• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    improved cell culture medium. the present invention relates to a cell culture medium with a high content of choline chloride. the cell culture media also comprise only moderate amounts of amino acids, in particular, the amount of glutamine in the cell culture media is limited. cell culture media with a high choline chloride content are particularly suitable for batch-fed cell culture, so cell viability remains at a higher level for a longer time and high polypeptide titrations, although limited amounts are used of amino acids.
    公开号:BR112012027434B1
    申请号:R112012027434-1
    申请日:2011-04-25
    公开日:2020-09-29
    发明作者:Wolfgang Ernst Gustav Budach；Helene C.Chassin；Kerstin Dorsch
    申请人:Novartis Ag；
    IPC主号:
    专利说明:

    Technical Field of the Invention
    [001] The present invention relates to the general field of biotechnology, particularly cell culture and its use for the production of polypeptides on an industrial scale.
    [002] The present invention provides cell culture media with a high content of choline chloride that allows the culture of cells with high cell viability over an extended period of time. The cell culture media according to the present invention also allows obtaining high polypeptide productivity and / or improved product quality, when used for the production of a polypeptide by recombinant polypeptide expression, using cell culture systems CHO, particularly on an industrial scale. Technical Background of the Invention
    [003] The preparation of polypeptides using recombinant technology has become a standard procedure over the past couple of decades. Access to the recombinant polypeptides by cloning the genes encoding the respective polypeptide followed by the subsequent transformation of suitable expression hosts with the gene to be expressed and the final production and purification of the obtained recombinant polypeptide product has provided access to a new class of substances biologically designed and produced therapies.
    [004] Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry, using recombinant DNA technology, followed by production processes developed in the field of bioengineering.
    [005] Such biological products include monoclonal antibodies, which have been developed for important treatment options in various fields of medicine, including autoimmune diseases, inflammatory disorders, immunosuppression, oncology or the like.
    [006] The development of such therapeutic substances of biological origin requires production on an industrial scale, thus providing access to large amounts of recombinant polypeptide. Preferred expression systems are mammalian cell cultures that are superior to most eukaryotic systems, based on insect, yeast or similar cells, or even traditional prokaryotic expression systems.
    [007] However, the culture of mammalian cells includes enormous challenges, especially on an industrial scale. Production facilities for culturing mammalian cells require complete optimization of many process conditions.
    [008] One of the most important process parameters for controlling the general production process is the medium in which the cells are grown. Appropriate cell culture media should provide cell cultures with all necessary nutrients, which is especially difficult if there are no components of animal origin, such as serum or proteins, for example, growth factors are added to the media.
    [009] In addition, mammalian cell cultures require particular supplementary components at different stages of the polypeptide production process. Therefore, cell culture media must provide the necessary substrates during a) initial growth and proliferation of host cells at lower densities, b) subsequent cultivation of cells at high density, c) the actual process of polypeptide formation in cultured cells.
    [0010] The general process for the production of recombinant polypeptide preferably comprises an expansion phase and a production phase. During the expansion phase, the host cells are cultured at high densities, using a growth medium in order to maximize subsequent polypeptide production later. During the production phase, the actual formation of the desired polypeptide in large quantities is achieved through the use of a production medium. In order to satisfy the specific metabolic requirements of cells at each stage of the general polypeptide production process, compositions of different media have been designed for the expansion and production phases, respectively. For example, the means of production often contain higher amounts of amino acids than the growth medium.
    [0011] Consequently, considerable efforts have been made in the past to develop cell culture media with special emphasis on their use for large-scale production of polypeptides. However, the continuous improvement of cell culture media is still an important objective, in order to further maximize polypeptide production in terms of product quality and quantitative yields.
    [0012] Many components of cell culture media have been investigated in the past in terms of their role in the production of polypeptides. Possible goals are inorganic salts, amino acids, carbon sources like glucose, or vitamins.
    [0013] For example, it has been shown that supplementation of compounds such as vitamins, choline chloride or amino acids can increase the viability and productivity of cells cultured under protein-free conditions (Kim do Y et. Al., Cytotechnology 2005, 47, 37-49).
    [0014] Choline chloride is a normal component of cell culture media, which serves as a phospholipid precursor for cells. After being collected and processed by the cells, it ends, in addition to phosphatidylethanolamine and phosphatidylinositol as one of the main phospholipids in the cell membranes called phosphatidyl choline.
    [0015] Cell culture media commonly used as D-MEM (Dulbecco's Modified Eagle Medium) and D-MEM / F-12 have been widely used for the growth of a wide range of mammalian cell lines. These media include amounts of choline chloride of 4 mg / L and 8.98 mg / L, respectively.
    [0016] Other commercially available media such as Ham's F-12 (commercially available from BioConcept) and MEM (commercially available from HyClone) also comprise small amounts of choline chloride of 13.96 mg / L and 56 mg / LT, respectively.
    [0017] US 6,180,401 describes an improved method for producing a polypeptide in the culture of animal cells. One objective is to increase the concentration of the final product. Several parameters are modified in order to maximize the yield of the product in the production phase, including glucose concentration, osmolality and glutamine concentration. US 6,180,401 discloses cell culture media, which have a choline chloride content of 50.86 mg / L.
    [0018] US 5,122,469 describes a culture medium for the propagation of a series of mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, and allows the cultivation of cells in high densities as monolayers or in suspension suitable for small and large scale propagation of mammalian cells. An additional advantage is improved product performance. The medium is a chemically defined culture medium containing high levels of certain amino acids. The choline chloride content is 50.86 mg / L.
    [0019] Only very few media with high choline chloride content are known in the prior art. Waymouth described a cell culture medium that can be used to culture the cell line of the mouse fibroblast connective tissue L929 (C. Waymouth, J. Natl. Cancer. Inst., 1959, 22, 1003-1017). This medium is a chemically defined synthetic serum-free medium and has a choline chloride content of 250 mg / L. This medium is commercially available under the name Waymouth Medium MB 752/1 (BioConcept and Sigma-Aldrich). The known applicability is limited to whole organ culture, the establishment of carcinoma cell lines from pleural effusions and the growth of potentially tumorigenic cells before their in vivo evaluation.
    [0020] WO 02/101019 describes two medium compositions with relatively high choline chloride content, 101.72 mg / L and 209.40 mg / L, respectively. These media were used to study the impact of glutamine and glutamate on the production of recombinant protein. However, both media still contained large amounts of glutamine.
    [0021] Information available from the prior art is limited as to the role of choline chloride content in cell culture media for the production of the polypeptide in question. US 6,048,728 briefly discusses the role of choline chloride content in cell culture media for the production of biological products using hybridoma cells. In the case of cells expressing antibody, the secretion of maximum amounts of antibodies was observed in media containing a choline supplement greater than 4 mg / L and preferably about 4 to 75 mg / L, together with the other reagents of the Primary Supplement. At these concentrations, choline is described as not being limiting and without apparent toxicity.
    [0022] Production cell culture media, especially those designed for use in large-scale industrial production of recombinant polypeptides require an increase in the amount of components, for example, amino acids.
    [0023] However, highly concentrated cell culture media show limited solubility of components from selected media. Limited solubility represents a technical disadvantage because highly concentrated media for large-scale production bear the risk of precipitation of individual components, for example, during the production phase and, especially, during storage. This can lead to variations in the composition of the media and a deterioration of cell culture conditions at the critical point of product formation.
    [0024] As another consequence, precipitation leads to an effective removal of precious components from the media from the actual production process. Additional recycling processes designed to overcome such drawbacks are technically difficult to perform and require additional effort in terms of resources and time. Less concentrated cell culture media, when equally effective in polypeptide production, would allow significant cost reductions to be achieved in industrial production processes.
    [0025] Considering the above challenges and the existing drawbacks, there is a continuing need in the field of industrial biotechnology for improved culture media that allow the production of recombinant polypeptides on an industrial scale with even higher yields, that is, increased specific productivity and general, and the increase in product quality. Improved cell culture media are especially desirable for improving productivity during the production phase.
    [0026] A specific technical objective of polypeptide production processes is to maintain the viability of the highest cells at the end of the production process, in order to maximize the final yield of the polypeptide, in particular due to the prolongation of production time. In addition, the reduction of aggregation of the formed recombinant polypeptide and the improved quality of the product, particularly in terms of post-translation modifications, such as the glycosylation pattern, are also an important technical objective.
    [0027] Finally, improved means of production for large-scale production of polypeptides are desirable, which contain reduced amounts of components while being equally effective, or even better in terms of cell growth, polypeptide productivity, quality of recombinant polypeptide and polypeptide functionality. Summary of the invention
    [0028] In order to solve the aforementioned technical challenges, the present invention provides cell culture media with a high choline chloride content, which leads to an unexpected improvement in specific cell productivity and cell viability, especially in later stages of biotechnological production processes. In addition, the quality of the recombinant product through the use of cell culture media can also be surprisingly improved. The cell culture media according to the present invention are especially suitable for use during the production phase. Therefore, the present invention allows for the production of recombinant polypeptide from CHO cells.
    [0029] Cell culture media can be used in particular as means of production, in order to achieve high cell growth, high viable cell densities and high polypeptide titration during the production phase. It has also been found that the quality of the product, in terms of less aggregation and / or better post-translational modification, such as improved glycosylation pattern of the recombinant product, can be improved by using the cell culture media according to the present invention.
    [0030] In the present invention, choline chloride is preferably used. However, other sources of choline, for example choline hydroxide, choline tartrate / bitartrate, choline sulfate, choline phosphate, or any other choline compound based on the use of a different counterion are also suitable for use in media cell culture according to the present invention. If these other choline compounds are used, their amount is preferably chosen in order to achieve the same molar choline concentration as is achieved using choline chloride in the concentration ranges and values indicated above, that is, the other choline salt it is preferably present in a concentration equivalent to the concentration of choline chloride as outlined. This also applies to the specific aspects and modalities mentioned below.
    [0031] According to the first aspect of the present invention, a cell culture medium is provided with a choline chloride content in the range of 60 mg / L to 2500 mg / L. The choline chloride content in the cell culture medium can be 80 mg / L or higher, alternatively 160 mg / L or higher, 200 mg / L or higher or 220 mg / L or higher. The content of choline chloride in the cell culture medium is limited to 2500 mg / L, alternatively 1000 mg / L, 840 mg / L, 500 mg / L or 300 mg / L. Choline chloride can be present in a concentration of about 240 mg / L.
    [0032] The cell culture medium according to the first aspect of the invention further comprises only a reduced content of amino acids expressed by a total amino acid concentration of 20 to 57 mmol / L. Alternatively, the total concentration of amino acids is greater than 25 mmol / L, greater than 30 mmol / L, greater than 35 mmol / L or even greater than 40 mmol / L. In addition, the total concentration of amino acids can be less than 54 mmol / L. The total concentration of amino acids can, for example, be about 51 mmol / L.
    [0033] In addition, the cell culture medium optionally comprises a reduced glutamine content. In particular, glutamine is present in a concentration of 500 to 1400 mg / L, alternatively 800 to 1400 mg / L or even 900 to 1200 mg / L.
    [0034] The amino acid content in the cell culture medium according to the first aspect of the present invention can optionally include the following amino acids in the following concentrations expressed in mmol / L: Arginine 4.0 - 6.0, preferably 4.5 - 5.5 Asparagine 3.0 - 6.0, preferably 4.0 - 5.5 Aspartic Acid 2.5 - 4.0, preferably 3.0 - 3.6 Glycine 0.3 - 0.8, preferably 0 , 5 - 0.7 Histidine 0.6 -1.0, preferably 0.7 - 0.9 Isoleucine 2.0 - 5.0, preferably 3.0 - 4.0 Leucine 3.0 - 7.0, preferably 3.5 - 6.0 Lysine 2.0 - 4.0, preferably 2.5 - 3.5 Methionine 1.0-1.5, preferably 1.2 -1.4 Phenylalanine 1.0 - 2.0, preferably 1.3-1.8 proline 2.5 - 6.0, preferably 3.0 - 5.5 serine 3.0 - 8.0, preferably 4.0 - 7.0 threonine 2.0 - 3.5 , preferably 2.5 - 3.1 Tryptophan 0.4 -1.0, preferably 0.5 - 0.8 Valine 2.5 - 5.0, preferably 3.0 - 4.5 Tyrosine 1.0 - 2, 0, preferably 1.2 - 1.8 Cystine 0.5 -1.0, preferably 0.6 - 0.8
    [0035] Cell culture media are preferably serum-free and protein-free. Preferably, they are also free of protein hydrolysates.
    [0036] According to a second aspect of the present invention, a process for the production of a recombinant polypeptide is provided comprising a production step, in which recombinant CHO cells are cultured in cell culture media according to the first aspect of the invention.
    The prepared recombinant polypeptide is, in particular, a recombinant antibody.
    [0038] In the process of the invention, the cells are preferably grown in a fed batch process. Brief Description of Drawings
    [0039] In Figure 1 to Figure 8, the three media are growth medium with low choline content (♦ (diamond), control 1), production medium (A (triangle), control 2) and growth medium with high choline content, ie low choline growth medium supplemented with an additional 200 mg / L choline chloride (■ (square)).
    [0040] Figure 1 shows normalized cell densities of viable cells expressing mAb1 grown in shake flasks as a function of time in three different cell culture media (Experiment 1).
    [0041] Figure 2 represents the viability of cells expressing mAb1 cultured in shake flasks as a function of time in three different cell culture media (Experiment 1).
    [0042] Figure 3 represents the normalized polypeptide titer obtained after culturing cells expressing mAb1 in shake flasks as a function of time for three different media (Experiment 1).
    [0043] Figure 4 illustrates normalized viable cell densities of cells expressing mAb2 grown in shake flasks as a function of time in three different cell culture media (Experiment 2).
    [0044] Figure 5 represents the viability of cells expressing mAb2 grown in shake flasks as a function of time in three different cell culture media (Experiment 2).
    [0045] Figure 6 represents the normalized polypeptide titer obtained after culturing cells expressing mAb2 in shake flasks as a function of time for three different media (Experiment 2).
    [0046] Figure 7 represents normalized viable cell densities of cells expressing mAb3 grown in shake flasks as a function of time in three different cell culture media (Experiment 3).
    [0047] Figure 8 represents the viability of cells expressing mAb3 cultured in shake flasks as a function of time in three different cell culture media (experiment 3).
    [0048] Figure 9 shows the normalized polypeptide titer obtained after culturing cells expressing mAb3 in shake flasks as a function of time for three different media (experiment 3).
    [0049] Figure 10 shows the aggregation rate of mAb3 produced after seven days of cultivation in shake flasks. The rate of aggregation is measured by size exclusion chromatography (SEC). The error bars represent standard deviations of three biological replicates.
    [0050] Figure 11 shows normalized cell densities of viable cells expressing mAb3 grown in a batch run fed to a bioreactor as a function of time in three different cell culture media (Experiment 4).
    [0051] Figure 12 illustrates the viability of cells expressing mAb3 grown in a batch run fed from a bioreactor as a function of time in three different cell culture media (Experiment 4).
    [0052] Figure 13 represents the normalized polypeptide titer obtained using cells that express mAb3 in a batch operation fed from a bioreactor as a function of time using three different media (Experiment 4).
    [0053] Figure 14 represents the normalized mAb3 antibody titer obtained after 13 days of cell culture using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0054] Figure 15 represents the viability of cells that express mAb3 on day 13 of cell culture using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0055] Figure 16 represents normalized cell densities of viable cells expressing mAb3 from day 3 (100%) to day 13 using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0056] Figure 17 represents the viability of cells that express mAb3 from day 3 to day 13, using the low choline growth medium supplemented with varying concentrations of choline chloride.
    [0057] Figure 18 shows the normalized development of mAb3 antibody titer from day 3 to day 13 of culture low choline growth medium supplemented with varying concentrations of choline chloride.
    [0058] Figure 19 represents normalized cell densities of viable cells expressing mAb4 from day 0 to day 17 using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0059] Figure 20 represents the viability of cells expressing mAb4 from day 0 to day 17 using the low choline growth medium supplemented with varying concentrations of choline chloride.
    [0060] Figure 21 represents the viability of cells expressing mAb4 on day 17 using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0061] Figure 22 represents the normalized mAb4 antibody titer from day 7 to day 17 using the low choline growth medium supplemented with varying concentrations of choline chloride.
    [0062] Figure 23 represents the concentration of mAb4 antibody normalized on day 17 using low choline growth medium supplemented with varying concentrations of choline chloride.
    [0063] Figure 24 shows the normalized average cell specificity (qP) of cells expressing mAb4 obtained within 17 days of cell culture, using low choline chloride growth medium supplemented with varying concentrations of choline chloride . Detailed Description of the Invention
    [0064] Conventional cell culture media that are used for cell culture and the subsequent production of polypeptides from these cell cultures contain only small amounts of choline chloride. Only very few cell culture media described in the prior art contain moderate or even high amounts of choline chloride. However, these media have not been systematically investigated in terms of the role of high amounts of choline chloride on specific cell productivity, cell growth and product quality, especially when used during the production phase.
    [0065] In accordance with the present invention, an unexpected improvement in cell specific productivity (or polypeptide expression) is observed when culturing CHO cells using cell culture media as the production medium comprising high amounts of chloride choline compared to media with lower amounts of choline chloride. It is shown here that even a growth medium when supplemented with high amounts of choline chloride can surprisingly be used as an efficient production medium for the cultivation of CHO cells during the production phase, thus obtaining large amounts of polypeptide, preferably polypeptide obtained by recombinant expression of polypeptide in cell cultures.
    [0066] Although choline chloride is preferably used in accordance with the present invention, other sources of choline, for example choline hydroxide, choline tartrate / bitartrate, choline sulfate, choline phosphate, or any other choline compound based on the use of a different counterion it is also equally suitable for use in cell culture media according to the present invention.
    [0067] The use of the cell culture medium according to the present invention for the production of polypeptides in general involves the cultivation of CHO cells for the recombinant expression of polypeptides. It is preferable that the cell culture medium is used for the large-scale production of polypeptides. Large-scale production of polypeptides refers to the quantities normally required for the industrial production of recombinant polypeptides used for the preparation of therapeutically active biopharmaceutical products. Cell cultures of at least 500 I in volume, at least 1000 I, at L 5000 or even greater volumes typically represent large scale production applications.
    [0068] The amount of choline chloride in the cell culture medium according to the present invention used for the production of polypeptide is significantly higher than the choline chloride content known from the cell culture media previously used for the large-scale polypeptide production.
    [0069] Therefore, the present invention is suitable for the production of a recombinant polypeptide using CHO cells that comprise high choline chloride content, such as 60 mg / L or higher, 80 mg / L or higher, 160 mg / L or higher , 200 mg / L or higher, or even 220 mg / L or higher. The content of choline chloride in the cell culture medium is limited to 2500 mg / L, 1000 mg / L, 840 mg / L, 500 mg / L or even 300 mg / L. Choline chloride can be present at a concentration of about 240 mg / L.
    [0070] The higher the concentration of choline chloride, the higher the costs for the medium. Thus, concentrations of choline chloride that are too high are disadvantageous from the cost point of view. In addition, the choline chloride content contributes to the osmolality of the medium. Choline chloride concentrations that are too high can be disadvantageous since they can lead, together with the other components of the media, to a total osmolality that is higher than desired. In particular, in batch-fed processes it is not desirable to use very high starting osmolality, as this may impose limitations on the feeding strategy.
    [0071] For the above reasons, the inventors believe that the ideal choline chloride concentrations are within the limits established as here.
    [0072] If choline compounds with the exception of choline chloride are used, they are used in equivalent concentrations. Equivalent concentrations means that the molar concentrations of choline are achieved, which are in the same ranges as that achieved when choline chloride is used in concentrations within the above ranges.
    [0073] According to the first aspect of the present invention, the cell culture medium comprises only a limited content of amino acids expressed by the total concentration of amino acids. More in particular, the cell culture medium according to the first aspect of the present invention is characterized by a total amino acid concentration of 20 to 57 mmol / L. The total concentration of amino acids can be above 25 mmol / L, above 30 mmol / L, above 35 mmol / L or even above 40 mmol / L. In addition, the total concentration of amino acids can be less than 54 mmol / L. The total concentration of amino acids can, for example, be about 51 mmol / L.
    [0074] At the same time, the concentration of choline chloride is as mentioned above, that is, in the range of 60 mg / L to 2500 mg / L, with preferred ranges and values also as mentioned above.
    [0075] The cell culture medium according to the first aspect of the present invention can be used in particular as a production medium in order to achieve high cell growth, high viable cell densities and high polypeptide titration during the phase of production. In addition, the highest quality product of the recombinant product is obtained, in particular in terms of less aggregation and / or better post-translation modifications, as an improved glycosylation standard.
    [0076] The role of the amino acid glutamine for the growth of cell cultures and the resulting polypeptide productivity has been the subject of extensive studies in recent years. It has been found that glutamine is not only an important element for polypeptide synthesis, but also constitutes a primary energy source for mammalian cells. In this way, high concentrations of glutamine were normally included in the cell culture media used for the production of polypeptide. Large amounts of glutamine in cell culture media are important for cell growth and polypeptide expression, particularly on an industrial scale.
    [0077] However, glutamine metabolism results in the breakdown of glutamine and the accumulation of ammonium ions, which is known as a by-product being toxic to cell growth and polypeptide production. Therefore, it is desirable to limit the amount of glutamine in cell cultures. Various glutamine replacement agents have been suggested in the prior art, for example, glutamic acid. However, it has been reported that replacing glutamine with glutamic acid in batch feed processes leads to less by-product formation, but also to lower productivity (Doverskog et. Al., J. Biotechnol., 1997, 59, 103- 115). Therefore, cell culture media containing reduced amounts of glutamine are desirable, while still allowing high cell growth and polypeptide productivity.
    [0078] It has been found that the addition of large amounts of choline chloride allows the use of media comprising reduced amounts of glutamine compared to some known media, especially during the production phase, although cell productivity remains largely unaffected.
    Thus, according to the first aspect of the present invention, a cell culture medium is provided, further comprising an optional amount of glutamine which is significantly reduced when compared to prior art media. Cell culture media can also be free of glutamine-substituting agents such as glutamic acid or the like. The cell culture medium optionally comprises glutamine in a concentration of 500 to 1400 mg / L, 800 to 1400 mg / L, or 900 to 1200 mg / L.
    [0080] At the same time, the concentration of choline chloride is as mentioned above, that is, in the range of 60 mg / L to 2500 mg / L, with preferred ranges and values also as mentioned above. In addition, the total concentration of amino acids in the cell culture medium is at the same time from 20 to 57 mmol / L, with preferred ranges and values also as mentioned above.
    [0081] According to another optional modification of the first aspect of the present invention, the respective amounts of individual amino acids are as defined below.
    [0082] Such moderate amounts of amino acids are even higher than the amounts of amino acids contained in conventional cell culture media, such as DMEM or RPMI, but at the same time significantly less than the amounts of amino acids contained in the means of production typical used for large-scale production.
    [0083] The increase in the amount of amino acids in means of production is considered important for high productivity and high product quality, especially when polypeptide production is carried out on a larger scale, or even on an industrial scale. It has now been found that the presence of high amounts of choline chloride makes it possible to limit the amounts of individual amino acids, especially in cell culture media used during the production phase.
    [0084] The content of individual amino acids in the cell culture medium according to this optional modification of the first aspect of the present invention comprises the following amino acids in the following amounts expressed in mmol / L: Arginine 4.0 - 6.0, preferably 4 , 5 - 5.5 Asparagine 3.0 - 6.0, preferably 4.0 - 5.5 Aspartic Acid 2.5 - 4.0, preferably 3.0 - 3.6 Glycine 0.3 - 0.8, preferably 0.5 - 0.7 Histidine 0.6 -1.0, preferably 0.7 - 0.9 Isoleucine 2.0 - 5.0, preferably 3.0 - 4.0 Leucine 3.0 - 7.0 , preferably 3.5 - 6.0 Lysine 2.0 - 4.0, preferably 2.5 - 3.5 Methionine 1.0-1.5, preferably 1.2 -1.4 Phenylalanine 1.0 - 2, 0, preferably 1.3 - 1.8 Proline 2.5 - 6.0, preferably 3.0 - 5.5 Serine 3.0 - 8.0, preferably 4.0 - 7.0 Threonine 2.0 - 3 , 5, preferably 2.5 - 3.1 Tryptophan 0.4 -1.0, preferably 0.5 - 0.8 Valine 2.5 - 5.0, preferably 3.0 - 4.5 Tyrosine 1.0 - 2.0, preferably 1.2 -1.8 Cystine 0.5 -1.0, preferable elly 0.6 - 0.8
    [0085] At the same time, the concentration of choline chloride is as mentioned above, that is, in the range of 60 mg / L to 2500 mg / L, with preferred ranges and values also as mentioned above. In addition, the total concentration of amino acids in the cell culture medium is at the same time from 20 to 57 mmol / L, with preferred ranges and values also as mentioned above.
    [0086] Due to the high choline chloride content, the respective amounts of amino acids may be significantly less than the amounts used in other cell culture media used for large-scale production of polypeptides. In other words, the addition of high amounts of choline chloride allows to significantly reduce the amount of most of the amino acids, without deteriorating cell growth, cell viability and polypeptide titration. This has the technical advantage that cell culture media with a lower concentration of most of the included amino acids can be used in order to avoid problems of precipitation of less soluble components of cell culture media. In addition, significant cost reductions with respect to cell culture media are achieved, although the overall quality and yield of the polypeptide product are not affected or can even be improved. As described below, the aggregation of the recombinant polypeptide product can be reduced by using cell culture media according to the present invention. In addition, better post-translation modifications were also obtained, such as improved glycosylation pattern or other quality attributes of the protein, such as lower aggregation of the recombinant polypeptide. In some cases, modification of the cell culture media, as disclosed by the present invention, further helps to increase cell viability and cell growth, as well as the resulting polypeptide titration.
    [0087] The term "cell culture medium" refers to an aqueous solution of nutrients that can be used for cell growth over an extended period of time. Typically, cell culture media include the following components: an energy source, which will generally be composed of a carbohydrate, preferably glucose, amino acids, preferably the basic set of amino acids, including all essential and non-essential amino acids , vitamins and / or other organic compounds that are required in low concentrations, free fatty acids and inorganic compounds including trace elements, inorganic salts, buffering compounds and nucleosides and bases.
    [0088] The term "growth medium" refers to a cell culture medium that is normally used during the expansion phase of the general production process. The expansion phase is the first period of the general cultivation / production process that is predominantly characterized by high cell growth and less polypeptide production. The expansion phase has the purpose of expanding the cells, which means the generation of an adequate number of cells that are in the exponential growth phase to inoculate a production bioreactor.
    [0089] The term "production medium" refers to a cell culture medium that is normally used during the production phase of the general production process. The production phase is a second phase of the general cultivation / production process, which aims to produce large quantities of product. During the production phase, cells must be maintained in a viable and productive manner for as long as possible.
    [0090] The use of cell culture media in the field of the pharmaceutical industry, for example, for the production of therapeutically active recombinant polypeptides in general does not allow the use of any material of animal origin, due to safety and contamination issues. Therefore, the cell culture medium according to the present invention is preferably a medium free of serum and / or proteins. The term "serum and / or protein-free medium" represents a totally chemically defined medium, containing no additives of animal origin such as tissue hydrolyzates, fetal bovine serum or the like. In addition, proteins, especially growth factors such as insulin, transferrin or the like, are also preferably not added to the cell culture according to the present invention. Preferably, the cell culture medium according to the present invention is also not supplemented with a hydrolyzed protein source such as soy, wheat or rice peptone or yeast hydrolyzate or the like.
    [0091] The cell culture medium according to the present invention can be used in various cell culture processes. Cell culture can be carried out in adherent culture, for example, in monolayer culture or, preferably, in suspension culture.
    [0092] The large-scale cultivation of cells can be used, for example, by the various fermentation processes established in industrial biotechnology. Batch and continuous cell culture processes, such as perfusion and chemostat, can be used using the cell culture media according to the present invention. Discontinuous processes, including repeated batch and repeated fed batch, are a preferred embodiment.
    [0093] Batch cell culture includes fed batch culture or single batch culture. The term "fed batch cell culture" refers to cell culture in which cells and cell culture media are supplied to the culture vessel initially and additional culture nutrients are fed continuously or in discrete increments to the culture during the culture process with or without periodic cell and / or product harvest before the end of the culture. The term "simple fed culture" refers to a procedure in which all components for cell culture, including cells and cell culture media, are supplied to the culture vessel at the beginning of the culture process.
    [0094] The cells cultured in cell culture medium according to the present invention are CHO cells.
    [0095] Polypeptides that can be produced from cell cultures and the cell culture media according to the present invention are not limited. Polypeptides can be recombinant or non-recombinant. The term "polypeptide" as used herein includes molecules composed of a chain of more than two amino acids linked by peptide bonds; molecules containing two or more such chains; molecules comprising one or more such chains being further modified, for example, by glycosylation. The term polypeptide is intended to encompass proteins.
    [0096] The preferred class of polypeptides produced by cell cultures and the cell culture media according to the present invention are recombinant antibodies.
    [0097] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), nanobodies, modified antibodies, antibody subunits, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments long enough to exhibit the desired biological activity. Monoclonal antibodies as used herein can be human antibodies.
    [0098] However, polypeptides other than antibodies can also be produced using cell cultures and the cell culture media according to the present invention, for example, polypeptides such as transmembrane proteins, receptors, hormones, growth factors, proteases, coagulation and anticoagulation proteins, inhibitory proteins, interleukins, transport factors, fusion proteins and the like. The cell culture medium can also be used for the production of viruses.
    [0099] Products obtained from such cell culture processes can be used for the preparation of pharmaceutical preparations. The term "pharmaceutical preparation" indicates a composition suitable or adapted for administration to a mammal, especially a human. In addition, the protein (s) according to the invention can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants, containers, carriers, diluents and vehicles.
    [00100] The presence of large amounts of choline chloride in the cell culture medium according to the present invention allows the reduction of the amino acid content in the cell culture medium without negatively affecting the ability of the medium to support cell growth at high densities and allowing high polypeptide titration at the same time. This effect is especially important for the production phase of the general production process.
    [00101] Most experiments revealed that even better performance parameters are achieved by the cell culture media according to the present invention when compared to conventional production media comprising higher concentrations of selected amino acids combined with only small amounts of chloride. hill.
    [00102] Although not linked to a certain theory, it is assumed that the concentration of the precursor phospholipid choline is linked to the amount of the essential component of the cell membrane phosphatidylcholine, which is, in addition to other phospholipids, necessary to preserve the integrity and the functionality of cell membranes. It can be assumed that cells growing in a medium with a low choline content are limited in this substrate during the cultivation process and thus in phosphatidylcholine, even if choline is a non-essential component of the media and the cells must be able to synthesize it independently. However, an inactive or limited pathway may be the reason why limited amounts of choline are available to produce sufficient amounts of phosphatidylcholine. This can lead to an "abnormal" composition of membranes, especially membranes of the endoplasmic reticulum and the Golgi complex. This could negatively influence the function of these membranes and reduce polypeptide expression rates or the transport of polypeptides within cells. Inhibition of polypeptide transport from the Golgi complex to the plasma membrane in CHO cells depleted of phosphatidylcholine has been shown by Testerink et al. (2009; Journal of Lipid Research, Vol. 50, 2182-2192). This defect can be rescued by adding exogenous phosphatidylcholine.
    [00103] Several important advantages result from the specific composition of the cell culture media according to the present invention.
    [00104] First, the reduction of the total content of amino acids in the cell culture medium according to the present invention allows the performance of cell culture processes with the same technical performance parameters, or even better, in terms of cell growth , viable cell densities, as well as polypeptide productivity, while at the same time, the reduction in the total content of amino acids or some selected amino acids in cell culture leads to a better overall economic balance of the production process, due to the reduction cost as it relates to the cell culture medium.
    [00105] Secondly, the cell culture media according to the present invention avoids the risk of precipitation of components contained in the cell culture medium, in particular hydrophobic amino acids contained in the medium in relatively high concentrations. This is advantageous, in particular, during storage of the media. For these components, reduced concentrations help to prevent deterioration of the supply with essential substrate during cell growth and polypeptide production. This is especially beneficial during the production phase of the general production process.
    [00106] Thirdly, the cell culture media according to the present invention allows the production of higher quality recombinant polypeptides. The aggregation of the polypeptide formed is reduced, cells are able to produce polypeptides with better post-translational modifications and glycosylation patterns are also improved.
    [00107] The aggregation of the polypeptide formed during recombinant expression is a technical problem that leads to reduced product yields. In addition, aggregation makes it more difficult to purify the product of the functionally active polypeptide.
    [00108] Therefore, it is desirable to reduce the aggregation of the polypeptide product formed as much as possible during the actual production process. It has been found that the cell culture media according to the present invention leads to reduced aggregation of recombinant polypeptide product when compared to typical production media.
    [00109] Many polypeptides are subject to post-translational modification, especially polypeptide glycosylation. The resulting polypeptides comprise covalently linked oligosaccharide chains. Glycosylation is known to be an important mediator of polypeptide functionality. Therefore, the ability of a host cell system used for the production of recombinant polypeptide to properly mimic endogenous glycosylation structures is an important aspect of product quality. The therapeutic efficiency of a recombinant polypeptide can be strongly affected by improper glycosylation of the polypeptide due to immunogenic properties and reduced half-life in vivo after administration of incorrectly glycosylated polypeptides.
    [00110] In general, mannosylation is considered a critical aspect in the production of recombinant polypeptide, especially in the field of recombinant antibodies. It is a general objective in the production of recombinant polypeptides to avoid high mannosylation of polypeptides. It is therefore an important objective to reduce as much as possible high mannosylation during the production of recombinant polypeptides.
    [00111] The cell culture media according to the present invention allows the production of recombinant polypeptides in a very low degree of high mannosylation. This technical effect is particularly significant with respect to typical means of production. For example, the relative amount of highly mannosylated recombinant polypeptide with high mannosylation of the total amount of recombinant polypeptide obtained from expression using the cell culture media according to the present invention is preferably reduced by about 50% compared to the means of production.
    [00112] Furthermore, it was surprisingly found that the recombinant polypeptides obtained using the cell culture media according to the present invention have higher percentages of β-galactosylation compared to the corresponding polypeptides obtained using growth media or media. conventional production methods.
    [00113] Another advantage of the medium with high concentrations of hill is that it allows having only one medium for the production and expansion phase, saving time and resources. Examples
    [00114] The experiments that follow are intended to further illustrate the invention, as defined in this patent application. Description of cell culture media
    [00115] The following three means are tested:
    [00116] Growth medium with low choline content, that is, with a choline chloride content of 40 mg / L (control 1);
    [00117] Means of production (control 2);
    [00118] Growth medium with high choline content, that is, growth medium with low choline content supplemented with an additional amount of choline chloride of 200 mg / L, resulting in a total choline chloride content of 240 mg / L.
    [00119] The first two media (low choline low growth and production medium) are used for comparison purposes only, while the third high choline chloride medium represents a medium according to the present invention.
    [00120] Low choline growth medium is a typical medium designed for the growth and proliferation of a cell culture. This medium allows the cultivation of cells until high cell densities are achieved, which is an important requirement for large-scale production of polypeptide. However, the growth medium with low choline content is not designed for the production of polypeptides from cell culture, because the content of many amino acids is low to moderate, considering the total concentration of amino acids in the medium of 51.1 mmol / L.
    [00121] The amino acid composition of the low choline growth medium is as follows:

    *) L-tyrosine and L-cystine are added to the medium to achieve the above concentrations of tyrosine and cystine.
    [00122] In contrast to the low choline growth medium, the second medium (production medium) is a useful medium for large-scale polypeptide production using cell cultures. This production medium contains larger amounts of most amino acids (the total concentration of amino acids in the medium is 90.50 mmol / L) when compared to the low choline growth medium, although the amounts of other components are basically identical .
    [00123] The amino acid composition of the production medium used is as follows:


    [00124] The choline chloride content in this comparative production medium is also significantly higher than the low choline growth medium. It is important to note that the means of production known from the prior art usually contain much smaller amounts of choline chloride. Therefore, the high choline chloride content in the comparative production medium should be considered as an important difference in relation to the known production media of the prior art, which normally do not differ from the known growth media in terms of their choline chloride content. .
    [00125] The third medium is a medium according to the present invention represented by the growth medium with high choline content, that is, the growth medium with low choline content supplemented with 200 mg / L of choline chloride, resulting in a total choline chloride content of 240 mg / L. With the exception of the higher choline chloride content, the third medium according to the present invention can still be considered as a typical growth medium.
    [00126] The examples demonstrate the significant improvement achieved by high amounts of choline chloride in growth media, such as the low choline growth medium, when used during the production phase, making such growth media high in choline chloride not only far superior to supplemented media with low choline chloride content in terms of cell growth, cell viability and polypeptide titration, but even superior to production media with equally high amounts of choline chloride in the same aspects. The addition of greater amounts of choline chloride helps to achieve better cell growth and viability and improved polypeptide titration in normal growth media. Experimental Configuration
    [00127] For the experiments, a parental CHO cell line is used, which is derived from the dhfr (+) CHO-K1 ATCC CCL-61 cell line (Kao et. Al, Genetics, 1967, 55, 513-524, Kao et. al., PNAS, 1968, 60, 1275-1281; Puck et. al., J. Exp. Med., 1958, 108, 945-959) by adapting protein-free, serum-free media conditions. Three aliquots of this parental cell line are transfected to express three different monoclonal antibodies, mAb1 mAb2, mAb3, respectively. Shake Bottle Experiments (Experiments 1 to 3)
    [00128] All nine shake flasks (three for each experiment) are run under the same conditions, except for the medium. The conditions involve a batch culture fed with two daily boluses starting on days 3 and 5, with a feed rate of 2 and 0.4% of the initial culture volume per day, a temperature change of 36.5 ° C at 33 ° C on day 5, 10% CO2 in the incubator, an agitation rate of 150 rpm (stroke radius = 25 mm). Cell growth / cell viability, as well as the resulting titer of expressed recombinant antibodies are determined. a) Experience 1
    [00129] Figures 1 and 2 show the results obtained for cells that express mAb1 cultured in Experiment 1 in terms of cell growth and viability.
    [00130] As illustrated in Figures 1 and 2, the medium of the invention with high choline chloride content (growth medium with high choline content) shows a 53% increase in maximum viable cell density and a slower decline in viability when compared to choline growth medium with low choline chloride content.
    [00131] Figure 3 shows the results obtained for the cells grown in Experiment 1, in terms of polypeptide titration.
    [00132] As illustrated by Figure 3, the titration of recombinant antibody polypeptide obtained in the medium of the invention with high choline chloride content (the growth medium with high choline content) shows a 330% increase in the polypeptide titration in the day 13 compared to the low choline growth medium, which has only low choline chloride content and represents a typical growth medium only.
    [00133] Figure 3 also shows that the medium of the invention with a high choline chloride content even allows to achieve a polypeptide titration that is slightly higher than the titration obtained from the production medium. b) Experience 2
    [00134] Figures 4 and 5 show the results obtained for cells expressing mAb2 grown in Experiment 2 in terms of cell growth and viability.
    [00135] As illustrated by Figures 4 and 5, the medium of the invention, with high choline chloride content (the growth medium with high choline content) has only little influence on cell growth, but gives significantly higher viability at the end of the process, when compared to the low choline growth medium, which has only low choline chloride content.
    [00136] Figure 6 shows the results obtained for cells cultured in Experiment 2 in terms of polypeptide titration.
    [00137] Figure 6 reveals an 85% increase in polypeptide titer on day 11 for the cell culture medium according to the present invention when compared to the low choline growth medium. Furthermore, the polypeptide titration obtained in the cell culture medium according to the present invention is even greater when compared to the polypeptide titration obtained in the production medium having an equally high choline chloride content. c) Experience 3
    [00138] Figures 7 and 8 show the results obtained for cells that express mAb3 cultured in Experiment 3 in terms of cell growth and viability.
    [00139] As illustrated by Figures 7 and 8, higher cell viability at the end of the cultivation process is obtained with the medium according to the present invention, when compared to the comparative cell culture medium (the growth medium with low choline content), which comprises only small amounts of choline chloride.
    [00140] Figure 9 shows the results obtained for cells cultured in Experiment 3 in terms of polypeptide titration.
    [00141] Figure 9 reveals that the polypeptide titration obtained in the cell culture medium according to the present invention is increased by 145% on day 11 when compared to the comparison medium, the low choline growth medium. The polypeptide titration for the cell culture medium according to the present invention is still slightly higher than the titration obtained using the production medium with high choline chloride content.
    [00142] It was further experimentally confirmed that the use of higher concentrations of choline chloride in the production medium did not result in the improvement of cell growth or polypeptide titration when compared to the use of the production medium with the normal amount of choline chloride. 240 mg / L.
    [00143] The following additional experiments are carried out in order to determine the influence of the cell culture medium on the quality of the polypeptide product. In particular, the product is analyzed to determine the influence of the media on aggregation and glycosylation.
    [00144] Figure 10 shows the percentage aggregation rate of the recombinant antibody product, in relation to the total amount of recombinant antibody. The rate of aggregation in the cell culture medium according to the present invention is reduced by more than 30% in relation to the production medium.
    [00145] The expression of mAb3 in different cell culture media, followed by analysis of the glycosylation pattern of the recombinant antibodies demonstrates that the growth medium according to the present invention with a high choline chloride content leads to a recombinant antibody with a low total amount of recombinant product comprising high mannosylation, which represents an unwanted glycosylation pattern. The use of a medium according to the invention leads to a reduction of more than 55% in terms of high mannosylation compared to the use of the production medium. Batch runs fed by Fed-batch bioreactor (Experiment 4)
    [00146] The experience to follow is the batch execution fed into the bioreactor. It corresponds to experience 3 above. Cells that express mAb3 were used. The conditions are as follows: initial volume 2L; continuous feeding of two different feeding solutions from days 3 and 5 with a feeding rate of 2 and 0.4% of the initial culture volume per day; a temperature change from 36.5 ° C to 33 ° C on day 5; pO2 = 30%, pH = 6.9 (Deadband 0.1); controlled with CO2 and 0.5 M NaOH; agitation rate of 300 rpm.
    [00147] Figures 11 and 12 show the results obtained for the density of viable cells and percentage of cell viability in Experiment 4 performed as batch execution fed into a bioreactor.
    [00148] With regard to cell growth and cell viability, the results of the batch run fed into the bioreactor are consistent with the results obtained in the shake flask experiments. That is, the batch run fed to the bioreactor basically confirms the results obtained in the shake flask experiments.
    [00149] As in the shake flask experiment, peak viable cell densities obtained in the production medium were slightly higher compared to the cell culture medium of the invention (the high choline growth medium), with high choline chloride content. However, the percentage of viability remains at a significantly higher level when the cell culture medium of the invention, that is, the medium supplemented with high choline chloride content is used in comparison to the non-supplemented growth medium having only low content choline chloride.
    [00150] Figure 13 shows the results obtained regarding the polypeptide titration in Experiment 4 performed as batch execution fed in a bioreactor.
    [00151] The polypeptide titration measured on day 11 for the invention medium of cells of the invention with high choline chloride content is increased by 170% when compared to the comparative growth medium having only small amounts of choline chloride.
    [00152] While the polypeptide titration obtained for cell culture medium of the invention with high choline chloride content is still superior to the polypeptide titration obtained using the production medium in the shake flask experiments for comparative purposes, the experiment in batch execution fed to the bioreactor reveals a slightly lower polypeptide titration in the cell culture medium of the invention with high choline chloride content when compared to the production medium. However, the polypeptide titration obtained in the bioreactor using the cell culture medium of the invention with a high choline chloride content is very high and still at a level comparable to the polypeptide titration obtained by use for the purpose of comparing the medium of production.
    [00153] Experiment 4 in the bioreactor further confirms that the cell culture medium of the invention with high choline chloride content represents an adequate alternative to conventional production means, allowing to achieve comparable titers of polypeptides, while the total amount of amino acids or the amounts of selected amino acids can be significantly reduced in the respective cell culture medium.
    [00154] Experiment 4 in the bioreactor also proves once again that the increase in the choline chloride content in a typical growth medium helps to achieve a great increase in the polypeptide titration when using the growth medium for the production of polypeptides . The addition of different concentrations of choline chloride to the growth medium
    [00155] In another set of experiments, choline chloride is added in various concentrations (40, 80, 120, 160, 200, 240, 500, 840, 1000, 3000 and 5000 mg / L in total) to the powder growth. The media thus obtained are used for the production of mAb3 and mAb4, respectively, in general as described above.
    [00156] Normalized antibody concentrations, normalized viable cell densities, and viability for mAb3 and, furthermore, for mAb4, the specific cell productivity normalized for the entire duration of the culture (13 resp. 17 days) are further illustrated from Figure 14 to Figure 24.
    [00157] As can be seen from these data, the lowest values in terms of viability and the concentration of antibody after 13 or 17 days in culture are obtained in growth medium with 40 mg / L of choline chloride in total. Concentrations of 3000 mg / L and more do not negatively affect the viability of the cells, but since the cells do not grow in such media, the antibody concentration remains below 1. Viability is highest for all higher choline concentrations at 40 mg / L. There appears to be a choline chloride concentration-dependent effect on viability. Higher concentrations of choline chloride in the medium resulted in greater viability at the end of cultivation.
    权利要求:
    Claims (8)
    [0001]
    1. A process for producing a recombinant polypeptide comprising a production phase, characterized by the fact that: recombinant CHO cells are grown in a cell culture medium, and the recombinant polypeptide is expressed, the cell culture medium being serum-free and protein-free, comprises 60 to 2500 mg / L of choline chloride or an equivalent amount of another choline salt, and has a total amino acid concentration of 20 to 57 mmol / L and in which the process comprises a temperature change from a first temperature to a lower second temperature.
    [0002]
    2. Process according to claim 1, characterized by the fact that the recombinant polypeptide is a recombinant antibody.
    [0003]
    3. Process according to claim 1 or 2, characterized by the fact that the cells are grown in a fed batch process.
    [0004]
    Process according to any one of claims 1 to 3, characterized in that the cell culture medium comprises 80 to 1000 mg / L of choline chloride or an equivalent amount of another choline salt.
    [0005]
    Process according to any one of claims 1 to 4, characterized in that the cell culture medium has a total amino acid concentration of 35 to 54 mmol / L.
    [0006]
    Process according to any one of claims 1 to 5, characterized in that the cell culture medium contains glutamine in a concentration of 500 to 1400 mg / L.
    [0007]
    Process according to any one of claims 1 to 6, characterized in that the content of glutamine in the cell culture medium is in an amount of 900 to 1200 mg / L.
    [0008]
    Process according to any one of claims 1 to 7, characterized in that the cell culture medium, as defined in claim 6 or 7, comprises the amino acids Arginine 4.0 to 6.0 mmol / L, Asparagine 3.0 to 6.0 mmol / L, Aspartic Acid 2.5 to 4.0 mmol / L, Glycine 0.3 to 0.8 mmol / L, Histidine 0.6 to 1.0 mmol / L, Isoleucine 2 , 0 to 5.0 mmol / L, Leucine 3.0 to 7.0 mmol / L, Lysine 2.0 to 4.0 mmol / L, Methionine 1.0 to 1.5 mmol / L, Phenylalanine 1.0 to 2.0 mmol / L, Proline 2.5 to 6.0 mmol / L, Serine 3.0 to 8.0 mmol / L, Threonine 2.0 to 3.5 mmol / L, Tryptophan 0.4 to 1 , 0 mmol / L, Valine 2.5 to 5.0 mmol / L, Tyrosine 1.0 to 2.0 mmol / L and Cystine 0.5 to 1.0 mmol / L.
    类似技术:
    公开号 | 公开日 | 专利标题
    BR112012027434B1|2020-09-29|PROCESS FOR PRODUCTION OF A RECOMBINANT POLYPEPTIDE UNDERSTANDING A PRODUCTION PHASE
    JP6603628B2|2019-11-06|Improved cell culture media
    KR102109463B1|2020-05-12|Cell culture medium comprising small peptides
    同族专利:
    公开号 | 公开日
    IL222453D0|2012-12-31|
    US20150218509A1|2015-08-06|
    MX2012012525A|2012-11-23|
    WO2011134921A1|2011-11-03|
    US8986957B2|2015-03-24|
    SG185037A1|2012-11-29|
    US9243224B2|2016-01-26|
    AU2011246504A1|2012-11-22|
    IL222453A|2018-11-29|
    EP2563903B1|2019-08-21|
    CN105331659B|2019-07-16|
    BR112012027434A2|2015-09-22|
    JP6113653B2|2017-04-12|
    AU2011246504B2|2013-09-26|
    CA2797140C|2018-01-23|
    RU2563353C2|2015-09-20|
    JP2013524826A|2013-06-20|
    CN102858953B|2015-09-09|
    JP2016052310A|2016-04-14|
    EP3599276A1|2020-01-29|
    RU2012150281A|2014-06-10|
    CA2797140A1|2011-11-03|
    KR101848945B1|2018-04-16|
    ES2755907T3|2020-04-24|
    CN105331659A|2016-02-17|
    KR20130098869A|2013-09-05|
    EP2563903A1|2013-03-06|
    US20130102032A1|2013-04-25|
    CN102858953A|2013-01-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4767704A|1983-10-07|1988-08-30|Columbia University In The City Of New York|Protein-free culture medium|
    US6048728A|1988-09-23|2000-04-11|Chiron Corporation|Cell culture medium for enhanced cell growth, culture longevity, and product expression|
    US5122469A|1990-10-03|1992-06-16|Genentech, Inc.|Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins|
    WO1992015670A1|1991-02-28|1992-09-17|Hyclone Laboratories, Inc.|Blended bovine sera cellular growth media and their methods of production and use|
    GB9118664D0|1991-08-30|1991-10-16|Celltech Ltd|Cell culture|
    USH1532H|1993-11-03|1996-05-07|Genetics Institute, Inc.|Adaption of mammalian cell lines to high cell densities|
    US5856179A|1994-03-10|1999-01-05|Genentech, Inc.|Polypeptide production in animal cell culture|
    EP2218775B1|1996-08-30|2015-01-28|Life Technologies Corporation|Method for producing a polypeptide in vitro in mammalian cells in a protein-free and serum-free culture medium|
    US20060286668A1|1999-04-30|2006-12-21|Invitrogen Corporation|Animal-cell culture media comprising non-animal or plant-derived nutrients|
    US6103529A|1996-10-10|2000-08-15|Life Technologies, Inc.|Animal cell culture media comprising peptides derived from rice|
    US6692961B1|1996-10-11|2004-02-17|Invitrogen Corporation|Defined systems for epithelial cell culture and use thereof|
    EP1434857B1|2001-10-02|2007-08-01|Novo Nordisk Health Care AG|Method for production of recombinant proteins in eukaryote cells|
    US20030096414A1|2001-03-27|2003-05-22|Invitrogen Corporation|Culture medium for cell growth and transfection|
    AU2002316230A1|2001-06-13|2002-12-23|Genentech, Inc.|Methods of culturing animal cells and polypeptide production in animal cells|
    EP1686173A4|2003-10-09|2006-11-22|Kyowa Hakko Kogyo Kk|Genomically modified cell neutralized to serum-free system|
    JP4740138B2|2003-10-10|2011-08-03|ノボノルディスクヘルスケアアクチェンゲゼルシャフト|Method for large-scale production of polypeptides in eukaryotic cells and culture vessels suitable therefor|
    US7335491B2|2004-08-27|2008-02-26|Wyeth Research Ireland Limited|Production of anti-abeta|
    TWI384069B|2004-08-27|2013-02-01|Pfizer Ireland Pharmaceuticals|Production of polypeptides|
    TWI364458B|2004-08-27|2012-05-21|Wyeth Res Ireland Ltd|Production of tnfr-lg|
    CA2585547A1|2004-10-29|2006-05-11|Centocor, Inc.|Chemically defined media compositions|
    KR101196103B1|2004-11-02|2012-11-01|아레스 트레이딩 에스.에이.|Serum-free cell culture medium for mammalian cells|
    TWI369401B|2005-07-05|2012-08-01|Ares Trading Sa|Serum-free culture medium for the production of recombinant gonadotropins|
    US20070231895A1|2005-11-02|2007-10-04|Lee Gene W|Methods for adapting mammalian cells|
    PL1974014T3|2006-01-04|2017-09-29|Baxalta Incorporated|Oligopeptide-free cell culture media|
    EP2064315B1|2006-11-03|2015-05-13|Wyeth LLC|Glycolysis-inhibiting substances in cell culture|
    FR2910024B1|2006-12-19|2009-03-27|Agronomique Inst Nat Rech|CHEMICALLY DEFINED SYNTHETIC CULTURE MEDIUM.|
    CA2687082C|2007-05-11|2014-01-14|Amgen Inc.|Improved feed media|
    AU2008309934B2|2007-10-12|2014-03-06|F. Hoffmann-La Roche Ag|Protein expression from multiple nucleic acids|
    CN102007207B|2008-04-18|2012-05-30|上海抗体药物国家工程研究中心有限公司|A concentrated culture solution and application method thereof|
    CN101381702A|2008-09-10|2009-03-11|吉林省赛达生物科技有限公司|Cell culture medium without animal component|
    MX2011003241A|2008-09-26|2011-04-21|Schering Corp|High titer antibody production.|
    PT2563904E|2010-04-26|2015-05-12|Novartis Ag|Improved cell culture medium|
    DK2563906T3|2010-04-26|2018-02-05|Novartis Ag|Method for culturing CHO cells|
    US8986957B2|2010-04-26|2015-03-24|Novartis Ag|Cell culture medium|US8986957B2|2010-04-26|2015-03-24|Novartis Ag|Cell culture medium|
    US9976163B2|2012-10-03|2018-05-22|Kyowa Hakko Kirin Co., Ltd|Method for preventing reduction of polypeptide by adding amino acid to culture solution|
    ES2633960T3|2012-10-15|2017-09-26|Bristol-Myers Squibb Company|Processes of mammalian cell culture for protein production|
    WO2015115849A1|2014-01-29|2015-08-06|주식회사 엘지생명과학|Method for controlling galactosylation of recombinant protein through optimization of culture medium|
    JPWO2015155975A1|2014-04-07|2017-04-13|東洋製罐グループホールディングス株式会社|Animal cell culture medium and culture vessel|
    EP2975050B1|2014-07-18|2019-05-29|Sandoz Ag|Quantification of misfolded TNFR2:Fc|
    EP3736326A1|2015-04-01|2020-11-11|Boehringer Ingelheim International GmbH|Cell culture medium|
    EP3303555B1|2015-05-29|2020-06-24|Merck Patent GmbH|Deep eutectic solvents and/or ionic liquids in cell culture media|
    EP3303554B1|2015-05-29|2020-06-24|Merck Patent GmbH|Deep eutectic solvents and/or ionic liquids as feed media|
    EP3314046B1|2015-06-25|2019-04-24|Hooke Bio Limited|Mechanical device for generating combinatorial library|
    US20180312811A1|2015-11-09|2018-11-01|Bristol-Myers Squibb Company|Methods to manipulate quality attributes of polypeptides produced in cho cells|
    KR20180047404A|2016-10-31|2018-05-10|삼성바이오에피스 주식회사|Process for reducing undesirable by-product in cell culture|
    CN106676058B|2016-12-09|2020-06-19|中农威特生物科技股份有限公司|BHK21 suspension cell high-density fed-batch culture method and application thereof in foot-and-mouth disease virus proliferation|
    GB201708655D0|2017-05-31|2017-07-12|Ucb Biopharma Sprl|Cell culture methods|
    WO2021090888A1|2019-11-05|2021-05-14|味の素株式会社|Method for producing protein|
    WO2021145321A1|2020-01-14|2021-07-22|味の素株式会社|High-density cell culture method|
    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-07-16| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|
    2020-01-21| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|
    2020-06-16| B09A| Decision: intention to grant|
    2020-09-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US32783710P| true| 2010-04-26|2010-04-26|
    US61/327.837|2010-04-26|
    PCT/EP2011/056509|WO2011134921A1|2010-04-26|2011-04-25|Improved cell culture medium|
    [返回顶部]