WO2023031878A1 - Surrogate dna to increase protein production by stably transfected mammalian cells - Google Patents

Abstract

The present disclosure relates to the use of surrogate DNA, such as salmon testis DNA or plasmid DNA, to increase protein production by stably transfected mammalian cells, such as Chinese Hamster Ovary (CHO) cells or human embryonic kidney (HEK) cells. Provided are stably transfected mammalian cells for production of proteins of interest, methods for producing proteins of interest in stably transfected mammalian cells, and compositions for stable transfection of mammalian cells to enable production of proteins of interest by the mammalian cells.

Description

SURROGATE DNA TO INCREASE PROTEIN PRODUCTION BY STABLY TRANSFECTED MAMMALIAN CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional application no. 63/239,991 filed September 2, 2021, the entire contents of which are incorporated herein by reference.

FIELD

[0002] The present disclosure relates to protein production in mammalian cells, particularly to methods and compositions for increased expression of recombinant proteins in mammalian cells, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

INTRODUCTION

[0003] Human or animal cells are routinely used in academia and industry to produce proteins. Proteins can be produced through transient or stable protein expression. For stable protein expression, generally a stable pool of cells which can be used for production is first generated, and/or cells within this pool are cloned to identify cell lines that are good producers. Either way, scientists are looking to increase the productivity of cells to reduce production costs.

[0004] Several ways are used to increase protein production from stably expressing cells, such as modifying the codons of the gene of interest, modifying the promoter, incorporating Scaffold/Matrix Attachment Region (S/MAR) or Ubiquitous Chromatin Opening Element (UCOE) elements, improving the cell culture media and feeds, and better selecting high expressing cells. Other methods to increase protein production from stable pools or cell lines are needed.

SUMMARY

[0005] The present inventors have surprisingly demonstrated that mixing surrogate DNA (also referred to as filler DNA or stuffer DNA) with an expression vector encoding a protein of interest increases production of the protein of interest by mammalian cells into which the mixture is stably introduced. Although the use of surrogate DNA for transient transfection of mammalian cells has been described previously (for example see Kichler et al, 2005; Carpentier et al, 2007; Kiseljak et al, 2011; Rajendra et al, 2011), the present inventors are not aware of any reports demonstrating that the use of surrogate DNA can lead to increased protein expression in stably transfected mammalian cells. Indeed, some published reports demonstrate a reduction in protein expression when surrogate DNA is used for transient transfection (e.g., see Rajendra et al, 2011).

[0006] Surrogate DNA, such as plasmid DNA or fragmented genomic DNA, was used by the present inventors in stable transfection of mammalian cells to increase protein production. The inclusion of surrogate DNA in the transfection mixture was unexpectedly found to increase productivity in stable cell lines. This effect was particularly pronounced for sheared salmon testis DNA, although similar effects were seen with other types of surrogate DNA, such as circular plasmid DNA, linearized plasmid DNA, and fragmented genomic DNA. The surrogate DNA used did not include a coding sequence for the protein of interest.

[0007] Accordingly, one aspect of the disclosure is a mammalian cell for production of a protein of interest, wherein the mammalian cell is produced by stable transfection of a mammalian host cell with a nucleic acid mixture, the nucleic acid mixture comprising at least one expression vector encoding the protein of interest and at least one surrogate DNA.

[0008] In an embodiment, the at least one expression vector is integrated into the genome of the mammalian cell.

[0009] In an embodiment, the protein of interest produced by the mammalian cell is an antibody or an antibody fragment. In a further embodiment, the antibody is Palivizumab.

[0010] In an embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0011] In an embodiment, the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

[0012] In an embodiment, the at least one surrogate DNA comprises salmon testis DNA. In an embodiment, the at least one surrogate DNA comprises fragmented salmon testis DNA. In an embodiment, the at least one surrogate DNA comprises sheared salmon testis DNA. [0013] In an embodiment, the molar ratio of the expression vector and the surrogate DNA in the nucleic acid mixture is within the range of 3:1 to 1:3. In an embodiment, the molar ratio of the expression vector and the surrogate DNA in the nucleic acid mixture is about 1: 1.

[0014] Another aspect of the disclosure is a method for producing a protein of interest in a mammalian cell, the method comprising: introducing into a mammalian host cell a nucleic acid mixture comprising at least one expression vector and at least one surrogate DNA; applying selective pressure to select for cells that stably carry the at least one expression vector; and culturing the cells selected in step b) under conditions for production of the protein of interest.

[0015] In an embodiment of the method, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0016] In an embodiment of the method, introducing the nucleic acid mixture into the mammalian host cell comprises transfection. In an embodiment, the transfection comprises transfection with polyethylenimine (PEI).

[0017] In an embodiment of the method, the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

[0018] In an embodiment of the method, the at least one surrogate DNA comprises fragmented salmon testis DNA.

[0019] In an embodiment of the method, the at least one surrogate DNA comprises sheared salmon testis DNA.

[0020] In an embodiment, the method further comprises isolating the produced protein of interest.

[0021] Another aspect of the disclosure is a composition for stable transfection of a mammalian cell to enable production of a protein of interest by the mammalian cell, the composition comprising at least one expression vector encoding the protein of interest and at least one surrogate DNA, wherein the ratio of the at least one expression vector and the at least one surrogate DNA in the composition is within the range of 3:1 to 1:3.

[0022] In an embodiment of the composition, the ratio of the at least one expression vector and the at least one surrogate DNA in the composition is about 1:1.

[0023] In an embodiment of the composition, the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

[0024] In an embodiment of the composition, the at least one surrogate DNA comprises fragmented salmon testis DNA. In an embodiment, the at least one surrogate DNA comprises sheared salmon testis DNA.

[0025] In an embodiment, the composition is for stable transfection of a Chinese Hamster Ovary (CHO) cell.

[0026] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages, objects and embodiments are expressly included within the scope of the present disclosure.

DESCRIPTION OF THE DRAWINGS

[0027] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0028] Figure 1 presents electrophoresis gels showing migration of surrogate DNAs tested. [0029] Figure 2 shows volumetric productivity of stable pools expressing the model antibody Palivizumab transfected or not with 50% sheared salmon testis DNA (stDNA).

[0030] Figure 3 shows titers of protein expressed by CHO cell pools co -transfected with 0%, 25%, or 50% of sheared salmon testis DNA. PLVZM = palivizumab, RTX = rituximab, MEDI- 573 = dusigitumab, CD200-Fc = CD200 human protein fused with Fc domain, and AlAT-His = alpha- 1 antitrypsin with 10 Histidine C-terminal tag.

[0031] Figure 4 shows volumetric productivity of CHO cell pools transfected with various surrogate DNAs. stDNA = salmon testis DNA, ssDNA = salmon sperm DNA, IFN-SAR = scaffold-attached regions from the human interferon beta, DNAzol™ gDNA = genomic CHO DNA extracted with DNAzol™ reagent and sonicated for 5-90 seconds, Zamenhof gDNA = genomic CHO DNA extracted using the method described in Zamenhof, 1957 (same protocol used by Sigma- Aldrich for stDNA extraction) and sonicated for 5-90 seconds.

[0032] Figure 5 shows volumetric productivity of three cell lines, CHO^55E1, CHO-3E7, and HEK293-6E, transfected with varying levels and types of surrogate DNA. stDNA = salmon testis DNA, VAconc = concatemers of DNA encoding viral RNA VA, pAdvantage = circular plasmid DNA from pAdvantage™, EBERconc = concatemers of DNA encoding Epstein-Barr viral RNA EBER-1, pLEX III = circular plasmid DNA from pLEX III.

[0033] Figure 6 shows volumetric productivity of pools transfected using Lipofectamine 3000™. stDNA = salmon testis DNA, pLEX III = circular plasmid DNA from pLEX III.

[0034] Figure 7 shows quantification of integrated gene copy numbers in CHO cell pools cotransfected with plasmids encoding CD200 human protein fused with Fc domain, dusigitumab, rituximab, or palivizumab; together with 0%, 25%, or 50% of sheared salmon testis DNA (stDNA). The plasmid encoding palivizumab was also co-transfected with 75% stDNA. CD200 = CD200 human protein fused with Fc domain, MEDI = dusigitumab, RTX = rituximab, PLVZM = palivizumab. DETAILED DESCRIPTION

[0035] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, 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 terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.

I. Definitions

[0036] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0037] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0038] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0039] All numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art. [0040] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified.

[0041] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of' or, when used in the claims, "consisting of' will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0042] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of’ and "consisting essentially of’ shall be closed or semiclosed transitional phrases, respectively.

[0043] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0044] The term “about” as used herein means plus or minus 10-15%, 5-10%, or optionally about 5% of the number to which reference is being made. In reference to measured values, the term “about” optionally refers to measurement error.

[0045] It should be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

II. Compositions of Matter

[0046] The inclusion of surrogate DNA in a transfection mixture was found to increase production of proteins of interest in stably transfected mammalian cells. Accordingly, provided herein are surrogate DNAs useful for increased expression of a protein of interest.

[0047] The term “surrogate DNA” as used herein refers to DNA that is other than the expression vector, which by itself does not lead to the expression of the protein of interest. For example, the surrogate DNA may be genomic DNA isolated from the cell line used for protein expression or isolated from another source of genomic DNA, such as a different cell type and/or a different organism. The surrogate DNA may alternately be a circular, linearized, or fragmented plasmid. Preferably a plasmid used as surrogate DNA is a different plasmid than the plasmid used to construct the expression vector and/or the plasmid used as surrogate DNA does not comprise the promoter used to drive expression of the protein of interest in the expression vector. If genomic DNA is employed as a surrogate DNA, the genomic DNA may be fragmented, for example by mechanical shearing, sonication, or enzymatic cleavage. Enzymatic cleavage may, for example, comprise digestion by one or more restriction endonucleases to produce blunt and/or cohesive ends.

[0048] The term “sheared” as used herein in reference to a nucleic acid molecule, such as DNA, that has been randomly fragmented. Methods for shearing nucleic acid molecules will be known to a person skilled in the art and include, but are not limited to, acoustic shearing, sonication, nebulization, centrifugal shearing, point-sink shearing, and needle shearing.

[0049] The term "nucleic acid molecule" and its derivatives, as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically double-stranded or a mixture of single- and double- stranded regions. In addition, the nucleic acid molecules can be composed of triplestranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning.

[0050] The term “protein” as used herein refers to a molecule comprising one or more than one polypeptide. The term “polypeptide” as used herein refers to a chain of two or more amino acid residues linked by peptide bonds. The term protein is intended to include linear polypeptides, as well as polypeptides that have folded into a three dimensional structure. A protein may be monomeric, dimeric, or multimeric. A protein that comprises more than one polypeptide may be homomeric or heteromeric. A protein may, but need not, further comprise one or more than one post-translational modification such as phosphorylation, glycosylation, ubiquitination, S- nitrosylation, methylation, N-acetylation, and/or lipidation.

[0051] As used herein, the term “antibody” refers to a full-length immunoglobulin molecule (e.g., an IgA, IgD, IgE, IgG, or IgM antibody). An antibody may be naturally occurring, or it may be formed using recombinant technology. An antibody typically comprises two heavy chains and two light chains. However, the term “antibody” also includes single domain antibodies and heavy chain only antibodies (hcAbs), such as those produced by sharks and camelids. [0052] As used herein, the term “antibody fragment” refers to a portion of an immunoglobulin molecule that is able to specifically bind an antigen. Examples of antibody fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, sFv and the like. The term antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the light chain variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“sFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic a hypervariable region.

[0053] The term “operably linked” as used herein refers to a relationship between two components that allows them to function in an intended manner. For example, where a nucleic acid molecule encoding a protein of interest is operably linked to a promoter, the promoter actuates expression of the nucleic acid molecule, resulting in production of the protein.

[0054] The term “promoter” or “promoter sequence” generally refers to a regulatory DNA sequence capable of being bound by an RNA polymerase to initiate transcription of a downstream (i.e. 3’) sequence to generate an RNA. Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters for the expression cassette will be known to the skilled person. In some embodiments, the promoter is an inducible promoter. Examples of inducible promoters include, without limitation, a tetracycline response element (TRE) (e.g. Tet-ON or Tet-OFF systems), po nA- inducible expression systems (Agilent Technologies), or cumate-inducible promoters such as CuO (System Biosciences). In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include human Ubiquitin C (UBC) promoter, human Elongation Factor 1 alpha (EFl A) promoter, human phosphoglycerate kinase 1 (PGK) promoter, simian virus 40 early promoter (SV40) promoter (GenBank accession number J02400.1), cytomegalovirus immediate-early promoter (CMV), chicken b- Actin promoter coupled with CMV early enhancer (CAG), EF1-HTLV hybrid promoter, and Chinese hamster EFl promoter (CHEF). [0055] The term “selectable marker” as used herein refers to an element in a nucleic acid construct that confers a selective advantage to cells harboring the nucleic acid construct. For example, the selectable marker may encode a protein that is expressed and confers resistance to a specific drug. Alternatively, the selectable marker may encode a protein that is expressed and is essential for cell viability under specific growth conditions. Suitable selectable markers are known to the skilled person. Examples of suitable drug-selectable markers include, without limitation, markers that confer neomycin resistance, hygromycin resistance, blasticidin resistance, zeocin resistance or puromycin resistance. Such markers are also referred to as resistance genes. Examples of genes required for growth under specific growth conditions include, without limitation, Glutamine Synthetase (GS) (GenBank accession number AY486122.1) and dihydrofolate reductase (DHFR).

[0056] The term “expression cassette” or “cassette” as used herein refers to a nucleic acid construct comprising a promoter sequence operably linked to a nucleic acid sequence encoding a protein of interest that allows expression of the protein of interest when the expression cassette is introduced into a suitable host cell, such as a mammalian host cell. The promoter may be any promoter that is suitable to actuate expression of the protein of interest by the host cell. Suitable promoters will be understood to one skilled in the art and include constitutive, cell-specific promoters, tissue-specific promoters, and inducible promoters. Examples of promoters suitable for expression in mammalian cells include human Elongation Factor 1 alpha (EF1A) promoter, simian virus 40 early promoter (SV40) promoter (GenBank accession number J02400.1), cytomegalovirus immediate-early promoter (CMV), chicken b- Actin promoter coupled with CMV early enhancer (CAG), rat aquaporin-5 (rAQP5) promoter and human NF-kappa B p65 promoter. The expression cassette may be comprised within a plasmid or other vector, such as Ad5.

[0057] A vector comprising an expression cassette, is referred to herein as an “expression vector”. An expression vector may further comprise a selectable marker to allow for selection of host cells comprising the expression vector. Examples of vectors suitable for expression in mammalian cells include pTT®l-242, pcDNA™l-6, pEFl-5, pUB, pCMV, pTK, pAd, pLentil-7, pBudCE 4.1, pZeoSV2, pShooter™, pBlue TOPO™ and pFRT/lacZeo 1 -2. III. Methods

[0058] A surrogate DNA, as described herein, may be mixed with an expression vector encoding a protein of interest, for the increased production of the protein of interest by mammalian cells into which the mixture is introduced. Accordingly, one aspect of the present disclosure is a method of increased production of a protein of interest, the method comprising: a) introducing into the mammalian cell a nucleic acid mixture, the nucleic acid mixture comprising at least one expression vector and at least one surrogate DNA; b) applying selective pressure to the cell to select for cells that stably carry the expression vector; and c) culturing the cell under conditions for production of the protein of interest.

[0059] Increased production, as used herein, refers to an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 250% of protein production compared to mammalian cells into which the expression vector is introduced in the absence of surrogate DNA.

[0060] The expression vector may be stably introduced into a mammalian host cell by any suitable method known in the art. In some embodiments, the nucleic acid construct is introduced into the host cell by transfection, including calcium phosphate transfection and electroporation/nucleofection. Suitable transfection reagents are well known in the art and may include, without limitation, cationic polymers such as polyethylenimine (PEI), cationic lipids such as Lipofectamine™ and related reagents (Invitrogen) and non-liposomal reagents such as Fugene® and related reagents (Promega). In some embodiments, the expression vector is introduced into the host cell by transfection using PEI.

[0061] When an expression vector is stably introduced into a mammalian host cell by transfection, this is referred to as “stable transfection” and the host cell is considered to be “stably transduced”. An expression cassette is considered to be “stably introduced” when the expression cassette is maintained throughout several rounds of cell division by virtue of being integrated into the genome of the host cell or being stably maintained by the host cell as a replicating episome. In some embodiments, the expression cassette is integrated into the genome of the host cell. [0062] Various mammalian host cells may be used in the methods for production of the protein of interest. Suitable cells are well known in the art and may include, without limitation, SP2/0 and NS/0 mouse cells, HT-1080 cells, PER.C6, HKB-11, CAP HuH-7, HeLa and HEL293 human cells and Chinese Hamster Ovary (CHO) cells of various subtypes including CHO-K1, CHO-DG44 and CHO-DXB11. In some embodiments, the cell is a CHO cell or a HEK293 cell. In some embodiments the host cell is a CHO^55E1 cell, a CHO-3E7 cell, or a HEK293-6E cell.

[0063] As described herein, the expression vector may comprise an inducible promoter. Accordingly, in some embodiments, the conditions for the production of the protein of interest comprise the addition of an inducing agent (also referred to as an effector molecule). Examples of inducible expression systems for expression in mammalian cells are known in the art (for example, see Kallunki et al., 2019) and include, but are not limited to, a tetracycline-controlled system, a cumate-controlled system, and inducible protein-protein interaction based chimeric systems. For example, where the inducible promoter is a cumate-inducible promoter, the conditions for the production of the protein of interest comprise the addition of cumate or a cumate analogue to the growth medium.

[0064] As demonstrated herein, the protein of interest may be an antibody comprising a heavy chain and a light chain. The antibody heavy chain and antibody light chain may be encoded by separate expression vectors or they may be encoded by two expression cassettes in the same expression vector. In some embodiments, the antibody is Palivizumab.

[0065] In some embodiments, the method further comprises collecting the cell and/or a cell medium containing the protein of interest, and optionally purifying the protein of interest from the collected cell and/or the cell medium. Purification methods are known in the art and will depend on the protein being purified.

[0066] The following non-limiting examples are illustrative of the present disclosure: IV. Examples

Example 1. Production of surrogate DNA

[0067] DNA sodium salt from salmon testes (stDNA) was purchased from Sigma- Aldrich and solubilized in PBS before sonication for a duration of 90 seconds to shear the stDNA. DNA from salmon sperm (ssDNA) was purchased from Thermo Fisher Scientific in a sheared form and ready - to-use solution. Genomic DNA from CHO cells was prepared using either DNAzol™ reagent or the protocol described in Zamenhof, 1957 (which is the same protocol used for extraction of stDNA by Sigma- Aldrich) and sonicated for 5, 30, 90, 180 or 270 seconds to shear the genomic DNA. Plasmid fragments were prepared by digesting circular plasmids pMK-RQ-IFN-SAR and pTT5-noCMV with restriction enzymes FspI, Bglll, Acil, Haelll and Pvul in order to generate blunt or cohesive DNA fragments of various sizes before purification by gel extraction. These plasmids are composed of sequences regularly found in expression plasmids but do not contain the promoter and the cassette used to express the protein of interest. VA concatemers were prepared by enzymatically digesting fragments from pAdvantage™ with EcoRI and Hindlll restriction enzymes and EBER concatemers by digestion of pLEX III plasmid with EcoRI and Avril s followed by ligation with T4 ligase and gel extraction. The size of each DNA was verified by migration on a 1% agarose gel (Fig. 1).

Example 2. Plasmid Constructs

[0068] Human codon-optimized sequences of CD200-Fc and Al AT were cloned in plasmids with one expression cassette (pTT®75) while MEDI-573, rituximab and palivizumab sequences were cloned in plasmids with two expression cassettes (pTT®96 and pTT®109) regulated by the cumate-inducible CR5 promoter. Glutamine synthetase (GS) gene expression is controlled by the SV40 promoter to allow selection of cells having stably integrated the plasmid using with methionine sulfoximine (MSX). Plasmids were amplified in E. coli DH5a and purified using a Fractogel® ion exchange chromatography resin. [0069] Table 1. Description of plasmids used to generate the stable pools

Example 3. Generation of Stable Pools

[0070] Experiments were performed using the CHO^BRI/55E1 cell line previously described in Poulain et al., 2017 that expresses both the reverse cumate transactivator (rcTA) and the cymene repressor (cymR). Cells were cultured in suspension in PowerCHO™-2 medium (Lonza, Walkersville, MD, USA) supplemented with 4 mM L-glutamine (Hyclone, Logan, UT, USA) and 0.1% Kolliphor® P 188 (Sigma- Aldrich, St Louis, MO, USA) in a humidified incubator at 37 °C with 5% CO2 under constant agitation at 120-rpm on a 25 -mm orbit Celltron™ shaker (Infers, Montreal, Canada). Exponentially growing cells were transfected at a density of 2.0 x 10⁶ cells/mL in CD DG44 (Life Technologies, Burlington, ON, Canada) supplemented with 4 rnM L-glutamine and 0.1% Kolliphor® P 188 using 1 pg/mL of total DNA (plasmid and filler DNA) and 5 pg/mL of linear PEI Max (Polysciences, Warrington, PA, USA). 24 hours post -transfection, cells were centrifuged and resuspended at a density of 0.5 x 10⁶ viable cells/mL in PowerCHO™-2 medium supplemented with 50 pM MSX (Sigma-Aldrich). Ligure 5 also shows results obtained using CHO-3E7 cells selected using 25 pM MSX and HEK293-6E cells selected using 2 pg/mL blasticidin. Cells were split with fresh media every other day until viability reached at least 95%. Example 4. Protein Expression

[0071] Stable pools were seeded at 0.7 x 10⁶ cells/mL in 125 mL flat-bottom flasks (Corning Incorporated) containing 20 mL of BalanCD® Growth A medium (Irvine Scientific, Irvine, CA, USA) supplemented with 50 pM MSX and placed in a 37 °C incubator under 5% CO2 on an orbital shaker operating at 120 rpm. Protein production was induced by adding 2 pg/mL of cumate when cell density reached 3-5 x 10⁶ cells/mL. For the next 14 days, temperature was reduced to 32°C and, every 2-3 days, cells were counted on a CEDEX™ automated cell counter (Roche Diagnostics, Mannheim, Germany) to assess viability, glucose was adjusted to a minimum of 17 mM and feed 12.7 (Irvine Scientific) was added up to 55% of initial volume. Fed-batch productions were stopped between 10-14 days, when viability decreased between 60-70%. Production medium samples were harvested and titers were evaluated (Fig. 2-6).

Example 5. Evaluation of Protein Expression

Evaluation of protein levels

[0072] Palivizumab, rituximab, MEDL573 and CD200-Fc titers shown in Fig. 2-6 and Tables 2- 3 were determined by protein-A high performance liquid chromatography using a 2.1 mmD x 30 mmH, 104 pL, POROS® A20 column. Before their injection on the column at a flow rate of 2 mL/min, samples were filtered by centrifugation at 8000-11,000 x g for 3 min through a Nanosep® MF GHP 0.45 pm filter. Elution was performed using 0.15 M NaCl, pH 2.0, and UV detection was done at 280 nm. Quantification was done by integrating the surface under the elution peak and comparing against an IgG standard curve. A1AT titers (shown in Fig. 3) were evaluated by purification of his-tag proteins using immobilized metal affinity chromatography (IMAC) and further quantification of purified proteins using a Nanodrop spectrophotometer.

[0073] Table 2: Impact of stDNA on the productivity of various recombinant proteins in CHO^55E1 stable pools

PLVZM = palivizumab, RTX = rituximab, MEDI-573 = dusigitumab, CD200-Fc = CD200 human protein fused with Fc domain, and AlAT-His = alpha-1 antitrypsin with 10 Histidine C- terminal tag.

[0074] Table 3: Impact of various DNA surrogates on the productivity of Palivizumab in CHO^55E1 stable pools

stDNA = salmon testis DNA, pMK-IFN/SAR = plasmid encoding human beta interferon scaffold attachment region, pTT®5 no CMV/FR = plasmid encoding elements necessary for plasmid replication and FR plasmid stabilizing sequence from EBNA-1, VAconc = concatemers of DNA encoding viral RNA VA, EBERconc = concatemers of DNA encoding epstein-Barr viral RNA EBER-1

Example 6. Transfection using Lipofectamine 3000™

[0075] Cells were grown at a density of 2.0xl0⁶ cells/mL and DNA was transfected using Lipofectamine™ 3000 according the manufacturer’s instructions. Cells were centrifuged and resuspended at 0.5 X10⁶ cells/mL in CD-DG44 with 4 mM L-glutamine. For each 3 mL transfection, 5 pg of DNA was mixed with 10 pL of P3000 reagent and the volume was completed to 150 pL with CD-DG44. To this mix was added 7.5 pL of Lipofectamine™ 3000 reagent diluted in 142.5 pL CD-DG44. After a 5 -minute incubation, 300 pL of the transfection mix was added to 2.7 mL of cells. PLVZM titers for each pool are shown in Figure 6. Example 7. Quantification of CD200-Fc, Dusigitumab, Rituximab, and Palivizumab integrated copies in the nucleus of cells co-transfected with various amounts of stDNA

[0076] 5.OxlO⁶ cells (from a frozen vial) were centrifuged and washed in cold PBS before extraction of genomic DNA using the Monarch DNA purification kit (NEB). 1 ug of genomic DNA was digested with EcoRI and BamHI enzymes to disrupt concatemers that may be present at the integration site(s). 10 or 25 ng of digested DNA was subjected to duplex digital droplet PCR (ddPCR) using one primer set specific for each transgene and second primer set specific for a reference gene known to be present as a single copy in the CHO cell genome (Bio-Rad QX200 ddPCR instrument). The reaction mix also includes a fluorescent- (FAM) labelled probe specific for the reference gene amplicon and a HEX labelled probe specific for the transgene amplicons. After droplet fluorescence was measured, copy numbers for the transgenes and reference gene were calculated for each sample using Bio-Rad Quantasoft software. Copy number per cell is calculated as the transgene copy number divided by the reference gene copy number for each sample.

[0077] For all plasmids, the integrated gene copy number increased as the amount of stDNA was increased up to 50%. No further increase in integrated gene copy number was observed when the Palivizumab-encoding plasmid (pTT®109-PLVZM-LC-HC-2.0) was co-transfected with 75% stDNA. Results are shown in Figure 7.

Example 8. Quantification of PLVZM transgene mRNA in the cytosol of cells co-transfected with various amounts of stDNA

RNA extraction

[0078] Cells are grown at a density of 2.0xl0⁶ cells/mE and induced with 2 pg/mE cumate. 2 days post-induction, 5. OxlO⁶ cells are centrifuged and washed in cold PBS before resuspension in 1 mF of the same lysis buffer for DNA extraction. Cell lysates are centrifuged and 200 pF of chloroform is added to supernatants. After centrifugation, the RNA is precipitated from the aqueous phase by adding 500 pF of isopropanol. Samples are incubated and centrifuged each for 10 minutes before washing the pellet 2 times with 1 mF of 75% ethanol. RNA is dissolved by letting the pellet dry shortly and by re-suspending in DEPC-treated water. To evaluate RNA concentration and purity, RNA is diluted 1/40 in DEPC-treated water and 2 pL is pipette on NanoDrop™ to measure the A260/280 ratio, considering that 1 OD at 260 equals 40 pg/ml RNA. cDNA synthesis and quantitative PCR ( qPCR )

[0079] cDNA synthesis and denaturation are performed by a using RT Platinum™ Taq 30 minutes at 50 C and 94°C for 2 minutes in IX Platinum™ Taq reaction buffer and 0.2 pM sense/anti-sense primers. For qPCR reactions, 50 pL of IX Platinum™ Taq buffer (with 0.2 pM sense/anti-sense primers, IX SYBR Green and IX Platinum™ Taq) and 50 ng of DNA are added in each well before plates were covered with a PCR plate transparent seal. 40 cycles of amplification are completed by alternating steps for denaturation (94°C, 15 seconds), annealing (54°C, 30 seconds) and extension (70°C, 30 seconds). DNA amplification and detection are performed using the MyiQ™ Single-Color Real-Time PCR Detection System (Bio-Rad) and BioRad® MyiQ™ software.

Statistical analysis

[0080] Data are expressed as mean ± standard deviation (SD) from at least three independent replicates. The p values are represented as: p > 0.05 = n.s., p < 0.05 = *, p < 0.01 = **, p < 0.001 *** ll statistical analyses are performed using GraphPad Prism® 7 software (San Diego, CA, USA).

[0081] The preceding examples have been provided to illustrate various aspects of the disclosure. However, these example are not intended to limit the disclosure, and a person of skill in the art will understand that modifications and substitutions are possible and included within the breadth of the present disclosure. The scope of the invention is defined by the appended claims, which are to be understood in view of common general knowledge and the teachings of the disclosure as a whole. References:

Carpentier, E., et al., Limiting factors governing protein expression following polyethylenimine- mediated gene transfer in HEK293-EBNA1 cells. J Biotechnol, 2007. 128(2): p. 268-280.

Kallunki T, Barisic M, Jaattela M, Liu B., How to Choose the Right Inducible Gene Expression System for Mammalian Studies?. Cells, 2019. 8(8):796.

Kichler, A., C. Leborgne, and O. Danos, Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines. J.Gene Med., 2005. 7(11): p. 1459-1467.

Kiseljak, D., et al., The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells. BMC proceedings, 2011. 5 Suppl 8: p. P33.

Poulain, A., Perret, S., Malenfant, F., Mullick, A., Massie, B., Durocher, Y. (2017), Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. Journal of Biotechnology, 255: p. 16-27.

Rajendra, Y., et al„ Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells. Biotechnol Bioeng, 2012. 109(9): p. 2271-8.

Zamenhof, S., Preparation and assay of deoxyribonucleic acid from animal tissue. Methods in Enzymology, 1957. 3: p. 696-704.

Claims

22 WHAT IS CLAIMED IS:

1. A mammalian cell for production of a protein of interest, wherein the mammalian cell is produced by stable transfection of a mammalian host cell with a nucleic acid mixture, the nucleic acid mixture comprising at least one expression vector encoding the protein of interest and at least one surrogate DNA.

2. The mammalian cell of claim 1, wherein the at least one expression vector is integrated into the genome of the mammalian cell.

3. The mammalian cell of claim 1 or 2, wherein the protein of interest is an antibody or an antibody fragment.

4. The mammalian cell of claim 3, wherein the antibody is Palivizumab.

5. The mammalian cell of any one of claims 1 to 4, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

6. The mammalian cell of any one of claims 1 to 5, wherein the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

7. The mammalian cell of any one of claims 1 to 5, wherein the at least one surrogate DNA comprises salmon testis DNA.

8. The mammalian cell of claim 7, wherein the at least one surrogate DNA comprises fragmented salmon testis DNA.

9. The mammalian cell of claim 8, wherein the at least one surrogate DNA comprises sheared salmon testis DNA.

10. The mammalian cell of any one of claims 1 to 9, wherein the molar ratio of the expression vector and the surrogate DNA in the nucleic acid mixture is within the range of 3: 1 to 1:3.

11. The mammalian cell of claim 10, wherein the molar ratio of the expression vector and the surrogate DNA in the nucleic acid mixture is about 1: 1.

12. A method for producing a protein of interest in a mammalian cell, the method comprising: a) introducing into a mammalian host cell a nucleic acid mixture comprising at least one expression vector and at least one surrogate DNA; b) applying selective pressure to select for cells that stably carry the at least one expression vector; and c) culturing the cells selected in step b) under conditions for production of the protein of interest.

13. The method of claim 12, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

14. The method of claim 12 or 13, wherein introducing the nucleic acid mixture into the mammalian host cell comprises transfection.

15. The method of claim 14, wherein the transfection comprises transfection with polyethylenimine (PEI).

16. The method of any one of claims 12 to 15, wherein the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

17. The method of any one of claims 12 to 16, wherein the at least one surrogate DNA comprises fragmented salmon testis DNA.

18. The method of any one of claims 12 to 16, wherein the at least one surrogate DNA comprises sheared salmon testis DNA.

19. The method of any one of claims 12 to 18, further comprising isolating the produced protein of interest.

20. A composition for stable transfection of a mammalian cell to enable production of a protein of interest by the mammalian cell, the composition comprising at least one expression vector encoding the protein of interest and at least one surrogate DNA, wherein the ratio of the at least one expression vector and the at least one surrogate DNA in the composition is within the range of 3:1 to 1:3.

21. The composition of claim 20, wherein the ratio of the at least one expression vector and the at least one surrogate DNA in the composition is about 1: 1.

22. The composition of claim 20 or 21, wherein the at least one surrogate DNA comprises circular plasmid DNA, linearized plasmid DNA, fragmented plasmid DNA, and/or fragmented genomic DNA.

23. The composition of claim 20 or 21, wherein the at least one surrogate DNA comprises fragmented salmon testis DNA.

24. The composition of claim 23, wherein the at least one surrogate DNA comprises sheared salmon testis DNA.

25. The composition of any one of claims 20 to 24, wherein the composition is for stable transfection of a Chinese Hamster Ovary (CHO) cell.