WO1999064560A9 - Procedes pour controler la stabilite d'une proteine ou d'une autre substance cellulaire secretee par des cellules dans un milieu de fermentation - Google Patents

Procedes pour controler la stabilite d'une proteine ou d'une autre substance cellulaire secretee par des cellules dans un milieu de fermentation

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Publication number
WO1999064560A9
WO1999064560A9 PCT/US1999/012862 US9912862W WO9964560A9 WO 1999064560 A9 WO1999064560 A9 WO 1999064560A9 US 9912862 W US9912862 W US 9912862W WO 9964560 A9 WO9964560 A9 WO 9964560A9
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WO
WIPO (PCT)
Prior art keywords
cells
sample
harvested
culture
protein
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PCT/US1999/012862
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English (en)
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WO1999064560A1 (fr
Inventor
Gerald R Carson
Linda Hammill
Original Assignee
Basf Ag
Gerald R Carson
Linda Hammill
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Filing date
Publication date
Application filed by Basf Ag, Gerald R Carson, Linda Hammill filed Critical Basf Ag
Publication of WO1999064560A1 publication Critical patent/WO1999064560A1/fr
Publication of WO1999064560A9 publication Critical patent/WO1999064560A9/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • An important aspect of the production of proteins or other cellular products by cells is the stability of gene expression under large scale culture conditions. This is particularly important for recombinant protein production, where expression of a recombinant vector can vary from cell to cell and over time. Moreover, this is also important in the stable expression of other cellular products that are the result of a biosynthetic pathway that has been recombinantly introduced into a given host cell.
  • the ideal production process has a robust level of expression which can be maintained through many cell generations without the selection pressure of antibiotics or other drugs. In practice, cell lines producing recombinant proteins can often lose their high levels of expression even when maintained under selection pressure. Therefore, the evaluation of a fermentation process typically includes monitoring for signs of expression instability.
  • a number of parameters have been used to monitor the condition of cells in fermenters. Examples include monitoring of glucose uptake, lactate accumulation, and oxygen consumption, each of which monitor the metabolism of the culture as a whole. Periodic viable cell counts can be performed to determine the growth rate of the population. Currently, periodic measurements of the specific productivity of a cell line are performed by assaying the amount of protein produced and the number of cells in the culture. Specific productivity, expressed as picograms of recombinant protein or an amount of a given cellular product produced per cell per day, thus can detect only the average loss of expression of the total population.
  • This invention provides a method for monitoring the stability of protein or cellular product secretion that allows for the testing of individual cell productivity within a large population of cells, such as cells within a fermenter culture.
  • the method of the invention allows for a more discriminating evaluation of the productivity of cells from fermenters cultures, since the method measures productivity of individual cells in the population.
  • the method of the invention provides rapid results and is relatively simple to perform.
  • the method of the invention generally involves harvesting a sample of the cells from a fermenter culture, incorporating cells from the sample into gel microdrops (GMDs), performing a GMD secretion assay to assess protein or cellular product secretion by individual cells in the sample and then repeating this procedure with a second sample of cells, harvested at a later time than the first sample of cells, to thereby monitor the stability of protein or cellular product secretion by the culture.
  • the procedure can be repeated with additional cell samples, harvested over time, to continue monitoring the stability of protein or small molecule secretion by the culture.
  • the invention provides a method for monitoring stability of protein secretion by cells in a fermenter culture, comprising: • (a) harvesting from a fermenter culture a first sample of cells that secrete a protein;
  • step (d) repeating steps (a) through (c) on a second sample of cells, wherein the second sample of cells is harvested from the fermenter culture at a time later than when the first sample of cells was harvested to thereby monitor stability of protein secretion by cells in the fermenter culture.
  • the method can further comprise step (e): repeating steps (a) through (c) on a third sample of cell/s, wherein the third sample of cells is harvested from the fermenter culture at a time later than when the second sample of cells was harvested.
  • the method can still further comprise step (g): repeating steps (a) through (c) on a fifth sample of cells, wherein the fifth sample of cells is harvested from the fermenter culture at a time later than when the fourth sample of cells was harvested.
  • the method of the invention can be used to monitor the stability of protein secretion by any type of cells that is suitable for recombinant protein production, including both eukaryotic and prokaryotic cells.
  • the method is used to monitor a culture of mammalian cells, such as CHO cells, COS cells, NS/O cells and the like.
  • the method is used to monitor a culture of yeast cells.
  • the protein whose production is monitored is an antibody secreted by the cells of the culture.
  • the protein whose production is monitored is a protein encoded by an expression vector incorporated into the cells (i.e., a recombinant protein).
  • the protein whose production is monitored is an antibody encoded by at least one expression vector incorporated into the cells (i.e., a recombinant antibody).
  • the method of the invention allows for the monitoring of large scale fermenter cultures.
  • the fermenter culture comprises at least 10 liters of culture medium.
  • the fermenter culture comprises at least 50 liters of culture medium.
  • the fermenter culture comprises at least 100 liters of culture medium.
  • Monitoring of the culture is achieved by assaying at least two samples of cells, wherein the second sample is harvested at a later time than the first sample.
  • the second sample of cells is harvested at least one day later than the first sample of cells.
  • the second sample of cells is harvested at least two days later than the first sample of cells.
  • the second sample of cells is harvested at least three days later than the first sample of cells.
  • the method can further comprise recovering a cell population with stable expression of the protein.
  • the invention provides a method for monitoring the stability of cellular product secretion by cells in a fermenter culture, comprising:
  • step (d) repeating steps (a) through (c) on a second sample of cells, wherein the second sample of cells is harvested from the fermenter culture at a time later than when the first sample of cells was harvested to thereby monitor the stability of cellular product secretion by cells in the fermenter culture.
  • the method can further comprise step (e): repeating steps (a) through (c) on a third sample of cells, wherein the third sample of cells is harvested from the fermenter culture at a time later than when the second sample of cells was harvested.
  • the method can still further comprise step (f): repeating steps (a) through (c) on a fourth sample of cells, wherein the fourth sample of cells is harvested from the fermenter culture at a time later than when the third sample of cells was harvested.
  • the method can still further comprise step (g): repeating steps (a) through (c) on a fifth sample of cells, wherein the fifth sample of cells is harvested from the fermenter culture at a time later than when the fourth sample of cells was harvested.
  • the method of the invention can be used to monitor the stability of cellular product secretion by any type of cell/ s that is suitable for recombinant cellular product production, including both eukaryotic and prokaryotic cells.
  • the method is used to monitor a culture of mammalian cells, such as CHO cells, COS cells, NS/O cells and the like.
  • the method is used to monitor a culture of fungal cells, preferably, yeast cells.
  • the method is used to monitor a culture of bacterial cells
  • the cellular product whose production is monitored is an antibiotic, preferably, a polyketide antibiotic, secreted by the cells of the culture.
  • the cellular product is an amino acid, preferably, an essential amino acid, such as, for example, histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, or arginine.
  • the cellular product is a vitamin, preferably, a B vitamin (e.g., B2, B6, or B12), vitamin C, vitamin K, or riboflavin.
  • the cellular product is a carbohydrate, for example, a saccharide, or a polymer thereof.
  • the cellular product whose production is monitored is a cellular product produced by an enzyme encoded by an expression vector incorporated into the cells.
  • the cellular product whose production is monitored is a antibiotic produced by an enzyme encoded by at least one expression vector incorporated into the cells.
  • the method of the invention allows for the monitoring of large scale fermenter cultures.
  • the fermenter culture comprises at least 10 liters of culture medium.
  • the fermenter culture comprises at least 50 liters of culture medium.
  • the fermenter culture comprises at least 100 liters of culture medium. Monitoring of the culture is achieved by assaying at least two samples of cells, wherein the second sample is harvested at a later time than the first sample.
  • the second sample of cells is harvested at least one day later than the first sample of cells. In another embodiment, the second sample of cells is harvested at least two days later than the first sample of cells. In yet another embodiment, the second sample of cells is harvested at least three days later than the first sample of cells.
  • the method can further comprise recovering a cell population with stable expression of the protein.
  • Figures 1A-1D are FACS histograms of the gel microdrop secretion assays of samples of a fermentation of the D8/E cell line, secreting the recombinant antibody D2E7.
  • Figure 1 A represents antibody secretion by the fermenter inoculum (AFI 615.75)
  • Figure IB represents antibody secretion by the first harvest (AFF 610A)
  • Figure 1C represents antibody secretion by the fourth harvest (AFF 610D)
  • Figure ID represents antibody secretion by the eighth harvest (AFF 61 OH).
  • Figures 2A-2B are bar graphs depicting the gene copy numbers for the D2E7 antibody heavy chain (Figure 2A) or the D2E7 antibody light chain ( Figure 2B) for cells harvested from the seed train and the fermenter of the D8/E cell culture.
  • Samples 615 0.6, 615 5.0 and 615 75 were taken from the fermenter seed inoculum culture as it was expanded from 0.6 liters to 5 liters and then to 75 liters.
  • Samples 610A, 610D and 61 OH were taken from the fermenter at the first, fourth and eight harvest.
  • Figures 3A-3B depict the antibody secretion profiles, as determined using the gel microdrop secretion assay, of cells from the end of the fermenter run 607 ( Figure 3 A) or the fermenter run 702 ( Figure 3B).
  • Figure 4 is a bar graph depicting the antibody productivity (expressed in picograms of antibody per cell in 24 hours) of high and low expressor subpopulations of the 702 fermenter run over a period of 4 weeks (for the low expressor subpopulation) or ten weeks (for the high expressor subpopulation).
  • the low expressor subpopulation (702hLo) is represented by the bar graphs on the left, whereas the high expressor subpopulation (702hHi) is represented by the bar graphs on the right.
  • Figures 5A-5B are bar graphs depicting the gene copy numbers for the D2E7 antibody heavy chain (Figure 5A) or the D2E7 antibody light chain (Figure 5B) for cells from the final harvest of the 607 fermenter run (607H) or the 702 fermenter run (702H) or for their sorted subpopulations that produce low antibody levels (607H low and 702H low, respectively) or high antibody levels (607H high and 702H high, respectively).
  • This invention pertains to methods for monitoring the stability of protein secretion (e.g., antibody secretion) from cells in fermenter cultures.
  • the invention provides a method for monitoring the stability of cellular product secretion, i.e., a non-protein based product (e.g., an antibiotic, an amino acid, a vitamin, or a carbohydrate) from a cell in a fermenter.
  • the methods of the invention allow for the testing of individual cell productivity within a large population of cells and thus allow for a more discriminating evaluation of the productivity of cells from fermenters cultures.
  • the invention is based, at least in part, on the use of gel microdrop encapsulation and gel microdrop secretion assays to monitor protein or cellular product secretion.
  • gel microdrop technology is known in the art, it has not heretofore been used to monitor the stability of protein or cellular product secretion over time by cells within fermenter cultures.
  • the appearance within a fermenter culture of a subpopulation of cells with lower productivity of, for example, a protein was detected using the gel microdrop secretion assay, by assaying sequential harvests of the culture over time. Subsequent analysis of the culture by Southern blot analysis confirmed the presence of lower productivity cells that had lower copy numbers of the expression vector encoding the protein.
  • the gel microdrop method of the invention enables one to detect the nature of this instability in a very short period of time (e.g.
  • the method of the invention can be used to recover a population with stable expression of the protein or cellular product of interest.
  • the term "protein” is intended to include any selected polypeptide of interest that is capable of being expressed in a cell.
  • the protein is secreted and may be, for example, an antibody, a cytokine, a growth factor, or a hormone.
  • the term "cellular product” is intended to include any non-protein based cellular product that a cell is capable of producing naturally or upon genetic manipulation.
  • the cellular product is an antibiotic, an amino acid (e.g., an essential amino acid), a vitamin, a carbohydrate, or any non-protein based molecule suitable for use as a pharmaceutical, nutrient, dietary supplement, or food additive (e.g., a flavoring, colorant, or preservative) that can be expressed from a cell.
  • the antibiotic is a polyketide antibiotic.
  • antibiotic is intended to include any substance produced by a cell that can inhibit the growth of or destroy a microorganism.
  • amino acid is intended to include any member of a group of organic compounds marked by the presence of an amino group (NH 2 ) and a carboxyl group (COOH).
  • the term is intended to include any amino acid found in nature (of which there are over 80) and preferably, those 20 amino acids necessary for protein synthesis, and thus, the growth of any living organism.
  • the amino acid may be an "essential amino acid” and this term is intended to include any amino acid required by an animal, e.g. , a human, that must be obtained from the diet.
  • vitamin is intended to include any group of organic substances other than proteins, carbohydrates, fats, minerals, and organic salts which are essential for normal metabolism, growth, and development of an organism, e.g. , a human.
  • the vitamin is a B vitamin (e.g., Bl, B2, B6, B 12, or a combination thereof) vitamin C, vitamin K, or riboflavin.
  • carbohydrate is intended to include a group of chemical substances containing only carbon, oxygen, and hydrogen, e.g., sugars, and also polymers thereof, e.g., glycogen, starches, dextrins, celluloses, alginate, curdlan, levan, phosphomannan, poly-beta-hydroxybutyrate, scleroglucan, and xanthans (see also Table 5).
  • fertilizer culture is intended to refer to a large scale cell culture (e.g., greater than 10 liters, more preferably greater than 50 liters and even more preferably greater than 100 liters) that is used to generate proteins (e.g., secreted proteins) or cellular products (e.g., an antibiotic, an amino acid, a carbohydrate, or a vitamin), which are then harvested for further use.
  • proteins e.g., secreted proteins
  • cellular products e.g., an antibiotic, an amino acid, a carbohydrate, or a vitamin
  • harvesting refers to removing or isolating a sample of cells from the fermenter culture.
  • gel microdrops refers to particles having a very small volume (e.g., 10" 10 to 10" 5 ml) and comprising at least one gel region, which provide a mechanical matrix capable of entrapping or surrounding (without necessarily contacting) a biological entity, such as an individual cell.
  • the GMD consists entirely of gel, in which case containment of the biological entity (e.g., cell) occurs by entrapment of the biological entity by the gel matrix.
  • suitable polymer gels for encapsulation include agarose, alginate, agar, carrageenan, polyacrylamide, collagen, gelatine and fibrinogen, with agarose being most preferred.
  • the diameter of the GMD typically is 0.2-1000 ⁇ m, more preferably 5-100 ⁇ m.
  • incorporating cells into gel microdrops refers to encapsulating cells within a gel microdrop, for example using procedures described herein.
  • gel microdrop sample refers to a sample of cells that have been incorporated (i.e., encapsulated) into gel microdrops.
  • gel microdrop secretion assay refers to an assay in which proteins or cellular products are secreted into the matrix of a GMD by a cell entrapped within the GMD, are labeled with a detectable label (e.g., a fluorescent label) and the GMD-associated label is detected (e.g., by flow cytometry).
  • a detectable label e.g., a fluorescent label
  • the GMD secretion assay is performed quantitatively ( . e., the amount of GMD- associated label is quantitated).
  • the term "cell” is intended to include any cell that expresses a desirable protein or cellular product, i.e., non-protein based molecule (or a combination thereof), and can be cultivated in a fermenter.
  • the cell is a eukaryotic cell such as a mammalian cell.
  • the cell is a eukaryotic cell such as a fungal cell or an insect cell (that is amenable, for example, for baculovirus expression).
  • the eukaryotic cell is a yeast cell.
  • the cell is a plant cell.
  • the cell is bacterial cell.
  • the cell may be a particular strain of any of the foregoing cells selected for the expression of a particular product, such as, for example, a particular antibiotic.
  • the cell may be altered by genetic manipulation, e.g., to incorporate a heterologous polynucleotide sequence/s, e.g., which can be transfected.
  • a polynucleotide sequence/s may encode a protein/s that is itself the desired product or, alternatively, may be an enzyme that is involved in a biosynthetic or catalytic pathway that liberates a desired cellular product, for example, an antibiotic, amino acid, vitamin, carbohydrate, etc.
  • the cell may be transformed with one or more vectors comprising such sequences and the polynucleotide sequences may encode one or more genes.
  • the term is intended to include progeny of the cell originally transfected. Techniques for culturing the above-mentioned cells are known in the art (see, e.g., Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990), Bacterial Cell Culture: Essential Data, Ball, A., John Wiley & Sons, (1997), Molecular and Cell Biology of Yeasts, Yarranton et al., Ed., Van Nostrand Reinhold, Pub., (1989); Yeast Physiology and Biotechnology, Walker, G., John Wiley & Sons, Pub., (1998); Baculovirus Expression Protocols, Richardson, C, Ed., Humana Press, Pub., (1998); Methods in Plant Molecular Biology: A Laboratory Course Manual, Maliga,
  • the invention provides a method for monitoring stability of protein or cellular product secretion by cells in a fermenter culture, comprising: (a) harvesting from a fermenter culture a first sample of cells that secrete a protein or cellular product; (b) incorporating cells from the first sample into gel microdrops (GMDs) to form a GMD sample;
  • GMDs gel microdrops
  • the Gel Microdrop (GMD) Composition is the Gel Microdrop (GMD) Composition
  • GMDs are formed by adding a conventional cell suspension containing a large number (e.g., millions) of cells to liquefied gel (e.g., 37 °C molten agarose), dispersing in a non-aqueous medium (e.g., mineral oil) and transiently cooling to cause gelation.
  • a non-aqueous medium e.g., mineral oil
  • the resultant gel microdrops then can be removed from the non- aqueous medium and suspended in an aqueous medium.
  • Poisson statistics as a guide, the size of GMDs which have a high probability of containing zero or one initial cell within the GMD can be obtained.
  • cells are incorporated into GMDs using a CellSys 100TM Microdrop Maker (One Cell Systems, Inc., Cambridge, MA), using the CelMixTM Emulsion Matrix and CelBioGelTM Encapsulation Matrix reagents supplied by the manufacturer (One Cell Systems, Inc., Cambridge, MA), according to the manufacturer's instructions. Detection Methods
  • cells secreting a protein are encapsulated in biotin-conjugated agarose and a biotinylated antibody directed against the protein secreted by the cell is linked in the gel matrix through streptavidin. This biotinylated antibody is used to retain the secreted protein within the GMD. The amount of bound protein is then quantified using a fluorescently-labeled secondary antibody and flow cytometry. Specific cells can be selected by fluorescence activated cell sorting (FACS) on the basis of the intensity of the GMD fluorescence.
  • FACS fluorescence activated cell sorting
  • fluorescent materials with which the secondary antibody can be labeled include fluorescein isothiocyanate, rhodamine, umbelliferone, fluorescein, dichlorotriazinyl-amine fluorescein, dansyl chloride and phycoerythrin.
  • fluorescent materials include fluorescein isothiocyanate, rhodamine, umbelliferone, fluorescein, dichlorotriazinyl-amine fluorescein, dansyl chloride and phycoerythrin.
  • antibodies against non-protein based cellular products for example, antibiotics, are also known in the art (see, for example, Pauillac, et al, Immunol. Methods, 164:165-73 (1993); Karkhanis, et al, J.Clin. Microbiol, 36:1414- 1418 (1998); Tsuchiya, et al, J.
  • the cellular product of interest e.g., an antibiotic, amino acid, vitamin, carbohydrate, etc.
  • the cellular product of interest is a hapten that can be conjugated to a protein resulting in a conjugate that can be used to immunize a mammal, for example, a mouse or rabbit.
  • GMD secretion assay utilizes a biotin-streptavidin system to trap the secreted protein or cellular product within the GMD and a fluorescently-labeled secondary antibody to allow for detection and quantitation of the entrapped protein
  • a biotin-streptavidin system to trap the secreted protein or cellular product within the GMD
  • a fluorescently-labeled secondary antibody to allow for detection and quantitation of the entrapped protein
  • the key feature of the GMD secretion assay is that proteins or cellular products are secreted into the matrix of the GMD by cells encapsulated by the GMD remain entrapped within the GMD and are labeled with a detectable label to allow for detection and quantitation of the secreted protein or cellular product.
  • prosthetic group complexes can be used to link the protein or cellular product within the matrix, such as a biotin-avidin system.
  • detectable substances can be used to label the protein to allow for its detection, including, for example, various enzymes, luminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase; an example of a luminescent material includes luminol; and examples of suitable radioactive material include I,
  • steps (a) through (c) of the method as described above are repeated on a second sample of cells, wherein the second sample of cells is harvested from the fermenter culture at a time later than when the first sample of cells was harvested.
  • the precise timing of harvesting i.e., the spacing of the first and second harvest, and any other subsequent harvests will depend upon the particular type of cells cultured, the type of protein or cellular product being monitored, and the nature of the instability or instabilities that may be anticipated to occur.
  • the second sample of cells is harvested at least one day later than the first sample of cells.
  • the second sample of cells is harvested at least two days later than the first sample of cells. In yet another embodiment, the second sample of cells is harvested at least three days later than the first sample of cells. In other embodiments, the second sample of cells is harvested at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours later than the first sample of cells. In still other embodiments, the second sample of cell is harvested at least 4, 5, 6, 7, 8, 9 or 10 days later than the first sample of cells.
  • steps (a) through (c) of the method as described above can be repeated on a third sample of cells, wherein the third sample of cells is harvested from the fermenter culture at a time later than when the second sample of cells was harvested.
  • the method further comprises repeating steps (a) through (c) on a fourth sample of cells, wherein the fourth sample of cells is harvested from the fermenter culture at a time later than when the third sample of cells was harvested.
  • the method further comprises repeating steps (a) through (c) on a fifth sample of cells, wherein the fifth sample of cells is harvested from the fermenter culture at a time later than when the fourth sample of cells was harvested. Steps (a) through (c) of the method can further be repeated on yet additional samples of cells harvested from the fermenter culture over time to allow for continuous monitoring of the culture over time.
  • the method of the invention can be performed using essentially any type of cell that secretes a protein or cellular product of interest and that can be cultured in fermenter cultures.
  • the cells are eukaryotic cells, such as mammalian cells (such as CHO cells, COS cells, NS/O cells) or fungal cells, e.g., yeast cells.
  • the cells are prokaryotic (e.g., E. coli cells).
  • the cells are insect cells (e.g., a baculoviral expression system for protein production).
  • the method of the invention can be applied in the monitoring of the stability of essentially any protein of interest secreted by the cultured cells, as long as the protein can be suitably detected by the GMD secretion assay (e.g., using an antibody specific for the protein).
  • the GMD secretion assay e.g., using an antibody specific for the protein.
  • a preferred protein whose secretion is assayed is an antibody.
  • proteins whose secretion can be assayed include proteins of clinical utility, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 etc.), interferons (e.g., a-, ⁇ - or ⁇ -IFN), growth factors (e.g., GM-CSF), other cytokines (e.g., TNF ⁇ ) erythropoietin, tissue plasminogen activator, streptokinase, clotting factors (e.g., Factor VIII, Factor IX), insulin, anti-angiogenesis factors, human growth hormone (HGH) and human chorionic gonadotropin (hCG).
  • interleukins e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 etc.
  • interferons e.g., a-,
  • soluble CD4 cell surface molecules that have been engineered to be secreted from the cell (i.e., by deletion of transmembrane and cytoplasmic domains), such as soluble CD4, and fusions of polypeptides with portions of immunoglobulin molecules (e.g., CTLA4- Ig).
  • the protein may be a protein that is naturally secreted by the cell (e.g., an antibody that is secreted by a hybridoma) or the cell may be genetically engineered to secrete the protein, in which case the protein typically is encoded by an expression vector incorporated into the cells.
  • the protein is comprised of more than one subunit, the individual subunits may be encoded by different vectors incorporated into the cell (i.e., the cell may carry more than one expression vector) or the subunits may be encoded by a single expression vector within the cell.
  • Recombinant DNA techniques are well known in the art for engineering a cell to express a secreted protein.
  • the invention is also designed to provide a method for detecting a wide range of other cellular products (i.e., non-protein based molecules) expressed by a cell.
  • non-protein based molecules are the cellular products of animal cells, plant cells, or microorganisms and include molecules within the chemical classes comprising alkaloids, amino acids, carbohydrates, esters, lipids, nucleic acids, organic acids or alcohols, polyketides, and non-ribosomal peptides.
  • the cellular product can be an antibiotic, an aroma, a pharmaceutical other than an antibiotic, an enzyme, an enzyme inhibitor, a flavor, a flavor enhancer, a hormone, a pesticide, a pigment, a surfactant, a vitamin, or any agent stated in Tables 1- 9.
  • hundreds of different animal cells, plant cells, and microorganisms produce cellular products such as these and any of these molecules may be used to manufacture a corresponding antibody for use in applying the methods of the invention described herein.
  • the invention is intended to include a method for identifying any cell that produces a cellular product of interest, preferably, e.g., antibiotics, polyketide antibiotics, amino acids, antifungals, vitamins, non-antibiotic pharmaceuticals, food additives (flavors, colorants, etc.), chemicals, and polymers that can be detected using an antibody-based assay.
  • a cellular product of interest preferably, e.g., antibiotics, polyketide antibiotics, amino acids, antifungals, vitamins, non-antibiotic pharmaceuticals, food additives (flavors, colorants, etc.), chemicals, and polymers that can be detected using an antibody-based assay.
  • such a cellular product may be, a product essential for life, i.e., a primary metabolite, such as, for example, an amino acid.
  • a primary metabolite such as, for example, an amino acid.
  • the invention is intended to include methods for detecting any cell capable of producing any of the twenty amino acids, for example, lysine or tryptophan, useful as nutritional supplements for either humans or animals, for example, livestock.
  • the invention is intended to include any of the essential amino acids, e.g., histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, or arginine.
  • the invention is also intended to encompass any amino acid (modified or unmodified) that are linked to produce, for example, an artificial sweetener, such as, e.g., aspartame, which is comprised of two amino acids, aspartic acid and phenylalanine.
  • the invention may be used to monitor the production of other cellular products such as vitamins.
  • the B vitamins for example, vitamin B2-aldehyde and vitamin B2-acid are produced by Schizophyllum commune, a Basidiomycete, and, for example, riboflavin, a reduction product of B2-aldehyde which can be catalyzed by Lactobacillus casei (Tachibanna et al. , J. Nutr. Sci. Vitaminol. 25:361-366 (1979); Tachibana et al, J. Nutr. Sci. Vitaminol, 26:419-426 (1980); Tachibana et al, J. Nwtr. Sci. Vitaminol, 28:335-342 (1982).
  • Other vitamins routinely produced by microorganism that may be subjected to the detection methods of the invention include vitamin C, vitamin B 12, vitamin A, and vitamin K.
  • the cellular product produced and capable of being detected by the methods of the invention is a secondary metabolite, i.e., a nonessential product produced by the cell, such as, e.g., an antibiotic.
  • a secondary metabolite i.e., a nonessential product produced by the cell
  • Antibiotics are some of the most desirable secondary metabolites produced by microorganisms and the ability to optimally culture such organisms is an advantage of the invention.
  • the method of the invention involves the detection of a cell/s producing an antibiotic, such as penicillin produced from, for example, a strain of Penic ⁇ llium crysogenum (Aldrige, S., New Scientist (1997)).
  • antibiotics include gentamicins produced by Micromonospora purpurea (Abou-Zeid et al, Naturwiss. 133:261-275 (1978)) and Daptomycin (Cubist Pharmaceuticals, Inc.) produced from Streptomyces roseosporus (Mchenny et ⁇ /., J Bacteriol, 180:143-151 (1998)).
  • Other preferred cellular products include penicillin G or V produced by Penic ⁇ llium chrysogenum, cephalosporin C produced by Cephalosproium acremonium, and any antibiotic produced by the species Streptomyces, for example, Streptomyces rimosus (High Tech Separation News, Business Communications Co., (1998)).
  • Table 2 Microbiological Production of Various Antibiotics by Fermentation
  • the method of the invention is capable of detecting a cellular product that is member of the chemical class of polyketides, such as, for example, polyketide antibiotics.
  • Polyketides are complex natural products that are built from simple carboxylic acid monomers and are produced by a number of microorganisms. A number of naturally-occurring polyketides are highly desirable, and commercially successful, bioactive molecules useful in a wide range of applications in human and animal health (see Table 3, adapted from Kosan Sciences, Inc, http://www.kosan.com). It is known in the art that cellular products of this chemical class can be made immunogenic such that antibodies that bind the molecule can be produced. Accordingly, the skilled artisan will appreciate that the invention may be used to detect the presence and production of any of the polyketide agents presented herein and, for example, as set forth in Table 3, below.
  • the invention also provides the ability to detect novel polyketides that are produced from cells that have been manipulated using genetic engineering. It is known in the art that the enzymes in the biosynthetic pathway involved in polyketide synthesis can be manipulated to produce various "unnatural" polyketide products (see, e.g., Hutchinson, C, Bio/Technology 12:375-380 (1994); Kennedy et al., Science 284:1368-1372 (1999) and Cane et al., Science 282:63-68 (1998)). Accordingly, the novel polyketide product, using techniques described herein, can be made into a immunogen suitable for raising antibodies that can bind to the novel or "unnatural” polyketide product. The method of the invention is then modified to incorporate this antibody in order to detect cell/s expressing the variant polyketide as described. Anti-Fungal Agents
  • the invention is suitable for detecting cells producing anti-fungal agents, for example, the sphingofungins, produced by Aspergillus fumigatus (ATCC 20857) (VanMiddlesworth et al, J. Antibiot. 45:861-867 (1992)).
  • anti-proliferative agent fumagillin also produced by Aspergillus fumigatus may be monitored using the methods of the invention (Casey et al, Am. J. ofOpth. 124:521-531 (1997)).
  • antifungal molecules for agricultural application are encompassed by the invention.
  • the bacteria Pseudomonas fluorescens and other Pseudomonas strains that produce the antibiotics 2,4- diacetylphloroglucinol and pheazine-1-carboxylic acid that inhibit the fungus, G. graminis, which destroys wheat crops, may be monitored using the methods of the invention (Stelljes, K., Agricultural Research, 47:10 (1999); Pesticide & Toxic Chemical News, Information Access Co., Pub. (1997)).
  • the invention is intended to include methods of detecting cells producing other cellular products such as non-antibiotic pharmaceuticals (see, e.g., Table 4).
  • the invention also encompasses the method of detecting cells expressing a carbohydrate, preferably, a desirable polymer comprising a carbohydrate (see, e.g. , Table 5).
  • the methods of the invention may be used for the detection of any other antibiotics, food pigments, food flavorings, alkaloids, herbicides, pesticides, or any other cellular product known in the art that can be produced by an animal cell, plant cell, or microorganism under culture conditions involving a fermenter and subject to being detected by an antibody
  • any other antibiotics, food pigments, food flavorings, alkaloids, herbicides, pesticides, or any other cellular product known in the art that can be produced by an animal cell, plant cell, or microorganism under culture conditions involving a fermenter and subject to being detected by an antibody
  • the cellular product to be detected by the methods of the invention is produced by a plant cell.
  • plant cells may be used as a source of various useful pharmaceuticals, nutrients, flavorings, colorants, and antimicrobials (see, e.g., Table 9; Engineering Plants for Commercial Products and Applications, Collins, G., Ed., Ann. N. Y. Acad. of Sciences, Pub., (1997); and Plant Cell and Tissue Culture for the Production of Food Ingredients, Fu, T-J., Ed., Plenum Pub., (1999)).
  • Table 9 Cellular Products from Plants and Their Application
  • totipotent plant cells which can be grown in liquid culture, e.g., in a fermenter.
  • such cells are isolated from the root tip or meristem and cultured into a callus which can then be divided into single cells and cultured in the presence of appropriate nutrients and hormones.
  • Such cells may be selected for various properties (e.g., using growth conditions), mutagenized to acquire certain enhanced properties (e.g., using radiation), or transformed using genetic engineering to express a desirable molecule, for example, a cellular product.
  • the method of the invention allows for the monitoring of large scale fermenter cultures.
  • the fermenter culture comprises at least 10 liters of culture medium. More preferably, the fermenter culture comprises at least 50 liters of culture medium. Even more preferably, the fermenter culture comprises at least 100 liters of culture medium.
  • monitoring of fermenter cultures of 1000 or more liters can be accomplished using the method of the invention.
  • a cell population with stable expression of the protein can be recovered. This stable, recovered population can then be further cultured, if desired.
  • FACS fluorescence activated cell sorting
  • D2E7*peaBJ a vector which directs the expression of the heavy and light chains of D2E7, was used to transfect CHO cells.
  • D2E7 is a recombinant human antibody against human tumor necrosis factor alpha, described further in PCT Publication WO 97/29131.
  • the vector carries the murine dhfr gene driven by the SV40 early promoter which functions as a selection and amplification marker (Kaufman, R.J. et al. (1985) Mol. Cell. Biol. 5:1750).
  • D8/E a CHO line transfected with the D2E7 expression vector, was amplified via methotrexate selection to increase the number of copies of the vector and therefore the level of antibody secretion.
  • CHO cells were incorporated into gel microdrops (GMDs) using a CellSys 101® microdrop maker (One Cell Systems, Inc.)
  • the cells' level of antibody secretion was determined using the hybridoma secretion assay protocol supplied by One Cell Systems Inc. and modified as follows. Cells washed once with cold phosphate buffered saline were encapsulated using 350 ⁇ L of CelBioGel biotin-conjugated agarose and lOO ⁇ L of pluronic solution (Gibco/Life Technologies).
  • the resulting GMDs were washed twice and incubated with streptavidin (Gibco) at 90 ⁇ g/ml, washed again and incubated with biotin-conjugated goat anti-human IgG (Organontechnika) or biotin-conjugated murine monoclonal against human IgG (Zymed). After washing, the GMD-encapsulated cells were returned to growth medium in a 37°C incubator with a 5% CO2 atmosphere for a period of time sufficient to allow an accumulation of secreted antibody within the drop.
  • GMDs were incubated with goat anti-human IgG conjugated with fluorescein isothiocyanate (FITC) or FITC-conjugated monoclonal against human IgG (Zymed). After washing the GMDs were analyzed by flow cytometry. Gates were chosen such that only GMDs containing cells were analyzed.
  • FITC fluorescein isothiocyanate
  • methotrexate at a final concentration of 500 nM was included in the cell culture medium until the inoculation of the 100 L. fermenter. No methotrexate was added to the cell culture medium used in the 100 L fermenter or in the 1000 L fermenter used for production of D2E7.
  • Subpopulations of cells identified using the antibody secretion assay were separated using a FACStar cell sorter.
  • Cell lines established via the outgrowth of cells from individual GMDs were cultured in spinner flasks in the same medium used in the fermenters. Samples were taken daily and assayed for the number of viable cells and for the concentration of human IgG.
  • Genomic DNA was extracted from the recovered cells according to the protocol described in Current Protocols in Molecular Biology; Ausubel, F.M., Brent, R., Moore, D.M., guitarist, R.E., Seidman, J.G., Smith, J.A., and K. Struhl eds; Wiley Interscience, N.Y., N.Y. (1990).
  • Ten micrograms of DNA isolated from each culture were digested with restriction endonucleases. Digested DNAs were subjected to agarose gel electrophoresis. Each gel was loaded with 5 ⁇ g of digested genomic DNA from each of the cell banks as well as a series of standards containing known amounts of D2E7 heavy chain and light chain DNA.
  • the standards were prepared as follows.
  • the D2E7 expression vector was digested with EcoRI and N ⁇ tl to release fragments carrying the heavy chain or light chain coding regions along with their upstream adenovirus promoters.
  • the digested plasmid was diluted to concentrations which allowed the loading of D2 ⁇ 7 D ⁇ A at levels equivalent to standard copy numbers.
  • These standards were D ⁇ As in amounts equivalent to 100, 20, 8, 4, or 2 copies of these genes per genome.
  • the resulting gels were blotted to nylon membranes and hybridized to radioactively-labeled probes as described in Current Protocols. Duplicate blots were hybridized with probes which consisted of either the D2E7 heavy or light chain DNA encoding the full length coding region.
  • DNA fragments were isolated from D2E7 expression vectors by restriction endonuclease digestion with Srfl and Not I. DNA fragments were isolated and purified by gel electrophoresis and subsequent extraction. Isolated DNA fragments were radioactively labeled using the Rediprime labeling system (Amersham Corp.). This system employs a random primer technology and nick translation reaction to produce probes with high specific activity. Blots were exposed to radioactive probes in hybridization solution, described in Current Protocols in
  • the D8/E cell line carrying an expression vector for the recombinant antibody D2E7, was inoculated into a fermenter culture and cultured for a total of six weeks, as described above. Eight harvests of aliquots of the fermenter culture were taken over time and the secretion of D2E7 by cells in the culture was assessed using the gel microdrop secretion assay described above.
  • the secretion profiles of the initial fermenter inoculum (AFI 615.75) and the first harvest (AFF 610A), fourth harvest (AFF 610D) and eighth harvest (AFF 610H), illustrated by FACS histogram, are shown in Figures 1 A- ID.
  • the smaller flanking peaks in the figures represent the negative and positive control samples, which were prepared in parallel with the secretion assay and show background fluorescence and the maximal signal attainable from the microdrops, respectively.
  • the results from the secretion assays depicted in Figures 1 A- ID show that the secretion profile of cells grown directly from the stored cell banks (i.e., the fermenter inoculum) differed from that of cells recovered from the final fermenter harvest. The latter showed a distinctly bimodal distribution of expression levels while the former showed a relatively uniform level of expression.
  • EXAMPLE 3 Analysis of Subpopulations of the Fermenter Culture While the copy number experiments described in Example 2 were not able to determine if a loss of vector copies had occurred, the data from the GMD secretion assay described in Example 1 suggested that a genetic instability existed in the D8/E cell line. To confirm this hypothesis, we set out to isolate the subpopulations which produce different levels of fluorescence in the assay. These subpopulations were tested for their rate of antibody production and for the number of vector copies in the genome.
  • sample 607H The FACS histogram for the last harvest of fermentation run 607 (referred to as sample 607H) is shown in Figure 3 A, whereas the FACS histogram for the last harvest of fermentation run 702 (referred to as sample 702H) is shown in Figure 3B.
  • the FACS histogram showed two subpopulations, one which appeared to represent a population of low expression cell lines (labeled as region R3 in Figures 3 A- 3B) and one representing a population of high level expression cell lines (labeled as region R2 in Figures 3 A-3B).
  • the FACStar Plus (Becton-Dickenson) was used to isolate representatives of each of the R2 and R3 populations to allow for the determination of each populations' productivity.
  • the 702H sort contained 1.3 X 10 5 cells for the low expression population and 1.5 X 10 5 cells for the high expression population.
  • 0.5 X 10 5 cells and 1.5 X 10 5 cells were collected by sorting for the low and high level expression populations, respectively.
  • Each pool of sorted cells was divided among 3 wells of a 6 well cell culture dish and grown in fermenter culture medium containing penicillin- streptomycin and gentamycin. Upon growth to confluence, cells were transferred into spinner flask culture in fermenter culture medium containing penicillin-streptomycin and gentamycin.
  • the cultures were tested for their level of antibody productivity.
  • the low and high producer lines were cultured in spinner flasks (50 ml volume) for 4 weeks with daily cell counts and samples taken for antibody productivity determinations by the gel microdrop secretion assay. All cultures were subcultured twice a week.
  • the high expression subpopulation of the 702H sample was grown in spinner culture for an additional 6 weeks (for a total often weeks) to test for expression stability.
  • the antibody productivity of the low expressor (702hLo) and high expressor (702hHi) subpopulations of the 702 culture are illustrated in the bar graph of Figure 4, in which the antibody productivity is expressed as picograms of antibody expressed per cell in 24 hours. Each productivity value is the average of the daily values over a four day period of growth.
  • the results shown in Figure 4 demonstrate that during the extended period of culture of the high producer cell line over a period often weeks, the high level of antibody production was maintained.
  • Productivity of the high expression subpopulation was greater than three-fold higher than that of the low expression cells.
  • the vector copy number in the 607H and 702H samples, and in the 607H low, 607H high, 702H low and 702H high subpopulations were determined by Southern blot analysis.
  • the GMD secretion assay method detects the appearance of a low producer subpopulation derived from the original producer line but which has lost copies of the expression vector from its genome.
  • the gel microdrop secretion assay has been used successfully to monitor the stability of recombinant antibody secretion from transfected CHO cells.
  • the Examples and the specification sufficiently teach that the monitoring of any secreted cellular product capable of being identified using an antibody, can be accomplished using the methods of the invention.
  • the cellular product may be expressed by any living cell, from a cell in a homogeneous or heterogeneous culture, and by a cell drawn from any environment or reaction vessel in which cells can be cultured and monitored. Such equivalents are intended to be encompassed by the invention and the following claims.

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Abstract

L'invention concerne des dispositifs et un procédé pour contrôler la stabilité d'une protéine ou d'une autre substance cellulaire sécrétée par des cellules dans un milieu de fermentation. Le procédé consiste à prélever dans un milieu de fermentation un premier échantillon de cellules sécrétant une protéine ou une autre substance cellulaire, à incorporer lesdites cellules dans des microgouttes de gel (GMD), et à mesurer la protéine ou l'autre substance cellulaire sécrétée par des cellules dans un plusieurs prélèvements GMD sur une certaine durée. La protéine sécrétée par les cellules peut être, par exemple, un anticorps, une cytokine ou un facteur de croissance. Le substance cellulaire sécrétée par les cellules peut être, par exemple, un antibiotique, un acide aminé ou un hydrate de carbone.
PCT/US1999/012862 1998-06-08 1999-06-08 Procedes pour controler la stabilite d'une proteine ou d'une autre substance cellulaire secretee par des cellules dans un milieu de fermentation WO1999064560A1 (fr)

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