Method of producing a recombinant protein in HEK-293 cells

Abstract

A transient protein expression system and kit, a composition for producing a recombinant protein in cultured cells, and a method for producing a recombinant protein in cultured cells. The method includes: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein; contacting the transfected 293 cells with at least one expression enhancer composition; and culturing the transfected 293 cells in the presence of the at least one expression enhancer composition for a period of time such that the recombinant protein is expressed.

Description

PATENT DOCKET NO. TP388599WO1METHOD OF PRODUCING A RECOMBINANT PROTEIN IN MAMMALIAN CELLS CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 681,658, filed August 9, 2024, and to U.S. Provisional Patent Application No.63 / 767,786, filed March 6, 2025, the disclosures of which are considered part of, and incorporated in their entireties by reference in the disclosure of this application. SEQUENCE STATEMENT

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on August 8, 2025, is named TP388599WO1, and is 512,697 bytes in size. FIELD

[0003] The present disclosure generally relates to the fields of transfection and cell culture. In particular, the present disclosure provides systems, methods and compositions for high yield expression of recombinant proteins in mammalian cells. BACKGROUND

[0004] Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and formulations of cell culture media vary depending upon the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient compositions.

[0005] Once a suitable culture medium for the growth of a particular cell type has been determined, it is frequently necessary to alter the cell in question so as to optimize the production of a desired biological substance. A critical step in the effective production and purification of biological substances is the introduction of nucleic acids for production of biological molecules (e.g., peptides, proteins, nucleic acids, and the like) into the cell in which the material will be produced. This can bePATENT DOCKET NO. TP388599WO1accomplished by a variety of methods. One widely used method to introduce macromolecules into a cell is transfection.

[0006] There remains a need in the art for cell mediums and transient transfection systems that permit the growth of eukaryotic cells in high density suspension culture while permitting automated, high-throughput transfection of the cells with a reduced amount of manipulation and reduced overall timeframe for producing an expression product. Such a transient transfection system will allow easier and more cost-effective and efficient production and purification of high quantities of commercially or scientifically important biological substances (e.g., viruses, recombinant proteins, biologics, recombinant antibodies, and the like). These and other needs are addressed by the present disclosure. SUMMARY

[0007] The present disclosure provides a transient transfection system and methodology that supports introduction by way of transfection and subsequent expression of one or more macromolecules (such as, e.g., expressible nucleic acids) into a eukaryotic cell in culture in a reduced timeframe. The present disclosure provides the cultivation and growth of cells subsequent to the introduction / transfection such that the expression product is generated in a reduced time and higher yield, and may be generated in an automated manner. The disclosure enables flexibility in producing an expression product in which an expression enhancer composition(s) is contacted with cells before, during, or after transfection to reduce the eventual time required to harvest a desired expression product (such as, e.g., recombinant protein) produced by transfected cells. Additionally, the growth of cells may optionally continue in the culture medium after transfection in the absence of the medium being supplemented with fresh medium. Conveniently, the system and methodology described herein may be performed in an automated manner using, for example, automated robotics, and optionally cells provided in a multi-well format for high-throughput processing.

[0008] In various aspects, the present disclosure provides a method for producing a recombinant protein in cultured cells, such as human embryonic kidney 293 cells (HEK-293 cells or 293 cells). In some embodiments, the method includes: transfecting cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2×106to about 2×107cells / ml; incubating the transfected cells for a first period of time being less than about 2 hours; contacting the transfected cells with one or more expression enhancer compositions; and culturing the transfected cells in thePATENT DOCKET NO. TP388599WO1presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0009] In some embodiments, the method includes: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2×106to about 2×107cells / ml; incubating the transfected 293 cells for a first period of time being less than about 2 hours; contacting the transfected 293 cells with one or more expression enhancer compositions; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0010] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the cells with a nucleic acid capable of expressing a recombinant protein; optionally incubating the transfected cells for a first period of time of less than about 2 hours; optionally contacting the transfected cells with an expression enhancer composition; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0011] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with at least one expression enhancer composition; transfecting the 293 cells with a nucleic acid capable of expressing a recombinant protein; contacting the transfected 293 cells with at least one expression enhancer composition; and culturing the transfected 293 cells in the presence of the at least one expression enhancer composition for a second period of time, wherein the recombinant protein is expressed.

[0012] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the cells with a nucleic acid capable of expressing a recombinant protein; incubating the transfected cells for a first period of time of less than about 2 hours; contacting the transfected cells with an expression enhancer composition; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0013] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107PATENT DOCKET NO. TP388599WO1cells / ml with one or more expression enhancer compositions; transfecting the 293 cells with a nucleic acid capable of expressing a recombinant protein; incubating the transfected 293 cells for a first period of time of less than about 2 hours; contacting the transfected 293 cells with an expression enhancer composition; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0014] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the cells with a nucleic acid capable of expressing a recombinant protein; incubating the transfected cells for a first period of time of less than about 15 minutes; contacting the transfected cells with an expression enhancer composition; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0015] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the 293 cells with a nucleic acid capable of expressing a recombinant protein; incubating the transfected 293 cells for a first period of time of less than about 15 minutes; contacting the transfected 293 cells with an expression enhancer composition; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a second period of time, wherein the recombinant protein is expressed.

[0016] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the cells with a nucleic acid capable of expressing a recombinant protein; contacting the transfected cells with an expression enhancer composition; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed.

[0017] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the 293 cells with a nucleic acid capable of expressing a recombinant protein; contacting the transfected 293 cells with an expression enhancer composition; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed.PATENT DOCKET NO. TP388599WO1

[0018] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the cells with a nucleic acid capable of expressing a recombinant protein; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed and optionally harvested.

[0019] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with one or more expression enhancer compositions; transfecting the 293 cells with a nucleic acid capable of expressing a recombinant protein; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed and optionally harvested.

[0020] In some embodiments, the method includes: transfecting cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2×106to about 2×107cells / ml; contacting the transfected cells with at least one expression enhancer composition, wherein the transfected cells are contacted with the at least one expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0021] In some embodiments, the method includes: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2×106to about 2×107cells / ml; contacting the transfected 293 cells with at least one expression enhancer composition, wherein the transfected 293 cells are contacted with the at least one expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0022] In some embodiments, the method includes: transfecting cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2×106to about 2×107cells / ml, and wherein the cells are transfected using an automated robotic assembly; contacting the transfected cells with at least one expression enhancer composition, wherein the transfected cells arePATENT DOCKET NO. TP388599WO1contacted with the at least one expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0023] In some embodiments, the method includes: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2×106to about 2×107cells / ml, and wherein the 293 cells are transfected using an automated robotic assembly; contacting the transfected 293 cells with at least one expression enhancer composition, wherein the transfected 293 cells are contacted with the at least one expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0024] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with at least one expression enhancer compositions; transfecting the cells; contacting the transfected cells with at least one expression enhancer composition; and culturing the transfected cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed, and wherein the contacting of the cells in the suspension culture, transfecting the cells, and / or the contacting of the transfected cells is performed using an automated robotic assembly. In some embodiments, the cells are provided in a multi-well format before transfecting the cells.

[0025] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with at least one expression enhancer compositions; transfecting the 293 cells; contacting the transfected 293 cells with at least one expression enhancer composition; and culturing the transfected 293 cells in the presence of the expression enhancer composition(s) for a period of time, wherein the recombinant protein is expressed, and wherein the contacting of the 293 cells in the suspension culture, transfecting the 293 cells, and / or the contacting of the transfected 293 cells is performed using an automated robotic assembly. In some embodiments, the 293 cells are provided in a multi-well format before transfecting the 293 cells.

[0026] In some embodiments, the method includes: contacting cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with at least one expression enhancer compositions; transfecting the cells; and culturing thePATENT DOCKET NO. TP388599WO1transfected cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed, and wherein the contacting of the cells in the suspension culture, transfecting the cells, and / or the contacting of the transfected cells is performed using an automated robotic assembly. In some embodiments, the cells are provided in a multi-well format before transfecting the cells.

[0027] In some embodiments, the method includes: contacting 293 cells in a suspension culture having a high density culture medium and a cell density of between about 2×106to about 2×107cells / ml with at least one expression enhancer compositions; transfecting the 293 cells; and culturing the 293 transfected cells in the presence of the at least one expression enhancer composition for a period of time, wherein the recombinant protein is expressed, and wherein the contacting of the 293 cells in the suspension culture, transfecting the 293 cells, and / or the contacting of the transfected 293 cells is performed using an automated robotic assembly. In some embodiments, the 293 cells are provided in a multi-well format before transfecting the 293 cells.

[0028] In various embodiments, the suspension culture is obtained from frozen cells which are thawed, suspended in culture medium, and cultured in suspension to a cell density of between about 2×106to about 2×107cells / ml before the cells are transfected. In some embodiments, the suspension culture is obtained from frozen 293 cells which are thawed, suspended in culture medium, and cultured in suspension to a cell density of between about 2×106to about 2×107cells / ml before the cells are transfected. In some embodiments, obtaining the suspension culture from frozen cells, culturing the cells in suspension, transfecting the cells, incubating the cells for a first period of time, and contacting the transfected cells with at least one expression enhancer composition is completed in less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, incubating the 293 cells for a first period of time, and contacting the transfected 293 cells with at least one expression enhancer composition is completed in less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, obtaining the suspension culture from frozen cells, culturing the cells in suspension, transfecting the cells, and contacting the transfected cells with at least one expression enhancer composition is completed in less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, and contacting the transfected 293 cells with at least one expression enhancer composition is completed in less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, the suspension culture is obtained without splitting the cells.PATENT DOCKET NO. TP388599WO1

[0029] In some embodiments, it is not necessary to remove, replenish or replace the medium used during the introduction / transfection of the cells from the presence of the cells to support the further growth thereof. In another preferred embodiment, after the introduction / transfection, growth of the cells and production of an expressed protein from the expressible nucleic acid can be accomplished in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which the introduction / transfection occurred. Using the medium of the present disclosure, it is not necessary to replenish, replace or supplement the medium after one has introduced nucleic acid into cells, and before cells into which nucleic acid has been introduced are further cultured to express the nucleic acid.

[0030] Transient expression is fast becoming the system of choice for rapid mammalian protein production. The flexibility of transient transfection enables a rapid realization time from concept to protein-in-hand and many different proteins can be produced simultaneously, or serially, optionally with the use of automated robotic assemblies and performed in a high-throughput manner. The next key advance in transient transfection technology is to approach or equal expression levels attained using stable expression systems without losing the speed and flexibility of the transient format. In some embodiments, the present disclosure provides a system that utilizes high density 293 cells to generate significantly higher expression levels of a desired expression product in less time. The expression system and methodology describe herein produce expression products in higher amounts with increased time-efficiency while reducing manual manipulation and cost. Further increasing the utility of the present disclosure is the inclusion of automated assemblies and formats that allow for high-throughput production and remote guidance by a technician.

[0031] To attain such high levels of protein expression, a cell culture system which includes an improved high density growth culture medium in combination with a population of suspension cells that are adapted for high density growth in such a media was developed that allows certain populations of mammalian cells to reach viable cell densities of up to 20x106cells / ml (more typically up to about 15x106cells / ml). These ultra-high density cultures enable transfection at higher cell densities than traditional protocols, significantly increasing the volumetric yield of protein. The addition of one or more expression enhancer compositions following or during transfection was also found to boost protein expression level to levels up to 10- to 12-fold higher than the expression levels seen with current commercially available transient transfection systems. Parental suspension culture mammalian cells were adapted for improved growth and viability characteristics under high density culture conditions, and were then further selected for increased protein production. The resulting highPATENT DOCKET NO. TP388599WO1density adapted cells have an increased growth rate, increased cell size, and increased specific productivity compared to the parental cell line. Finally, the transfection method was optimized through the use of one or more transfection reagents that may include a cationic lipid component that are used in combination with one or more expression enhancer compositions to further increase overall protein yield.

[0032] When all of these improvements were combined into a single expression system, protein levels were increased up to 10-fold for both IgG and non-IgG recombinant proteins in less time as compared to the commercially available Expi293™ Expression System and FreeStyle™ 293 Expression System. Additionally, protein functionality was demonstrated to be comparable for several proteins expressed in the high yield expression system of the present disclosure when compared to the commercially available Expi293™ Expression System and FreeStyle™ 293 Expression System. Together, these results indicate that significant increases in functional protein yields can be attained using a transient mammalian expression system that incorporates numerous advances in protein expression technology into a single, easy to use format.

[0033] The present disclosure also provides a method of cultivating eukaryotic cells comprising: (a) contacting the cells with the cell culture medium of the present disclosure; (b) maintaining the cells under conditions suitable to support cultivation of the cells in culture; and (c) optionally expressing a nucleic acid to form a protein product, wherein the nucleic acid is optionally introduced into eukaryotic cells by transfection using an automated robotic assembly.

[0034] The present disclosure further provides a recombinant protein production system. In embodiments, the system includes one or more of the following: 293 cells, a cell culture medium capable of supporting high density suspension culture of the 293 cells with a cell density of between about 2x106to about 2x107cells / ml, a transfection reagent, and an expression enhancer composition, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 150 mM and / or sodium proprionate at a concentration of at least about 1500 mM.

[0035]

[0036] The present disclosure also provides a kit for the cultivation and transfection of cells in vitro, the kit comprising the cell culture medium of the present disclosure, and optionally further comprising one or more of: one or more agents for the introduction of at least one molecule into a cell, one or more macromolecules, at least one cell, and instructions for culturing the at least one cell in culture and / or for introducing at least one macromolecule into at least one cell in culture.PATENT DOCKET NO. TP388599WO1

[0037] The present disclosure also provides a composition including the cell culture medium of the present disclosure and one or more of: a eukaryotic cell, an agent(s) for the introduction of at least one macromolecule into the cell, and one or more macromolecules. Other embodiments of the present disclosure will be apparent to one of ordinary skill in light of the following drawings and description of the disclosure, and of the claims. BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 is a diagram depicting the workflow of the commercially available Expi293™ Expression System (‘Expi293’; Thermo Fisher Scientific; Catalog No. A14525). Notably, the workflow requires the cells to be split and then further incubated the day before transfection (Day -1 of transfection step). Additionally, complexation must be performed using two tubes in which an expressible nucleic acid encoding a desired protein is added to a first tube including half of the total volume of complexation media and a transfection reagent is added to a second tube including half of the total volume of complexation media. The contents of the two tubes are then combined in a single tube to perform the complexation reaction. Also, the transfection reagent does not remain stable in the complexation media for a sufficient length of time that is amenable for automated liquid handling and / or processing.

[0039] FIG.2 is a diagram depicting an embodiment of the workflow of the 293 expression system of the disclosure. The 293 expression system of the disclosure follows a simplified and flexible workflow protocol as compared to the commercially available Expi293™ Expression System. Notably, the workflow of the 293 expression system of the present disclosure does not require cells to be split and incubated the day before transfection (Day -1 of transfection step), complexation may be performed in a single tube or using two tubes (two tubes are required with the commercially available Expi293™ Expression System), and expression enhancer may be added immediately after transfection thereby reducing the time required to perform the workflow and harvest produced protein of interest. In such embodiments, the 293 expression system of the disclosure has a streamlined and reduced time workflow in which harvestable protein is typically produced in 5 days or less so that support from a technician (e.g., to add and / or replace media and like) is not needed on a weekend.

[0040] FIG. 3 is a diagram depicting an embodiment of an automated workflow of the 293 expression system of the disclosure. High throughput protein production may be performed using multi-well plates in an automated manner (e.g., use of an automated robotic liquid handler).PATENT DOCKET NO. TP388599WO1Expressible nucleic acid encoding desired protein(s) diluted in complexation media is first added to wells of the plate. Transfection reagent diluted with complexation media is subsequently added to the wells. The nucleic acid / transfection reagent mixture is then incubated for a short time (e.g., about 5 minutes) to perform the complexation reaction and form nucleic acid / transfection reagent complexes. Cells are then immediately added to the wells to allow transfection of the cells. Expression enhancer is then added to the wells immediately following transfection or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-transfection.

[0041] FIG.4 is a bar graph showing average protein yield of 11 different monoclonal antibodies and human IgG protein obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T0 Protocol (T0).

[0042] FIG.5 is a bar graph showing average protein yield of 41 different monoclonal antibodies obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0043] FIG. 6 is a bar graph showing average protein yield of secreted non-antibodies obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0044] FIG.7 is a bar graph showing average protein yield of secreted antibodies obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0045] FIG.8 is a bar graph showing average protein yield of membrane proteins obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0046] FIG.9 is a bar graph showing average protein yield of difficult-to-express proteins (e.g., chimeric antibodies) obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0047] FIG. 10 is a bar graph showing average protein yield of secreted viral proteins obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0048] FIG. 11 is a bar graph showing average protein yield of intracellular proteins obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).PATENT DOCKET NO. TP388599WO1

[0049] FIG. 12 is a bar graph showing average protein yield of extracellular domain proteins obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18).

[0050] FIG.13 is a bar graph showing average protein yield of different protein classes obtained using the commercially available Expi293™ Expression System (Expi293) and the 293 expression system of the present disclosure following the T18 Protocol (T18). DETAILED DESCRIPTION

[0051] The present disclosure provides improved an improved system for the growth of both eukaryotic and prokaryotic cells to produce a desired protein. The system, and components thereof, supports introduction of an expressible nucleic acid (e,g., macromolecule) encoding a desired protein into cells in culture (suspension or otherwise) to produce a high yield of expression product in a reduced amount of time, cost, and labor, as compared to conventional systems and methodology. The protein expression system of the present disclosure provides increased protein yields as compared to convention systems and allows for expression of challenging and typically low-yield proteins. Additionally, the expression system of the present disclosure allows for production of a desired protein in less time and with fewer steps as compared to convention systems and provides higher protein yields at less cost. Further, the expression system of the present disclosure is suitable for small-scale protein production, as well as use with automated platforms and for large-scale protein production.

[0052] Definitions

[0053] In the description that follows, a number of terms used in cell culture and recombinant DNA technology are utilized extensively. In order to provide a clear and more consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0054] The term “introduction” of a macromolecule or compound into culture refers to the provision of the macromolecule or compound into the culture medium.

[0055] The term “introduction” of a macromolecule or compound into at least one cell refers to the provision of a macromolecule or compound to a cell, such that the macromolecule or compound becomes internalized in the cell. For example, a macromolecule or compound can be introduced into a cell using transfection, transformation, injection, and / or liposomal introduction, and may also bePATENT DOCKET NO. TP388599WO1introduced into a cell using other methods known to those of ordinary skill in the art. Preferably, a macromolecule or compound is introduced into a cell by liposomal introduction. The macromolecule is preferably a protein, peptide, polypeptide, or nucleic acid. The macromolecule may be a protein. Alternatively, the macromolecule may be a peptide. Alternatively, the macromolecule may be a polypeptide. The macromolecule may also be a nucleic acid.

[0056] The term “macromolecule,” as used herein, encompasses biomolecules. In one embodiment, the term macromolecule refers to nucleic acid. In a preferred embodiment, the term macromolecule refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). More preferably, the term macromolecule refers to DNA. More preferably, the term macromolecule refers to complementary DNA (cDNA). A macromolecule can be charged or uncharged. A DNA molecule is an example of a charged macromolecule. In some instances, the term “macromolecule”, as used herein, may be used interchangeably with the term “expressible nucleic acid”.

[0057] The term “transfection” is used herein to mean the delivery of nucleic acid, protein or other macromolecule to a target cell, such that the nucleic acid, protein or other macromolecule is expressed or has a biological function in the cell.

[0058] The term “expressible nucleic acid” as used herein includes both DNA and RNA without regard to molecular weight, and the term “expression” means any manifestation of the functional presence of the nucleic acid within the cell including, without limitation, both transient expression and stable expression. Functional aspects include inhibition of expression by oligonucleotides or protein delivery. An expressible nucleic acid encoding, for example, a protein, may be exogenously introduced into the cell (i.e., via transfection) or endogenously reside in the cell. Further, an expressible nucleic acid may be linear (e.g., linear expression cassette) or circular (e.g., a circular plasmid).

[0059] The term “expression of nucleic acid” and their equivalents refer to the replication of the nucleic acid in a cell, to transcription of DNA to messenger RNA, to translation of RNA to protein, to post-translational modification of protein, and / or to protein trafficking in the cell, or variations or combinations thereof.

[0060] The term “ingredient” refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the growth or proliferation of cells. The terms “component,” “feed”, “nutrient” and “ingredient” can be used interchangeably and are all meant to refer to such compounds. Typical ingredients that are used in cell culture media include amino acids, salts, metals, sugars (e.g., glucose and / or galactose), lipids, nucleic acids, hormones,PATENT DOCKET NO. TP388599WO1vitamins, fatty acids, proteins and the like. Other ingredients that promote or maintain cultivation of cells ex vivo can be selected by those of skill in the art, in accordance with the particular need. Media of the present disclosure can include one or more components selected from the group consisting of bovine serum albumin (BSA) or human serum albumin (HSA), a one or more growth factors derived from natural (animal) or recombinant sources such as epidermal growth factor (EGF) or fibroblast growth factor (FGF), one or more lipids, such as fatty acids, sterols and phospholipids, one or more lipid derivatives and complexes, such as phosphoethanolamine, ethanolamine and lipoproteins, one or more proteins, one or more and steroid hormones, such as insulin, hydrocortisone and progesterone, one or more nucleotide precursors; and one or more trace elements.

[0061] The term “cell” as used herein refers includes all types of eukaryotic and prokaryotic cells. In preferred embodiments, the term refers to eukaryotic cells, especially mammalian cells. In certain exemplary though non-limiting embodiments, the term “cell” is meant to refer to human 293 cells, or a clonal derivative thereof, such as, e.g., a 293 clonal derivative that can grow in suspension. Particularly preferred are clonal derivatives of 293 cells that can grow, proliferate and be transfected in suspension culture, in particular those clonal derivatives that can be cultured at high density (e.g., greater than about 2x106cells / ml, more preferably greater than about 3x106cells / ml, or even optionally greater than about 4x106cells / ml). An example of such a clonal derivative 293 cell line is EXPI293™F cells. In other exemplary though non-limiting embodiments, the term “cell” is meant to refer to a CHO cell.

[0062] As used herein, the term “high density” when used in the context of culturing cells in accordance with the present disclosure, and of methods of the disclosure employing same for the purpose of conducting transfection workflows, generally refers to a known cell line, or a clonal derivative of a known cell line, that can be grown or cultured in an appropriate cell culture medium to densities of greater than about 1x106cells / ml, more preferably greater than about 2x106cells / ml, most preferably greater than about 3x106cells / ml, or even optionally greater than about 4x106cells / ml, or more up to about 20x106cells / ml, while still retaining the ability to be transfected at high efficiency and are able to express a target protein at high levels (e.g., levels exceeding 200 µg / ml to up to about 1 mg / ml or more).

[0063] The phrase “high density culture medium” is used herein to refer to any culture medium capable of sustaining the growth of mammalian cells, preferably cells growing in suspension, at densities of up to about 2x107cells / ml while maintaining viability of said cells in excess of about 80% and further, maintaining the ability of said suspension cells to be efficiently transfected andPATENT DOCKET NO. TP388599WO1express high amounts of recombinant protein. The “high density culture medium” used in the practice of the present disclosure may vary between different applications and uses, and may depend on the nature of the cell line being used, the desired protein being transiently expressed, the nature of the transfection modality selected for transfer of the expression vector into cells, and the amount and nature of any expression enhancers added to the system as described below. Nevertheless, preferred “high density culture medium” contemplated for use in the present transient expression systems and methods will typically be serum-free, protein-free, allow the cultivation and growth of suspension cells to a density of up to about 2x107cells / ml, more typically between about 2x106cells / ml to about 1x107cells / ml, and will further enable the yield of protein produced in the transient expression system to exceed at least 200 µg / mL of cell culture up to 2 mg / mL of cell culture, more typically between about 500 µg / ml of cell culture to about 1 mg / mL of cell culture. Ideally, the high density culture medium used in accordance with the present disclosure will facilitate the transfection of cells at densities in the range of about 1x106to about 20x106cells / ml, about 2x106to about 2x106cells / ml, or about 2.5x106to about 6x106cells / ml. Exemplary high density culture media suitable for use in the practice of the present disclosure include, though are not limited to, HuMEC Basal Serum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC / iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106, Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™ 293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154 CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™ -II Complete Medium, CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™ Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and any derivatives or modifications thereof.

[0064] In certain preferred though non-limiting embodiments, a high density culture media may be CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, GIBCO® FREESTYLE™ 293 Expression Medium, EXPI293™ ExpressionPATENT DOCKET NO. TP388599WO1Medium, or a like medium, or a modified version thereof. The above listed exemplary high density culture media may be particularly suitable for the high density growth, propagation, transfection and maintenance of CHO cells, a CHO cell variant, 293 cells, a 293 cell variant, CapT cells, a CapT cell variant, or any other cells adapted for use in a high density culture system.

[0065] The phrase “cells adapted for high density culture” is meant to refer to a cell lineage or a (non-clonal) population of cells derived from the same parental cell lineage that has been adapted to grow at high density in a high-density culture medium while retaining cell viability at or above about 80%. Such cells may be isolated or selected out from the parental population of cells by maintaining the cells at high density over>40, >50, >60, >70, or >80 sequential passages and gradually replacing the proportion of growth medium with the desired high-density culture medium. Optionally, during the process, different pools of cells may be individually propagated and subjected to the selection procedure while simultaneously assessing transfection efficiency and or protein expression efficiency, so that non-clonal population of cells may be selected that can be sustained and grown at high density, transfected with high efficiency, and express high levels of a desired recombinant protein. While it will be readily apparent to the skilled practitioner that a variety of cell types and lineages may be subjected to this selection procedure, it has been determined that cell lineages derived from CHO cells, cell lineages derived from 293 fibroblast cells, and cells derived from CapT cells are particularly amenable to the selection process for being adapted to high density growth conditions. Ideally, cells that are adapted to high density growth culture and amenable for use in the present disclosure will also be capable of being transfected at high efficiency and / or capable of expressing recombinant protein at yield exceeding at least about 200 µg / mL of cell culture up to about 2 mg / mL of cell culture, more typically between about 500 µg / ml of cell culture to about 1 mg / mL of cell culture. Ideally, cells adapted for high density culture used in accordance with the present disclosure are capable of being sustained and transfected at densities in the range of about 1x106to about 20 x106cells / ml, about 2x106to about 2x106cells / ml, or about 2.5x106to about 6x106cells / ml.

[0066] By “cell culture” or “culture” is meant the maintenance of cells in an artificial, in vitro environment.

[0067] By “cultivation” is meant the maintenance of cells in vitro under conditions favoring growth and / or differentiation and / or or continued viability. “Cultivation” can be used interchangeably with “cell culture.” Cultivation is assessed by number of viable cells / ml culturePATENT DOCKET NO. TP388599WO1medium. Cultivation after introduction of a macromolecule preferably includes production of a product, for example, a protein product on a virus.

[0068] The term “replenishing, replacing, or supplementing medium” as used herein refers to adding a volume of fresh cell culture medium to medium that was already present in culture and / or replacing medium that was already present in culture with fresh medium, and / or supplementing medium already present in culture with new medium. Fresh medium is medium that does not contain the one or more macromolecules or compounds to be introduced into at least one cell or medium that has not been in contact with cells to support their growth on cultivation. The skilled artisan can determine whether there is an advantage from or a need to remove and / or replenish, replace or supplement medium by monitoring cell growth and / or viability by techniques known in the art, such as cell counting (manual or automated), trypan blue exclusion, production of protein or other substance, alamar blue assay, presence or concentration of one or more metabolic products, cell adhesion, morphological appearance, analysis of spent medium, etc. One or a combination of monitoring techniques can be used to determine whether the medium needs to be to support growth, introduction of at least one macromolecule and / or cultivation after introduction of at least one macromolecule.

[0069] “Recombinant protein” refers to protein that is encoded by a nucleic acid that is introduced into a host cell. The host cell expresses the nucleic acid. The term “expressing a nucleic acid” is synonymous with “expressing a protein from an RNA encoded by a nucleic acid. “Protein” as used herein broadly refers to polymerized amino acids and illustrative example of which include peptides, polypeptides, proteins, lipoproteins, glycoproteins, membrane proteins, antibodies including fragments thereof, chimeric antibodies, bispecific antibodies, secreted non-antibodies, enzymes, toxoids, multidomain proteins, intracellular proteins, fusion proteins, Fc-fusion proteins, viral proteins (e.g., structural proteins, nonstructural proteins, regulatory proteins, and accessory proteins). It will be appreciated that any of these illustrated proteins may be a difficult-to-express protein (DEP). It will be appreciated that any of these illustrated proteins may be secreted or non-secreted.

[0070] The term “protein yield” refers to the amount of protein expressed by cultured cells, and can be measured, for example, in terms of grams of protein produced / ml medium. If the protein is not secreted by the cells, the protein can be isolated from the interior of the cells by methods known to those of ordinary skill in the art. If the protein is secreted by the cells, the protein can be isolated from the culture medium by methods known to those of ordinary skill in the art. The amount ofPATENT DOCKET NO. TP388599WO1protein expressed by the cell can readily be determined by those of ordinary skill in the art. The protein may be a recombinant protein.

[0071] A “protein product” is a product associated with production or an action by a protein. A protein product may be a protein. A protein product may also be a product resulting from action of a protein by one or more other substances to produce a product. An example of such action is enzymatic action by a protein.

[0072] By “suspension culture” is meant cell culture in which the majority or all of cells in a culture vessel are present in suspension, and the minority or none of the cells in the culture vessel are attached to the vessel surface or to another surface within the vessel. Preferably, “suspension culture” has greater than 75% of the cells in the culture vessel are in suspension, not attached to a surface on or in the culture vessel. More preferably, a “suspension culture” has greater than 85% of the cells in the culture vessel are present in suspension, not attached to a surface on or in the culture vessel. Even more preferred is a “suspension culture” with greater than 95% of the cells in the culture vessel present in suspension, not attached to a surface on or in the culture vessel.

[0001] The system of the present disclosure is suitable for monolayer or suspension culture, transfection, and cultivation of cells, and for expression of protein in cells in monolayer or suspension culture. In some embodiments, the system of the present disclosure is for suspension culture, transfection, and cultivation of cells, and for expression of protein product in cells in suspension culture.

[0073] By “culture vessel” is meant any container, for example, a glass, plastic, or metal container, that can provide an aseptic environment for culturing cells.

[0074] The phrases “cell culture medium,” “tissue culture medium,” “culture medium” (plural “media” in each case) and “medium formulation” refer to a nutritive solution for cultivating cells or tissues. These phrases can be used interchangeably.

[0075] The term “combining” refers to the mixing or admixing of ingredients.

[0076] Derivative of a molecule includes some compounds that comprise the base molecule, but have additional or modified side groups. Preferably, a “derivative” can be formed by reacting the base molecule with only 1, but possibly 2, 3, 4, 5, 6, etc. reactant molecules. A single step reaction is preferred, but multi-step, e.g., 2, 3, 4, 5, 6, etc. reactions are known in the art to form derivatives. Substitution, condensation and hydrolysis reactions are preferred and may be combined to form the derivative compound. Alternatively, a derivative compound may be a compound that preferably inPATENT DOCKET NO. TP388599WO11, but possibly 2, 3, 4, 5, 6, etc. reactions can form the base compound or a substitution or condensation product thereto.

[0077] A cell culture medium is composed of a number of ingredients and these ingredients can vary from medium to medium. Each ingredient used in a cell culture medium has its unique physical and chemical characteristics. Compatibility and stability of ingredients are determined in part by the “solubility” of the ingredients in aqueous solution. The terms “solubility” and “soluble” refer to the ability of an ingredient to form and remain in solution with other ingredients. Ingredients are thus compatible if they can be maintained in solution without forming a measurable or detectable precipitate.

[0078] By “compatible ingredients” is also meant those media components which can be maintained together in solution and form a “stable” combination. A solution containing “compatible ingredients” is said to be “stable” when the ingredients do not precipitate, degrade or decompose substantially such that the concentration of one or more of the components available to the cells from the media is reduced to a level that no longer supports the optimum or desired growth of the cells. Ingredients are also considered “stable” if degradation cannot be detected or when degradation occurs at a slower rate when compared to decomposition of the same ingredient in a 1X cell culture media formulation. For example, in 1X media formulations glutamine is known to degrade into pyrolidone carboxylic acid and ammonia. Glutamine in combination with divalent cations are considered “compatible ingredients” since little or no decomposition of the glutamine can be detected over time in solutions or combinations in which both glutamine and divalent cations are present, see, e.g., U.S. Pat. No.5,474,931. Thus, the term “compatible ingredients” as used herein refers to the combination of particular culture media ingredients which, when mixed in solution either as concentrated or 1X formulations, are “stable” and “soluble.”

[0079] The term “1X formulation” is meant to refer to any aqueous solution that contains some or all ingredients found in a cell culture medium at working concentrations. The “1X formulation” can refer to, for example, the cell culture medium or to any subgroup of ingredients for that medium. The concentration of an ingredient in a 1X solution is about the same as the concentration of that ingredient found in a cell culture formulation used for maintaining or cultivating cells in vitro. A cell culture medium used for the in vitro cultivation of cells is a 1X formulation by definition. When a number of ingredients are present, each ingredient in a 1X formulation has a concentration about equal to the concentration of each respective ingredient in a medium during cell culturing. For example, RPMI-1640 culture medium contains, among other ingredients, 0.2 g / L L-arginine, 0.05PATENT DOCKET NO. TP388599WO1g / L L-asparagine, and 0.02 g / L L-aspartic acid. A “1X formulation” of these amino acids contains about the same concentrations of these ingredients in solution. Thus, when referring to a “1X formulation,” it is intended that each ingredient in solution has the same or about the same concentration as that found in the cell culture medium being described. The concentrations of ingredients in a 1X formulation of cell culture medium are well known to those of ordinary skill in the art. See, for example, Methods For Preparation of Media, Supplements and Substrate For Serum- Free Animal Cell Culture Allen R. Liss, N.Y. (1984), Handbook of Microbiological Media, Second Ed., Ronald M. Atlas, ed. Lawrence C. Parks (1997) CRC Press, Boca Raton, Fla. and Plant Culture Media, Vol. 1: Formulations and Uses E. F. George, D. J. M. Puttock, and H. J. George (1987) Exegetics Ltd. Edington, Westbury, Wilts, BA134QG England each of which is incorporated by reference herein in its entirety. The osmolarity and / or pH, however, can differ in a 1X formulation compared to the culture medium, particularly when fewer ingredients are contained in the 1X formulation.

[0080] A “10X formulation” is meant to refer to a solution wherein the concentration of each ingredient in that solution is about 10 times more than the concentration of each respective ingredient in a medium during cell culturing. For example, a 10X formulation of RPMI-1640 culture medium can contain, among other ingredients, 2.0 g / L L-arginine, 0.5 g / L L-asparagine, and 0.2 g / L L- aspartic acid (compare 1X formulation, above). A “10X formulation” can contain a number of additional ingredients at a concentration about 10 times that found in the 1X culture formulation. As will be readily apparent, “25X formulation,” “50X formulation,” “100X formulation,” “500X formulation,” and “1000X formulation” designate solutions that contain ingredients at about 25-, 50- , 100-, 500-, or 1000-fold concentrations, respectively, as compared to a 1X cell culture formulation. Again, the osmolarity and pH of the medium formulation and concentrated solution can vary.

[0081] The term “trace element” or “trace element moiety” refers to a moiety which is present in a cell culture medium in only very low (i.e., “trace”) amounts or concentrations, relative to the amounts or concentrations of other moieties or components present in the culture medium. In the present disclosure, these terms encompass Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Cu1+, Cu2+, Fe2+, Fe3+, Ge4+, Se4+, Br-, I-, Mn2+, F-, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+ and Zr4+ and salts thereof. For example, the following salts can be used as trace elements in the culture media of the disclosure: AgNO3, AlCl3•6H2O, Ba(C2H3O2)2, CdSO4•8H2O, CoCl2•6H2O, Cr2(SO4)3•1H2O, GeO2, Na2SeO3, H2SeO3, KBr, KI, MnCl2•4H2O, NaF, Na2SiO3•9H2O, NaVO3, (NH4) 6Mo7O24•4H2O, NiSO4•6H2O, RbCl, SnCl2, and ZrOCl2•8H2O. Suitable concentrations of tracePATENT DOCKET NO. TP388599WO1element moieties can be determined by one of ordinary skill in the art using only routine experimentation.

[0082] The term “amino acid” refers to amino acids or their derivatives (e.g., amino acid analogs), as well as their D- and L-forms. Examples of such amino acids include glycine, L- alanine, L- asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, N-acetyl cysteine.

[0083] A “chemically defined” medium is one in which each chemical species and its respective quantity is known prior to its use in culturing cells. A chemically defined medium is made without lysates or hydrolysates whose chemical species are not known and / or quantified. A chemically defined medium is one preferred embodiment of the medium of the present disclosure.

[0084] The terms “serum-free culture conditions” and “serum-free conditions” refer to cell culture conditions that exclude serum of any type. These terms can be used interchangeably.

[0085] A “serum-free medium” (sometimes referred to as “SFM Medium”) is a medium that contains no serum (e.g., fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, etc.) and is generally designated by the letters SFM. Exemplary though non-limiting serum- free media familiar to the skilled artisan include HuMEC Basal Serum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC / iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106, Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial- SFM, GIBCO® FREESTYLE™ 293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154 CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™ -II Complete Medium, CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™ Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and any derivatives or modifications thereof.

[0086] The phrase “protein-free” culture media refers to culture media that contain no protein (e.g., no serum proteins such as serum albumin or attachment factors, nutritive proteins such asPATENT DOCKET NO. TP388599WO1growth factors, or metal ion carrier proteins such as transferrin, ceruloplasmin, etc.). Preferably, if peptides are present, the peptides are smaller peptides, e.g., di- or tri-peptides. Preferably, peptides of deca-peptide length or greater are less than about 1%, more preferably less than about 0.1%, and even more preferably less than about 0.01% of the amino acids present in the protein free medium.

[0087] The phrase “low-protein” culture media as used herein refers to media that contain only low amounts of protein (typically less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1%, of the amount or concentration of total protein found in culture media containing standard amounts of protein, such as standard basal medium supplemented with 5-10% serum).

[0088] The term “animal derived” material as used herein refers to material that is derived in whole or in part from an animal source, including recombinant animal DNA or recombinant animal protein DNA. In some embodiments, media contain no animal material. In some embodiments, media are xeno-free. In some embodiments, media are serum-free. In some embodiments, media are albumin-free.

[0089] The term “expression enhancer” generally refers to one or more liquid (preferably aqueous) additives used to supplement a culture medium formulation in accordance with the presently described disclosure, said additives being selected to improve the yield of expressed protein produced in a transient protein expression system in accordance with the presently described embodiments. The term encompasses any one or more of several compounds that affect cell cycle progression, inhibit apoptosis, slow cell growth and / or promote protein production. In the context of the present disclosure, the term “expression enhancers” generally refers to any one or more compounds added to a transient transfection system, the presence of which enhances or promotes expression of a target protein by a factor of at least 2 fold up to about 10-fold above the expression level seen in the absence of such expression enhancer(s). Exemplary expression enhancers suitable for use with the presently described embodiments include, though are not limited to, additives such as a histone deacetylase (HDAC) inhibitor, sodium proprionate, egg lecithin, lithium acetate, trichostatin hydroxyurea, nocodazole-DMSO, NaCl, caffeine, dimethyl sulfoxide (DMASO), sugars including galactose and / or glucose, amino acid mixtures, butyric acid, or any combination thereof. In embodiments, the HDAC inhibitor is apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and / or valproic acid. In embodiments, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.PATENT DOCKET NO. TP388599WO1

[0090] The optimal concentration of each specific expression enhancer may vary according to individual characteristics of the expression system and the requirements of the user, and the determination of what constitutes an optimal concentration of any one or more expression enhancer in a given experimental scenario is well within purview of a practitioner having ordinary skill level in the art. By way of example only, in some embodiments, the optimal final concentration ranges of valproic acid (VPA) used in a culture, e.g., suspension culture, in the practice of the present disclosure may be in the range of about 0.20 mM to about 25 mM. More preferably, the final concentration of VPA may be in the range of about 0.25 mM to about 24 mM, about 0.26 mM to about 23 mM, 0.27 mM to about 23 mM, 0.28 mM to about 23 mM, 0.29 mM to about 22 mM, about 0.30 mM to about 21 mM, about 0.31 mM to about 20 mM, about 0.32 mM to about 19 mM, about 0.33 mM to about 17 mM, about 0.34 mM to about 18 mM, about 0.35 mM to about 17 mM, about 0.36 mM to about 16 mM, about 0.37 mM to about 15 mM, about 0.40 mM to about 14 mM, about 0.41 mM to about 13 mM, about 0.42 mM to about 12 mM, about 0.43 mM to about 11 mM, about 0.44 mM to about 10 mM, about 0.45 mM to about 9 mM, about 0.46 mM to about 8 mM, about 0.47 mM to about 7 mM, about 0.48 mM to about 6 mM, about 0.49 mM to about 5 mM, about 0.50 mM to about 4 mM, about 0.50 mM to about 4 mM, about 0.55 mM to about 3 mM, 0.6 mM to about 2.5 mM or 2.0 to about 2.5 mM. In some preferred though non-limiting embodiments, the final concentration of VPA used in the practice of the present disclosure may be between about 0.15 mM to about 2.5 mM, about 0.16 mM to about 2.5 mM, about 0.17 mM to about 2.5 mM, about 0.18 mM to about 2.5 mM, about 0.19 mM to about 2.5 mM, about 0.20 mM to about 2.5 mM, about 0.25 mM to about 2.5 mM, about 0.30 mM to about 2.5 mM, about 0.40 mM to about 2.5 mM, about 0.50 mM to about 2.5 mM, about 0.60 mM to about 2.5 mM, about 0.70 mM to about 2.5 mM, about 0.80 mM to about 2.5 mM, about 0.90 mM to about 2.5 mM or about 0.10 mM to about 2.5 mM. In some preferred though non-limiting embodiments, the final concentration of VPA used in the practice of the present disclosure may be between about 0.20 to about 2.5 mM, about 0.21 to about 2.4 mM, about 0.22 to about 2.3 mM, about 1.0 to about 2.4 mM, about 1.5 to about 2.4 mM, about 1.7 to about 2.3 mM, about 1.8 to about 2.2 mM, about 1.9 to about 2.1 mM, about 1.95 to about 2.1 mM, about 2.0 to about 2.1 mM, about 1.1 to about 2.0 mM, about 1.1 to about 1.8 mM, about 1.1 to about 1.6 mM, about 1.2 to about 1.4 mM, or about 1.2 to about 1.3 mM,. In some preferred though non-limiting embodiments, the final concentration of VPA in a culture, e.g., suspension culture, used in the practice of the present disclosure may be at least about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM,PATENT DOCKET NO. TP388599WO1about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 4.0 mM, about 5.0 mM or greater.

[0091] In further embodiments, the optimal final concentration ranges of sodium proprionate (NaPP) used in a culture, e.g., suspension culture, in the practice of the present disclosure may be in the range of about 0.2 mM to about 100 mM. In certain preferred though non-limiting embodiments, the optimal final concentration of NAPP may be in the range of about 0.5 to about 80 mM, about 0.4 mM to about 70 mM, about 0.5 mM to about 60 mM, about 0.6 mM to about 50 mM, about 0.7 mM to about 40 mM, about 0.8 mM to about 30 mM, about 0.9 mM to about 25 mM, about 1 mM to about 24 mM, about 2 mM to about 23 mM, about 3 mM to about 22 mM, about 4 mM to about 21 mM, about 10 mM to about 20 mM, about 15 mM to about 20 mM, about 16 mM to about 20 mM, or about 17 mM to about 19 mM. In certain preferred though non-limiting embodiments, the optimal final concentration of NAPP may be in the range of about 1 mM to about 25 mM, about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about 4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 8 mM to about 9 mM, about 9 mM to about 10 mM, about 10 mM to about 11 mM, about 11 mM to about 12 mM, about 12 mM to about 13 mM, about 13 mM to about 14 mM, about 14 mM to about 15 mM, about 15 mM to about 16 mM, about 16 mM to about 17 mM, about 17 mM to about 18 mM, about 18 mM to about 19 mM, or about 19 mM to about 20 mM. In certain preferred though non- limiting embodiments, the optimal final concentration of NAPP in a culture, e.g., suspension culture, used in the practice of the present disclosure may be about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 10.5 mM, about 11 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about 17 mM, about 17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, or about 20 mM.

[0092] In further embodiments, the optimal final concentration of lithium acetate (LiAc) used in the practice of the present disclosure may be in the range of about0.25 to about 25 mM, about 0.26 mM to about 20 mM, about 0.27 mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29 mM to about 5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4 mM, about 0.35 mMPATENT DOCKET NO. TP388599WO1to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3 mM.

[0093] In further embodiments, the optimal final concentration of butyric acid used in the practice of the present disclosure may be in the range of about 0.25 to about 25 mM, about 0.26 mM to about 20 mM, about 0.27 mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29 mM to about 5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4 mM, about 0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3 mM.

[0094] An “expression enhancer composition” used in accordance with the present disclosure may be added to the culture medium prior to transfection, during transfection, or after transfection prior to harvesting the cells and / or expression product, such as recombinant protein. In some embodiments an expression enhancer composition includes one or more expression enhancers. In embodiments, the expression enhancer composition includes one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, egg lecithin, lithium acetate, trichostatin hydroxyurea, nocodazole- DMSO, NaCl, and caffeine. In embodiments, the HDAC inhibitor is apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and / or valproic acid. In embodiments, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid. Expression enhancers may be added to a culture medium sequentially, or as a cocktail, prior to transfection, during transfection, or after transfection.

[0095] The optimal concentration of each specific expression enhancer on an expression enhancer composition may vary according to individual characteristics of the expression system and the requirements of the user, and the determination of what constitutes an optimal concentration of any one or more expression enhancer in a given experimental scenario is well within purview of a practitioner having ordinary skill level in the art. By way of example only, in some embodiments, the optimal final concentration ranges of valproic acid (VPA) used in an expression enhancer composition, in the practice of the present disclosure may be in the range of about 0.5 mM to about 500 mM. More preferably, the concentration of VPA in the expression enhancer composition may be in the range of about 0.5 mM to about 475 mM, about 0.6 mM to about 450 mM, 0.7 mM to about 425 mM, 0.8 mM to about 400 mM, 0.9 mM to about 400 mM, about 1.0 mM to about 400 mM, about 25 mM to about 400 mM, about 50 mM to about 400 mM, about 75 mM to about 400 mM, about 100 mM to about 400 mM, about 125 mM to about 400 mM, about 150 mM to about 400 mM,PATENT DOCKET NO. TP388599WO1about 175 mM to about 375 mM, about 200 mM to about 350 mM, about 200 mM to about 325 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, or about 250 mM to about 275 mM. In some non-limiting embodiments, the concentration of VPA in an expression enhancer composition, used in the practice of the present disclosure may be at least about 0.1 mM, about 1.0 mM, about 10 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, or greater.

[0096] In further embodiments, the optimal final concentration ranges of sodium proprionate (NaPP) used in an expression enhancer composition used in the practice of the present disclosure may be in the range of about 0.2 M to about 10 M. In certain preferred though non-limiting embodiments, the optimal final concentration of NAPP in an expression enhancer composition may be in the range of about 0.5 to about 9 M, about 0.5 M to about 8 M, about 0.5 M to about 7 M, about 0.5 M to about 6 M, about 0.5 M to about 5 M, about 0.5 M to about 4 M, about 1 M to about 4 M, about 1 M to about 3 M, about 1.5 M to about 2.75 M, about 1.5 M to about 2.5 M, about 1.75 M to about 2.5 M, or about 2 M to about 2.5 M. In certain non-limiting embodiments, the optimal final concentration of NAPP in an expression enhancer composition, used in the practice of the present disclosure may be at least about 1 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, or greater.

[0097] In some non-limiting embodiments described herein, an expression enhancer composition includes valproic acid at a concentration of about 200 mM – 300 mM, sodium propionate at a concentration of about 0.5 M – 10 M, glucose at a concentration of about 700 mM – 1,000 mM, valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentration of about 0.5 M – 10 M, valproic acid at a concentration of about 200 mM – 300 mM admixed with glucose at a concentration of about 700 – 1,000 mM, sodium propionate at a concentration of about 0.5 M – 10 M admixed with glucose at a concentration of about 700 – 1,000 mM, or valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentration of about 0.5 M – 10 M and glucose at a concentration of about 700 – 1,000 mM. In some embodiments, an expression enhancer composition includes valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentration of about 2.0 M – 2.5 M, and optionally glucose at a concentration of about 700 – 1,000 mM.PATENT DOCKET NO. TP388599WO1

[0098] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. Certain vectors used in accordance with the practice of disclosure described herein may be well-known vectors used in the art, such as, e.g., pcDNA 3.3 and pcDNA 3.4, or a modified version thereof. Non-limiting examples of the types of modification to a vector that may be suitable in the practice of the present disclosure include, though are not limited to, modification such as the addition of modification of one or more enhancers, one or more promoters, one or more ribosomal binding sites, one or more origins of replication, or the like. In certain preferred though non-limiting embodiments, and expression vector used in the practice of the present disclosure may include one or more enhancer elements selected to improve expression of the protein of interest in the present transient expression system. The selected enhancer element may be positioned 5’ or 3’ to the expressible nucleic acid sequence used to express the protein of interest. A particularly preferred though non-limiting enhancer element is the woodchuck hepatitis post- transcriptional regulatory element (WPRE).

[0099] As used herein, the phrase “nucleic acid capable of expressing a recombinant protein,” “nucleic acid having a sequence capable of producing an expressed protein,” “nucleic acid having a sequence capable of expressing a protein,” or “expression vector containing a genetic sequence capable of producing an expressed protein” generally refers to a vector as defined above which is capable to accommodating an expressible nucleic acid sequence having at least one open-reading frame of a desired protein of interest (said protein of interest being selected by the user of the present disclosure) in additional to one or more nucleic acid sequences or elements that are required toPATENT DOCKET NO. TP388599WO1support the expression thereof in a cell or in a cell-free expression system. Such additional nucleic acid sequences or elements that may be present in an expression vector as defined herein may include, one or more promoter sequences, one or more enhancer elements, one or more ribosomal binding sites, one or more translational initiation sequences, one or more origins of replication, or one or more selectable markers. A variety of nucleic acid sequences or elements serving this purpose are familiar to the skilled artisan, and the selection of one or more thereof for use in the practice of the present disclosure is well within the purview of the skilled practitioner.

[0100] The terms “polynucleotide” and “nucleic acid” as used herein refers to any nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In preferred embodiments, “nucleic acid” refers to DNA, including genomic DNA, complementary DNA (cDNA), and oligonucleotides, including oligo DNA. In certain preferred though non-limiting embodiments, “nucleic acid` refers to genomic DNA and / or cDNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms ofPATENT DOCKET NO. TP388599WO1ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl- , 2'-O-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR', CO, or CH2 (“formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0101] “Oligonucleotide,” as used herein, generally refers to short, generally single-stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0102] As used herein, the phrase “first period of time”, when used in the context of a method for transiently transfecting cells in accordance with the methods of the disclosure described herein generally refers to the time interval between transfecting a population of cells with an expressible nucleic acid and the addition of one or more expression enhancers to the transfected cells. Typically, a first period of time will be in the range of about 0 minutes to about 2 hours. In certain preferred though non-limiting embodiments, a first period of time may be in the range of about 0 to about 120 min, about 0.2 to about 120 min, about 0.4 to about 120 min, about 0.6 to about 120 min, about 0.8 to about 120 min, about 1 to about 120 min, about 2.5 to about 120 min, about 5 to about 120 min, about 10 to about 120 min, about 15 to about 120 min, about 20 to about 120 min, about 30 to about 120 min, about 45 to 120 min, about 60 to about 120 min, about 1 to about 60 min, about 1 to about 45 min, about 1 to about 30 min, about 1 to about 20 min, about 1 to about 15 min, about 1 to about 10 min, or about 1 to about 5 min. In other preferred to non-limiting embodiments, a first period of time may be up to about 1 min, up to about 5 min, up to about 10 min, up to about 15 min, up to about 20 min, up to about 25 min, up to about 30 min, up to about 35 min, up to about 40 min, up to about 45 min, up to about 50 min, up to about 55 min, up to about 60 min, up to about 80 min, up to about 100 min, or up to about 120 min.PATENT DOCKET NO. TP388599WO1

[0103] As used herein, the phrase “second period of time”, when used in the context of a method for transiently transfecting cells in accordance with the methods of the disclosure described herein generally refers to the time interval between the addition of one or more expression enhancers and either the addition of one or more additional enhancers, or the harvesting of the transfected cells and purification or isolation of the protein expressed therein. Typically, a second period of time will be in the range of about 10 hrs to about 10 days, though other time intervals may be used if determined to be optimal for the protein being expressed. In some preferred though non-limiting embodiments, the second period of time may be in the range of 2 hrs to 5 days, 2.5 hrs to 4 days, about 3 to about 90 hrs, about 4 to about 85 hr, about 5 to about 80 hrs, about 6 to about 75 hrs, about 7 to about 70 hrs, about 8 to about 65 hrs, about 9 to about 60 hrs, about 10 to about 55 hrs, about 11 to about 50 hrs, about 12 to about 45 hrs, about 13 to about 40 hrs, about 14 to about 35 hrs, about 15 to 30 hrs, about 16 to about 24 hrs, about 17 to about 24 hrs, about 18 to about 24 hrs, about 19 to about 24 hrs, about 20 to about 24 hrs, about 21 to about 24 hrs, about 22 to about 24 hrs or about 23 to about 24 hrs. In other preferred to non-limiting embodiments, a second period of time may be up to about 15 hrs, up to about 16 hrs, up to about 17 hrs, up to about 18 hrs, up to about 19 hrs, up to about 20 hrs, up to about 21 hrs, up to about 22 hrs, up to about 23 hrs, up to about 24 hrs, up to about 25 hrs, up to about 26 hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrs or up to about 30 hrs.

[0104] As used herein the phrase “third period of time”, when used in the context of a method for transiently transfecting cells in accordance with the methods of the disclosure described herein generally refers to the time interval between the addition of at least a first expression enhancer and at least a second expression enhancer. The time interval between the addition of a first and second expression enhancer may be on the order of seconds to days, though in some embodiments such first and second expression enhancer may be added essentially simultaneous, or may optionally be provided in a single formulation.

[0105] As used herein the terms “complexation reaction,” “complexation media,” “complexation buffer” or the like, generally refer to a physiologically acceptable culture media or reaction in which a nucleic acid is complexed to a transfection reagent formulation, such as to form a transfection complex for example. Typically, a nucleic acid that is to be introduced into a cell for the purpose of expressing a protein is first complexed with a suitable transfection reagent (such as, e.g., a cationic lipid formulation) to lipid / nucleic acid complexes or aggregates.

[0106] By “transition element” or “transition metal” (which can be used interchangeably) is meant an element in which an inner electron valence shell, rather than an outer shell, is only partially filled,PATENT DOCKET NO. TP388599WO1such that the element acts as a transitional link between the most and least electropositive in a given series of elements. Transition elements are typically characterized by high melting points, high densities, high dipole or magnetic moments, multiple valencies, and the ability to form stable complex ions. Examples of such transition elements useful in the present disclosure include scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), rubidium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), cadmium (Cd), lanthanum (La), hafnium (Hf), tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), mercury (Hg), and actinium (Ac). Of particular interest as a transition element for use in culture media compositions, including those of the present disclosure, are ions, chelates, salts, and complexes of iron (Fe2+ or Fe3+).

[0107] A variety of techniques and reagents are available for the introduction of macromolecules into a target cell in a process known as “transfection”. Commonly used reagents include, for example, calcium phosphate, DEAE-dextran and lipids. For examples of detailed protocols for the use of reagents of these types, numerous references texts are available for example, Current Protocols in Molecular Biology, Chapter 9, Ausubel, et al. Eds., John Wiley and Sons, 1998. Additional methods for transfecting cells are known in the art, and may include electroporation (gene electrotransfer), sono-poration, optical transfection, protoplast fusion, impalefection, magnetofection, or viral transduction.

[0108] A “reagent for the introduction of macromolecules” into cells or a “transfection reagent” is any material, formulation or composition known to those of skill in the art that facilitates the entry of a macromolecule into a cell. For example, see U.S. Pat. No.5,279,833. In some embodiments, the reagent can be a “transfection reagent” and can be any compound and / or composition that increases the uptake of one or more nucleic acids into one or more target cells. A variety of transfection reagents are known to those skilled in the art. Suitable transfection reagents can include, but are not limited to, one or more compounds and / or compositions comprising cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged dendrimers and fractured dendrimers, cationic β-cyclodextrin containing polymers (CD-polymers), DEAE-dextran and the like.

[0109] Cationic polymers such as polyethyleneimine (PEI) include, but are not limited to those described in Internation Pub. Nos. WO2009074970, WO2020089342 and WO2021023796, which are incorporated herein by reference in their entireties. In some embodiments, the transfection reagentPATENT DOCKET NO. TP388599WO1is a polymeric amine based transfection reagent. In some embodiments, the transfection reagent includes PEI, linear PEI, or a derivative of PEI.

[0110] In some embodiments, a reagent for the introduction of macromolecules into cells can comprise one or more lipids which can be phospholipids, cationic lipids and / or neutral lipids. Preferred lipids include, but are not limited to, cholesterol, cholesterol derivatives, 1,2- dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1-oleoyl-2-hydroxy-sn-glycero-3- phosphoethanolamine, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), N, N’-(aminopropyl)-N,N’-bistetradecyl-1,4-damino-2,3- butandiol (DMS), 1,2-Bis(oleoyloxy)-3-(4'-trimethylammonio) propane (DOTAP), 1,2-dioleoyl-3- (4'-trimethylammonio) butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4'-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2- dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), O,O'-didodecyl-N- [p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylam- monium chloride, spermine conjugated to one or more lipids (for example, 5-carboxyspermylglycine dioctadecylamide (DOGS), N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tet-rapalmitylspermine (TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde (DPPES)), lipopolylysine (polylysine conjugated to DOPE), Bis(3-{N-3-aminopropyl-N-palmityl}amino-2-hydroxypropyl)- piperazine, TRIS (Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids (TFAs) and / or peptides such as trilysyl-alanyl-TRIS mono-, di-, and tri-palmitate, (3β-[N--(N',N'- dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), N-(α -trimethylammonioacetyl)- didodecyl-D-glutamate chloride (TMAG), dimethyl dioctadecylammonium bromide (DDAB), 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin- iniumtrifluoroacetate (DOSPA) and combinations thereof.

[0111] Those skilled in the art will appreciate that certain combinations of the above mentioned lipids have been shown to be particularly suited for the introduction of nucleic acids into cells for example a 3:1 (w / w) combination of DOSPA and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. under the trade name LIPOFECTAMINE™, a 1:1 (w / w) combination of DOTMA and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1 (M / M) combination of DMRIE and cholesterol is available from Life Technologies Corporation, Carlsbad, Calif. under the trade name DMRIE-C reagent, a 1:1.5PATENT DOCKET NO. TP388599WO1(M / M) combination of TM-TPS and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. under the trade name CellFECTIN® and a 1:2.5 (w / w) combination of DDAB and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. under the trade name LipfectACE®. Additional lipid combinations include DOPE and cholesterol and optionally DMS, or DMS and cholesterol and optionally DOPE, or DOPE and DMS and optionally cholesterol. In addition to the above-mentioned lipid combinations, other formulations comprising lipids in admixture with other compounds, in particular, in admixture with one or more peptides and proteins optionally including nuclear localization sequences, are known to those skilled in the art, such as those in Table 1. Additional peptides are illustrated in, for example, international application no. PCT / US99 / 26825, published as WO 00 / 27795, both of which are incorporated by reference herein in their entireties.

[0112] In embodiments, a peptide is a naturally occurring or non-naturally occurring membrane- penetrating peptide. In embodiments, a peptide comprises a naturally occurring or non-naturally occurring membrane-penetrating peptide sequence. Non-limiting examples of suitable membrane- penetrating peptides and peptide sequences are provided in U.S. Pat. No. 9,856,496, which is incorporated herein by reference in its entirety. In embodiments, a peptide is a fusogenic peptide, a cell-penetrating peptide, a nuclear localization peptide, a cell surface adhesion peptide, or a plant virus movement peptide. In embodiments, a peptide comprises a fusogenic peptide sequence, a cell- penetrating peptide sequence, a nuclear localization peptide sequence, a cell surface adhesion peptide sequence, or a plant virus movement peptide sequence. Non-limiting examples of suitable fusogenic, cell-penetrating, nuclear localization, cell surface adhesion, and plant virus movement peptides and peptide sequences are provided in U.S. Pat. Application Publication No. 2017 / 0253888, which is incorporated herein by reference in its entirety.

[0113] In embodiments, a transfection reagent includes one or more peptides from Table 1.

[0114] Table 1: Peptides SEQ ID NO Peptide SequencePATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 8 WRRRRNRRPTSYGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 44 GLLEELLELLEELWEELLEGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 80 GLFEALLELLESLWELLLEAWYGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 116 KTPKKAKKPKTPKKAKKPPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 152 GACLQHKSMPCGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 188 AARSPSYYRYDYPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 224 KPDVRSYTITGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 260 LLEFTSARYIRLPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 296 RDFTKATNIRLRFLRPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 332 TWYKIAFQRNRKPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 368 VRWGMQQIQLVVPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 404 TNLRIKFVKLHTPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 440 DGRGDSVAYGPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 476 RLVSYNGILFFLPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 512 STSKRKRGDDANWSKRTTKKKPSS H Q R D S S S V EPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence NKRYNCWASFSDLERDCNGVYGNITKNALLVYYCWLSDAQSKASTYVSPATENT DOCKET NO. TP388599WO1SEQ ID NO Peptide Sequence 575 DIAFRAPTVKILSKQFTDRDVDFSHVGYGKWERKLIRSASTVKYGL [, . term “nucleic acid binding moiety” as used herein refers to a compound or molecule capable binding to nucleic acid. In some embodiments, the binding molecule is capable of noncovalently binding to nucleic acid, while in other embodiments, the binding molecule links covalently to a cell binding adhesion sequence, a plant virus movement protein or peptide fragments, a nuclear localization sequence, transfection enhancer, and / or a fusion agent. The binding molecule can include, but is not limited to spermine, spermine derivative, spermidine, histones or fragments thereof, protamines or fragments thereof, HMG proteins or fragments thereof, poly-lysine, poly-arginine, poly-histidine, polyamines and cationic peptides, nucleic acid intercalaters, protein nucleic acid sequences or aptamers. In addition, this includes but is not limited to analogs or derivatives of the above compounds. Non limiting examples are the cationic peptides that are repeats of lysine or arginine, for example a sequence having between 8-20 lysine residues (K8-K20) (SEQ ID NO:583) or between 8-20 arginine residues (R8-R20) (SEQ ID NO:584).

[0116] In some embodiments, peptides may be attached to a second molecule, such as a binding molecule by covalent bonding, or are connected to the binding molecule via a spacer. The term “spacer,” or “linker,” which are used interchangeably herein, as used herein refers to a chemical structure that links two molecules to each other. In some embodiments, the spacer binds each molecule on a different part of the spacer molecule. In other embodiments, the spacer is a hydrophilic moiety and comprises about 6 to 30 carbon atoms. In other embodiments, the spacer comprises a polyether, for example —CH2—O—(CH2—CH2—O—)iCH2—. In other embodiments, the spacer comprises a hydrophilic polymer, for example [(gly)i(ser)j]k (SEQ ID NO: 585). In these formulae i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In some embodiments, the spacerPATENT DOCKET NO. TP388599WO1is a peptide of sequence APYKAWK (SEQ ID NO:505). In other embodiments, the spacer is a sequence that is degraded in vivo by a peptidase.

[0117] Lipid aggregates such as liposomes have been found to be useful as agents for the delivery of macromolecules into cells. In particular, lipid aggregates comprising one or more cationic lipids have been demonstrated to be extremely efficient at the delivery of anionic macromolecules (for example, nucleic acids) into cells. One commonly used cationic lipid is N- [1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Liposomes comprising DOTMA alone or as a 1:1 mixture with dioleoylphosphatidylethanolamine (DOPE) have been used to introduce nucleic acids into cells. A 1:1 mixture of DOTMA:DOPE is commercially available from Life Technologies Corporation, Carlsbad, Calif. under the trade name of LIPOFECTIN™. Another cationic lipid that has been used to introduce nucleic acids into cells is 1,2-bis(oleoyl-oxy)- 3-3-(trimethylammonia) propane (DOTAP). DOTAP differs from DOTMA in that the oleoyl moieties are linked to the propylamine backbone via ether bonds in DOTAP whereas they are linked via ester bonds in DOTMA. DOTAP is believed to be more readily degraded by the target cells. A structurally related group of compounds wherein one of the methyl groups of the trimethylammonium moiety is replaced with a hydroxyethyl group are similar in structure to the Rosenthal inhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J. Biol. Chem.233:2202-2206.). The RI has stearoyl esters linked to the propylamine core. The dioleoyl analogs of RI are commonly abbreviated DOR1- ether and DOR1-ester, depending upon the linkage of the lipid moiety to the propylamine core. The hydroxyl group of the hydroxyethyl moiety can be further derivatized, for example, by esterification to carboxyspermine.

[0118] Another class of compounds which has been used for the introduction of macromolecules into cells comprise a carboxyspermine moiety attached to a lipid (see, Behr, et al., (1989) Proceedings of the National Academy of Sciences, USA 86:6982-6986 and EPO 0 394 111). Examples of compounds of this type include dipalmitoylphosphatidylethanolamine 5- carboxyspermylamide (DPPES) and 5-carboxyspermylglycine dioctadecylamide (DOGS). DOGS is commercially available from Promega, Madison, Wis. under the trade name of TRANSFECTAM™.

[0119] A cationic derivative of cholesterol (3β-[N--(N',N'-dimethylaminoethane)-carbamoyl] cholesterol, DC-Chol) has been synthesized and formulated into liposomes with DOPE (see Gao, et al., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells. The liposomes thus formulated were reported to efficiently introduce DNA into the cells with a low level of cellular toxicity. Lipopolylysine, formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBAPATENT DOCKET NO. TP388599WO11065:8-14), has been reported to be effective at introducing nucleic acids into cells in the presence of serum.

[0120] Other types of cationic lipids that have been used to introduce nucleic acids into cells include highly packed polycationic ammonium, sulfonium and phosphonium lipids such as those described in U.S. Pat. Nos. 5,674,908 and 5,834,439, and international application no. PCT / US99 / 26825, published as WO 00 / 27795. One particularly preferred though non-limiting transfection reagent for delivery of macromolecules in accordance with the present disclosure is LIPOFECTAMINE 2000™ which is available from Life technologies (see U.S. international application no. PCT / US99 / 26825, published as WO 00 / 27795). Another preferred though non- limiting transfection reagent suitable for delivery of macromolecules to a cell is EXPIFECTAMINE™. Other suitable transfection reagents include LIPOFECTAMINE™ RNAiMAX, LIPOFECTAMINE™ LTX, OLIGOFECTAMINE™, Cellfectin™, INVIVOFECTAMINE™, INVIVOFECTAMINE™ 2.0, and any of the lipid reagents or formulations disclosed in U.S. Patent Appl. Pub. No. 2012 / 0136073 (incorporated herein by reference thereto). A variety of other transfection reagents are known to the skilled artisan and may be evaluated for the suitability thereof to the transient transfection systems and methods described herein.

[0121] The present disclosure is directed to a high-yield transient transfection system that supports (a) the introduction of at least one macromolecule, preferably an expressible nucleic acid molecule, into eukaryotic cells in culture, (b) the cultivation of cells into which at least one macromolecule is introduced, and optionally (c) the production of recombinant protein product or expression of the nucleic acid in cells into which at least one macromolecule is introduced, wherein medium containing the macromolecule does not need to be removed from the culture and replaced with fresh medium after introduction of at least one macromolecule into cells and prior to cultivation and production of protein product or expression of nucleic acid.

[0122] The transient transfection system of the present disclosure, and the use thereof in accordance with the methods described herein, results in the rapid and reproducible expression of high levels of a protein of interest in a cell culture system. Typically, the present transient transfection systems and methods are capable of producing recombinant expressed protein at levels in the range of about 200 µg protein / L of culture to about 2 g protein / L of culture, depending on the individual expression characteristics of the desired recombinant protein and cell type used. Using the transient transfection system and methods provided for herein, a user may obtain levels of expressed proteinPATENT DOCKET NO. TP388599WO1that are about 2-fold to up to about 20-fold in excess of what is currently obtainable using standard commercially available transient transfection systems. Using the transient transfection system and methods provided for herein, a user may obtain levels of expressed protein that is about 2.5-fold, about 3-fold, about 3.5-fold, about 4- fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, bout 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, or up to about 10-fold or greater than that seen with contemporary transient expression systems. For example, using the present transient transfection system to produce a recombinant protein, a user may obtain a protein yield between about 2-fold up to about 10-fold higher than the protein yield obtained using a commercially available transient transfection system optimized for production of recombinant protein in suspension cells, such as, e.g., Expi293™ Expression System and FreeStyle™ 293 Expression System.

[0123] Using the system of the present disclosure, which system includes, among other elements, at least a high density culture medium, at least a population of suspension cells adapted for high density growth, optionally one or more expression vectors, optionally one or more transfection reagents, and optionally one or more expression enhancers, it is not necessary to replenish, replace or supplement the medium after one has introduced at least one macromolecule into at least one cell, and before cells into which at least one macromolecule has been introduced are further cultured to produces protein product or express a nucleic acid. In the system of the present disclosure, the medium is ideally a serum-free medium and / or a chemically defined medium and / or protein free or substantially low protein medium, and / or a medium that does not contain animal derived components, or a medium having combinations of these features.

[0124] In one non-limiting aspect of the disclosure, with respect to the introduction of compounds or macromolecules (e.g., nucleic acid) into cells in culture, the high yield culture medium of the present disclosure facilitates higher cell transfection efficiency than can typically be obtained using presently available transient transfection systems. In another related though non-limiting aspect of the disclosure, the system also does not require transfecting the cells in a smaller volume than cells are to be cultured in after transfection. In yet another related though non-limiting aspect of the present disclosure, the system facilitates higher cell viability than presently available transient transfection systems. In yet a further related though non-limiting aspect still, the system facilitates higher cell density (i.e., cells / ml of culture medium) than presently available transient transfection systems. In another related though non-limiting aspect of the present disclosure, the system facilitates a higher level of recombinant protein expression in cells in culture than presently available transientPATENT DOCKET NO. TP388599WO1transfection systems. Preferably, though not necessarily, the same volume of medium can be used for to introduce at least one macromolecule into a cell and subsequent cultivation without having to replace, remove, supplement or replenish the medium in which the transfection of the cells has occurred. Alternatively, the cells are divided or medium volume is increased from about 2, about 5, about 8 or about 10 times.

[0125] The components of the system of the present disclosure are intended to be used to introduce at least one macromolecule or to transfect and culture cells in any volume of culture medium. Such introduction is may be accomplished in 0.1 to 10 times the amount of medium used to culture cells to be transfected. In some embodiments, the cell culture volume is greater than about one milliliter. In some embodiments, the cell culture volume is from about 50, 75, 100, 125, 150, 175 or 200 µl to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 100 liters. In some embodiments, the cell culture volume is from about 2 ml to about 50 liters, most preferably from about 5 ml to about 5 liters. In some embodiments, the cell culture volume is from about 100 ml to about 50 liters. In some embodiments, the cell culture volume is from about 500 ml to about 50 liters. In some embodiments, the cell culture volume is from about 500 ml to about 25 liters. In some embodiments, the cell culture volume is from about 500 ml to about 10 liters. In some embodiments, the cell culture volume is from about 500 ml to about 5 liters. In some embodiments, the cell culture volume is from about 500 ml to about 1 liter. In some embodiments, the cell culture volume is about 1, 10, 100, 500 or 1000 µl. In some embodiments, the cell culture volume is about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 liters. In some embodiments, the cell culture volume is about 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100, 250, 500, 750, or 1000 ml

[0126] In the components of the system of the present disclosure, the medium optionally does not contain compounds that can interfere with introduction of macromolecules or transfection, e.g., polyanionic compounds such as polysulfonated and / or polysulfated compounds. Preferably, the medium does not contain dextran sulfate.

[0127] The components of the system of the present disclosure permit the introduction of compounds or macromolecules (particularly macromolecules, for example nucleic acids, proteins and peptides) into the cultured cells (for example by transfection) without the need to change the medium. In one preferred embodiment, the present disclosure provides a medium for the cultivation and transfection of eukaryotic cells.PATENT DOCKET NO. TP388599WO1

[0128] Using the system of the present disclosure, those of ordinary skill in the art can introduce macromolecules or compounds (e.g., nucleic acid) into cells in culture. Preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into at least about 20 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 20 to about 100 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 30 to about 100 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 50 to about 100 percent of the cells. Practically, the macromolecule or compound might be introduced into about 20% to about 90% of the cells, about 20% to about 80% of the cells, about 30% to about 60, 70, 80 or 90% of the cells, about 20, 30, 40 or 50% to about 70, 75, 80, 85, 90, 95 or 98% of the cells, etc. Even about 60, 70, 75 or 80 to about 90% or close to 100% of the cells may contain the introduced molecule or compound.

[0129] In embodiments of the components of the system of the present disclosure, one or more undesirable components (i.e., one or more serum components, one or more undefined components, one or more protein components and / or one or more animal derived components) have been substituted or replaced in one or more functions by one or more replacement compounds. Replacement compounds of the disclosure may optionally include one or more metal binding compounds and / or one or more transition element complexes, said complexes comprising one or more transition elements or a salts or ions thereof, in a complex with one or more metal-binding compounds. Preferably, the medium is capable of supporting the cultivation of a cell in vitro in the absence of one or more naturally derived metal carriers, such as transferrin, or other animal derived proteins or extracts. The metal binding compound can be in a complex with a transition metal prior to addition of the metal binding compound to the medium. In other embodiments, the metal binding compound is not in a complex with a transition metal prior to addition of the metal binding compound to the media. Preferably, the medium of the present disclosure does not contain transferrin and / or does not contain insulin.

[0130] The present disclosure also relates to a cell culture medium obtained by combining a medium with one or more replacement compounds. Preferably, the medium can be a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or can be a medium lacking animal derived components. The medium preferably does not contain transferrin and / or does not contain insulin. In some preferred embodiments, the medium can be capable of supporting the cultivation of a cell in vitro and / or can permit the introduction ofPATENT DOCKET NO. TP388599WO1macromolecules into the cell. In some embodiments, one or more of the replacement compounds can be a metal binding compound and / or can be a transition element complex, said complex comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound. Preferred transition elements, metal-binding compounds, and transition element complexes for use in this aspect of the disclosure include those described in detail herein.

[0131] Replacement compounds of the present disclosure can facilitate the delivery of transition metals to cells cultured in vitro. In preferred embodiments, the replacement compounds can deliver iron and replace transferrin. A preferred replacement compound is a hydroxypyridine derivative. Preferably, the hydroxypyridine derivative is selected from the group consisting of 2- hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3- hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3- hydroxypyrid-4-one, 1-methyl-3- hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, and pyridoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, 2-hydroxy-nicotinic acid. Most preferably, the hydroxypyridine derivative is 2- hydroxypyridine-N-oxide.

[0132] The replacement compounds of the present disclosure can be used with any media, including media for cultivating or growing eukaryotic and / or prokaryotic cells, tissues, organs, etc. Such media include, but are not limited to, CD FORTICHO™ Medium, Expi293™ Expression Media, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Ham's F-10, Ham's F-12, αMinimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM). Other media that are commercially available (e.g., from Life Technologies Corporation, Carlsbad, Calif.) or that are otherwise known in the art can be equivalently used in accordance with the present disclosure including, but not limited to, 293 SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium, CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAX™ supplemented media, Grace's insect cell media, HEPES buffered media, Richter's modified MEM, IPL-41 insect cell medium, Leibovitz's L-15 media, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle's Medium (MEM), Medium NCTC- 109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752 / 1 media, William's Media E, protein free hybridoma medium II (PFHM II), AIM V media, Keratinocyte SFM, defined Keratinocyte SFM, STEMPRO® SFM, STEMPRO® complete methylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-A medium, Hibernate.TM. A medium, Hibernate E medium, Endothelial SFM, HumanPATENT DOCKET NO. TP388599WO1Endothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 TI SFM, EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium, AMINOMAX-C100 complete medium, AMINOMAX-C 100 basal medium, PB-MAX™ karyotyping medium, KARYOMAX bone marrow karyotyping medium, KNOCKOUT D-MEM and CO2 independent medium. The above media are obtained from manufacturers known to those of ordinary skill in the art, such as JRH, Sigma, HyClone, and BioWhittaker. Additional examples of media suitable for use in the practice of the present disclosure can be found in U.S. Pat. Nos.5,135,866 and 5,232,848 as well as in international publications nos. WO 88 / 02774, WO 98 / 15614, WO 98 / 08934 and European Patent No.0282942, the entireties of which are specifically incorporated herein by reference.

[0133] The present disclosure also provides a method for introducing macromolecules into cells, comprising culturing cells in a medium of the disclosure and contacting the cells in the medium with one or more macromolecules under conditions causing the macromolecules to be taken up by one or more of the cells. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or can be a medium lacking animal derived components. Preferred cells include eukaryotic cells. More preferably, the cells are mammalian cells. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin. In some preferred embodiments, the medium permits the growth and transfection of the cell in the same medium. In some embodiments, the macromolecules can comprise one or more nucleic acids and conditions causing the nucleic acid molecules to be taken up by the cells include contacting the nucleic acid with a reagent which causes the nucleic acid to be introduced into one or more cells.

[0134] The present disclosure also provides a composition comprising a culture medium of the disclosure and a cell. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components. Preferred cells include eukaryotic cells. More preferably, the cells are mammalian cells. Most preferred are suspension cells derived from 293 fibroblasts. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin. Preferably, the medium supports the growth and transfection of the cell in the same medium, more preferably, the medium supports the growth and cultivation of mammalian cells expressing a recombinant protein, where said medium does not have to be replenished, replaced or otherwise supplemented after the introduction of an expressible nucleic acid therein for the purposes of producing a recombinant protein.PATENT DOCKET NO. TP388599WO1

[0135] The present disclosure also provides compositions comprising a culture medium of the present disclosure and one or more reagents for the introduction of macromolecules into one or more cells. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin. Preferably, the medium contains a transfection reagent and the macromolecules are nucleic acids. The macromolecules might also be proteins and / or peptides. In some embodiments, the reagent comprises one or more lipids of which one or more can be cationic lipids. More preferably, the reagent comprises a mixture of neutral and cationic lipids. In some embodiments, the reagent comprises one or more peptides and / or proteins which can be provided alone or in admixture with one or more lipids, such as one or more cationic lipids and / or one or more neutral lipids.

[0136] The present disclosure also provides compositions comprising a culture medium of the disclosure and one or more macromolecules to be introduced into a cell. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin. The macromolecules can be, for example, nucleic acids and / or proteins and / or peptides and can be uncomplexed or can be in the form of a complex with one or more reagents for the introduction of macromolecules into cells. Preferably, the macromolecules are nucleic acids and can be in the form of a complex with one or more transfection reagents.

[0137] The present disclosure also provides a composition comprising at least one component (or combination thereof) of a system of the present disclosure, such as, at least one culture medium, at least one cell, at least one macromolecule, at least one transfection reagent for introducing at least one macromolecule into at least one cell, at least one expression enhancer, a complexation buffer, and any combination thereof. Preferably, the cells are eukaryotic cells. More preferably, the cells are mammalian cells. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin. In some preferred embodiments, the reagent is a transfection reagent and the macromolecules are nucleic acids, for example RNA and / or DNA. Alternatively, the macromolecules are proteins and / or peptides.PATENT DOCKET NO. TP388599WO1

[0138] In some embodiments, the transfection reagent comprises one or more lipids of which one or more can be cationic lipids. More preferably, the transfection reagent comprises a mixture of neutral and cationic lipids. In some embodiments, the transfection reagent comprises one or more peptides and / or proteins which can be provided alone or in admixture with one or more lipids. In preferred embodiments, the transfection reagent complexes with the macromolecule to introduce the macromolecule into the cell.

[0139] The present disclosure also provides kits for the culture and transfection of cells comprising at least one container comprising a component of the system of the disclosure. Such kits may include at least one culture medium for the culture and transfection of cells, at least one component (or combination thereof) of a culture medium of the present disclosure, at least one cell, at least one macromolecule, at least one transfection reagent for introducing at least one macromolecule into at least one cell, at least one expression enhancer, a complexation buffer, and instructions for using the kit to introduce at least one macromolecule into at least one cell.

[0140] Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components. The medium can comprise one or more replacement compounds and preferably does not contain transferrin and / or does not contain insulin and / or does not contain an animal growth factor. The medium can comprise one or more replacement compounds that can be metal binding compounds and / or can comprise one or more complexes comprising one or more replacement compounds. In some embodiments, the medium can comprise one or more complexes, said complex comprising one or more transition elements or salts or ions thereof complexed one or more replacement compounds which can be metal-binding compounds. In some embodiments, said medium is capable of supporting the cultivation of a cell in vitro and permits transfection of cells cultured therein. In some embodiments, kits of the disclosure can further comprise at least one container comprising a lipid for transfecting cells. In some embodiments, the kits of the disclosure can comprise at least one container comprising a nucleic acid.

[0141] According to one aspect of the disclosure, a transition element is preferably selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, rubidium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, and actinium, or salts or ions thereof, and is preferably an iron salt. Suitable iron salts include, but are not limited to, FeCl3, Fe(NO3)3 or FeSO4 or other compounds that contain Fe+++PATENT DOCKET NO. TP388599WO1or Fe++ ions. Preferred replacement compounds include, but are not limited to, metal-binding compounds. See, for example, international patent application no. PCT / US00 / 23580, Publication No. WO 01 / 16294.

[0142] Metal binding compounds of the present disclosure include any macromolecules which can interact with or bind with transition elements and facilitate their uptake by cells. Such interaction / binding can be covalent or non-covalent in nature. The metal-binding compound used in this aspect of the disclosure is preferably selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)amino-methylbenzene, ethylenediamine-N,N'-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g., ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nicotinic acid-N- oxide, 2- hydroxy-nicotinic acid, mono-, bis-, or tris-substituted 2,2'-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid derivative, deferoxamine, ferrioxamine, iron basic porphine and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α- hydroxycarboxylic acid, a polyethylenecarbamate, ethyl maltol, 3-hydroxy-2-pyridine, and IRC011. In one preferred embodiment, the metal-binding compound is a polyol such as sorbitol or dextran, and particularly sorbitol. In a related embodiment, the metal-binding compound is a hydroxypyridine derivative, such as 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3- hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1- hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid- 4-one, 1-methyl-3-hydroxypyrid-2-one, 3- hydroxy-2(1H)-pyridinone, ethyl maltol or pyridoxal isonicotinyl hydrazone, and is preferably 2- hydroxypyridine-N-oxide. In particularly preferred embodiments according to this aspect of the disclosure, the transition metal complex can be a sorbitol-iron complex or 2-hydroxypyridine-N- oxide-iron complex. The metal binding compounds of the present disclosure can also bind divalent cations such as Ca++ and Mg++.

[0143] The disclosure relates to cell culture media comprising one or more replacement compounds which can be metal-binding compounds and further comprising one or more ingredients selected from the group of ingredients consisting of at least one amino acid (such as L-alanine, L- arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L- histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L- threonine, L-tryptophan, L-tyrosine or L-valine, N-acetyl-cysteine), at least one vitamin (such as biotin, choline chloride, D-Ca++-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine or vitamin B12), at least one inorganic salt (such as a calcium salt, CuSO4, FeSO4, Fe(NO3)3, FeCl3, KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl,PATENT DOCKET NO. TP388599WO1NaHCO3, Na2HPO4, Na.2SO4, a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt, ZnCl2, ZnSO4 or other zinc salts), adenine, ethanolamine, D-glucose, one or more cytokines, heparin, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, PLURONIC F68, and thymidine.

[0144] The culture media of the present disclosure can optionally include one or more buffering agents. Suitable buffering agents include, but are not limited to, N-[2-hydroxyethyl]- piperazine-N'- [2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate and other buffering agents suitable for use in cell culture applications. A suitable buffering agent is one that provides buffering capacity without substantial cytotoxicity to the cells cultured. The selection of suitable buffering agents is within the ambit of ordinary skill in the art of cell culture.

[0145] According to the disclosure, a medium suitable for use in forming the cell culture media of the disclosure can comprise one or more ingredients, and can be obtained, for example, by combining one or more ingredients selected from the group consisting of adenine, ethanolamine, D- glucose, heparin, a buffering agent, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, thymidine, L-alanine, L- arginine, L-asparagine, L- aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L- histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- tyrosine, L-valine, N-acetyl-cysteine, biotin, choline chloride, D- Ca++-pantothenate, folic acid, i- inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, Pluronic F68, recombinant insulin, a calcium salt, CuSO4, FeSO4, FeCl3, Fe(NO3)3, KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO3, Na2HPO4, Na2SO4, a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt, ZnCl2, ZnSO4 or other zinc salts, wherein each ingredient is added in an amount which supports the cultivation of a cell in vitro.

[0146] The disclosure is also directed to a cell culture medium comprising ingredients selected from ethanolamine, D-glucose, HEPES, insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68, putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca++-pantothenate, folic acid, i- inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, one or more calcium salts, Fe(NO3) 3, KCl, one or more magnesium salts, one or more manganese salts, NaCl, NaHCO3, Na2HPO4, one or more selenium salts, one or more vanadium salts and one or more zinc salts, wherein each ingredient is present inPATENT DOCKET NO. TP388599WO1an amount which supports the suspension cultivation of a mammalian epithelial cell in vitro. The disclosure is also directed to such media which can optionally further comprise one or more supplements selected from the group consisting of one or more cytokines, heparin, one or more animal peptides, one or more yeast peptides and one or more plant peptides (most preferably one or more of rice, aloevera, soy, maize, wheat, pea, squash, spinach, carrot, potato, sweet potato, tapioca, avocado, barley, coconut and / or green bean, and / or one or more other plants), e.g., see international application no. PCT / US97 / 18255, published as WO 98 / 15614.

[0147] The media provided by the present disclosure can be protein-free, and can be a 1X formulation or concentrated as, for example, a 10X, 20X, 25X, 50X, 10X, 500X, or 1000X medium formulation.

[0148] The media of the disclosure can also be prepared in different forms, such as dry powder media (“DPM”), a granulated preparation (which requires addition of water, but not other processing, such as adjusting pH), liquid media or as media concentrates.

[0149] The basal medium that is a medium useful only for maintenance, but not for growth or production of product, can comprise a number of ingredients, including amino acids, vitamins, organic and inorganic salts, sugars and other components, each ingredient being present in an amount which supports the cultivation of a mammalian epithelial cell in vitro.

[0150] In the system of the present disclosure, the culture medium can be used to culture a variety of cells. Preferably, the medium is used to culture eukaryotic cells. More preferably, the medium is used to culture plant and / or animal cells. More preferably, the medium is used to culture mammalian cells, fish cells, insect cells, amphibian cells or avian cells. More preferably, the medium is used to culture mammalian cells. More preferably, the medium may be used to culture mammalian cells, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CapT cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver,PATENT DOCKET NO. TP388599WO1kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CHO cells, CV- 1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN2O5 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK- (Mouse L) cells, NCTC clones 2472 and 2555, SCC- PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, CII cells, and Jensen cells, or derivatives thereof). Most preferably, the medium is used to culture mammalian cells selected from the group consisting of 293 cells, 293 F cells or derivatives thereof, PER-C6 cells or derivatives thereof, CHO cells or derivatives thereof, CapT cells or derivatives thereof, COS-7L cells or derivatives thereof and Sp2 / 0 cells or derivatives thereof, or any other suspension cell line or derivative capable of being cultured at high cell density as defined above. More preferably, the medium is used to culture 293 cells or a modified 293 cell line specifically adapted for optimal growth in the cell culture medium that forms the basis of the present disclosure. In some preferred though non-limiting aspects, the medium is used to culture cells in suspension.

[0151] Cells supported by the medium of the present disclosure can be derived from any animal, preferably a mammal, and most preferably a mouse or a human. The cells cultivated in the present media can be normal cells or abnormal cells (i.e., transformed cells, established cells, or cells derived from diseased tissue samples).

[0152] The present disclosure also provides methods of cultivating mammalian epithelial or fibroblast cells using the culture medium formulations disclosed herein, comprising (a) contacting the cells with the cell culture media of the disclosure; and (b) cultivating the cells under conditions suitable to support cultivation of the cells. In some embodiments, the methods of the present disclosure can optionally include a step of contacting the cultured cells with a solution comprising one or more macromolecules (preferably comprising one or more nucleic acids) under conditions causing the introduction of one or more of the macromolecules into one or more of the cells. Preferably, cells cultivated according to these methods (which can include any of the cells described above) are cultivated in suspension.PATENT DOCKET NO. TP388599WO1

[0153] In some aspects, a transient transfection system may include a high density culture medium suitable for the growth and propagation of cultured mammalian cells at densities in the range of about 1x106to about 20x106cells / ml, more preferably in the range of about 2x106to about 6x106. Any culture medium may be used in the practice of the present disclosure, with the proviso that the culture medium employed is capable of sustaining the growth of mammalian cells, preferably cells growing in suspension, at densities of up to about 2x107cells / ml while maintaining viability of said cells in excess of about 80% and further, maintaining the ability of said suspension cells to be efficiently transfected and express high amounts of recombinant protein. The high density culture medium used in the practice of the present disclosure may vary between different applications and uses, and may depend on the nature of the cell line being used, the desired protein being transiently expressed, the nature of the transfection modality selected for transfer of the expression vector into cells, and the amount and nature of any expression enhancers added to the system as described below. Nevertheless, preferred high density culture medium contemplated for use in the present transient expression systems and methods will typically be serum-free, protein-free, allow the cultivation and growth of suspension cells to a density of up to about 2x107cells / ml, more typically between about 2x106cells / ml to about 1x107cells / ml, and will further enable the yield of protein produced in the transient expression system to exceed at least 200 µg / mL of cell culture up to 2 mg / mL of cell culture, more typically between about 500 µg / ml of cell culture to about 1 mg / mL of cell culture. Ideally, the high density culture medium used in accordance with the present disclosure will facilitate the transfection of cells at densities in the range of about 1x106to about 20x106cells / ml, about 2x106to about 2x106cells / ml, or about 2.5x106to about 6x106cells / ml.

[0154] Particularly preferred high density growth media suitable for the practice of the present disclosure may be a chemically defined medium in which each chemical species and its respective quantity is known prior to its use in culturing cells. The selected chemically defined medium may optionally be made without cellular or tissue lysates or hydrolysates whose chemical species are not known and / or quantified.

[0155] In some aspects of the present disclosure a particularly suited type of medium for the practice of the present disclosure is a serum-free medium (sometimes referred to as “SFM Medium”) being entirely devoid of, e.g., fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, and the like. Exemplary though non-limiting serum-free media familiar to the skilled artisan include HuMEC Basal Serum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC / iPSCPATENT DOCKET NO. TP388599WO1Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106, Medium, 131, Medium, 154, Medium,

[0156] 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial- SFM, GIBCO® FREESTYLE™ 293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154 CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™ -II Complete Medium, CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™ Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and any derivatives or modifications thereof.

[0157] In some aspects of the present disclosure a particularly suited type of medium for the practice of the present disclosure is a protein-free medium (sometimes referred to as “PFM Medium”) being entirely devoid of protein (e.g., no serum proteins such as serum albumin or attachment factors, nutritive proteins such as growth factors, or metal ion carrier proteins such as transferrin, ceruloplasmin, etc.). Preferably, if peptides are present, the peptides are smaller peptides, e.g., di- or tri-peptides. Preferably, peptides of deca-peptide length or greater are less than about 1%, more preferably less than about 0.1%, and even more preferably less than about 0.01% of the amino acids present in the protein free medium.

[0158] Ideally, both serum-free and protein-free media contemplated for use with the present disclosure will further be devoid of any animal derived material, or any material that is derived in whole or in part from an animal source, including recombinant animal DNA or recombinant animal protein DNA.

[0159] Exemplary high density culture media suitable for use in the practice of the present disclosure include, though are not limited to, HuMEC Basal Serum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC / iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106, Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial- SFM, GIBCO® FREESTYLE™ 293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154 CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium,PATENT DOCKET NO. TP388599WO1STEMPRO™-34 Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™ -II Complete Medium, CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and any derivatives or modifications thereof. In certain preferred though non- limiting embodiments, a high density culture media may be CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, GIBCO® FREESTYLE™ 293 Expression Medium, EXPI293™ Expression Medium, or a like medium, or a modified version thereof. The above listed exemplary high density culture media may be particularly suitable for the high density growth, propagation, transfection and maintenance of CHO cells, a CHO cell variant, 293 cells, a 293 cell variant, CapT cells, a CapT cell variant, or any other cells adapted for use in a high density culture system. Optionally, a user may wish to formulate a new culture medium having the properties described herein, or may opt instead to reformulate or modify existing culture media.

[0160] In some aspects, a high density growth medium may be selected from the list Such media include, but are not limited to, CD FORTICHO™ Medium, Expi293™ Expression Media, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Ham's F-10, Ham's F-12, α-Minimal Essential Medium (α- MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM). Other media that are commercially available (e.g., from Life Technologies Corporation, Carlsbad, Calif.) or that are otherwise known in the art can be equivalently used in accordance with the present disclosure including, but not limited to, 293 SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium, CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAX™ supplemented media, Grace's insect cell media, HEPES buffered media, Richter's modified MEM, IPL-41 insect cell medium, Leibovitz's L-15 media, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle's Medium (MEM), Medium NCTC- 109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752 / 1 media, William's Media E, protein free hybridoma medium II (PFHM II), AIM V media, Keratinocyte SFM, defined Keratinocyte SFM, STEMPRO® SFM,PATENT DOCKET NO. TP388599WO1STEMPRO® complete methylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-A medium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, Human Endothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 TI SFM, EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium, AMINOMAX-C100 complete medium, AMINOMAX-C 100 basal medium, PB-MAX™ karyotyping medium, KARYOMAX bone marrow karyotyping medium, KNOCKOUT D-MEM and CO2 independent medium. The above media are obtained from manufacturers known to those of ordinary skill in the art, such as JRH, Sigma, HyClone, and BioWhittaker. Additional examples of media suitable for use in the practice of the present disclosure can be found in U.S. Pat. Nos.5,135,866 and 5,232,848 as well as in international publications nos. WO 88 / 02774, WO 98 / 15614, WO 98 / 08934 and European Patent No.0282942, the entireties of which are specifically incorporated herein by reference. Optionally, a user may wish to formulate a new culture medium having the properties described herein, or may opt instead to reformulate or modify existing culture media.

[0161] The disclosure further provides compositions comprising the culture media of the present disclosure, which optionally can further comprise one or more mammalian epithelial or fibroblast cells, such as those described above, particularly one or more 293 cells, 293 F cells, PER-C6 cells, CHO cells, CapT cells, COS-7L cells and Sp2 / 0 cells, or any derivatives thereof.

[0162] In some aspects of the disclosure, the high yield transient transfection system of the present disclosure may include one or more cells or cell lines that are or have been adapted to grow under high density condition without substantial loss in their viability, ability to be efficiently transfected, or their ability to express high levels of recombinant protein. Preferably, a cell are cell line suitable for use in the present disclosure growth and propagation of cultured mammalian cells at densities in the range of about 1x106to about 20x106cells / ml, more preferably in the range of about 2x106to about 6x106. Any cell line may be used, without limitation, provided the cell line are capable of growing under high density conditions as defined above, while maintaining their viability at high density in excess of about 80%, and retaining their ability to transfect at high efficiency and express recombinant protein at levels up to about 2 g / L of culture. The identification of such a cell line is well within the purview of the skilled artisan, and such a person can identify a suitable cell line for use in the present disclosure without departing from the spirit and scope thereof. The cells adapted for high density culture may be a cell lineage or a (non-clonal) population of cells derived from the same parental cell lineage which have been adapted to grow at high density in a high density culture medium while retaining cell viability at or above about 80%. Such cells may be isolated or selectedPATENT DOCKET NO. TP388599WO1out from the parental population of cells by maintaining the cells at high density over>40, >50, >60, >70, or >80 sequential passages and gradually replacing the proportion of growth medium with the desired high density culture medium. Optionally, during the process, different pools of cells may be individually propagated and subjected to the selection procedure while simultaneously assessing transfection efficiency and or protein expression efficiency, so that non-clonal population of cells may be selected that can be sustained and grown at high density, transfected with high efficiency, and express high levels of a desired recombinant protein. While it will be readily apparent to the skilled practitioner that a variety of cell types and lineages may be subjected to this selection procedure, it has been determined that cell lineages derived from CHO cells, cell lineages derived from 293 fibroblast cells, and cells derived from CapT cells are particularly amenable to the selection process for being adapted to high density growth conditions. Ideally, cells that are adapted to high density growth culture and amenable for use in the present disclosure will also be capable of being transfected at high efficiency and / or capable of expressing recombinant protein at yield exceeding at least 200 about µg / mL of cell culture up to about 2 mg / mL of cell culture, more typically between about 500 µg / ml of cell culture to about 1 mg / mL of cell culture. Ideally, cells adapted for high density culture used in accordance with the present disclosure are capable of being sustained and transfected at densities in the range of about 1x106to about 20x106cells / ml, about 2x106to about 2x106cells / ml, or about 2.5x106to about 6x106cells / ml.

[0163] By way of non-limiting example, cells or cell lines that may be adapted for high density culture according to the embodiments described herein may include cell such as cultured eukaryotic cells, more preferably, cultured plant and / or animal cells, more preferably, cultured mammalian cells, fish cells, insect cells, amphibian cells or avian cells. In certain preferred though non limiting embodiments, cells or cell lines that may be adapted for high density culture according to the embodiments described herein may include culture mammalian cells, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CapT cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC 256 cells, MH1C1 cells, XCPATENT DOCKET NO. TP388599WO1cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast- like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC- 5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV- BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN2O5 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK- (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, CII cells, and Jensen cells, or derivatives thereof). Most preferably, the medium is used to culture mammalian cells selected from the group consisting of 293 cells, 293 F cells or derivatives thereof, PER-C6 cells or derivatives thereof, CHO cells or derivatives thereof, CapT cells or derivatives thereof, COS-7L cells or derivatives thereof and Sp2 / 0 cells or derivatives thereof, or any other suspension cell line or derivative capable of being cultured at high cell density as defined above. More preferably, the medium is used to culture 293 cells or a modified 293 cell line specifically adapted for optimal growth in the cell culture medium that forms the basis of the present disclosure. In some preferred though non-limiting aspects of the present disclosure, the cells adapted for use in high-density culture are suspension cells, or adherent cells that have been adapted to grow in suspension.

[0164] Cells supported by the medium of the present disclosure can be derived from any animal, preferably a mammal, and most preferably a mouse or a human. The cells cultivated in the present media can be normal cells or abnormal cells (i.e., transformed cells, established cells, or cells derived from diseased tissue samples).

[0165] Cells adapted to high density cultured in accordance with the embodiments described herein may optionally express one or more expression-enhancing proteins. As used herein, the term “expression enhancing protein” refers to any protein expressed by a cell; the expression of the protein enhances the expression of a recombinant protein. The expression of an expression- enhancing protein by a cell line or populations of cells may be stable or transient, for the purposes of the present embodiments. A variety of such expression-enhancing proteins are known in the art, and may includePATENT DOCKET NO. TP388599WO1proteins such as, e.g., PKBa, Bcl-xL, P21, P18, AKT, and the like. In some aspects of the disclosure, the high yield transient transfection system of the present disclosure may include one or more expression vectors for transiently expressing a recombinant protein of interest. The expression vector may be provided already containing an expressible nucleic acid (such as, e.g., a positive control to assess expression efficiency when compared to an optimized control protein), or alternatively, the expression vector may be provided in a form whereby the user may easily insert an expressible nucleic acid containing an open-reading frame of a protein of interest, such that the protein of interest can be expressed recombinantly and at high efficiency in the cells.

[0166] For recombinant production of a protein of interest, an expressible nucleic acid encoding the protein is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the protein may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0167] Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0168] Signal Sequence Component

[0169] A protein of interest may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.

[0170] Origin of Replication

[0171] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2µ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.PATENT DOCKET NO. TP388599WO1Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

[0172] Selection Gene Component

[0173] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0174] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0175] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0176] For example, cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co- transformed nucleic acid. A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) may be used.

[0177] Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. The GS selection / amplification system may be used in combination with the DHFR selection / amplification system described above.

[0178] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.4,965,199.PATENT DOCKET NO. TP388599WO1

[0179] A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

[0180] In addition, vectors derived from the 1.6 µm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio / Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio / Technology, 9:968-975 (1991).

[0181] Promoter Component

[0182] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding a protein of interest. A variety of promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0183] Protein transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0184] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector isPATENT DOCKET NO. TP388599WO1disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

[0185] Enhancer Element Component

[0186] Transcription of a DNA encoding a protein of interest in accordance with the present disclosure by higher eukaryotes is often increased or enhanced by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Often, though not exclusively, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody-encoding sequence, but is preferably located at a site 5' from the promoter. Additional enhancers are known in art, and may include, for example, enhancers obtained or derived from mammalian or viral genes. One particularly preferred enhancers contemplated for use herein is the woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0187] Transcription Termination Component

[0188] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

[0189] In some aspects, an expression vector well-suited for the practice of the present disclosure may be any of the well-known vectors used in the art, such as, e.g., pcDNA 3.3 and pcDNA 3.4, or a modified version thereof. Non-limiting examples of the types of modification to a vector that may be suitable in the practice of the present disclosure include, though are not limited to, modification such as the addition of modification of one or more enhancers, one or more promoters, one or morePATENT DOCKET NO. TP388599WO1ribosomal binding sites, one or more origins of replication, or the like. In certain preferred though non-limiting embodiments, and expression vector used in the practice of the present disclosure may include one or more enhancer elements selected to improve expression of the protein of interest in the present transient expression system. The selected enhancer element may be positioned 5’ or 3’ to the expressible nucleic acid sequence used to express the protein of interest. A particularly preferred though non-limiting enhancer element is the woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0190] In one preferred though non-limiting embodiment, an expression vector used in accordance with the presently described disclosure may be a pcDNA vector, or particularly, a pcDNA 3.3 vector or pcDNA 3.4 vector, more particularly a variant of a pcDNA 3.3 vector or pcDNA 3.4 vector. The vector may optionally include an enhanced promoter, such as, e.g., and enhanced CMV promoter. Optionally, the vector may include an Adeno T+M region, optionally an SV40ori site, optionally an SV40 splice donor / acceptor site, or optionally a woodchuck hepatitis post- transcriptional regulatory element (WPRE).

[0191] In some aspects of the disclosure, the high yield transient transfection system of the present disclosure may include one or more expression enhancers. An expression enhancer can be an aqueous solution containing one or more compounds that increase expression of a recombinant protein in a transient expression system. A variety of expression enhancers are known in the art, and any one or more may be used in the practice of the present disclosure without limitation.

[0192] Generally, the one or more expression enhancers are contacted with a population of protein-expressing cells during or after said cells have been transfected with an expressible nucleic acid or expression vector. When two or more expression enhancer are used, each expression enhancer may be contacted with the cells at substantially the same time, or alternatively the expression enhancers may be contacted with the protein-expressing cells sequentially, optionally after a period of time has passed between contacting the cells with a first expression enhancer and contacting the cells with a second expression enhancer.

[0193] While it will be readily appreciated by the skilled artisan that any number of expression enhancers may be used in the practice of the present disclosure, without limitation, and the identification of what constitutes a suitable expression enhancer for use in the present embodiments is well within the purview of such a person, a variety of exemplary though non- limiting expression enhancers will be described below, though it is to be understood that the recitation thereof does notPATENT DOCKET NO. TP388599WO1limit the scope of suitable expressions that may be contemplated for use in the practice of the present disclosure.

[0194] In some aspects, one or more expression enhancers may include liquid (preferably aqueous) additives used to supplement a culture medium formulation in accordance with the presently described embodiments, said additives being selected to improve the yield of expressed protein produced in a transient protein expression system in accordance with the presently described embodiments. One or more expression enhancers may include one or more of several compounds that impact cell cycle progression, inhibit apoptosis, slow cell growth and / or promote protein production. In the context of the present disclosure, the term “expression enhancers” generally refers to any one or more compounds added to a transient transfection system, the presence of which enhances or promotes expression of a target protein by a factor of at least 2 fold up to about 10-fold above the expression level seen in the absence of such expression enhancer(s). Exemplary expression enhancers suitable for use with the presently described embodiments include, though are not limited to, additives such as valproic acid (VPA, acid and sodium salt), sodium propionate, lithium acetate, dimethyl sulfoxide (DMSO), sugars including glucose and galactose, amino acid mixtures, or butyric acid, or any combinations of the aforementioned. In some embodiments, the expression enhancer is free, or substantially free of galactose. The optimal concentration of each specific expression enhancer may vary according to individual characteristics of the expression system and the requirements of the user, and the determination of what constitutes an optimal concentration of any one or more expression enhancer in a given experimental scenario is well within purview of a practitioner having ordinary skill level in the art.

[0195] In some non-limiting embodiments described herein, “293 Enhancer” generally refers to an expression enhancer composition including valproic acid at a concentration of about 200 mM – 300 mM, sodium propionate at a concentration of about 0.5 M – 10 M, glucose at a concentration of about 700 mM – 1,000 mM, valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentration of about 0.5 M – 10 M, valproic acid at a concentration of about 200 mM – 300 mM admixed with glucose at a concentration of about 700 – 1,000 mM, sodium propionate at a concentration of about 0.5 M – 10 M admixed with glucose at a concentration of about 700 – 1,000 mM, or valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentration of about 0.5 M – 10 M and glucose at a concentration of about 700 – 1,000 mM. In some embodiments, an expression enhancer composition includes valproic acid at a concentration of about 200 mM – 300 mM admixed with sodium propionate at a concentrationPATENT DOCKET NO. TP388599WO1of about 2.0 M – 2.5 M, and optionally glucose at a concentration of about 700 – 1,000 mM. Expression enhancer compositions may be added to a culture medium sequentially, or as a cocktail.

[0196] In some aspects of the disclosure, the high yield transient transfection system of the present disclosure may include one or more reagents for the introduction of macromolecules into the cultured cells (said reagents being commonly referred to as “transfection reagents”). A transfection reagent used in accordance with the presently described embodiments can be any compound or other chemical modality for introducing a biological molecule, particularly a nucleic acid molecule, into a cell whereby the nucleic acid may exert a biological function, or in the case of an expressible nucleic acid, where a gene or protein encoded by said expressible nucleic acid can be expressed. A variety of suitable transfection reagents are known in the art, and any one or more may be used in the practice of the present disclosure without limitation.

[0197] The present disclosure is directed, in part, to a high-yield transient transfection system that supports (a) the introduction of at least one macromolecule, preferably an expressible nucleic acid molecule, into eukaryotic cells in culture, (b) the cultivation of cells into which at least one macromolecule is introduced, and optionally (c) the production of recombinant protein product or expression of the nucleic acid in cells into which at least one macromolecule is introduced, wherein medium containing the macromolecule does not need to be removed from the culture and replaced with fresh medium after introduction of at least one macromolecule into cells and prior to cultivation and production of protein product or expression of nucleic acid. The transient transfection system of the present disclosure, and the use thereof in accordance with the methods described herein, results in the rapid and reproducible expression of high levels of a protein of interest in a cell culture system. Typically, the present transient transfection systems and methods are capable of producing recombinant expressed protein at levels in the range of about 200 µg protein / L of culture to about 2 g protein / L of culture, depending on the individual expression characteristics of the desired recombinant protein and cell type used. Using the transient transfection system and methods provided for herein, a user may obtain levels of expressed protein that are about 2-fold to up to about 20-fold in excess of what is currently obtainable using standard commercially available transient transfection systems. Using the transient transfection system and methods provided for herein, a user may obtain levels of expressed protein that is about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5- fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, bout 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, or up to about 10-fold or greater than that seen with contemporary transient expression systems. For example, using the present transient transfectionPATENT DOCKET NO. TP388599WO1system to produce a recombinant protein, a user may obtain a protein yield between about 2-fold up to about 10-fold higher than the protein yield obtained using a commercially available transient transfection system optimized for production of recombinant protein in suspension cells, such as, e.g., Expi293™ Expression System and FreeStyle™ 293 Expression System.

[0198] The present disclosure further relates to methods for expressing high levels of a protein of interest. Methods of the disclosure may include cultivating mammalian cells (particularly those described above and most particularly 293 cells, 293 F cells, PER-C6 cells, CHO cells, CapT cells, COS-7L cells and Sp2 / 0 cells, or any derivatives thereof) in suspension comprising (a) obtaining a mammalian cell to be cultivated in suspension; and (b) contacting the cell with the culture media of the disclosure under conditions sufficient to support the cultivation of the cell in suspension, transfecting the cultured cells with an expressible nucleic acid encoding a protein of interest, contacting the transfected cells with one or more expression enhancers, culturing the transfected cells under conditions permissive to the expression of the protein of interest for a defined period of time, and harvesting the cells.

[0199] The present disclosure further relates to methods of producing a polypeptide, and to polypeptides produced by these methods, the methods comprising (a) obtaining a cell, preferably a mammalian cell described above and most preferably a 293 cells, 293 F cells, PER-C6 cells, CHO cells, CapT cells, COS-7L cells and Sp2 / 0 cells, or any derivatives thereof; (b) contacting the cell with a solution comprising a nucleic acid encoding the polypeptide under conditions causing the introduction of the nucleic acid into the cell; and (c) cultivating the cell in the culture medium of the disclosure under conditions favoring the expression of the desired polypeptide by the cell.

[0200] A method for expressing a recombinant protein in according with the present disclosure may include obtaining a culture of cells in a high density culture medium. The cells are preferably a suspension culture of 293 cells, 293 F cells, PER-C6 cells, CHO cells, CapT cells, COS-7L cells or Sp2 / 0 cells, or any derivatives thereof, which cells have been adapted for growth in high density medium. While it will be readily appreciated by the skilled artisan that any volume of cell culture may be used in the practice of the present disclosure, the culture will typically be from about 200 µl to 100 liters, more preferably, the cell culture volume is from about 2 ml to about 50 liters, most preferably from about 5 ml to about 5 liters. In some aspects, the cell culture volume can be from about 100 ml to about 50 liters. More preferably, the cell culture volume is from about 500 ml to about 50 liters. More preferably, the cell culture volume is from about 500 ml to about 25 liters. More preferably, the cell culture volume is from about 500 ml to about 10 liters. More preferably, the cellPATENT DOCKET NO. TP388599WO1culture volume is from about 500 ml to about 5 liters. More preferably, the cell culture volume is from about 500 ml to about 1 liter. In some embodiments, the cell culture volume can be up to about 100 liters, up to about 95 liters, up to about 90 liters, up to about 85 liters, up to about 80 liters, up to about 75 liters, up to about 70 liters, up to about 65 liters, up to about 60 liters, up to about 55 liters, up to about 50 liters, up to about 45 liters, up to about 40 liters, up to about 35 liters, up to about 30 liters, up to about 35 liters, up to about 20 liters, up to about 15 liters, up to about 10 liters, up to about 9 liters, up to about 8 liters, up to about 7 liters, up to about 6 liters, up to about 5 liters, up to about 4 liters, up to about 2 liters or up to about 1 liter.

[0201] In one embodiment, the cell culture may be maintained at a cell density of between about 1.5x106cells / ml to about 20x106cells / ml, or any concentration, concentration range or sub-range encompassed therein.

[0202] To express a protein in cells in accordance with the presently described disclosure, the cells will typically be diluted into a fresh volume of medium. The optimal dilution can vary, though for illustrative purposes, the density of cells diluted into a fresh volume of medium can be between 0.5x106cells / ml to about 10x106cells / ml, more preferably 1x106cells / ml to about 5x106cells / ml, more preferably, 1.5x106cells / ml to about 3x106cells / ml.

[0203] In some embodiments, following dilution of the cells into a fresh volume of culture medium, the cells can be cultured in said volume for a period of time, prior to being transfected with an expressible nucleic acid. Optionally, the cells can be cultured for up to 2 days, more preferably up to about a day and a half, most preferably, up to about a day. Optionally, the cells can be cultured in the fresh volume of medium until the density of the cells cultured therein has increased by up to about 100%, more preferably up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60% up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20% or up to about 15%.

[0204] In some embodiments, cells may be transfected with a macromolecule, such as an expressible nucleic acid or an expression vector after the cells have been cultured in the high density growth media for a period of time as described above. The precise sequence of steps a user undertakes to accomplish the introduction of the expression vector into the cells may vary, depending on the specific transfection reagent selected, the cell line, the media and various other experimental parameters, as will be readily recognized by a practitioner having ordinary skill level in the art. By way of example only, in the case where a lipid-based transfection system is selected (in particular, aPATENT DOCKET NO. TP388599WO1transfection system having at least one cationic lipid), the transfection reagent will first be contacted with the nucleic acid in an aqueous solution to form lipid-DNA complexes in a process known informally as “complexation” or a “complexation reaction” as defined above and incorporated herein. Such a reaction will typically be accomplished in a separate reaction vessel from that in which the cells are being cultured.

[0205] In some embodiments, complexation may be performed in a single tube. In some embodiments, a reagent including an expressible nucleic acid encoding the desired protein is added to a tube containing complexation buffer thereby diluting the nucleic acid. A non-diluted (e.g., neat) transfection reagent is subsequently added to the tube. The tube is admixed and a complexation reaction occurs forming transfection complexes for transfection of cells. In some embodiments, a non-diluted transfection reagent is added to a tube containing complexation buffer thereby diluting the transfection reagent. An expressible nucleic acid encoding the desired protein is subsequently added to the tube. The tube is admixed and a complexation reaction occurs forming transfection complexes for transfection of cells. In some embodiments, before the nucleic acid is added to the tube containing the transfection reagent and complexation buffer, the transfection reagent remains stable for greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more.

[0206] In some embodiments, complexation may be performed using two tubes. In such embodiments, an expressible nucleic acid encoding the desired protein is added to a tube containing a half volume complexation buffer thereby diluting the nucleic acid. In a second tube, transfection reagent is added to a second half volume of complexation buffer. The fluid volumes are then combined after an amount of time. In some embodiments, before the fluid contents of the two tubes are combined, the transfection reagent remains stable for greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or more.

[0207] Notably, the ability of the transfection reagent to remain stable in the complexation buffer for an amount of time before addition of nucleic acid to form transfection reagent / nucleic acid complexes for transfection, allows the workflow to be performed in an automated manner (e.g., with the assistance of an automated robotic assembly such as a robotic liquid handler) to facilitate high throughput processing including parallel processing. In related embodiments using automated processes, fluid volumes may be increased, either in one tube or two tube complexation, to facilitate high throughput processing and ensure volumetric ratios of components are maintained to reliably form transfection complexes in increased volumes used to transfect cells in embodiments of the disclosure.PATENT DOCKET NO. TP388599WO1

[0208] In some embodiments, following the formation of transfection complexes (e.g., lipid- nucleic acid complexes) in the complexation step described above, the transfection complexes can be contacted with the cultured cells. After contacting the cells with the transfection complexes, the cells can be cultured in the presence of the transfection complexes for a first period of time. The duration of the first period of time will vary according to the nature of the cells, the transfection reagent used, and a variety of other factors know to those skilled in the art. The phrase “first period of time”, when used in the context of a method for transiently transfecting cells in accordance with the methods of the disclosure described herein generally refers to the time interval between transfecting a population of cells with an expressible nucleic acid and the additional of one or more expression enhancers to the transfected cells. Typically, a first period of time will be in the range of about 0 min to about 2 days, or any ranges or sub-ranges encompassed therein. Typically, a first period of time will be in the range of about 0 minutes to about 2 hours. In certain preferred though non-limiting embodiments, a first period of time may be in the range of about 0 to about 120 min, about 0.2 to about 120 min, about 0.4 to about 120 min, about 0.6 to about 120 min, about 0.8 to about 120 min, about 1 to about 120 min, about 2.5 to about 120 min, about 5 to about 120 min, about 10 to about 120 min, about 15 to about 120 min, about 20 to about 120 min, about 30 to about 120 min, about 45 to 120 min, about 60 to about 120 min, about 1 to about 60 min, about 1 to about 45 min, about 1 to about 30 min, about 1 to about 20 min, about 1 to about 15 min, about 1 to about 10 min, or about 1 to about 5 min. In other preferred to non-limiting embodiments, a first period of time may be up to about 1 min, up to about 5 min, up to about 10 min, up to about 15 min, up to about 20 min, up to about 25 min, up to about 30 min, up to about 35 min, up to about 40 min, up to about 45 min, up to about 50 min, up to about 55 min, up to about 60 min, up to about 80 min, up to about 100 min, or up to about 120 min. In some embodiments, after contacting the cells with the transfection complexes, the cells can be cultured for a first period of time of less than about 0.5.1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 min.

[0209] In one highly preferred though non-limiting embodiment, the culture medium is not replaced, supplemented or replenished following the introduction of the transfection complexes to the cells, and for the duration of the first period of time.

[0210] In some embodiments, the transfected cells in culture may be contacted with one or more expression enhancers following the first period of time. An expression enhancer can be an aqueous solution containing one or more compounds that increase expression of a recombinant protein in aPATENT DOCKET NO. TP388599WO1transient expression system. A variety of expression enhancers are known in the art, and any one or more may be used in the practice of the present disclosure without limitation.

[0211] In some embodiments, the cells in culture may be contacted with one or more expression enhancers before contacting the cells with the transfection complexes.

[0212] In some embodiments, the cells in culture may be contacted with one or more expression enhancers before contacting the cells with the transfection complexes and again contacting the cells with one or more expression enhancers following the first period of time.

[0213] Generally, the one or more expression enhancers are contacted with a population of protein-expressing cells during or after said cells have been transfected with an expressible nucleic acid or expression vector. However, one or more expression enhancers may be contacted with a population of cells before said cells have been transfected with an expressible nucleic acid or expression vector. Additionally, one or more expression enhancers may be contacted with a population of cells before said cells have been transfected with an expressible nucleic acid or expression vector, and one or more additional expression enhancers may be contacted with the cells during or after transfection of the cells. It will be understood that when two or more expression enhancers are used, each expression enhancer may be contacted with the cells at substantially the same time, or alternatively the expression enhancers may be contacted with the protein-expressing cells sequentially, optionally after a period of time has passed between contacting the cells with a first expression enhancer and contacting the cells with a second expression enhancer.

[0214] In some embodiments, one or more expression enhancers may include liquid (preferably aqueous) additives used to supplement a culture medium formulation in accordance with the presently described embodiments, said additives being selected to improve the yield of expressed protein produced in a transient protein expression system in accordance with the presently described embodiments. One or more expression enhancers may include one or more of several compounds that impact cell cycle progression, inhibit apoptosis, slow cell growth and / or promote protein production. In the context of the present disclosure, the term “expression enhancers” generally refers to any one or more compounds added to a transient transfection system, the presence of which enhances or promotes expression of a target protein, and in some embodiments increases the expression level of a recombinant protein by a factor of at least 2 fold up to about 10-fold above the expression level seen in the absence of such expression enhancer(s). Exemplary expression enhancers suitable for use with the presently described embodiments are described herein and include, though are not limited to, additives such as valproic acid (VPA, acid and sodium salt), sodium propionate,PATENT DOCKET NO. TP388599WO1lithium acetate, dimethyl sulfoxide (DMSO), sugars including glucose and / or galactose, amino acid mixtures, or butyric acid, or any combinations of the aforementioned. The optimal concentration of each specific expression enhancer may vary according to individual characteristics of the expression system and the requirements of the user, and the determination of what constitutes an optimal concentration of any one or more expression enhancer in a given experimental scenario is well within purview of a practitioner having ordinary skill level in the art.

[0215] Expression enhancers compositions may be added to a culture medium sequentially, or as a cocktail. In some embodiments, when two or more expression enhancers are used, the two or more expression enhancers can be contacted with the transfected cultured cells substantially simultaneously, or alternatively the transfected cultured cells can first be contacted with a first expression enhancer, and after a second period of time, the transfected cultured cells can be contacted with the second expression enhancer. In one aspect, the “second period of time”, when used in the context of a method for transiently transfecting cells in accordance with the methods of the disclosure described herein generally refers to the time interval between the addition of one or more expression enhancers and either the addition of one or more additional enhancers, or the harvesting of the transfected cells and purification or isolation of the protein expressed therein.

[0216] Typically, a second period of time will be in the range of about 10 hrs to about 10 days, though other time intervals may be used if determined to be optimal for the protein being expressed. In some preferred though non-limiting embodiments, the second period of time may be in the range of 2 hrs to 5 days, 2.5 hrs to 4 days, about 3 to about 90 hrs, about 4 to about 85 hr, about 5 to about 80 hrs, about 6 to about 75 hrs, about 7 to about 70 hrs, about 8 to about 65 hrs, about 9 to about 60 hrs, about 10 to about 55 hrs, about 11 to about 50 hrs, about 12 to about 45 hrs, about 13 to about 40 hrs, about 14 to about 35 hrs, about 15 to 30 hrs, about 16 to about 24 hrs, about 17 to about 24 hrs, about 18 to about 24 hrs, about 19 to about 24 hrs, about 20 to about 24 hrs, about 21 to about 24 hrs, about 22 to about 24 hrs or about 23 to about 24 hrs. In other preferred to non-limiting embodiments, a first period of time may be up to about 15 hrs, up to about 16 hrs, up to about 17 hrs, up to about 18 hrs, up to about 19 hrs, up to about 20 hrs, up to about 21 hrs, up to about 22 hrs, up to about 23 hrs, up to about 24 hrs, up to about 25 hrs, up to about 26 hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrs or up to about 30 hrs.

[0217] After an appropriate amount of time has elapsed, the user can harvest the cells and optionally purify, or otherwise isolate the expressed recombinant protein.PATENT DOCKET NO. TP388599WO1

[0218] The method of the present disclosure allows a user to transiently express a recombinant protein in accordance with the embodiments described above without having to replace, supplement or otherwise replenish the culture medium during the process. The methods described herein allow the user express up to about 2 g / L of cultured cells. In some embodiments, the user can express up to about 1.9 g, up to about 1.8 g, up to about 1.7 g, up to about 1.6 g, up to about 1.5 g, up to about 1.4 g, up to about 1.3 g, up to about 1.2 g, up to about 1.1 g, or up to about 1 g of recombinant protein for every liter of cultured cells.

[0219] The present disclosure is also directed to compositions, particularly a high density cell culture media as defined above, optionally comprising one or more replacement compounds. The disclosure is also directed to methods of use of such compositions, including, for example, methods for the cultivation of eukaryotic cells, particularly animal cells, in vitro. The disclosure also relates to compositions comprising such culture media and one or more cells, especially those cells specifically referenced herein, and to kits comprising one or more of the above- described compositions. The disclosure also relates to expression vectors comprising one or more expressible nucleic acid sequences in combination with one or more promoters, enhancers, and other elements required for expressing said expressible nucleic acid in a cultured cells, as defined above and incorporated herein. The disclosure also relates to compositions comprising one or more expression enhancer compositions, especially those selected to enhance expression of said expressible nucleic acid in a cultured cell by at least a factor or 2- to 2.5 fold. Optionally, the expression enhancers can be a combination of two or more expression enhancers co-formulated or provided separately. The disclosure also relates to transfections reagents, especially those optimized to facilitate the delivery of one or more nucleic acid molecules to the interior of a cultured cell. The disclosure also relates to kits comprising one or more of the above-described compositions, vectors, expression enhancers, transfection reagents, and the like, and to kits comprising one or more of the above-described compositions, especially those cells specifically referenced herein.

[0220] In another aspect, the disclosure relates to a kit for the cultivation of cells in vitro. The kit comprise one or more containers, wherein a first container contains the culture medium of the present disclosure. The kit can further comprise one or more additional containers, each container containing one or more supplements selected from the group consisting of one or more cytokines, heparin, one or more animal or animal-derived peptides, one or more yeast peptides and one or more plant peptides (which are preferably one or more peptides from rice, aloevera, soy, maize, wheat, pea, squash,PATENT DOCKET NO. TP388599WO1spinach, carrot, potato, sweet potato, tapioca, avocado, barley coconut and / or green bean, and / or one or more other plants).

[0221] The kit of the present disclosure can further comprise one or more containers comprising a nucleic acid and / or a reagent that facilitates the introduction of at least one macromolecule, e.g., a nucleic acid into cells cultured in the media of the present disclosure, i.e., a transfection reagent. Preferred transfection reagents include, but are not limited to, cationic lipids and the like.

[0222] A kit according to one aspect of the disclosure can comprise one or more of the culture media of the disclosure, one or more replacement compounds, which can be one or more metal binding compounds, and / or one or more transition element complexes, and can optionally comprise one or more nucleic acids and transfection reagents. Kits according to another aspect of the disclosure can comprise one or more cell culture media (one of which can be a basal medium) and optionally one or more replacement compounds. The kit of the present disclosure can also contain instructions for using the kit to culture cells and / or introduce macromolecules or compounds (e.g., nucleic acid, such as DNA), into cells.

[0223] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and can be made without departing from the scope of the disclosure or any embodiment thereof. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the disclosure. EXAMPLES The following Examples are provided to illustrate certain aspects of the disclosure and to aid those of skill in the art in practicing the disclosure. These Examples are in no way to be considered to limit the scope of the disclosure or appended claims in any manner.

[0224] Example 1: 293 expression system

[0225] Exemplary, non-limiting guidelines and protocols for the 293 expression system provided herein.

[0226] General guidelines for growing and maintaining 293 cells: • All solutions and equipment that come in contact with the cells must be sterile. Always use proper aseptic technique and work in a laminar flow hood.PATENT DOCKET NO. TP388599WO1• In embodiments, the 293 cells are a robust cell line adapted to high-density growth conditions with a doubling time of approximately 23–24 hours during log phase growth. • For general maintenance of cells, passage the 293 cells when they reach a density of approximately 4–6×106viable cells / mL (i.e., early log-phase growth), typically every 3–4 days. • Cells that are subcultured at densities outside of this early log-phase growth window may show longer doubling times and lower titers over time. Modify the initial seeding density to attain the target cell density of 4–6×106viable cells / mL at the time of subculturing. • For all cell manipulations, mix the cells by gentle swirling; avoid vigorous shaking / pipetting. • Reagents and equipment to determine viable cell density and percent viability are well known and commercially available (for example, without limitation, a hemocytometer with trypan blue or a Vi-Cell™ XR Cell Viability Analyzer (Thermo Fisher Scientific). Log phase cultures should be >95% viable. • When thawing or subculturing cells, transfer cells into pre-warmed medium.

[0227] Guidelines for thawing cells: 1. Add 30 mL of pre-warmed 293 Culture Medium to a 125-mL Erlenmeyer shaker flask. 2. Remove the vial of 293 cells from liquid nitrogen and swirl gently in a 37°C water bath for 1 to 2 minutes to thaw the cells rapidly until only a small amount of ice remains. Do not submerge the vial in the water. 3. Just before the cells are completely thawed, decontaminate the vial with 70% ethanol before opening it in a laminar flow hood. 4. Gently invert the cell vial to mix the contents. Uncap the cell vial and transfer 50 μL of cells into 450 μL of Ca2+ / Mg2+free PBS for viability and viable cell density determination, for example, by trypan blue dye exclusion assay. 5. Trypan blue may interact with components in cell culture media leading to aggregation, that can be misinterpreted as dead cells using typical cell counting instruments and algorithms. Dilution with PBS is not required during routine cell culture maintenance. 6. Using a 1-, 2-, or 5‑mL pipette, gently transfer the remaining cell volume drop wise to the shake flask containing the pre-warmed culture medium prepared in step 1.PATENT DOCKET NO. TP388599WO17. Incubate the cells in in a 37°C incubator with ≥80% relative humidity and 8% CO2 on an orbital shaker platform. The use of non-humidified incubators is not recommended due to the significant loss of volume in the culture flasks by evaporation. 8. Set the shake speed to 125 ±5 rpm for shakers with a 19‑mm shaking diameter, 120 ±5 rpm for shakers with a 25-mm shaking diameter and 95 ±5 rpm for shakers with a 50‑mm shaking diameter. 9. Three to four days post-thaw, determine the viable cell density and percent viability. Cell viability should ≥90% with a viable cell density >1 × 106viable cells / mL. 10. If the viability is <90% on days 3 to 4 post-thaw, cells may be cultured for up to an additional 3 days in order to reach the desired viability. Cells should not be subcultured until viable cell density reaches >1 × 106viable cells / mL. 11. For subsequent routine cell culture maintenance, subculture cells every 3 to 4 days when the viable cell density reaches 4 to 6 × 106viable cells / mL according to Table 2. 12. Do not subculture cells before reaching early log phase growth of ≥4 × 106cells / mL. Similarly, do not let cells overgrow above ≥6.5 × 106cells / mL. Modify the initial seeding density to attain target cell density of 4–6 × 106viable cells / mL at the time of subculturing.

[0228] Guidelines for sub-culturing 293 cells: 1. Determine viable cell density and percentage viability. Use the viable cell density to calculate the volume of cell suspension required to seed a new shake flask according to the recommended seeding densities in Table 2 and the recommended culture volumes in Table 3. Table 4 provides recommended settings for the Vi-Cell™ XR Cell Viability Analyzer. Table 2 Sub-culture timing Recommended seeding densityTable 3 Flask size Culture volume (mL) Shake SpeedPATENT DOCKET NO. TP388599WO1125 mL 30 mL 250 mL 60 mL 125 ± 5 rpm (19mm shaking diameter)Table 4 Parameter Value Parameter Value2. Transfer the calculated volume of cells to fresh, pre-warmed 293 Culture Medium in a shake flask. 3. Incubate flasks in a 37°C incubator with ≥80% relative humidity and 8% CO2 on an orbital shaker platform until cultures reach a density of 4–6×106viable cells / mL. Note: Cells that are subcultured at densities outside of this early log-phase growth window may show longer doubling times and lower titers over time. Modify the initial seeding density to attain the target cell density of 4–6×106viable cells / mL at the time of subculturing.PATENT DOCKET NO. TP388599WO14. Repeat Steps 1–3 to maintain or expand the cells for transfection.

[0229] Transfection and protein expression protocols: 1. Subculture and expand 293 cells until the cells reach a density of approximately 4–6 × 106viable cells / mL. 2. Three days prior to transfection (Day −3), split the 293 cell culture from Step 1 to a final density of 0.8 – 1.0×106viable cells / mL and allow the cells to grow for three days. Alternatively, two days prior to transfection (Day −2), split the 293 cell culture from Step 1 to a final density of 1.6 –1.8×106viable cells / mL and allow the cells to grow for two days. Note: The cells should reach a density of approximately 6.0–7.0×106viable cells / mL for transfection. If they do not, adjust the seeding density. 3. Day 0: Determine viable cell density and percent viability. The cells should have reached a density of approximately 6.0–7.0×106viable cells / mL. Viability should be 95–99% to proceed with transfection. Avoid cells reaching a density above 8.0×106viable cells / mL. 4. Dilute the cells from Step 3 to a final density of 5 × 106viable cells / mL with fresh 293 Culture Medium, pre-warmed to 37°C. Swirl the flasks gently to mix the cells. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2in air on an orbital shaker (for suggested shake speeds, see Table 3). Note: Discard the remaining cells; do not re-use high-density cells for routine subculturing.

[0230] T18 Protocol (T18) 5. Prepare 293 Transfection Reagent / plasmid DNA complex as follows. Refer to Table 5 for volumes. Note: Total plasmid DNA of 1.0 µg per mL of culture volume to be transfected is appropriate for most proteins. One Tube Transfection: a) Gently invert the 293 Transfection Reagent bottle 4–5 times to mix. b) Dilute plasmid DNA with cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801). Mix by swirling the tube and / or by gentle inversion. c) Immediately add the 293 Transfection Reagent to diluted plasmid DNA. Mix by swirling the tube and / or by gentle inversion.PATENT DOCKET NO. TP388599WO1Note: 293 Transfection Reagent diluted in Opti-PlexTMComplexation Buffer / plasmid DNA remains stable for at least 30 minutes which allows for automated processing. OR Two Tube Transfection: a) Gently invert the 293 Transfection Reagent bottle 4–5 times to mix. b) Add plasmid DNA to half volume of cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801) in Tube 1. Mix by swirling the tube and / or by gentle inversion. c) Add 293 Transfection Reagent to half volume of cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801) in Tube 2. Mix by swirling the tube and / or by gentle inversion. Note: 293 Transfection Reagent diluted in Opti-PlexTMComplexation Buffer remains stable for at least 30 minutes before addition to Tube 1 which facilitates automated processing. d) Add volume of Tube 2 to Tube 1. Mix by swirling the tube and / or by gentle inversion. 6. Incubate 293 Transfection Reagent / plasmid DNA complexes (from Step 5c (One Tube) or from Step 5d (Two Tube)) at room temperature for 5 minutes, and then slowly transfer the solution to the starter flask from Step 4, swirling the flask gently during addition. 7. Incubate the cells in a 32°C or 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker (for suggested shake speeds, see Table 3). 8. On the day after transfection (Day 1, 16-18 hours post-transfection), add 293 Enhancer to the flask (see Table 5 for volumes), gently swirling the flask during addition. Return the flask to the 32°C or 37°C incubator with a humidified atmosphere of 8% CO2 with shaking. 9. Optimal time to harvest protein will depend on the specific properties of the proteins being expressed. Typical harvest times range from 4-7 days post transfection for secreted proteins and 2-3 days for membrane and intracellular proteins.

[0231] T0 Protocol (T0) One Tube Transfection: 5. Prepare 293 Transfection Reagent / plasmid DNA complex as follows. Refer to Table 6 for volumes. Note: Total plasmid DNA of 1.0 µg per mL of culture volume to be transfected is appropriate forPATENT DOCKET NO. TP388599WO1most proteins. a) Gently invert the 293 Transfection Reagent bottle 4–5 times to mix. b) Dilute plasmid DNA with cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801). Mix by swirling the tube and / or by gentle inversion. c) Immediately add the 293 Transfection Reagent to diluted plasmid DNA. Mix by swirling the tube and / or by gentle inversion. Note: 293 Transfection Reagent diluted in Opti-PlexTMComplexation Buffer / plasmid DNA remains stable for at least 30 minutes which allows for automated processing. OR Two Tube Transfection: a) Gently invert the 293 Transfection Reagent bottle 4–5 times to mix. b) Add plasmid DNA to half volume of cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801) in Tube 1. Mix by swirling the tube and / or by gentle inversion c) Add 293 Transfection Reagent to half volume of cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801) in Tube 2. Mix by swirling the tube and / or by gentle inversion. Note: 293 Transfection Reagent diluted in Opti-PlexTMComplexation Buffer remains stable for at least 30 minutes before addition to Tube 1 which allows for automated processing. d) Add volume of Tube 2 to Tube 1. Mix by swirling the tube and / or by gentle inversion. 6. Incubate 293 Transfection Reagent / plasmid DNA complexes (from Step 5c (One Tube) or from Step 5d (Two Tube)) at room temperature for 5 minutes, and then slowly transfer the solution to the starter flask from Step 4, swirling the flask gently during addition. 7. Incubate the cells in a 32°C or 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker (for suggested shake speeds, see Table 3). 8. Immediately after transfection, add 293 Enhancer to the flask (see Table 6 for volumes), gently swirling the flask during addition. Incubate the cells in a 32°C or 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker (for suggested shake speeds, see Table 3). 9. Optimal time to harvest protein will depend on the specific properties of the proteins beingPATENT DOCKET NO. TP388599WO1expressed. Typical harvest times range from 4-7 days post transfection for secreted proteins and 2-3 days for membrane and intracellular proteins. Table 5: Recommended volumes for T18 Protocol (T18) transfection at various scales 96 deep 24 deep 125 mL Vessel type well plate well plate flask1 L flask2.8 L flask 5 L flask10LmL L thespecific laboratory equipment used. [2] Assuming a plasmid DNA stock concentration of 1mg / mL and a final concentration of 1.0 μg plasmid DNA per mL. [3] Volume of Opti-PlexTMComplexation Buffer used to dilute plasmid DNA. Table 6: Recommended volumes for T0 Protocol (T0) transfection at various scales 96 deep 24 deep 125 mL kPATENT DOCKET NO. TP388599WO1Number of cells615 × 10615 × 10712 × 1084.09 10required5 × 10× 10 1.0 × 10 L L L the[2] Assuming a plasmid DNA stock concentration of 1mg / mL and a final concentration of 1.0 μg plasmid DNA per mL. [3] Volume of Opti-PlexTMComplexation Buffer used to dilute plasmid DNA.

[0232] Example 2. Comparison of protein production using 293 expression system of the disclosure and conventional Expi293TMExpression System

[0233] Protein production in a transient 293 expression system as provided herein was compared to the commercially available Expi293TMExpression System (Thermo Fisher Scientific; Catalog No. A14525).

[0234] Recombinant monoclonal antibodies and human IgG1 were transiently expressed using the 293 expression system as described herein following the T0 Protocol (T0) described in Example 1 and using the Expi293™ Expression System according to manufacturer protocol (Thermo Fisher Scientific; Catalog No. A14525). Transient transfections were performed with plasmid DNA encoding the desired proteins in pcDNA3.4 expression vector (Thermo Fisher Scientific; Catalog No. A14697). Total plasmid DNA of 1.0 micrograms per mL of cell culture volume was used for thePATENT DOCKET NO. TP388599WO1transfection reactions. It is noted that a linear DNA encoding a desired protein may be used and resultant protein expression is expected to be comparable to use of a circular plasmid DNA encoding a desired protein.

[0235] For the 293 expression system of the present disclosure, a transfection reagent including a cationic lipid (293 Transfection Reagent) and 293 cells were used. The 293 cells were grown to a density of approximately 6.0-7.0 x 106viable cells / ml, with a viability of about 95-99%. These cells were brought to a final density of about 5 x 106viable cells / ml with fresh culture medium and incubated in a 37ºC on an orbital shaker while the transfection reagent / plasmid DNA complex was prepared. Notably, the cells were not split and further incubated the day before transfection to bring the cells to a final density of about 5 x 106viable cells / ml.

[0236] Plasmid DNA encoding the desired recombinant protein was diluted in cold Opti-PlexTMComplexation Buffer (Thermo Fisher Scientific; Catalog No. A4096801) and gently mixed. 293 Transfection Reagent was immediately added to the diluted plasmid DNA and the solution was gently mixed. The 293 Transfection Reagent / plasmid DNA complexes were incubated at room temperature for about 5 minutes and then added to the prepared 293 cells.

[0237] Following the T0 Protocol (T0) described in Example 1, immediately after the transfection, 293 Enhancer was added to the 293 cell culture. The transfected cells were incubated at 37ºC on an orbital shaker. The expressed proteins were harvested at day 7 and quantified by ForteBio OctetTMor ELISA.

[0238] For the commercially available Expi293™ Expression System, according to manufacturer protocol, Expi293F™ cells were grown to a density of approximately 3.0-5.0x106viable cells / ml. On the day prior to transfection (Day -1), the Expi293F cell culture was split to a final density of 2.5- 3x106viable cells / ml and the cells were allowed to grow overnight. On the next day (Day 0), the cells reached a density of approximately 4.5-5.5x106viable cells / ml, with a viability of 95-99%. These cells were brought to a final density of 3x106viable cells / ml with fresh Expi293™ Expression Medium and incubated at 37ºC on an orbital shaker while the transfection reagent / plasmid DNA complex was prepared.

[0239] Plasmid DNA encoding the recombinant protein was diluted in Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific; Catalog No. 31985062) and gently mixed. The transfection reagent of the commercially available Expi293TMExpression System, ExpiFectamine™ 293 Reagent was diluted with Opti-MEM™ I Reduced Serum Medium, gently mixed and incubated at room temperature for 5 minutes. After the 5 minute incubation, the diluted ExpiFectamine™ 293PATENT DOCKET NO. TP388599WO1Reagent was added to the diluted plasmid DNA, and the mixture was gently mixed. The ExpiFectamine™ 293 Reagent / plasmid DNA complexes were incubated at room temperature for 10- 20 minutes and then added to the Expi293F™ cells. The cells were incubated at 37ºC on an orbital shaker.

[0240] On the day after transfection (Day 1, 18-22 hours post-transfection), ExpiFectamine™ 293 Enhancers 1 and 2 of the commercially available Expi293TMExpression System were added to the transfected cell culture and the flask was returned to the 37ºC incubator with shaking. The expressed proteins were harvested at day 7 and quantified by ForteBio OctetTMor ELISA.

[0241] Results

[0242] Exemplary data comparing transient expression of 11 different monoclonal antibodies with the 293 expression system of the present disclosure following the T0 Protocol (T0) with the commercially available Expi293™ Expression System is shown in FIG. 4. Compared to the Expi293™ Expression System, the 293 expression system of the present disclosure generated about equivalent or higher titers of recombinant monoclonal antibodies and human IgG protein in less time following the reduced timeframe T0 Protocol (T0) in which 293 Enhancer is added immediately following transfection. This equivalent or higher protein production was obtained using the present 293 expression system with a simplified and reduced time workflow as compared to that of the commercially available Expi293™ Expression System.

[0243] FIG. 5 shows protein titers of 41 recombinant monoclonal antibodies using the 293 expression system of the present disclosure following the T18 Protocol (T18) and using the commercially available Expi293™ Expression System (same post transfection protein harvest time). An overall average increase of protein titers of greater than 4X was observed using the 293 expression system of the present disclosure as compared to the commercially available Expi293™ Expression System with production of over 70% of the antibodies being greater than 1 g / L.

[0244] Exemplary data comparing transient expression of secreted non-antibodies using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.6 (same post transfection protein harvest time). As shown in FIG.6, the 293 expression system of the present disclosure increased expression of a diverse array of secreted non-antibodies by an average of 10X, with improvements up to 20X as compared to the commercially available Expi293™ Expression System.

[0245] Exemplary data comparing transient expression of secreted antibodies using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commerciallyPATENT DOCKET NO. TP388599WO1available Expi293™ Expression System is shown in FIG.7 (same post transfection protein harvest time). As shown in FIG.7, the 293 expression system of the present disclosure increased expression of secreted antibodies by an average of 4X as compared to the commercially available Expi293™ Expression System.

[0246] Exemplary data comparing transient expression of membrane proteins using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.8 (same post transfection protein harvest time). As shown in FIG.8, the 293 expression system of the present disclosure increased expression of membrane proteins by an average of 4X as compared to the commercially available Expi293™ Expression System.

[0247] Exemplary data comparing transient expression of difficult-to-express proteins (chimeric antibodies) using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.9 (same post transfection protein harvest time). As shown in FIG. 9, the 293 expression system of the present disclosure increased expression of chimeric antibodies up to 11X as compared to the commercially available Expi293™ Expression System.

[0248] Exemplary data comparing transient expression of secreted viral proteins using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.10 (same post transfection protein harvest time). As shown in FIG.10, the 293 expression system of the present disclosure increased expression of secreted viral proteins by an average of 6X, with improvements up to 11X, as compared to the commercially available Expi293™ Expression System.

[0249] Exemplary data comparing transient expression of intracellular proteins using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.11 (same post transfection protein harvest time). As shown in FIG.11, the 293 expression system of the present disclosure increased expression of intracellular proteins by an average of 3.5X as compared to the commercially available Expi293™ Expression System.

[0250] Exemplary data comparing transient expression of extracellular domain proteins using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.12 (same post transfection protein harvest time). As shown in FIG. 12, the 293 expression system of the present disclosurePATENT DOCKET NO. TP388599WO1increased expression of extracellular domain proteins by an average of 13X as compared to the commercially available Expi293™ Expression System, and a canine IgG protein by 11X as compared to the commercially available Expi293™ Expression System.

[0251] Exemplary data comparing transient expression of different protein classes using the 293 expression system of the present disclosure following the T18 Protocol (T18) with the commercially available Expi293™ Expression System is shown in FIG.13 (same post transfection protein harvest time).

[0252] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In case of conflict, the specification herein, including definitions, will control. Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.

[0253] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description. While certain embodiments and details have been included herein and in the attached disclosure for purposes of illustrating embodiments of the present disclosure, it will be apparent to those skilled in the art that various changes in the systems, methods, compositions, and kits disclosed herein may be made without departing from the scope of the disclosure or of the disclosure, which is defined in the appended claims. All changes which come within the meaning and range of equivalency of the following claims are to be embraced within their scope.

[0254] Some embodiments of the disclosure are represented by the following clauses:

[0255] Clause 1. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2x106to about 2x107cells / ml; incubating the transfected 293 cells for a first period of time, the first period of time being less than about 2 hours; contacting the transfected 293 cells with an expression enhancer composition; andPATENT DOCKET NO. TP388599WO1culturing the transfected 293 cells in the presence of the expression enhancer composition for a second period of time, wherein the recombinant protein is expressed.

[0256] Clause 2. The method of clause 1, wherein the suspension culture is obtained by culturing the 293 cells without splitting the cells.

[0257] Clause 3. The method of any one of clauses 1 or 2, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106to about 2x107cells / ml before transfecting.

[0258] Clause 4. The method of clause 3, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, incubating the 293 cells for the first period of time, and contacting the transfected 293 cells with the expression enhancer composition is completed in less than about 7-12 days.

[0259] Clause 5. The method of clause 1, wherein the first period of time is less than about 30, 15, 5 or 1 minute.

[0260] Clause 6. The method of clause 1, wherein the second period of time is about 10 hours to about 10 days.

[0261] Clause 7. The method of clause 1, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

[0262] Clause 8. The method of clause 7, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting the nucleic acid with the transfection reagent; or (iii) diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

[0263] Clause 9. The method of clause 8, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

[0264] Clause 10. The method of any one of clauses 1-9, wherein transfecting the 293 cells is performed using an automated robotic assembly.

[0265] Clause 11. The method of any one of clauses 1-10, wherein transfecting the 293 cells comprises providing the 293 cells in a multi-well format and contacting the 293 cells with the nucleic acid.

[0266] Clause 12. The method of any one of clauses 1-11, wherein the method is automated.PATENT DOCKET NO. TP388599WO1

[0267] Clause 13. The method of clause 1, further comprising harvesting transfected 293 cells.

[0268] Clause 14. The method of clause 1, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

[0269] Clause 15. The method of clause 1, wherein the 293 cells have been adapted for growth under high density conditions.

[0270] Clause 16. The method of clause 15, wherein the 293 cells are 293 F cells.

[0271] Clause 17. The method of clause 1, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

[0272] Clause 18. The method of clause 17, wherein the transfection reagent comprising a cationic lipid and a peptide.

[0273] Clause 19. The method of any one of clauses 1-18, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

[0274] Clause 20. The method of clause 19, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

[0275] Clause 21. The method of clause 19, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0276] Clause 22. The method of clause 21, wherein the expression enhancer composition comprises valproic acid.

[0277] Clause 23. The method of clause 22, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM.

[0278] Clause 24. The method of clause 22, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

[0279] Clause 25. The method of clause 19, wherein the expression enhancer composition comprises sodium propionate.

[0280] Clause 26. The method of clause 25, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

[0281] Clause 27. The method of clause 25, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.PATENT DOCKET NO. TP388599WO1

[0282] Clause 28. The method of clause 21, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

[0283] Clause 29. The method of clause 1, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

[0284] Clause 30. The method of clause 1, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

[0285] Clause 31. The method of clause 1, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

[0286] Clause 32. The method of clause 1, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.

[0287] Clause 33. The method of clause 1, wherein the cell density of the transfection step is between about 3x106to about 20x106cells / ml.

[0288] Clause 34. The method of clause 1, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

[0289] Clause 35. The method of clause 1, wherein the high density culture medium is a serum- free / protein-free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

[0290] Clause 36. The method of clause 1, further comprising isolating the expressed recombinant protein.

[0291] Clause 37. The method of clause 1, wherein the 293 cells express one or more expression enhancing proteins.

[0292] Clause 38. The method of clause 37, wherein the 293 cells transiently express one or more expression enhancing proteins.

[0293] Clause 39. The method of clause 37, wherein the 293 cells stably express one or more expression enhancing proteins.

[0294] Clause 40. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2x106to about 2x107cells / ml;PATENT DOCKET NO. TP388599WO1contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0295] Clause 41. The method of clause 40, wherein the suspension culture is obtained by culturing the 293 cells without splitting the cells.

[0296] Clause 42. The method of any one of clauses 40 or 41, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106 to about 2x107 cells / ml before transfecting.

[0297] Clause 43. The method of clause 42, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, and contacting the transfected 293 cells with expression enhancer composition is completed in less than about 7-12 days.

[0298] Clause 44. The method of clause 40, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 1 to about 5 minutes after transfecting.

[0299] Clause 45. The method of clause 44, wherein the period of time is about 10 hours to about 10 days.

[0300] Clause 46. The method of clause 40, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

[0301] Clause 47. The method of clause 46, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting the nucleic acid with the transfection reagent; (iii) or diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

[0302] Clause 48. The method of clause 47, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

[0303] Clause 49. The method of any one of clauses 40-48, wherein transfecting the 293 cells is performed using an automated robotic assembly.PATENT DOCKET NO. TP388599WO1

[0304] Clause 50. The method of any one of clauses 40-49, wherein transfecting the 293 cells comprises providing the 293 cells in a multi-well format and contacting the 293 cells with the nucleic acid.

[0305] Clause 51. The method of any one of clauses 40-50, wherein the method is automated.

[0306] Clause 52. The method of clause 40, further comprising harvesting transfected 293 cells.

[0307] Clause 53. The method of clause 40, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

[0308] Clause 54. The method of clause 40, wherein the 293 cells have been adapted for growth under high density conditions.

[0309] Clause 55. The method of clause 54, wherein the 293 cells are 293 F cells.

[0310] Clause 56. The method of clause 40, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

[0311] Clause 57. The method of clause 56, wherein the transfection reagent comprising a cationic lipid and a peptide.

[0312] Clause 58. The method of any one of clauses 40-57, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

[0313] Clause 59. The method of clause 58, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

[0314] Clause 60. The method of clause 58, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0315] Clause 61. The method of clause 60, wherein the expression enhancer composition comprises valproic acid.

[0316] Clause 62. The method of clause 61, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM.

[0317] Clause 63. The method of clause 61, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

[0318] Clause 64. The method of clause 58, wherein the expression enhancer composition comprises sodium propionate.PATENT DOCKET NO. TP388599WO1

[0319] Clause 65. The method of clause 64, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

[0320] Clause 66. The method of clause 64, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.

[0321] Clause 67. The method of clause 58, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

[0322] Clause 68. The method of clause 40, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

[0323] Clause 69. The method of clause 40, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

[0324] Clause 70. The method of clause 40, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

[0325] Clause 71. The method of clause 40, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.

[0326] Clause 72. The method of clause 40, wherein the cell density of the transfection step is between about 3x106to about 20x106cells / ml.

[0327] Clause 73. The method of clause 40, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

[0328] Clause 74. The method of clause 40, wherein the high density culture medium is a serum- free / protein-free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

[0329] Clause 75. The method of clause 40, further comprising purifying the expressed recombinant protein.

[0330] Clause 76. The method of clause 40, wherein the cells express one or more expression enhancing proteins.

[0331] Clause 77. The method of clause 76, wherein the cells transiently express one or more expression enhancing proteins.

[0332] Clause 78. The method of clause 76, wherein the cells stably express one or more expression enhancing proteins.PATENT DOCKET NO. TP388599WO1

[0333] Clause 79. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2x106to about 2x107cells / ml, and wherein the 293 cells are transfected using an automated robotic assembly; contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0334] Clause 80. The method of clause 79, wherein the 293 cells are obtained by culturing the 293 cells without splitting the cells.

[0335] Clause 81. The method of any one of clauses 79 or 80, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106to about 2x107cells / ml before transfecting.

[0336] Clause 82. The method of clause 81, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, and contacting the transfected 293 cells with the expression enhancer composition is completed in less than about 7-12 days.

[0337] Clause 83. The method of clause 79, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 1 to about 5 minutes after transfecting.

[0338] Clause 84. The method of clause 83, wherein the period of time is about 10 hours to about 10 days.

[0339] Clause 85. The method of clause 79, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

[0340] Clause 86. The method of clause 85, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting thePATENT DOCKET NO. TP388599WO1nucleic acid with the transfection reagent; or (iii) diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

[0341] Clause 87. The method of clause 86, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

[0342] Clause 88. The method of any one of clauses 79-88, wherein the method is automated.

[0343] Clause 89. The method of clause 79, further comprising harvesting transfected 293 cells.

[0344] Clause 90. The method of clause 79, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

[0345] Clause 91. The method of clause 79, wherein the 293 cells are provided in a multi-well format before transfecting.

[0346] Clause 92. The method of clause 83, wherein the 293 cells are 293 F cells.

[0347] Clause 93. The method of clause 79, wherein transfecting the 293 cells comprises contacting the 293 cells with a transfection reagent comprising a cationic lipid or peptide.

[0348] Clause 94. The method of clauses 93, wherein the transfection reagent comprising a cationic lipid and a peptide.

[0349] Clause 95. The method of any one of clauses 79-94, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

[0350] Clause 96. The method of clause 95, wherein the HDAC inhibitor is selected from apicidin,

[0351] belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

[0352] Clause 97. The method of clause 95, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0353] Clause 98. The method of clause 97, wherein the expression enhancer composition comprises valproic acid.

[0354] Clause 99. The method of clause 98, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM.

[0355] Clause 100. The method of clause 98, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

[0356] Clause 101. The method of clause 95, wherein the expression enhancer composition comprises sodium propionate.PATENT DOCKET NO. TP388599WO1

[0357] Clause 102. The method of clause 101, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

[0358] Clause 103. The method of clause 101, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.

[0359] Clause 104. The method of clause 95, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

[0360] Clause 105. The method of clause 79, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

[0361] Clause 106. The method of clause 79, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

[0362] Clause 107. The method of clause 79, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

[0363] Clause 108. The method of clause 79, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.

[0364] Clause 109. The method of clause 79, wherein the cell density of the transfection step is between about 3x106to about 20x106cells / ml.

[0365] Clause 110. The method of clause 79, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

[0366] Clause 111. The method of clause 79, wherein the high density culture medium is a serum- free / protein-free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

[0367] Clause 112. The method of clause 79, further comprising purifying the expressed recombinant protein.

[0368] Clause 113. The method of clause 79, wherein the cells express one or more expression enhancing proteins.

[0369] Clause 114. The method of clause 113, wherein the cells transiently express one or more expression enhancing proteins.

[0370] Clause 115. The method of clause 113, wherein the cells stably express one or more expression enhancing proteins.PATENT DOCKET NO. TP388599WO1

[0371] Clause 116. The method of any preceding clause, wherein the recombinant protein is a difficult-to-express protein (DEP).

[0372] Clause 117. The method of clause 116, wherein the DEP is a membrane protein, antibody, enzyme, toxoid, viral protein or multidomain protein.

[0373] Clause 118. A recombinant protein production system comprising: 293 cells; a cell culture medium capable of supporting high density suspension culture of the 293 cells with a cell density of between about 2x106to about 2x107cells / ml; a transfection reagent; and an expression enhancer composition, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 150 mM and / or sodium proprionate at a concentration of at least about 1500 mM.

[0374] Clause 119. The system of clause 118, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 175 mM, 200 mM, 225 mM or 250 mM.

[0375] Clause 120. The system of clause 118, wherein the expression enhancer composition comprises sodium proprionate at a concentration of at least about 1750 mM, 2000 mM or 2200 mM.

[0376] Clause 121. The system of clause 118, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 200 mM and sodium proprionate at a concentration of at least about 2000 mM.

[0377] Clause 122. The system of any one of the preceding clauses, further comprising glucose.

[0378] Clause 123. The system of clause 122, wherein the glucose is at a concentration of at least about 600 mM, 700 mM or 800 mM.

[0379] Clause 124. The system of any one of the preceding clauses, wherein the transfection reagent comprises a cationic lipid or a peptide.

[0380] Clause 125. The system of clause 124, wherein the transfection reagent further comprises a cationic lipid and a peptide.

[0381] Clause 126. The system of any one of the preceding clauses, wherein the system further comprises a complexation buffer.

[0382] Clause 127. The system of any one of the preceding clauses, wherein the system further comprises a nucleic acid capable of expressing a recombinant protein.

[0383] Clause 128. A suspension culture composition comprising: 293 cells in a suspension culture having a high density culture medium and having a cellPATENT DOCKET NO. TP388599WO1density of between about 2x106to about 2x107cells / ml; and glucose at a concentration of greater than about 55 mM, 60 mM, 65 mM or 70 mM in the suspension culture.

[0384] Clause 129. The composition of clause 128, further comprising a transfection complex.

[0385] Clause 130. The composition of clause 128 or clause 129, further comprising an expression enhancer.

[0386] Clause 131. The composition of claim 129 or clause 130, wherein the transfection complex is generated by a transfection reagent comprising a cationic lipid or peptide.

[0387] Clause 132. The composition of clauses 131, wherein the transfection reagent comprises a cationic lipid and a peptide.

[0388] Clause 133. The composition of any one of clauses 128-132, wherein the expression enhancer comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), galactose, an amino acid, butyric acid, or any salts or any combinations thereof.

[0389] Clause 134. The composition of clause 133, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

[0390] Clause 135. The composition of clause 134, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0391] Clause 136. The composition of clause 135, wherein the expression enhancer comprises valproic acid.

[0392] Clause 137. The composition of clause 135, wherein the concentration of valproic acid is at least about 1.0 mM.

[0393] Clause 138. The composition of clause 133, wherein the expression enhancer comprises sodium propionate.

[0394] Clause 139. The composition of clause 138, wherein the concentration of sodium proprionate is at least about 10 mM.

[0395] Clause 140. The composition of clause 128, wherein the expression enhancer comprises valproic acid and sodium proprionate.

[0396] Clause 141. The composition of clause 140, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM.

[0397] Clause 142. A suspension culture composition comprising:PATENT DOCKET NO. TP388599WO1293 cells in a suspension culture having a high density culture medium and having a cell density of between about 2x106to about 2x107cells / ml; and an expression enhancer, wherein the expression enhancer comprises valproic acid at a concentration of at least about 1.0 mM and / or sodium proprionate at a concentration of at least about 10 mM.

[0398] Clause 143. The composition of clause 142, wherein the expression enhancer comprises valproic acid and sodium proprionate.

[0399] Clause 144. The composition of clause 143, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM.

[0400] Clause 145. The composition of any one of clauses 142-144, further comprising glucose at a concentration of greater than about 55 mM, 60 mM, 65 mM or 70 mM.

[0401] Clause 146. The composition of any one of clauses 142-145, further comprising a transfection complex.

[0402] Clause 147. The composition of clause 146, wherein the transfection complex is generated using a transfection reagent comprising a cationic lipid or peptide.

[0403] Clause 148. The composition of clause 147, wherein the transfection reagent comprises a cationic lipid and a peptide.

[0404] Clause 149. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2x106to about 2x107cells / ml; contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 24 hours after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

[0405] Clause 150. The method of clause 149, wherein the transfected 293 cells are contacted with the expression enhancer composition about 16-22 hours after transfecting.

[0406] Clause 151. The method of clause 149, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,PATENT DOCKET NO. TP388599WO113, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 hour, or wherein the first period of time is less than about 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or about 1 minute.

[0407] Clause 152. The method of clause 149, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

[0408] Clause 153. The method of clause 152, wherein the transfection reagent comprising a cationic lipid and a peptide.

[0409] Clause 154. The method of any one of clauses 149-153, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

[0410] Clause 155. The method of clause 154, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

[0411] Clause 156. The method of clause 154, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0412] Clause 157. The method of clause 156, wherein the expression enhancer composition comprises valproic acid.

[0413] Clause 158. The method of clause 157, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM before contacting the transfected 293 cells.

[0414] Clause 159. The method of clause 157, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM before contacting the transfected 293 cells.

[0415] Clause 160. The method of clause 154, wherein the expression enhancer composition comprises sodium propionate.

[0416] Clause 161. The method of clause 160, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M before contacting the transfected 293 cells.

[0417] Clause 162. The method of clause 160, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M before contacting the transfected 293 cells.PATENT DOCKET NO. TP388599WO1

[0418] Clause 163. The method of clause 155, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM before contacting the transfected 293 cells and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M before contacting the transfected 293 cells.

[0419] Clause 164. The method of any one of the preceding clauses, wherein the expression enhancer composition further comprises glucose.

[0420] Clause 165. The method of clause 164, wherein the glucose is at a concentration of at least about 600 mM, 700 mM or 800 mM in the expression enhancer composition before contacting the transfected 293 cells.

[0421] Clause 166. The method of clause 164 or clause 165, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM during culturing of the transfected 293 cells.

[0422] Clause 167. The method of clause 166, wherein the concentration of glucose is greater than about 55 mM, 60 mM, 65 mM or 70 mM during culturing of the transfected 293 cells.

[0423] Clause 168. The method of any one of clauses 149-167, wherein transfecting is automated.

[0424] Clause 169. The method of clause 168, wherein transfecting and contacting the transfected 293 cells with the expression enhancer is automated.

[0425] Clause 170. The method of clause 169, wherein transfecting and / or contacting the transfected 293 cells with the expression enhancer is performed using a robotic assembly.

Claims

PATENT DOCKET NO. TP388599WO1CLAIMS WHAT IS CLAIMED IS:

1. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2x106to about 2x107cells / ml; incubating the transfected 293 cells for a first period of time, the first period of time being less than about 24 hours; contacting the transfected 293 cells with an expression enhancer composition; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a second period of time, wherein the recombinant protein is expressed.

2. The method of claim 1, wherein the suspension culture is obtained by culturing the 293 cells without splitting the cells.

3. The method of any one of claims 1 or 2, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106to about 2x107cells / ml before transfecting.

4. The method of claim 3, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, incubating the 293 cells for the first period of time, contacting the transfected 293 cells with the expression enhancer composition, and incubating the transfected 293 cells is completed in less than about 2-12 days.

5. The method of claim 1, wherein the first period of time is less than about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 hour, or wherein the first period of time is less than about 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or about 1 minute.PATENT DOCKET NO. TP388599WO16. The method of claim 1, wherein the second period of time is about 10 hours to about 10 days.

7. The method of claim 1, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

8. The method of claim 7, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting the nucleic acid with the transfection reagent; or (iii) diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

9. The method of claim 8, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

10. The method of any one of claims 1-9, wherein transfecting the 293 cells is performed using an automated robotic assembly.

11. The method of any one of claims 1-10, wherein transfecting the 293 cells comprises providing the 293 cells in a multi-well format and contacting the 293 cells with the nucleic acid.

12. The method of any one of claims 1-11, wherein transfecting, incubating and / or contacting is automated.

13. The method of claim 1, further comprising harvesting transfected 293 cells.

14. The method of claim 1, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

15. The method of claim 1, wherein the 293 cells have been adapted for growth under high density conditions.PATENT DOCKET NO. TP388599WO116. The method of claim 15, wherein the 293 cells are 293 F cells.

17. The method of claim 1, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

18. The method of claim 17, wherein the transfection reagent comprising a cationic lipid and a peptide.

19. The method of any one of claims 1-18, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

20. The method of claim 19, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

21. The method of claim 19, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

22. The method of claim 21, wherein the expression enhancer composition comprises valproic acid.

23. The method of claim 22, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM.

24. The method of claim 22, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

25. The method of claim 19, wherein the expression enhancer composition comprises sodium propionate.PATENT DOCKET NO. TP388599WO126. The method of claim 25, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

27. The method of claim 25, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.

28. The method of claim 21, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

29. The method of claim 1, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

30. The method of claim 1, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

31. The method of claim 1, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

32. The method of claim 1, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.

33. The method of claim 1, wherein the cell density of the transfection step is between about 3x106to about 20x106cells / ml.

34. The method of claim 1, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

35. The method of claim 1, wherein the high density culture medium is a serum-free / protein- free chemically defined culture medium capable of promoting the growth of transfected cells at cellPATENT DOCKET NO. TP388599WO1densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

36. The method of claim 1, further comprising isolating the expressed recombinant protein.

37. The method of claim 1, wherein the 293 cells express one or more expression enhancing proteins.

38. The method of claim 37, wherein the 293 cells transiently express one or more expression enhancing proteins.

39. The method of claim 37, wherein the 293 cells stably express one or more expression enhancing proteins.

40. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, the suspension culture having a cell density of between about 2x106to about 2x107cells / ml; contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

41. The method of claim 40, wherein the suspension culture is obtained by culturing the 293 cells without splitting the cells.

42. The method of any one of claims 40 or 41, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106to about 2x107cells / ml before transfecting.PATENT DOCKET NO. TP388599WO143. The method of claim 42, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, contacting the transfected 293 cells with expression enhancer composition, and incubating the transfected 293 cells is completed in less than about 2-12 days.

44. The method of claim 40, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 1 to about 5 minutes after transfecting.

45. The method of claim 44, wherein the period of time is about 10 hours to about 10 days.

46. The method of claim 40, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

47. The method of claim 46, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting the nucleic acid with the transfection reagent; (iii) or diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

48. The method of claim 47, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

49. The method of any one of claims 40-48, wherein transfecting the 293 cells is performed using an automated robotic assembly.

50. The method of any one of claims 40-49, wherein transfecting the 293 cells comprises providing the 293 cells in a multi-well format and contacting the 293 cells with the nucleic acid.

51. The method of any one of claims 40-50, wherein the transfecting and / or contacting is automated.PATENT DOCKET NO. TP388599WO152. The method of claim 40, further comprising harvesting transfected 293 cells.

53. The method of claim 40, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

54. The method of claim 40, wherein the 293 cells have been adapted for growth under high density conditions.

55. The method of claim 54, wherein the 293 cells are 293 F cells.

56. The method of claim 40, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

57. The method of claim 56, wherein the transfection reagent comprising a cationic lipid and a peptide.

58. The method of any one of claims 40-57, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

59. The method of claim 58, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

60. The method of claim 58, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

61. The method of claim 60, wherein the expression enhancer composition comprises valproic acid.

62. The method of claim 61, wherein the concentration of valproic acid in the expressionPATENT DOCKET NO. TP388599WO1enhancer composition is in the range of about 0.20 mM to about 500 mM.

63. The method of claim 61, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

64. The method of claim 58, wherein the expression enhancer composition comprises sodium propionate.

65. The method of claim 64, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

66. The method of claim 64, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.

67. The method of claim 58, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

68. The method of claim 40, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

69. The method of claim 40, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

70. The method of claim 40, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

71. The method of claim 40, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.

72. The method of claim 40, wherein the cell density of the transfection step is between aboutPATENT DOCKET NO. TP388599WO13x106to about 20x106cells / ml.

73. The method of claim 40, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

74. The method of claim 40, wherein the high density culture medium is a serum-free / protein- free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

75. The method of claim 40, further comprising purifying the expressed recombinant protein.

76. The method of claim 40, wherein the cells express one or more expression enhancing proteins.

77. The method of claim 76, wherein the cells transiently express one or more expression enhancing proteins.

78. The method of claim 76, wherein the cells stably express one or more expression enhancing proteins.

79. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2x106to about 2x107cells / ml, and wherein the 293 cells are transfected using an automated robotic assembly; contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 15 minutes after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.PATENT DOCKET NO. TP388599WO180. The method of claim 79, wherein the 293 cells are obtained by culturing the 293 cells without splitting the cells.

81. The method of any one of claims 79 or 80, wherein the suspension culture is obtained from frozen 293 cells, and wherein the frozen 293 cells are thawed, suspended in the culture medium and cultured in suspension to a cell density of between about 2x106to about 2x107cells / ml before transfecting.

82. The method of claim 81, wherein obtaining the suspension culture from frozen 293 cells, culturing the 293 cells in suspension, transfecting the 293 cells, contacting the transfected 293 cells with the expression enhancer composition, and incubating the transfected 293 cells is completed in less than about 2-12 days.

83. The method of claim 79, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 1 to about 5 minutes after transfecting.

84. The method of claim 83, wherein the period of time is about 10 hours to about 10 days.

85. The method of claim 79, wherein transfecting comprises contacting the nucleic acid with a transfection reagent to form a transfection complex before contacting the nucleic acid with the 293 cells.

86. The method of claim 85, wherein transfecting further comprises: (i) diluting the nucleic acid with a complexation buffer before contacting the nucleic acid with the transfection reagent; (ii) diluting the transfection reagent with the complexation buffer before contacting the nucleic acid with the transfection reagent; or (iii) diluting the transfection reagent and the nucleic acid with the complexation buffer before contacting the nucleic acid with the transfection reagent.

87. The method of claim 86, wherein the transfection reagent remains stable for at least about 2 hours in the complexation buffer before contacting with the nucleic acid.

88. The method of any one of claims 79-88, wherein the method is automated.PATENT DOCKET NO. TP388599WO189. The method of claim 79, further comprising harvesting transfected 293 cells.

90. The method of claim 79, wherein the high density culture medium is not replaced, replenished, or supplemented with fresh media following transfection.

91. The method of claim 79, wherein the 293 cells are provided in a multi-well format before transfecting.

92. The method of claim 83, wherein the 293 cells are 293 F cells.

93. The method of claim 79, wherein transfecting the 293 cells comprises contacting the 293 cells with a transfection reagent comprising a cationic lipid or peptide.

94. The method of claims 93, wherein the transfection reagent comprising a cationic lipid and a peptide.

95. The method of any one of claims 79-94, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid, or any salts or any combinations thereof.

96. The method of claim 95, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

97. The method of claim 95, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

98. The method of claim 97, wherein the expression enhancer composition comprises valproic acid.PATENT DOCKET NO. TP388599WO199. The method of claim 98, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM.

100. The method of claim 98, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM.

101. The method of claim 95, wherein the expression enhancer composition comprises sodium propionate.

102. The method of claim 101, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M.

103. The method of claim 101, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M.

104. The method of claim 95, wherein the expression enhancer composition comprises a combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M.

105. The method of claim 79, wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L.

106. The method of claim 79, wherein the volume of the suspension culture is in the range of about 25 mL to about 50 L.

107. The method of claim 79, wherein the volume of the suspension culture is in the range of about 100 mL to about 1 L.

108. The method of claim 79, wherein the volume of the suspension culture is in the range of about 200 mL to about 500 mL.PATENT DOCKET NO. TP388599WO1109. The method of claim 79, wherein the cell density of the transfection step is between about 3x106to about 20x106cells / ml.

110. The method of claim 79, wherein the cell density of the transfection step is in the range of about 2x106to about 6x106.

111. The method of claim 79, wherein the high density culture medium is a serum-free / protein- free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5x106cells / ml with cell viability remaining in excess of 80%.

112. The method of claim 79, further comprising purifying the expressed recombinant protein.

113. The method of claim 79, wherein the cells express one or more expression enhancing proteins.

114. The method of claim 113, wherein the cells transiently express one or more expression enhancing proteins.

115. The method of claim 113, wherein the cells stably express one or more expression enhancing proteins.

116. The method of any preceding claim, wherein the recombinant protein is a difficult-to- express protein (DEP).

117. The method of claim 116, wherein the DEP is a membrane protein, antibody, enzyme, toxoid, viral protein or multidomain protein.

118. A recombinant protein production system comprising: 293 cells; a cell culture medium capable of supporting high density suspension culture of the 293 cells with a cell density of between about 2x106to about 2x107cells / ml; a transfection reagent; andPATENT DOCKET NO. TP388599WO1an expression enhancer composition, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 150 mM and / or sodium proprionate at a concentration of at least about 1500 mM.

119. The system of claim 118, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 175 mM, 200 mM, 225 mM or 250 mM.

120. The system of claim 118, wherein the expression enhancer composition comprises sodium proprionate at a concentration of at least about 1750 mM, 2000 mM or 2200 mM.

121. The system of claim 118, wherein the expression enhancer composition comprises valproic acid at a concentration of at least about 200 mM and sodium proprionate at a concentration of at least about 2000 mM.

122. The system of any one of the preceding claims, further comprising glucose.

123. The system of claim 122, wherein the glucose is at a concentration of at least about 600 mM, 700 mM or 800 mM.

124. The system of any one of the preceding claims, wherein the transfection reagent comprises a cationic lipid or a peptide.

125. The system of claim 124, wherein the transfection reagent further comprises a cationic lipid and a peptide.

126. The system of any one of the preceding claims, wherein the system further comprises a complexation buffer.

127. The system of any one of the preceding claims, wherein the system further comprises a nucleic acid capable of expressing a recombinant protein.

128. A suspension culture composition comprising:PATENT DOCKET NO. TP388599WO1293 cells in a suspension culture having a high density culture medium and having a cell density of between about 2x106to about 2x107cells / ml; and glucose at a concentration of greater than about 55 mM, 60 mM, 65 mM or 70 mM in the suspension culture.

129. The composition of claim 128, further comprising a transfection complex.

130. The composition of claim 128 or 129, further comprising an expression enhancer.

131. The composition of claim 129 or 130, wherein the transfection complex is generated by a transfection reagent comprising a cationic lipid or peptide.

132. The composition of claims 131, wherein the transfection reagent comprises a cationic lipid and a peptide.

133. The composition of any one of claims 128-132, wherein the expression enhancer comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), galactose, an amino acid, butyric acid, or any salts or any combinations thereof.

134. The composition of claim 133, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

135. The composition of claim 134, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

136. The composition of claim 135, wherein the expression enhancer comprises valproic acid.

137. The composition of claim 135, wherein the concentration of valproic acid is at least about 1.0 mM.PATENT DOCKET NO. TP388599WO1138. The composition of claim 133, wherein the expression enhancer comprises sodium propionate.

139. The composition of claim 138, wherein the concentration of sodium proprionate is at least about 10 mM.

140. The composition of claim 128, wherein the expression enhancer comprises valproic acid and sodium proprionate.

141. The composition of claim 140, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM.

142. A suspension culture composition comprising: 293 cells in a suspension culture having a high density culture medium and having a cell density of between about 2x106to about 2x107cells / ml; and an expression enhancer, wherein the expression enhancer comprises valproic acid at a concentration of at least about 1.0 mM and / or sodium proprionate at a concentration of at least about 10 mM in the suspension culture.

143. The composition of claim 142, wherein the expression enhancer comprises valproic acid and sodium proprionate.

144. The composition of claim 143, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM.

145. The composition of any one of claims 142-144, further comprising glucose at a concentration of greater than about 55 mM, 60 mM, 65 mM or 70 mM.

146. The composition of any one of claims 142-145, further comprising a transfection complex.

147. The composition of claim 146, wherein the transfection complex is generated using a transfection reagent comprising a cationic lipid or peptide.PATENT DOCKET NO. TP388599WO1148. The composition of claim 147, wherein the transfection reagent comprises a cationic lipid and a peptide.

149. A method for producing a recombinant protein in cultured 293 cells, the method comprising: transfecting 293 cells in a suspension culture having a high density culture medium with a nucleic acid capable of expressing a recombinant protein, wherein the suspension culture has a cell density of between about 2x106to about 2x107cells / ml; contacting the transfected 293 cells with an expression enhancer composition, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 24 hours after transfecting; and culturing the transfected 293 cells in the presence of the expression enhancer composition for a period of time, wherein the recombinant protein is expressed.

150. The method of claim 149, wherein the transfected 293 cells are contacted with the expression enhancer composition about 16-22 hours after transfecting.

151. The method of claim 149, wherein the transfected 293 cells are contacted with the expression enhancer composition less than about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 hour, or wherein the first period of time is less than about 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or about 1 minute.

152. The method of claim 149, wherein the 293 cells are transfected with a transfection reagent comprising a cationic lipid or peptide.

153. The method of claim 152, wherein the transfection reagent comprising a cationic lipid and a peptide.

154. The method of any one of claims 149-153, wherein the expression enhancer composition comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium proprionate, caffeine, lithium acetate, dimethyl sulfoxide (DMSO), glucose, galactose, amino acid mixtures, butyric acid,PATENT DOCKET NO. TP388599WO1or any salts or any combinations thereof.

155. The method of claim 154, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

156. The method of claim 154, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

157. The method of claim 156, wherein the expression enhancer composition comprises valproic acid.

158. The method of claim 157, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 0.20 mM to about 500 mM before contacting the transfected 293 cells.

159. The method of claim 157, wherein the concentration of valproic acid in the expression enhancer composition is in the range of about 200 mM to about 300 mM before contacting the transfected 293 cells.

160. The method of claim 154, wherein the expression enhancer composition comprises sodium propionate.

161. The method of claim 160, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.2 M to about 50 M before contacting the transfected 293 cells.

162. The method of claim 160, wherein the concentration of sodium proprionate in the expression enhancer composition is in the range of about 0.5 M to about 10 M before contacting the transfected 293 cells.

163. The method of claim 155, wherein the expression enhancer composition comprises aPATENT DOCKET NO. TP388599WO1combination of valproic acid and sodium proprionate, and wherein the concentration of valproic acid is in the range of about 200 mM to about 300 mM before contacting the transfected 293 cells and the concentration of sodium proprionate is in the range of about 0.5 M to about 10 M before contacting the transfected 293 cells.

164. The method of any one of the preceding claims, wherein the expression enhancer composition further comprises glucose.

165. The method of claim 164, wherein the glucose is at a concentration of at least about 600 mM, 700 mM or 800 mM in the expression enhancer composition before contacting the transfected 293 cells.

166. The method of claim 164 or claim 165, wherein the concentration of valproic acid is at least about 1.0 mM and the concentration of sodium proprionate is at least about 10 mM during culturing of the transfected 293 cells.

167. The method of claim 166, wherein the concentration of glucose is greater than about 55 mM, 60 mM, 65 mM or 70 mM during culturing of the transfected 293 cells.

168. The method of any one of claims 149-167, wherein transfecting is automated.

169. The method of claim 168, wherein transfecting and contacting the transfected 293 cells with the expression enhancer is automated.

170. The method of claim 169, wherein transfecting and / or contacting the transfected 293 cells with the expression enhancer is performed using a robotic assembly.

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