Effect of glucose concentration and pH on lactic acid formation was investigated in batch culture... more Effect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.
Summary Cultivation of a model hybridoma cell line in Dulbecco's modification of Eagle&am... more Summary Cultivation of a model hybridoma cell line in Dulbecco's modification of Eagle's minimal essential medium (DMEM) at various glucose and glutamine levels showed that maxmium cell growth was obtained in a broad range of glucose/glutamine ratios of 0.6 to 6.0. A new parameter called the viability index is proposed to quantify cell viability. A strong correlation between viability index
Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture ... more Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1x106 cells/ml. Concentrations of monoc[onal antibody (MAb) reached 140 rag/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during acdve exponential growth and not during the stationary phase.
Un medio de cultivo celular que comprende entre 7 mM a 30 mM de leucina, entre 7 mM a 30 mM de li... more Un medio de cultivo celular que comprende entre 7 mM a 30 mM de leucina, entre 7 mM a 30 mM de lisina, entre 7 mM a 30 mM de treonina, entre 7 mM a 30 mM de prolina y entre 7 mM a 30 mM de valina.
L'invention concerne des procedes permettant de produire une proteine en culture cellulaire c... more L'invention concerne des procedes permettant de produire une proteine en culture cellulaire comprenant un compose anti-senescence, tel que l'antioxydant carnosine. Selon l'invention, les cellules cultivees dans un milieu de culture cellulaire comprenant un compose anti-senescence presentent une viabilite et une productivite accrues. De plus, les cultures cellulaires cultivees en presence d'un compose anti-senescence presentent des niveaux reduits d'agregats de poids moleculaire eleve dans le milieu de culture cellulaire.
The present invention provides a novel method of reducing protein misfolding and aggregation in t... more The present invention provides a novel method of reducing protein misfolding and aggregation in the cell culture by growing the cell culture at a reduced temperature and/or reduced pH. As a result, the quality of the protein produced in the cell culture is greatly improved. Thus, the present invention facilitates improvements in the efficacy of therapeutic proteins produced in cell culture.
La presente invention concerne un systeme ameliore de production a grande echelle de proteines et... more La presente invention concerne un systeme ameliore de production a grande echelle de proteines et/ou de polypeptides dans une culture de cellules. Selon la presente invention, des cellules exprimant la proteine ou le polypeptide recherche sont cultivees dans un milieu qui comprend une substance inhibant la glycolyse. En plus et/ou a titre d'alternative, des cellules exprimant la proteine ou le polypeptide recherche sont cultivees dans un milieu dans lequel la glutamine est limitee. L'utilisation d'un tel systeme permet la production de niveaux eleves de proteine ou de polypeptide et diminue l'accumulation de dechets metaboliques indesirables tels que le lactate. Les proteines et les polypeptides exprimes conformement a la presente invention peuvent etre utilises de maniere avantageuse pour la preparation de compositions commerciales pharmaceutiques, immunogenes, agricoles ou autres.
In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a b... more In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage. Abbreviations: CHO-Chinese hamster ovary; rhM-CSF-recombinant human macrophage colony stimulating factor; RIA-radioimmunoassay; RP-HPLC-reverse phase high performance liquid chromatography.
Un procedimiento de produccion de una proteina en un cultivo celular que comprende las etapas de:... more Un procedimiento de produccion de una proteina en un cultivo celular que comprende las etapas de: Cultivar celulas de mamifero transformadas con un gen que codifica una proteina de interes, el cual gen se expresa en condiciones de cultivo celular, en un medio de cultivo celular que comprende un compuesto anti senescencia seleccionado de entre carnosina, acetil-carnosina, homocarnosina, anserina, y β-alanina; en el que el compuesto anti senescencia se encuentra a una concentracion de entre aproximadamente 5 mM a aproximadamente 100 mM, y mantener el cultivo en condiciones y durante un tiempo suficiente para permitir la expresion de la proteina; en el que el cultivo celular muestra una caracteristica de cultivo celular mejorada que difiere de una caracteristica de cultivo celular correspondiente que podria observarse en condiciones por lo demas identicas en un medio por lo demas identico que carece del compuesto anti senescencia; en el que la caracteristica de cultivo mejorada se sele...
A method for producing BMP-2 comprising the steps of: i) culturing a suitable host cell comprisin... more A method for producing BMP-2 comprising the steps of: i) culturing a suitable host cell comprising a DNA molecule encoding a BMP-2 in a culture medium comprising iron at a concentration of at least 2.25 uM, a polyanionic compound, and if pyridoxal is present, represents less than 55% of the molar concentration of vitamin B6 in the culture medium; and ii) recovering the protein of interest, wherein the host cell is cultured in a bioreactor having a capacity of at least 160 l, wherein the culture medium further comprises copper at a concentration of at least 10 nM; and wherein the suitable host cell is a CHO cell.
A method for producing an antibody against an Aβ fragment of the amyloid precursor protein in a l... more A method for producing an antibody against an Aβ fragment of the amyloid precursor protein in a large-scale cell culture culture comprising the steps of: providing a cell culture comprising; mammalian cells containing a gene encoding the antibody, whose gene is expressed in a cell culture condition; and a medium containing glutamine and having a characteristic medium selected from the group consisting of: (i) a cumulative amount of amino acids per unit volume greater than about 70 mM, (ii) a cumulative molar ratio of glutamine with respect to asparagine accumulated of less than about 2, (iii) a cumulative molar ratio of glutamine with respect to the total accumulated amino acids of less than about 0.2, (iv) a molar ratio of accumulated inorganic ions with respect to the total accumulated amino acids between approximately 0.4 to 1, (v) a cumulative amount of glutamine and asparagine combined per unit volume greater than approximately 16 mM, and combinations thereof; maintaining said ...
Effect of glucose concentration and pH on lactic acid formation was investigated in batch culture... more Effect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.
Summary Cultivation of a model hybridoma cell line in Dulbecco's modification of Eagle&am... more Summary Cultivation of a model hybridoma cell line in Dulbecco's modification of Eagle's minimal essential medium (DMEM) at various glucose and glutamine levels showed that maxmium cell growth was obtained in a broad range of glucose/glutamine ratios of 0.6 to 6.0. A new parameter called the viability index is proposed to quantify cell viability. A strong correlation between viability index
Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture ... more Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1x106 cells/ml. Concentrations of monoc[onal antibody (MAb) reached 140 rag/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during acdve exponential growth and not during the stationary phase.
Un medio de cultivo celular que comprende entre 7 mM a 30 mM de leucina, entre 7 mM a 30 mM de li... more Un medio de cultivo celular que comprende entre 7 mM a 30 mM de leucina, entre 7 mM a 30 mM de lisina, entre 7 mM a 30 mM de treonina, entre 7 mM a 30 mM de prolina y entre 7 mM a 30 mM de valina.
L'invention concerne des procedes permettant de produire une proteine en culture cellulaire c... more L'invention concerne des procedes permettant de produire une proteine en culture cellulaire comprenant un compose anti-senescence, tel que l'antioxydant carnosine. Selon l'invention, les cellules cultivees dans un milieu de culture cellulaire comprenant un compose anti-senescence presentent une viabilite et une productivite accrues. De plus, les cultures cellulaires cultivees en presence d'un compose anti-senescence presentent des niveaux reduits d'agregats de poids moleculaire eleve dans le milieu de culture cellulaire.
The present invention provides a novel method of reducing protein misfolding and aggregation in t... more The present invention provides a novel method of reducing protein misfolding and aggregation in the cell culture by growing the cell culture at a reduced temperature and/or reduced pH. As a result, the quality of the protein produced in the cell culture is greatly improved. Thus, the present invention facilitates improvements in the efficacy of therapeutic proteins produced in cell culture.
La presente invention concerne un systeme ameliore de production a grande echelle de proteines et... more La presente invention concerne un systeme ameliore de production a grande echelle de proteines et/ou de polypeptides dans une culture de cellules. Selon la presente invention, des cellules exprimant la proteine ou le polypeptide recherche sont cultivees dans un milieu qui comprend une substance inhibant la glycolyse. En plus et/ou a titre d'alternative, des cellules exprimant la proteine ou le polypeptide recherche sont cultivees dans un milieu dans lequel la glutamine est limitee. L'utilisation d'un tel systeme permet la production de niveaux eleves de proteine ou de polypeptide et diminue l'accumulation de dechets metaboliques indesirables tels que le lactate. Les proteines et les polypeptides exprimes conformement a la presente invention peuvent etre utilises de maniere avantageuse pour la preparation de compositions commerciales pharmaceutiques, immunogenes, agricoles ou autres.
In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a b... more In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage. Abbreviations: CHO-Chinese hamster ovary; rhM-CSF-recombinant human macrophage colony stimulating factor; RIA-radioimmunoassay; RP-HPLC-reverse phase high performance liquid chromatography.
Un procedimiento de produccion de una proteina en un cultivo celular que comprende las etapas de:... more Un procedimiento de produccion de una proteina en un cultivo celular que comprende las etapas de: Cultivar celulas de mamifero transformadas con un gen que codifica una proteina de interes, el cual gen se expresa en condiciones de cultivo celular, en un medio de cultivo celular que comprende un compuesto anti senescencia seleccionado de entre carnosina, acetil-carnosina, homocarnosina, anserina, y β-alanina; en el que el compuesto anti senescencia se encuentra a una concentracion de entre aproximadamente 5 mM a aproximadamente 100 mM, y mantener el cultivo en condiciones y durante un tiempo suficiente para permitir la expresion de la proteina; en el que el cultivo celular muestra una caracteristica de cultivo celular mejorada que difiere de una caracteristica de cultivo celular correspondiente que podria observarse en condiciones por lo demas identicas en un medio por lo demas identico que carece del compuesto anti senescencia; en el que la caracteristica de cultivo mejorada se sele...
A method for producing BMP-2 comprising the steps of: i) culturing a suitable host cell comprisin... more A method for producing BMP-2 comprising the steps of: i) culturing a suitable host cell comprising a DNA molecule encoding a BMP-2 in a culture medium comprising iron at a concentration of at least 2.25 uM, a polyanionic compound, and if pyridoxal is present, represents less than 55% of the molar concentration of vitamin B6 in the culture medium; and ii) recovering the protein of interest, wherein the host cell is cultured in a bioreactor having a capacity of at least 160 l, wherein the culture medium further comprises copper at a concentration of at least 10 nM; and wherein the suitable host cell is a CHO cell.
A method for producing an antibody against an Aβ fragment of the amyloid precursor protein in a l... more A method for producing an antibody against an Aβ fragment of the amyloid precursor protein in a large-scale cell culture culture comprising the steps of: providing a cell culture comprising; mammalian cells containing a gene encoding the antibody, whose gene is expressed in a cell culture condition; and a medium containing glutamine and having a characteristic medium selected from the group consisting of: (i) a cumulative amount of amino acids per unit volume greater than about 70 mM, (ii) a cumulative molar ratio of glutamine with respect to asparagine accumulated of less than about 2, (iii) a cumulative molar ratio of glutamine with respect to the total accumulated amino acids of less than about 0.2, (iv) a molar ratio of accumulated inorganic ions with respect to the total accumulated amino acids between approximately 0.4 to 1, (v) a cumulative amount of glutamine and asparagine combined per unit volume greater than approximately 16 mM, and combinations thereof; maintaining said ...
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