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WO1988001643A1 - Procede de culture cellulaire - Google Patents

Procede de culture cellulaire Download PDF

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Publication number
WO1988001643A1
WO1988001643A1 PCT/US1987/002089 US8702089W WO8801643A1 WO 1988001643 A1 WO1988001643 A1 WO 1988001643A1 US 8702089 W US8702089 W US 8702089W WO 8801643 A1 WO8801643 A1 WO 8801643A1
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WO
WIPO (PCT)
Prior art keywords
cell
glucose
cell line
time
interest
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1987/002089
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English (en)
Inventor
David P. Sours
Bradley G. Andersen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Endotronics Inc
Original Assignee
Endotronics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Endotronics Inc filed Critical Endotronics Inc
Publication of WO1988001643A1 publication Critical patent/WO1988001643A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte

Definitions

  • the present invention relates to cell culturing, and in particular, it relates to the control of glucose delivery and pH and lactate maintenance in a continuous culturing system.
  • Continuous culturing systems have been found to be highly successful in culturing cells for achieving both high cell densities and for providing environmental conditions to cells, such as mammalian cells. Mammalian cells, in turn, have recently been employed to produce useful biological products, such as insulin, interferons and ' other proteins or particles.
  • ACUSYST-P cell culturing system manufactured by Endotronics, Inc. of Coon Rapids, Minnesota.
  • This system employs the use of hollow fiber cartridges wherein a medium containing nutrients and other factors is delivered through the lumens of the hollow fibers and cells are cultured in an extracapillary space (ECS) between the hollow fibers and the inside surface of the cartridge ⁇ walls.
  • ECS extracapillary space
  • Nutrients and other factors pass through the semipermeable membrane wall of the hollow fibers into the ECS while waste products pass from the ECS through the semipermeable membrane walls into the media and are carried away. If the cell line produces a useful biological product, this is retained in the cell culturing space due to the selected porosity of the hollow fiber membrane.
  • Such continuous cell culturing systems provide glucose and maintain pH and lactate at set points that have been emperically determined for a cell line of interest and then kept constant throughout the growth phase of the cell, the production phase of the cell line and the death phase of the cell line.
  • Static culturing systems which were in use well prior to the ' hollow fiber continuous culturing systems, provide glucose and maintain pH and lactate using a cruder approach that involves removing media from the static culturing vessel, at a selected time interval, such as once a day, and then replacing that media.
  • the present invention includes a method of culturing cells wherein set point values for pH, glucose and lactate are determined for optimum growth and maintenance of a cell line of interest and the cell line of interest is then cultured in a continuous culture system using those set points. In addition, separate glucose delivery and pH and lactate maintenance set points are determined and used for maximum cell production if the cell line of interest is used to produce a biological product.
  • the method includes culturing a cell line of interest in a static culture and obtaining data for pH, glucose and lactate levels, cell count and biological product concentration, all with respect to time.
  • Values for pH, glucose and lactate are determined at the maximum growth rate of the cell line of interest, the greatest increase in cell count, the maximum cell count and at a point of maximum production per cell of the biological product. These values are then used as set points to control pH, lactate and glucose in a continuous culturing system, such as a hollow fiber cartridge system, maximizing growth and providing conditions for optimum maintenance of the cells and, if desired, production of a biological product by the. cells.
  • pH level is maintained in the continuous culturing system corresponding to a pH value occurring at a point in time of greatest percentage increase in cell number in the static culture of the cell line of interest.
  • Glucose and lactate in the continuous culturing system are maintained at concentrations corresponding to glucose and lactate concentrations at a point in time of greatest quantitative increase in cell number in the static culture of the cell line of interest.
  • the pH set point is changed when the cell count is. at a maximum to a pH level corresponding to a pH value occurring at the maximum cell count in the static culture of the cell line of interest.
  • the glucose and lactate setpoints are changed in the continuous culturing system to concentrations corresponding to glucose and lactate concentrations at a time where greatest production per cell occurs in the static culture of the cell line of interest.
  • Figures 1 and 2 are graphic views of kinetic data from an AFP-27 murine hybridoma cell line.
  • Figure 3 is a graphic view of the meta- bolic growth parameters with respect to time using the growth phase and the production phase set points of the present invention on the AFP-27 murine hybri ⁇ doma cell line.
  • Figure 4. is a . graphic view comparing * meta- bolic growth parameters of the AFP-27 murine hybri- ' - do a cell line between the process of the present invention and a prior art process.
  • Figure 5 is a graphic representation of IgG production per day comparing the process of the present invention with the prior art process in an ACUSYST-P.
  • the present invention includes a method of culturing cells in a continuous culturing system such as a perifusion system used in culturing cells in hollow fiber cartridges .
  • the method of the present invention provides set point values for glucose, lactate and pH that maximize cell growth and set points for maintenance of and production by the cells if a useful biological product is to be produced by the cells.
  • the method of the present invention includes, determining continuous culturing set points for pH, glucose and lactate from data generated by static culturing of the cell line of interest. Values for pH,- glucose delivery and lactate production are then determined at the time of maximum growth rate of the cells, greatest increase in cell count, maximum cell count and greatest production of biological products on a per cell basis.
  • maximum growth rate is meant the greatest percentage increase in cell number at a point in time. The growth rate is determined by the following equation:
  • greater increase in cell count is meant the greatest numerical increase in cell count at a particular point in time. The greatest increase in cell count is found where the slope of a curve defined by a cell count curve generated by the static culture data of the cell line of interest is the highest. The slope is calculated by inserting the time value into the first derivative of the curve representing cell count.
  • the ' continuous culture process is divided into- two par-ts, the growth phase and the production phase.
  • growth phase is meant that phase of culturing wherein the cells are increasing in number, the growth phase ending at a point wherein a curve defining the increase in cell number over time reaches a minimum slope.
  • production phase is meant that phase occurring immediately after the growth phase wherein the cells are placed in an environment using glucose, lactate and pH levels for the specific purpose of maintenance of the cell population and for the production of a biological product and not for cell growth or proliferation.
  • the pH value occurring at the time period wherein maximum growth rate occurs in the static culture is used as a pH set point in the continuous culturing system.
  • the pH is maintained as close as possible to " that pH value for ensuring maximum rate of growth of the cell -line of interest.
  • Glucose delivery and lactate production are maintained at levels which are based on values either occurring at the time of maximum growth rate or at the time of greatest increase in cell count. If the cell count in the continuous system at the maximum growth rate of the static culture is too small for proper consumption of the glucose level, then the glucose value and lactate value issued ,at the point in time where greatest increase in .cell number occurs in the static culture. .'Although the glucose level at the greatest increase in cell count is typically the choice, this is a matter of discretion depending on the particular cell line of * interest.
  • the pH, glucose and lactate set points are changed at a point in time corresponding to maximum cell number in the continuous cell culturing system (i.e., cell count is at a maximum) .
  • a pH set point is chosen based on the pH value occurring at the point in time where the slope of the cell count curve is at a minimum in the. static culturing system.
  • Set point values for glucose and lactate are chosen based on their values when the production per cell in the static culture is at a maximum. These values occur in the static culturing system typically at a point in time after the cell count has reached a maximum.
  • the data resulting from the assays was used by a computer program to generate curves using the cubic spline curve-fitting method.
  • the curves were used to determine the maximum growth rate, greatest increase in cell count, maximum cell count and greatest production rates per cell number for each hour of the static culture.
  • FIG. 1 A typical hybridoma cell "fingerprint" from this computer model is illustrated in Figure 1.
  • cells grown in a static culture follow a predictable pattern of an initial lag phase and an exponential growth phase, slowing to a preliminary stationary phase, a stationary phase and a subsequent death phase.
  • Glucose consumption as illustrated by the glucose curve follows an initial lag phase, followed by an exponential phase concomitant with the cell growth phase.
  • Lactate is produced in approximately a 1:1 ratio to glucose consumption. Lactate level is controlled by increasing or decreasing medium flow.
  • pH is initially in the range of 7.35 to 7.45 and steadily decreases with time. Late in the death phase, pH shows a slight increase.
  • product accumulates proportionately to cell growth, but continues to increase even during the death phase.
  • the increase in product concentration during the death phase is associated with increased production per cell, not with intracellular product released by lysed cells.
  • the factors to which the 'shift from stationary phase to death phase is commonly attributed are low nutrient levels, high metabolic waste products, low pH levels or cellular-produced feedback inhibition.
  • the exact mechanism causing cell population to shift into the death phase is probably an intricate combination of all these factors and perhaps other factors, as yet undefined.
  • Amino Acid Data for AFP-27 Cells Grown in McCoy 5A Media Cell/ml is (x 10*5), amino acid data is in micromolar
  • the pH value for the continuous culture set point was selected at the apex (minimum slope of the curve) of the cell count curve.
  • the metabolic growth parameters, glucose uptake rate and lactate uptake rate showed that if the lactate, glucose and pH levels were controlled as indicated previously, the metabolic rates increased exponentially. Since metabolic rates are primarily dependent on a viable cell number, it is believed that the cell population in the hollow fiber cartridge increased at a similar rate. Thus, the doubling time of the cells in the hollow fiber cartridge system was about 22 hours, compared to 18 hours in the static culture system.
  • Figure 5 illustrates the difference in production rates for the runs of Figure 4.
  • the process of the present invention there was an increase in production of about 100% over the cells that were cultured using the prior art set points.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Des cellules sont cultivées dans un système de culture en continu, dans lequel l'apport de glucose et le maintien du pH sont basés sur des valeurs déterminées dans une culture statique de la ligne cellulaire étudiée. Dans le but d'obtenir une croissance maximale de la ligne cellulaire dans le système de culture en continu, on y maintient un niveau de pH correspondant à une valeur pH apparaissant au moment de la plus forte augmentation en pourcentage du nombre de cellules dans une culture statique de la ligne cellulaire étudiée. On maintient les niveaux de glucose et de lactate dans le système de culture en continu à des niveaux correspondant au moment de la plus forte augmentation quantitative du nombre de cellules dans la culture statique de la ligne cellulaire étudiée. Si cette dernière est une ligne cellulaire utilisée pour fabriquer un produit biologique utile, on modifie alors la valeur pH de consigne, à un comptage cellulaire maximal, en une valeur correspondant à la valeur pH apparaissant au moment où le comptage cellulaire est à un niveau maximal dans la culture statique, et on modifie les valeurs de consigne de glucose et de lactate en des valeurs correspondant à un moment où la production du produit biologique par cellule par unité de temps est la plus forte dans la culture statique.
PCT/US1987/002089 1986-08-29 1987-08-21 Procede de culture cellulaire Ceased WO1988001643A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90227786A 1986-08-29 1986-08-29
US902,277 1986-08-29

Publications (1)

Publication Number Publication Date
WO1988001643A1 true WO1988001643A1 (fr) 1988-03-10

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387840A1 (fr) * 1989-03-14 1990-09-19 Hitachi, Ltd. Méthode et appareil pour contrôler les conditions de culture pour des cellules animales
US5856179A (en) * 1994-03-10 1999-01-05 Genentech, Inc. Polypeptide production in animal cell culture
EP1609853A1 (fr) 1995-06-06 2005-12-28 F.Hoffmann-La Roche Ag Procéde de regulation de la sialylation de proteines produites par une culture de cellules de mammiferes
US8309347B2 (en) 2007-03-05 2012-11-13 Terumo Bct, Inc. Cell expansion system and methods of use
US8691565B2 (en) 2008-03-05 2014-04-08 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US8906688B2 (en) 2007-04-13 2014-12-09 Terumo Bct, Inc. Cell expansion system and methods of use
US9617506B2 (en) 2013-11-16 2017-04-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US9677042B2 (en) 2010-10-08 2017-06-13 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10077421B2 (en) 2014-04-24 2018-09-18 Terumo Bct, Inc. Measuring flow rate
US10577576B2 (en) 2012-08-20 2020-03-03 Terumo Bct, Inc. System for expanding cells
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941662A (en) * 1971-06-09 1976-03-02 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Apparatus for culturing cells
US4444882A (en) * 1980-11-26 1984-04-24 Hitachi, Ltd. Process and apparatus for controlling cultivation of microorganisms
US4468455A (en) * 1980-12-02 1984-08-28 Phillips Petroleum Company Cell culture control

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941662A (en) * 1971-06-09 1976-03-02 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Apparatus for culturing cells
US4444882A (en) * 1980-11-26 1984-04-24 Hitachi, Ltd. Process and apparatus for controlling cultivation of microorganisms
US4468455A (en) * 1980-12-02 1984-08-28 Phillips Petroleum Company Cell culture control

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5304483A (en) * 1989-03-14 1994-04-19 Hitachi, Ltd. Controlling cultivation conditions for animal cells
EP0387840A1 (fr) * 1989-03-14 1990-09-19 Hitachi, Ltd. Méthode et appareil pour contrôler les conditions de culture pour des cellules animales
US5856179A (en) * 1994-03-10 1999-01-05 Genentech, Inc. Polypeptide production in animal cell culture
US6180401B1 (en) 1994-03-10 2001-01-30 Genentech, Inc. Polypeptide production in animal cell culture
EP1609853A1 (fr) 1995-06-06 2005-12-28 F.Hoffmann-La Roche Ag Procéde de regulation de la sialylation de proteines produites par une culture de cellules de mammiferes
US9260698B2 (en) 2007-03-05 2016-02-16 Terumo Bct, Inc. Cell expansion system and methods of use
US8309347B2 (en) 2007-03-05 2012-11-13 Terumo Bct, Inc. Cell expansion system and methods of use
US8785181B2 (en) 2007-03-05 2014-07-22 Terumo Bct, Inc. Cell expansion system and methods of use
US8906688B2 (en) 2007-04-13 2014-12-09 Terumo Bct, Inc. Cell expansion system and methods of use
US9428729B2 (en) 2008-03-05 2016-08-30 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US10577582B2 (en) 2008-03-05 2020-03-03 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US8691565B2 (en) 2008-03-05 2014-04-08 Terumo Bct, Inc. Method of reseeding adherent cells grown in a hollow fiber bioreactor system
US10870827B2 (en) 2010-10-08 2020-12-22 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US9677042B2 (en) 2010-10-08 2017-06-13 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US9725689B2 (en) 2010-10-08 2017-08-08 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11746319B2 (en) 2010-10-08 2023-09-05 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10669519B2 (en) 2010-10-08 2020-06-02 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10577576B2 (en) 2012-08-20 2020-03-03 Terumo Bct, Inc. System for expanding cells
US10633625B2 (en) 2013-11-16 2020-04-28 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US9617506B2 (en) 2013-11-16 2017-04-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US10557112B2 (en) 2013-11-16 2020-02-11 Terumo Bct, Inc. Expanding cells in a bioreactor
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US10077421B2 (en) 2014-04-24 2018-09-18 Terumo Bct, Inc. Measuring flow rate
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11999929B2 (en) 2016-06-07 2024-06-04 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion

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