Cell culture media and feed development for mammalian cells

Abstract

Development of media and feeds for CHO cells The present invention provides a DNA array-based method for assessing the effect of at least one test component of a cell culture medium on at least one phenotype of interest in a test mammalian cell line cultured in the cell culture medium comprising the test component, comprising: (a) measuring a test methylation profile of one or more preselected methylation sites within the DNA of a test cell line; (b) comparing the test methylation profile obtained from (a) with at least one control methylation profile obtained from the same mammalian cell line cultured in a cell culture medium that does not contain the test component. and a significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test cell has the phenotype of interest and that the test component does not affect the phenotype of interest; a significant difference between the test methylation profile of (a) and the control methylation profile indicates that the test cell has a phenotype of interest and that the test component affects the phenotype of interest; (c) The test methylation profile obtained from (a) (i) at least one first control methylation profile obtained from a first mammalian control cell line exhibiting at least one phenotype of interest; and / or (ii) at least one second control methylation profile obtained from a second mammalian control cell line that does not exhibit the phenotype of interest; Process to compare with and The control cell line was not in contact with the test component. a significant similarity between the test methylation profile of (a) and the first or second control methylation profile indicates that the test cell has a phenotype of interest or does not have a phenotype of interest, respectively; wherein a difference between the test methylation profile of (a) and the first or second control methylation profile indicates that the test cell does not have a phenotype of interest or that the test cell has a phenotype of interest.

Description

[Technical Field]

[0001] The present invention relates to an epigenetics-based method, i.e., a DNA methylation-based method, for quantitatively and qualitatively assessing the influence of a cell culture medium / feed or its components on at least one phenotype of interest, such as cell viability, cell performance and / or production of a protein of interest in mammalian cells, as well as on cell stability before, during or after the actual production of said protein. Specifically, a DNA methylation array can be used to measure the differential methylation of promoters and / or CpG sites in mammalian cells in the presence of at least one component of the cell culture medium, thereby providing insight into the effect of said component on the quantitative and qualitative production of a protein of interest by mammalian cells. [Background technology]

[0002] Mammalian cells are used in research and for the production of recombinant proteins, including therapeutic proteins (e.g., monoclonal antibodies). These mammalian cells are typically grown and cultured in cell media containing serum or protein hydrolyzed components (e.g., peptone, tryptone). These components contain growth factors and a variety of other uncharacterized components that are beneficial for cell growth and culture. However, they also contain uncharacterized components that inhibit growth or adversely affect recombinant protein production. They may also be the source of undesired mutations. Typically, most mammalian cell lines exhibit high initial protein expression, but production levels decline as the culture period increases. This results in lower process yields, impacting timelines and increasing costs. Changes in the cell culture environment can lead to changes in cellular behavior and protein productivity of production cell lines. Cell culture media provide sufficient nutrients to all cells to achieve optimal growth, high productivity, and quality. Optimizing the appropriate media is crucial for cell line development and bioprocessing. Media composition and optimization have a significant impact on cell health, metabolism, protein production, and quality. For example, several studies have concluded that media composition broadly influences protein quality attributes, such as glycosylation patterns, aggregation, and charge variants. The composition of individual media components and their relative concentrations can significantly alter media performance. However, the impact of media optimization is not always uniform because different cell lines producing various recombinant proteins can respond in different ways to a given media composition. Therefore, media optimization is a topic of ongoing research to improve cell growth, protein productivity, and quality. Media optimization typically involves multiple optimization rounds, analyzing spent media for individual component utilization and monitoring the impact of supplements on the desired culture outcome. Media are composed of numerous components, each of which can be combined in numerous concentration-dependent combinations. As a result, the optimization process is cumbersome, highly complex, restrictive and time consuming. To reduce the number of physical experiments, mathematical models such as design of experiments (DOE) have been developed to predict the outcome of media formulations. However, these algorithms require data input from cell culture systems. Therefore, when starting a media development project from scratch, it can take several months to optimize the appropriate formulation. Chinese hamster ovary (CHO) cells have been known as the mainstay of industrial production of recombinant therapeutic proteins since 1987 and are widely used in biopharmaceutical manufacturing. Approximately 70% of recombinant biopharmaceutical proteins and monoclonal antibodies approved since 2016 have been produced in CHO cells. The advantages of using CHO cells for biopharmaceutical manufacturing include their resistance to genetic manipulation, ease of adaptability to manufacturing process scale, rapid growth rate, and ability to perform human-compatible post-translational modifications. However, biopharmaceutical manufacturing systems using CHO cells face a bottleneck due to a decline in protein productivity over time. Summary of the Invention [Problem to be solved by the invention]

[0003] In the current bioproduction market, there is a strong demand for efficient and robust analytical methods to monitor medium composition and improve medium formulation optimization and development. Such methods can meet the diverse metabolic needs of various clones and cell lines. Unfortunately, most media currently used on the market are not fully optimized due to a lack of robust analytical tools. Therefore, there is still a need for such robust analytical tools to optimize cell media for mammalian cell lines, especially CHO cells.

[0004] FIG. 1 is a graph showing the results of principal component analysis (PCA) of the identified 122 differentially methylated regions (DMRs). FIG. 2 is a graph showing the results of principal component analysis (PCA) of the identified 289 differentially methylated regions (DMRs). Figure 3 is a diagram of the cell culture workflow in a medium adaptation experiment. FIG. 4 is a graph showing the results of a PCA analysis of a medium-adaptation experiment in which all CpG sites were methylated. FIG. 5 is a graph showing the results of a PCA analysis of a medium adaptation experiment with differential methylation of CpG sites. [Means for solving the problem]

[0005] The present invention addresses these problems by providing a method for distinguishing the effect of one cell culture medium component from the effect of other components on cells cultured in the cell culture medium using DNA methylation patterns. This method according to any of the embodiments of the present invention is not only accurate and reliable, but also saves the time, cost, and effort required to measure the effect of a particular cell culture medium or its components on a cell's desired phenotype, such as the overall health and / or performance of the cell in the short or long term. In particular, the method according to any of the embodiments of the present invention can be used to measure and / or predict the long-term effect of a cell culture medium or its components on the stability, growth, and protein production capacity of cells cultured in the cell culture medium without long-term monitoring of the cell or cell population. In particular, the cells may be mammalian cells. The method according to any of the embodiments of the present invention also provides a method for predicting cell performance based on the DNA methylation profile of the cells. In particular, the method according to any of the embodiments of the present invention also provides a method for monitoring the effect of cell culture medium type, its components, or regimen on the current or future performance of the cells. Furthermore, the method according to any of the embodiments of the present invention further provides a means for managing cell culture operations by determining the appropriate cell culture medium and / or its components for culturing the cells in order to obtain the best performance and / or desired prototype from the cells. Improved control allows for optimization of the performance of the cells and the heterologous proteins produced therefrom.

[0006] The present invention is based on the discovery that components of cell culture media can alter a cell's epigenome through epigenetics. In particular, the ability to adapt to the environment and maintain adapted biological patterns depends on epigenetic mechanisms, including DNA methylation. More specifically, the present invention is based on the discovery that cell culture media can also alter a cell's epigenetic mechanisms, including DNA methylation patterns, and that these patterns can be inherited by various products derived from the cell.

[0007] The inventors have unexpectedly discovered that this property can be exploited to identify a genomic "epigenetic fingerprint" unique to a cell culture medium component, which can improve the overall cellular stability and / or performance of not only a single cell receiving that component, but all cells grown in the cell culture medium or its components. Based on these discoveries, the present invention provides a means for identifying the specific short- and long-term effects that all components of a cell culture medium have on the overall cellular stability and / or performance of cells cultured in that medium. In particular, the methods of any embodiment of the present invention may be used to determine whether a particular component of a cell culture medium has a positive or negative impact on the overall cellular stability and / or performance of cells. For example, component X in a cell culture medium may improve the overall cellular stability and / or performance of cells cultured in the cell culture medium in the short and / or long term, resulting in cells with relatively better cellular stability and / or performance. In another example, component Y in a cell culture medium may deteriorate the existing overall cell stability and / or performance of cells cultured in the cell culture medium, resulting in relatively poor cell stability (i.e., cell exhaustion and low cell viability) and / or performance of the cells. More specifically, the method according to any embodiment of the present invention may be used to determine whether a particular component of a cell culture medium or the cell culture medium itself has a positive or negative impact on the overall cell stability and / or performance of the cells themselves. In this way, the method according to any embodiment of the present invention may be used to accurately, reliably, and quickly measure the specific effect of a component in a cell culture medium on cells, and based on these results, it can be determined whether the component should be included in the cell culture medium of the cells or removed from the cell culture medium in which the cells are cultured.

[0008] According to one aspect of the present invention, there is provided a DNA array-based method for assessing the effect of at least one test component of a cell culture medium on at least one phenotype of interest in a test mammalian cell line cultured in the cell culture medium comprising the test component, comprising: (a) measuring a test methylation profile of one or more preselected methylation sites within the DNA of a test cell line; (b) comparing the test methylation profile obtained from (a) with at least one control methylation profile obtained from the same mammalian cell line cultured in a cell culture medium that does not contain the test component. and a significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test cell has the phenotype of interest and that the test component does not affect the phenotype of interest; A method is provided in which if the test methylation profile of (a) and the control methylation profile are significantly different, this indicates that the test cell has a phenotype of interest and the test component affects the phenotype of interest.

[0009] As used herein, the term "phenotype of interest" in relation to mammalian cells refers to cells that exhibit at least one characteristic selected from the group consisting of optimal heterologous protein production, phenotypic uniformity, protein quality, optimal carbohydrate metabolism, optimal amino acid metabolism, optimal lipid metabolism, optimal cell viability, and combinations thereof. In particular, the phenotype of interest refers to a characteristic exhibited by a mammalian cell according to any embodiment of the present invention that is beneficial to cell survival, the suitability of the cell for protein production, and the overall protein production of the cell. In particular, the "phenotype of interest" is not limited to protein productivity, but can also assess optimal conditions for heterologous protein production, phenotypic uniformity, protein quality, optimal carbohydrate metabolism, optimal amino acid metabolism, optimal lipid metabolism, and / or optimal cell viability.

[0010] As used herein, the term "suitability" refers to a mammalian cell line that is suitable for optimal heterologous protein production. In one example, a mammalian cell line can be considered suitable for optimal heterologous protein production before a transgene is introduced into the cells. In this case, the mammalian cell line can have at least one desired phenotype or characteristic that allows the cell line to grow well, easily incorporate a transgene of interest, and, after incorporating the transgene, optimally produce the protein that is the product of the transgene of interest as a heterologous protein. These desired characteristics or phenotypes include at least optimal glucose consumption, growth rate, lactate production, ammonia accumulation, etc. If a mammalian cell line is confirmed to exhibit at least one of these desired phenotypes, the mammalian cell line can be considered suitable for optimal heterologous protein production when a transgene of interest is introduced into the cells.

[0011] In another example, a mammalian cell line may be suitable for optimal heterologous protein production after a transgene has been introduced into the cells. In this case, the mammalian cell line is genetically modified using methods known in the art, and a transgene is introduced into the cells. The genetically modified cells can optimally produce the protein, which is the translation product of the transgene, as a heterologous protein. In this example, the mammalian cell line may have at least one desired phenotype that enables the genetically modified cell line to have good viability and optimally produce the desired protein. These desired phenotypes may include cell viability (viability), protein productivity (in terms of protein quantity and quality), phenotypic uniformity, cell exhaustion, etc. Therefore, the method according to any aspect of the present invention may be used with a mammalian cell line that has been genetically modified (i.e., a transgene has been introduced into the cell line) or a mammalian cell line that has not yet been genetically modified. In either case, the mammalian cell line is used for heterologous protein production.

[0012] As used herein, the term "transgene" refers to a gene that is taken from the genome of one organism and inserted into the genome of another organism by artificial techniques used for genetic modification. For example, a human gene is artificially introduced into the genome of a mammalian cell to produce at least one protein of interest, particularly a therapeutic protein.

[0013] As used herein, the term "therapeutic protein" refers to a genetically engineered version of a naturally occurring human protein. Examples of therapeutic proteins include therapeutic antibodies, anticoagulants, blood factors, bone morphogenetic proteins, artificial scaffolding proteins, enzymes, growth factors, hormones, interferons, interleukins, etc.

[0014] As used herein, the term "cell viability" refers to the ability of cells to survive and undergo cell proliferation. Cell viability is a measure of the proportion of living cells in a cell population. Cell proliferation refers to an increase in cell number due to cell division. Assays commonly used to test cell viability include the BrdU cell proliferation assay, the MTT cell proliferation assay, trypan blue counting, and the ATP cell viability assay.

[0015] As used herein, the term "cell exhaustion" refers to a state in which cells have lost the ability to perform metabolic activities, including heterologous protein production. Cell exhaustion can be measured by metabolite detection assays.

[0016] As used herein, the term "phenotypic homogeneity" refers to a state in which all cells in a cell population exhibit the same phenotype under certain conditions.

[0017] As used herein, "heterologous protein production" refers to the production of a protein that is not endogenous to a cell. This refers to the expression of a gene or part of a gene, particularly a transgene, in a host mammalian cell that does not naturally express that gene. Assays commonly used to quantify heterologous protein production include enzyme-linked immunosorbent assays (ELISAs), chromatography, and bioprocess analyzers. As used herein, the term "host cell" refers to a cell line for expressing a heterologous protein. For example, CHO cells are a primary host for the production of various therapeutic proteins.

[0018] As used herein, the term "optimal heterologous protein production" refers to mammalian cells capable of high-level protein production, particularly in industrial or large-scale production of recombinant proteins. The protein is typically a functional protein not naturally present in wild-type mammalian cells. In particular, for optimal heterologous protein production, mammalian cell lines minimize metabolic burden and toxic effects on the cells. More specifically, "optimal heterologous protein production" refers to high-level protein production in which a mammalian cell line, e.g., CHO cells, not only produces a protein of interest at a high yield, but also maintains consistent protein production over the production period (i.e., long-term culture), thereby consistently maintaining the quality of the protein produced. In particular, for mammalian cells according to any embodiment of the present invention to be capable of "optimal heterologous protein production," the cells must exhibit at least one or more of the following target phenotypes: phenotypic uniformity, protein productivity, and protein quality. More specifically, for "optimal heterologous protein production," mammalian cells may have phenotypic uniformity and protein productivity, or phenotypic uniformity and protein quality, or protein productivity and protein quality, or phenotypic uniformity, protein productivity and protein quality.

[0019] As used herein, the term "protein productivity" refers to a measure of protein production per viable cell at a single titer point. It is calculated by dividing the titer (mg) by the viable cell density (VCD or cells / mL), with the final measurement expressed as protein per cell (mg / cell).

[0020] The term "protein quality" refers to post-translational modifications that determine a protein's efficacy and function. These modifications typically include phosphorylation, glycosylation, ubiquitination, methylation, acetylation, and protein folding. For example, protein glycosylation is an important quality characteristic that regulates the efficacy, stability, and half-life of therapeutic proteins. Protein quality can be measured using immunoprecipitation techniques, biochemical assays, mass spectrometry (MS), and other methods.

[0021] As used herein, the term "carbohydrate metabolism" refers to nearly all biochemical processes responsible for the metabolic production, degradation, and interconversion of carbohydrates within cells. This involves multiple pathways, such as glycolysis, gluconeogenesis, glycogenolysis, and glycogenogenesis. For example, glycolysis is one of the major metabolic pathways in CHO cells. Through glycolysis, CHO cells consume glucose as the primary carbon source for energy production and produce lactic acid as the most common metabolic by-product. In particular, the term "optimal carbohydrate metabolism" refers to the ideal or best carbohydrate metabolism that CHO cells are capable of performing.

[0022] Similarly, as used herein, the term "amino acid metabolism" refers to the entire biochemical process responsible for the metabolic production, degradation, and interconversion of amino acids within cells. Amino acids are the basic building blocks of proteins and constitute all proteinaceous materials in cells, including the cytoskeleton, protein components of enzymes, receptors, and signaling molecules. In addition, amino acids are utilized for cell growth and maintenance. For example, glutaminolysis is an important metabolic pathway in CHO cells. Glutaminolysis is a common pathway by which CHO cells absorb organic nitrogen for biomass synthesis while releasing ammonium as a major byproduct. In particular, the term "optimal amino acid metabolism" refers to the ideal or best amino acid metabolism that CHO cells can perform.

[0023] As used herein, the term "lipid metabolism" refers to the synthesis and degradation of lipids within cells, including the breakdown or storage of fat as an energy source and the synthesis of structural and functional lipids. Lipids are major components of cell membranes, function as second messengers in intercellular communication, and are involved in signal transduction, transport, and secretion. Lipids are also important sources of energy through β-oxidation and the tricarboxylic acid (TCA) cycle. Lipid metabolism can have a significant impact on cell growth. For example, the processes of triacylglycerol synthesis and degradation in CHO cells can significantly affect overall cellular metabolism and viability. In particular, the term "optimal lipid metabolism" refers to the ideal or best amino acid metabolism that CHO cells can perform.

[0024] Carbohydrate, amino acid, and lipid metabolism can be measured by metabolite detection assays, HPLC, and bioprocess analyzers, as described at least in Coulet, M. et al., Cells (2022), 11, 1929; Fan Y. et al., Biotechnol Bioeng (2015) 112(3):521-535; and Ali AS et al., Biotechnol J. (2018);13(10):e1700745.

[0025] The terms "methylation profile," "methylation pattern," "methylation state," or "methylation status" are used herein to describe the state, status, or condition of methylation of a genomic sequence, and refer to characteristics associated with the methylation of DNA fragments at specific genomic loci. These characteristics include, but are not limited to, whether any cytosine (C) residues in a DNA sequence are methylated, the location of methylated C residues, the proportion of methylated C residues in specific residue regions, and inter-allelic differences in methylation due to differences in allelic origin, etc.

[0026] The term "methylation status" refers to the status of a particular methylation site (i.e., methylated vs. unmethylated), meaning that a residue or methylation site is methylated or unmethylated. Based on the methylation status of one or more methylation sites, a methylation profile can be measured. Thus, the term "methylation profile" or "methylation pattern" refers to the relative or absolute concentration of methylated or unmethylated C residues in a particular residue region in the genomic material of a biological sample. For example, if a cytosine (C) residue that is normally unmethylated in a DNA sequence is methylated, it can be referred to as "hypermethylated." On the other hand, if a cytosine (C) residue that is normally methylated in a DNA sequence is unmethylated, it can be referred to as "hypomethylated." Similarly, if a cytosine (C) residue in a DNA sequence (e.g., DNA derived from a sample nucleic acid from a test subject) is methylated compared to another sequence from a different region or a different individual (e.g., compared to a standard nucleic acid such as a normal nucleic acid or control sequence), the sequence is considered to be hypermethylated relative to other sequences. On the other hand, if a cytosine (C) residue in a DNA sequence is unmethylated compared to another sequence from a different region or a different individual, the sequence is considered to be hypomethylated compared to other sequences. These sequences are called "differential methylation." The measurement of the level of differential methylation can be performed by various methods known to those skilled in the art. One method is, but is not limited to, using the bisulfite method to measure the methylation level of each CpG site measured.

[0027] The term "hypermethylation" refers to an average methylation state corresponding to an increased abundance of 5-mCyt at one or more CpG dinucleotides in a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0028] The term "hypomethylation" refers to an average methylation state corresponding to a decreased abundance of 5-mCyt at one or more CpG dinucleotides in a DNA sequence of a test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0029] As used herein, the term "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl group on a nucleotide base, which is not present in a typical nucleotide base that is normally recognized. For example, cytosine does not have a methyl group on the pyrimidine ring in its normal form, but 5-methylcytosine has a methyl group at the 5th position of the pyrimidine ring. Thus, although cytosine is not considered a methylated nucleotide in its normal form, 5-methylcytosine can be considered a methylated nucleotide. As another example, thymine has a methyl group at the 5th position of the pyrimidine ring, but in this specification, thymine is not considered a methylated nucleotide when present in DNA. Typical nucleotide bases in DNA are thymine, adenine, cytosine, and guanine. Typical bases in RNA are uracil, adenine, cytosine, and guanine. Correspondingly, a "methylation site" is a position where methylation may occur in a nucleic acid region of a gene of interest. For example, a site containing CpG is a methylation site, and cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide having a methyl group attached to a nucleotide position that can be methylated. These methylated nucleotides are usually found in nature, and to date, they are mainly found in dinucleotide CpG sequences, although methylated cytosine, which is also found in CpNpG and CpNpN sequences, is considered to be the most common. In principle, other naturally occurring nucleotides can also be methylated, but they are not considered in any aspect of the present invention.

[0030] A "control methylation profile" can be defined based on multiple training samples using multivariate analysis methods such as principal component analysis and multidimensional scaling.

[0031] In particular, a control methylation profile according to any of the embodiments of the present invention is a compilation of two or more CpG sites obtained from at least one control mammalian cell line exhibiting at least one phenotype of interest. In one example, different CpG sites are collected from a single control mammalian cell line exhibiting at least one phenotype of interest. In another example, different CpG sites are collected from multiple cell lines, each cell line exhibiting at least one phenotype of interest. Thus, a control methylation profile according to any of the embodiments of the present invention may not be a naturally occurring methylation profile obtained from a single mammalian cell line, but may be an artificial profile obtained by combining related CpG sites obtained from different control mammalian cell lines, each exhibiting at least one phenotype of interest.

[0032] As used herein, "CpG site" or "methylation site" refers to a nucleotide in a nucleic acid (DNA or RNA) that is susceptible to methylation, either by naturally occurring events in vivo or by events introduced in vitro to chemically methylate the nucleotide. Some of these sites may be hypermethylated or hypomethylated in cells. In some cases, a CpG site may not be considered completely hypermethylated or hypomethylated, but a value may be provided that provides a measure of the methylation of the CpG site. Thus, methylation may be quantified and not necessarily completely hypermethylated or hypomethylated.

[0033] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that includes one or more nucleotides that are methylated.

[0034] In this specification, "CpG island" refers to the segment of DNA sequence that contains functionally or structurally deviant CpG density.For example, Yamada et al. have described a set of criteria for measuring CpG island: CpG island must be at least 400 nucleotides in length, have a GC content of more than 50%, and an OCF / ECF ratio of more than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266).On the other hand, some literature has more leniently defined CpG island as a sequence that is at least 200 nucleotides in length, has a GC content of more than 50%, and an OCF / ECF ratio of more than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745).

[0035] In particular, if a methylation difference is detected in the test cells, i.e., if the cells show absolute hypermethylation or hypomethylation, or at least a quantitative methylation difference, at at least one CpG site compared to a control (i.e., a CHO cell line with at least one phenotype of interest), the test cells may also contain the phenotype of interest and be capable of optimal heterologous protein production. More specifically, if the CpG sites in the test cells show the same methylation status as the corresponding CpG sites in the control cells or control methylation profile, the test cells may express the phenotype of interest and be capable of optimal heterologous protein production. Overall, this platform provides an opportunity to detect a wide range of DNA methylation states in CHO cells and correlate them with industry-relevant parameters essential for the development of at least one biopharmaceutical.

[0036] In particular, in step (a) of the method according to any embodiment of the present invention, the methylation status of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 CpG sites is measured. A person skilled in the art will be able to determine the number of CpG sites that need to be used in step (a) of any embodiment of the present invention. Even more particularly, in step (a) of the method according to any embodiment of the present invention, the methylation status of at least two CpG sites is measured.

[0037] As used herein, the term "epigenetic changes" refers to chemical (e.g., methylation) or protein (e.g., histone) changes that occur in the body of a gene or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can leave imprints on genes that are passed on from one generation to the next.

[0038] As used herein, the term "significantly similar," particularly in the context of comparing methylation profiles (e.g., comparing a test profile (from a test subject) with a control profile), refers to similarity observed by statistical means (i.e., using bioinformatics) and / or visual observation. Significant similarity is observed, for example, when a test profile overlaps with a control profile defined by multiple training samples through multivariate analysis methods such as principal component analysis or multidimensional scaling. In particular, a test profile is significantly similar to a given control profile when more than 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the methylation pattern / profile overlaps with that of the control profile. The significance of similarity decreases when a test profile is similar to multiple control profiles, such as two, three, or all of the control profiles.

[0039] As used herein, the term "genomic material" refers to nucleic acid molecules or fragments of the genome of a mammalian cell or cell line. In particular, such nucleic acid molecules or fragments are DNA, RNA, or hybrids thereof, and most preferably, molecules of the DNA genome of a CHO cell or cell line.

[0040] As used herein, a "DNA sample" refers to DNA extracted from cells according to any aspect of the present invention using methods known in the art.

[0041] As used herein, the term "preselected methylation sites" refers to methylation sites selected from genes or regions that showed the highest methylation variation during training of the method, and that meet certain quality criteria, such as a minimum sequence coverage of 5x or more and five or more eligible CpG sites. Additionally, genes with average methylation levels below 0.1 or above 0.9 can be excluded due to their limited dynamic range. In particular, the preselected methylation sites are associated with at least one phenotype of interest in the test cell line.

[0042] As used herein, the term "cell culture medium" is used interchangeably with the term "cell culture medium" or "fermentation broth" when cells are cultured in a fermenter or bioreactor. Specifically, cell culture medium refers to a medium for culturing mammalian cells, particularly in a buffered medium (e.g., pH about 7.0, particularly pH 7.3-6.6, more particularly pH 7.0), containing a minimum of essential nutrients and components, such as vitamins, trace elements, salts, bulk salts, amino acids, lipids, and carbohydrates. The cell culture medium may be a basal cell culture medium or a basal cell culture medium to which additives may be added. Because arrays according to any embodiment of the present invention are developed from experimentally identified functional CpG sites, all components and / or additives of the culture medium may be considered "components" of the cell culture medium.

[0043] As used herein, the term "basal medium" or "basal cell culture medium" refers to a cell culture medium for culturing mammalian cells. It is used to culture cells from the initiation of cell culture and is not used as an additive to other media, although various (test) components may be added to the medium. The basal medium serves as a base for adding additional additives or feed media as needed during the culture, i.e., cell culture. The basal cell culture medium is provided from the beginning of the cell culture process. Generally, basal cell culture media provide nutrients such as a carbon source, amino acids, vitamins, bulk salts (e.g., sodium chloride or potassium chloride), various trace elements (e.g., manganese sulfate), pH buffer, lipids, and glucose. Major bulk salts are usually provided only in the basal medium, and the final osmolality of the cell culture medium should not exceed approximately 280-350 mOsm / kg to ensure that the cell culture can grow and proliferate under appropriate osmotic stress.

[0044] As used herein, the term "feed" or "feed medium" refers to a nutrient concentrate / concentrated nutrient composition used as a feed in mammalian cell culture. It is provided as a "concentrated feed medium" to avoid dilution of the cell culture broth. A feed medium typically contains high concentrations of most, but not all, components of a basal cell culture medium. Generally, a feed medium replaces nutrients consumed during cell culture, such as amino acids and carbohydrates, while salts and buffers are less important and are typically added to the basal medium. A feed medium is typically added to a (basal) cell culture medium / fermentation broth in fed-batch culture. However, a feed can be added in various ways, including continuous addition, bolus addition, or perfusion-related techniques (e.g., chemostat or hybrid perfusion systems). Each component of a feed or feed medium, and the specific concentration of each component, may fall within the definition of "test component" as used herein. The feed rate is understood as the average feed rate over the entire feeding period. In particular, feed medium is added daily, but may be added more frequently, such as twice a day, or less frequently, such as every other day. Nutrient additions are typically made during culture (i.e., from day 0 onwards). In contrast to basal medium, feeds consist of highly concentrated nutrient solutions (e.g., greater than 6x) and provide all the same components as basal medium, except for "osmotically active compounds" such as major bulk salts (e.g., NaCl, KCl, NaHCO3, MgSO4, Ca(NO3)2).

[0045] Cell culture media (basal and / or feed media) may be serum-free, chemically defined, or chemically defined and protein-free. As used herein, "serum-free media" refers to cell culture media for in vitro cell culture that does not contain animal-derived serum. Serum-free media are preferred because serum may contain animal-derived contaminants such as viruses, and serum is ill-defined and subject to batch-to-batch variation. Basal and feed media according to any embodiment of the present invention may be serum-free.

[0046] As used herein, "chemically defined media" refers to cell culture media with known components that are suitable for in vitro cell culture. More specifically, the media do not contain any additives, such as animal serum, plant, yeast, or animal-derived hydrolysates. Hydrolysates may be included only if all components have been analyzed, their exact composition is known, and reproducible preparation is possible. The basal and feed media according to the present invention are preferably chemically defined media.

[0047] As used herein, the term "commercially available medium / medium system" refers to a commercially available cell culture medium with a completely known composition. These media serve as a control for the medium of the present invention because precise nutrient composition is required. Examples of commercially available media include DMEM:F12 (1:1), DMEM, HamsF12, and RPMI. The feed media of the commercially available media used herein were prepared as 12-fold concentrates of bulk salt-free basal media. The term "commercially available medium system" refers to a system comprising a commercially available basal cell culture medium (e.g., DMEM:F12 (1:1), DMEM, HamsF12, RPMI) and a feed medium that is a concentrated basal medium (e.g., 12-fold concentrate) of the respective bulk salt-free or reduced bulk salt-free basal medium.

[0048] The term "cell culture medium" according to any embodiment of the present invention may refer to any one of the above cell culture media for mammalian cell culture. A test component may then be added to the cell culture medium, and the effect of the test component on the mammalian cells may be measured using a method according to any embodiment of the present invention. In particular, the components added to the cell culture medium may be selected from the group consisting of only amino acids, small peptides, buffers, carbon-based energy sources such as carbohydrates (e.g., glucose, mannose), inorganic salts or ions, serum (or its essential components, such as growth factors, hormones, lipids, proteins, and trace elements), vitamins, and minerals.

[0049] As used herein, the term "amino acid" refers to the 20 naturally occurring amino acids encoded by the universal genetic code, typically L-amino acids (i.e., L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine). Amino acids (e.g., glutamine and / or tyrosine) may be provided as dipeptides for improved stability and / or solubility, preferably including L-alanine (L-ala-x) or L-glycine extensions (L-gly-x), such as glycylglutamine and alanylglutamine. Additionally, cysteine ​​may also be provided as L-cystine. As used herein, the term "amino acid" includes all of the various salts thereof, such as L-arginine monohydrochloride, L-asparagine monohydrate, L-cysteine ​​hydrochloride monohydrate, L-cystine dihydrochloride, L-histidine monohydrochloride dihydrate, L-lysine monohydrochloride, and hydroxyl L-proline, L-tyrosine disodium dihydrate.

[0050] Suitable buffering agents include, but are not limited to, Hepes, phosphate buffers (e.g., potassium phosphate monobasic, potassium phosphate dibasic, and / or sodium phosphate dibasic anhydrous, and sodium phosphate monobasic), phenol red, sodium bicarbonate, and / or sodium bicarbonate.

[0051] The terms "cell cultivation" or "cell culture" include cell culture and fermentation processes at any scale (e.g., from microtiter plates to large-scale industrial bioreactors, i.e., from sub-mL scale to over 10,000 L scale), any process mode (e.g., batch, fed-batch, perfusion, continuous), any process control mode (e.g., uncontrolled systems, fully automated systems, and controlled systems with control of pH, temperature, oxygen content, etc.), and any type of fermentation system (e.g., single-use systems, stainless steel systems, glassware systems). In a preferred embodiment of the present invention, the cell culture is a mammalian cell culture and is a batch or fed-batch culture.

[0052] As used herein, the term "fed-batch" refers to cell culture in which cells are continuously or periodically fed with a nutrient-containing feed medium. Feeding can begin immediately after the initiation of cell culture, or more typically on the first, second, or third day after initiation. Feeding can occur according to a pre-established schedule, such as daily, every two, or every three days. Alternatively, cell growth, nutrients, or toxic by-products can be monitored during culture, and feeding can be adjusted accordingly. Common monitoring methods for animal cell culture are described in the experimental section below. In general, the following parameters are often measured routinely: viable cell concentration, product concentration, metabolites such as glucose or lactic acid (an acidic waste metabolite that lowers pH and results from intracellular glucose conversion), pH, osmolality (an indicator of salt content), and ammonium (a growth inhibitor that adversely affects growth rate and reduces viable cell mass). Compared to batch culture (culture without feeding), fed-batch culture can achieve higher product titers. Typically, fed-batch culture is stopped at some point, and the cells and / or target protein in the culture medium are harvested and, if necessary, purified.

[0053] As used herein, the term "test" in conjunction with the term cell refers to a subject that is subjected to a method according to any embodiment of the present invention and that serves as the basis for the analytical application of the present invention. Accordingly, a "test cell" or a "test profile" refers to a cell that is tested according to the present invention, or a profile obtained or generated in connection therewith. Conversely, the term "control" refers to a primarily pre-measured subject that is used for comparison with a test subject. For example, the term "control cell" refers to a cell that is used for comparison with or as a control for a "test cell." Similarly, the terms "sample" and / or "test cell DNA sample" used according to any embodiment of the present invention refer to a subject that may be subjected to a method according to any embodiment of the present invention. In particular, the sample may be any DNA sample obtained from a test cell that may be subjected to a method according to any embodiment of the present invention, where a DNA methylation profile is first measured, and then the test methylation profile is compared to a control (a control methylation profile from a control cell that may or may not exhibit a phenotype of interest) to measure the effect of selected components of the cell on the phenotype of interest of the cell.

[0054] In the present specification, the term "comprising" is interpreted as encompassing both "including" and "consisting of," both meanings being specifically intended, and thus encompassing individually disclosed aspects of the present invention. In this specification, "and / or" is interpreted as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" is interpreted as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. In the context of this specification, the terms "about" and "approximately" indicate a range of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. These terms typically indicate a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the indicated numerical value. As a person skilled in the art would understand, the specific deviation of a numerical value for a particular technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally be subject to greater such deviations than artificial or engineered technical effects. Where an indefinite or definite article (e.g., "a", "an", "the") is used to refer to a singular noun, the plural of that noun is also included unless otherwise stated.

[0055] As used herein, the term "performance" refers to the protein production capacity of a cell (i.e., phenotypic uniformity, protein productivity, and protein quality). The term "general stability" of a cell refers to the state of the cell, such as cell viability, survival rate, vitality, and cell exhaustion.

[0056] The terms "vitality" and "viability" are used interchangeably and refer to the proportion of viable cells in a cell culture as measured by methods known in the art, such as trypan blue exclusion using the Cedex instrument (Innovatis AG, Bielefeld) based on automated microscopic cell counting. However, many other methods exist for measuring viability, including fluorometric methods (such as those based on propidium iodide), calorimetric methods, or enzymatic methods used to reflect the energy metabolism of living cells (e.g., LDH lactate dehydrogenase, or methods using certain tetrazolium salts such as Alamar Blue, MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), or TTC (tetrazolium chloride)).

[0057] As used herein, the term "mammalian cells" refers to cells of any animal belonging to the order Mammalia, including cells of mice, rats, monkeys, guinea pigs, dogs, minipigs, humans, cattle, sheep, pigs, goats, horses, donkeys, mules, hamsters, cats, dolphins, elephants, and the like. Mammalian cells also include established or immortalized cell lines. In particular, immortalized cell lines are capable of producing proteins, particularly therapeutic proteins. More specifically, immortalized cell lines can be therapeutic immortalized cell lines. For example, mammalian cells according to any embodiment of the present invention can be CHO cell lines, which refer to immortalized Chinese hamster ovary cell lines derived from Cricetulus griseus. In particular, the CHO cell line may be selected from the group comprising only CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO-DXB11 (ATCC), ExpiCHO-S™ cells (Thermo Fisher Scientific), FreeStyle™ CHO-S™ cells (Thermo Fisher Scientific), CHO1-15 [subscript 500] (ATCC), Agarabi CHO (ATCC), and CHOK1SV cells including all variants (e.g., POTELLIGENT®, Lonza, Slough, UK), CHOK1SV GS-KO (glutamine synthetase knockout) cells including all variants (e.g., XCEED™, Lonza, Slough, UK). Mammalian cells may be derived from baby hamster kidney fibroblasts (BHK (ATCC CCL-10) or Vero cells (ATCC CCL-81). Exemplary human cells include human embryonic kidney (HEK) cells such as HEK293 (ATCC CRL-1573), HEK 293T (ATCC CRL-3216), HeLa cells (ATCC CCL-2), NS0 cells (ECACC 85110503), or Sp2 / 0 cells (ATCC CRL-1581). Mammalian cells according to any embodiment of the invention may include mammalian cell cultures, which may be either adherent or suspension cell cultures.

[0058] The method according to any embodiment of the present invention is a DNA array, particularly a DNA methylation array. The array enables a high-throughput and robust method for measuring semi-quantitative / quantitative DNA methylation information using a small amount of target extracted DNA sample. These custom-designed arrays may use Illumina iScan and Infinium platform technology, or equivalent technologies, which allow for the placement of, for example, 100,000 beads covalently attached to DNA methylation probes on each chip. Each probe represents one CpG methylation site at the end of the probe sequence. Before hybridization on the array chip, the DNA sample undergoes bisulfite conversion, amplification, fragmentation, precipitation, and resuspension steps. On the chip, the DNA hybridizes to a bead at each CpG site, allowing methylation changes at each site to be unambiguously detected by single-nucleotide extension. This is particularly advantageous because the array format is simple and the array results are accurate and highly reproducible. Compared with other prior art methods of performing WGS / WGBS to identify differential methylation, the customized DNA methylation array according to any of the embodiments of the present invention may be used to assess DNA methylation, and compared with known methods in the prior art, the method according to any of the embodiments of the present invention is more efficient and accurate. In particular, the DNA methylation array according to any of the embodiments of the present invention is based on the deduction of methylation values ​​from multiple CpG sites (i.e., differentially methylated regions, dynamic regions, variably methylated regions) throughout the CHO cell genome and regulatory regions within the CHO cell genome.

[0059] Furthermore, compared to conventional sequencing, which can take weeks to generate data, this array technology has a much faster turnaround time. The volume and complexity of the data generated is less than with sequencing, resulting in a lower computational burden. This reduces the computational time required to obtain interpretable results from experimental groups. Overall, microarray technology is approximately 10 times faster and 10 times cheaper than conventional sequencing, yet is capable of quantifying methylation levels at specific CpG sites.

[0060] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which can be prepared synthetically or biosynthetically. The probe molecules within an array can be identical or different from one another. Arrays can take a variety of forms, including libraries of soluble molecules, libraries of compounds immobilized on resin beads, silica chips, or other solid supports.

[0061] In particular, arrays provide a convenient platform for simultaneously analyzing a large number of CpG sites, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 100,000, or more sites or loci. In particular, arrays contain a plurality of different probe molecules that are attached to a substrate or spatially distinguishable within the array. Examples of arrays that can be used in accordance with any embodiment of the present invention include slide arrays, silicon wafer arrays, liquid arrays, bead arrays, and the like. As an example, the array technology used in accordance with any embodiment of the present invention combines a compact array platform, a high degree of assay multiplexing, and scalable automation for sample and data processing.

[0062] In particular, an array according to any embodiment of the present invention may be an array of arrays (also referred to as a composite array) having multiple individual arrays configured to process multiple samples simultaneously. Examples of composite arrays and the technology behind them are disclosed at least in U.S. Pat. No. 6,429,027 and U.S. Patent Application Publication No. 2002 / 0102578. A composite array substrate has multiple individual array locations, each with multiple probes, and each array location physically separated from other assay locations on the same substrate to prevent fluid contacting one array location from contacting another array location. Each array location may have multiple different probe molecules attached to the substrate either directly or via rigid particles in the wells (also referred to herein as beads in the wells).

[0063] By way of example, the array substrate can be a fiber optic bundle or array of bundles, such as those described in U.S. Patent Nos. 6,023,540, 6,200,737, and / or 6,327,410. In fiber optic bundles or arrays of bundles, the probes are attached to the fibers directly or via beads. One of ordinary skill in the art would readily be able to determine which substrate is most suitable for an array according to any embodiment of the present invention. WO 2004 / 110246 further discloses other substrates that can be used in arrays according to any embodiment of the present invention, as well as methods for attaching beads to the substrate.

[0064] For example, the surface of the substrate can have physical modifications to allow probe attachment or to generate array locations.For example, the surface of the substrate can be modified to include chemically modified sites that are useful for covalently or non-covalently attaching probe molecules or particles with probe molecules attached thereto.Probes can be attached by any of a variety of methods known in the art, such as inkjet printing, spotting techniques, photolithography synthesis, or mask-based printing.WO 2004 / 110246 discloses these techniques in more detail.

[0065] By way of example, an array according to any embodiment of the invention can be a bead array in which beads are immobilized on a solid support, such as those commercially available from Illumina, Inc. (San Diego, Calif.). Bead arrays useful according to any embodiment of the invention can also be in a fluidic format, such as the fluid stream of a flow cytometer or similar device. Commercially available fluidic formats for identifying beads include those used in Luminex's XMAP™ technology and Lynx Therapeutics' MPSS™ method.

[0066] As used herein, the terms "solid support," "support," and "substrate" are used interchangeably to refer to a material or group of materials having a rigid or semi-rigid surface. In many instances, at least one surface of the solid support is substantially flat, although in some instances it may be desirable to physically separate synthesis regions for different compounds, e.g., using wells, raised areas, pins, etched grooves, etc.

[0067] Arrays or microarrays according to any embodiment of the invention may be very high density arrays, e.g., about 10,000,000 probes / cm 2 ~approximately 2,000,000,000 probes / cm 2 , or approximately 100,000,000 probes / cm 2 ~approximately 1,000,000,000 probes / cm2 High density arrays are particularly useful according to any aspect of the invention because they can include a large number of CpG sites on the array.

[0068] Arrays according to any aspect of the invention can be used to analyze or assess multiple loci, simultaneously or sequentially as needed. In one example, multiple different probe molecules can be attached to a substrate or spatially separated within the array. Each probe is typically unique to a particular locus and can be used to distinguish the methylation state of that locus.

[0069] The term "probe molecule" or "probe", as used interchangeably herein, refers to a surface-immobilized molecule that can be recognized by a specific target. The probes used in the array can be specific to the methylated alleles of CpG sites, the unmethylated alleles of CpG sites, or both, or the methylated alleles of non-CpG sites, the unmethylated alleles of non-CpG sites, or both.

[0070] As used herein, the term "target" refers to a molecule that has affinity for a particular probe molecule. Targets can be naturally occurring or artificial molecules. They can be used in their natural state or as aggregates. Targets can be attached to a binding member either covalently or non-covalently, directly or via a specific binding substance. Examples of targets that can be used in accordance with any embodiment of the present invention include methylated and unmethylated CpG sites. Targets are sometimes referred to in the art as antiprobes. Although the term "target" is used herein, it does not imply any difference in meaning.

[0071] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a sequence or primer-binding site on a single-stranded nucleic acid to be amplified. Complementary nucleotides are generally A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand are optimally aligned and compared, and pair with at least about 80%, usually at least about 90%-95%, and more preferably about 98%-100% of the nucleotides of the other strand, with appropriate nucleotide insertions or deletions. Fully complementary means 100% complementarity throughout the length of the sequence. For example, a 25-base probe is fully complementary to a target if all 25 bases of the probe are complementary to the contiguous 25-base sequence of the target and there are no mismatches between the probe and the target throughout the length of the probe.

[0072] The method according to any aspect of the present invention may further comprise the additional step of: (c) comparing the test methylation profile obtained from step (a) with (i) at least one first control methylation profile obtained from a first mammalian control cell line exhibiting at least one phenotype of interest; and / or (ii) at least one second control methylation profile obtained from a second mammalian control cell line that does not exhibit the phenotype of interest; Process to compare with and The control cell line was not in contact with the test component. a significant similarity between the test methylation profile of step (a) and the first or second control methylation profile indicates that the test cell has a phenotype of interest or does not have a phenotype of interest, respectively; A difference between the test methylation profile of step (a) and the first or second control methylation profile indicates that the test cell does not have the phenotype of interest, or that the test cell has the phenotype of interest.

[0073] In particular, the first control methylation profile is a collection of CpG sites obtained from at least one control cell line that exhibits at least one phenotype of interest, and the second control methylation profile is a collection of CpG sites obtained from at least one control cell line that does not exhibit at least one phenotype of interest.

[0074] The control methylation profile, specifically the first and second control methylation profiles, is a "predetermined control profile" used to refer to a typical or standard methylation profile of the genomic material of a mammalian control cell line exhibiting at least one phenotype of interest. In one example, a predetermined control profile may be used in connection with control cells that have demonstrated good protein production (i.e., the control cells are capable of high quantitative and qualitative protein production). In particular, the term "predetermined control profile" may be used herein in connection with control cells, where the control animal has good protein production and / or general stability, and the control cells have optimal carbohydrate metabolism, optimal amino acid metabolism, optimal lipid metabolism, optimal cell viability, or combinations thereof, compared to baseline values ​​for cells of the same species as the control cells.

[0075] As used herein, the term "baseline" in relation to a phenotype of interest refers to various aspects of cells when the cells are cultured in a cell culture medium that does not contain one or more optional additives. That is, it refers to the phenotype of interest when the cells are cultured in a basal medium. A panel of predetermined control profiles for control cells can also include profiles from various samples that exhibit various phenotypes of interest or combinations thereof. Each of these samples can have its own predetermined methylation control profile, which also constitutes part of the panel of predetermined control profiles.

[0076] According to a further aspect of the present invention, there is provided a DNA array-based method for assessing the effect of at least one test component of a cell culture medium on the production of at least one biosimilar from an immortalized test cell line, comprising: The biosimilar is significantly similar to the innovator protein produced by an immortalized control cell line that is the same cell line as the test cell line; (a) measuring a first test methylation profile from DNA obtained from an immortalized test cell line cultured in a cell culture medium containing a test component; (b) measuring a second methylation profile from DNA obtained from the immortalized test cell line cultured in a cell culture medium that does not contain the test component; (c) comparing the test methylation profile obtained from steps (a) and (b) with a control methylation profile obtained from an immortalized control cell line. and A method is provided in which a significant similarity between the test methylation profile of step (a) and the control methylation profile, and a dissimilarity between the test methylation profile of step (b) and the control methylation profile, indicates that the two cell lines produce a biosimilar and that the test component positively affects the production of a biosimilar from the immortalized cell line.

[0077] As used herein, the term "biosimilar" refers to a recombinant protein produced by genetically modified mammalian cells that is highly similar to an original biopharmaceutical reference product and shares quality, safety, and efficacy with that reference product. In particular, the product produced is phenotypically / epigenetically similar to the reference product. The term "biosimilar" is more clearly explained at least in A. Ishii-Watabe et al., (2019) Drug Metab. Pharmacokinet. 34(1):64-70, and Wolff-Holz, E. et al., (2019) BioDrugs 33, 621-634.

[0078] Information on DNA methylation patterns in cell lines could provide a clearer specification profile for product release in mammalian cells, serve as "copyright" protection from biosimilar developers, and potentially become a "gold standard" for the regulatory processes required for biosimilar development.

[0079] As used herein, the term "innovator protein" refers to a wild-type protein, i.e., a protein that occurs in nature.

[0080] According to a further aspect of the present invention, there is provided a DNA array-based method for evaluating the effect of at least one test component of a cell culture medium on the production of at least one bioidentical from an immortalized test cell line, comprising: Bioidenticals are significantly similar to Innovator proteins produced by an immortalized control cell line that is the same cell line as the test cell line; (a) measuring a first test methylation profile from DNA obtained from an immortalized test cell line cultured in a cell culture medium containing a test component; (b) measuring a second methylation profile from DNA obtained from the immortalized test cell line cultured in a cell culture medium that does not contain the test component; (c) comparing the test methylation profile obtained from steps (a) and (b) with a control methylation profile obtained from an immortalized control cell line. and A method is provided in which a significant similarity between the test methylation profile and the control methylation profile in step (a) and a dissimilarity between the test methylation profile and the control methylation profile in step (b) indicates that the two cell lines produce a bioidentical and that the test component positively affects the production of a bioidentical from the immortalized cell line.

[0081] As used herein, the term "bioidentical" refers to a recombinant protein produced by genetically modified mammalian cells and having a molecular structure identical to that of the original biopharmaceutical reference product. The term "bioidentical" is more clearly explained at least in Stanczyk FZ et al., Climacteric. 2021;24:38-45.

[0082] Mammalian cells, particularly CHO cells, capable of producing biosimilar or bioidentical proteins have a CpG methylation profile that is significantly similar to or identical to a control profile of mammalian cells of the same type as the test mammalian cells, particularly a control profile of a parent clone capable of producing a wild-type protein, particularly a protein most similar to a therapeutic protein. In another example, mammalian cells producing biosimilar or bioidentical proteins have a methylation profile of selected regions (e.g., but not limited to, hypomethylated regions (LMRs), partially methylated domains (PMDs), differentially methylated regions (DMRs), differentially methylated points (DMPs), etc.) that is significantly similar to or identical to a control profile of mammalian cells, particularly a control profile of a parent clone capable of producing a wild-type protein, particularly a protein most similar to a therapeutic protein. In another example, mammalian cells producing biosimilar or bioidentical proteins have a significantly higher CpG methylation distribution (e.g., beta value distribution) compared to other mammalian cells. In yet another example, mammalian cells producing biosimilar or bioidentical proteins have no or minimal partial methylation at each site compared to other cells, particularly where the heterologous protein is a monoclonal antibody and / or a therapeutic protein.

[0083] A hypomethylated region (LMR) is a genomic region in which less than 60% of the CpGs within the region are methylated. More specifically, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the CpGs within an LMR are methylated. Any method known in the art may be used to identify or detect LMRs in genomic DNA. Well-known methods include the use of programs such as MmethylSeekR. In particular, LMRs in genomic DNA have at least three consecutive CpGs and no single nucleotide polymorphisms (SNPs) at any CpG position. More specifically, LMRs in genomic DNA are identified based on at least the methods disclosed in Burger, L., (2013) Nucleic Acids Research, 41(16): e155 and / or Stadler, M., (2011) Nature 480, pp. 490-495. LMRs are known to have an average methylation rate ranging from 10% to 50%, are CG-sparse regions that do not overlap with CpG islands, tend to be enriched in H3K4me1, DHS, and p300 / CBP, and / or are located primarily distal to promoters in intergenic or intronic regions. In particular, LMRs have the following characteristics: - The average methylation rate is between 10% and 50%. - Low CG density area, - enrichment of lysine 4 monomethylated histone H3 (H3K4me1), DNase I hypersensitive sites (DHS), and transcriptional coactivators CREB-binding protein (CPB) and p300; - located primarily distal to the promoter of an intergenic or intronic region, and / or - No single nucleotide polymorphisms (SNPs) at any CpG positions.

[0084] Hypomethylated regions (LMRs) represent a key feature of the dynamic methylome. LMRs are localized decreases in the DNA methylation landscape and represent CpG-poor distal regulatory regions that often reflect the binding of transcription factors and other DNA-binding proteins. LMRs were first reported in mice (Stadler et al. (2011) Nature: 480, 490-95). The evolutionary conservation of LMRs outside of mammals remains unclear.

[0085] Differentially methylated regions (DMRs) are genomic regions whose methylation status differs across multiple biological samples, such as tissues, cells, or individuals. These are genomic regions that differ between phenotypes. Statistical power is likely to be higher when neighboring DMRs are considered together [Gu H et al. (2010) Nat Methods 2010;7:133-6]. DMR lengths can range from hundreds to thousands of bases [Rakyan et al. (2011) Nat Rev Genet 12:529-41, 2011; Bock C (2012) Nat Rev Genet 2012;13:705-19].

[0086] DMRs can occur throughout the genome, but have been particularly identified around gene promoter regions, within gene bodies, and in intergenic regulatory regions. There are two types of regions: predefined and user-defined. Regions with special biological significance, such as CpG islands, CpG shores, and UTRs, are predefined. Many traditional statistical tests, such as t-tests and Wilcoxon rank-sum tests, can be performed at the region level. User-defined regions apply criteria such as a fixed region length, a fixed number of significant and adjacent CpG sites, and a significant and smoothed effect size estimate.

[0087] Partially methylated domains (PMDs) are extended regions of DNA with reduced average DNA methylation levels. PMDs cover gene-poor, transcriptionally inactive regions and tend to be heterochromatin-rich.

[0088] Differentially methylated regions (DMPs) are CpG sites with different DNA methylation status among various biological samples and are thought to be functional regions that may be involved in gene transcription regulation.

[0089] According to a further aspect of the present invention, there is provided the use of a DNA methylation array for determining the effect of at least one test component of a cell culture medium on the production of a mammalian cell line exhibiting at least one phenotype of interest.

[0090] According to a further aspect of the present invention, there is provided a DNA methylation array for determining the effect of at least one test component of a cell culture medium on the production of a mammalian cell line exhibiting at least one phenotype of interest.

[0091] According to another aspect of the present invention, there is provided a method for developing a DNA array-based test system for determining whether a test component of a cell culture medium can produce a test mammalian cell line capable of optimal heterologous protein production, comprising: (a) measuring a first test methylation state of one or more preselected methylation sites from genomic material obtained from a test CHO cell line cultured in a cell culture medium containing a test component; (b) measuring a second test methylation state of one or more preselected methylation sites from genomic material obtained from the test CHO cell line cultured in a cell culture medium that does not contain the test component; (c) selecting, from the preselected methylation sites, for each phenotypic parameter or phenotype of interest, a control panel of methylation sites characterized by a specific and distinct methylation differential profile; (d) obtaining a test system by assigning a control methylation profile for each phenotypic parameter or phenotype of interest; and By comparing the test methylation profile obtained from steps (a) and (b) with the control methylation profile obtained in step (c), a method is provided in which it is possible to determine whether the test mammalian cell line has optimal heterologous protein production capacity and whether the test component has a positive, negative, or no effect on the optimal heterologous protein production capacity of the mammalian cell line. [Brief explanation of the drawings]

[0092] [Figure 1] FIG. 1 is a graph showing the results of principal component analysis (PCA) of the identified 122 differentially methylated regions (DMRs). [Figure 2] FIG. 2 is a graph showing the results of principal component analysis (PCA) of the identified 289 differentially methylated regions (DMRs). [Figure 3] Figure 3 is a diagram of the cell culture workflow in a medium adaptation experiment. [Figure 4] FIG. 4 is a graph showing the results of a PCA analysis of a medium-adaptation experiment in which all CpG sites were methylated. [Figure 5] FIG. 5 is a graph showing the results of a PCA analysis of a medium adaptation experiment with differential methylation of CpG sites. [Example]

[0093] The above describes preferred embodiments, and those skilled in the art will recognize that changes or modifications may be made in design, configuration, or operation without departing from the scope of the claims. For example, these modifications are intended to be included within the scope of the claims.

[0094] Experimental Example 1 Oxidative stress in CHO cell cultures Wet Lab Method In this experiment, the transgenic CHO cell line Agarabi CHO (ATCC® CRL-3440™) was cultured in CD FortiCHO medium supplemented with L-glutamine (8 mM) at 37°C, 8% CO2, and 130 RPM. Six flasks were maintained in batch cultures for 7 days. Three flasks were technical replicates for the control set, and the remaining three flasks were technical replicates for the treatment set. On day 0, flasks were seeded with 3E5 viable cells / mL. To induce oxidative stress, hydrogen peroxide was added to the treatment set every 48 hours to a final concentration of 120 μM. Cell count, cell viability, and heterologous protein production were measured every two days, and cell pellets were harvested from both the control and treatment sets on day 7. Induction of oxidative stress in CHO cells by hydrogen peroxide treatment resulted in a decrease in growth rate and cell viability and a slight increase in heterologous protein production in the treated set compared to the control set.

[0095] Genomic DNA was purified from the harvested cell pellets using the DNeasy Blood & Tissue Kit (Qiagen) and quantified using PicroGreen or NanoDrop™ 2000. Genomic DNA (500 ng) from the control and treatment sets was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Libraries were sequenced by a third party on the NovaSeq platform, which generated 125 GB of data per sample.

[0096] calculation method The raw sequencing data were subjected to quality control (fastqc)1, sequencing adapter trimming (TrimGalore)2, and alignment using Bismark.3 The CMV promoter combined with the CHOK1-GS (Cricetulus griseus) genome was used as a control genome.

[0097] Bismark was also used to remove duplicate reads from the alignment output and extract methylation counts. SNPs were excluded, and only counts with a minimum coverage of 10x were used for downstream analysis. As a result, 3,711,013 CpG sites were identified in the hydrogen peroxide-treated sample.

[0098] Because regulated methylation targets are mostly clustered in short regions, we performed modified single-link clustering of methylation sites using DMRfinder 4. When the maximum distance between CpG sites was 100 bp, 1,728,014 genomic regions were found in the hydrogen peroxide-treated samples.

[0099] Methylation differential analysis Methylation differential analysis was performed between the control and treatment groups using MethylKit5. Logistic regression was used to measure methylation differentials across all regions, and FDR correction was performed using the sliding linear model (SLIM)6 method. Regions with an FDR-corrected p-value of less than 0.05 and methylation changes greater than 25% between groups were identified as differentially methylated regions (DMRs). In the hydrogen peroxide-treated samples, 122 DMRs were identified, as shown in Table 1. Principal component analysis (PCA) is a dimensionality reduction technique that highlights variation within a dataset. PCA analysis of DMRs is shown in Figure 1.

[0100] Preliminary results indicate that DMRs are involved in epigenetic changes of oxidative stress and may be used as markers in future studies.

[0101] Table 1: List of differentially methylated regions (DMRs) identified in hydrogen peroxide-treated samples. [Table 1]

[0102] Experimental Example 2 Adaptation of CHO cells with medium additives Wet Lab Method In this experiment, the transgenic CHO cell line Agarabi CHO (ATCC® CRL-3440™) was adapted to CD FortiCHO medium supplemented with L-glutamine (8 mM) and human insulin-like growth factor 1 (IGF-1) (1 mg / L) at 37°C, 8% CO2, and 130 RPM for 2 weeks. Six flasks were maintained in batch culture for 7 days. Three flasks were technical replicates of the control set (no IGF-1 adaptation) and the remaining three flasks were technical replicates of the IGF-1 adaptation set. On day 0, flasks were seeded with 3E5 viable cells / mL, and insulin growth factor (1 mg / L) was added to the adaptation set. Cell count, cell viability, and protein production were measured every 2 days, and cell pellets were harvested from both the control and treatment sets on day 7. Adaptation of CHO cells with IGF-1 did not significantly affect the growth rate and viability, but heterologous protein production doubled compared to the control set.

[0103] Genomic DNA was purified from the harvested cell pellets using the DNeasy Blood & Tissue Kit (Qiagen) and quantified using PicroGreen or NanoDrop™ 2000. Genomic DNA (500 ng) from the control and adaptive sets was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Library sequencing was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample.

[0104] calculation method The raw sequencing data were subjected to quality control (fastqc)1, sequencing adapter trimming (TrimGalore)2, and alignment using Bismark.3 The CMV promoter combined with the CHOK1-GS (Cricetulus griseus) genome was used as a control genome.

[0105] Bismark was also used to remove duplicate reads from the alignment output and extract methylation counts. SNPs were excluded, and only counts with a minimum coverage of 10x were used for downstream analysis. As a result, 4,244,091 CpG sites were identified in the IGF-1-adapted sample.

[0106] Because regulated methylation targets are mostly clustered in short regions, we performed modified single-link clustering of methylation sites using DMRfinder 4. With a maximum distance between CpG sites of 100 bp, we found 2,048,904 genomic regions in the IGF-1-adapted samples.

[0107] Methylation differential analysis MethylKit5 was used to perform differential methylation analysis between the control and adaptive groups. Logistic regression was used to measure methylation differences across all regions, and FDR correction was performed using the sliding linear model (SLIM) method. Regions with an FDR-corrected p-value of less than 0.05 and greater than 25% methylation change between groups were identified as differentially methylated regions (DMRs). In the IGF-1-adapted sample, 289 DMRs were identified, as shown in Table 2. Principal component analysis (PCA) is a dimensionality reduction technique that highlights variation within a dataset. PCA analysis of DMRs is shown in Figure 2.

[0108] Preliminary results indicate that DMRs are involved in epigenetic changes in IGF-1 adaptation and may serve as markers in future studies.

[0109] Experimental Example 3 Wet Lab Method Media adaptation of Humira cells Initially, Humira431 cells (A*STAR Bioprocessing Technology Institute) were cultured in EX-CELL Advanced CHO medium (Sigma-Aldrich, 14366C). At passage 28 (P28), Humira431 cells were transferred to new medium, CDFortiCHO (ThermoFisher), and cultured for four passages over two weeks. Control Humira431 cells were continuously cultured in EX-CELL Advanced CHO medium. At passage 32 (P32), adapted and control Humira431 cells were split into three flasks each to create biological replicates and cultured for seven days. Viable cell density (VCD) was measured over the seven-day period. On day 7, medium and cell pellets were collected from both adapted and control flasks for Cedex analysis and genomic DNA (gDNA) isolation (Figure 3).

[0110] DNA extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen), including RNAase treatment according to the manufacturer's instructions. DNA quantity was measured using the PicoGreen assay. DNA quality was assessed using a NanoDrop (Thermo Scientific) to ensure an A260 / A280 ratio of 1.8 or less. A small aliquot of each sample was then analyzed on an agarose gel to confirm that each sample contained high-molecular-weight DNA.

[0111] Bisulfite conversion and BeadChip analysis Genomic DNA samples were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ kit (Zymo Research), and methylation levels were then quantified using a customized Methylation BeadChip kit (Illumina), which allows for the quantitative analysis of over 50,000 methylation sites across the genome at single-nucleotide resolution.

[0112] Data Processing Customized chip array data processing was performed in R version 4.1.2 using Sesame version 1.14.2. DNA methylation levels at each site were calculated as methylation β values. β values ​​are defined as methylation signal / (methylation signal + unmethylation signal). This can be calculated using the getBetas function. The SeSAMe pipeline (Zhou et al., 2018) was used to generate normalized β values ​​and for quality control. Low-intensity detection calling and generation (based on p-values) were performed using pOOBAH. Background subtraction based on normal exponential deconvolution using out-of-band probe noob (Triche et al., 2013) was also implemented, along with additional bleed-through subtraction, if necessary.

[0113] result Plotting the first two CpG principal components before and after differential methylation analysis revealed significant clustering of samples. Differentially methylated positions (DMPs) were able to effectively cluster CHO samples by media adaptation from control samples. A list of identified DMPs is shown in Table 3. Figures 4 and 5 show PCA analyses using all methylated CpG sites and differentially methylated sites, respectively.

[0114] Table 2a: List of differentially methylated regions (DMRs) identified in IGF-1 adapted samples [Table 2a]

[0115] Table 2b: List of differentially methylated regions (DMRs) identified in IGF-1 adapted samples [Table 2b]

[0116] Table 3: Differentially methylated probes (CpG sites) identified between control and adapted samples Table 3

Claims

1. 1. A DNA array-based method for assessing the effect of at least one test component of a cell culture medium on at least one phenotype of interest in a test mammalian cell line cultured in a cell culture medium comprising said test component, comprising: (a) measuring a test methylation profile of one or more preselected methylation sites within the DNA of said test cell line; (b) comparing the test methylation profile obtained from step (a) with at least one control methylation profile obtained from the same mammalian cell line cultured in a cell culture medium that does not contain the test component. and a significant similarity between the test methylation profile and the control methylation profile of step (a) indicates that the test cell has the phenotype of interest and that the test component does not affect the phenotype of interest; a significant difference between the test methylation profile and the control methylation profile of step (a) indicates that the test cell has the phenotype of interest and that the test component affects the phenotype of interest; (c) comparing the test methylation profile obtained from step (a) with (i) at least one first control methylation profile obtained from a first mammalian control cell line exhibiting at least one phenotype of interest; and / or (ii) at least one second control methylation profile obtained from a second mammalian control cell line that does not exhibit the phenotype of interest; Process to compare with and the control cell line has not been contacted with the test component; a significant similarity between the test methylation profile of step (a) and the first or second control methylation profile indicates that the test cell has the phenotype of interest or does not have the phenotype of interest, respectively; A method wherein a difference between the test methylation profile in step (a) and the first or second control methylation profile indicates that the test cell does not have the desired phenotype or that the test cell has the desired phenotype.

2. The method of claim 1 , wherein the preselected methylation sites are associated with at least one phenotype of interest in the test cell line.

3. 3. The method of claim 1 or claim 2, wherein the desired phenotype is selected from the group consisting of phenotypic uniformity, protein nutritional value, optimal carbohydrate metabolism, optimal amino acid metabolism, optimal lipid metabolism, optimal heterologous protein production, optimal cell viability, and combinations thereof.

4. 4. The method of claim 1, wherein the first control methylation profile is a collection of one or more CpG sites from at least one control cell line that exhibits at least one phenotype of interest, and the second control methylation profile is a collection of one or more CpG sites from at least one control cell line that does not exhibit at least one phenotype of interest.

5. 5. The method of any one of claims 1 to 4, wherein the components of the cell culture medium are selected from the group consisting of only amino acids, small peptides, buffers, carbohydrates, inorganic salts, serum or fractions thereof, vitamins, and minerals.

6. 5. The method of any one of claims 1 to 4, wherein the mammalian cell line is derived from a mammal selected from the group consisting of mouse, rat, guinea pig, dog, minipig, human, cow, sheep, pig, goat, horse, donkey, mule, and hamster.

7. The method according to any one of claims 1 to 6, wherein the mammalian cells are immortalized cell lines.

8. 8. The method of claim 7, wherein the immortalized cell line is selected from the group consisting of only CHO, BHK, Vero, HEK293, HEK 293T, HeLa cells, NS0 cells, Sp2 / 0 cells, and derivatives thereof.

9. a DNA array-based method for evaluating the effect of at least one test component of a cell culture medium on the production of at least one biosimilar from an immortalized test cell line, wherein the biosimilar is significantly similar to an innovator protein produced by an immortalized control cell line that is the same cell line as the test cell line; (a) measuring a first test methylation profile from DNA obtained from the immortalized test cell line cultured in the cell culture medium containing the test component; (b) measuring a second methylation profile from DNA obtained from the immortalized test cell line cultured in cell culture medium that does not contain the test component; (c) comparing the test methylation profile obtained from step (a) and step (b) with a control methylation profile obtained from an immortalized control cell line. and wherein a significant similarity between the test methylation profile and the control methylation profile in step (a) and a dissimilarity between the test methylation profile and the control methylation profile in step (b) indicate that the two cell lines produce biosimilars and that the test component has a positive effect on the production of biosimilars from immortalized cell lines.

10. a DNA array-based method for evaluating the effect of at least one test component of a cell culture medium on the production of at least one bioidentical from an immortalized test cell line, said bioidentical being significantly similar to an innovator protein produced by an immortalized control cell line that is the same cell line as said test cell line; (a) measuring a first test methylation profile from DNA obtained from the immortalized test cell line cultured in the cell culture medium containing the test component; (b) measuring a second methylation profile from DNA obtained from the immortalized test cell line cultured in cell culture medium that does not contain the test component; (c) comparing the test methylation profile obtained from step (a) and step (b) with a control methylation profile obtained from an immortalized control cell line. and A significant similarity between the test methylation profile and the control methylation profile in step (a) and a difference between the test methylation profile and the control methylation profile in step (b) indicate that the two cell lines produce bioidenticals and that the test component has a positive effect on the production of bioidenticals from the immortalized cell line.

11. The method according to any one of claims 1 to 10, wherein the DNA methylation array is a bead array.

12. The use of a DNA methylation array to determine the effect of at least one test component of a cell culture medium on the production of a mammalian cell line that exhibits at least one phenotype of interest.

13. A DNA methylation array for determining the effect of at least one test component of a cell culture medium on the production of a mammalian cell line exhibiting at least one phenotype of interest.

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