TH115319A - TNFR-Ig production - Google Patents

Abstract

DC60 protein and / or a-beta production system in mass cell culture at Has been improved Especially In one characteristic medium or I) Accumulated amino acid concentrations above 70 mM approx., Ii) Molar ratio of accumulated glutamine to accumulated aspartame is approximately less than 2, iii) Mo ratios. Lar Of accumulated glutamine against total amino acids that are less than approximately 0.2; iv) Molar ratio Of the accumulated inorganic ions against the accumulated amino acid approximately 0.4 to 1, or v) the total concentration of accumulated glutamine. And asparagene accumulation between approximately 16 and 36 mM was provided. The use of such systems facilitates a high level of beta production and reduces the accumulation of some undesirable factors such as ammonium and / or lactate. In addition, the culture method, which includes the normal half temperature change Including a temperature reduction when cultured to approximately 20-80% of the maximum cell density was provided as an alternative. Or other than this The present invention provides a method of characteristics that, after achieving the highest degree There will be a constant decrease in lactate and / or ammonium levels in culture.

Claims (7)

Disclaimer (all) which will not appear on the advertisement page. : ------ 22/03/2018 ------ (OCR) Page 1 of 14 pages Disclaimer 1. Production method TNFR-Ig in cell culture in mass production which Assembled included With the steps of Arrangement, cell culture, which includes A mammalian cell containing a gene encoded as TNFR-Ig, in which the gene is expressed. Under the conditions of Shell culture and glutamine-containing media. And has the special characteristics of the medium that Selected from a group consisting of: (0 amino acid content per unit volume greater than 70 mM, (ii) the molar ratio of cumulative glutamine to cumulative aspartine less than 2, (iii)). Molar ratio The accumulated glutamine to total amino acids was less than 0.2, (iv) the molar ratio of the ions. Accumulated inorganic amino acids between 0.4 and 1, (v) cumulative total content of glutamine and aspartame per unit volume greater than 16 mM and their combinations. Such cultures were maintained in the insetting phase under the set culture conditions. This is a period of time sufficient for such cells to multiply to the density of the Live in the range of 20% - 80% of the highest living cell density possible if culture It was preserved under the preservation conditions of the first set. Changes at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR-Ig is accumulated in cell culture. 2. TNFR-Ig production method in cell culture in \ 'mass production consisting of Included steps of Arrangement, cell culture, which includes A mammalian cell containing a gene encoded as TNFR-Ig, in which the gene is expressed. Under the conditions of cell culture and culture media containing accumulated amino acids per unit volume greater than 70 mM and such medium containing glutamine. And there are two The special characteristics of the injectable media are selected from a group that includes: (0 Molar Ratio Of cumulative glutamine per cumulative aspartate less than 2, (ii) the molar ratio of cumulative glutamine to Total amino acids accumulated at less than 0.2, (iii) Molar ratio of accumulated inorganic ions to acid P 2 of 14 p. Total amino accumulated between 0.4 to 1, (iv) total accumulated volume. Of glutamine and aspartame per unit Volumes greater than 16 mM and their combination Such cultivation was maintained in the initial growth phase under set culture conditions. One is a period of time sufficient for the cell to reproduce to the density of the affected shell. Live in the range of 20% - 80% of the highest living cell density is possible if cultured. It was preserved under one of the cultured conditions. Changes at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR-Ig is accumulated in cell culture. 3. Method for producing TNFR-Ig in cell culture in mass-composite production. With the steps of A cell culture arrangement which includes The mammalian cells containing the genes coded as TNFR-Ig were expressed. Under the conditions of cell culture and culture media containing accumulated molar ratios to accumulated aspartame less than 2, and such medium contained glutamine. And there are two such culture media The characteristics of the culture media were selected from the group consisting of: It consists of accumulated amino acids per unit volume greater than 70 mM, (ii) the molar ratio of The accumulated glutamine to total amino acids was less than 0.2, (iii) the molar ratio of the ioninanthol. Accumulated to amino acids accumulated between 0.4 and 1, (iv) cumulative total amounts of glutamine and asparagene per unit volume greater than 16 mM and their combinations. Such cultures were maintained in the insetting phase under the set culture conditions. Y \ 'n is a period of time sufficient for the cell to multiply to the density of the Live in the range of 20% - 80% of the living cell density as high as possible if cultured It was preserved under the preservation conditions of the first set. Changes at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR-Ig is accumulated in cell culture. 4. The production method of TNFR-Ig in cell culture is in the mass production of \ 'total composition. With the steps of page 3 of 14 pages, the cell culture arrangement includes A mammalian cell containing a gene encoded as TNFR-Ig, where the gene is expressed. Under the conditions of cell culture and culture media containing accumulated molar ratio of glutamine to total amino acids of less than 0.2, and such medium containing glutamine. And there are two such culture media. Characteristics of the culture media selected from. Groups consisting of: (i) Song culture medium. It consists of accumulated amino acids per unit volume greater than 70 mM, (ii) the molar ratio of The accumulated inorganic asparagene was less than 2, (iii) the molar ratio of the accumulated inorganic ions. (Iv) cumulative total amounts of glutamine and aspartine per unit volume greater than 16 mM and their combinations Such cultures were maintained in the insetting phase under the set culture conditions. It is a period of time sufficient for the cell to multiply to the density of the Live in the range of 20% - 80% of the maximum viable cell density if cultured. It was preserved under the preservation conditions of the first set. Changes at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR-Ig is accumulated in cell culture. 5. Production method TNFR-Ig in cell culture in mass-composite production. With the steps of The preparation of cell culture includes A mammalian cell containing a gene encoded as TNFR-Ig, the zong gene is expressed. Under the conditions of cell culture and culture medium containing the molar ratio of accumulated inorganic ions to amino acids. The total accumulated between 0.4 and 1 and the above medium contains glutamine. And there are two such culture media. The special characteristics of the culture media were selected from the group consisting of: It consists of accumulated amino acids per unit volume greater than 70 mM, (ii) the molar ratio of Cumulative glutamine per cumulative aspartate less than 2, (iii) molar ratio of cumulative glutamine to Total accumulated amino acids less than 0.2, (iv) cumulative total content of glutamine and aspartame per unit. Volumes greater than 16 mM and their rod aggregation, page 4 of the number of 14 pages, such culture retention in the initial growth phase under set cultures conditions. One is a period of time sufficient for the cell to multiply to the density of the Live in the range of 20% - 80% of the highest living shell density possible if cultured. It was preserved under one set of cultured conditions. Changes at least some of the culture conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR. -Ig is accumulated in cell culture 6. Method for producing TNFR-Ig in cell culture in the mass production of \ 'total composition. With the steps of A cell culture arrangement which includes A mammalian cell containing a gene encoded as TNFR-Ig, in which the gene is expressed. Under the conditions of cell culture and culture media containing accumulated amounts of glutamine. And Aspara Gene per unit Volumes greater than 16 mM and the above medium containing glutamine And there are two The characteristics of the culture media selected from the group consisting of: (0 Culture media which It consists of accumulated amino acids per unit volume greater than 70 mM, (ii) the molar ratio of Accumulated glutamine per aspartame is less than 2, (iii) the molar ratio of accumulated glutamine to amorphous acids. The total accumulated amino acid is less than 0.2, (iv) the molar ratio of the accumulated inorganic ions to the total amino acid accumulated between 0.4 and their combination. Such cultures were maintained in the insetting phase under the set culture conditions. This is a period of time sufficient for the cell to reproduce up to the density of the shell. Live in the range of 20% - 80% of the highest living cell density possible if culture It was preserved under the preservation conditions of the first set. At least one change in the culture conditions In order for the second set of cultures conditions to be applied, the preservation of such cultures was a second time under the second set of conditions and as a second period to allow TNFR-Ig to accumulate in cell culture 7. Method of claim 1, whereby such shell culture conditions in the process At least one change in culture conditions was selected from a group consisting of: (i) temperature, (ii) pH, (iii) osmolarity, (iv) degree of chemical induction. 8. Method of claim 1, where the initial glutamine concentration of such culture medium is less than or equal to 13 mM. 9. Method Clause 1, where the initial glutamine concentration of such media is less than or equal to 10 mM 51 0. Method of claim 1 where the glutamine concentration Start of the above medium Is less than or equal to 7 mM 1 1. Method of claim 1 where the initial glutamine concentration of the culture medium was Is less than or equal to 4 mM 1 2. Method of claim 1 where the total cumulative volume per unit volume of glutamine of Such medium is less than or equal to 13 mM 1 3. Method of claim 1 where the total cumulative amount per unit volume of glutamine Such culture media is less than or equal to 10 mM 1 4. Method of claim 1 where the cumulative total volume per unit volume of glutamine Such culture media is less than or equal to 7 mM 1 5. Method of claim 1 whereby the cumulative total volume per unit volume of glutamine Such medium is less than or equal to 4 mM 1 6. Method of claim 1 where glutamine is provided only "in" in the starting medium at Beginning of cell culture 1 7. Method of claim 1 where the dissolved iron concentration in the culture medium is greater than 5 mm 1 8. Method of claim 1 by At the density of the live cells of the culture The viability of claim 1 is measured on the basis of period 1 9. The viability of said aquaculture is measured at intervals of 2 0. The method of claim 1, where The said state of such culture is measured. On the basis of period 2 1. Method of claim 1, whereby the ammonium level of the said culture was Measured on the basis of period 2 2. Method of claim 1 whereby the aforementioned titer of such culture is measured on Periodic Fund 2 3. Method of claim 1 where the osmolarity of such culture is measured on Period Fundamentals 2 4. Method of Clause 18-23 where such measure is taken every day page 6 of the number 14 Page 2 5. Method of Clause 18-23. 1 whereby the initial density of the mammalian cells Therefore, it is at least 2x1 or 2 shells / ml 2 6. Method of claim 1 wherein the initial density of mammalian cell Such is at least 2X103 cells / ml. 52 7. Method of claim 1 whereby the initial density of mammalian cells. Such is at least 2x104 cells / ml. 2. 8. Method of claim 1 whereby the initial density of mammalian cells. Such is at least 2x105 cells / ml 2. 9. Method of claim 1 whereby the initial density of mammalian cells. Such is at least 2x106 cells / ml 3. 0. Method of claim 1, whereby the initial density of mammalian cells. Such is at least 5x106 cells / ml. 3. 1. Method of claim 1 whereby the initial density of mammalian cells. Such as at least 1 Oxl 06 shell / ml 3 2. Method of claim 1 where the preparation process includes at least 1000 liters of aquaculture. Claim No. 1, where the preparation process includes at least 2500 liters of aquaculture preparation 3 4. Method of claim 1 where the preparation process includes preparation of at least 2500 liters of culture. At least 5000 liters of culture 3 5. Method of claim 1 where the preparation process includes at least 8000 liters of aquaculture 3 6. Method of claim 1 Where the preparation process includes the preparation of at least 10 000 liters of culture 3 7. Method of claim 1, where the preparation process includes at least 12,000 liters of preparation Culture 3 8. Method of claim 1 where the condition of the above set consists of the temperature range at which The condition of claim 1, where the condition of the first set consists of the temperature range at 1, where 32 to 40 degrees Celsius 4 0. Method of claim 1 where the condition of the first set consists of the temperature range that 34 to 38 degrees Celsius. Page 7 of number 14 Page 4 1. Method of claim 1 where the condition set above includes the temperature range at The condition of this set consists of the temperature range at 36 to 37 ° C. 4 2. Method of claim 1. The condition of the second set consists of the temperature range at 37 ° C. 4 3. Method of claim 1. The second condition is 25 to 41 degrees Celsius. 4 4. Method of claim 1 where the condition of the second set consists of the temperature range that The second condition is 27 to 38 degrees Celsius 4 5. Method of claim 1 where the condition of the second set consists of the temperature range that The second condition is 29 to 35 degrees Celsius. 4 6. Method of claim 1 where the condition of the second set consists of the temperature range that The second condition is 29 to 33 degrees Celsius. 4 7. Method of claim 1 where the condition of the second set consists of the temperature range that The second set of conditions is 30 to 32 degrees Celsius. 4 8. Method of claim 1 where the condition of the second set consists of the temperature range at The second is at 31 degrees Celsius. 4 9. The method of claim 1 is supplemented by a second change procedure to From at least one of the above-mentioned changes in cultivation conditions, which consisted of Change at least one culture condition, so that the third set condition is applied to culture 5 0. The method of claim 49 where the second transformation step consists of Change at least one of the cultured conditions selected from a group consisting of: (0 temperature, (ii) pH, (iii) osmolarity, (iv) degree of chemical induction). 5 1. Method of claim 49 where the third conditional set consists of the temperature range. The third condition is 25 to 40 degrees Celsius 5 2. Method of claim 49 where the condition of the third set consists of the temperature range. The third condition is 27 to 37 ° C 5 3. Method of claim 49 where the condition of the third set consists of the temperature range. The third condition is 29 to 34 degrees Celsius 5 4. Method of claim 49 where the condition of the third set consists of the temperature range. Third, which is 30 to 32 degrees Celsius 5 5. Method of claim 1 where the period is between 0-8 days 5 6. The method of claim that 1 where the period is between 1-7 days, page 8, number 14, page 5 7. Method of claim 1, where the time period is between 2-6 days 5 8. Method Clause 1, where the duration is 3-5 days. 5. 9. Method of claim 1, where the forward period is 4 days. 6 0. Clause 1. Clause 1 is held with a 5-day duration. 6 1. Method of claim 1 where the period is 6 days. 6 2. Method of claim 1, whereby the time period is 6 days. Total of one such period And the second period The said treatment is at least 5 days. 6 3. Method of claim 1, where in the process of preserving the said culture is The second period, the exchange level decreases from the lactate level in culture to the maximum level 6 4. The method of claim 1, where in the culture preservation process is The second period, the ammonium level was further reduced from the cultured ammonium level to the highest level 6 5. Clause 1 method where the total content of TNFR-Ig produced was At least 1.5 times higher than the amount of TNFR-Ig produced under the same conditions. Or in culture media 6 6. Method of claim 1 where the total content of the said TNFR-Ig produced is at least 2 times higher than the TNFR-Ig content. Ig produced under the same conditions. Or in culture media that 6 7. Method of claim 1, where cell culture is additionally provided. Supplementary Ingredients 6 8. Method of claim 67 whereby the ancillary components are selectable from the group that is Contains hormones and / or growth factors, specific ions (such as sodium, chloride, calcium, magnesium and phosphate), buffers, vitamins, nucleosides or nucleotides, micronutrients. (Inorganic compounds normally present at large final concentrations), amino acids, lipids or glucose 6 9. TNFR-Ig production method in cell culture in the production of \ 'quantity. Total With the steps of Arrangement of cell culture, which includes Mammal cells containing the genes encoded as TNFR-Ig, whose genes were expressed. Under the conditions of shell culture and the specified media containing glutamine. And there are at least two special characteristics Of the culture medium selected from a group containing: i) the initial amino acid concentration greater than 70 mM, ii) the molar ratio of glutamine to aspartine less than 2, iii). The molar ratio of Glutamine to total amino acids that is less than 0.2, iv) the molar ratio of inorganic ions to total amino acids between 0.4 to 1 and v) total glutamine concentrations and Asparagene greater than 16 mM page 9 of the 14 pages, such culture was maintained in the initial growth phase under the cultured conditions. The period of time that is sufficient for such cell reproduction is in the range of 20% - 80% of the maximum living cell density possible if such culture is maintained. Under the condition Cultured set at the maturity Changes at least some of the culture conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR. -Ig accumulated in cell culture 7 1. Initial amino acid concentration greater than 70 mM, ii) Molar ratio of glutamine to aspartine less than 2, iii) ratio. Molar of Glutamine to total amino acids less than 0.2, iv) the molar ratio of inorganic ions to amino acids. The total concentrations of glutamine and aspartame were greater than 16 mM. Such cultures were maintained in the initial growth phase under cultured conditions. The period of time that is sufficient for such cell reproduction is in the range of 20% - 80% of the maximum living cell density possible if such culture is maintained. Under the condition Culture set at the nong To change at least one culture condition, so that the second set of cultivation conditions was applied, such preservation was a second period under the second set condition and the second period to allow TNFR-Ig is accumulated in shell culture 7 1. Production method TNFR-Ig in cell culture in mass-composite production. With the steps of The preparation of cell culture includes Mammal cells containing the TNFR-Ig gene encoded as genes were expressed. Under the conditions of the designated cell culture and culture media, which consist of a tarmacet. And there are at least four unique characteristics of The medium was selected from a group containing: i) amino acid concentrations starting at more than 70 pages 10 of the 14 mM, ii) the molar ratio of glutamine to aspartine at Less than 2, iii) the molar ratio of Glutamine to total amino acids is less than 0.2, iv) the molar ratio of total inorganic ions \ 'total amino acids between 0.4 to 1 and v) total glutamine concentration. Amine and asparagene at greater than 16 mM, such cultivation was maintained in the initial growth phase under the cultured conditions. It is a sufficient time for the shell to reproduce in the range of 20% - 80% of the maximum viable cell density possible if the culture is properly fertilized. Kept under Cultured set at the maturity At least one change in the culture conditions, so that the second set of cultivation conditions were applied, the preservation of such culture was maintained for a second period under the second set condition and the second period. For TNFR-Ig to accumulate in cell culture 7 1. Concentration Amino acids starting at greater than 70 mM, ii) the molar ratio of glutamine to aspartine is less than 2, iii) the molar ratio of glutamine to total amino acids at Less than 0.2, iv) the molar ratio of The ionic ion to total amino acids is between 0.4 and 1 and v) the total concentration of glutamine and Asparagene at greater than 16 mM, such cultured retention in the initial growth phase under set culture conditions. The period of time that is sufficient for such cell reproduction is in the range of 20% - 80% of the maximum living cell density possible if such culture is maintained. Under the condition Cultured set at the maturity Changes at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second and second set conditions to allow TNFR-Ig is accumulated in cell culture. 7 3. Production method TNFR-Ig in cell culture mass production, which includes With the steps of The reconstructive cell culture arrangement consists of 11 pages of the 14 pages. Mammal cells containing the TNFR-Ig gene, which are expressed in genes, are carried out. Under the conditions of cell culture and culture media containing glutamine. The cumulative total content of glutamine and asparagene per unit volume was greater than 16 mM. Such cultured storage in the initial growth phase under set-up culture conditions was observed. The first is a period of time sufficient for the cell to reproduce in the range of 20% - 80% of the highest living cell density possible if the culture is maintained under the condition. The Culture set one Changing at least one of the cultures conditions, so that the second set of cultures conditions were applied, the preservation of such cultures was a second time under the second set condition and the second period to provide TNFR- Ig accumulation in Shell culture 7 4. Method of claim 1 where the above medium is composed of culture medium. Name contains glutamine And the characteristics of the culture medium were selected from groups containing: (0 amino acid concentrations greater than 70 mM, (ii) molar \ 'ratio of glutamine). Initial glutamine per aspartate is less than 2, (iii) molar ratio of glutamine intrinsic to amino acids. Total intracutaneously less than 0.2, (iv) the molar ratio of the intrinsic anonin to total amino acids. Beginning between 0.4 and 1, (v) total glutamine concentrations begin. And asparagene starts at over 16 mM and their combination 7 5. Method of either Clause 1-6 or 69-74 where the exchange level is greater than the observed level. Can be under the same conditions in the medium that Same without the special characteristics of such culture medium The ammonia level was greater than the observed level under the same conditions in the medium at The same without the specific characteristics of the culture medium and the total amount of TNFR-Ig produced is at least as high as the observed quantity under conditions that 7 6. Method of claim 1, where such culture is not supplemented by the constituents of the same culture media. 7 7. Method of claim 1, without which the culture was not supplemented with additional glutamine. During the aforementioned TNFR-Ig production period 7 8. Method of claim 1 where the glutamine concentration in such cultures decreased. Noticeably less Before the transition to the second set of cultures conditions, page 12 of the number 14, page 7 9. Method of claim 1, wherein the concentration of glutamine in such cultures decreased. Noticeably less At the same time as the transition to the second set of cultures, Kang said, 8 0. Method of claim 1 where glycyl glutamine was replaced by glutamine in the culture. Lieng Kang said 8 1. Method of claim 1, wherein culture medium consisted of (0 amino acid content per unit volume greater than 70 mM, (ii) the molar ratio of glutamine). Continue to accumulate Asparagene accumulated less than 2, (Wed) the molar ratio of accumulated glutamine to total amino acids was less than 0.2, (iv) the molar ratio of accumulated inorganic ions. (V) the cumulative total content of gutamine and aspartine per unit volume greater than 16 mM 8 2. Method of claim 1 where food There were (i) accumulated amino acids per unit volume greater than 70 mM, (ii) molar ratio of accumulated glutamine to acids. The total amino accumulated less than 0.2, (iii) the molar ratio of the accumulated anionic ions to the amino acids. The cumulative total is between 0.4 and the sludge (iv) the cumulative total content of glutamine and aspartame per unit. Volumes greater than 16 mM 8 3. Method of claim 1 wherein the cumulative total content of histidine, isoleucine, lucene, methionine, t! Nylalanine, Tryptophan, thyrosine and proline per unit volume in feed Kungung is greater than 25 mM 8 4. Method of claim 1 where the cumulative total content of histidine, isoleucine, ru) zine, methionine, t! Lanine, tryptophan, thyrosine and proline per unit volume in feed Kangung is greater than 35 mM 8 5. Method of claim 1 where total intrinsic amounts of histidine, isoleucine, lucene, methionine, T! Lanine, tryptophan, thyrosine and proline per unit volume in feed Kungung is greater than 25 mM. 8 6. Method of claim 1 where total initial concentrations of histidine, isoleucine, lucene, methionine, T! Lanine, tryptophan, thyrosine and proline per unit volume in feed The said species is greater than 35 mM. 8 7. Method of claim 1 wherein the said culture has the characteristics of the food. The cultures were selected from groups containing: (0 cumulative total volume of histidine per unit volume greater than 1.7 mM (ii) cumulative total volume of isoleucine per unit volume greater than 3.5 mM (iii) volume. Total cumulative volume of rubzine per unit volume greater than 5.5 mM (iv) Cumulative total volume of mithionine per unit volume greater than 2.0 mM page 13 of the number 14 page (v) Cumulative total volume of phenylalanine. Per unit volume greater than 2.5 rnM (vi) Cumulative total volume of proline per unit volume greater than 2.5 mM (vii) Cumulative total tryptophan per unit volume greater than 1.0 mM (viii) Cumulative total volume Of thyrosine per unit volume greater than 2.0 mM and (ix) the cumulative total volume of proline per unit volume greater than 2.5 mM. Specialties of food Variable cultures were obtained from groups consisting of: (0 isolucine intrinsic volume per unit volume greater than 1.7 mM (ii) isolein initiated per unit volume greater than 3.5 mM (iii) volume. Intrinsic to lucene per unit volume greater than 5.5 mM (iv) initial volume of methionine per unit volume greater than 2.0 mM (v) pinillalanine initiation per unit volume greater than 2.5. mM (vi) Proline intrinsic volume per unit volume greater than 2.5 mM (vii) Tryptophan initiation per unit volume greater than 1.0 mM (viii) Thyroxine initiation per unit volume. Volumes greater than 2.0 mM (ix) Proline intrinsic volume per unit volume greater than 2.5 mM 8 9. Method of claim 1 where \ 'the total accumulated volumes of Xr per unit volume in food. The above culture was greater than 7 mM 9 0. Method of claim 1 wherein the total accumulated content of Sron per unit volume in the food. Such cultures were greater than 10 mM. 9 1. Method of claim 1 where \ 'the cumulative total volume of asparagene per unit volume in Such culture media was greater than 8 mM. 9 2. Method of claim 1 whereby the cumulative total content of asparagene per unit volume in Such culture media is greater than 12 mM. 9 3. Method of claim 1 wherein the total initial volume of asparagene per unit volume in Such medium is greater than 8 mM 9 4. Method of claim 1 where \ 'the total initial volume of asparagene per unit volume in Such culture media was greater than 12 mM 9 5. Method of claim 1 whereby the accumulated total content of phosphorus per unit volume in The above culture medium was greater than 2.5 mM 9 6. Method of claim 1 where the total accumulated content of phosphorus per unit volume in Such culture media is greater than 5 mM, page 14 of the number 14, page 9 7. Clause 1 Method, where the total accumulated volumes of the gutamate per unit volume in / J. Less than 1 mM 9 8. Claim Method 1 where \ 'the cumulative total volume of calcium pantothenate per unit. The volume in the culture medium is greater than 8 mg / L 59 9. Method of claim 1 where the total cumulative amount of calcium pantothenate per unit The volume in the culture medium is greater than 20 mg / L 10 0. Claim Method 1 where \ 'the cumulative total volume of nicotinamide per unit volume. In the medium above 7 mg / L 10. 1. Method of claim 1 whereby the cumulative total volume of nicotinamide per unit volume In the medium above 25 mg / L 10. 2. Method of claim 1 where \ 'the cumulative total volume of pyridoxine. And pyridoxol The per unit volume of such culture media is greater than 5 mg / L 10. 3. Method of claim 1 where \ 'the cumulative total volume of pyridoxine. And pyridoxol The per unit volume of such culture media is greater than 35 mg / L 10. 4. Method of claim 1 whereby the cumulative total volume of riboflavin per unit volume In such medium is greater than 1.0 mg / L 10. 5. Method of claim 1, where \ 'cumulative total volume of riboflavin per unit \' volume. In the medium above 2.0 mg / L 10. 6. Method of claim 1 whereby the cumulative total content of thiamine hydrochloride per The unit volume in such culture medium is greater than 7 mg / L 10. 7. Method of claim 1 whereby the cumulative total content of thiamine hydrochloride per The unit volume in such culture medium is greater than 35 mg / L ------------