CN117025538A - High-efficiency expression method of recombinant nerve growth factor - Google Patents
High-efficiency expression method of recombinant nerve growth factor Download PDFInfo
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- CN117025538A CN117025538A CN202310802822.0A CN202310802822A CN117025538A CN 117025538 A CN117025538 A CN 117025538A CN 202310802822 A CN202310802822 A CN 202310802822A CN 117025538 A CN117025538 A CN 117025538A
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12N2510/00—Genetically modified cells
Abstract
The application relates to a high-efficiency expression method of recombinant nerve growth factor, in particular to a method for improving the expression quantity of the recombinant nerve growth factor by adding succinic acid in the cell culture process, wherein the culture can be batch culture, fed-batch culture or continuous culture. The method of the application can increase the yield of the recombinant nerve growth factor expressed by the cells by at least 100 percent, and has important clinical and commercial values.
Description
Technical Field
The application belongs to the technical field of bioengineering, and particularly relates to a method for improving recombinant protein expression, in particular to a method for improving recombinant beta-NGF protein expression by adding succinic acid in a cell culture process.
Background
Nerve growth factor (never growth factor, NGF) is a class of neurotrophic factors that are primarily involved in the regulation of neuronal growth, maintenance, proliferation and survival. NGF was the earliest neurotrophic factor discovered and was first isolated from mouse sarcoma cells in 1953 by Rita Levi-Montalcini, which has led to the development of neuroscience, cell biology and developmental biology, and has opened up a new area for research in neurology. For this reason, levi-Montalcini was also awarded a Nobel medical physiology prize in 1986.
The complete NGF consists of a complex of 130-kDa consisting of the alpha, beta and gamma subunits in a 2:1:2 ratio, non-covalently, this NGF also being known as proNGF (NGF precursor). Wherein the beta subunit is an active subunit of NGF, and consists of two peptide chains containing 118 amino acids through non-covalent bonds, and the gamma subunit is serine proteinase which can cut the N end of the beta subunit, so that the protein is activated, and NGF with functions is generated. The amino acid sequence of human full length NGF (hNGF) can be found in Genbank accession number NP-002497.2 or UniProtKB accession number P01138. Human full length NGF typically comprises 241 amino acids, with NGF signal peptide moieties at positions 1-18, leader peptide moieties at positions 19-121, and mature NGF at positions 122-241. Full length NGF can be converted to mature NGF by a cleavage process.
Studies have shown that NGF is primarily involved in regulating neuronal growth, maintenance, proliferation and survival, and is also critical for survival and maintenance of sympathetic and sensory neurons. NGF has basic nutrition supporting effect on nerve cells and tissues, and also can promote nerve cell differentiation and determine the direction of axon extension. Has decisive regulation and control effects on promoting brain development, nervous system growth, nerve regeneration and injury repair.
The NGF therapeutic drugs currently marketed in China are mainly murine NGF (mNGF), and are widely applied to clinic and mainly used for treating n-hexane toxic peripheral neuropathy, and it is also reported that mNGF is used for treating other toxic peripheral neuropathy, diabetic peripheral neuropathy, alzheimer's disease, parkinson's disease, facial neuritis, nerve injury and other nervous system diseases.
Although mNGF has 85% homology with human NGF (hNGF) in amino acid sequence, mNGF belongs to foreign protein for human body, has certain immunogenicity, and simultaneously, can be purified from mouse mandibular gland, and has a certain risk of mouse-derived virus pollution. With the prolongation of human life, the incidence of diabetic peripheral neuritis, alzheimer's disease, parkinson's disease and other neurological lesions gradually increases in recent years, and the development and marketing of hNGF drugs have become necessary. However, the content of hNGF in the human body periphery is low, and it is almost impossible to obtain a large amount of hNGF from natural sources, so recombinant expression of hNGF using a bioengineering technique is the most feasible method, namely, using a genetic engineering technique, constructing eukaryotic expression vector transfected cells (commonly used CHO cells), screening cell lines having a high expression level for culture, and collecting the product after termination of culture. Such methods are widely used and are conventional in the art. For example, construction of eukaryotic expression vectors for human β -NGF fusion proteins and expression studies (medical examination and clinic; 2022 (004): 033) thereof are described in patent CN108034678A, US20210009649A1 and literature, and NGF yields of about 80 to 602mg/L have been achieved.
Methods for producing sufficient protein using cell culture in commercial production are largely divided into three types: batch culture, fed-batch culture and continuous culture. In batch culture methods, all nutrients are provided in the initial medium, and as the nutrients continue to decrease, metabolic inhibitors such as lactic acid increase, failing to place the cells under an optimal condition and thus being somewhat limited in application. Fed-batch culture refers to a culture method in which fresh medium is intermittently or continuously fed during the culture until the reaction is completed, and the reaction system is taken out, which can avoid the inhibition of cells by the lack of cells and products in metabolic processes and certain nutrients. Continuous culture is a culture method in which the culture medium is continuously poured in the culture process, and waste can be removed, nutrition can be provided and products can be harvested.
Currently, recombinant expression of β -hNGF using expression systems such as mammalian cells, insect cells, and E.coli has been studied. Mammalian cell surfaces are used because of their ability to produce properly folded and assembled heterologous proteins, as well as their ability to post-translationally modify. At present, a culture method for efficiently expressing hNGF is not available, and the large-scale production and clinical and pharmaceutical applications of hNGF are greatly limited.
Disclosure of Invention
To solve the above problems, the present application provides a method of culturing mammalian cells expressing a recombinant protein, the method comprising: culturing the mammalian cells in a medium comprising succinic acid or a salt thereof, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
The present application also provides a method for increasing the expression level of a recombinant protein in a mammalian cell expressing the recombinant protein, the method comprising: culturing the mammalian cells in a medium comprising succinic acid or a salt thereof, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
In some embodiments, the method comprises adding the succinic acid or salt thereof in a concentration of 2.0 to 50.0mM in the medium.
In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, preferably a CHO-DG44 cell, CHO-K1 cell, CHO DXB11 cell, CHO-S cell, CHO GS deficient cell or a derivative of any of these cells.
In some embodiments, the method is selected from batch, fed-batch or continuous culture, preferably batch and fed-batch culture, more preferably fed-batch culture.
In some embodiments, the recombinant Nerve Growth Factor (NGF) and fragments thereof are recombinant human Nerve Growth Factor (NGF) and fragments thereof, preferably the fragments of recombinant nerve growth factor are selected from the group consisting of β subunit of nerve growth factor (β -NGF).
In some embodiments, the method further comprises the step of isolating the recombinant protein.
In some embodiments, the method increases the expression level of the recombinant protein by at least 100% as compared to a culture method without succinic acid.
The present application further provides a culture medium for increasing the expression amount of a recombinant protein of a mammalian cell expressing the recombinant protein, and comprising succinic acid or a salt thereof at a concentration of 2.0 to 50.0mM, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
In some embodiments, the medium further comprises a serum-free medium.
Finally, the present application provides the use of succinic acid or a salt thereof for increasing the expression level of a recombinant protein in a mammalian cell expressing the recombinant protein, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
The application greatly improves the expression level of hNGF in CHO cells by adding succinic acid in the cell culture process, provides a method capable of efficiently expressing hNGF, and has important clinical and commercial values.
Drawings
FIG. 1 shows the change over time of the viable cell density (VCD, indicated by solid line) and the cell viability (VIA, indicated by broken line) of CHO cells expressing NGF by means of fed-batch culture. The abscissa represents days, and the left ordinate represents Viable Cell Density (VCD) in 10 6 cell/mL, the ordinate on the right represents the cell Viability (VIA), expressed as a percentage%. The open boxes indicate that no succinic acid was added, and the remaining icons are shown in the figure, indicating that succinic acid was added at different concentrations.
FIG. 2 shows a bar graph of the expression level of β -NGF protein in the presence of medium supplemented with succinic acid at various concentrations. The abscissa represents the concentration of succinic acid added differently, and the ordinate represents the expression amount of beta-NGF protein in mg/L. Wherein Empty means that succinic acid was not added.
Detailed Description
The present application will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the application in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this application belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
As used herein, the terms "comprising," "including," "having," "can," "containing," and variations thereof are generally intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The term "consisting of … …" generally indicates that no additional component (or, as such, feature, integer, step, etc.) can be present.
As used herein, the term "about" may be used to encompass a change from a particular value of ±10% or less, a change of ±5% or less, a change of ±1% or less, a change of ±0.5% or less, or a change of ±0.1% or less.
As used herein, the term "nerve growth factor (never growth factor, NGF)" is a class of secreted proteins that are primarily involved in the regulation of neuronal growth, maintenance, proliferation and survival. The amino acid sequence of human full-length NGF can be found in Genbank accession number NP-002497.2 or UniProtKB accession number P01138. The term as used herein is not limited to human NGF and encompasses all species orthologs of human NGF. The term encompasses precursor forms of NGF (pro-form), NGF precursor (pro NGF), full length NGF, and any form of NGF produced by intracellular processes. The term also encompasses naturally occurring variants of NGF, such as splice variants, allelic variants and isoforms.
As used herein, the term "β -NGF" refers to the β subunit of NGF. The beta subunit is an active subunit of NGF, has a molecular weight of 26-kDa, is the only bioactive component in NGF precursors, and consists of two peptide chains containing 118 amino acids through non-covalent bonds. When used in the present application, the fragment of NGF may be β -NGF, or a fragment comprising β -NGF, for example a fragment comprising a cleavage or hydrolysis site that can be cleaved to produce β -NGF.
As used herein, the term "wild-type" generally refers to a sequence of amino acids or nucleic acids that naturally occur within a species or population (e.g., human, mouse, rat, cell, etc.).
The term "recombinant protein" as used herein refers to a protein molecule expressed using a recombinant DNA molecule, such as a protein of interest, which is exogenous to the cell producing the polypeptide.
As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that may or may not contain a plasmid or vector comprising a nucleic acid molecule encoding a recombinant protein described herein, or that is capable of expressing a recombinant protein described herein. As used herein, the term "mammalian cell" is a cell line suitable for the production of recombinant proteins. As used herein, the term "cell culture" refers to a population of cells suspended in a culture medium under conditions suitable for cell survival and/or growth, and may comprise a combination of the culture medium and the population of cells suspended therein.
As used herein, the term "succinic acid (SUC)", also known as succinic acid or ethane dicarboxylic acid, formula C 4 H 6 O 4 Is a dicarboxylic acid, which is suitable for various applications.
As used herein, the term "succinate" refers chemically to a salt or ester of succinic acid. Both "succinate" and "succinic acid" refer to the same compound, both of which exist in a form that depends on the pH of the solution. Thus, the terms "succinate" and "succinic acid" are used interchangeably herein.
As used herein, the term "alkali metal salt of succinic acid" refers to salts of six metallic elements of group ia of the periodic table except hydrogen (H), namely lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), francium (Fr), and succinic acid anions.
As used herein, the term "alkaline earth metal salt of succinic acid" refers to six elements of group iia elements of the periodic table of elements, including salts of beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), and succinic acid anions.
As used herein, the term "batch culture" is a method of culturing in a closed system with a limited amount of nutrient input at a time.
As used herein, the term "fed-batch culture" is a culture process that provides additional components to the culture, such as cell nutritional supplements that are depleted during the culture, either continuously or intermittently at some point after the start of the culture process.
As used herein, the term "continuous culture" refers to a culture method in which fresh medium is added to a fermentation vessel at a certain rate while the culture liquid is discharged at the same rate, thereby maintaining the liquid amount in the fermentation vessel constant and allowing a cell culture to grow in an approximately constant state.
As used herein, the term "culture medium" is a cell culture medium that is cultured to contain the desired recombinant protein expression system.
As used herein, the term "initial medium" is a medium used for batch culture prior to addition of the fed-batch medium in fed-batch or continuous culture.
As used herein, the term "feed medium" is the medium that will be provided to the fermentor when fed-batch or continuous culture is performed. The feed medium may contain all or part of the components necessary for cell growth. The feed medium may include all or part of the components and proportions of the initial medium, or all or part of the components or proportions thereof may be altered, or components and proportions entirely different from the initial medium may be used. Any suitable medium for the culture of the corresponding host cells known in the art may be used as the Feed medium of the present application, such as commercially available EX-Cell Advanced CHO Feed1 (Sigma), EX-Cell Cellvento 4Feed (Merck), efficiency Feed A+ (Gibco), efficiency Feed B+ (Gibco), efficiency Feed C+ (Gibco), cell boost 7a (Hyclone), cell boost 7B (Hyclone), balanCD Feed4 (Irvine), CHO Max FA (Mich organism), CHO Max FB (Mich organism), CHO FX (Mich organism), CHO FB (Mich organism) or combinations thereof. The person skilled in the art can choose the appropriate feed medium addition concentration and timing depending on the cell growth conditions during the culture.
As used herein, the term "ELISA" is simply referred to as an Enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay). The method comprises the steps of combining known antigen or antibody on the surface of a solid phase carrier, then incubating the antigen or antibody marked (coupled) by enzyme, and developing by a chromogenic substance, wherein the color development depth is in direct proportion to the content of a substance to be detected, and the substance can be observed by naked eyes. In ELISA experiments, there are three necessary reagents: known antigens or antibodies (for binding to a solid support); enzyme-labeled antibodies or antigens (markers); color developer (for color development). Four types of ELISA experiments are common: direct ELISA, indirect ELISA, sandwich ELISA, and competition ELISA.
As used herein, the term "viable cell density (Viable Cell Density, VCD)" refers to the number of cells present in a given volume of medium. The viable cell density can be measured by any method known to those skilled in the art. Preferably, the viable cell density is measured using an automated cell counter such as BioProfile. The unit "cells/mL" is used herein. One of ordinary skill in the art will appreciate that one of many methods for determining cell viability is included in the present application. For example, dyes (e.g., trypan blue) that do not pass through living cell membranes but can pass through the damaged membrane of dead or dying cells can be used to determine cell viability.
As used herein, the term "Viability" refers to the ratio of the total number of viable cells per milliliter of cell fluid to the total number of cells, expressed in units "%".
As used herein, the term "protein expression" refers to the process by which a polynucleotide sequence is successfully transcribed and translated to express a detectable level of an amino acid sequence or protein.
As used herein, the term "protein expression amount" refers herein to the amount of NGF protein per L of medium detected using an ELISA method, expressed in units of "mg/L".
As used herein, the term "increase", "increase" generally means 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 120%, at least 140%, at least 160%, at least 180%, at least 200% compared to a control cell or control method.
In one aspect of the present application, there is provided a method of culturing mammalian cells expressing a recombinant protein, the method comprising: culturing the mammalian cells in a medium comprising succinic acid or a salt thereof.
In some embodiments, such a method comprises the steps of:
1) Constructing a cell, preferably a mammalian cell, such as a CHO cell, containing the recombinant protein expression vector;
2) The above cells are inoculated into a culture vessel, and a medium containing succinic acid or a salt thereof is added to the culture vessel to perform cell culture.
Optionally, a fed-batch culture is performed by adding a feed medium during the culture; or batch culturing without adding fed-batch culture medium, and collecting NGF protein product in cell suspension after the final culturing; or continuously culturing, adding fresh culture medium into the culture container at a certain speed, simultaneously flowing out culture solution at the same speed, and collecting the product. In some embodiments, the succinic acid or salt thereof is added at a concentration of 2.0 to 50.0 mM. Preferably, the concentration of succinic acid or salt thereof is 2.0mM, 4.0mM, 8.0mM, 10.0mM, 12.0mM, 14.0mM, 16.0mM, 18.0mM, 20.0mM, 22.0mM, 24.0mM, 26.0mM, 28.0mM, 30.0mM, 32.0mM, 34.0mM, 36.0mM, 38.0mM, 40.0mM, 42.0mM, 44.0mM, 46.0mM, 48.0mM, 50.0mM or any value therebetween. Further preferably, the concentration of succinic acid or a salt thereof is 8 to 32mM.
Cells for use in the present application include cells capable of expressing a recombinant protein described herein, such as nerve growth factor NGF or a subunit thereof. In some embodiments, the cells may be obtained by transfecting the cells in vitro with a suitable vector.
In some embodiments, the mammalian cell is a cell line suitable for the production of recombinant proteins, preferably growth factor proteins, further preferably nerve growth factor NGF, more preferably β -hNGF.
In some embodiments, the mammalian cell is a rodent cell, such as a chinese hamster ovary cell (CHO cell). They are suitable for serial passage in cell culture and do not include primary non-transformed cells or cells that are part of an organ structure. In some embodiments, the mammalian cells are BHK21, BHK TK, CHO-K1, CHO-S cells, CHO-DXB11 (also known as CHO-DUKX or DuxB 11), and CHO-DG44 cells or derivatives/progeny of any of such cell lines. Particularly preferred are CHO-DG44, CHO-K1 and BHK21, and even more preferred are CHO-DG44 and CHO-K1 cells; most preferred are CHO-DG44 cells. In some embodiments, the mammalian cells may also be murine cells, such as murine myeloma cells, e.g., NS0 and Sp2/0 cells or derivatives/progeny of any of such cell lines. In some embodiments, derivatives/offspring of these cells, other mammalian cells (including but not limited to human, mouse, rat, monkey, and rodent cell lines) may also be used herein, particularly for the production of recombinant proteins.
In some embodiments, the mammalian cell may further comprise one or more expression cassettes encoding a recombinant protein.
In some embodiments, the recombinant protein is selected from recombinant Nerve Growth Factor (NGF) and fragments thereof, particularly recombinant human nerve growth factor and fragments thereof, preferably the recombinant protein is the β subunit of nerve growth factor (β -NGF). In some embodiments, the recombinant protein may also be selected from the group consisting of growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin a chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulants such as protein C; atrial natriuretic (atrial natriuretic) factor; a lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factors-alpha and-beta; enkephalinase; RANTES (factor regulated by activation, expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; muellerian inhibitory substances; relaxin a chain; relaxin B chain; a relaxin source; a mouse gonadotrophin-related peptide; microbial proteins such as beta-lactamase; a DNase; igE; cytotoxic T-lymphocyte-associated antigens (CTLA), such as CTLA-4; inhibin; an activin; vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors; protein a or D; a rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophic factors-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6); platelet Derived Growth Factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF) such as TGF- α and TGF- β, including TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, or TGF- β 5; insulin-like growth factors-I and-II (IGF-I and IGF-I I); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; an osteoinductive factor; an immunotoxin; bone Morphogenic Proteins (BMP); interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSF), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (IL), e.g., IL-1 through IL-10; superoxide dismutase; a T cell receptor; surface membrane proteins; decay accelerating factors; viral antigens such as, for example, part of the AIDS envelope; a transport protein; homing the recipient; address elements; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor associated antigens such as HER2, HER3 or HER4 receptor; and fragments of said polypeptides.
In some embodiments, the NGF is not limited to human NGF and comprises all orthologs of human NGF. Including but not limited to precursor forms of NGF (pro-form), NGF precursor (pro NGF), full length NGF, β -NGF, and any form of NGF produced by intracellular processes. Variants of naturally occurring NGF, such as splice variants, allelic variants and isoforms, are also included.
In some embodiments, cells containing an expression vector encoding a recombinant protein of interest, such as those described in CN108034678A, WO2022026468A1, US20210009649A1, are constructed using methods conventional in the art, and then cultured in a medium containing succinic acid or salts thereof.
In some embodiments, the methods of the application further comprise the step of isolating the recombinant protein. The method of the present application is capable of increasing the expression level of a recombinant protein by at least 100%, at least 120%, at least 140%, at least 150%, at least 160%, at least 180%, or at least 200% as compared to a culture method without succinic acid.
In a second aspect, the application also provides a medium comprising succinic acid or a salt thereof at a concentration of 2.0 to 50.0 mM. Preferably, the concentration of succinic acid or salt thereof is 2.0mM, 4.0mM, 8.0mM, 10.0mM, 12.0mM, 14.0mM, 16.0mM, 18.0mM, 20.0mM, 22.0mM, 24.0mM, 26.0mM, 28.0mM, 30.0mM, 32.0mM, 34.0mM, 36.0mM, 38.0mM, 40.0mM, 42.0mM, 44.0mM, 46.0mM, 48.0mM, 50.0mM or any value therebetween. Further preferably, the concentration of succinic acid or a salt thereof is 8 to 32mM.
The succinate salt that can be used includes alkali metal salts or alkaline earth metal salts of succinic acid, such as lithium (Li), sodium (Na), potassium (K), magnesium (Mg), calcium (Ca) salts, and the like.
The culture medium of the application can be used for increasing the recombinant protein expression quantity of the mammalian cells expressing the recombinant protein. For example, the culture medium of the present application may be used in fed-batch culture, batch culture or continuous culture to culture mammalian cells expressing recombinant proteins, thereby increasing the recombinant protein expression level of the mammalian cells.
In some embodiments, the medium further comprises a serum-free medium.
According to the application, succinic acid is added in the fed-batch culture stage, so that the highest hNGF yield can be improved to about 2900mg/L, and the expression level of hNGF in CHO cells is greatly improved.
Examples and figures are provided below to aid in the understanding of the application. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the application in any way. The actual scope of the application is set forth in the following claims. Throughout the specification, references to "one embodiment," "an embodiment," "one example," or "an example" mean: a particular feature, structure, or characteristic described in connection with the embodiment or example is included within at least one embodiment of the application. Thus, the appearances of the phrases "in one embodiment," "in an example," or "in an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and/or sub-combination in one or more embodiments or examples. It will be understood that any modifications and variations may be made without departing from the spirit of the application.
Examples
EXAMPLE 1 NGF expression with addition of succinic acid at various concentrations
The cell culture procedure was as follows:
1) CHO cells containing recombinant expression vectors expressing wild-type human NGF beta subunit (β -NGF) were constructed using conventional methods as described previously:
the coding nucleic acid sequence of wild human beta-NGF is synthesized by conventional means, and the nucleic acid sequences are cloned into a vector pUC-GW-Kan (Jin Weizhi Biotechnology Co., ltd.) respectively, to prepare recombinant plasmid pUC-GW-Kan-beta-NGF. An expression plasmid for the protein of interest is then constructed. In brief, the expression vector pMGT (Hanteng organism) described in CN2022100228281 is digested with HindIII and BamHI respectively to obtain recombinant plasmid pUC-GW-Kan-beta-NGF, and the recovered target fragment beta-NGF is connected with linearized expression vector pMGT to obtain expression plasmid pMGT-beta-NGF of beta-NGF protein.
Chinese hamster ovary CHO cells were used as host cells for the expression of β -NGF proteins. The constructed expression plasmid pMGT (+) -beta-NGF was transfected into CHO cells, respectively. Culturing until the activity rate is recovered to more than 90%, obtaining a stable cell pool which can stably express beta-NGF protein, and carrying out liquid nitrogen freezing for resuscitation.
2) Resuscitating the CHO cell pool expressing beta-NGF, inoculating to CD CHO Fusion serum-free medium, and passaging for 3 times until the cell activity rate is recovered to more than 96%;
3) The cells were then mixed at 0.5X10 6 Inoculating cells/mL into shake flasks, using an Ex-cell Advanced fermentation medium as an initial medium, and adding different amounts of succinic acid according to different groups respectively to make the final concentration of succinic acid be 2, 4, 8, 16 and 32mM;
4) Continuing to culture cells, adding Cell boost 7a+7b feed medium every other day from day 3, sampling every day during the culture period, and detecting the Viable Cell Density (VCD) and the Cell Viability (VIA) respectively by using a Vi-Cell cytometer;
5) Stopping culturing until the cell activity is lower than 85% or culturing for 14 days;
6) After harvesting the cell suspension and centrifuging, the supernatant is subjected to ELISA detection of NGF (R & D DY256, system, human beta-NGF DuoSet ELISA) to determine the expression level of beta-NGF, and if necessary, the supernatant may be diluted and subjected to ELISA detection.
Comparative example 1 NGF expression without succinic acid addition
The experimental procedure of comparative example 1 was the same as example 1, the only difference being that succinic acid was not added in step 3).
Experimental results
As shown in FIG. 1, the cell viability was maintained at 85% or more for 14 days of culture at a final concentration of 8mM in comparison with comparative example 1, exceeding that of comparative example 1 for at least 3 days. The final concentration of succinic acid was 16mM, which gives the highest viable cell density, and the difference from comparative example 1 was significant.
FIG. 2 shows that NGF expression levels increased significantly at final concentrations of succinic acid of 8mM,16mM and 32mM relative to comparative example 1 to 3.2-fold, 2.78-fold and 2.3-fold, respectively.
From this, it was found that addition of succinic acid to the medium can promote cell growth, prevent apoptosis, and significantly promote expression of β -NGF protein. In addition, the experimental result shows that the succinic acid with the final concentration of 8mM-32mM has obviously improved beta-NGF expression level, and the yield is more than 2000mg/L, wherein the fed-batch culture method with the final concentration of 8mM succinic acid is used, the maintenance time of the cell viability of CHO cells is longest, the beta-NGF expression level is highest, and the optimal concentration of succinic acid for fermenting and culturing NGF expression is reached to 2929.5 mg/L.
The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the application can be made without departing from the spirit of the application, which should also be considered as disclosed herein.
Claims (10)
1. A method of culturing mammalian cells expressing a recombinant protein, the method comprising: culturing the mammalian cells in a medium comprising succinic acid or a salt thereof, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
2. A method of increasing the expression level of a recombinant protein in a mammalian cell expressing the recombinant protein, the method comprising: culturing the mammalian cells in a medium comprising succinic acid or a salt thereof, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
3. The method of claim 1 or 2, wherein the succinic acid or salt thereof is added at a concentration of 2.0 to 50.0 mM.
4. The method of claim 1 or 2, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell, preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO DXB11 cell, a CHO-S cell, a CHO GS deficient cell or a derivative of any of these cells.
5. The method of claim 1 or 2, wherein the cultivation is selected from batch cultivation, fed batch cultivation or continuous cultivation, preferably batch cultivation and fed batch cultivation, more preferably fed batch cultivation.
6. The method of claim 1 or 2, wherein the recombinant Nerve Growth Factor (NGF) and fragments thereof are recombinant human nerve growth factor and fragments thereof, and/or the fragments of recombinant nerve growth factor are selected from the group consisting of or comprising the β subunit of nerve growth factor (β -NGF).
7. The method of claim 1 or 2, further comprising the step of isolating the recombinant protein.
8. The method of claim 2, wherein the method increases the expression level of the recombinant protein by at least 100% as compared to a culture method without succinic acid.
9. A culture medium for increasing the expression level of a recombinant protein in a mammalian cell expressing the recombinant protein and comprising succinic acid or a salt thereof at a concentration of 2.0 to 50.0mM, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof; preferably, the medium comprises a serum-free medium.
10. Use of succinic acid or a salt thereof for increasing the expression level of a recombinant protein in a mammalian cell expressing the recombinant protein, wherein the recombinant protein is selected from the group consisting of recombinant Nerve Growth Factor (NGF) and fragments thereof.
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