high-mannose oligosaccharides similar to Pichia derived proteins, and they have acidic optimal pH ranges which are found in lysosomes.
  • 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 produced by otlier expression systems, such as CHO, will not be required to expose mannose moieties.
  • Otlier 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 INA)(galactosamine-6-sulfatase), MPS INB (beta-D-galactosidase), and
  • Other proteins that may be produced in accordance with the present invention include lysosomal acid lipase.
  • 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 lysosomal enzymes by cell-surface receptors in addition to mannose-6-phosphate receptors
  • 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.
  • 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.
  • the process utilizes expression vectors comprising a constitutive promoter, such as the GAPDH promoter, operably linked to a coding DNA sequence.
  • constitutive promoters such as the CMN promoter, the adenoviral major late promoter, and ubiquitin promoters, as well as inducible promoters, such as the alcohol oxidase promoter (Ellis et al., Mol. Cell. Biol.
  • the preferred coding D ⁇ A sequences include any therapeutic protein for which activity and targeting are not adversely impacted by high-mannose glycosylation.
  • the coding D ⁇ A sequences comprise a sequence encoding a protein which is desired to be targeted to macrophages.
  • preferred coding D ⁇ A sequences include those sequences encoding, glucocerebrosidase and acid sphingomyelinase, for the treatment of patients with Gaucher's Disease and ⁇ iemann-Pick Disease, respectively.
  • D ⁇ A 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 INA); beta-D-galactosidase (MPS INB); and arylsulfatase B (MPS NI).
  • a cD ⁇ A 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.
  • 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 ⁇ iemann-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 INA); beta-D- galactosidase (for MPS INB); and arylsulfatase B (for MPS NI).
  • P. pastoris cells were made competent and transformed by electroporation as previously described (Becker et al., 1991) with slight modifications.
  • P. pastoris strains X33 and SMD1168 (Invitrogen) were grown to OD 600 of 0.5-0.8. in a 50 ml culture, pelleted and resuspended in 10 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.
  • test tubes 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 OD 600 and a fresh 5 ml YPD was inoculated with 2.5 x 10 6 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.
  • YPD medium 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, KH2PO4 11.8 g,
  • 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 OD 600 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 liter deionized water): Glucose 40 g, H3PO4 (85%) 26.7 ml,
  • trace salts solution 4.35 ml; (trace salts solution(per liter deionized water): Fe2(S ⁇ 4) • 7H2O 65
  • the cells were grown batchwise until the initial glucose was depleted ( ⁇ 24 hours) and the wet cell weight (WCW) was ⁇ 80-100 g/L.
  • WCW wet cell weight
  • 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 water): 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).
  • the continuous feed medium contained (per liter deionized water): D-glucose 300 g, H3PO4 (85%>) 13.35
  • 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 [U/ml] to protein units [mg/ml]. g. SDS Page and Gel Staining
  • Casamino acids have been shown to protect proteins from proteolytic degradation when 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 enzymes under fed batch conditions and that rh-Chitinase can be stabilized by addition of casimino acids.
  • FIG. 4a shows rh-Chitinase and growth data of the constitutive clone (pGAPZ ⁇ -SMD 1168) in a continuous mode. Medium was exchanged at a rate of 1.0 NND. 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 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 enzyme(s) 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.
  • pGAPZ ⁇ -X33 Clone 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 NND. The feeding rate was ramped up slowly to 1.2 NND 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 (-400 g/L WCW) and rh-Chitinase concentration plateaued on day 9 ( ⁇ 300 mg/L). The culture was continuously fed with 30% glucose feed medium, as discussed in the present description of the invention, at a rate of 1.2 NND for an additional 24 days.
  • This continuous system provides not only for greatly enhanced production of 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 of proteins which previously could not be produced in methylotrophic Pichia due to proteolytic degradation and/or economic reasons.
  • D-glucose 20 g
  • soy peptone Type IN, Sigma, MO
  • yeast extract yeast extract
  • yeast nitrogen base w/o amino acids
  • Bioreactor cultivations were performed in a 21 L stirred tank reactor (STR) with 15 L working volume (CF 3000, Chemap AG, Switzerland) and a height/diameter ratio of 2.0.
  • the medium used for bioreactor batch cultivations contained (per liter deionized water): D-glucose 40 g; H3PO4 (85%) 26J ml; K2SO4 18.2 g;
  • 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 OD 600 of ⁇ 450 was attained and the reactor was switched to continuous mode at a medium feed rate of 7 ml/min, which was increased to ⁇ 11 ml/min after 24 h.
  • 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).
  • 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 p0 2 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.
  • the oxygen demand of the process in terms of k L a and oxygen transfer rate (OTR) can be estimated as follows.
  • dc 02 L / dt 0 and consequently
  • the oxygen uptake rate (OUR) can be determined via oxygen mass balance derived from exhaust analysis: 1* P X 02,in -M-W 02 X 02,out (1 " X 02,in " X C02,in)
  • a correlation for k L a using the parameters power input per volume (PN L ), volume related aeration rate (F/V L ), and viscosity ( ⁇ ) can be described as:
  • P/N L idem
  • OTR idem.
  • N large N smaU (d small / d large ) " (1 )
  • rh-LAL lysosomal acid lipase : rh-LAL was expressed in a 15L continuous culture with Pichia pastoris SMD 1168
  • LAL activity was measured via fluorometric assay (similar assay published by: Grabowski, J Biol Chem, 270, 27766 (1995)).
  • Example 4 Pichia Expression of rh-GCR (Glucocerebrosidase): rh-GCR was expressed in a 1.5L continuous culture with Pichia pastoris X33 (prototrophic strain) using the constitutive GAP promoter. Under steady-state conditions, a maximum volumetric productivity (NPR) of 466 [U/L day] was attained at a volumetric turnover rate of 1.2 [volume/volume day] (NND). GCR activity in the supernatant was 388 [U/L] and the wet cell weight (WCW) 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%o by oxygen sparging).
  • NPR volumetric productivity
  • DOT dissolved oxygen tension

Abstract

L'invention porte sur un procédé de fermentation continu qui a été mis au point dans la levure Pichia pastoris (P.pastoris) afin de produire des quantités élevées de protéines humaines recombinées. Des niveaux d'expression élevés sont apparus durant la production continue de l'enzyme par la levure P.pastoris à l'aide d'un agent promoteur constitutif dans un fermenteur de volume de travail de 1,5 litres en utilisant du glucose ou du glycérol comme source de carbone. La fermentation peut être prolongée sur une longue période avec la possibilité d'obtenir un excellent niveau de concentration de protéines à état stable et de densités cellulaires. Aucune dégradation protéolytique de l'enzyme n'a été constatée au cours du mode continu de fermentation.
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US7951557B2 (en) 2003-04-27 2011-05-31 Protalix Ltd. Human lysosomal proteins from plant cell culture
NZ576986A (en) 2004-01-30 2010-12-24 Shire Pharmaceuticals Ireland Ltd Production and purification of recombinant arylsulfatase A
WO2005099748A1 (fr) * 2004-04-13 2005-10-27 Cilian Ag Enzymes lysosomiales de recombinaison presentant un motif de glycosylation typique de ciliates destinees a la therapie
CA2644642C (fr) 2006-04-04 2016-01-26 Zymenex A/S Procede de concentration d'un polypeptide
ES2660667T3 (es) 2007-05-07 2018-03-23 Protalix Ltd. Biorreactor desechable a gran escala
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BR112015015948B1 (pt) 2013-01-09 2022-05-17 Takeda Pharmaceutical Company Limited Método de purificação da proteína arilsulfatase a (asa) recombinante
GB201421343D0 (en) * 2014-12-02 2015-01-14 VIB VZW and Universiteit Gent Improved production of lipase in yeast
CN116334052B (zh) * 2023-04-14 2023-12-29 上海腾瑞制药股份有限公司 一种巴曲酶的发酵培养基及发酵方法

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CN103320334B (zh) * 2013-06-26 2015-12-23 中国农业科学院饲料研究所 Chi92蛋白的新用途以及表达Chi92蛋白的菌株

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