WO2020086408A1 - A high-yield perfusion-based transient gene expression bioprocess - Google Patents

Abstract

High efficiency methods are disclosed for producing a heterologous protein using transient gene expression in host cells. The heterologous protein can then be isolated from the host cells.

Description

A HIGH-YIELD PERFUSION-BASED TRANSIENT GENE EXPRESSION BIOPROCESS

CROSS REFERENCE TO RELATED APPLICATIONS

This claim the benefit of U.S. Provisional Application No. 62/751,204, filed October 26, 2018, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

This is related to the field of transient gene expression, specifically to the use of a perfusion- based system for transient gene expression of a heterologous protein, such as, but not limited to, a vaccine or an antibody.

BACKGROUND

Transient gene expression (TGE) technology is an established biomanufacturing process to meet high recombinant protein production needs (Khan, Adv Pharm Bull, 3(2), 257-263.

doi: 10.568 l/apb.20l3.042, 2013; Wright, Hum Gene Ther, 20(7), 698-706.

doi: l0.l089/hum.2009.064, 2009). TGE bioprocesses have been reported for producing monoclonal antibodies (mAh) and virus-like-particles (VLPs) using cell lines such as Chinese hamster ovary (CHO) (Baldi et al., Biotechnol Lett, 29(5), 677-684. doi: 10. l007/s 10529-006-9297, 2007; Pham et al., Mol Biotechnol, 34(2), 225-237. doi: lOT385/MB:34:2:22, 2006; Rajendra et al., Biotechnol Bioeng, 112(5), 977-986. doi:l0.l002/bit.255l4, 2015; Tait et al., Biotechnol Bioeng, 88(6), 707-721. doi:l0.l002/bit.2026, 2004; Ye et al., Biotechnol Bioeng, 103(3), 542-551.

doi:l0.l002/bit.22265, 2009) and Human embryonic kidney (HEK) 293 cells (Ansorge et al., J Gene Med, 11(10), 868-876. doi:l0.l002/jgm.l370, 2009; Cervera et al., 2 Biotechnol Bioeng, 112(5), 934-946. doi:l0.l002/bit.25503, 2015; Jager et al., BMC Biotechnol, 13, 52.

doi:l0.1186/1472-6750-13-52, 2013; Jain et al., Protein Expr Purif, 134, 38-46.

doi:l0.l0l6/j.pep.20l7.03.0l8, 2017; Swiech et al., BMC Biotechnol, 11, 114. doi: 10.1186/1472- 6750-11-114, 2011). The TGE bioprocess typically uses fed-batch strategy that keeps all the cells and protein product in the same vessel throughout the production run. However, such a fed-batch process has intrinsic limitations. First, waste products like cell debris or other undesirable small molecules accumulate in the vessel during the run and have potential to disrupt cell growth, protein production, and the stability of the generated protein of interest. In addition, necessary media exchange and/or cell concentration must be performed outside the culturing vessel, which requires increased handling and poses higher risks of contamination· Furthermore, even with the addition of feeds in the fed-batch process, the production length is limited by the decline in cell viability.

Therefore, a need remains for better methods for large scale TGE production methods.

SUMMARY OF THE DISCLOSURE

High efficiency methods are disclosed for producing a heterologous protein using transient gene expression in host cells. The disclosed methods utilize a perfusion bioreactor.

These methods include: a) culturing the host cells in a perfusion bioreactor at a density of 5.5 X l0⁶to 8.5 X 10⁶ viable cells/ml wherein the cells are in an exponential phase of growth, wherein the bioreactor comprises a growth medium; b) replacing the growth medium in the perfusion bioreactor with a transfection medium using perfusion; c) concentrating the host cells in the transfection medium using perfusion to a cell density of 13.5 X l0⁶to 17.5 X 10⁶ viable cells/ml; d) transfecting the host cells with a vector encoding the heterologous protein in the absence of aeration and perfusion in the bioreactor, wherein host cells express the heterologous protein in the bioreactor; e) replacing the transfection medium in the perfusion bioreactor with an expression medium using perfusion, wherein the perfusion begins 1 to 4 days following step b); and f) culturing the host cells expressing the heterologous protein in the bioreactor at a perfusion rate of 25 pL/cell/day to 100 pL/cell/day in the expression medium at a constant total volume. The heterologous protein can then be isolated from the host cells.

The heterologous protein can be any protein of interest. In some embodiments, the host cells are mammalian, such as, but not limited to, human host cells.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Flow chart for exemplary methods for fed-batch (A) and perfusion-based (B) TGE bioprocesses. The fed-batch TGE bioprocess included cell growth, centrifugation, media exchange, cell concentration, transfer to the bioreactor, transfection, and expression with daily supplementation. In comparison, the perfusion-based TGE bioprocess included cell growth, media exchange, cell concentration, transfection, and expression all done in the same vessel.

FIGS. 2A-2G. Viable Cell Density (VCD) and viability (A), titer (B), and metabolite profiles (C-G) from fed-batch TGE bioprocess. Average from three independent runs show a slight cell growth with decreasing viability but reasonable transient gene production during the expression (circle). Despite similar trends in metabolite profiles, the cells with microbubble condition during transfection (square) generated much lower, if any, products than that from without microbubble condition. Data presented as Mean ± SD.

FIGS. 3A-3F. VCD and viability (A) and metabolite profiles (B-F) during cell growth. Average data show gradual VCD increase with sustainable viability and metabolite profiles.

Asterisk indicates the point of perfusion initiation for media exchange and cell concentration (A). Data presented as Mean ± SD.

FIGS. 4A-4H. VCD (A), viability (B), titer (C) and metabolite profiles (D-H) from perfusion bioprocess starting at day 4. The data show 100 pL/cell/day perfusion from day 4 with (diamond) or without (circle) feeding increase VCD while maintaining the viability and produce high titer with reasonable metabolite levels. In comparison, 25 pL/cell/day perfusion from day 4 (triangle) was insufficient to increase VCD and to maintain the viability and the metabolite levels. Asterisk indicates the point of perfusion initiation media supplementation (A, B and C). Data presented as Mean ± SD.

FIGS. 5A-5H. VCD (A), viability (B), titer (C) and metabolite profiles (D-H) from perfusion bioprocess starting at day 1. The data show 100 (circle) but not 25 (triangle) pL/cell/day perfusion from day 1 increases VCD while maintaining the viability and produces high titer. In comparison, the absence of perfusion (square) stalls the cell growth, viability and production after day 4. Whereas glucose (D), glutamine (E), glutamate (F), lactate (G), and NH₄ (H) profiles with the perfusion-based processes were maintained at certain levels (circle), those were depleted or not sustained without perfusion (square). Asterisk indicates the point of perfusion initiation for media supplementation (A, B and C). Data presented as Mean ± SD.

FIGS. 6A-6B. Comparison of cell specific productivity, Qp (A) and consumable cost per milligram (B) between the TGE bioprocesses with or without ATF perfusion. Whereas the Qp at Day 3 post-transfection (p- value = .1974, two-tailed t-test) was not significantly different, the Qp at day 4 (* indicates p- value = .0246, two-tailed t-test) and day 5 post- transfection (** indicates p- value = .0168, two-tailed t-test) were significantly different between the bioprocesses suggesting a sustainable cellular level productivity benefits from the ATF perfusion strategy. Data presented as mean ± SD. In addition, the 9-days ATF perfusion-based bioprocess (100 pL/cell/day from Day 4 post-transfection) showed -70% reduction in consumable cost per milligram compared to the 4- days baseline bioprocess. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Disclosed herein is a versatile method that uses a perfusion bioreactor for production of a heterologous protein using transient gene expression in cell culture. The development of the disclosed process included optimization of conditions for cell growth and expression of the heterologous protein in the perfusion bioreactor. Conditions were assessed for transfection mediated by polyethylenimine (PEI), but are applicable to numerous transfection methods and cell types. The use of the methods disclosed herein results in a high product yield, and can be used for the production of many products, including, but not limited to, vaccines and antibodies.

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference , published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

About: Unless context indicated otherwise,“about” refers to plus or minus 5% of a reference value. For example,“about” 100 refers to 95 to 105.

Aeration: The process by which a gas, such as air, is circulated through, mixed with or dissolved in a liquid or substance.

Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, small molecule, organic compound, inorganic compound, or other molecule of interest. Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject). In some embodiments, an agent is a protein.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term“subject” includes both human and veterinary subjects, for example, non-human primates, dogs, cats, horses, rabbits, pigs, mice, rats, and cows. Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), an antigenic fragment thereof, or a dimer or multimer of the antigen. The term“antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. , bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, Fiab'h; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g. , Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010).

Bioreactor: Any culture vessel used for the growth of a mammalian cell culture. The bioreactor can be of any size so long as it is useful for the culturing of mammalian cells. A bioreactor can be at least 30 ml and may be at least 1 , 10, 100, 250, 500, 1 00, 2500, 5000, 8000, 10,000, 12,0000 liters or more, or any intermediate volume. The internal conditions of the bioreactor, including but not limited to pH and temperature, are typically controlled during the culturing period. A“perfusion bioreaclor” has an inlet and outlet that provides for the provision of medium and the removal of waste or spent medium from the bioreactor at a specified flow rate. The medium is provided, and the waste is removed, at a specified continuous flow rate when the perfusion system is activated. The term also connotes that cells are retained or separated from the effluent (exit) stream and maintained to accumulate in the bioreactor. In a“Fed-batch bioreactor,” there is a process of (a) adding nutrient media in bolus feeds to the bioreactor at designated time points, or (b) adding glucose (or another single nutrient) to the bioreactor as the glucose (or other single nutrient) is consumed at designated time point, without using a continuous flow. Thus, a fed- batch bioreactor is distinct from a perfusion bioreactor.

Cell Cal tore: The maintenance of cells in an artificial, in vitro environment that favors growth and survival. Suspension cell culture is a cell culture in which the majority or ail of cells in a bioreactor, such as a culture vessel, are present in suspension (freely floating in liquid phase media), and the minority (or none) of the cells are attached to a surface. In several embodiments, a suspension culture has greater than 75%, 85%, or 95% of the cells in suspension, and thus not attached to a surface on or in the bioreactor. The term "batch culture” refers to a method of culturing cells in which all the components that will ultimately he used in culturing the cells, including the medium as well as the cells themselves, are provided at the beginning of the culturing process. A batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified. The term "fed-batch culture" refers to a method of culturing cells, in a fed-batch bioreactor, in which additional components are provided to the culture at some defined time point(s) subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells that have been depleted during the culturing process. A fed- batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified. Media is not perfused into a fed-batch culture.

The term "perfusion culture" refers to a method of culturing cells in which additional fresh medium is provided, continuously at a defined rate over some period of time, to the culture, and simultaneously spent medium is removed. The fresh medium typically provides nutritional supplements for the cells that have been depleted during the culturing process. Protein product, which may be present in the spent medium, is optionally purified. Perfusion also allows for removal of cellular waste produets /flushing) from the cell culture growing in the bioreact.or.

Implicit in this terminology is that cell are retained in the culture system and not allowed to be lost through the exit stream.

Cell culture medium, tissue culture medium, culture medium (plural“media”): A nutritive solution for cultivating cells or tissues. These phrases can be used interchangeably. A “chemically defined” cell culture medium is one in which each chemical species and its respective quantity is known prior to its use in culturing cells. A chemically defined cell culture medium is made without lysates or hydrolysates whose chemical species are not known and/or quantified. The terms“serum-free culture conditions” and“serum-free conditions” refer to cell culture conditions that exclude serum of any type. These terms cart be used interchangeably. Cell culture media include growth media, transfection media, and expression media.

Cell density: A number of cells present in a given volume of medium. The term "viable cell density" as used herein refers to the number of live cells present in a given volume of medium under a given set of experimental conditions.

Cell viability: Hie ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term also refers to that portion of cells that are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time. Expand: A process by which the number or amount of cells in a cell culture is increased due to cell division. Similarly, the terms“expansion” or“expanded” refers to this process. The terms "proliferate," "proliferation," or "proliferated" may be used interchangeably with the words "expand," "expansion", or "expanded." Typically, during an expansion phase, the cells do not differentiate to form mature cells, but divide to form more cells.

Expansion or Growth medium: A synthetic set of culture conditions with the nutrients necessary to support the growth (cell proliferation/expansion) of a specific population of cells. In one embodiment, the cells are stem cells, such as iPSCs. Growth media generally include a carbon source, a nitrogen source and a buffer to maintain pH. In one embodiment, growth medium contains a minimal essential media, such as DMEM, supplemented with various nutrients to enhance stem cell growth. Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum.

Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

Growth factor: A substance that promotes cell growth, survival, and/or differentiation. Growth factors include molecules that function as growth stimulators (mitogens), factors that stimulate cell migration, factors that function as chemotactic agents or inhibit cell migration or invasion of tumor cells, factors that modulate differentiated functions of cells, factors involved in apoptosis, or factors that promote survival of cells without influencing growth and differentiation. Examples of growth factors are a fibroblast growth factor (such as FGF-2), epidermal growth factor (EGF), cilliary neurotrophic factor (CNTF), and nerve growth factor (NGF), and actvin-A.

Growth phase: The time period of a culture process that is the period of exponential cell growth (the log phase) where cells are generally rapidly dividing.

Heterologous: With regard to a protein, a heterologous protein is not naturally produced by a specified cell type. Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term“host cell” is used.

Isolated: An“isolated” biological component, such as a nucleic acid, protein or organelle that has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, /._<?., chromosomal and extra- chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Similarly, an“isolated” cell has been substantially separated, produced apart from, or puified away from other cells of the organism in which the cell naturally occurs. Isolated cells can be, for example, at least 99%, at leat 98%, at least 97%, at least 96%, 95%, at least 94%, at least 93%, at least 92%, aor at least 90% pure.

Mammal: This term includes both human and non-human mammals. Examples of mammals include but are not limited to: humans and veterinary and laboratory animals, such as pigs, cows, goats, cats, dogs, rabbits and mice.

Marker or Label: An agent capable of detection, for example by ELISA,

spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy, Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of markers include, but are not limited to, radioactive isotopes, nitorimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

In some embodiments, the marker is a fluorophore (“fluorescent label”). Fluorophores are chemical compounds, which when excited by exposure to a particular wavelength of light, emits light (/._<?., fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or“color.” Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 l. Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-690 l. In other embodiments, the marker is a protein tag recognized by an antibody, for example a histidine (His)-tag, a

hemagglutinin (HA)-tag, or a c-Myc-tag.

Microbubbles: Small, gas-filled bubbles, typically between 0.5pm and lOpm in diameter.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Perfusion: In a bioreactor for the cultivation of mammalian cells, medium is perfused through the bioreactor at a specified rate while the cell mass is contained within the bioreactor by means of a cell retention device. In a suspension culture system, the cell retention device can be a filter, but numerous other methods can be employed, such as sonic separation, inclined plane settling, external centrifuges, internal filters such as spinning or oscillating, external hydrocyclones, etc. Fresh culture media is provided to the cells in the bioreactor. As the cell mass continues to grow and increase in number and mass, the rate of perfusion can be increased to remove metabolic byproducts and supply necessary nutrients. The perfusion rate can remain at a constant rate, or can be increased step-wise at several rates. In some embodiments, a specific ratio of perfusion medium volume per time to cell number is maintained (CSPR, or cell-specific perfusion rate, often in nanoliters/cell/day), see Ozturk, Cytotechnology 2: 3-16, 1996 and Konstantinov et ak, Adv.

Biochem. Eng/Biotechnol. 101: 75-98, 2006. In some embodiments, a perfusion rate can control the concentration of glucose or L-lactate (Konstantinov et ak, Biotechnol. Prog, 12(1): 100-9, 1996 and Ozturk et ak, Biotechnol. Bioeng. 53(4): 372-8), 2006, or can be based on oxygen uptake rate measurements (Feng et ak, J. Biotechnol. 20; 122(4): 422-430, 2006). See also PCT Publication No. WO 2016/196261A1.

Protein: A polypeptide in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred in nature. The term protein is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

Substantially purified protein as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.

Protein yield: 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.

Replenishing, replacing, or supplementing medium: Adding a volume of fresh cell culture medium to bioreactor that already includes medium and/or replacing medium that was already present in the bioreactor with fresh medium, and/or supplementing medium already present in culture with new medium. Fresh medium is medium that has not previously been in contact with the cells of interest in the bioreactor, and does not contain one or more macromolecules that are waste products (e.g., lactic acid) produced by the cells when they are cultivated.

Purified: The term“purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified cell preparation is one in which the cells are more pure than the cells in its natural environment. For example, a preparation of a cells is purified such that the cells represents at least 50% of the total population. A purified population of cells is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% pure, or free other cell types.

Recombinant: A recombinant nucleic acid or polypeptide molecule is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis of polypeptide or nucleic acid molecules, or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. A vector may also include a sequence encoding for an amino acid motif that facilitates the isolation of the desired protein product such as a sequence encoding maltose binding protein, c-myc, or GST.

Viral antigen: A virus or portion of the virus which can induce an immune response in a subject against said antigen.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms“a,”“an,” and“the” include plural referents unless context clearly indicates otherwise. Similarly, the word“or” is intended to include“and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are

approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term“comprises” means“includes.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The term“about” indicates a variation of 5 percent or less. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

II. Methods

Methods are disclosed herein from the production of a heterologous protein. These methods include seeding cells of interest into a perfusion bioreactor. In some embodiments, the perfusion is alternating tangential flow (ATF). In other embodiments, the perfusion in the perfusion cell culture is tangential flow. The internal conditions of the bioreactor, for example, but not limited to pH and temperature, are typically controlled during the culturing period. The bioreactor can be composed of any material that is suitable for holding mammalian cell cultures suspended in media under the culture conditions of the present invention, including glass, plastic or metal. In some embodiments, the perfusion in the perfusion cell culture uses filtration devices such as hollow fiber and open channel filters. In one non-limiting example, the bioreactor includes an ATF filtration system with a hollow fiber filter.

The bioreactor can be of any volume. Suitable bioreactors include, but are not limited to, those of 15 ml to about 40, 000 liters. These include bioreactors used in laboratory and commercial settings. In some embodiments, the bioreaction can have a volume of 1 to 50 liters, such as 2 to 10 liters, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 liters. In one embodiment, the bioreactor as a volume of 2-5 liters, such as about 3 liters.

In further embodiments, the bioreactor can have a volume of less than 4000 liters, less than 3750 liters, less than 3500 liters, less than 3250 liters, less than 3000 liters, less than 2750 liters, less than 2500 liters, less than 2250 liters, less than 2000 liters, less than 1750 liters, less than 1500 liters, less than 1250 liters, or less than 1000 liters. In some embodiments, the bioreactor is about 50 to about 20,000 liters, about 50 to about 15,000 liters, about 50 to about 10,000 liters, about 50 to about 3570 liters, about 50 to about 3500 liters, about 50 to about 3000 liters, about 50 to about 2500 liters, about 50 to about 2000 liters, about 50 to about 1500 liters, about 50 to about 1000 liters, about 50 to 500 liters, about 100 to about 10,000 liters, about 100 to about 5000 liters, or about 100 to about 4000 liters. In certain embodiments, the bioreactor is 4000 liters, 3750 liters, 3500 liters, 3250 liters, 3000 liters, 2750 liters, 2500 liters, 2250 liters, 2000 liters, 1750 liters, 1500 liters, 1250 liters, 1000 liters, 750 liters, 500 liters, 250 liters, 200 liters, 100 liters, or 50 liters. In other embodiments, the bioreactor is between about 200 liters to about 20,000 liters, about 200 liters to about 15,000 liters, about 200 liters to about 12,000 liters, about 200 liters to about 10,000 liters, about 200 liters to about 8,000 liters, about 200 liters to about 5,000 liters, about 1000 liters to about 20,000 liters, about 1000 liters to about 20,000 liters, about 1000 liters to about 15,000 liters, about 1000 liters to about 12,000 liters, about 1000 liters to about 10,000 liters, about 1000 liters to about 8,000 liters, or about 1000 liters to about 5,000 liters. In other embodiments, the volume of the bioreactor is about 200 liters, about 500 liters, about 800 liters, about 1000 liters, about 1200 liters, about 1500 liters, about 2000 liters, about 2500 liters, about 5000 liters, about 7500 liters, about 10,000 liters, about 12,000 liters, about 15000 liters, about 18000 liters, or about 20,000 liters. In some embodiments, the volume of the N culture vessel is less than or equal to about 20,000 liters.

The bioreactor is a perfusion bioreactor, such as an alternating tangential flow (ATF) bioreactor or a tangential follow bioreactor. The internal conditions of the bioreactor, for example, but not limited to pH and temperature, are typically controlled during the culturing period.

A diagram of an exemplary process is shown in Fig. 1. The steps in the process are discussed in more detail below. A. Initial Culture and Growth Media

The cells are in the exponential phase of growth. The methods can include culturing the cells in a perfusion bioreactor to a density of 5.5 X l0⁶to 8.5 X 10⁶ wherein the cells are in an exponential phase of growth, wherein the bioreactor comprises a growth medium. The cells can be grown to a density of about 6 X l0⁶to 7.5 X 10⁶ cells, such as about 6.5 X l0⁶ to about 7.5 X 10⁶ cells/ml, such as about 6.5 X 10⁶, 6.6 X 10⁶, 6.7 X 10⁶, 6.8 X 10⁶, 6.9 X 10⁶, 7.0 X 10⁶, 7.1 X 10⁶, 7.2 X 10⁶, 7.3 X 10⁶, 7.4 X 10⁶, or 7.5 X 10⁶ cells/ml.

In some embodiments, the cells are cultured for 1-10 days to reach the exponential phase of growth, such as 2-9 days or 3-8 days. In some non-limiting examples, the cells are grown for 3-7 days, including, but not limited to, 4-6 days, or 4-5 days. Suitable non-limiting examples are 2, 3,

4, 5, 6, 7, or 8 days. Cell density can be measured using a hemocytometer, a Coulter counter, fluorescence activated cell sorting, or Cell density examination (CEDEX). Viable cell density may be determined by staining a culture sample with Trypan blue.

In some embodiments, the cells are grown to the desired cells density without the use of perfusion.

In some embodiments, for cell growth, the media can be replaced using perfusion. In some embodiments, the perfusion rate is 25 pL/cell/day to 100 pL/cell/day. In other embodiments, the perfusion rate is about 50 pL/cell/day to about 100 pL/cell per day. In further embodiments, the perfusion rate is about 75 pL/cll per day to about 100 pL/cell/day. The perfusion rate can be about 25, 50, 75, 100 pL/cell/day. In other embodiments, the perfusion rate in the perfusion cell culture is 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 90, 95, 100 pL/cell/day.

The cells are eukaryotic. Although in certain embodiments the cell culture comprises mammalian cells, one skilled in the art will understand that it is possible to reeombinantiy produce polypeptides of interest in lower eukaryotes such as yeast. One skilled in the art would know that the culture conditions for yeast cultures will differ from the culture conditions of mammalian cells, and will understand how these conditions will need to be adjusted in order to optimize cell growth and/or protein production. The use of animal ceils encompasses use of invertebrate,

non mam alian vertebrate (for example, avian, reptile and amphibian), and mammalian cells. Non- limiting examples of invertebrate cells insect cells, such as Spodopterafrugiperda (caterpillar), Aedes aegypli (mosquito), Aedes alhopictus (mosquito), Drosophila melanogaster (fmitfly), and Botnbyx rnori (silk moth).

In some embodiments, the ceils are mammalian ceils. The cells can be human or veterinary, such as mouse, monkey, rabbit, rat, etc. Numerous mammalian ceil lines are suitable host cells for recombinant expression of polypeptides of interest. Mammalian host cell lines include, for example, PER.C6, TM4, VERO076, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, 293T, A431, 3T3, CV-1, C3H10T1/2, Colo205, 293, HeLa, L cells, HL-60, FRhL-2, 11937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-I, PC12, Mix, murine myelomas (e.g., SP2/0 and NSO) and C2C12 cells, as well as primate cell lines, normal diploid cells, and cell strains derived from in vitro culture of primary tissue and primary explants. Other mammalian cells of use are CHO cells, HEK-293 cells, VERO cells, NSO cells, PER.C6 cells. Sp2/0 cells, MDCK cells, BHK, MDBK cells, and COS cells. The cells can be stem cells, including embryonic stem cells, induced pluripotent stem cells, and hematopoietic stem cells. The stem cells can be totipotent, pluripotent, or multipotent. Any eukaryotic cell that is capable of expressing the polypeptide of interest may be used in the disclosed cell culture methods. Numerous cell lines are available from commercial sources such as the American Type Culture Collection (ATCC)

Non·· limiting examples of mammalian cells include B ALB/c mouse myeloma line (NSO/l, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands));

monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et aL, i. Gen Virol , 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese h mster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proe. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et aL, Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Hie cells are also grown in an environment conducive to cell growth, such as with regard to pH and C0₂ concentration. For example, most mammalian cells, e.g., CHO cells or 293 cells, grow well within the range of about 35° C to 39° C, preferably at 37° C, whereas insect cells are typically cultured at 21 C. The conditions can include, for example, specific C0₂ concentrations, such as, hut not limited to, about 5% C0₂. The C0₂ concentration for growing specific cell types is known in the art. The pH can be monitored and maintained, for example, at about 6 to about 8, such as about 7 to about 7 5, such as about 7.2 to about 7.4 The temperature, pH and CO2 concentration can be maintained at all steps of the process. In some embodiments, the cells are mammalian, and the pH is about 7 4 to about 7.6, and the C0₂ is about 5%.

Tiie cells tire grown in a growth medium suitable for expanding the cells in number. The medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. The medium can also contain components that enhance growth and/or survival above the minimal rate, including hormones and growth factors. The medium can be supplemented with factors, such as L-glutamine, amino acids, sodium bicarbonate, poloxamer, and/or salts such as sodium chloride. In some embodiments, the medium contains, for example, inorganic salts, carbohydrates (such as sugars such as glucose, galactose, maltose or fructose), amino acids, vitamins (such as B group vitamins (for example, B12), vitamin A vitamin E, riboflavin, thiamine and biotin), fatty acids and lipids (such as cholesterol and steroids), proteins and peptides (such as albumin, transferrin, fibronectin and letuin), and trace elements (such as zinc, copper, selenium and tricarboxylic acid

intermediates). Optionally the medium can include serum (such as fetal bovine serum, newborn calf serum, horse serum or human serum), hydrolysates (hydrolyzed proteins derived from plant or animal sources), and combinations thereof. See U.S. Published Application No. 2016/0289633, incorporated by reference herein.

In some embodiments, the medium includes serum. In other embodiments, the medium can be a“defined medium” or“chemically defined medium” that is a serum-free medium that contains no serum, and no serum proteins, hydrolysates or serum fractions.

In some embodiments, the medium of use is free of animal-derived components and all components have a known chemical structure. A defined medium can comprise recombinant glycoproteins or proteins, for example, but not limited to, hormones, cytokines, interleukins and other signaling molecules. Suitable mediums, include, but are not limited to, Dulbecco’s Modified Essential Medium (DMEM), RPMI, CDM4HEK293 media, and other commercially available media. The cells can he grown in chemically defined medium with supplements, such as hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), additional salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamyem), trace elements, glucose or an equivalent energy source.

Commercially available media such as 5 x -concentrated DMEM/F12 (Invitrogen), CD OPTICHO® feed (invitrogen), CD EFFICIENTFEED® (Invitrogen), CELL BOOST® (HyClone), BALANCD® CHO Feed (Irvine Scientific), BD RECHARGE® (Becton Dickinson),

CELLVENTO® Feed (EMD Millipore), EX-CELL CHOZN FEED® (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ACTICHO© (PAA/GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), Roswell Park Memorial institute (RPMI)-1640 (Sigma), and Duibeeco's Modified Eagle’s Medium (DMEM, Sigma) can be used in growth media. In addition any of the media described in Ham and Wallace, (1979) Meth. Enz., 58:44; Barnes and Sato, (1980) Anal. Biochem., 102:255; U.S. Pat. Nos.

4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; PCT Publication No. WO 90/03430; and WO 87/00195, see also U.S. Published Application No. 20160289633, can be used as growth media.

In some embodiments, the cells are HEK-293 cells, and the medium is CDM4HEK293 media (SH3A3770.02, HyClone, Chicago, IL). In one non-limiting example, the medium is supplemented with sodium bicarbonate, and a poloxamer, such as, but not limited to Poloxamer 188, L-glutamine, and sodium chloride. An exemplary medium is disclosed in the examples section below.

In certain embodiments, conditions may be periodically monitored. As non-limiting example, it may be beneficial to monitor temperature, pH, cell density, cell viability, lactate levels, ammonium levels, or osmolarity. Numerous techniques are well known in the art that will allow one of ordinary skill in the art to measure these conditions. For example, HPLC can be used to determine the levels of lactate and/or ammonium.

B. Replacement of Growth Medium with Transfection Medium, and Transfection

After the cells are grown to the desired density and are in an exponential phase of growth, the growth medium in the bioreactor is replaced with a transfection medium using perfusion, and the cells are transfected with a vector encoding a heterologous protein of interest. The transfection medium is any medium that allows introduction of a vector encoding the heterologous nucleic acid. In some embodiments, the growth medium and the transfection medium are the same. In other embodiments, the grown medium and the transfection medium are different. Under most circumstances, the medium used for transfection does not support the growth of the cells, but the transfection medium can be used for the purpose of introducing nucleic acids into the cells.

In some embodiments, the cells are maintained at the same temperature, pH and CO2 concentration of use with the growth medium. However, these parameters can also be varied as necessary for high efficiency transfection. One of skill in the art can readily modify these features appropriately. The transfection medium can be any medium known to those of skill in the art that is of use for the introduction of nucleic acids into host cells. Exemplary high density culture media include, though are not limited to, HuMEC Basal Serum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC/iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium,

ESSENTL4L™~8 Medium, Medium 254, Medium, 106, Medium, 131 , Medium, 154, Medium,

171 , Medium 171 , Medium 200, Medium 231, HeptoZYME-SFM (Thermo Fisher), Human Endotheli al- 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, AMIN QM AX™ -II Complete Medium, CD FORTICHO™ Medium, CD CHO ACT 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 modifications thereof. In some 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 form thereof. The above listed exemplary high-density culture media may he 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 mammalian cells of use in the disclosed methods.

In some embodiments, the transfection medium is serum free. In a specific non-limiting example, the transfection medium is serum free GIBCO® FREESTYLE™ 293 Expression

Medium, such as for HEK-293 cells.

The cells are concentrated in the transfection medium, using perfusion, to a cell density of about 13.5 X l0⁶to about 17.5 X 10⁶ cells/ml. In some embodiments, the cells are concentrated to a cell density of 14 X l0⁶to about 17.5 X 10⁶ cells/ml. In other embodiments, the cells are concentrated to a cell density of about 15 X l0⁶ to about 17.5 X 10⁶ cells/ml. In further

embodiments, the cells are concentrated to a cell density of about 16 X l0⁶to about 17.5 X 10⁶ cells/ml. In yet other embodiments, the cells are concentrated to a cell density of 17 X 10⁶ to about 17.5 X 10⁶ cells/ml. The cells can be concentrated to a cell density of about 13.5 X 10⁶ cells/ml, 14 X 10⁶ cells/ml, 14.5 X 10⁶ cells/ml, 15 X 10⁶ cells/ml, 15.5 X 10⁶ cells/ml, 16 X 10⁶ cells/ml, 16.5 X 10⁶ cells/ml, 17 X 10⁶ cells/ml, or 17.5 X 10⁶ cells/ml. The cells can be concentrated using filtration in the perfusion bioreactor. The cells can be concentrated in the absence of medium replacement. Thus, no new transfection medium is provided during concentration.

Following concentration, the cells are then transfected with a vector encoding the heterologous protein in the absence of aeration or perfusion in the bioreactor, such that cells express the heterologous protein in the bioreactor. For the absence of aeration, the gas flow to the bioreactor can be turned off. The process of transfection can occur over about 1 to about 4 hours, such as about 1, 2, 3, or 4 hours. The process of transfection can occur, for example for about 2 to about 3 hours. Thus, the cells are maintained in the absence of aeration for this time.

For expression, a polynucleotide sequence encoding the heterologous protein is operably linked to transcriptional control sequences including, for example a promoter and a polyadenylation signal. A promoter is a polynucleotide sequence recognized by the transcriptional machinery of the host cell (or introduced synthetic machinery) that is involved in the initiation of transcription. A polyadenylation signal is a polynucleotide sequence that directs the addition of a series of nucleotides on the end of the mRNA transcript for proper processing and trafficking of the transcript out of the nucleus into the cytoplasm for translation.

Exemplary promoters include viral promoters, such as cytomegalovirus immediate early gene promoter (“CMV”), herpes simplex vims thymidine kinase (“tk”), SV40 early transcription unit, polyoma, retroviruses, papilloma virus, hepatitis B vims, and human and simian

immunodeficiency viruses. Other promoters are isolated from mammalian genes, including the immunoglobulin heavy chain, immunoglobulin light chain, T-cell receptor, HLA DQ a and DQ b, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II, HLA-DRa, b-actin, muscle creatine kinase, prealbumin (transthyretin), elastase I, metallothionein, collagenase, albumin, fetoprotein, b-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), al- antitrypsin, H2B (TH2B) histone, type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human semm amyloid A (SAA), troponin I (TNI), platelet-derived growth factor, and dystrophin, and promoters specific for keratinocytes, and epithelial cells.

The promoter can be either inducible or constitutive. An inducible promoter is a promoter which is inactive or exhibits low activity except in the presence of an inducer substance. Examples of inducible promoters include, but are not limited to, MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, a-2-macroglobulin, MHC class I gene h-2kb, HSP70, proliferin, tumor necrosis factor, or thyroid stimulating hormone gene promoter. In some embodiments, the promoter is a constitutive promoter that results in high levels of transcription upon introduction into a host cell in the absence of additional factors. Optionally, the transcription control sequences include one or more enhancer elements, which are binding recognition sites for one or more transcription factors that increase transcription above that observed for the minimal promoter alone.

It may be desirable to include a polyadenylation signal to effect proper termination and polyadenylation of the gene transcript. Exemplary polyadenylation signals have been isolated from bovine growth hormone, SV40 and the herpes simplex virus thymidine kinase genes. Any of these or other polyadenylation signals can be utilized in the context of the vectors described herein.

The polynucleotides encoding the heterologous polypeptide include a recombinant DNA, which is incorporated into a vector. The polynucleotides can be ribonucleotides,

deoxyribonucleo tides, or modified forms of either nucleotide. The term includes single and double forms of DNA. Generally, it is advantageous to transfect cells with a vector encoding the heterologous protein of interest operably linked to the transcriptional control sequences, such as, but not limited to, a promoter.

A viral gene delivery system can be an RNA-based or DNA-based viral vector. However, other delivery systems are also of use, such as a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian vims 40 (SV40)-based episomal vector, a bovine papilloma vims (BPV)-based vector, or a lentiviral vector. Suitable vectors for transfection include, but are not limited to retroviral vectors, adenoviral vectors, adeno- associated viral vectors, lentiviral vectors, plasmids and Sendai virus, amongst others. Plasmids have been designed with a number of goals in mind, such as achieving regulated high copy number and avoiding potential causes of plasmid instability in bacteria, and providing means for plasmid selection that are compatible with use in mammalian cells, including human cells. There are dual requirements of plasmids for use in human cells. First, they are suitable for maintenance and fermentation in E. coli, so that large amounts of DNA can be produced and purified. Second, they are safe and suitable for use in human patients and animals. The first requirement calls for high copy number plasmids that can be selected for and stably maintained relatively easily during bacterial fermentation. The second requirement calls for attention to elements such as selectable markers and other coding sequences. In some embodiments plasmids that encode a heterologous protein are composed of: (1) a high copy number replication origin, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) transcription termination sequences, including the tyrosinase enhancer and (4) a multicloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors that are known in the art for inducing a nucleic acid encoding a protein. These include, but are not limited to, the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No.

7,989,425; and U.S. Patent No. 6,416,998, which are incorporated herein by reference. The vector can also encode a marker.

Viral vectors can be utilized for the introduction of nucleic acids, including polyoma, SV40 (Madzak et ak, 1992, J. Gen. Virol., 73: 15331536), adenovirus (Berkner, 1992, Cur. Top.

Microbiol. Immunol., 158:39-6; Berliner et ak, 1988, Bio Techniques, 6:616-629; Gorziglia et ak, 1992, J. Virol., 66:4407-4412; Quantin et ak, 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et ak, 1992, Cell, 68:143-155; Wilkinson et ak, 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et ak, 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et ak,

1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et ak, 1990, Gene, 89:279-282), herpes viruses including herpes simplex virus (HSV) and Epstein Barr Vims (EBV) (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et ak, 1992, J. Virol., 66:29522965; Fink et ak, 1992, Hum. Gene Ther. 3:11- 19; Breakfield et ak, 1987, Mol. Neurobiok, 1:337-371; Fresse et ak, 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et ak, 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11: 18- 22; I. Frolov et ak, 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377), human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses of avian (Brandyopadhyay et ak, 1984, Mol. Cell Biol., 4:749-754; Petropouplos et ak, 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et ak, 1985, Mol. Cell Biol., 5:431-437; Sorge et ak, 1984, Mol. Cell Biol., 4:1730-1737; Mann et ak, 1985, J. Virol., 54:401-407), and human origin (Page et ak, 1990, J. Virol., 64:5370-5276; Buchschalcher et ak, 1992, J. Virol., 66:2731-2739). Baculovirus ( Autographa californica multinuclear polyhedrosis vims; AcMNPV) vectors can be used. Vectors can be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.). Suitable vectors are disclosed, for example, in U.S. Published Patent Application No. 2010/0247486, which is incorporated herein by reference. In specific non-limiting examples, the vectors are retrovims vectors (for example, lentivirus vectors), measles vims vectors, alphavims vectors, baculovims vectors, Sindbis vims vectors, adenovirus and poliovirus vectors. In some embodiments, the vector encodes the heterologous protein of interest (see below), and a marker. Markers include, but are not limited to, fluorescence proteins (for example, green fluorescent protein or red fluorescent protein), enzymes (for example, horse radish peroxidase or alkaline phosphatase or firefly/renilla lucif erase or nanoluc), or other proteins. A marker may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mRNA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the marker of the cell type of interest. The markers can also be identified by a biochemical or enzyme assay or biological response that depends on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to a promoter and/or an enhancer.

Transfection can be accomplished by any method available to those of skill in the art.

Methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody, labeled antibody, or functional fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.

Transfection reagents are disclosed, for example, in PCT Publication No. WO

2013/166339A1, incorporated herein by reference. 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 b-cyclodextrin containing polymers (CD- polymers), DEAE-dextran and the like. In some embodiments, a reagent for the introduction of macromolecules into cells can comprise one or more lipids which can be cationic lipids and/or neutral lipids. Preferred lipids include, but are not limited to, N-[l-(2,3- dioleyloxy)propylj-N,N,N-trimethylamonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE),l,2-Bis(oleoyloxy)-3-(4'-trimethylammonio) propane (DOTAP), l,2-dioleoyl-3-(4’- trimethylammonio) butanoyl-sn-glycerol (DOTB), 1 ,2-dioleoyl- 3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4'-trimethylammonio)butanoate (Cho^'TB), cetyltrimetbylammonium bromide (CTAB), l,2-dioleoyl-3-dimethyl-hydroxyefhyl ammonium bromide (DORI), 1,2- dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dimyristyloxypropyl- 3 -dimethyl -hydroxyethyl ammonium bromide (DMRIE), 0,0'-didodecyl-N-[p(2- trimethylammonioethyloxy)benzoyl]-N,N,N-frimethylatn- monium chloride, spermine conjugated to one or more lipids (for example, 5- carboxyspermylglycine dioctadecylamide (DOGS), N,N^I,Nⁿ,N^m- tetramethyl-N,N^!,N^H,N^ra- tet- rapalmitylspermine (TM-TPS) and

dipalmitoylphasphatidylethanolamine 5- carboxyspermylaminde (DPPES)), lipopolylysine

(polylysine conjugated to DOPE), TRIS (Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids (TFAs) and/or peptides such as trilysyl-alanyl-TRIS mono-, di-, and tri- palmitate, (3b-[N— (N',N'- dimethylaminoethanej-carbamoyl] cholesterol (DC-Choi), N-(a - trimethyiammonioacetyi)- didodecyl-D-glutamate chloride (TMAG), dimethyl

dioctadecylammonium bromide (DDAB), 2,3-dioleyloxy-N-[2(spermme-carboxamido)ethyl]-N,N- dimethyl-l-propanamin- ini umtrifluoroacetate (DOSPA) and combinations thereof.

A cationic derivative of cholesterol (3 -[N-(NjN'-dimeihylaminoethane)- carbamoyl] cholesterol, DC-Choi) has been synthesized and formulated into liposomes with DOPE (see Gao, et ah, (1991) BBRC 179(i):280--285) and used to introduce DNA into ceils. 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) BBA 1065:8-14), has been reported to be effective at introducing nucleic acids into cells in the presence of serum.

Other types of cationic lipids that have been used to introduce nucleic acids into cells include highly packed polyeationie ammonium, sulfonium and phosphonium lipids such as those described in U.S. Pat. Nos. 5,674,908 and 5,834,439, and PCX Publication No. WO 00/27795. One transfection reagent for delivery of macromolecules is LIPOFECT AMINE 2000™ which is available from Life technologies (see U.S. international application no. PCT/US99/26825, published as WO 00/27795).

In certain non-limiting example, the transfection regent is PEI. The PEI to vector ratio can be 1: 1 , 1 :2, 1:3, 1 :4, 1:5, 5:1, 4: 1, 3: 1, or 2: 1.

C. Replacement of the Transfection Medium with a Growth Medium for Production of the

Protein

Following transfection, the transfection medium in the perfusion bioreactor is replaced with an expression medium. The transfection medium can be replaced with the expression medium using perfusion. The cells expressing the heterologous protein are cultured in the bioreactor in the expression medium at a constant total volume for about 1 to about 4 days using perfusion. The perfusion can be initiated at about 1 to about 4 days following transfection. The perfusion can be initiated about 1-2, 2-3, or 3-4 days following transfection. In some embodiments, perfusion is initiated about 1, 2, 3, or 4 days following transfection. The cells are then cultured in the expression medium using perfusion.

The expression medium can be any expression medium known in the art. In some embodiments, the expression medium and the transfection medium are different. In other embodiments, the expression medium and the transfection medium are the same. The expression medium can be serum- free.

The expression medium can be any medium known to those of skill in the art that is of use for production of proteins in host cells. Exemplary high density culture media include, though are not limited to, HuMEC Basal Serum tree 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, Mediu 200PRF, Medium 131, Essential™~6 Medium, STEMPRO™-34 Medium, GIBCO® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMIN OM AX™ -P Complete Medium, CD FQRTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-9QQ™ 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. 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.

The expression medium can include supplements, such as amino acids, growth factors, cytokines and dextran sulfate. In some embodiments, the expression medium includes

supplements, such as hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamyein), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. In some embodiments the nutrient media is serum-free media, a protein-free media, or a chemically defined media. Any other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art.

In some embodiments, the expression medium includes valproic acid, such as about 1 mM to about 5 mM valproic acid, such as about 3.5 mM to about 4.5 mM valproic acids, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mM valproic acids. In a non-limiting example, the expression medium includes 4 M valproic acid.

In a specific non-limiting example, the transfection medium is serum free GIBCO® FREESTYLE™ 293 Expression Medium, such as for HEK-293 cells. The expression medium further includes dextran sulfate, and/or a commercially available supplement, such as CELL BOOST™ 5 supplement, which provides as lipids, amino acids, vitamins, and growth factors.

In some embodiments, the perfusion rate for the expression medium is 25 pL/cell/day to 100 pL/cell/day. In other embodiments, the perfusion rate is about 50 pL/cell/day to about 100 pL/cell per day. In further embodiments, the perfusion rate is about 75 pL/cll per day to about 100 pL/cell/day. The perfusion rate can be about 25, 50, 75, or 100 pL/cell/day. In other embodiments, the perfusion rate in the perfusion cell culture is 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 90, 95, or 100 pL/cell/day.

In some embodiments, the method produces the recombinant protein at an accumulated titer of about 125-175 mg/L at four days following transfection.

In other embodiments, the method produces the recombinant protein at an accumulated titer of about 160 mg/L at four days following transfection. In further embodiments, the method produces the recombinant protein at an accumulated titer of about 200-300 mg/L at nine days following transfection. In yet other embodiments, the method produces the recombinant protein at an accumulated titer of about 270 mg/L at nine days following transfection.

In further embodiments, the heterologous protein is purified from the culture at about 3 to about 10 days following transfection, such as bout 4 to about 9 days following transfection. The heterologous protein can be purified about 3, 4, 5, 6, 7, 8, 9, or 10 days following transfection.

The heterologous protein can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, immunoaffinity purification, or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all incorporated herein by reference).

For immunoaffinity chromatography, the protein can be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support. Alternatively, affinity tags such as an influenza coat sequence, poly-histidine, or glutathione- S -transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column. Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin can be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process. Protease inhibitors can be used when cells must be lysed in order to isolate and purify the expressed polypeptide or protein. One of ordinary skill in the art will appreciate that the exact purification technique will vary depending on the character of the protein to be purified, the type of the cells from which the polypeptide or protein is expressed, and the composition of the medium in which the cells were grown.

II. Exemplary Heterologous Proteins that Can Be Produced by the Disclosed Methods

A variety of heterologous proteins can be produced using the disclosed methods. Exemplary proteins are listed below. However, this disclosure should not be construed to be limiting. One of skill in the art readily understands that nucleic acids encoding the following, and degenerate variants thereof, are of use in the disclosed methods. For any protein listed below, variants, such as conservative variants, and modified forms of these proteins can be produced. Glycosylated and non-glycosylated forms of the heterologous protein can be produced, depending on the host cells utilized in the process, and the modifications made to the protein sequence.

A. Antibodies

Numerous antibodies are currently in use or under investigation as pharmaceutical or other commercial agents. Any antibody that can be expressed in a host cell can be used in accordance with the present methods. In one embodiment, the antibody to be expressed is a monoclonal antibody. The antibodies can be in the form of full length antibodies, or in the form of fragments of antibodies, e.g., Fab, F(ab')2, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, for example, Published U.S. Patent application No 2005/0079574 and Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010.

Particular antibodies can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences. Numerous methods are available for obtaining antibodies, particularly human antibodies.

One exemplary method for identifying antibodies of interest includes screening protein expression libraries, such as phage or ribosome display libraries. Phage display is described, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.

WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; and PCT Publication No. WO 90/02809. The display of Fab's on phage is described, e.g., in U.S. Patent No. 5,658,727; U.S. Patent No. 5,667,988; and U.S. Patent No. 5,885,793.

In addition to the use of display libraries, other methods can be used to obtain an antibody. For example, a protein or a peptide thereof can be used as an antigen in a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen- specific monoclonal antibodies derived from the genes with the desired specificity can be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, U.S. Published Patent Application No. 2003/0070185, PCT Publication No. WO 96/34096, and PCT Publication No. WO 96/33735. In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized. Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207, Oi et al. (1986) BioTechniques 4:214, and by U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 5,859,205; and U.S. Patent No. 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, can be obtained from a hybridoma producing an antibody against a predetermined target, as described above, from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector. In one embodiment, the expression vector comprises a polynucleotide encoding a glutamine synthetase polypeptide. (See, e.g., Porter et al., Biotechnol. Prog 26(5): 1446-54 (2010).)

The antibody can include a human Fc region, e.g., a wild- type Fc region or an Fc region that includes one or more alterations. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237. Antibodies can have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation. For example, antibodies can have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260. Antibodies can also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, see, for example, U.S.

Published Application No. U.S. 2005/0037000.

In other embodiments, the antibody can be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used in this context, "altered" means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites can be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art.

The antibodies can be in the form of full length antibodies, or in the form of fragments of antibodies, e.g., Fab, F(ab')2, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, e.g., U.S. Patent Publication No. 2005-0079574 and Davies et al. (1996) Protein Eng. 9(6):53l-7.

In another embodiment, the antibody can be a human, humanized, CDR-grafted, chimeric, mutated, affinity matured, deimmunized, synthetic or otherwise in vitro-generated antibody, and combinations thereof.

In some embodiments, the heavy and light chains of the antibody can be substantially full- length. The encoded heterologous protein can include at least one or two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment). In yet other embodiments, the antibody has a heavy chain constant region. The antibody can be an IgGi, IgCT, IgCT, IgG₄, IgM, IgAi, IgA₂, IgD, or IgE. In some embodiments, the heavy chain constant region is human or a modified form of a human constant region. In another embodiment, the antibody has a light chain constant region chosen from, e.g., kappa or lambda, particularly, kappa (e.g., human kappa).

B. Receptors

The heterologous protein produced using the disclosed methods can be a receptor.

Receptors are typically trans-membrane glycoproteins that function by recognizing an extra-cellular signaling ligand. The receptor can be any type of receptor, include, but not limited to, a receptor for a hormone, growth factor, or neurotransmitter.

In some embodiments, the receptor can have a kinase domain in addition to the ligand recognizing domain, which initiates a signaling pathway by phosphorylating target intracellular molecules upon binding the ligand, leading to developmental or metabolic changes within the cell. In one embodiment, the receptor is modified so as to remove the transmembrane and/or intracellular domain(s), in place of which there can optionally be attached an Ig-domain. In one embodiment, the receptor is a receptor tyrosine kinase (RTK). The RTK family includes receptors involved with a variety of functions numerous cell types (see, e.g., Yarden and Ullrich, Ann. Rev. Biochem.

57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254, 1990). Non-limiting examples of RTK receptors include members of the fibroblast growth factor (FGF) receptor family, members of the epidermal growth factor receptor (EGF) family, platelet derived growth factor (PDGF) receptor, tyrosine kinase with immunoglobulin and EGF homology domains- 1 (TIE-l) and TIE-2 receptors (Sato et ak, Nature 376(6535):70-74, 1995) and c-Met receptors. Other non-limiting examples are fetal liver kinase 1 (FLK-l) (also referred to as kinase insert domain-containing receptor (KDR) (Terman et ak, Oncogene 6:1677-83, 1991) or vascular endothelial cell growth factor receptor 2 (VEGFR-2)), fins-like tyrosine kinase-l (Flt-l) (DeVries et al. Science 255; 989-991, 1992;

Shibuya et ak, Oncogene 5:519-524, 1990), also referred to as vascular endothelial cell growth factor receptor 1 (VEGFR-l), neuropilin-l, endoglin, endosialin, and Axl.

The heterologous protein can be a G-protein coupled receptor. A G-protein coupled receptor (GPCR) has seven transmembrane domains; upon binding of a ligand to a GPCR, a signal is transduced within the cell which results in a change in a biological or physiological property of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels which are modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs. These genes and gene-products are potential causative agents of disease.

The GPCR protein superfamily contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species. The superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the 2-adrenergic receptor and represented by over 200 unique members; Family II, the recently characterized parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family in mammals; Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptors such as STE2. Any of these can be the heterologous protein.

C. Growth Factors and Other Signaling Molecules

The disclosed methods can also be used to produce a heterologous protein that is a growth factor or signaling molecule. Growth factors are secreted by cells and bind to and activate receptors on other cells, initiating a metabolic or developmental change in the receptor cell.

Non-limiting examples of mammalian growth factors and other signaling molecules include cytokines; epidermal growth factor (EGF); platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs) such as a-FGF and b-FGF; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF- beta 5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD- 19;

erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon- alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M- CSF, GM-CSF, and G-CSF. Cytokines include interleukins (ILs), e.g., IL-l to IL-10.

The heterologous protein can be a hormone, such as insulin, proinsulin, glucan, somatostatin, follicle stimulating hormone, calcitonin, luteinizing hormone. The heterologous protein can be an anti-clotting factors such as Protein C. The heterologous protein can be atrial natriuretic factor or a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA). The heterologous protein can be bombesin, thrombin, hemopoietic growth factor, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MIP-l -alpha), mullerian-inhibiting substance, relaxin A-chain, relaxin B-chain, prorelaxin, mouse gonadotropin-associated peptide. The heterologous protein can be a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin NT)-3, -4, -5, or -6, or a nerve growth factor.

D. Clotting Factors

In some embodiments, the heterologous protein is a clotting factor. A clotting factor is a molecule, or analog thereof, that prevents or decreases the duration of a bleeding episode in a subject with a hemostatic disorder. For example, a clotting factor can be a full-length clotting factor, a mature clotting factor, or a chimeric clotting factor.“Clotting activity” is the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode. Examples of clotting factors can be found in U.S. Patent No. 7,404,956, which is herein incorporated by reference.

In one embodiment, the clotting factor is Factor VIII, Factor IX, Factor XI, Factor XII, fibrinogen, prothrombin, Factor V, Factor VII, Factor X, Factor XIII or von Willebrand Factor. The clotting factor can be a factor that participates in the extrinsic pathway. The clotting factor can be a factor that participates in the intrinsic pathway. Alternatively, the clotting factor can be a factor that participates in both the extrinsic and intrinsic pathway.

The clotting factor can be a human clotting factor or a non-human clotting factor. The clotting factor can be chimeric clotting factor, for example wherein the clotting factor includes a portion of a human clotting factor and a portion of a non-human clotting factor, such as, but not limited to, a porcine clotting factor.

In some embodiments, the clotting factor is a Factor VIII protein. A functional FVIII protein can be a fusion protein, such as, but not limited to, a fusion protein comprising a fully or partially B -domain deleted FVIII, at least a portion of an immunoglobulin constant region, such as an Fc domain. A number of functional FVIII variants have been constructed and can be produced using the methods disclosed herein. See PCT Publication No. WO 2011/069164, PCT Publication No. WO 2012/006623, PCT Publication No. WO 2012/006635, or PCT Publication No. WO 2012/006633 A2 for specific FVIII proteins.

In some embodiments, the recombinant FVIII protein includes a heterologous moiety. In one embodiment, the heterologous moiety can be a half-life extending moiety. Examples of the heterologous moieties include, but are not limited to, an immunoglobulin constant region or a fragment thereof, such as an Fc region or an FcRn binding partner, a VWF molecule, albumin, albumin binding polypeptide, Fc, PAS, the .beta subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding small molecules, or combinations thereof.

The heterologous protein can be a long-acting or long-lasting FIX polypeptide, such as a chimeric polypeptide comprising a FIX polypeptide and an FcRn binding partner. In one embodiment, the FIX polypeptide is a human, bovine, porcine, canine, feline, or murine FIX polypeptids. The full length polypeptide and polynucleotide sequences of FIX are known, as are many functional variants, e.g., fragments, mutants and modified versions. The clotting factor can also include a FIX protein or any variant, analog, or functional fragments thereof. A great many functional FIX variants are known, see, for example, PCT Publication No. WO 02/040544 A3, which discloses mutants that exhibit increased resistance to inhibition by heparin and PCT

Publication No. WO 03/020764 A2, which discloses FIX mutants with reduced T cell

immunogenicity, and PCT Publication No. WO 2007/149406 A2, which discloses functional mutant FIX molecules that exhibit increased protein stability, increased in vivo and in vitro half- life, and increased resistance to proteases The heterologous protein can be a FIX protein that includes a non-functional mutation, see PCT Publication No. WO 09/137254 A2.

In some embodiments, the clotting factor is a mature form of Factor VII or a variant thereof. Factor VII (FVII, F7; also referred to as Factor 7, coagulation factor VII, serum factor VII, serum prothrombin conversion accelerator, SPCA, proconvertin and eptacog alpha) is a serine protease that is part of the coagulation cascade. FVII includes a Gla domain, two EGF domains (EGF- 1 and EGF-2), and a serine protease domain (or peptidase Sl domain) that is highly conserved among all members of the peptidase Sl family of serine proteases, such as for example with chymotrypsin. FVII occurs as a single chain zymogen, an activated zymogen-like two-chain polypeptide and a fully activated two-chain form.

Exemplary FVII variants include those with increased specific activity, e.g., mutations that increase the activity of FVII by increasing its enzymatic activity (Kcat or Km). Such variants have been described in the art and include, for example, mutant forms of the molecule as described for example in Persson et al. 2001. PNAS 98:13583; Petrovan and Ruf 2001. J. Biol. Chem. 276:6616; Persson et al. 2001 J. Biol. Chem. 276:29195; Soejima et al. 2001. J. Biol. Chem. 276:17229; Soejima et al. 2002. J. Biol. Chem. 247:49027. Exemplary mutations include V158D-E296V- M298Q. High specific activity variants of FIX are also known in the art. For example, Simioni et al. (2009 N. E. Journal of Medicine 361:1671) describe an R338L mutation. Chang et al. (1988 JBC 273:12089) and Pierri et al. (2009 Human Gene Therapy 20:479) describe an R338A mutation. Other mutations are known in the art and include those described, e.g., in Zogg and Brandstetter. 2009 Structure 17: 1669; Sichler et al. 2003. J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. FEBS Lett 412:295. The contents of these references are incorporated herein by reference.

Patents or applications disclosing examples of clotting factors that can be produced using the disclosed methods include U.S. Patent No. 7,404,945, U.S. Patent No. 7,348,004, U.S. Patent No. 7,862,820, U.S. Patent No. 8,329,182, U.S. Patent No. 7,820,162.

E. Viruses and Vaccines for Infectious Agents

The disclosed methods can be used for production of viruses and viral proteins. Thus, in some embodiments, the heterologous protein is a viral protein. The present disclosure is directed to the production of proteins. In some embodiments, the protein is present as part of a virus. Thus, in some embodiments, viruses and viral particles can be produced using the disclosed methods. In some embodiments, the vector is a viral vector, and the heterologous protein is a viral protein. In further embodiments, the heterologous protein is from the same vims as the vector. In one non limiting example, the viral protein is an influenza virus protein, and the vector is an influenza virus vector. In more embodiments, the heterologous protein is from a different virus that the vector. In one non- limiting example, the viral protein is an influenza vims protein, and the vector is an adenovims vector. In additional embodiments, the methods utilize a plasmid encoding a viral protein.

In some embodiment, a vims is utilized in the disclosed methods, wherein the virus infects the cultured cells. In some embodiments, the cells are mammalian, and the vimses is from the genera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flavivimses, arenaviruses, herpesvimses, poxviruses, coronaviruses and adenoviruses. The virus used can be a wild- type virus, an attenuated virus, or a recombinant virus. In addition, instead of actual virions being used to infect the cells with a vims, an infectious nucleic acid can be a viral vector including a viral protein. In one embodiment, the virus produced is an influenza virus. In another embodiment, the viral protein is a hemagglutinin (HA) protein. The viral antigen can be a whole inactivated vims, a split vims, a modified vims, viral proteins, such as proteins, like haemagglutinin or neuraminidase, or envelope proteins.

In some embodiments the vims is an enveloped virus. Enveloped vimses, include, but are not limited to, flavivimses, togaviruses, retroviruses, coronavimses, filovimses, rhabdoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses, arenavimses, hepadnaviruses, herpesvimses, and poxviruses. In other preferred embodiments, the vimses are flaviruses, coronaviruses, orthomyxoviruses, or togaviruses. Exemplary enveloped viruses are influenza, including strains of influenza A, B or C, West Nile, and Ross River viruses (RRV.)

The virus can be an enveloped RNA virus. Enveloped RNA viruses, include, but are not limited to, flaviviruses, togaviruses, retroviruses, coronaviruses, filoviruses, rhabdoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses, and arenaviruses. The virus can be an orthomyxovirus, for example, an influenza virus strain. Influenza vims strains may have varying combinations of hemaglutianin and neuraminidase surface proteins.

In another example, the vims is a togavims, for example an alphavirus such as the RRV). The vims can be a coronavims, including the Severe Acute Respiratory Syndrome (SARS) vims. In other embodiments, the virus is a flavivirus, including Japanese Encephalitis, tick borne encephalitis (TBE), Dengue fever vims, yellow fevers vims, West Nile Vims and hemorrhagic fever vims.

The vims can be a poxvirus, such as an orthopox-vims (such as vaccinia or modified vaccinia Ankara viruses), or an avipoxvirus.

The vims can be a lentivirus, such as, but not limited to a human immunodeficiency vims (HIV), such as HIV-l or HIV2. The disclosed methods can be used to produce a vaccine, such as a gp4l, pgl20, or gpl60 protein.

In some embodiments, the disclosed methods are of use for protein of vimses or viral antigens of Retroviridae Picornaviridae (for example, polio vimses, hepatitis A vims;

enteroviruses, human coxsackie viruses, rhinovimses, echovimses); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, equine encephalitis viruses, rubella vimses); Flaviridae (for example, dengue viruses, encephalitis vimses, yellow fever viruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (for example, vesicular stomatitis vimses, rabies viruses); Filoviridae (for example, ebola vimses); Paramyxoviridae (for example, parainfluenza viruses, mumps vims, measles vims, respiratory syncytial virus); Orthomyxoviridae (for example, influenza viruses); Bungaviridae (for example, Hantaan viruses, bunga vimses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever vimses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvo vimses); Papovaviridae (papilloma vimses, polyoma viruses); Adenoviridae (most adenoviruses);

Herpesviridae (herpes simplex vims (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovims (CMV), herpes vimses); Poxviridae (variola vimses, vaccinia vimses, pox viruses); and

Iridoviridae (such as African swine fever virus); and unclassified viruses (for example, the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

The disclosed methods are no limited to the production of viruses and viral antigens. The disclosed methods are also of use for producing bacterial antigens and fungal antigens.

Examples of fungal infections include but are not limited to: aspergillosis; thrush (caused by Candida albicans), cryptococcosis (caused by Cryptococcus), and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Specific examples of bacterial pathogens include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. ( such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis an d Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari an d Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum an d Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae an d Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropatho genic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli an d uropathogenic E. coli ) Enterococcus sp. (such as

Enterococcus faecalis an d Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia an d Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis,

Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus an d Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi an d Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis an d Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp.,

Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,

Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium,

Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, an d Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica an d Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides.

Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris an d Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri an d Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes,

Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis an d Salmonella typhimurium), Serratia sp. (such as Serratia marcesans an d Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as

Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin- resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline -resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim- resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4

Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum an d Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio

alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela an d Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis ) and Xanthomonas maltophilia among others.

Bacterial antigens suitable for use in the disclosed methods include proteins,

polysaccharides, lipopolysaccharides, and outer membrane vesicles which may be isolated, purified or derived from a bacterium. In addition, bacterial antigens include bacterial lysates and inactivated bacteria formulations. Bacteria antigens can be produced by recombinant expression. Bacterial antigens preferably include epitopes which are exposed on the surface of the bacteria during at least one stage of its life cycle. Bacterial antigens include but are not limited to antigens derived from one or more of the bacteria set forth above as well as the specific antigens examples identified below.

Neiserria gonorrhoeae antigens include Por (or porin) protein, such as PorB (see, e.g. , Zhu et al. (2004) Vaccine 22:660-669), a transferring binding protein, such as TbpA and TbpB (see, e.g. , Price et al. (2004) Infect. Immun. 7l(l):277-283), an opacity protein (such as Opa), a reduction- modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see, e.g. , Plante et al. (2000) J. Infect. Dis. 182:848-855); WO 99/24578; WO 99/36544; WO 99/57280; and WO

02/079243, all of which are incorporated by reference).

Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba and C (agents of trachoma, a cause of blindness), serotypes Li, L3 (associated with Lymphogranuloma venereum), and serotypes, D-K. Chlamydia trachomas antigens also include antigens identified in WO 00/37494; WO 03/049762; WO 03/068811; and WO 05/002619 (all of which are incorporated by reference), including PepA (CT045), LcrE (CT089), Art (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), MurG (CT761), CT396 and CT761, and specific combinations of these antigens. Treponemapallidum (Syphilis) antigens include TmpA antigen.

In some embodiments, the antigen is from an infectious agent that causes a sexually transmitted disease (STD). Such antigens can provide for prophylactis or therapy for STDs such as chlamydia, genital herpes, hepatitis (such as HCV), genital warts, gonorrhea, syphilis and/or chancroid (see PCT Publication No. WO 00/15255, which is incorporated by reference). Antigens may be derived from one or more viral or bacterial STDs. Viral STD antigens for use in the invention may be derived from, for example, HIV, herpes simplex virus (HSV-I and HSV-2), human papillomavirus (HPV), and hepatitis (HCV). Bacterial STD antigens for use in the invention may be derived from, for example, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponemapallidum, Haemophilus ducreyi, E. coli, and Streptococcus agalactiae.

F. Tumor Antigens

The disclosed methods can be used for production of a heterologous protein that is a tumor antigen. Exemplary tumor antigens (antigens produced by tumor cells that can stimulate tumor- specific T-cell immune responses) include one or more of the following RAGE-l, tyrosinase, MAGE-l, MAGE-2, NY-ESO-l, Melan-A/MART-l, glycoprotein (gp) 75, gplOO, beta-catenin, preferentially expressed antigen of melanoma (PRAME), MUM-l, Wilms tumor (WT)-l, carcinoembryonic antigen (CEA), and PR-l. Additional tumor antigens are known in the art (for example see Novellino et al, Cancer Immunol. Immunother. 54(3): 187-207, 2005) and are described below. Tumor antigens are also referred to as“cancer antigens.” The tumor antigen can be any tumor-associated antigen, which are well known in the art and include, for example, carcinoembryonic antigen (CEA), b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin- reactive AFP, thyroglobulin, RAGE-l, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, macrophage colony stimulating factor, prostase, prostate- specific antigen (PSA), PAP, NY-ESO-l, LAGE-la, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumor antigen- 1, MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. A list of selected tumor antigens and their associated tumors are shown below.

Exemplary tumors and their tumor antigens

The disclosure is further illustrated by the following non-limiting Examples.

EXAMPLES

Disclosed herein is a versatile perfusion system to a TGE bioprocess for production of an exemplary product, namely a universal influenza vaccine, Hl stabilized-stem nanoparticle (Hl-ss- NP). Hl -ss-NP is structurally defined as a combination of eight trimers of the highly conserved hemagglutinin (HA) stem region on a ferritin nanoparticle and has been shown to elicit immune responses to heterosubtypic influenza virus in mice and ferret models (Yassine et al., 2015). The implication of these animal models can be significant since this universal vaccine may replace iterative yearly vaccination for preventing the seasonal flu. For further efficacy examination of this vaccine in human clinical trials, early-stage TGE bioprocess development was required. The development of the TGE bioprocess included optimization of conditions for cell growth in the bioreactor. In addition, conditions were assessed for transfection mediated by polyethylenimine (PEI), and expression for an extended period. The use of the methods disclosed herein result in a high product yield, and can be used for the production of other products, such as, but not limited to, vaccines and antibodies. Example 1

Materials and Methods

Cells and Material: Serum-free suspension-adapted HEK 293 cell line (VRC 293) from a cGMP master cell bank was used for manufacturing clinical materials. Cells were cultured in CDM4HEK293 media (SH3A3770.02, HyClone, Chicago, IL) supplemented with 0.2 % sodium bicarbonate (BDH9280, VWR, Radnor, PA), 0.1 % poloxamer 188 (P1169, Spectrum, New Brunswick, NJ), 0.875 g/L of L-glutamine (2078-06, JT Baker, Center Valley, PA), and 4.605 g/L of sodium chloride (SO 160, Spectrum, New Brunswick, NJ). For expression media, 7 g/L of Cell Boost 5 supplement (SH30865.01, HyClone, Chicago, IL) and 0.2 g/L of dextran sulfate (D4911- 10G, Sigma, St. Louis, MO) was added to the growth media. For expression feed, a solution of 62.5 g/L dextrose anhydrous, 14.6 g/L of L-glutamine, 60 g/L of Cell Boost 5 supplement was prepared. Cells were maintained in 3L Erlenmeyer flasks (431252, Coming, Steuben, NY) in an incubator (AJ125, Infors HT, Bottmingen, Switzerland) maintained at 37 °C with 130 rpm, 5 % C02 and 80 % humidity. Cell count and viability were determined using Vicell XR instrument (Beckman, Fullerton, CA). Hl-ss-NP plasmid DNA (VRC 3928, Aldervron, Fargo, ND) was used for the transfection.

Transient Transfection: Cationic polymer polyethylenimine (PEI) with linear MW 25,000 (23966, PolySciences, Warrington, PA) was used to mediate the anionic plasmid DNA interaction with the anionic cell surface for further endocytosis. Briefly, the CDM4HEK293 culture media was exchanged with FREESTYLE™ 293 (12338-002, Invitrogen, Carlsbad, CA), and the HEK 293 cells were concentrated to a target density of ~20 x 10⁶ cells/ml afterwards (Sun et ak, Biotechnol Bioeng, 99(1), 108-116. doi: l0.l002/bit.2l537, 2008). Then these cells were incubated with 20 mg/ml plasmid DNA for 5 min followed by 40 mg/ml PEI (Tait et ak, 2004) for 2 hours (hrs) without pH and dissolved oxygen (DO) controls. The pH and DO controllers were turned off during the 2 hrs of transfection because a zero-microbubble condition was preferred for the success of the transfection. Based on previous experience, the polarity of the microbubble interfered with the electrostatic interaction between plasmid DNA and PEI that will eventually get endocytosed by the cell. After 2 hrs of transfection, expression media was added to dilute the cells to a target density of -10 x 10⁶ cells/ml.

Fed-batch and Perfusion System: For the fed-batch TGE bioprocess, cells were cultured in shake flasks, spun down using a centrifuge (kSep400, Satorius-Stedim, Bohemia, NY), resuspended in FREESTYLE™ 293 media at a target density, and transferred to the bioreactor. Next the plasmid DNA and PEI were added for transient transfection. After the transfection duration, the cells were diluted and monitored for 4 days. During the expression of the transient gene, 4 mM valproic acid (VP A) (1069-66-5, MP Biomedicals, Santa Ana, CA), expression feed, and 6 mM L- glutamine was added at day 1, 2, and 3, respectively, to promote less cell aggregation and supplement the necessary nutrients. A summarized fed-batch TGE bioprocess protocol is shown in Fig. 1A.

For the perfusion-based TGE bioprocess, an alternating tangential flow (ATF) filtration system (Refine Technology) with a 50 kDa hollow fiber filter (F2:RF50PS, Repligen, Waltham, MA) was used. Pump setting of 0.7 LPM during both exhausting and filling was used to retain the cells while filtrating the cell culture waste and unwanted small molecules. At the same time, fresh media was replenished with the guidance of a level controller feedback loop to maintain a constant volume in the reactor, allowing for the transiently transfected cells to express improved yields for the protein of interest.

To perform a media exchange, the perfusion system ran at 5 VVD overnight l-day prior to transfection using FREESTYLE™ 293 media. During this period, almost 97% of the media was exchanged from culture media to FREESTYLE™ 293. Next, the cells were concentrated to the target density using the perfusion system without any media replacement. Once the cells were concentrated to a reduced volume, the perfusion system was turned off and the cells were transfected with the plasmid DNA and PEI. After the transfection duration, the cells were diluted to a target density using expression media, and the pH and DO controls were turned on to the original set points. To suppress epigenetic effects in recombinant production (Backliwal et ak, 2008), 4mM valproic acid (VP A) was added 24 hrs post-transfection. Finally, perfusion was turned on either at day 1 or 4 post-transfection at a rate of 25 or 100 pL/cell/day. The average viable cell density (VCD), viability, titer, and metabolites were monitored during the 9 days of culture. A summarized perfusion-based TGE bioprocess protocol is shown in Fig. 1B.

For both the fed-batch and perfusion vessels, 3L bioreactors (Applikon, Foster City, CA) were maintained with set points of 37 °C, 7.1 ± 0.2 pH, 50 % DO, 295 rpm (for culture) or 350 rpm (for transfection and expression). The stir speed was increased to 350 rpm during transfection to increase the probability of DNA and PEI interaction with the cell membrane.

Ion exchange and size exclusion chromatography: The concentration of the Hl-ss-NP was determined by running the sample in both ion exchange (TSK-Gel Q-STAT, Tosoh Biosciences, Griesheim, Germany) and size exclusion chromatography (SRT SEC-500, Sepax, Newark, DE) columns. Negatively charged particles having similar size to Hl-ss-NP in cell culture supernatant were retained from the ion exchange column with 2X PBS. Unwanted molecules were eluted by 1M NaCl. The titer was determined by comparing total Hl-ss-NP peak areas of UV absorbance with a standard calibration curve.

Example 2

Fed-batch TGE Process

A fed-batch TGE bioprocess was developed that consisted of external cell culture, media exchange, cell concentration, and transfer to a bioreactor for transfection and expression (Fig. 1A). Despite being labor intensive and having inherently higher contamination risks during the centrifugation steps, the developed process yielded reliable cell growth with sufficient viability and titer. With the use of the daily feeding strategy, the variable cell density (VCD) increased by 38% on day 1 and thereafter decreased (filled circle, Fig. 2A) until day 4. The cultures maintained high viability of over 80 % throughout the ran (open circle, Fig. 2A). The fed-batch bioprocess produced an average titer of -106.7 ± 29.7 mg/L on day 4 (Fig. 2B).

Elimination of the microbubble formation from the pH and DO sparging controllers during the incubation of DNA and PEI was important for transfection. When microbubbles were present during the transfection, the culture produced substantially lower titer (filled square, Fig. 2B) despite the comparable cell growth (filled square, Fig. 2A). Interestingly, the viability of cells with microbubbles during the transfection decreased (opened square, Fig. 2A) much faster, probably due to toxic contributions from PEI which failed to form complex with plasmid DNA in the presence of the microbubbles. However, these microbubble effect was not as detrimental in metabolite profiles (Fig. 2C-G). Despite the stable level of production using the fed-batch process, there was short expression time span and high contamination risk.

Example 3

Perfusion-based TGE Process

To improve the TGE production, a perfusion-based strategy was integrated into the fed- batch process. In contrast to the fed-batch TGE bioprocess, the perfusion-based process allowed the cells to be cultured in the bioreactor from the beginning. These cells were washed,

concentrated, transfected and expressed all within the same vessel (Fig. 1B). During four days of initial culture in the bioreactor, the VCD reached -7-8 x 10⁶ cells/ml (filled circle, Fig. 3A) with the cell viability maintaining above 95 % (opened circle, Fig. 3A) while successfully maintaining the metabolites levels (Fig. 3B-F). A day prior to transfection, the perfusion system was used to initiate a media exchange followed by cell concentration on the day of transfection. By implementing the perfusion system at this step, the risk of contamination during media exchange and manual spin down of the cells in the fed-batch TGE bioprocess was eliminated. After overnight media exchange and concentration, the cells were then transfected and cultured for expression of the transient gene with continuous supplement of new media via the perfusion system.

To improve the 4-day fed-batch TGE bioprocess, the effect of starting the perfusion system at day 4 during expression, after the 4-days of regular feeding strategy (opened diamond, Fig. 4A- C), was tested. The effect was significant that the VCD started to increase again at day 4 and at day 9 reached an average of 16.9 ± 4.2 x 10⁶ cells/ml (open diamond, Fig. 4A) with cell viability of 80 % (open diamond, Fig. 4B). Importantly, the protein titer resulted in 339.5 ± 96.1 mg/L at day 9 (open diamond, Fig. 4C). It was also tested whether the supplement from the perfusion system at day 4 can override 4-days of feeding strategy with either 100 or 25 pL/cell/day perfusion rate. Surprisingly, 100 pL/cell/day perfusion starting from day 4 resulted in similar VCD (opened circle, Fig. 4A), viability (opened circle, Fig. 4B) and titer (opened circle, Fig. 4C) to the pervious data. This evidenced perfusion supplement can override the daily feeding strategy. In addition, 25 pL/cell/day perfusion starting from day 4 resulted poorly in VCD (opened triangle, Fig. 4A) and viability (opened triangle, Fig. 4B) but produced comparable titer of 324.5 mg/L at day 9 (open triangle, Fig 4C), which is still significantly greater than the fed-batch results. The metabolite profile showed 25 to 100 pL/cell/day, perfusion was required to sufficiently maintain glucose (opened circle vs. triangle, Fig. 4D) and glutamine (opened circle vs. triangle, Fig. 4E) levels while suppressing lactate (opened circle vs. triangle, Fig. 4G) and NH4 (opened circle vs. triangle, Fig. 4H) levels.

To further improve the process, it was determined if the effect of the perfusion system can be initiated earlier than day 4 after transfection to reduce the delay time and to benefit earlier production during the expression stage. The effect of starting the perfusion system at day 1 of expression was tested, thus eliminating the feeding strategy to make the process simpler. As a control, a process with no perfusion during the expression resulted in an average peak VCD of 12.7 ± 0.6 x 10⁶ cells/ml with viability of 89 % at day 4 (filled square, Fig. 5A and B) but decayed significantly thereafter to 7.5 ± 5.8 x 10⁶ cells/ml with viability of 58 % at day 6. Furthermore, the titer was only 147.6 ± 14.5 mg/L at day 6 (filled square, Fig. 5C). In comparison, with 100 pL/cell/day perfusion staring at day 1, the VCD increased gradually, reaching an average of 28.4 ± 3.6 x 10⁶ cells/ml at day 9 (filled circle, Fig. 5A) with high cell viability (> 85 %) (filled circle, Fig. 5B) throughout the run. The protein titer reached an average of 269.1 ± 76.4 mg/L at day 9 (filled circle, Fig. 5C), which was 2.5 times that of the 4-day fed-batch process. In contrast, the25 pL/cell/day perfusion starting at day 1 (filled triangle, Fig. 5A-C) sustained the VCD and viability better than no perfusion but resulted in marginal increase in titer of 192.2 mg/L at day 9.

The benefit of having perfusion starting from day 1 was evident in the metabolite profile. Whereas the process without perfusion resulted in significant reduction in daily measure of metabolite levels by day 4 (opened square, Fig. 5D-H), the process with 25 and 100 pL/cell/day perfusion maintained adequate levels throughout the ran (opened circle vs triangle, Fig. 5D-H). The 100 pL/cell/day perfusion ran maintained metabolites at the following averages: glucose > 2.1 g/L (Fig. 5D), glutamine > 2.0 mM (Fig. 5E), glutamate > 1.8 mM (Fig. 5F), and lactate < 1.4 g/L level (Fig. 5G). In addition, the perfusion-based processes maintained a low level of NH₄ (< 4.1 mM) which was not suppressed in the absence of the perfusion system (Fig. 5H). A minimum of 25 pL/cell/day perfusion was necessary to maintain metabolites in certain level (Fig. 5E-H).

Overall, the run with 25 to 100 pL/cell/day perfusion starting at day 1 without any feeding resulted in continuous cell growth with high viability and increased titer with sufficient level of metabolites during the expression.

Example 4

Productivity and Cost Comparison Between Fed-batch vs. Perfusion-based TGE Bioprocess

The benefits from ATF perfusion-based TGE bioprocess with a perfusion rate of 100 pL/cell/day starting at Day 1 post-transfection can be understood by plotting the cell specific productivity (Qp), which is the daily increment of the titer divided by the cell number at a given day. Interestingly, both bioprocesses with or without ATFperfusion showed similar peak Qp at Day 3 as 4.97 ± 0.56 and 4.05 ± 0.94 pg/cell/day, respectively (Fig. 6A). However, the trend of decline in Qp afterwards showed a significant difference between the two bioprocesses (Fig. 6A). Whereas the bioprocess without ATF perfusion catastrophically decreased its Qp to the level of 1.17 ± 0.75 pg/cell/day at Day 4 and almost zero at Day 5, the bioprocess with ATF perfusion marginally declined to the level of 2.98 ± 0.73 pg/cell/day at Day 4 and sustained production until Day 9.

It has been suggested that perfusion-based bioprocesses are more cost effective than fed- batch processes (Pollock et ak, Biotechnol Bioeng, 110(1), 206-219. doi: l0.l002/bit.24608, 2013). To further demonstrate the benefit of a perfusion-based TGE bioprocess to a TGE fed-batch process, the financial aspects were considered. In terms of consumable cost during a single run, a 9-day perfusion-based 3L bioreactor process (100 pL/cell/day from day 1) costs ~$3,200 which included the costs for alternating tangential flow (ATF) filter, culture media and FREESTYLE™ 293 transfection media. This cost was 16 % lower than the cost for the 4-day fed-batch 3L bioreactor process which was $3,800. Including costs for centrifugation single-use inserts, culture media and FREESTYLE™ 293 media. For an enhanced cost comparison of the two processes, these consumable costs were divided by the average titer of the bioprocess to derive a representative value of consumable cost per milligram. Whereas the consumable cost per milligram was $22.3 for a 4-day fed-batch process, it was $7.4 for 9-day perfusion-based process (100 pL/cell/day from day 1) which was a 67 % saving (Fig. 6B). The significant benefits of perfusion-based TGE bioprocess, including high productivity, robustness, and less contamination risk, can be transferred to a large scale in biomanufacturing production and provide enhanced financial benefits.

A TGE bioprocess was developed that uses a perfusion system that resolves the current fed- batch limitations. This process was used for Hl-ss-NP vaccine production and can easily be applied to other products. The components of this development were two-fold: 1) the conditions for PEI mediated gene transfection in the bioreactor without microbubbles; and 2) the

implementation of the perfusion-based system for a single-system transfection operation, combining the steps of cell concentration, waste clearance, culturing/media replenishing, and protein expression within a single vessel. The disclosed bioprocess achieved high productivity, robustness, cost efficiency and less contamination risk.

Multiple transfection can be conducted in the same bioreactor by repeatedly exchanging the media and concentrating the cells with perfusion system (Cervera et ak, Biotechnol Bioeng, 112(5), 934-946. doi:l0.l002/bit.25503, 2015). In addition, the perfusion rate can be varied during the ran to readily supplement the necessary nutrient needs as cells continuous to grow. The disclosed methods can be used for rapid protein expression, vaccine development and drug manufacturing.

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for producing a heterologous protein, comprising:

a) culturing host cells in an a growth medium in a perfusion bioreactor at a density of 5.5 X l0⁶to 8.5 X 10⁶ viable cells/ml wherein the host cells are in an exponential phase of growth; b) replacing the growth medium with a transfection medium using perfusion;

c) concentrating the host cells in the transfection medium using perfusion to a cell density of 13.5 X l0⁶ to 17.5 X 10⁶ viable cells/ml;

d) transfecting the host cells with a vector encoding the heterologous protein in the absence of aeration and perfusion in the bioreactor, wherein the host cells express the heterologous protein in the bioreactor;

e) replacing the transfection medium in the perfusion bioreactor with an expression medium using perfusion that begins 1 to 4 days following step d);

f) culturing the host cells expressing the heterologous protein in the expression medium in the bioreactor at a perfusion rate of 25 pL/cell/day to 100 pL/cell/day at a constant total volume, and

g) isolating the heterologous protein, thereby producing the heterologous protein.

2. The method of claim 1, wherein the vector is a plasmid or a viral vector.

3. The method of claim 1 or claim 2, wherein the heterologous protein is a vaccine, an scFv, a heavy chain domain of an antibody, or a light chain domain of an antibody.

4. The method of any one of claims 1-3, wherein transfecting the host cells comprises contacting the host cells with the vector and with polyethylenimine (PEI).

5. The method of claim 4, wherein the host cells are contacted with the vector and the PEI at a ratio around 1:2.

6. The method of any one of claims 1-5, wherein the method produces the recombinant protein at an accumulated titer of about 125-175 mg/L at four days following step b.

7. The method of any one of claims 1-5, wherein the method produces the recombinant protein at an accumulated titer of about 160 mg/L at four days following step b.

8. The method of any one of claims 1-5, wherein the method produces the recombinant protein at an accumulated titer of about 200-300 mg/L at nine days following step b.

9. The method of claim 8, wherein the method produces the recombinant protein at an accumulated titer of about 270 mg/L at nine days following step b.

10. The method of any one of claims 1-9, wherein the host cells are mammalian host cells.

11. The method of claim 10, wherein the host cells are human host cells.

12. The method of any one of claims 1-11, wherein the growth medium and the expression medium are the same.

13. The method of any one of claims 1-12, wherein transfecting the host cells in the absence of aeration minimizes formation of microbubbles in the transfection medium.

14. The method of any one of claims 1-13, wherein the method further comprises adding valproic acid to the transfection medium following transfecting the host cells with the vector in step d.

15. The method of any one of claims 1-14, further comprising monitoring cell viability, protein titer, and/or metabolites following transfecting the host cells with the vector in step d.

16. The method of any one of claims 1-15, wherein the perfusion is alternating tangential flow perfusion.

17. The method of any one of claims 1-16, wherein the growth medium, the transfection medium, and/or the expansion medium are serum free.

18. The method of any one of claims 1-17, wherein the perfusion rate is 25 pL/cell/day in step d.

19. The method of claim 18, wherein the perfusion rate is 100 pL/cell/day in step d.

20. The method of any one of claims 3-19, wherein the vaccine is a viral vaccine.

21. The method of claim 20, wherein the viral vaccine is an influenza virus vaccine, a papilloma vims vaccine, a human immunodeficiency vims vaccine, a rotavims vaccine, or an adenovims vaccine.