Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

Definitions

• This invention relates to methods of regulating and reducing the build-up of cellular waste products in cell culture systems, with concomitant synthesis of nutrients.
• Continuous growth of cultures may be achieved by removing a portion of the culture and replacing it with fresh medium at regular intervals. This serves to remove waste products and replenish needed nutrients.
• fluid containing cells and media is continuously withdrawn from the cell culture container and replaced with fresh medium.
• cells may be confined to the cell culture vessel such that only medium is withdrawn while fresh medium is added.
• the components of a cell culture system are not depleted uniformly.
• the perfused media may be depleted of three or four nutrients, it may contain adequate amounts of other components. The removal of these useful components by perfusion is uneconomical.
• Perfusion is associated with other disadvantages.
• perfusion systems are susceptible to plugging by cells which must be separated from spent culture media. This is a significant concern for high density cultures since prodigious amount of fluids must be exchanged, sometimes greater than one culture volume per day.
• cell products must be isolated from the large volumes of spent culture media generated, and after products are isolated, large volumes of spent culture media must be disposed of.
• a multienzyme system permits the synthesis of nutrients, such as amino acids, and reduction of waste products, such as ammonia and lactic acid in cell culture systems.
• lactate dehydrogenase converts lactate to pyruvate in the presence of the co-factor NAD + .
• Leucine dehydrogenase converts ⁇ -keto acids to corresponding amino acids in the presence of NADH, which is generated by the lactate dehydrogenase reaction.
• the co-factor recycles continuously in the presence of adequate levels of substrates.
• the present invention provides a mechanism for converting deleterious waste products into nutrients otherwise depleted in the cell culture system. It can be applied to systems for maintaining the levels of amino acids and other components necessary for continuous growth of cells in culture. As a result, the productive life-span of cultured cells is prolonged.
• Figure 1 illustrates a representative multienzyme system. Detailed Description of the Invention
• a reduction in waste products and a concomitant synthesis of amino acids and/or other nutrients in a cell culture system can be achieved using a multienzyme system.
• cell culture system is meant a system of growing cells in vitro in a culture vessel.
• the system is not limited to any particular culture vessel configuration or cell type, in a preferred embodiment, mammalian cells are grown in liquid culture media.
• contacting in reference to the cell culture medium, is meant the exposure of cell culture medium to the multienzyme system of the invention such that the enzymes of the multienzyme system (a) catalyze reactions between at least one component of the medium, such as a waste product, and a component of the multienzyme system, such as an amino acid precursor, and (b) regenerate the recycling co-factor of the multienzyme system.
• the enzymes of the multienzyme system (a) catalyze reactions between at least one component of the medium, such as a waste product, and a component of the multienzyme system, such as an amino acid precursor, and (b) regenerate the recycling co-factor of the multienzyme system.
• nicotinamide adenine dinucleotide is a co-factor.
• NAD + is capable of conversion to NADH by electron transfer.
• NAD + is generated by the action of leucine dehydrogenase on an amino acid precursor to form an amino acid. Lactic dehydrogenase converts lactate to pyruvate, and by this reaction regenerates the co-factor as NADH.
• nutrient is meant any biological compound essential for growth and/or division of cells in culture.
• amino acids are nutrients.
• Non-amino acid compounds, such as pyruvate are also nutrients.
• nutrient precursor is meant a compound capable of enzymatic conversion into a nutrient.
• the amino acid precursors ⁇ -ketoisocaproate, ⁇ -ketoisovalerate and ⁇ - keto- ⁇ -methylvalerate are precursors of, respectively, leucine, valine and isoleucine. One or more of these precursors can be employed in the multi-enzyme system.
• multienzyme system is meant a plurality of cooperatively functioning biological components capable of achieving the synthesis of at least one nutrient, utilizing at least one waste product and at least one nutrient precursor.
• the multienzyme system of this invention contains lactate dehydrogenase, leucine dehydrogenase, ADP, KCl, NAD + , ⁇ -ketoisocaproate, ⁇ - ketoisovalerate and ⁇ -keto- ⁇ -methylvalerate.
• Enzymes useful in the multienzyme system of the invention can be tested and selected on the basis of the following eight factors: 1) Toxicity and required concentrations of enzymes and co-factors. At working concentrations, the required molecules must not be toxic or inhibitory to cell growth and/or to the synthesis of biological products of the cells. 2) Enzyme activity in cell culture media. Some enzymes are known to be inhibited by various amino acids which are major components of cell culture media.
• Physiological environment is important. Many mammalian cell cultures require media of essentially neutral pH and temperatures of 37 ⁇ 2°C, a point at which many native enzymes are unstable.
• One method of selecting a particular enzyme for use in the multienzyme system is to first identify in a cell culture the accumulation of an undesirable waste product. Once this product is identified, enzymes are selected that will convert the waste product into another form, preferably a nutrient source for the cell culture.
• the enzymes may be used in combination with an appropriate co-factor, preferably a co-factor capable of continuously recycling in the presence of adequate levels of substrates. The identity of the co-factor will be determined by the requirements of the enzymes chosen and their particular substrates.
• FIG. 1 A representative multienzyme system is illustrated in Figure 1. This system meets the requirements enumerated above for use in a cell culture system.
• the system employs two enzymes, lactic dehydrogenase and leucine dehydrogenase, and a recycling co-factor (NAD + /NADH).
• Lactate dehydrogenase converts lactate to pyruvate in the presence of NAD + .
• Leucine dehydrogenase converts ⁇ -ketoacids ( ⁇ -ketoisocaproate, ⁇ -ketoisovalerate, and ⁇ -keto- ⁇ -methyl valerate) into corresponding amino acids (leucine, valine, and isoleucine, respectively) in the presence of NADH, which is generated by the lactate dehydrogenase reaction.
• the co-factor recycles continuously as long as adequate levels of lactate, ammonia, and an amino acid precursor are present.
• the multienzyme system achieves the synthesis of amino acids with concomitant decreases in the waste products ammonia and lactic acid.
• alanine can be produced by the reductive amination of pyruvate
• glutamine can be produced from glutamic acid by the action of glutamine synthetase.
• the form of the invention is not limited to a soluble solution or microencapsulated enzymes.
• the invention can be utilized in conjunction with a container unit, wherein some or all of the cell culture medium is exposed to the multienzyme system within the container.
• the multienzyme system can be placed within a hollow fiber unit, within a compartment separated by a semipermeable membrane. Cell culture (media and cells) passes through the center of the hollow fibers, and waste products from the media diffuse across the semipermeable membrane to contact the enzyme system.
• the waste products are converted into nutrients, such as amino acids, by the action of the multienzyme system, and these nutrients pass "through the membrane to the cell culture.
• the container unit may be included as a component of a kit for convenient use with a cell culture system.
• the container unit of the kit may contain the enzymes of the multienzyme system. Other compartments of the multienzyme system, specifically a nutrient precursor and a recycling co-factor, can be supplied separately.
• the kit would also provide a means for contacting the cell culture with the multienzyme system.
• the kit also provides a container having cell culture medium.
• the container means is a hollow fiber unit, and the enzymes are contained within a first compartment.
• One or more nutrient precursors and a recycling co-factor are added to the compartment containing the enzymes.
• the cell culture is contacted with the multienzyme system through a second compartment which is separated from the first compartment by the semi-permeable membrane.
• the second compartment receives cell culture media, or media and cells, from the main culture vessel through, for example, a hollow tube.
• the first compartment of the container unit may contain the enzymes of the multienzyme system, and, in addition, one or more nutrient precursors and/or a recycling cofactor.
• kits which hold, respectively, enzymes, co-factor (s) and nutrient precursors.
• the enzymes may be provided in a microencapsulated form.
• the components are added to the culture system when appropriate on the basis of the growth conditions of the culture.
• the multienzyme system can be conveniently incorporated into virtually any cell culture system.
• the invention is not intended to be limited to use with known cell culture systems, and it is envisioned that as new and improved mechanical cell culture systems and apparatus are developed, spent cell culture media can be regenerated using the multienzyme system of the invention.
• tissue culture medium for use or to formulate this as a concentrate in tissue culture medium which is then diluted into tissue culture media to provide the indicated final concentrations. (This may be desired if several types of cells or conditions are to be tested simultaneously.) It is also possible to adjust concentrations of reactants depending upon cell type, the desired rate of depletion of culture nutrients, and the rate of generation of waste products.
• concentrations of reactants depending upon cell type, the desired rate of depletion of culture nutrients, and the rate of generation of waste products.
• Cells should have viabilities greater than 90% and be in late log phase. It is preferable to avoid lag-phase cells.
• Table 2 illustrates the synthesis of amino acids in a PBS aqueous phase (no cells) over 168 hours. As can be seen, isoleucine, leucine and valine increased significantly over this time period..
• Tables 3 and 4 illustrate production levels of synthesized amino acids produced in a pH and oxygen-controlled bioreactor at high cell density, with enzymes free in solution (Table 3) and microencapsulated (Table 4). Both experiments demonstrate that amino acid levels were increased by enzymatic means in cell culture and that microencapsulation is a viable alternative for separating enzymes from the cellular milieu.
• Tables 5 and 6 illustrate the results of a high density 12 well tray assay.
• the presence of the enzyme mixture correlated with significantly higher cellular viability, monoclonal antibody production and less ammonia and lactic acid than without the enzyme mixture.
• the data illustrate that amino acid levels, as well as concentrations of lactic acid and ammonia, correlated with viability and monoclonal antibody production.

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• 1990
  • 1990-10-05 US US07/593,930 patent/US5106747A/en not_active Expired - Lifetime
• 1991
  • 1991-09-06 EP EP91916631A patent/EP0551305A1/en not_active Withdrawn
  • 1991-09-06 CA CA002092413A patent/CA2092413A1/en not_active Abandoned
  • 1991-09-06 WO PCT/US1991/006380 patent/WO1992006177A1/en not_active Ceased
  • 1991-09-06 JP JP3515358A patent/JPH06501611A/ja active Pending

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