Mammalian cell line models and related methods

Abstract

The invention provides models and methods useful for optimizing cell lines. The invention provides methods and computer readable medium or media containing such methods. Such a computer readable medium or media can comprise commands for carrying out a method of the invention. The methods of the invention can be utilized to model improved characteristics of a cell line, for example, improved product production, improved growth, improved culture characteristics, and the like.

1 . A computer readable medium or media having stored thereon computer executable commands for performing the steps of:
 (a) providing a data structure relating a plurality of reactants to a plurality of reactions from a cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a constraint set for said plurality of reactions for said data structure;   (c) providing an objective function, wherein said objective function is uptake rate of two or more nutrients, wherein said two or more nutrients are carbon sources; and   (d) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said data structure, wherein said at least one flux distribution is predictive of a culture condition for said eukaryotic cell.   
     
     
         2 . The computer readable medium or media of  claim 1 , wherein said objective function further comprises product formation, energy synthesis, biomass production, or a combination thereof. 
     
     
         3 . The computer readable medium or media of  claim 1 , wherein said objective function further comprises decreasing byproduct formation. 
     
     
         4 . The computer readable medium or media of  claim 1 , wherein said culture condition is selected from optimized culture medium for said cell, optimized cell culture process, optimized cell productivity, and metabolic engineering of said cell. 
     
     
         5 . The computer readable medium or media of  claim 4 , wherein optimized cell productivity is selected from increased biomass production and increased product yield. 
     
     
         6 . The computer readable medium or media of  claim 1 , wherein said culture condition is reduced scale up variability. 
     
     
         7 . The computer readable medium or media of  claim 1 , wherein said culture condition is reduced batch to batch variability. 
     
     
         8 . The computer readable medium or media of  claim 1 , wherein said culture condition is reduced clonal variability. 
     
     
         9 . The computer readable medium or media of  claim 1 , wherein said culture condition is improved cell growth, viable cell density or cell productivity in exponential growth phase or stationary phase. 
     
     
         10 . The computer readable medium or media of  claim 1 , wherein said data structure comprises a reaction network. 
     
     
         11 . The computer readable medium or media of  claim 1 , wherein said data structure comprises a plurality of reaction networks. 
     
     
         12 . The computer readable medium or media of  claim 1 , wherein said cell is derived from an animal, plant or insect. 
     
     
         13 . The computer readable medium or media of  claim 12 , wherein said cell is a mammalian cell. 
     
     
         14 . The computer readable medium or media of  claim 13 , wherein said cell is a mammalian cell selected from Chinese Hamster Ovary (CHO), BHK, NS0, SP2/0, 3T3, Hybridoma, C127, HEK293, PER.C6, HepG2, HeLa, MRCS, W138, MDCK, Vero, and COS. 
     
     
         15 . The computer readable medium or media of  claim 1 , wherein said cell produces a product selected from an exogenous growth factor, monoclonal antibody, hormone, cytokine, fusion protein, enzyme, vaccine, virus, anticoagulant, and nucleic acid. 
     
     
         16 . The computer readable medium or media of  claim 1 , wherein said data structure comprises a set of linear algebraic equations. 
     
     
         17 . The computer readable medium or media of  claim 1 , wherein at least one reactant in said plurality of reactants or at least one reaction in said plurality of reactions is annotated with an assignment to a subsystem or compartment. 
     
     
         18 . The computer readable medium or media of  claim 17 , wherein at least a first substrate or product in said plurality of reactions is assigned to a first compartment and at least a second substrate or product in said plurality of reactions is assigned to a second compartment. 
     
     
         19 . A method for predicting a culture condition for a eukaryotic cell from a multicellular organism, comprising performing the steps on the computer readable medium or media of  claim 1 , said steps comprising:
 (a) providing a data structure relating a plurality of reactants to a plurality of reactions from a cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a constraint set for said plurality of reactions for said data structure;   (c) providing an objective function, wherein said objective function is uptake rate of two or more nutrients, wherein said two or more nutrients are carbon sources; and   (d) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said data structure, wherein said at least one flux distribution is predictive of a culture condition for said eukaryotic cell.   
     
     
         20 - 36 . (canceled) 
     
     
         37 . A computer readable medium or media having stored thereon computer executable commands for performing the steps of:
 (a) providing a first data structure relating a plurality of reactants to a plurality of reactions from a cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a constraint set for said plurality of reactions for said first data structure;   (c) providing an objective function, wherein said objective function is uptake rate of two or more nutrients, wherein said two or more nutrients are carbon sources;   (d) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said data structure;   (e) deleting a reaction from said data structure to generate a second data structure and repeating steps (b) through (d);   (f) optionally repeating step (e) by deleting a different reaction, wherein said at least one flux distribution determined with said second data structure is predictive of a reaction required for cell growth or cell viability, thereby identifying a target selectable marker reaction or reactant.   
     
     
         38 . The computer readable medium or media of  claim 37 , further comprising computer executable commands for performing the steps of:
 (g) providing said second data structure;   (h) providing one or more extracellular substrates or products corresponding to one or more reactions of said one or more extracellular exchange reactions to said second data structure to generate a third data structure;   (i) providing a constraint set for said plurality of reactions for said third data structure;   (j) providing an objective function, wherein said objective function is cell viability or growth; and   (k) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said third data structure, wherein said at least one flux distribution determined with said third data structure is predictive of an extracellular substrate or product that complements the target selectable marker reaction or reactant, thereby identifying a selectable marker reaction or reactant.   
     
     
         39 . (canceled) 
     
     
         40 . A method for identifying a target selectable marker for a cell, comprising performing the steps on the computer readable medium or media of  claim 37 , said steps comprising:
 (a) providing a first data structure relating a plurality of reactants to a plurality of reactions from a cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a constraint set for said plurality of reactions for said first data structure;   (c) providing an objective function, wherein said objective function is uptake rate of two or more nutrients, wherein said two or more nutrients are carbon sources;   (d) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said data structure;   (e) deleting a reaction from said data structure to generate a second data structure and repeating steps (b) through (d);   (f) optionally repeating step (e) by deleting a different reaction, wherein said at least one flux distribution determined with said second data structure is predictive of a reaction required for cell growth or cell viability, thereby identifying a target selectable marker reaction or reactant.   
     
     
         41 . The method of  claim 40 , further comprising:
 (g) providing said second data structure;   (h) providing one or more extracellular substrates or products corresponding to one or more reactions of said one or more extracellular exchange reactions to said second data structure to generate a third data structure;   (i) providing a constraint set for said plurality of reactions for said third data structure;   (j) providing an objective function, wherein said objective function is cell viability or growth; and   (k) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said third data structure, wherein said at least one flux distribution determined with said third data structure is predictive of an extracellular substrate or product that complements the target selectable marker reaction or reactant, thereby identifying a selectable marker reaction or reactant.   
     
     
         42 . (canceled) 
     
     
         43 . A computer readable medium or media having stored thereon computer executable commands for performing the steps of:
 (a) providing a first data structure relating a plurality of reactants to a plurality of reactions from a non-contaminated cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a second data structure relating a plurality of reactants to a plurality of reactions from a contaminated cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (c) providing a constraint set for said plurality of reactions for said first and second data structures;   (d) providing an objective function, wherein said objective function is uptake rate of one or more nutrients, wherein said two or more nutrients are carbon sources;   (e) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said first data structure;   (f) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said second data structure;   (g) comparing the at least one flux distribution determined for said first data structure with the at least one flux distribution determined for said second data structure, wherein a difference in the at least one flux distribution for said first and second data structures is predictive of a biomarker for a contaminant of said cell culture.   
     
     
         44 . (canceled) 
     
     
         45 . A method for predicting a biomarker for a contaminant of a cell culture of a eukaryotic cell from a multicellular organism, comprising performing the steps on the computer readable medium or media of  claim 43 , said steps comprising:
 (a) providing a first data structure relating a plurality of reactants to a plurality of reactions from a non-contaminated cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (b) providing a second data structure relating a plurality of reactants to a plurality of reactions from a contaminated cell, each of said reactions comprising a reactant identified as a substrate of the reaction, a reactant identified as a product of the reaction and a stoichiometric coefficient relating said substrate and said product, wherein said plurality of reactions comprises one or more extracellular exchange reactions;   (c) providing a constraint set for said plurality of reactions for said first and second data structures;   (d) providing an objective function, wherein said objective function is uptake rate of one or more nutrients, wherein said two or more nutrients are carbon sources;   (e) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said first data structure;   (f) determining at least one flux distribution that minimizes or maximizes the objective function when said constraint set is applied to said second data structure;   (g) comparing the at least one flux distribution determined for said first data structure with the at least one flux distribution determined for said second data structure, wherein a difference in the at least one flux distribution for said first and second data structures is predictive of a biomarker for a contaminant of said cell culture.   
     
     
         46 . (canceled) 
     
     
         47 . A computer readable medium or media having stored thereon computer executable commands for performing the steps of:
 (a) providing a data structure relating a plurality of reactants to a plurality of reactions, wherein said plurality of reactants and plurality of reactions are selected from a selection of reactants and reactions as shown in Table 23 for a Chinese hamster ovary (CHO) cell, Table 21 for a hybridoma cell, and Table 22 for an NS0 cell;   (b) providing a constraint set for said plurality of reactions for said data structure; and   (c) determining at least one flux distribution that minimizes or maximizes an objective function when said constraint set is applied to said data structure, wherein said at least one flux distribution is predictive of a physiological function of said CHO cell, hybridoma cell or NS0 cell.   
     
     
         48 - 49 . (canceled)