HK1144204A - Method for generating stable cell lines expressing high levels of a protein of interest - Google Patents

Description

Methods for generating stable cell lines expressing high levels of a protein of interest

Technical Field

The present invention relates to the industrial production of proteins. More specifically, the present invention relates to a method for obtaining cells stably expressing a protein of interest even when cultured without selective pressure. DHFR was used as a surrogate marker. Selection of transfected cells is not based on resistance to toxic compounds, but on fluorescence as measured by FACS using fluorescent MTX.

Background

Introduction of heterologous genes into animal host cells and screening for expression of the introduced genes is a lengthy and complex process. There are often a number of obstacles that need to be overcome: (i) constructing a large expression vector; (ii) transfection and selection of clones with long term expression; and (iii) screening for heterologous proteins of interest with high expression rates.

1. Selection of clones expressing heterologous genes

1.1.Screening of transformants

Clones incorporating the gene of interest are selected using a selection marker resistant to the selection pressure. Most selectable markers are resistant to antibiotics such as neomycin, kanamycin, hygromycin, gentamycin, chloramphenicol, puromycin, zeocin, or bleomycin.

When a cell clone expressing a gene of interest is to be generated from an expression vector, the host cell is typically transfected with a plasmid DNA vector that encodes the protein of interest and a selectable marker in the same vector. Many times the capacity of the plasmid is limited and the selectable marker has to be expressed on a second plasmid that is co-transfected with the plasmid comprising the gene of interest.

Stable transfection by typical methods results in random integration of the expression vector into the genome of the host cell. Using selective pressure, e.g. applying antibiotics to the culture medium, will remove all such cells: they cannot be integrated with vectors containing selection markers resistant to the respective antibiotics or selection pressure. If the selectable marker is on the same vector as the gene of interest, or if the selectable marker is on a second vector, co-integrated with the vector containing the gene of interest, the cell will express both the selectable marker and the gene of interest.

1.2.Screening for high-level production cells

Once the transformants are obtained, the cells are recloned and high level producer cells (high producer) are selected.

One possible method of selecting high-level producer cells is to directly quantify the expression of the protein of interest using, for example, ELISA. However, high-level producer cells that express surrogate markers that can be readily assayed are typically first screened. When a surrogate marker is used, the protein of interest and the surrogate marker are typically on the same vector, and the expression levels of both proteins are correlated.

An easy and convenient method is to re-clone the cells using Fluorescence Activated Cell Sorting (FACS) and to quantify the expression level of a fluorescent surrogate marker such as jellyfish or sea cucumber Green Fluorescent Protein (GFP).

Therefore, the use of FACS screening in combination with selection pressure to select for high-level producer cells is widely used in the art (see, e.g., Gubin et al, 1999; Yoshikawa et al, 2001; DeMaria et al, 2007).

For example, Yoshikawa et al (2001) disclose an improved method for selecting CHO cells amplified from high-level production genes by FACS. Cells were selected for resistance to MTX and expanded, and then high-level production cells were screened by FACS using the f-MTX/DHFR system.

The selection pressure is generally removed after selection of the transformants in which the nucleic acid encoding the protein of interest and the selection marker are integrated into the genome. High-level producer cells are then selected by FACS without any selective pressure (see, e.g., Gubin et al, 1997).

In any case, transformants are first selected under selection pressure. In fact, attempts to select transfected cells without selective pressure have proven unsuccessful (see, e.g., Migliaccio et al 2000).

Otto et al (2005) discloses a method for recloning cells without the use of selection pressure. However, the aim of this method is to reclone cells that have been previously transfected. In addition, selection of cells by FACS was based on the expression level of ZS green protein. Since the gene encoding the protein is cloned from the sea anemone florae fungus, it may cause poisoning and safety problems in the production process.

2. Limitations associated with selection of pressure

Although selection pressure is widely used to screen for high-level production cells, there are still many problems with the use of selection pressure.

When the selection pressure is removed, expression often becomes very unstable, or expression is lost. Therefore, only a small number of the original transformants can provide high levels and stable long-term expression, and it takes much time to identify these clones in a large number of candidate populations. Typically, high level expression candidates are isolated and then cultured under no selective pressure. In such cases, it is excluded because most of the originally selected candidates lost the expression of the gene of interest after the selection pressure was removed. Therefore, it is advantageous to culture the candidates under no selection pressure after the initial phase of selection for stable transfection, and then to screen for expression of the gene of interest.

In addition, the selection pressure of the added drug is correlated with multiple gene copy number events that are randomly integrated or amplified. Multiple replicative number events were associated with gene expression instability, probably due to the gradual loss of replicative number over time in the absence of selective pressure in the medium. The result is a low titer bioproduction fermentation.

Therefore, finding a novel and efficient method to isolate high-level production cells in which a heterologous protein of interest is stably expressed, becomes extremely useful in the field of industrial production of therapeutic proteins.

Disclosure of Invention

The present invention results from the discovery of a method for establishing a cell line capable of stably expressing high levels of a protein of interest. The method is based on the use of DHFR as a surrogate marker, and is characterized by the absence of the need to select cells based on drug resistance. Example 1 discloses such a method of the invention. The method comprises selecting cells based on DHFR expression, wherein DHFR expression is determined by fluorescent labeling and FACS analysis, without first selecting transfected cells using toxic compounds. As shown in examples 2 and 3, the cell lines selected by the method stably express high levels of the protein of interest.

Accordingly, in a first aspect the present invention relates to a method of screening for cells expressing a protein of interest (POI), comprising the steps of:

a) cells were transfected with the following:

(i) a nucleic acid encoding the protein of interest; and

(ii) a nucleic acid encoding DHFR;

b) measuring the expression of DHFR using a fluorescent compound that binds to DHFR; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high DHFR expression;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

The second aspect of the present invention relates to a method of screening for cells expressing a protein of interest, comprising the steps of:

a) transfecting a cell with a nucleic acid encoding the protein of interest:

b) determining the expression of the protein of interest using a fluorescent antibody that binds to the protein of interest; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high expression of the protein of interest;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

In a third aspect the present invention relates to a method for obtaining a cell line expressing a protein of interest, said method comprising the steps of:

a) selecting cells according to any of the methods of the invention described above; and

b) establishing a cell line from at least one of said cells.

The fourth aspect of the present invention relates to a method for producing a protein of interest, said method comprising the steps of:

a) culturing the cell line obtained according to the above method under conditions allowing the expression of the protein of interest; and

b) collecting the protein of interest.

A fifth aspect of the invention relates to the use of DHFR for screening cells, or obtaining cell lines, expressing a protein of interest, characterized in that the selection of said cells or cell lines is not based on resistance to MTX, nor on the metabolic advantage of the absence of Hypoxanthine and Thymidine (HT).

Drawings

FIG. 1 compares the productivity of cell lines obtained by the method of the invention with cell lines obtained by conventional drug selection methods. The Mean Fluorescence Intensity (MFI) is the result of the fluorescent MTX forming a complex with DHFR. The acquisition and analysis of these stable cells is described in examples 1 and 2. PRE-Round 1 refers to the cells prior to the first FACS sorting. PRE-Round 2 refers to the cells prior to performing the second FACS sorting. POST-Round 2 refers to the cells after the second FACS sorting.

FIG. 2 shows stability studies of cells selected using the methods of the invention. The protein of interest studied was a variant of human chorionic gonadotropin (hCG). Specific productivity was recorded as picograms per cell per day (pcd). Population Doubling Levels (PDL) refer to cumulative mitotic events. Population doubling time is a measure of growth.

Detailed Description

The present invention results from the discovery of a method for establishing cell lines. Surprisingly, the method does not use drug resistance to select for stable cells (example 1). The cells isolated by the method express the protein of interest at levels close to those of conventional methods based on drug resistance (example 2). Because FACS methods produce recombinant stable cells using growth media without drugs, there is no pressure to integrate or express multiple replicative numbers of the gene of interest. Thus, the instability caused by the absence of drug stress is no longer a problem. Specific productivity has been observed to be completely stable at population doublings greater than 50 (example 3). Thus, the present invention provides an efficient method for isolating a protein of interest that stably expresses the protein.

1. Method of the invention

A first aspect of the invention relates to a method of screening for cells expressing a protein of interest (POI), comprising the steps of:

a) cells were transfected with the following:

(i) a nucleic acid encoding the protein of interest; and

(ii) a nucleic acid encoding DHFR;

b) measuring the expression of DHFR using a fluorescent compound that binds to DHFR; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high DHFR expression;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

The term "DHFR" refers to a polypeptide that belongs to a member of the dihydrofolate reductase family (EC 1.5.1.3) and that is capable of catalyzing the following enzymatic reactions,

5, 6, 7, 8-tetrahydrofolate + NADP⁺7, 8-dihydrofolate + NADPH

The nucleic acid encoding DHFR may be of any origin. For example, it may be derived from bacteria such as E.coli (Miller et al, 2005). The DHFR is preferably derived from a eukaryotic cell. More preferably it is derived from a mammal. It is preferably from a mouse (Subramani et al, 1981). In one example, the species in which the DHFR gene is cloned is the same as the transfected cells.

The DHFR may correspond to a wild-type DHFR polypeptide or a mutant thereof, as long as the mutant retains the ability to catalyze the above reaction. Mutant DHFR polypeptides with improved kinetic parameters are known in the art.

The term "transfected cells" is understood to mean the introduction of a recombinant nucleic acid (e.g., a vector) into a cell.

Among a population of cells, cells that exhibit "relatively high DHFR expression" are those that exhibit higher levels of DHFR expression than other cells. For example, cell No.1 expressed 10mg/L DHFR and cell No.2 expressed 1mg/L DHFR. In this example, cell No.1 exhibited relatively high DHFR expression.

The term "screening" as used herein refers to testing or examining a particular trait in a large number of cells.

The term "selecting" as used herein refers to selecting certain specific cells in a group of cells.

The term "toxic compound" refers to any such compound: because the compound kills or inhibits the growth of untransfected cells, untransfected cells cannot be cultured in the presence of the compound. Examples of such compounds include, for example, MTX, puromycin, neomycin, kanamycin, hygromycin, gentamycin, chloramphenicol, zeocin, and bleomycin.

In a preferred embodiment of the invention, the selection of cells is not based on resistance to toxic compounds, nor on metabolic advantage between steps (a) and (b).

The term "metabolic advantage" refers to the ability of a transfected cell to grow in the absence of a compound that is obligatory to the growth of an untransfected cell. For example, CHO cells containing a gene encoding glutamine synthetase (CS) can be grown in the absence of glutamine, while CHO cells containing a gene encoding DHFR can be grown in the absence of thymidine and/or Hypoxanthine (HT). If the transfected cells do not contain (or contain a defective) glutamine synthetase or DHFR gene, the transfected cells acquire a metabolic advantage that they can be selected by culturing the cells in the absence of glutamine or hypoxanthine, respectively.

Fluorescent compounds that bind to DHFR are known in the art and include compounds that can covalently bind to DHFR, such as fluorescently labeled folic acid analogs. Such fluorescently labeled folate analogs include fluorescent methotrexate (f-MTX) and fluorescent trimethoprim (f-TMP) (Miller et al, 2005).

Any conventional means of measuring fluorescence may be used to measure DHFR expression, such as fluorescence microscopy, Fluorescence Activated Cell Sorting (FACS), and the like. It is most desirable to measure DHFR expression using FACS.

To measure the expression of DHFR in step (b) of the method of the invention, the cells are cultured in the presence of a fluorescent compound that binds DHFR. Although such fluorescent compounds that bind DHFR may be toxic to cells, the time for cell culture is short and does not kill any cells. Indeed, DHFR-deficient cells need to be cultured in the presence of MTX or f-MTX for more than 24 hours for drug selection to occur. Therefore, this culturing step is not a step of selecting cells based on resistance to fluorescent compounds that bind DHFR.

In preferred embodiments of the invention, the cells are cultured in the presence of a fluorescent compound that binds DHFR for less than 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, or 2 hours. More preferably, the cells are cultured in the presence of a fluorescent compound that binds DHFR for about 20 hours or about 4 hours.

Any cell is suitable for performing the methods of the invention. The cells may be primary cells or established cell lines from a variety of eukaryotes, including plant and animal cells. Preferably, the cell is a eukaryotic cell. More preferably, the cell is a mammalian cell. Preferably, the cell is a CHO cell, a human cell, a mouse cell or a hybridoma cell. Preferably, the cells are CHO-DUKX cells (Urlaub and Chasin, 1980).

In a first example, the cell is DHFR-deficient (e.g., CHO-DUKX or CHO-DG 44). Within the framework of this embodiment, any raw DHFR⁺The cells can be processed to become DHFR deficient.

In a second embodiment, the cell is DHFR⁺(i.e., its genome includes a functional endogenous DHFR gene). In fact, even when the cell is DHFR⁺Stable integration of the complementing replication of the DHFR gene can also be measured in this cell (see e.g.Connors et al 1988).

In a preferred embodiment, the nucleic acid encoding the protein of interest and the nucleic acid encoding DHFR are on the same vector to be transfected into the cell in step (a). Alternatively, the nucleic acid encoding the protein of interest and the nucleic acid encoding DHFR may be on separate vectors, which are co-transfected together into the cell in step (a).

When the nucleic acid encoding the protein of interest and the nucleic acid encoding DHFR are on the same vector, the vector may comprise at least two promoters, one of which drives expression of the nucleic acid encoding the protein of interest and the other of which drives expression of the nucleic acid encoding DHFR. Alternatively, the nucleic acid encoding the protein of interest and the nucleic acid encoding DHFR may be driven by the same promoter, while the vector comprises an internal ribosome insertion site (IRES) or a 2A sequence (de Felipe et al, 2006) between the nucleic acids.

The term "promoter" as used herein refers to a DNA region that: its function is to control the transcription of one or more DNA sequences, structurally identified as other DNA sequences whose presence depends on the binding site of the DNA's RNA polymerase or whose presence interacts to regulate the function of the promoter. Functional expression promoting fragments of a promoter are short or truncated promoter sequences which retain activity as a promoter. The activity of the promoter may be determined by any assay known in the art, for example, the reporter assay using DHFR as a reporter (Seliger and McElroy, 1960; Wood et al, 1984; de Wet et al, 1985), or fromThe method of (1). "enhancer region" refers to a region of DNA that functions to increase transcription of one or more genes. More specifically, the term "enhancer" as used herein refers to a DNA regulatory element capable of enhancing, increasing, improving or modifying the expression of a gene, regardless of its position and orientation relative to the gene to be expressed, and may enhance, increase, improve or modify the expression of more than one promoter.

In a preferred embodiment, the vector of the invention comprises at least one promoter of the mouse CMV immediate early region. This promoter may be, for example, the mCMV IE1 gene promoter ("IE 1 promoter"), as is known, for example, from WO 87/03905. The promoter may also be the mCMV IE2 gene promoter ("IE 2 promoter"), the mCMV IE2 gene itself being known, for example, from Messerle et al (1991). The IE2 promoter and IE2 enhancer regions are described in detail in WO 2004/081167. Preferably, the vector of the invention comprises at least two promoters for the mouse CMV immediate early region. Preferably, the two promoters are the IE1 and IE2 promoters.

In a preferred embodiment, the vector of the invention comprises at least two promoters of the mouse CMV immediate early region, one of which drives expression of the polypeptide of the invention and the other of which drives expression of the protein of interest.

According to the invention, the protein of interest may be any polypeptide which it is desired to produce. The protein of interest can be applied in the pharmaceutical, agricultural integrated enterprise or furniture fields of research laboratories. Preferred proteins of interest are used in the pharmaceutical field.

For example, the protein of interest can be, e.g., a naturally secreted protein, a normal cytoplasmic protein, a normal transmembrane protein, or a human or humanized antibody. When the protein of interest is a normal cytoplasmic protein or a normal transmembrane protein, it is preferred that the protein be processed so that it becomes soluble. The polypeptide of interest may be of any origin. Preferred polypeptides of interest are from humans.

In a preferred embodiment, the protein of interest is selected from the group consisting of: chorionic gonadotropin, follicle stimulating hormone, luteinizing hormone-chorionic gonadotropin, thyroid stimulating hormone, human growth hormone, interferons (e.g., interferon beta-1 a, interferon beta-1 b), interferon receptors (e.g., interferon gamma receptor), TNF receptors p55 and p75, interleukins (e.g., interleukin-2, interleukin-11), interleukin binding proteins (e.g., interleukin-18 binding protein), anti-CD 11a antibodies, erythropoietin, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, pituitary peptide hormones, menopausal gonadotropins, insulin-like growth factors (e.g., somatomedin-C), keratinocyte growth factor, glial cell line-derived neurotrophic factor, thrombomodulin, Basic fibroblast growth factor, insulin, blood coagulation factor VIII, somatotropin, bone morphogenetic protein-2, platelet derived growth factor, hirudin, erythropoietin (epoietin), recombinant LFA-3/IgG1 fusion protein, glucocerebrosidase, monoclonal antibodies, and muteins, fragments, soluble forms, functional derivatives, fusion proteins thereof.

Preferably, the monoclonal antibodies are directed against the following proteins: CD3 (e.g., OKT3, NI-0401), CD11a (e.g., efalizumab), CD4 (e.g., zanolimumab, TNX-355), CD20 (e.g., ibritumomab tiuxetan, rituximab, tositumomab, ocrelizumab, ofatumumab, IMMU □, TRU-015, AME-133, GA-101), CD23 (e.g., lumiximab), CD22 (e.g., epratuzumab), CD25 (e.g., basiliximab, daclizumab), Epidermal Growth Factor Receptor (EGFR) (e.g., panitumumab, cetuximab, zalutumumab, MDX-214), CD30 (e.g., MDX-060), cell surface glycoprotein CD52 (e.g., emulizumab), CD80 (e.g., vlgalib-0401), blood platelet alpha-antigen (e.g., TNF-2- α -2), interleukin alpha-beta-2 (e), interleukin alpha-beta-2 (e.g., interleukin-alpha-beta-2), interleukin alpha-beta-2 (e.g., TNF-beta-2), interleukin-2, interleukin-alpha-beta-2, e, e.g., interleukin-alpha-beta-2, interleukin-beta-2, and so as a, VEGF (e.g., bevacizumab, ranibizumab), immunoglobulin E (IgE) (e.g., omalizumab), HER-2/neu (e.g., trastuzumab), Prostate Specific Membrane Antigen (PSMA) (e.g., 111 In-Carrocumab pentapeptide (capromab pendetide), MDX-070), CD33 (e.g., gemtuzumab ozogamicin), GM-CSF (e.g., 002 KB, MT203), GM-CSF receptor (e.g., CAM-3001), EpCAM (e.g., adelimumab), IFN- γ (e.g., NI-0501), IFN- α (e.g., MEDI-545/MDX-1103), RANKL (e.g., denosumab), hepatocyte growth factor (e.g., AMG102), IL-15 (e.g., AMG 714), TRAIL (e.g., AMG 655), insulin-like growth factor receptor (e.g., AMG 479, R1507), IL-4-13 (e.g., AMG 1), and IL-3 (e.g., AMG 6332) receptors, CTLA-4 (e.g., ipilimumab).

Any number of cells can be screened according to the invention. For example, at least 10, 100, 1 ' 000, 10 ' 000, 100 ' 000, 1 ' 000 ' 000, 5 ' 000 ' 000, 10 ' 000 ' 000, 20 ' 000 ' 000, 30 ' 000 ' 000, 40 ' 000 ' 000, 50 ' 000 ' 000, 60 ' 000 ' 000, 70 ' 000 ' 000, 80 ' 000 ' 000, 90 ' 000 ' 000, 100 ' 000 ' 000 cells can be screened for fluorescence.

The population selected after completion of step (c) may be subjected to steps (b) (i.e.measurement of DHFR expression by fluorescence) and (c) (i.e.selection of the most fluorescent cells). For example, it may be repeated at least 2, 3, 4, 5 or 10 times. This may be done under conditions that change or maintain between the selection steps. Changing conditions includes, for example, changing culture conditions, such as medium composition or physicochemical parameters.

In a preferred embodiment, the cells selected after the last iteration of step (c) exhibit stable expression of the protein of interest in the absence of any drug selection, at a Population Doubling Level (PDL) of at least 10, 20, 30, 45 or 50.

In a preferred embodiment, the specific productivity (pcd) lost by the selected cells after the last iteration of step (c) is no more than 20%, 15%, 10%, 5% or 1% after a PDL of 15. Preferably, the cell loses no more than 20%, 15%, 10%, 5% or 1% specific productivity (pcd) after a PDL of 50. Preferably, the cell loses no more than 10% specific productivity (pcd) after a PDL of 50. Preferably, the cells do not lose any specific productivity (pcd) after a PDL of 50.

In a particular embodiment of the invention, the cells selected after the end of step (c) are subjected to a further screening comprising the steps of: (i) transfecting a cell with a nucleic acid encoding the protein of interest; (ii) measuring the expression of the protein of interest using a fluorescent antibody that binds to the protein of interest; and (iii) selecting from about 0.001% to about 25% of the cells tested in step (b) based on relatively high expression of the protein of interest.

After the last selection based on fluorescence (i.e. the last repetition of step (c)), the expression level of the protein of interest in the selected cells can be further measured (step d). The expression level of the protein of interest can be measured using any method known in the art, such as ELISA, FACS, northern blot or RT-PCR.

Then, about 0.001% to about 25% of the cells tested in step (d) can be selected based on relatively high expression of the protein of interest (step e). For example, cells exhibiting relatively high expression of the protein of interest in about 0.001%, 0.005%, 0.01%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% to about 15%, 18%, 20%, or 25% can be selected.

Another aspect of the invention relates to a method for obtaining a cell line expressing a protein of interest, said method comprising the steps of:

a) screening cells according to the method described above; and

b) establishing a cell line from said cell.

The term "cell line" as used herein refers to a specific cell type that is capable of being grown in a laboratory. Cell lines are generally capable of growing in permanently established cell culture media and proliferate indefinitely as long as there is adequate fresh medium and space. Methods for establishing cell lines from isolated cells are well known to those skilled in the art.

In a preferred embodiment, the cell line is a stable cell line, i.e. a cell line that does not lose more than 20%, 15%, 10%, 5% or 1% of its specific productivity (pcd) at a Population Doubling Level (PDL) of at least 10, 20, 30, 45 or 50 without any drug selection. Preferably, the cell line loses no more than 10% specific productivity (pcd) after a PDL of 50. Preferably, the cell line does not lose any specific productivity (pcd) after a PDL of 50.

Another aspect of the invention relates to a method for producing a protein of interest, said method comprising the steps of:

a) culturing the cells obtained according to the above method under conditions allowing the expression of the protein of interest; and

b) collecting the protein of interest.

The conditions which allow expression of the protein of interest can be readily determined by one skilled in the art using standard methods. For example, the conditions disclosed in example 3.3.1 may be used.

In a preferred embodiment, the above method for producing a protein of interest further comprises a step of purifying the protein of interest. Purification can be carried out by any technique known to those skilled in the art. If the protein of interest is used in the pharmaceutical field, the protein of interest is preferably formulated as a pharmaceutical composition.

Yet another aspect of the invention relates to the use of DHFR for screening cells expressing a protein of interest, characterized in that the selection of said cells is not based on resistance to MTX, nor on the metabolic advantage in the absence of hypoxanthine and thymidine.

Yet another aspect of the invention relates to the use of DHFR to obtain a cell line expressing a protein of interest, characterized in that the selection of said cell line is not based on resistance to MTX, nor on the metabolic advantage in the absence of thymidine. Preferably, the cell line stably expresses the protein of interest.

Alternatively, fluorescence of transfected cells can be measured using fluorescent antibodies that bind to the protein of interest instead of using fluorescent compounds that bind to DHFR. Typically, such a method of screening for cells expressing a protein of interest comprises the steps of:

a) transfecting a cell with a nucleic acid encoding the protein of interest:

b) determining the expression of the protein of interest using an antibody that binds to the protein of interest; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high expression of the protein of interest;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

All other steps are the same as the steps in the above method using DHFR.

2. Advantages of the invention over the prior art

The present invention provides a selection method for obtaining stable cells based on FACS, completely without the need to select for resistance to drugs.

In the method, the process of screening for high-level producing cells by FACS is combined with the process of selecting stable cells. Thus, the screening method has the advantage of reducing the number of process steps.

The method can also select stable cell lines without selective pressure. In fact, it has been found that the expression of a protein of interest in selected cells is stable when the population doubling number is greater than 50 under drug-free selection pressure. This is extremely advantageous for the industrial production of proteins.

In addition, the gene expression can be maintained stably without using drug cells, and a drug is not required in the production process. Finally, DHFR is a naturally occurring protein that occurs naturally in eukaryotic cells such as CHO cells. In other words, the cell lines obtained using the method of the invention express only two recombinant proteins: a recombinant protein of interest and a recombinant DHFR protein, wherein the gene for the DHFR protein is in any case naturally present in the cell. This is a clear difference from cell lines that include bacterial and/or cyanobacterial markers (e.g., GFP or ZS-Green). In particular, the expression of DHFR in cell lines producing the protein of interest does not present toxicity problems as is the case with, for example, GFP or ZS-Green as surrogate markers. Therefore, the cell lines obtained using the method of the present invention are safe in the production process.

Having fully described the invention, those skilled in the art can now make the same work within a wide range of equivalent parameters, concentrations, and conditions without undue experimentation, without departing from the spirit and scope of the invention.

While the invention has been described in conjunction with specific embodiments, it will be understood that other modifications may be made. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All documents cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patents, or other documents, are incorporated by reference herein. Including all data, tables, graphics, and text presented in the cited documents. In addition, the contents of the references cited within the references cited herein are also incorporated by reference in their entirety.

In general, reference to known method steps, conventional method steps, known methods, or conventional methods is not an admission that any aspect, statement, or embodiment of the invention is disclosed, suggested, or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation and without departing from the general concept of the present invention. Therefore, such variations and/or modifications are intended to be within the scope and range of equivalents of the disclosed embodiments, as described and guided herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present invention can be interpreted by the skilled artisan in light of the guidance presented and described herein, in combination with the general knowledge of one of ordinary skill in the art.

Examples

Example 1: scheme(s)

1.1.Cells, culture media and plasmids

The stable expression of the protein of interest in the Chinese hamster ovary cell line derived from CHO-DUKX-B11 was investigated. Prior to transfection, the host cells were adapted to grow in serum-free high-density suspension in chemically synthesized medium (hereinafter "growth medium") from PROCHO-5 medium (Bio-Whittaker/Cambrex, East Rutherford, N.J.) supplemented with 2% sodium Hypoxanthine and Thymidine (HT). All reagents were supplied by Invitrogen corp, Carlsbad, CA., unless otherwise indicated. The transfected cells were cultured in a disposable shake flask containing the growth medium described above, placed at 37 ℃ and 5% CO₂In the incubator, the flask was shaken at 125 rpm.

Transfection involves the application of a linear plasmid DNA combination encoding the alpha and beta subunits of the inactivated heterodimeric hormone human chorionic gonadotropin (hCG). The expression vector (hereinafter referred to as D-. alpha.) contains the gene of interest fused at its N-terminus to the signal sequence of human growth hormone, and is initiated by the metallothionein 1 promoter (MTT-1). It also contains the wild-type mouse DHFR gene driven by the sv-40 early promoter and a gene resistant to ampicillin. D-alpha vectors are described in detail in Kelton et al (1992).

1.2.Transfection

mu.L of lipofectin (DMRIE-C) and DNA (15. mu.g total, subunit volume ratio of 1: 1) were mixed with 1.5mL of growth medium, respectively, and the resulting mixtures were incubated at room temperature for 10 minutes, then combined, and further incubated at room temperature for 30 minutes to produce DNA-plasmid complexes. After centrifugation at 800rpm for 5 minutes, 2 million host cells for transfection were harvested and the cell pellet resuspended in 3mL of growth medium in a 25mL LT flask. Adding plasmid/DNA mixture to cells at 37 deg.C with 5% CO₂And (5) standing and culturing. After 4 hours, growth medium was added, to make up to 40mL, and the cells were transferred to a 250mL shake flask. At 37 deg.C, 5% CO₂The cell culture medium was maintained with shaking at 125rpm in the environment.

1.3.First FACS Classification

At 48 hours post-transfection, the cell number was counted and 2 million cells were collected and centrifuged as described above. The cell pellet was resuspended in 20 mM PHOROCHO-5 + supplemented with 4mM L-glutamine and 5. mu.M fluorescein MTX (f-MTX) and cultured at 37 ℃ in 5% CO₂Shaking at 125rpm in dark overnight.

The following day, the labeled cells were pelleted by centrifugation, washed twice with 5mL of frozen Phosphate Buffer (PBS) + 0.5% Pluronic acid + 25. mu. M Hepes buffer. The cells were then resuspended in 2mL of wash buffer and kept on ice until fluorescence was assessed using a FACS Aria flow cytometer (BD Biosciences, San Jose, CA) set to excite Fluorescein Isothiocyanate (FITC) at 488nM line using an argon laser. Sheath pressure (sheath pressure) was set at a low 25psi and a pore size of 100 μm.

The FACS gating process consists of a first gate based on light scattering events representing the physical parameters of the cells (selection population 1, named P1), forward and side scatter (FSC-H/SSC-H dot curves). Parameters were set according to the manufacturer's manual (BDFACS Aria instruction manual, version 336951Rev a., Aug 2003). P1 was then gated (gate) into the quadrant of the FSC-W/FITC-A dot curve. Quadrant 1(Q1) contained positive fluorescent cells (indicating DHFR expression due to vector insertion) and low FSC-W (indicating evaluation of single cells).

The host cells were loaded into a FACS Aria flow cytometer, optimizing the pressure settings: scatter events 100,000, mean fluorescence intensity < 200. Drop delay (drop delay) was determined using the Accudrop technique (BD FACS Aria operating manual).

The host cells passed through a single cell gate (P1) and the population was then displayed in a quadrant gate (Q1-Q4). The minimum threshold for the x-axis quadrant gate was set as the fluorescence baseline. Q1 corresponds to the desired sort gate, and no background events are found in Q1. To select for cells expressing the gene of interest, transfected cells were pooled, reloaded into the FACS Aria flow cytometer, and gated as described above. Analysis of the desired population Q1 was consistent with separation of the device (FITC) using a factor of 19.1 (compared to P1), with the population accounting for 3.1% of the population. The single cells of the Q1 gate were sorted and pooled into a collection tube containing 2mL of growth medium supplemented with 1% dialyzed fetal bovine serum and 2X penicillin streptomycin to prevent cell infection ("post-sort medium"). The cells were pelleted by centrifugation, resuspended in 5mL of post-sort media, and rested in a T25 flask until confluent. The medium was then trypsinized, expanded and placed in shake flasks and cultured as described above.

The classified population "Q1" was cultured in the post-classification medium for approximately 14 days. Before performing the second round of classification, the expression was analyzed.

To characterize expression of the selected cell pool, 1 million cells/mL were seeded in 10mL of selection medium, conditioned medium was generated, and cultured in a single shake flask at 37 ℃ for 24 hours. Centrifugation was carried out at 800rpm for 5 minutes, the conditioned medium was removed, and the supernatant was stored at 4 ℃ for analytical characterization. Protein expression was quantified using the DSL hCG ELISA and with reference to manufacturer's specifications and kit standards (Diagnostic Systems Laboratories, Webster, TX).

Specific productivity was calculated by the following equation:

Q_sp(p/c/d)＝(P₂-P₁)×ln(N_t/N_o)/(T₂-T₁)(N_t-N_o)

P₁: initial titer (Peak)

P₂: final titer (Peak)

N_t: final population size (cell number)

N_o: initial population size (cell number)

T₁: initial time (sky)

T2: harvesting time (sky)

The average specific productivity of the cells prior to the second classification was 0.2 pcd.

1.4.Second FACS Classification

To isolate the highest expressing cells, the cell population resulting from the first classification (Q1) was expanded, labeled with f-MTX for 4 hours, and subjected to another round of evaluation and classification as described above. Analysis of the desired population Q1 was consistent with separation of the device (FITC) using a factor of 19.1 (compared to P1), with the population accounting for 3.1% of the population. Analysis of the sorted population Q1-Q1 was consistent with separation of the device (FITC) using a factor of 8.4 (compared to P1), with the population accounting for 1.3% of the population.

After cell sorting, the cells were placed in a T25 flask containing 5mL of post-sorting medium without HT, allowed to stand, then expanded and placed in a shake flask, and cultured as described above. After the final classification, expression analysis was performed on the cell pool (cellpool), resulting in an average specific cell productivity of 3.2 pcd.

Example 2: comparison of Mean Fluorescence Intensity (MFI) of FACS-and drug-selected cells

As a control, drug-selected cell lines were established as follows: at 48 hours post-transfection, cells were transferred to selection medium (0.5. mu.M methotrexate in PROCHO-5 medium supplemented with 4mM L-glutamine). Every two to three days, cells were passaged by centrifugation and resuspended in fresh selection medium to a final density of 5X 10⁵Individual cells/mL. After approximately three weeks, the cells showed signs of restoration of growth. The CHO cell pool was considered stable when the growth rate was constant and the survival rate was greater than 90%.

The cells were labeled and the fluorescence at the different processing points (for FACS-selected cell lines: before first, before and after second sorting; for drug-selected cell lines: after the selection process) was evaluated by FACS as described above. The FITC-A histogram shows a single cell gate (P1).

A significant shift in MFI was observed, as transfected cells were subjected to multiple rounds of FACS selection (1a-1 c).

FACS selection of stable CHO cells generated in (1c) and grown in media without drug selection pressure showed a similar profile to that of the drug selection cell line (1d) grown in the presence of 0.5uM MTX. The results in FIG. 1 show that the method of the invention results in high levels of production of recombinant cell lines.

Example 3: evaluation of stability

Cells were cultured in post-sort medium (no drug selection) to a population doubling length of 50 in order to study the stability of gene expression of FACS-selected stable cell lines. Expression analysis was performed weekly to determine specific cell productivity. The results are shown in FIG. 2.

The cells of the final classification stably expressed hCG protein after the Population Doubling Length (PDL) reached 50, without any loss of average specific productivity. Cells maintained a steady and consistent growth rate with an average doubling time of 27 hours.

In conclusion, the method of the present invention allows the selection of stable high-level production recombinant cell lines that can be used in the study or production of proteins.

Reference to the literature

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Claims (18)

1. A method of screening for cells expressing a protein of interest (POI) comprising the steps of:

a) cells were transfected with the following:

(i) a nucleic acid encoding the protein of interest; and

(ii) a nucleic acid encoding dihydrofolate reductase (DHFR);

b) measuring the expression of DHFR using a fluorescent compound that binds to DHFR; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high DHFR expression;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

2. A method of screening for cells expressing a protein of interest comprising the steps of:

a) transfecting a cell with a nucleic acid encoding the protein of interest:

b) determining the expression of the protein of interest using a fluorescent antibody that binds to the protein of interest; and

c) selecting from about 0.001% to about 25% of the cells tested in step b) based on relatively high expression of the protein of interest;

wherein the selection of said cells is not based on resistance to toxic compounds between steps a) and b).

3. The method of claim 1, wherein said DHFR expression is measured using a Fluorescence Activated Cell Sorter (FACS).

4. The method of claim 1 or 3, wherein the fluorescent compound that binds to DHFR is fluorescent methotrexate (f-MTX) or fluorescent trimethoprim (f-TMP).

5. The method of any of the preceding claims, wherein the cell is selected from the group consisting of a human cell, a CHO cell, a mouse cell, and a hybridoma cell.

6. The method of any of the preceding claims, wherein the cell is a DHFR-deficient cell.

7. The method of claim 6, wherein the cell is a CHO-DUKX cell.

8. The method of any one of claims 1 and 3 to 7, wherein the nucleic acid encoding the protein of interest and the nucleic acid gene encoding DHFR are on the same vector, which is transfected into the cell of step (a).

9. The method of claim 8, wherein said vector comprises at least two promoters, one of which drives expression of said nucleic acid encoding a protein of interest and the other of which drives expression of said nucleic acid encoding DHFR.

10. The method of claim 8, wherein said nucleic acid encoding a protein of interest and said nucleic acid encoding DHFR are driven by the same promoter, and wherein said vector comprises an internal ribosome insertion site (IRES) or 2A sequence located between said nucleic acids.

11. The method of any preceding claim, wherein steps (b) and (c) are repeated at least 2, 3, 4 or 5 times.

12. A method according to any of the preceding claims, characterized in that the method further comprises the step of:

d) measuring the expression level of the protein of interest in the selected cells after completion of the last step (c).

13. The method of claim 12, further comprising the steps of:

e) selecting from about 0.001% to about 25% of the cells tested in step (d) based on relatively high expression of the protein of interest.

14. A method of obtaining a cell line expressing a protein of interest, the method comprising the steps of:

a) screening cells according to the method of any one of the preceding claims; and

b) establishing a cell line from at least one of said cells.

15. A method of producing a protein of interest, the method comprising the steps of:

a) culturing a cell line obtained according to the method of claim 14 under conditions that allow expression of the protein of interest; and

b) collecting the protein of interest.

16. The method of claim 15, further comprising the step of purifying the protein of interest.

17. The method of claim 16, further comprising the step of formulating the protein of interest into a pharmaceutical composition.

Use of DHFR in:

(i) screening for cells expressing a protein of interest; or

(ii) Obtaining a cell line expressing a protein of interest;

characterized in that the selection of said cells or cell lines is not based on resistance to MTX nor on the metabolic advantage obtained when culturing in the absence of Hypoxanthine and Thymidine (HT).