TH69081A - Method for obtaining cell strains in protein-free medium and cell strains obtained from that method - Google Patents

Abstract

DC60 (14/01/47) This invention relates to a method for extracting clones of the resulting mammalian cells. Been adjusted to thrive in pet food Without serum and protein The method includes a two-step adaptation process in order to thrive in that state. This invention revealed To the critical concentration of proteins, which cells must grow in In order to obtain the ability to survive in the absence of serum and protein, when cells grow at critical concentrations, subsequent reduction in concentrations will There is no impact on life and growth. Or the time of multiplication, the critical concentrations of proteins are specific to the cell species. This was revealed that it opened up a stable mud in the food. Cultured without serum and protein for at least 40 generations. Additionally, the clones revealed in this invention have Expression of the recombinant product The cell clone revealed in this invention produces humanized hR3 antibody, EGF-R antibody, humanized T1 hT antibody, humanized T1 hT antibody, chimeric. T3Q antibody, T3Q antibody, or a fragment of these antibodies The invention involved a method to extract clones of derived mammalian cells. Been adjusted to thrive in pet food That is free from serum and protein The method includes a two-step adaptation process in order to thrive in that state. This invention revealed To the critical concentration of proteins, which cells must grow in In order to obtain the ability to survive in the absence of serum and protein, when cells grow at critical concentrations, subsequent reduction in concentrations will There is no impact on life and growth. Or the time of multiplication, the critical concentrations of proteins are specific to the cell species. This was revealed that it opened up a stable mud in the food. Cultured without serum and protein for at least 40 generations. Additionally, the clones revealed in this invention have Expression of the recombinant product The cell clone revealed in this invention produces humanized hR3 antibody, EGF-R antibody, humanized T1 hT antibody, humanized T1 hT antibody, chimeric. T3Q antibody, T3Q antibody, or a fragment of these antibodies

Claims (9)

Disclaimer (all) which will not appear on the advertisement page: edit 27/10/2015 1. Methods for obtaining recombinant selmeiloma species. Growth was achieved in a serum and protein-free feed consisting of two steps I. Step one, where the viability and viability of the species was between 80 and 100% and the cells were cultured in Cell culture with sequential reduction in protein concentration until reaching Critical concentrations of proteins, where at that concentration, cell viability and growth drop to 0 percent and II. The second step where, as soon as that protein concentration, where cell growth is possible and the point Begins to slow down protein concentrations Until the cell is raised to life and Initial cell growth And the initial doubling time 2. Method for claim 1 where the first step consists of the following steps: i. Plant cells in three holes of a six-well cell culture dish with recombinant strains. Mammal cell myeloma was administered using a standardized cellular feed with initial protein concentrations, where cell density ranged from 1 to 5 x 105 cells / ml and after 48 hours substituted half of the liquid. Above the cells with a new protein-free feed, thus making the concentration Of the final protein, which is 50% of the initial state i i. Every 48 hours, the cellulose fluid is completely replaced with a new cell culture diet with Protein concentration, which is 50% of the primal condition ii i. Cultured cells until convergence in food containing 50% of initial protein concentration i v. Cells from step iii were planted in at least three wells. Wells at density in the range of 1 to 5 x 105 cells / ml. In cultured diets with protein concentrations which were 50 percent of the initial condition, after 48 hours replaced half of the supersonic fluid with new protein-free cultures, resulting in the final protein concentration which was 50 percent of the condition. Previously, v. Every 48 hours the supercellular fluid was completely replaced with a new feed containing The protein concentration, which is 50 percent of the preceding state, v i. Cultures the cells until convergence in the food with 50 percent of the previous protein concentration, and vi i.Repeat the steps from (iv) to ( vi), where during each cycle the concentration of Protein to 50 percent of the concentration of the previous cycle, until it reaches the concentration Of proteins which cause cell death ------------------------------------------------ 1. Methods for obtaining adapted mammal cell species. Grow in food without Serum and protein It consists of two steps I. Step one where the species' viability and maturity is between 80 and 100% and the cells are cultured in Cell food with sequential reduction of protein concentration up to critical protein concentration. Where at that concentration, viability and growth fell to 0% II. The second step where, when the critical concentration was pre-determined, then the pre-critical value was set to the prior protein concentration, where the cell growth Possible And this is the point Begins to slow down the protein concentration until the cells are fed Life and growth of the original cell And the initial doubling time 2. Methods set out in claim 1 where the first step consists of: The procedure is as follows: i.Plant the cells in the 3 wells of the six-well cell culture dish with recombinant cells using a standard cell culture (with initial protein concentration). Cell density should be from 1 to 5 x 10 5 cells / ml. After 48 hours, replace half of the Liquid above the cell with new food that It was protein-free, thus making the final protein concentration 50 percent of the initial state i i. For each 48 hours, the supercellular fluid was completely replaced by a new cell culture with protein concentration of 50 percent of the initial state. ii i. Cells were cultured until convergence in this protein concentration condition. i v. cells from step iii were planted in at least three wells at densities ranging from 1 to 5 x 10 5 cells / ml in the feed. After 48 hours, half of the supersonic fluid was raised with a new protein-free medium, which raised the final protein concentration to 50 percent of the previous condition, v. Each. In 48 h, the supercellular fluid was completely replaced with a new cell culture medium with a protein concentration of 50% of the preceding state v i. Cultured cells until convergence in this protein concentration state vi i. Repeat the steps. Episodes from (iv) to (vi) during each cycle reduce the protein concentration to 50 percent of the protein concentration. The intensity of the previous cycle Repeat this method until the protein concentration is reached which is causing cell death. 3. The procedure set out in claim 1, where the second step consists of: The procedure is as follows: vii i. Grow cells from live and growing cells of 80% or higher. Raised in protein concentrations Pre-crisis in at least three wells at a density range of 2 to 6 x 10 5 cells / ml Raising cells in the condition of a concentration of The pre-crisis protein and after 48 hours replaced 25 percent of the supersonic fluid with a new protein-based culture, thereby making the final protein concentration 75 percent of the pre-crisis protein concentration i x. Each 48 hours. Completely replaced Complete with food Raising new cells with a protein concentration of 75% of the pre-critical protein concentration x. Culturing cells until convergence in this protein concentration condition x i. Cells were grown from the (x) procedure in at least three wells at Dense thickness in the range of 2 to 6 x 10 5 cells / ml. In a feast The protein concentration was 75 percent of the pre-critical protein concentration after 48 hours, replacing 25 percent of the supersonic fluid. With the new protein-based feed, the final protein concentration was therefore made to 75 percent of the previous step concentration xi i. Complete with food Cultivate new cells with protein concentration to 75% of the concentration in the (x) step xii i. Cultivate cells until convergence in this protein concentration condition xi v. Repeat steps from (xi) to ( xiii) During each cycle, reduce Protein concentration to be 75 percent of The concentration of the previous cycle was repeated until the protein concentration was reached. Which does not cause a loss of presence Cell's life and prosperity And reducing the time of population increase Doubled in any way When the cells are transferred to the feed With lower protein concentrations And these cells can grow Without any loss of viability and cell viability and reducing the time of population multiplication to two. Before the first transfusion, the cells were assumed to be a non-critical step and the cells were directly transplanted into a protein-free medium (0 mg / ml protein concentration). 4. Methods set out in any of the Claims 1 to 3 in which serum feed and Protein is a component Initially, the cells consisted of between 5 and 10 percent of the serum of the pregnant calf. 5. Means as set out in any of the Claims 1 to 4, which cell breeds are pets. With enhanced milk in serum and protein fed diets, myeloma 6.The method defined in claim 5, where the Myeloma is the NSO cell strain. 7. Methods defined in claim 6, in which the aforementioned NSO cell strain consists of a sequence that defines the recom code. Binant peptide or recombinant protein 8. Methods defined in claim 7, in which the sequence defines the recombinant polypeptide or recombinant code. Then defines a code for recombinant Antibodies or pieces of That antibody 9. Mammal cell species obtained by means of Any one claim Of claim 1 to 8, which cell strains were optimized for growth in serum and protein-free diets 1 0. The mammal cell species as defined in claim 9, the cell species is Myeloma 1. 1. The mammal cell species as defined in claim 10, the cell species is NSO 1 cell. 2. The mammalian cell strains as defined in claim 11, where the NSO cell strains consist of a sequence coded recombinant, tiptide, or recombinant protein 1. 3. The mammalian cell species as defined in claim 12, the order of which the recombinant polypept code is defined. Tide or recombinant that protein Determine the code for the recombinant antibody or fragment of that antibody. 4. The mammalian cell species as defined in claim 13, wherein the sequence defines a recombinant code. Humanized antibody, type Antibody-EGF-Rh R3, or fragment of that antibody 1 5. The mammalian cell species as defined in claim 13, wherein the sequence defines the recombinant code. Humanized antibody type AnticCD6, T1 hT antibody or fragment of that antibody 1 6. The mammal cell species as defined in claim 13, where the sequence defines the chimeric cipher. Combinant Antit-CD3, T3Q Antibody or Fragment of That antibody 1 7. Use of the methods set forth in one claim. Any of the claims 1 through 8 to get the animal cell species Breastfeeding That have been adjusted to thrive in food culture Without serum and protein 1 8. Humanized monoclonal antibodies against EGF-R hR3 or that antibody fragment expelled by cell strains obtained by means of one of the claims of the claim. 1 to 8 1 9. Humanized monoclonal antibodies against CD6, T1 hT antigens, or antipit fragments that are excreted by cell strains obtained by the method of one of the claims Hold rights 1 to 8 2 0. Humanized monoclonal antibodies against CD3, T3Q antigen, or that antibody fragment excreted by cell strains obtained by the method of one of claim 1. To 8