WO2020257942A1 - Procédé pour augmenter les rendements et l'activité spécifique de certaines protéines de recombinaison dans des cellules de mammifère par la co-expression de furine pleine longueur - Google Patents

Procédé pour augmenter les rendements et l'activité spécifique de certaines protéines de recombinaison dans des cellules de mammifère par la co-expression de furine pleine longueur Download PDF

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WO2020257942A1
WO2020257942A1 PCT/CA2020/050893 CA2020050893W WO2020257942A1 WO 2020257942 A1 WO2020257942 A1 WO 2020257942A1 CA 2020050893 W CA2020050893 W CA 2020050893W WO 2020257942 A1 WO2020257942 A1 WO 2020257942A1
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furin
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bmp
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WO2020257942A8 (fr
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Sean A.F. Peel
Cameron M.L. Clokie
Aileen J.J. ZHOU
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    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/67—General methods for enhancing the expression
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21075—Furin (3.4.21.75)

Definitions

  • the present invention relates to a system and method for increasing yields and the specific activity of certain recombinant proteins in mammalian cells by co-expressing full- length Furin in those cells.
  • recombinant proteins for therapeutic use including hBMP-2 and hBMP-7, have been successfully produced recombinantly (Demain and Vaishnav 2009; Leader et al. 2008).
  • Some well recognized therapeutic recombinant proteins include insulin (for diabetes), Factors VII, VIII, and IX (for blood clotting disorders), luteinizing hormone (for infertility), erythropoietin (for anemia and renal disease), lysozyme (for metabolic disorder), platelet derived growth factor (for diabetic ulcers), and a large number of monoclonal antibodies (to treat a variety of different conditions including arthritis, osteoporosis, and cancer).
  • the systems include single cell expression systems which use bacteria, yeasts, baculovirus infected insect cells, mammalian cells, and multi-cellular expression systems that use transgenic plants or animals to produce transgenic proteins.
  • the systems include single cell expression systems which use bacteria, yeasts, baculovirus infected insect cells, mammalian cells, and multi-cellular expression systems that use transgenic plants or animals to produce transgenic proteins.
  • Mammalian cell expression has become the dominant recombinant protein production system to produce recombinant proteins for clinical applications. More than half of therapeutic proteins approved and on the market are produced using mammalian cells (Zhu 2012).
  • a major advantage of mammalian cells is that they are able to synthesize proteins which are very similar to native human proteins in terms of physical, biochemical and physiological properties, due to the proper protein folding, assembly, processing, and posttranslational modifications. As the result, the quality and efficacy of the proteins produced from mammalian systems are typically superior to that of the same proteins expressed in bacteria, yeasts or insects.
  • HEK-293 human embryonic kidney
  • N0 mouse myeloma
  • BHK baby hamster kidney
  • PER.C6 PER.C6 cell line which was derived from human embryonic retinal cells.
  • Mammalian expression systems are relatively costly to maintain compared to other expression systems and typically have lower recombinant protein yields. Further, while typical yields of recombinant monocolonal antibodies in mammalian cells are in the g/L range with specific productivities of 90 pg/cell/day (Wurm 2004), for other proteins reported yeilds are lower. For example it has been reported that yields for rhBMP-2 and rhBMP-7 remain low (Roe et al. 2004; Swencki-Underwood et al.
  • rhBMP-2 and rhBMP-7 are in the 10 to 30 mg/L range with an estimated specific productivity between 2 to 4 pg/cell/day, (Peel, S., personal communication).
  • pro-forms of proteins have received intensive scientific attention because of the roles pro-domains have in the maturation and processing of the precursor proteins (Harrison et al. 201 1).
  • the pro-domain of a protein is well known to confer latency to the mature protein.
  • pro-proteins can also affect the secretion of recombinant pro-proteins.
  • In-frame deletion of the pro-peptide of BMP-2 yielded a polypeptide that was not secreted from the cell (Israel et al. 1992).
  • Dick et al. constructed a mutated BMP-7 in which the amino acid residues Val and Gly were exchanged (Val to Gly) in the pro-domain; it was found that this dramatically reduced the amount of pro-domain and the mature protein secreted into the conditioned medium (Dick et al. 2000).
  • ProBMP-2 contains two proprotein convertase (PC) recognition sites (S1 and S2).
  • a mammalian cell is transfected with both the protein of interest and with full-length furin to increase yeilds of the recombinant protein of interest.
  • selected clones of mammalian cells that have been transfected with the protein of interest and undergone mutliple rounds of gene amplification, then undergo a second round of transfection with full-length furin to further increase protein yeilds.
  • mammalian cells are first transfected with full-length furin and they are screened to identify clones with the appropriate level of furin activity to generate a parent cell-line that can be used to transfect with the recombinant protein of interest where increased furin activity would be beneficial.
  • clones are selected for use following transfection with full length furin based on the level of furin expressed.
  • Figure 1 Schematic diagram of full-length and truncated PCs.
  • PCs human proprotein convertases
  • sFurin soluble Furin
  • sPC7 soluble PC7
  • Figure 2 FACS of CHO cells post PC transfection.
  • Doublets were discriminated using forward scatter and size scatter height and width parameters (FSC-H versus FSC-W).
  • FSC-H forward scatter and size scatter height and width parameters
  • B Furin-tGFP
  • C sFurin-tGFP
  • D no DNA plasmids (as control) were sorted on the basis of their tGFP expressions.
  • FIG. 3 Transient overexpression of PC-tGFP fusion protein on rhBMP-2 yield.
  • BMP-2 secreted by CHO cells expressing membrane-bound PCs: Furin, PC4, PC6B, and soluble PCs: sFurin, sPC7, PC6A, and PACE4 sorted into tGFP negative (Neg), low- (Low), mid- (Mid), and high-positive (High) groups. If the transfection efficiency of a PC was low, the cells were sorted into Neg, Low, and High positive groups (i.e. PC6A) or simply Neg and Pos groups (i.e. PC4 and PACE4). All data are shown as mean ⁇ SE. At least three sets of independent experiments were carried out for each of the PCs (N 3). Results also showed that cells over expressing lower levels of Furin produced more rhBMP-2 than cells overexpressing high levels of Furin
  • Figure 4 Overexpression of PCs with or without tGFP tag on mature rhBMP-2 yield.
  • Furin overexpressed in (A) conditioned media and in (B) cell lysates was detected by Western blot analysis probed with anti-furin antibody and quantified by furin ELISA. The amount of Furin and sFurin was normalized to the protein content of the cells.
  • NC negative control: cells not overexpressing Furin or sFurin; Furin: full-length furin (794 aa); sFurin: soluble Furin lacking the transmembrane and cytoplasmic domains (715 aa). Data are shown as mean ⁇ standard deviation (SD) (* P ⁇ 0.05, ** P ⁇ 0.01).
  • FIG. 7 BMP processing in untransfected and Furin and sFurin overexpressing cells
  • BMP-2 proteins secreted in (A) conditioned media and in (B) cell lysates were characterized by Western blot analysis and quantified by ELISA.
  • BMP-2 concentration was normalized to the protein content of the cells.
  • NC negative control: cells not overexpressing Furin or sFurin; Furin: full-length furin (794 aa); sFurin: soluble Furin lacking the transmembrane and cytoplasmic domains (715 aa). Data are shown as mean ⁇ SD (* P ⁇ 0.05).
  • Figure 8 The effect of extracellular furin treatment on BMP-2 yield.
  • CHO cells were cultured with cell culture media with or without added furin for 48 h, after which the BMP-2 in the conditioned media and the cell lysates were measured by BMP-2 ELISA.
  • the system and method of the invention can be used for increasing yeilds of difficult to express recombinant proteins that undergo processing by pro-protein convertases
  • the system and method of the invention can be used for increasing the specific activity of recombinant proteins that undergo incomplete processing by proprotein convertases in mammalian expression systems with insufficient proprotein convertase activtiy.
  • the system and method of the invention can be used to generate a parent cell line that can be used for production of recombinant proteins that undergo processing by proprotein convertases.
  • the term“recombinant” means a protein produced by a transiently transfected, stably transfected, or transgenic host cell or animal as directed by an expression construct containing the cDNA for that protein.
  • the term“recombinant” also encompasses pharmaceutically acceptable salts of such a polypeptide
  • polypeptide or“protein” refers to a polymer of amino acid monomers that are alpha amino acids joined together through amide bonds. Polypeptides are therefore at least two amino acid residues in length, and are usually longer. Generally, the term“peptide” refers to a polypeptide that is only a few amino acid residues in length. A polypeptide, in contrast with a peptide, may comprise any number of amino acid residues. Hence, the term polypeptide included peptides as well as longer sequences of amino acids.
  • bone morphogenetic protein or “bone morphogenic protein” or “BMP” are used interchangeably and refer to any member of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor beta (TGFp) superfamily of growth and differentiation factors, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1 , OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-1 1 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15.
  • the terms“bone morphogenetic protein” and“BMP” also encompass allelic variants of BMPs, function conservative variants of BMPs, and mutant BMPs that retain BMP activity.
  • allelic variants of BMPs include allelic variants of BMPs, function conservative variants of BMPs, and mutant BMPs that retain BMP activity.
  • the BMP activity of such variants and mutants may be confirmed by any of the methods well known in the art (see the section Assays to measure BMP activity, below).
  • the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP- 8 or BMP-9.
  • the BMP is BMP-2, BMP-4 or BMP-7.
  • the BMP is a mammalian BMP (e.g., mammalian BMP-2 or mammalian BMP-7).
  • the BMP is a human BMP (hBMP) (e.g. hBMP-2 or hBMP-7).
  • full-length Furin also called Furin or PACE
  • FURIN FURIN gene
  • PC human proprotein convertases
  • Furin NM_002569
  • PACE4 NM_138322
  • PC4 NM_017573
  • PC6A NM_006200
  • PC6B NM_001 190482
  • PC7 NM_004716
  • Soluble forms of Furin sFurin
  • PC7 sPC7
  • CHO-DG44 Chinese hamster ovarian (CHO-DG44) cells stably expressing hBMP2 gene were cultured with chemically defined medium CD OptiCHOTM (Invitrogen, Burlington, ON, Canada) plus 2 mM GlutMAXTM I (Invitrogen) and methotrexate (MTX), in 50 ml TubeSpin bioreactors (TPP, Trasdingen, Switzerland) on an orbital shaker rotating at 250 rpm. The cells were incubated at 37°C with 5% C02. CHO cells were transfected and then sorted, as described below.
  • CD OptiCHOTM Invitrogen, Burlington, ON, Canada
  • 2 mM GlutMAXTM I Invitrogen
  • MTX methotrexate
  • the transfected cells were cultured for 48 h and the conditioned media was harvested and centrifuged (500g for 5 min) to remove any debris.
  • the cells were lysed (CelLytic M, Sigma-Aldrich, St. Louis, MO), homogenized, and the cell lysates were collected.
  • CHO cells were cultured in CD OptiCHO medium without MTX.
  • the cDNA plasmids was transfected into the cells via electroporation using Amaxa cell line nucleofector kit V (Lonza, Basel, Switzerland), following manufacturer’s instructions. Briefly, every 1x10 6 CHO cells were transfected with 2.5 pg of the plasmid and cultured in a 24-well plate (at 1x10 6 cells per well) for 24 hr before cell sorting. Fluorescence-activated cell sorting (FACS)
  • PC proprotein convertase
  • the concentration of mature rhBMP-2 secreted in the conditioned media or in the cell lysates of the PC transfected cells was quantified by BMP-2 immunoassay (R&D Systems, Mineapolis, MN) as described previously (Zhou et al. 2012a). Briefly, the conditioned media samples were loaded into a 96-well microplate pre-coated with monocloncal anti-BMP2 antibodies. After 2hr incubation, the captured rhBMP-2 proteins were detected using polyclonocal anti-BMP2 antibodies. Recombinant human BMP-2 was used to generate the standard curve. Similarly, the amount of furin in the conditioned media and the cell lysates were determined using a furin DuoSet ELISA Development System (R&D Systems), following manufacturer’s instructions.
  • the conditioned media and the cell lysate samples were resolved by 4-12% LDS- PAGE (Invitrogen) and blotted to a nitrocellulose membrane (Invitrogen).
  • the membrane was blocked in 5% fat-free milk and probed with anti-BMP-2 (Abeam, Cambridge, MA; 2 pg/ml), anti-Furin (R&D Systems; 2 pg/ml) or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abeam; 1 pg/ml) antibodies, followed by horseradish peroxidase- linked secondary antibodies.
  • a murine myogenic cell line C2C12 (American Type Culture Collection, ATCC, Manassas, VA) was seeded at 2.5x10 4 cells per well in 500 pi a-minimum essential medium (a-MEM, Invitrogen) plus 10% (v/v) fetal bovine serum (FBS, Invitrogen) in 48-well plates. After 24 h culture, the media was replaced with 500 pi of test samples per well. After 48 h incubation, the cells were lysed (CelLytic M, 200 mI/well; Sigma-Aldrich, St.
  • a-MEM a-minimum essential medium
  • FBS fetal bovine serum
  • alkaline phosphatase (ALP) enzymatic activity was determined using paranitrophenyl phosphate (pNPP) (Sigma-Aldrich) as previously described (Zhou et al. 2012b).
  • the ALP level was normalized to the amount of cellular protein, measured by Bradford assay.
  • BMP-2 expressing CHO cells were seeded in a 24-well plate at 5x10 4 cells per well and cultured with 1 ml of fresh medium spiked with 0, 4, 40, or 400 ng/ml of recombinant human furin (R&D Systems) under standard cell culture conditions. After 48 h incubation, the conditioned media were collected and cell lysates were prepared (as described in the previous section BMP-2 bioactivity assay), and the amount of BMP-2 in the samples was measured by BMP-2 ELISA. In addition, some conditioned media from CHO cell culture were pipetted into a 96-well plate at 200 mI per well.
  • Recombinant human furin (R&D Systems) was added to the conditioned media at 0, 16, 80, 400, or 2000 ng/ml and incubated at 37°C for 24 h. Following the incubation, the conditioned media were collected and the amount of BMP-2 was measured by BMP-2 ELISA.
  • DuoSet ® rhBMP-2 ELISA kit (R&D Systems, cat #DY335)
  • Anti-ProBMP-2 antibody (R&D Systems, MAB2260 or Abeam, ab 14933)
  • Block plates by adding 300 pL of recommended Blocking Buffer to each well. Incubate at room temperature for a minimum of 1 hour.
  • the blocking buffer can be aspirated after step 3 and the plates can be dried under vacuum.
  • the plates can be stored at 4 to 8° C for at least 2 months.
  • the conditioned media of the transfected cells was collected 24 h post sorting and the amount of BMP-2 secreted was measured by ELISA.
  • PCs can be membrane-bound or soluble depending on whether they have a transmembrane domain in their sequences ( Figure 1).
  • PC6 has two naturally occurring variants: soluble PC6A and membrane-bound PC6B.
  • PACE4 does not contain a transmembrane domain and hence is naturally soluble.
  • the soluble furin (sFurin) and PC7 (sPC7) were constructed by deleting their transmembrane domains and the cytoplasmic tails from the full-length PCs. Cells overexpressing any one of the soluble PCs did not show any significant impact on the amount of mature BMP-2 secreted (Figure 3B).
  • the alkaline phosphatase (ALP) level was measured in the C2C12 cell line to assess the biological activity of mature BMP-2 expressed by cells overexpressing Furin in vitro.
  • ALP alkaline phosphatase
  • myogenic C2C12 cells express very low levels of ALP.
  • ALP activity of the C2C12 cells increases in a dose-dependent manner (Katagiri et al. 1994; Zhou et al. 2012b). This was observed when the mature BMP-2 secreted by cells overexpressing Furin were tested, indicating that the secreted BMP-2 is biologically active in vitro (Figure 5).
  • CHO cells were transfected with Furin or sFurin tagged with tGFP. Twenty-four hours post transfection, positively transfected and negative cells (cells that did not take up PC plasmid and hence expressed little or no GFP) were sorted and cultured for 48 h. After incubation, the conditioned media and cell lysates were collected and analyzed by Western blot.
  • BMP-2 ELISA showed significantly increased amount of extracellular (P ⁇ 0.05, ANOVA) and intracellular BMP-2 (P ⁇ 0.05) in cells overexpressing Furin than those overexpressing sFurin or the untransfected cells ( Figure 7B). Cells overexpressing sFurin expressed similar amount of BMP-2 to the untransfected cells extracellularly and intracellularly.
  • transmembrane and the cytoplasmic domains of furin have been found to be dispensable for the functional activity of furin.
  • High levels of expression of full-length recombinant furin have resulted in the natural secretion of a truncated furin, called“shed” furin, lacking those C-terminal domain (aa 683 to 794).
  • Mutant form of soluble furin constructed by deleting the transmembrane domain and cytoplasmic tail (aa 716-794) has been shown to still exhibit activity.
  • proBMP-2 When we incubated the purified proBMP-2 secreted by CHO cells with a commercially available recombinant soluble human furin (purchased from R&D Systems), proBMP-2 was cleavage into mature BMP-2 over 24 h in vitro (data not shown). Hence, in theory the soluble sFurin should be able to process proBMP-2.
  • HEK 293 Flp-ln cell lines and CHO DG44 cells expressing rhBMP-2 or varients were created.
  • HEK cells were stratified into 3 groups: FRT parent cell line; wild type BMP (WT); or mutant BMP (ms1).
  • CHO m713 cells express the equivalent of the BMP WT preform - they are a 7X amplified wild type gene.‘CHO m501 is similar, but the gene is only 5X amplified.
  • CHO EGFP expresses green fluorescent protein and acts as a control since it is not overexpressing BMP2.
  • CHO cells were grown with CD OptiCHO medium (Invitrogen) in T75 flasks at 37°C.
  • CelLytic M Cell Lysis Reagent Sigma-Aldritch
  • the mixture was homogenized using PowerGen 125 (Fischer-Scientific) and then centrifuged at 16,000 x g for 10 min. The supernatant was collected and stored at -80°C.
  • PC Proprotein Convertase
  • PC activity was determined by measuring the concentration of a fluorescent, cleaved product produced after incubation of a flourogenic substrate in the presence of Furin.
  • Cell lysates were diluted in 1 mM CaCI2, 0.5%(w/v) Brij-35 in 25mM Tris.
  • 50uL of fluorogenic BOC-RVRR-AMC substrate (Bachem) was added to 50uL of each cell lysate dilution, in duplicate and incubated at 37°C.
  • Stock rhFurin (R&D Systems) was treated identically as a positive control.
  • AMC 7- amino-4-methyl-coumarin
  • the purpose of this experiment is to determine the specific amount of Furin in HEK and CHO cell lysates.
  • 96 well plates were coated overnight with 100pL per well of 2pg/mL mouse anti-human Furin antibody (capture antibody) in PBS.
  • the plates were washed 3 times with 400uL wash buffer (0.05% Tween 20 in 1XPBS) then blocked for 1 hour with 300mI_ of reagent diluent (1 % fraction V BSA in 1XPBS). Plates were washed again, then incubated for 2 hours with 100mI_ of diluted sample or rhFurin standard.
  • Samples were undiluted or 3 fold dilutions of HEK and CHO cell lysate in reagent diluent.
  • the plates were washed after incubation and coated with 100mI_ of 100ng/ml_ biotinylated goat anti-human Furin antibody (detection antibody).
  • a 2 hour incubation period passed before another wash step, and successive addition of 100mI_ of streptavidin-HRP.
  • 100mI_ of substrate 50% tetramethylbenzidine, 50% hydrogen peroxide
  • 50mI_ of 2N H2S04 was added to stop the reaction. Readings were taken using a spectrophotometer at wavelengths 450 and 540. Furin levels were normalized by total protein amount for analysis.
  • Statistical analysis was performed using one-way analysis of variance (ANOVA) or a paired t test. Post hoc analyses used Dunnett’s and Tukey’s multiple comparison tests. Microsoft Excel 2010 and IBM PASW 18 softwares were used for graphical and statistical representation. Critical P is as denoted, but typically P ⁇ 0.05 was considered significant.
  • Furin ELISA results for each cell line variant demonstrated no significant difference between cell lines from the same parent (HEK or CHO) ( Figure 9). However, there is a significant difference HEK to CHO cell lines when combining results from all CHO vs all HEK cell lines (ANOVA, P ⁇ 0.05).
  • PC activity assay data revealed a significant difference between HEK and CHO cell lysates (Figure 10). PC activity was similar, with no significant difference between variants of the same cell line (P ⁇ 0.05). Notably, there is no difference in PC activity between cell lines containing a control vector, versus those that contain a BMP vector. For this reason, all HEK cell line variants may be considered one group, and likewise all CHO cell line variants may be considered a separate group. Rank ordered ANOVA demonstrated that a significant difference does exist between these two cell lines (P ⁇ 0.001).

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Abstract

Procédé pour augmenter le rendement d'une protéine de recombinaison d'intérêt comprenant la transfection d'une cellule de mammifère avec à la fois la protéine d'intérêt et la furine pleine longueur.
PCT/CA2020/050893 2019-06-28 2020-06-26 Procédé pour augmenter les rendements et l'activité spécifique de certaines protéines de recombinaison dans des cellules de mammifère par la co-expression de furine pleine longueur Ceased WO2020257942A1 (fr)

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US9873892B2 (en) * 2014-08-12 2018-01-23 Baxalta Incorporated Production of fully processed and functional factor X in a furin-secreting mammalian expression system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9873892B2 (en) * 2014-08-12 2018-01-23 Baxalta Incorporated Production of fully processed and functional factor X in a furin-secreting mammalian expression system

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BINGO M ET AL.: "Recombinant Factor X using a chimeric construct containing the prothrombin propeptide under co-expression of furin leads to high yield and matured protein expressions in 293T Cells", RINSHO BYORI, vol. 67, 2019, pages 24 - 31, ISSN: 0047-1860 *
LIU J ET AL.: "Improved Expression of Recombinant Human Factor IX by Co-expression of GGCX, VKOR and Furin", PROTEIN J., vol. 33, 2014, pages 174 - 183, XP055453954, DOI: 10.1007/s10930-014-9550-5 *
MINYUB CHA, NARA HAN , JIA PI , YONGSU JEONG , KWANGHEE BAEK AND JAESEUNG YOON: "Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells", J. MICROBIOL. BIOTECHNOL., vol. 27, no. 7, 2017, pages 1281 - 1287, XP055776680, ISSN: 1751-7915 *
WILBERS R HP. ET AL.: "Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transfor ming growth factor-bl into a biologically active protein", PLANT BIOTECHNOLOGY J., vol. 14, 2016, pages 1695 - 1704, XP055675257, ISSN: 1467-7652 *
ZHOU, JING JING AILEEN: "Investigation of Post-translational Strategies to Enhance Recombinant Human Bone Morphogenetic Protein-2 Production in Mammalian Cell Culture Systems", THESIS, 2014, University of Toronto, XP055776686 *

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