Methods of assessing protein production in CHO cells

Abstract

The present invention relates to a method for determining the suitability of at least one Chinese hamster ovary (CHO) test cell line for optimal heterologous protein production, said method comprising: (a) determining a test methylation profile from genomic material obtained from said CHO test cell line; and (b) comparing the test methylation profile obtained from (a) to a reference methylation profile, where the reference methylation profile comprises a methylation status of more than one CpG site from at least one CHO reference cell line exhibiting at least one phenotype of interest for optimal heterologous protein production, wherein a significant similarity of the test methylation profile of (a) to the reference methylation profile indicates that the CHO test cell line is suitable for optimal heterologous protein production, and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and determined using an array based on DNA methylation beads.

Description

Field of the Invention

[0001] The present invention relates to an epigenetics-based method for quantitatively and qualitatively evaluating target protein production and cell stability in CHO cells before, during, or after actual protein production. In particular, measurement of differential methylation of promoters and / or CpG sites in CHO cells can provide insights into the quantitative and qualitative production of target proteins by CHO cells. Background of the Invention Chinese hamster ovary (CHO) cells have been known since 1987 as the mainstay of industrial production of recombinant therapeutic proteins and are thus widely used in biologic production. Approximately 70% of all recombinant biopharmaceutical proteins and all monoclonal antibodies approved since 2016 have been manufactured in CHO cells. Several advantages of using CHO for biologic production include tolerance to genetic manipulation, ease of adaptation to manufacturing method scale, rapid growth rate, and the ability to perform post-translational modifications compatible with humans. However, the biologic production system in CHO faces a bottleneck due to the loss of protein productivity over time.

[0003] Initial protein expression from cell lines is high, but production decreases during extended culture. This results in reduced method yields, affects the timeline, and increases costs. Changes in the cell culture environment can lead to alterations in cell behavior and protein productivity of producer cell lines. Several reasons for productivity loss in CHO cells include the accumulation of a large number of genomic variations during extended culture, loss of transgenes, and epigenetic regulation of transgene insertion sites, among others. In particular, the integration sites of viral promoters are susceptible to transcriptional regulation via epigenetic regulation such as histone modification and DNA methylation. In particular, the DNA methylation status of viral promoters is an important factor in protein production or expression stability in producer CHO cells. An increase in DNA methylation in the promoter leads to transgene silencing at the transcriptional level. Variability in protein production in CHO cells has been associated with the regulation of the cytomegalovirus major immediate early and enhancer (CMV) promoter and the simian virus 40 (SV40) promoter mediated by DNA methylation, which are the most commonly used promoters for recombinant protein production in CHO cells.

[0004] Current methods for determining the suitability of CHO clones for target protein production are not only time-consuming but also not very accurate for selecting clones or cells for optimal protein production.

[0005] Furthermore, CHO clones with the same genes can still result in heterogeneous phenotypes, thus causing instability, inefficiency, and economic losses during the production of heterologous proteins on an industrial scale. The method of only using phenotypic analysis to compare and select CHO clones cannot guarantee consistency over time. The genotypic comparison of CHO clones cannot define how genes are differentially expressed to adapt to environmental conditions. As shown by Wippermann A et al., Appl Microbiol Biotechnol. January 2014; 98(2):579 - 89, the supplementation of butyrate, which is known to enhance the specific productivity of CHO cells, also leads to changes in epigenetic silencing events.

[0006] Therefore, there is a need in the art for an effective and affordable tool to globally evaluate and regulate CHO metabolism and protein production. There is also a need in the art for methods to select and maintain the same CHO population to improve the speed, quality, efficiency, and consistency of production. Brief Description of the Drawings Figure 1 It is a graph showing the principal component analysis (PCA) results of the 122 identified differentially methylated regions (DMRs).

[0008] Figure 2 It is a graph showing the principal component analysis (PCA) results of the 289 identified differentially methylated regions (DMRs).

[0009] Figure 3 It is a graph showing the viable cell counts of control CHO Humira431 cells and hyperosmolarity - treated CHO Humira431 cells. On day 3, sodium chloride was added to the hyperosmolarity - treated CHO Humira431 cells. From day 3 onwards, a stagnation in viable cell counts was seen in the hyperosmolarity - treated CHO Humira431 cells, as opposed to the control CHO Humira431 cells, which continued to increase until day 10, at which point the viable cell counts started to enter a stagnant period.

[0010] Figure 4It is a graph showing the heterologous protein productivity of CHO Humira431 cells and control CHO Humira431 cells treated with high osmolality on day 7 of fed-batch culture. It was found that the CHO Humira431 cells treated with high osmolality produced 86.5 pg / cell to 90.4 pg / cell of heterologous protein, as opposed to the control CHO Humira431 cells, which produced 40.4 pg / cell to 41.3 pg / cell of heterologous protein. Thus, it was found that adding sodium chloride to the CHO Humira431 cells treated with high osmolality resulted in increased heterologous protein productivity on day 7 of fed-batch culture.

[0011] Figure 5 It is a graph showing the classification of 6 clones based on heterologous protein productivity on days 9, 11, and 14 of fed-batch culture. Based on productivity, clones 2C9, 3D11, and 2H2 were classified as low producers, clone 10A8 was classified as a medium producer, and clones 7H9 and 8F8 were classified as high producers.

[0012] Figure 6 It is a PCA plot showing the clustering of groups based on protein productivity.

[0013] Description of the Invention The present invention solves the above problems by providing a method that not only identifies genetically identical CHO clones or cell lines but also confirms that these clones and / or cell lines are phenotypically homogeneous, thus ensuring stability, efficiency, and reduction of economic losses during the heterologous protein production process, especially on an industrial scale. In particular, the method according to any aspect of the present invention uses methylation patterns and the conservation of these methylation patterns in CHO clones and / or cell lines for selecting and maintaining an identical CHO population to improve the speed, quality, efficiency, and consistency of heterologous protein production. Since genotypic comparison of CHO clones cannot define how genes are differentially expressed to adapt to environmental conditions, and phenotypic analysis alone cannot guarantee consistency over time, epigenetic methods, especially DNA methylation, provide a state-of-the-art technique for selecting CHO clones that are not only genetically identical but also epigenetically and thus phenotypically identical for improved heterologous protein production. The method according to any aspect of the present invention allows the use of DNA methylation as a tool to improve the quantitative and qualitative protein production from CHO cells. Altering the DNA methylation pattern on the viral promoter driving transgene expression will transcriptionally increase protein expression in CHO cells.

[0014] According to one aspect of the present invention, there is provided a method for determining the suitability of at least one Chinese hamster ovary (CHO) test cell line for optimal heterologous protein production, the method comprising: (a) Determine a test methylation profile from genomic material obtained from said CHO test cell line; and (b) Compare the test methylation profile obtained from (a) with a reference methylation profile, wherein the reference methylation profile comprises the methylation status of more than one CpG site from at least one CHO reference cell line, said at least one CHO reference cell line exhibiting at least one phenotype of interest for optimal heterologous protein production, wherein a significant similarity of the test methylation profile of (a) compared to the reference methylation profile indicates that the CHO test cell line is suitable for optimal heterologous protein production, and wherein the test methylation profile and the reference methylation profile are determined from CpG sites of the CHO cell genome and using a DNA methylation bead-based array.

[0015] Epigenetic techniques thus provide solutions for the quantitative and qualitative analysis of protein production. In particular, the reference methylation profile may include environment-specific CpG sites or dynamic CpG sites, i.e., sites that appear to have a key role in several environmental conditions; CpG sites in viral promoters (CMV and SV40 promoters) and / or CpG sites from candidate gene regulatory regions of pathways important for certain key biological processes in CHO cells (e.g., metabolism-associated genes, protein production-associated genes, cell growth / division-associated genes, and methylation-associated genes). More particularly, the test and reference methylation profiles are from CpG sites, where the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome.

[0016] The term 'CHO cell genome' herein refers to the genomic DNA of CHO cells, which does not include the DNA of viruses used to introduce foreign DNA into the cells, particularly CMV and SV40. In particular, the CHO cell genome may represent a cell having a genomic composition in the form as naturally seen in the wild. The term may also include genes added to the CHO genome by genetic modification (i.e., in terms of improved protein production, etc.), but does not necessarily include or exclude the viral genes and promoters that have been used to introduce genes into the CHO genome. Thus, the term "CHO cell genome" may exclude viral genes and promoters, and / or may include endogenous or homologous genes of CHO cells and / or genetically modified endogenous or homologous genes of CHO cells and / or intergenic genes, the DNA found between the genes of CHO cells.

[0017] The method according to any aspect of the present invention can be used to quantify the methylation level of any of these CpG sites in CHO cells, particularly in test CHO cells. The information can then be used to phenotypically evaluate, assess, and enhance CHO cells under various cell culture conditions. More particularly, machine learning models can be used to analyze the generated quantitative and qualitative methylation data. Even more particularly, compared to the "trial and error" method currently used, the method according to any aspect of the present invention can be used to design optimal cell culture conditions in a predictive and precise manner, especially with respect to the selection of a suitable CHO cell line. This thus allows for the online and direct control of the manufacturing process, thereby enhancing the robustness and thus the overall quality of the molecule produced by CHO cells.

[0018] A CHO cell line refers to an immortalized Chinese hamster ovary cell line (CHO) derived from the Chinese hamster (Cricetulus griseus). In particular, the CHO cell line can be selected from CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO-DXB11 (ATCC), ExpiCHO-S TM cells (Thermo Fisher Scientific), FreeStyle TM CHO-S TM cells (Thermo Fisher Scientific), CHO 1-15[subscript 500] (ATCC), and Agarabi CHO (ATCC).

[0019] As used herein, the term 'fitness' refers to a CHO cell line suitable for optimal heterologous protein production. In one example, a CHO cell line can be considered suitable for optimal heterologous protein production before introducing a transgene into the cell. In such a case, the CHO cell line can have phenotypic parameters or characteristics that enable the cell line to grow well and allow for easy uptake of the transgene of interest, and after uptake of the transgene, allow for optimal heterologous protein production, where the protein is the product of the transgene of interest. These characteristics or phenotypic parameters include at least optimal glucose consumption, growth rate, lactate production, ammonia accumulation, etc. When it is confirmed that a CHO cell line exhibits at least one of these phenotypic parameters, the CHO cell line can be considered suitable for optimal heterologous protein production when introducing the transgene of interest into the cell.

[0020] In another example, a CHO cell line can be considered suitable for optimal heterologous protein production after a transgene has been introduced into the cells. In such a case, the CHO cell line is genetically modified using methods known in the art to introduce the transgene into the cells, and the genetically modified cells are capable of optimal heterologous protein production, where the protein is the product of translation of the transgene. The CHO cell line in this example can have at least one phenotype of interest that enables the genetically modified cell line to have good viability and optimal production of the target protein. These phenotypes of interest can include cell viability (survivability), protein productivity (in terms of the quantity and quality of the protein), phenotypic homogeneity, cell exhaustion, etc. Thus, the methods according to any aspect of the present invention can be used on a CHO cell line that has been genetically modified (i.e., a transgene has been introduced into the cell line) or a CHO cell line that has not been genetically modified. In both cases, the CHO cell line is used for heterologous protein production.

[0021] As used herein, the term 'transgene' refers to a gene that has been removed from the genome of one organism and inserted into the genome of another organism using artificial techniques employed in genetic modification. For example, a human gene is artificially introduced into the genome of a CHO cell for the production of at least one protein of interest, particularly a therapeutic protein.

[0022] As used herein, the term "therapeutic protein" refers to a genetically engineered form of a naturally occurring human protein. Examples of therapeutic proteins include antibody-based drugs, anticoagulants, blood factors, bone morphogenetic proteins, artificially engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, etc.

[0023] As used herein, the term 'cell viability' refers to the ability of a cell to be alive and undergo cell proliferation. Cell viability is a measure of the proportion of live cells in a population. Cell proliferation refers to an increase in the number of cells attributable to cell division. Assays commonly used to test cell viability include BrdU cell proliferation assay, MTT cell proliferation assay, trypan blue cell counting, and ATP cell viability assay.

[0024] As used herein, the term 'cell exhaustion' refers to a state in which a cell loses its ability to carry out metabolic activities, including heterologous protein production. Cell exhaustion can be determined by metabolite detection assays.

[0025] As used herein, the term 'phenotypic homogeneity' refers to a state in which all cells in a population exhibit the same phenotype under a certain condition.

[0026] As used herein, the term 'heterologous protein production' refers to the production of a protein that is not endogenous to the cell. It refers to the expression of a gene or a portion of a gene, particularly a transgene in a host CHO cell that does not naturally express said gene. Assays commonly used to quantify heterologous protein production include enzyme-linked immunosorbent assay (ELISA), chromatography, and bioprocess analyser. As used herein, the term 'host cell' refers to a cell system used for the expression of a heterologous protein. For example, CHO cells are the primary host for the production of various therapeutic proteins.

[0027] The term 'optimal heterologous protein production' as used herein refers to CHO cells that are capable of high-level protein production, particularly in the context of industrial or large-scale production of recombinant proteins, where the protein is typically a functional protein not naturally present in wild-type CHO cells. In particular, for optimal heterologous protein production, the CHO cell line has minimized the metabolic burden and toxic effects on the cell. More particularly, 'optimal heterologous protein production' refers to high-level protein production where the CHO cell line not only produces a high yield of the protein of interest but also continuously maintains protein production over the production period (i.e., an extended culture period), such that the quality of the protein produced is also consistent and maintained. In particular, for a CHO cell according to any aspect of the present invention to be capable of 'optimal heterologous protein production', the cell must exhibit at least one or more of the following phenotypes of interest: phenotypic homogeneity, protein productivity, and protein quality. More particularly, for 'optimal heterologous protein production', the CHO cell may include phenotypic homogeneity and protein productivity, or phenotypic homogeneity and protein quality, or protein productivity and protein quality, or phenotypic homogeneity, protein productivity, and protein quality.

[0028] As used herein, the term 'protein productivity' refers to a measure of the amount of protein produced 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), and the final measurement is expressed as the amount of protein per cell (mg / cell).

[0029] The term 'protein quality' refers to the post-translational modifications of a protein that determine the efficacy and function of the protein. Modifications typically include phosphorylation, glycosylation, ubiquitination, methylation, acetylation, protein folding, etc. For example, protein glycosylation is a key quality attribute that regulates the efficacy, stability, and half-life of therapeutic proteins. Protein quality can be determined using immunoprecipitation-based techniques, biochemical assays, mass spectrometry (MS), etc.

[0030] As used herein, the terms "methylation profile", "methylation pattern", "methylation status", or "methylation state" are used to describe the condition, situation, or circumstance of genomic sequence methylation, and such terms refer to the characteristics of a DNA segment at a particular genomic locus related to methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residue in the DNA sequence is methylated, the position of one or more methylated C residues, the percentage of methylated C at any particular residue sequence segment, and allelic differences in methylation attributable to, for example, differences in allelic origin.

[0031] The term "methylation state" refers to the state (i.e., methylated versus non-methylated) of a particular methylation site, which means that the residue or methylation site is methylated or not methylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined. Thus, the term "methylation profile" or also "methylation pattern" refers to the relative or absolute concentration of methylated C residues or non-methylated C residues at any particular residue sequence segment in the genomic material of a biological sample. For example, if one or more cytosine (C) residues that are generally not methylated in a DNA sequence are methylated, it can be called "hypermethylated"; while if one or more cytosine (C) residues that are generally methylated in a DNA sequence are not methylated, it can be called "hypomethylated". Similarly, if one or more cytosine (C) residues in a DNA sequence (e.g., the DNA of a sample nucleic acid from a test subject) are methylated compared to another sequence from a different region or from a different individual (e.g., relative to a normal nucleic acid or a standard nucleic acid of a reference sequence), the sequence is considered hypermethylated compared to the other sequence. On the other hand, if one or more cytosine (C) residues in a DNA sequence are not methylated compared to another sequence from a different region or from a different individual, the sequence is considered hypomethylated compared to the other sequence. These sequences are called "differentially methylated". The measurement of the level of differential methylation can be carried out in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation level of individual queried CpG sites determined by bisulfite sequencing.

[0032] The term "hypermethylation" refers to the average methylation state corresponding to the increased presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0033] The term "hypomethylation" refers to the average methylation status corresponding to the presence of a decrease in 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0034] As used herein, "methylated nucleotide" or "methylated nucleobase" refers to the presence of a methyl moiety on a nucleobase, where the methyl moiety is not normally present in a recognized canonical nucleobase. For example, cytosine in its normal form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Thus, cytosine in its normal form may not be considered a methylated nucleotide, while 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this disclosure, when thymine is present in DNA, it may not be considered a methylated nucleotide. The canonical nucleobases of DNA are thymine, adenine, cytosine, and guanine. The canonical bases of RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a position in a nucleic acid region of a target gene where methylation has the potential to occur. For example, a position containing CpG is a methylation site, where cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to a position of the nucleotide that is prone to methylation. These methylated nucleotides typically occur in nature and, so far, mainly in the context of the dinucleotide CpG, but also methylated cytosines in the context of CpNpG- and CpNpN-sequences may be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be considered for any aspect of the present invention.

[0035] As used herein, the term "significantly similar" particularly in the context of a comparison of methylation profiles (e.g., a comparison between a test profile (from one or more test subjects) and a reference profile) refers to similarity observed by statistical methods (i.e., by using bioinformatics) and / or also by visual inspection. For example, significant similarity is observed if the test profile overlaps with a reference profile defined by multiple training samples by multivariate statistical methods such as principal component analysis or multi-dimensional scaling. In particular, if more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the methylation patterns / profiles overlap with the reference profile, the test profile is significantly similar to the predetermined reference profile. Similarity of the test profile to more than one, e.g., two, three, or even all reference profiles reduces the significance of the similarity.

[0036] As used herein, the term "genomic material" refers to nucleic acid molecules or fragments of the genome of a CHO cell or cell line. In particular, such nucleic acid molecules or fragments are DNA or RNA or hybrids thereof, and most preferably molecules of the DNA genome of a CHO cell or cell line.

[0037] As used herein, a "DNA sample" refers to DNA extracted from cells according to any aspect of the invention using methods known in the art.

[0038] The 'bisulfite treatment' of genomic DNA, which is used interchangeably with the term 'bisulfite modification', refers to treating genomic DNA with a deaminating agent such as bisulfite, which can be used to treat all DNA, methylated or non-methylated. In particular, the term "bisulfite" as used herein includes any suitable type of bisulfite, such as sodium bisulfite, or other chemical reagents capable of chemically converting cytosine (C) to uracil (U) without chemically modifying methylated cytosine and thus can be used to differentially modify DNA sequences based on the DNA methylation status, e.g., U.S. Patent Publication US2010 / 0112595. As used herein, a reagent that "differentially modifies" methylated or non-methylated DNA includes any reagent that modifies methylated and / or unmethylated DNA in a process that produces distinguishable products from methylated and non-methylated DNA, thereby allowing identification of the DNA methylation status. Such processes can include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatments (e.g., cleavage by methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one that can cleave or digest a DNA molecule with much higher efficiency when the DNA is methylated, while an enzyme that preferentially cleaves or digests non-methylated DNA exhibits significantly higher efficiency when the DNA is non-methylated.

[0039] Thus, prior to performing step (a) according to any aspect of the invention, the genomic DNA contained in / obtained from or extracted from the cells is first subjected to bisulfite treatment.

[0040] Alternative methods available in the art can be used in place of bisulfite treatment. Those skilled in the art will understand which other methods to use. In one example, TET-assisted pyridine borane sequencing (TAPS) can be used to detect 5mC and 5hmC (Yibin Liu et al., Nature Biotechnology, 37:424-429 (2019)).

[0041] As used herein, the term "test" when used in conjunction with the term "cell" refers to a cell that is subjected to a method according to any aspect of the present invention and that is the basis for an analytical application of the present invention. Thus, a 'test cell' is a CHO cell or a group of CHO cells that is tested according to any aspect of the present invention, or a profile obtained or generated in that context. In contrast, the term "reference" or 'control' shall denote a normally pre-determined entity that is used for comparison with a test entity. In particular, a 'test cell' refers to a cell whose suitability for optimal homologous protein production is being tested, where the methylation status must be determined, while a 'control' or'reference' refers to a cell or its methylation profile that is known to exhibit optimal homologous protein production.

[0042] As used herein, a "CpG site" or "methylation site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation by an event that occurs naturally in vivo or by an event that chemically methylates a nucleotide in vitro. In a cell, some of these sites may be hypermethylated, while some may be hypomethylated. In some cases, a CpG site may not be considered to be completely hypermethylated or hypomethylated, but a value that is a measure of the methylation of the CpG site may be given. Thus, methylation can be quantified and may not always be an absolute case of hypermethylation or hypomethylation.

[0043] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0044] As used herein, a "CpG island" describes a DNA sequence segment that contains a CpG density that is functionally or structurally distinct from the norm. For example, Yamada et al. have described a set of criteria for determining CpG islands: it must be at least 400 nucleotides in length, have a GC content greater than 50% and an OCF / ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Others have less stringently defined a CpG island as a sequence that is at least 200 nucleotides in length, has a GC content greater than 50% and an OCF / ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745).

[0045] In particular, when differential methylation is detected in a test cell, i.e., the cell shows absolute hypermethylation or hypomethylation or at least quantitative differential methylation at at least one CpG site compared to a reference (i.e., from a CHO cell line having at least one phenotype of interest), then the test cell also contains the phenotype of interest and may be capable of optimal heterologous protein production. More particularly, when a CpG site shows the same methylation status in a test cell as the corresponding CpG site in a reference cell or reference methylation profile, the test cell expresses the phenotype of interest and may be capable of optimal heterologous protein production. Overall, the platform gives us the opportunity to detect the broad DNA methylation status in CHO cells and correlate it with industrially relevant parameters, which is crucial for at least the development of biopharmaceuticals.

[0046] In particular, in the method according to any aspect of the 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 determined in step (a). A person skilled in the art will be able to determine the number of CpG sites to be used in step (a) according to any aspect of the invention. Even more particularly, the methylation status of at least two CpG sites is determined in step (a) of the method according to any aspect of the invention.

[0047] As used herein, the term 'epigenetic change' refers to chemical (e.g., methylation) or protein (e.g., histone) changes that occur to the gene body or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can imprint genes passed from one generation to the next.

[0048] In particular, a reference methylation profile according to any aspect of the invention is a compilation of more than one CpG site from at least one CHO reference cell line, the at least one CHO reference cell line showing at least one phenotype of interest for optimal heterologous protein production. In one example, different CpG sites are collected from a single reference CHO cell line showing at least one phenotype of interest for optimal heterologous protein production. In another example, different CpG sites are collected from more than one cell line, where each cell line shows at least one phenotype of interest for optimal heterologous protein production. A reference methylation profile according to any aspect of the invention may thus not be the naturally occurring methylation profile from a single CHO cell line, but an artificial profile obtained by combining relevant CpG sites from different reference CHO cell lines, each having at least one phenotype of interest for optimal heterologous protein production.

[0049] The phenotypes of interest for optimal heterologous proteins can be selected from phenotypic homogeneity, protein productivity, and protein quality.

[0050] According to a further aspect of the present invention, there is provided a method for selecting at least one CHO cell comprising a phenotype of interest from a population of CHO cells derived from a parental clone, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO cells, and (b) comparing the test methylation profile of (a) with a reference methylation profile from a parental clone exhibiting the phenotype of interest, wherein a significant similarity between the test methylation profile and the reference methylation profile of (b) indicates that the cell has the phenotype of interest of the parental clone; and wherein the phenotype of interest is selected from phenotypic homogeneity, protein productivity, and protein quality, and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a bead-based DNA methylation array.

[0051] As used herein, the term 'parental clone' refers to a cell line derived from a host cell (CHO cell line) in which a transgene has been integrated into the genome. As used herein, the term'subclone' in relation to a parental clone refers to a clonal cell line derived from the parental clone that has the same genotype but a different phenotype due to epigenetic changes.

[0052] The method used according to the aspect of the present invention is to select at least one CHO cell that is genetically and phenotypically identical or significantly similar to the parental clone in at least one bioreactor. In particular, during cell replication in the bioreactor of the parental clone, a phenotypic plurality typically occurs. As used herein, the term 'phenotypic plurality' refers to the variation in phenotypes present within a cell population, particularly CHO cells, under a certain specific condition without any change in genotype. The method according to the aspect of the present invention allows the selection of at least one clone with minimal variation from an original / established parental clone from a phenotypically heterogeneous population of CHO cells, which may also exhibit a phenotype of interest (e.g., production of at least one human-like protein). In particular, by comparing the distribution of CpG site methylation (e.g., β-value distribution) in the clonal population of the bioreactor, CHO cells that are identical or significantly similar to the parental clone can be identified. CHO cells that are identical or significantly similar to the parental CHO cell line may have the same methylation profile. The partially methylated clonal population may also exhibit cell-to-cell variation.

[0053] Similarly, the method used according to the aspects of the present invention is to select at least one CHO cell or clone population having selectivity for protein productivity and a specific methylation profile. In the example, the selected CHO cells have the same methylation profile as the parental clone, wherein the parental clone exhibits protein productivity. The reference methylation profile in this context thus refers to the methylation profile of the parental clone having protein productivity.

[0054] In another example, the method used according to the aspects of the present invention is to select at least one CHO cell or clone population having selectivity for protein quality and a specific methylation profile. Protein quality can be measured based on desired glycosylation / glycan backbone, etc. In the example, the selected CHO cells have the same methylation profile as the parental clone, wherein the parental clone exhibits protein quality. The reference methylation profile in this context thus refers to the methylation profile of the parental clone having protein quality.

[0055] According to a further aspect of the present invention, there is provided a method of identifying at least one CHO test cell line capable of producing at least one biosimilar of a heterologous protein relative to that produced by a CHO reference cell line, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO test cell line, and (b) comparing the test methylation profile of (a) with a reference methylation profile of the CHO reference cell line, wherein a significant similarity between the test methylation profile of (a) and the reference methylation profile indicates that the two cell lines produce biosimilars, and wherein the test methylation profile and the reference methylation profile are derived from CpG sites of the CHO cell genome and are determined using a bead-based array for DNA methylation.

[0056] As used herein, the term 'biosimilar' refers to a recombinant protein produced by genetically modified CHO cells that is highly similar to the original biotherapeutic reference product and shares quality, safety, and efficacy with the 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.

[0057] Information on the DNA methylation patterns of cell lines can lead to a clearer specification profile of product release in CHO cells, can serve as "copyright" protection for biogeneric developers, and can be developed as a potential "gold standard" for the regulatory processes required for biogeneric development.

[0058] According to yet another aspect of the present invention, there is provided a method for identifying at least one CHO test cell line capable of producing at least one bio-identical of a heterologous protein relative to that produced by a CHO reference cell line, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO test cell line, and (b) comparing the test methylation profile of (a) with a reference methylation profile of the CHO reference cell line, wherein when the test methylation profile of (a) is the same as the reference methylation profile, it indicates that the two cell lines produce bio-identicals, and wherein the test methylation profile and the reference methylation profile are determined from CpG sites of the CHO cell genome and using a DNA methylation bead-based array.

[0059] As used herein, the term 'bio-identical' refers to a recombinant protein produced by a genetically modified CHO cell that has the same molecular structure as the original biotherapeutic reference product. The term 'bio-identical' is more clearly explained at least in Stanczyk FZ et al., Climacteric. 2021; 24:38-45.

[0060] CHO cells capable of producing biogenerics or bio-identical proteins have CpG methylation profiles that are significantly similar or identical, respectively, to the reference profiles from CHO cells, particularly parental clones that are capable of producing proteins that are most similar to wild-type proteins, particularly therapeutic proteins. In another example, CHO cells producing biogenerics or bio-identical proteins have methylation profiles of selected regions (such as but not limited to low-methylated regions (LMRs) / partially methylated domains (PMDs) / differentially methylated regions (DMRs) / differentially methylated positions (DMPs)) that are significantly similar or identical to the reference profiles from CHO cells, particularly parental clones that are capable of producing proteins that are most similar to wild-type proteins, particularly therapeutic proteins. In another example, CHO cells producing biogenerics or bio-identical proteins have a significantly higher CpG methylation distribution (e.g., beta value distribution) compared to other CHO cells. In yet another example, CHO cells producing biogenerics or bio-identical proteins have no or minimal partial methylation at each site compared to other cells. In particular, the heterologous protein is a monoclonal antibody and / or a therapeutic protein.

[0061] Hypomethylated regions (LMRs) are regions in the genome where fewer than 60% of the CpGs are methylated. More particularly, fewer than 50%, 40%, 30%, 20% or 10% of the CpGs in LMRs are methylated. Any method known in the art can be used to identify or detect LMRs in genomic DNA. Well-known methods include using a program such as MethylSeekR. In particular, LMRs in genomic DNA have at least three consecutive CpGs and no single nucleotide polymorphisms (SNPs) at any of the CpG positions. Even more particularly, LMRs in genomic DNA are identified based on methods disclosed at least in Burger, L., (2013) Nucleic Acids Research, 41(16):e155 and / or Stadler, M., (2011) Nature 480, 490-495. LMRs are known to have an average methylation ranging from 10% to 50%; be low CG density regions that do not overlap with CpG islands; tend to be enriched in H3K4me1, DHSs and p300 / CBP; and / or be mainly located distally to promoters within intergenic or intronic regions. In particular, LMRs: - have an average methylation ranging from 10% to 50%, - are low CG density regions; - are enriched in histone H3 monomethylated at lysine 4 (H3K4me1), DNase I hypersensitive sites (DHSs) and the transcriptional co-activator CREB-binding protein (CPB) and p300; - are mainly located distally to promoters within intergenic or intronic regions; and / or - have no single nucleotide polymorphisms (SNPs) at any of the CpG positions.

[0062] Hypomethylated regions (LMRs) represent a key feature of the dynamic methylome. LMRs are local decreases in the DNA methylation landscape and represent CpG-poor distal regulatory regions, which often reflect the binding of transcription factors and other DNA-binding proteins. LMRs were first described in mice (Stadler et al. (2011) Nature:480, 490-95). The evolutionary conservation of LMRs outside mammals remains unexplored.

[0063] Differentially methylated regions (DMRs) are genomic regions that have different methylation states in multiple biological samples such as tissues, cells, individuals, etc. These are genomic regions where there are differences between phenotypes. When considering adjacent DMPs as a whole together, the statistical power may be greater [Gu H et al. (2010) Nat Methods 2010; 7:133 - 6]. The length range of DMRs can be from several hundred to several thousand base pairs [Rakyan et al. (2011) Nat Rev Genet 12:529 - 41, 2011, Bock C (2012) Nat Rev Genet 2012; 13:705 - 19].

[0064] DMRs can occur throughout the genome but have been specifically identified around the promoter regions of genes, within gene bodies, and at intergenic regulatory regions. There are two types of regions: predefined or user - defined. Regions with special biological significance such as CpG islands, CpG shores, UTRs, etc., are predefined. Many traditional statistical tests, including t - tests and Wilcoxon rank - sum tests, can be performed at the region level. For user - defined regions, criteria such as fixed region length, fixed number of significant and adjacent CpG sites, significant and smooth estimated effect size, etc.

[0065] Partially methylated domains (PMDs) are extended regions in the genome that show a reduced average DNA methylation level. They include gene - poor and transcriptionally inactive regions and tend to be heterochromatic.

[0066] Differentially methylated positions (DMPs) are CpG sites that have different DNA methylation states across different biological samples and are considered as potential functional regions related to gene transcription regulation.

[0067] According to a further aspect of the invention, there is provided a method for assessing one or more phenotypic parameters of at least one test CHO cell line, the method comprising the steps of: (a) determining the test methylation status of one or more pre - selected methylation sites from genomic material obtained from the test CHO cell line; (b) determining the test methylation profile of the test CHO cell line based on the methylation status determined in (a); and (c) comparing the test methylation profile determined in (b) with at least one predetermined reference methylation profile, wherein each of the predetermined reference methylation profiles is specific to a reference CHO cell line having at least one phenotypic parameter; Wherein if the tested methylation profile is significantly similar to one of the predetermined reference methylation profiles, the tested CHO cell line has phenotypic parameters that are similar or preferably identical to those of a reference CHO cell line having the predetermined reference methylation profile, and wherein the tested methylation profile and the reference methylation profile are derived from CpG sites of the CHO cell genome and determined using a bead-based DNA methylation array.

[0068] In particular, the phenotypic parameters are selected from: - Optimal carbohydrate metabolism - Optimal amino acid metabolism - Optimal lipid metabolism - Optimal protein productivity; and - Optimal cell viability As used herein, the term 'carbohydrate metabolism' refers to almost all or all of the biochemical processes responsible for the metabolic formation, breakdown, and interconversion of carbohydrates in a cell. It involves multiple pathways such as glycolysis, gluconeogenesis, glycogenolysis, and glycogenesis. For example, glycolysis is one of the key metabolic pathways in CHO cells. Through glycolysis, CHO cells consume glucose as the main carbon source for energy production and produce lactate as the most common metabolic byproduct. In particular, the term 'optimal carbohydrate metabolism' refers to the ideal or best possible carbohydrate metabolism by CHO cells.

[0069] Similarly, as used herein, the term 'amino acid metabolism' refers to the entire biochemical process responsible for the metabolic formation, breakdown, and interconversion of amino acids in a cell. Amino acids are the basic building blocks of proteins and constitute all the protein material of a cell, including the cytoskeleton, the protein components of enzymes, receptors, and signaling molecules. In addition, amino acids are also used for cell growth and maintenance. For example, glutaminolysis is a key metabolic pathway in CHO cells. Glutaminolysis is a ubiquitous pathway in CHO cells that assimilates organic nitrogen for biomass synthesis while releasing ammonium as the main byproduct. In particular, the term 'optimal amino acid metabolism' refers to the ideal or best possible amino acid metabolism by CHO cells.

[0070] As used herein, the term 'lipid metabolism' refers to the synthesis and degradation of lipids in a cell, including the breakdown or storage of fats for energy and the synthesis of structural and functional lipids. Lipids are the main components of cell membranes, act as second messengers in cell communication, and are involved in signaling, transport, and secretion. Lipids are also an important energy source through beta-oxidation and the tricarboxylic acid (TCA) cycle. Lipid metabolism can have a significant impact on cell growth. For example, the process of synthesis and degradation of triglycerides in CHO cells can greatly affect overall cell metabolism and viability. In particular, the term 'optimal lipid metabolism' refers to the ideal or best possible amino acid metabolism by CHO cells.

[0071] Carbohydrate, amino acid, and lipid metabolism can be determined by metabolite detection assays, HPLC, and a bioprocess analyzer. These methods are further disclosed, 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.

[0072] As used herein, the term "pre - selected methylation site" refers to a methylation site selected from a gene or region that showed the highest degree of methylation variation during method training and meets certain quality criteria, such as considering a minimum sequencing coverage of ≥5x and ≥5 qualified CpG sites. Additionally, genes with an average methylation level of <0.1 or >0.9 can be excluded due to their limited dynamic range. Multivariate statistical methods, such as principal component analysis or multidimensional scaling analysis, can be used to define a "reference methylation profile" based on multiple training samples.

[0073] As used herein, and particularly in the context of a comparison of methylation profiles (e.g., a comparison between a test profile (from one or more test subjects) and a reference profile), the term "significantly similar" shall mean a similarity observed by statistical methods (i.e., by using bioinformatics) and / or also by visual inspection. For example, a significant similarity is observed if the test profile overlaps with a reference profile defined by multivariate statistical methods such as principal component analysis or multidimensional scaling analysis through multiple training samples. In particular, if more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the methylation patterns / profiles overlap with the reference profile, the test profile is significantly similar to the predetermined reference profile. The similarity of the test profile to more than one, e.g., two, three, or even all reference profiles reduces the significance of the similarity.

[0074] As used herein, the term "predetermined reference profile" refers to a typical or standard methylation profile of genomic material of a CHO cell line having specific characteristics depending on the context in which the term is used. In one example, for a method of determining a CHO cell line that exhibits at least one phenotypic parameter according to any aspect of the present invention, the phenotypic parameter conferring the potential for optimal heterologous protein production, the term "predetermined reference profile" refers to a typical or standard methylation profile of genomic material of a CHO cell line that exhibits one or more phenotypic parameters selected from optimal glucose consumption, optimal growth rate, optimal lactate production, and optimal ammonia accumulation. The predetermined reference profile can be obtained from one or more reference CHO cell lines each expressing one or more phenotypic parameters.

[0075] The method according to the aspects of the present invention attempts to create a methylation profile for a CHO cell line having the potential for optimal heterologous protein production because the cell line can exhibit cell viability, fitness, low cell exhaustion, and good metabolic readings. In particular, the method according to the aspects of the present invention provides a prognostic methylation profile of an ideal parental cell line prior to the introduction of a transgene.

[0076] According to yet another aspect of the present invention, there is provided a method for developing a test system for determining whether a test CHO cell line is capable of optimal heterologous protein production, the method comprising the steps of: (a) determining the test methylation status of one or more preselected methylation sites from genomic material obtained from the test CHO cell line; (b) selecting a methylation site reference group from the preselected methylation sites, the methylation site reference group being characterized by a specific and particular differential methylation profile for each phenotypic parameter or phenotype of interest; (c) obtaining a test system by assigning a reference methylation profile to each phenotypic parameter or phenotype of interest; and wherein comparison of the test methylation profile obtained from the test sample with the reference methylation profile obtained in (c) allows confirmation of whether the test CHO cell line is capable of optimal heterologous protein production, and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a bead-based array for DNA methylation.

[0077] The term'methylation site reference group' refers to specific and particular CpG sites or regions used to form a reference methylation profile.

[0078] According to yet another aspect of the present invention, there is provided a method for determining whether a CHO cell line is robust, stable, and capable of optimal heterologous protein production prior to introducing a transgene into the cell, the method comprising the steps of: (a) Determine the methylation profile from genomic material obtained from said CHO cell line; and (b) Compare the methylation profile of (a) with a reference methylation profile of a CHO cell line that is robust, stable, and capable of optimal heterologous protein production, wherein a significant similarity between the test methylation profile of (a) and the reference methylation profile indicates that the CHO cell line is robust, stable, and capable of optimal heterologous protein production, and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a bead-based DNA methylation array.

[0079] The DNA methylation profile of step (a) according to any aspect of the present invention is determined using a DNA methylation-based array. In particular, a bead-based DNA methylation array. The array according to any aspect of the present invention is advantageous because it enables an understanding of the genomic stability of the CHO cell line, enabling better control of the manufacturing / process development / product development / scaling up / validation process, thereby contributing to the selection of a better CHO cell line for industrial applications.

[0080] DNA-methylation-based arrays allow a high-throughput and robust method to determine semi-quantitative / quantitative DNA-methylation information from a small sample of the DNA of interest. These custom designed arrays can use Illumina iScan and Infinium platform technologies or their equivalents, which allow, for example, 100,000 different bead types covalently bound to DNA-methylation probes on each chip. Each probe represents a CpG methylation site at the end of the probe sequence. The DNA sample undergoes bisulfite conversion, amplification, fragmentation, precipitation, and resuspension steps before hybridization on the array chip. Once on the chip, the DNA hybridizes with the beads at each CpG site, enabling the methylation changes at each site to be specifically detected by single nucleotide extension. This is particularly advantageous because the array-based method is simple, and the results of the methylation-based array are accurate and reproducible.

[0081] Furthermore, compared to traditional sequencing that can take weeks to generate data, the array technology has a much shorter turnaround time. Compared to sequencing, the amount and complexity of the data generated are smaller, making it less computationally intensive. This allows faster computation to obtain interpretable results from experimental groups. Overall, the microarray technology is about 10x faster and about 1 / 10 the price of traditional sequencing, while still being able to quantify the methylation level of specific CpG sites.

[0082] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which can be synthesized or prepared biosynthetically. The probe molecules in an array can be identical or different from one another. An array can take a variety of forms, e.g., a library of soluble molecules; a library of compounds bound to resin beads, silicon chips, or other solid supports.

[0083] In particular, DNA methylation-based 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, 1000, 5000, 10,000, 100,000 or more sites or loci. In particular, an array includes a plurality of different probe molecules, which can be attached to a substrate or otherwise spatially distinguishable in the array. Examples of arrays that can be used in any aspect of the present invention include slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays, etc. In one example, the array technology used in any aspect of the present invention combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.

[0084] In particular, an array according to any aspect of the present invention can be an array of arrays, also referred to as a composite array, which has a plurality of individual arrays configured to allow simultaneous processing of multiple samples. Examples of composite arrays and the technology behind them are disclosed at least in US 6,429,027 and US 2002 / 0102578. The substrate of a composite array can include a plurality of individual array positions, each having a plurality of probes and each physically separated from other assay positions on the same substrate, such that fluid contacting one array position is prevented from contacting another array position. Each array position can have a plurality of different probe molecules, which are directly attached to the substrate or attached to the substrate via rigid particles in a well (also referred to herein as beads in a well).

[0085] In one example, the array substrate can be an optical fiber bundle or an array of bundles, as described in US6,023,540, US6,200,737, and / or US6,327,410. The optical fiber bundle or array of bundles can have probes directly attached to the fibers or via beads. Those skilled in the art will be able to readily determine which substrate will be most suitable for an array according to any aspect of the present invention. WO2004110246 further discloses other substrates and methods of attaching beads to substrates that can be used for an array according to any aspect of the present invention.

[0086] In one example, the surface of the substrate can be physically altered to enable attachment of probes or generation of array positions. For example, the surface of the substrate can be modified to contain chemically modified sites for covalently or non-covalently attaching probe molecules or particles having attached probe molecules. The probes can be attached using any of a variety of methods known in the art, including inkjet printing methods, spotting techniques, photolithographic synthesis methods, or printing methods using a mask. These techniques are more fully disclosed in WO2004110246.

[0087] In one example, a DNA methylation-based array according to any aspect of the invention can be a bead-based array, where the beads are associated with a solid support such as those commercially available from Illumina, Inc. (San Diego, Calif.). A bead array useful according to any aspect of the invention can also be in a fluid form, such as a fluid stream of a flow cytometer or similar device. Commercially available fluid forms for differentiating beads include, for example, those used in the XMAP(TM) technology from Luminex or the MPSS(TM) method from Lynx Therapeutics.

[0088] As used herein, the terms "solid support", "support", and "substrate" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many examples, at least one surface of the solid support will be substantially flat, although in some examples it may be desirable to physically separate the synthesis regions of different compounds using, for example, pores, raised areas, pins, etched trenches, etc.

[0089] A DNA methylation array according to any aspect of the invention can be a very high density array, e.g., having from about 10,000,000 probes / cm 2 to about 2,000,000,000 probes / cm 2 or from about 100,000,000 probes / cm 2 to about 1,000,000,000 probes / cm 2 Those. High density arrays are particularly useful according to any aspect of the invention for including a large number of CpG sites on the array.

[0090] A DNA methylation array according to any aspect of the invention can be used to simultaneously or sequentially analyze or evaluate such multiple loci as desired. In one example, multiple different probe molecules can be attached to the substrate or otherwise spatially differentiated in the array. Each probe is generally specific for a particular locus and can be used to distinguish the methylation state of the locus.

[0091] As used herein, the term "probe molecule" refers to a surface - immobilized molecule that can be recognized by a specific target. The probes used in the array can be specific for the methylated allele of a CpG site, the unmethylated allele of a CpG site, or both.

[0092] As used herein, the term "target" refers to a molecule that has an affinity for a given probe molecule. Targets can be naturally - occurring or artificial molecules. Also, they can be used in their unaltered state or as aggregates. Targets can be attached to the binding member covalently or non - covalently, either directly or via a specific binding substance. Examples of targets that can be used in any aspect of the present invention are methylated and unmethylated CpG sites. In the art, targets are sometimes referred to as anti - probes. When using the term target herein, no difference in meaning is intended.

[0093] As used herein, the term "complementary" refers to hybridization or base - pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double - stranded DNA molecule or between an oligonucleotide primer and a primer - binding site on a single - stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are generally A and T (or A and U), or C and G. Two single - stranded RNA or DNA molecules are generally considered to be complementary when at least about 80% of the nucleotides of one strand pair with the nucleotides of the other strand when optimally aligned and compared with appropriate nucleotide insertions or deletions, typically at least about 90% to 95%, and more preferably about 98 to 100%. Complete complementarity refers to 100% complementarity over the length of the sequence. For example, a 25 - base probe is completely complementary to a target when all 25 bases of the probe are complementary to an adjacent 25 - base sequence of the target with no mismatches over the length of the probe.

[0094] According to another aspect of the present invention, there is provided a method for determining the regulation of transgene expression in at least one CHO cell line genetically modified with a transgene, the method comprising the steps of: - measuring the methylation level of at least one CpG site of at least one promoter of the transgene, wherein the promoter is a viral promoter; and wherein a DNA methylation bead - based array is used to determine the DNA methylation level.

[0095] According to another aspect of the present invention, there is provided a method for determining the regulation of transgene expression in at least one CHO cell line genetically modified with a transgene, the method comprising the steps of: - measuring the methylation level of at least one CpG site of at least one promoter of the transgene, wherein the promoter is selected from cytomegalovirus (CMV) and simian virus 40 (SV40); and A DNA methylation bead-based array is used to determine the DNA methylation level.

[0096] As used herein, the term "promoter" or "gene promoter", which may be used interchangeably with the terms 'regulatory region' or 'regulatory sequence', refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to 1.5 kb downstream relative to the transcription start site (TSS), or an adjacent portion thereof. In particular, the 'regulatory region' refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to 0.5 kb downstream relative to the TSS. In some instances, the 'regulatory region' refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to the downstream edge of the CpG island, which overlaps with the region from 1.5 kb upstream to 1.5 kb downstream of the TSS (and in such cases may extend even further beyond 1.5 kb downstream), and an adjacent portion thereof. Changes in DNA methylation on the gene promoter responsible for protein glycosylation can lead to an improvement in protein quality. Protein glycosylation is a key quality attribute that regulates the efficacy, stability, and half-life of therapeutic proteins. Due to regulatory concerns, it is desirable to obtain a consistent glycoform profile in protein production. Thus, DNA methylation can act as a tool to globally regulate CHO metabolism and protein production.

[0097] According to a further aspect of the invention, there is provided the use of DNA methylation profiling for identifying at least one suitable insertion site or region in the genome of a CHO cell line for introducing at least one transgene. In particular, using the information of the CHO epigenome, suitable transgene insertion sites based on methylation patterns can be identified as the best 'hotspots' for transgene expression. For example, specific LMRs can be identified in the genome of a CHO cell line for targeted insertion of, for example, at least one transgene, since highly methylated sites will be silenced and will not be as productive for transgene expression (TIS analysis). In another example, the CMV promoter and surrounding repetitive elements can also be identified as hotspots for transgene insertion using methylation profiling.

[0098] Methylation profiling can also be used to screen for and select suitable promoters for use in CHO cells that result in optimal transgene expression. In particular, methylation data can be obtained from different promoters and transgene insertion sites and compared to select the best-performing promoters that can lead to improved transgene expression. In particular, the array according to any aspect of the invention can be used to monitor the activity (expression or silencing / imprinting) of a transgene by quantifying the DNA methylation level of the transgene promoter.

[0099] According to yet another aspect of the present invention, there is provided an array based on DNA methylation beads, which at least comprises: - a plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a plurality of CpG sites of CHO cells, wherein the CpG sites of the CHO cells are from the CHO genome and can be selected from at least one CpG in Tables 5a - 5f.

[0100] These CpG sites are environment-specific CpG sites (i.e., dynamic CpG sites), and CpG sites found in the promoters and the genes themselves of metabolism-related genes, protein production-related genes, cell growth and division-related genes, and epigenetic-related genes.

[0101] 'Environment-specific CpG sites', also known as dynamic CpG sites in the context of CHO cells, refer to CpG sites that are differentially methylated in different CHO cell lines. The cell lines used in the analysis include CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO-DXB11 (ATCC), ExpiCHO-S TM cells (Thermo FisherScientific), FreeStyle TM CHO-S TM cells (Thermo Fisher Scientific), CHO 1-15 500 (ATCC) and Agarabi CHO (ATCC).

[0102] As used herein, a'metabolism-related gene' in the context of CHO cells refers to genes related to several metabolic pathways, such as glycolysis, the TCA cycle, the pentose phosphate pathway, the malate-aspartate shuttle, amino acid metabolism, lactate metabolism, cholesterol biosynthesis, nucleotide biosynthesis, nucleotide sugar biosynthesis, etc. Several examples of such genes include Hk2, Pgk1, Idh3a, Pgm1, and Pdha1. Those skilled in the art will readily be able to identify the genes found in CHO cells belonging to the said category.

[0103] As used herein, a 'protein production-related gene' used in the context of CHO cells refers to genes related to cellular processes such as DNA replication and repair, mRNA transcription, mRNA translation, post-translational modification, and protein folding and export. Several examples of such genes include Gatb, Sec61a2, Ube2e3, Exosc1, Dna2, Pold1, etc. Those skilled in the art will readily be able to identify other genes found in CHO cells belonging to the said category.

[0104] As used herein, the term "cell growth and division associated genes" in the context of CHO cells refers to genes associated with cellular processes such as cell cycle regulation, cytoskeleton-related elements, cell signaling, nucleotide metabolism, and cell death. Several examples of such genes include Camk1, Cd82, Cdk4, Col1a1, and Ctsb. Again, a person skilled in the art will readily be able to identify other genes found in CHO cells belonging to said category.

[0105] As used herein, the term "epigenetic associated genes" in the context of CHO cells refers to genes associated with epigenetic modifications such as DNA methylation pathways, DNA demethylation pathways, folate and methionine cycles, and histone modifications. Several examples of such genes include Hat1, Shmt1, Bhmt, Dnmt1, and Ehmt1. A person skilled in the art will readily be able to identify other genes found in CHO cells belonging to said category.

[0106] As used herein, the term "viral promoter" in the context of CHO cells refers to at least the promoters and enhancers of cytomegalovirus (CMV) and simian virus 40. Viral promoters are typically rich in CpG sites, which makes them more susceptible to DNA methylation and thus inhibits protein expression.

[0107] The methods according to any aspect of the present invention can also be used to predict whether a CHO test cell is capable of optimal heterologous protein production. Examples The foregoing describes preferred embodiments which, as will be understood by those skilled in the art, can be subject to variations or modifications in design, construction, or operation without departing from the scope of the claims. Such variations are, for example, intended to be encompassed by the scope of the claims.

[0109] Example 1 Oxidative Stress in CHO Cell Culture Wet-Lab Method For said experiment, the transgenic CHO cell line Agarabi CHO ( CRL-3440 TM) Grow at a shaking speed of 130 RPM at 37 °C, 8% CO2 in CD FortiCHO medium supplemented with 8 mM L-glutamine. The batch culture of 6 flasks was maintained for 7 days, where 3 flasks represent the technical replicates of the control group and 3 flasks represent the technical replicates of the treatment group. On day 0, the flasks were inoculated with 3E5 viable cells / mL, and to induce oxidative stress, hydrogen peroxide was added to the treatment group every 48 hours at a final concentration of 120 μM. Cell count, cell viability, and heterologous protein production were measured every 2 days, and cell pellets of both the control and treatment groups were collected on day 7. Introduction of oxidative stress in CHO cells by treatment with hydrogen peroxide led to a decreased growth rate and cell viability compared to the control group, and thus there was a slight increase in heterologous protein productivity for the treatment group.

[0110] Purify genomic DNA from the collected cell pellets using the DNeasy Blood and Tissue Kit (Qiagen) and quantify using PicroGreen or NanoDrop TM 2000. Genomic DNA (500 ng) from the control and treatment groups was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample.

[0111] Computational Method Quality control (fastqc)1, sequencing adapter trimming (TrimGalore)2, and alignment with Bismark3 were performed on the raw sequencing data. The CMV promoter combined with the CHOK1-GS (Chinese hamster) genome was used as the reference genome. Bismark was also used to remove duplicate reads and extract methylation counts from the alignment output. SNPs were filtered out, and only counts with a minimum coverage of 10x were used for downstream analysis, which resulted in 3,711,013 CpG sites for the hydrogen peroxide-treated samples. Since regulatory methylation targets most commonly cluster into short regions, DMRfinder4 was used to perform modified single-linkage clustering of methylation sites. With a maximum distance between CpG sites of 100 bp, 1,728,014 genomic regions were found for the hydrogen peroxide-treated samples.

[0112] Differential Methylation Analysis Differential methylation analysis was performed using MethylKit5 between the control and treatment groups. Logistic regression was used to determine differential methylation across all regions, and the false discovery rate (FDR) correction was performed using the sliding linear model (SLIM)6 method. Regions with an FDR-corrected p-value < 0.05 and a methylation change greater than 25% between groups were identified as differentially methylated regions (DMRs), of which there were 122 for the hydrogen peroxide-treated samples, shown in Table 1. Principal component analysis (PCA) is a dimensionality reduction technique that emphasizes variation in a dataset. Figure 1 PCA analysis of the DMRs is shown.

[0113] Preliminary results indicate that DMRs play a role in the epigenetic changes of oxidative stress, which could potentially be used as markers for future studies.

[0114] Example 2 Adaptation of CHO cells to culture medium additives Wet-Lab Method For the experiment, the transgenic CHO cell line Agarabi CHO ( CRL-3440 TM ) was adapted for 2 weeks at 37 °C, 8% CO2 with a shaking speed of 130 RPM in CD FortiCHO medium supplemented with 8 mM L-glutamine and 1 mg / L human insulin-like growth factor 1 (IGF-1). Batch cultures of 6 flasks were maintained for 7 days, where 3 flasks represented technical replicates of the control group (without IGF-1 adaptation) and 3 flasks represented technical replicates of the IGF-1-adapted group. On day 0, the flasks were inoculated with 3E5 viable cells / mL, and 1 mg / L insulin growth factor was added to the adapted group. Cell counts, cell viability, and protein production were measured every 2 days, and cell pellets from both the control and treatment groups were collected on day 7. Adaptation of CHO cells to IGF-1 had no significant effect on growth rate and viability; however, heterologous protein productivity doubled compared to the control group.

[0115] Genomic DNA was purified from the collected cell pellets using the DNeasy Blood and Tissue Kit (Qiagen) and quantified using PicroGreen or NanoDrop TM 2000. Genomic DNA (500 ng) from the control and adapted groups was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample.

[0116] Computational Method Quality control (fastqc)1, sequencing adapter trimming (TrimGalore)2, and alignment with Bismark3 were performed on the raw sequencing data. The CMV promoter in combination with the CHOK1-GS (Chinese hamster) genome was used as the reference genome. Bismark was also used to remove duplicate reads and extract methylation counts from the alignment output. SNPs were filtered out, and only counts with a minimum coverage of 10x were used for downstream analysis, which resulted in 4,244,091 CpG sites for the IGF-1 adapted samples. Since regulatory methylation targets most commonly cluster into short regions, DMRfinder4 was used for modified single-linkage clustering of methylation sites. With a maximum distance between CpG sites of 100bp, 2,048,904 genomic regions were found for the IGF-1 adapted samples.

[0117] Differential Methylation Analysis Differential methylation analysis was performed between the control and adapted groups using MethylKit5. Logistic regression was used to determine differential methylation across all regions, and the FDR correction was performed using the sliding linear model (SLIM)6 method. Regions with an FDR-corrected p-value < 0.05 and a methylation change between groups greater than 25% were identified as differentially methylated regions (DMRs), which were 289 for the IGF-1 adapted samples and are listed in Table 2. Principal component analysis (PCA) is a dimensionality reduction technique that emphasizes variation in a dataset. Figure 2 The PCA analysis of the DMRs is shown.

[0118] Preliminary results indicate that DMRs play a role in the epigenetic changes of IGF-1 adaptation, which could potentially be used as markers for future studies.

[0119] Example 3 Detection of the quality of heterologous proteins from CHO cells Wet-Lab Method For the experiment, the transgenic CHO cell line Humira431 clone (obtained from A*Star BTI) was grown at a shaking speed of 150 RPM at 37 °C, 8% CO2 in EX-Cell Advanced Fed-batch medium supplemented with 6 mM L-glutamine. Six bottles of fed-batch cultures were maintained for 11 days, where three bottles represented technical replicates of the control group (C1, C2, C3), and three bottles represented technical replicates of the treatment group (T1, T2, T3). On day 0, the bottles were inoculated with 3E5 viable cells / mL, and the cultures were fed with Advanced CHO Feed 1 on days 3, 5, 7, 9, and glucose was replenished to 6 g / l with 45% glucose when glucose dropped below 3 g / l. To induce high osmolality in the cell culture medium, a concentrated sodium chloride solution was added to the treatment group on day 3, resulting in an increase in the osmolality of the medium from 320 mOsm / kg to 480 mOsm / kg. Cell counts, cell viability, and heterologous protein production were measured every 2 days, and cell pellets of both the control and treatment groups were collected on day 7. Inducing high osmolality in the CHO cell culture medium by sodium chloride led to a decreased growth rate ( Figure 3 a), an increase in heterologous protein productivity ( Figure 3 b), and alterations in the relative abundance of each N-glycan modification (Table 3) in the treatment group compared to the control group. Alterations in the relative abundance of each N-glycan symbolize changes in the quality of the heterologous protein.

[0120] DNA Extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen) according to the manufacturer's instructions, including RNase treatment. The DNA quantity was measured by PicoGreen assay and the DNA quality was evaluated via NanoDrop (Thermo Scientific) to ensure an A260 / 280 ratio ≤ 1.8. Then, a small amount of the sample was also analyzed by automated electrophoresis on a TapeStation (Agilent) to ensure that each sample contained high molecular weight DNA.

[0121] Sequencing Analysis Genomic DNA (500 ng) from the samples was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample with 20X coverage.

[0122] Data Processing: Processing and Analysis of Sequencing Data: Quality control (fastqc)1, sequencing adapter trimming (TrimGalore)2, and alignment with Bismark3 were performed on the raw sequencing data.

[0123] Bismark was also used to remove duplicate reads and extract methylation counts from the alignment output. SNPs were filtered out, and only the counts with a minimum coverage of 10x were used for downstream analysis.

[0124] Then, the methylation ratios of the control (C1) and treated (T1) samples were extracted. Then, the sites with a methylation difference of 30% were filtered. (Table 4) These methylated sites can indicate differences in protein quality between the samples.

[0125] Table 3 Comparison of the percentage of N-glycan modification abundances between control (C1) and treated (T1).

[0126] Table 4a CpG sites of the genes from Table 3 with a methylation difference of 30% or greater.

[0127] Table 4b CpG sites of the genes from Table 3 with a methylation difference of 30% or greater.

[0128] Table 4c CpG sites of the genes from Table 3 with a methylation difference of 30% or greater.

[0129] Example 4 Detection of the amount of heterologous protein from CHO cells Wet-Lab Method For the experiment, five transgenic CHO clones (obtained from A*Star BTI) were grown at a shaking speed of 225 RPM at 37 °C, 8% CO2 in EX-Cell Advanced Fed-Batch Medium supplemented with 6 mM L-glutamine. The five transgenic CHO cell lines included low producers (3D11, 2C9, 2H2), medium producer (10A8), and high producers (8F8, 7H9). On day 0, the flasks were inoculated with 3E5 viable cells / mL, and the cultures were fed with Cell Boost 7a on days 3, 5, 7, 9, 11, and glucose was topped up to 6 g / l with 45% glucose when it dropped below 2 g / l. The fed-batch cultures of the 6 clones were maintained for 14 days. Cell counts, cell viability, and heterologous protein production were measured every 2 days, and cell pellets were collected on day 9. As Figure 5 shown in

[0130] DNA Extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen) according to the manufacturer's instructions, including RNase treatment. The DNA quantity was measured by PicoGreen assay and the DNA quality was evaluated via NanoDrop (Thermo Scientific) to ensure an A260 / 280 ratio ≤ 1.8. Small amounts of samples were then also analyzed by automated electrophoresis on a TapeStation (Agilent) to ensure that each sample contained high molecular weight DNA.

[0131] Bisulfite Conversion and BeadChip Analysis The genomic DNA samples were then subjected to bisulfite conversion using the EZ DNA Methylation-Gold TM Kit (ZymoResearch). The methylation levels were then quantified using our custom methylation BeadChip kit (Illumina), which can quantitatively analyze over 50,000 methylation sites throughout the genome at single nucleotide resolution. After bisulfite conversion, the samples were processed through a three-day workflow, including sample amplification, fragmentation, precipitation, hybridization to the BeadChip, and X-stain according to the Infinium HD Methylation Assay (Methylation Assay) (Illumina, document #15019519 v07), and then imaged on an iScan (Illumina), where intensity files for calculating β values were generated.

[0132] Data Processing: Processing of Beadchip Data: Customized chip array data processing was performed in R version 4.1.2 using sesame version 1.14.2. The DNA methylation level at each locus was calculated as the methylation beta-value. The beta-value is defined as the methylated signal / (methylated signal + unmethylated signal). It can be calculated using the getBetas function. The SeSAMe pipeline (Zhou et al., 2018) was used to generate normalized beta-values and for quality control. pOOBAH was used for low-intensity detection calling and generation (based on p-values). Background subtraction based on normal-exponential deconvolution using out-of-band probes noob (Triche et al., 2013) was also implemented, and optionally with additional bleed-through subtraction.

[0133] After obtaining the beta-values, control probes were filtered out from the data frame. CpG sites with NA beta-values were also removed from the data frame. To obtain the differentially methylated positions (DMPs) between high protein productivity clones (7H9 & 8F8) and low protein productivity clones (2C9, 2H2 & 3D11), sample 10A8 was excluded from the beta-value data frame before extracting the DMPs. After filtering out 10A8, the dml and dmr functions from the sesame package were used to extract the DMPs between high and low protein productivity clones. The dmr function would result in a data frame, and to obtain more statistically significant DMPs, only DMPs with Pr(|t|) < 0.05 were retained, while the rest were removed from the data frame. This resulted in 901 remaining CpG sites (after removing probes with NA), and the PCA plot of these sites was plotted using prcomp followed by the autoplot function. These references are shown in Table 5.

Claims

1. A method for determining the suitability of at least one Chinese hamster ovary (CHO) test cell line for optimal heterologous protein production, the method comprising: (a) determining a test methylation profile from genomic material obtained from the CHO test cell line; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile, wherein the reference methylation profile comprises the methylation status of more than one CpG site from at least one CHO reference cell line, the at least one CHO reference cell line exhibiting at least one phenotype of interest for optimal heterologous protein production, wherein a significant similarity of the test methylation profile of (a) compared to the reference methylation profile indicates that the CHO test cell line is suitable for optimal heterologous protein production, wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a DNA methylation bead-based array.

2. The method according to claim 1, wherein the reference methylation profile is a compilation of more than one CpG site from at least one CHO reference cell line, the at least one CHO reference cell line exhibiting at least one phenotype of interest for optimal heterologous protein production.

3. The method according to any one of the preceding claims, wherein the phenotype of interest for optimal heterologous protein is selected from phenotypic homogeneity, protein productivity, and protein quality.

4. A method for selecting at least one CHO cell comprising a phenotype of interest from a population of CHO cells derived from a parental clone, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO cells, and (b) comparing the test methylation profile of (a) with a reference methylation profile from the parental clone exhibiting the phenotype of interest, wherein a significant similarity between the test methylation profile of (b) and the reference methylation profile indicates that the cell has the phenotype of interest of the parental clone; wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a DNA methylation bead-based array; and wherein the phenotype of interest is selected from phenotypic homogeneity, protein productivity, and protein quality.

5. A method for identifying at least one CHO test cell line capable of producing at least one biosimilar of a heterologous protein produced by a CHO reference cell line, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO test cell line, and (b) comparing the test methylation profile of (a) with the reference methylation profile of the CHO reference cell line, wherein a significant similarity between the test methylation profile of (a) and the reference methylation profile indicates that the two cell lines produce biosimilars; and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a DNA methylation bead-based array.

6. A method for identifying at least one CHO test cell line capable of producing at least one biological equivalent of a heterologous protein relative to that produced by a CHO reference cell line, the method comprising the steps of: (a) determining a test methylation profile from genomic material obtained from the CHO test cell line, and (b) comparing the test methylation profile of (a) with a reference methylation profile of the CHO reference cell line, wherein when the test methylation profile of (a) is the same as the reference methylation profile, it indicates that the two cell lines produce biological equivalents; and wherein the test methylation profile and the reference methylation profile are determined from CpG sites of the CHO cell genome and using a DNA methylation bead-based array.

7. A method for evaluating one or more phenotypic parameters of at least one test CHO cell line, the method comprising the steps of: (a) determining the test methylation status of one or more preselected methylation sites from genomic material obtained from the test CHO cell line; (b) determining a test methylation profile of the test CHO cell line based on the methylation status determined in (a); and (c) comparing the test methylation profile determined in (b) with at least one predetermined reference methylation profile, wherein each of the predetermined reference methylation profiles is specific for a reference CHO cell line having at least one phenotypic parameter; wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, the test CHO cell line has a similar or preferably the same phenotypic parameter as the reference CHO cell line having the predetermined reference methylation profile; and wherein the test methylation profile and the reference methylation profile are determined from CpG sites of the CHO cell genome and using a DNA methylation bead-based array.

8. The method according to claim 7, wherein the phenotypic parameter is selected from: optimal carbohydrate metabolism, optimal amino acid metabolism, optimal lipid metabolism, optimal protein productivity, and optimal cell viability.

9. A method for developing a test system for determining whether a test CHO cell line is capable of performing optimal heterologous protein production, the method comprising the steps of: (a) determining the test methylation status of one or more preselected methylation sites from genomic material obtained from the test CHO cell line; (b) selecting a methylation site reference group from the preselected methylation sites, the methylation site reference group being characterized by a specific and distinct differential methylation profile for each phenotypic parameter or phenotypic of interest; (c) obtaining a test system by assigning a reference methylation profile for each phenotypic parameter or phenotypic of interest; and wherein comparison of the test methylation profile obtained from a test sample with the reference methylation profile obtained in (c) allows confirmation of whether the test CHO cell line is capable of performing optimal heterologous protein production; and wherein the test methylation profile and the reference methylation profile are determined from CpG sites of the CHO cell genome and using a DNA methylation bead-based array.

10. A method for determining whether a CHO cell line is robust, stable and capable of optimal heterologous protein production before introducing a transgene into the cells, the method comprising the steps of: (a) determining the methylation profile from genomic material obtained from the CHO cell line; and (b) comparing the methylation profile of (a) with a reference methylation profile of a CHO cell line that is robust, stable and capable of optimal heterologous protein production, wherein a significant similarity between the test methylation profile of (a) and the reference methylation profile indicates that the CHO cell line is robust, stable and capable of optimal heterologous protein production; and wherein the test methylation profile and the reference methylation profile are from CpG sites of the CHO cell genome and are determined using a bead-based DNA methylation array.

11. The method according to any one of the preceding claims, wherein the CpG sites comprise at least one of the CpG sites provided in Tables 5a - 5f.

12. A method for determining the regulation of transgene expression in at least one CHO cell line genetically modified with a transgene, the method comprising the steps of: - measuring the methylation level of at least one CpG site of at least one viral promoter of the transgene, and wherein a bead-based DNA methylation array is used to determine the DNA methylation level.

13. A bead-based DNA methylation array, which at least comprises: - a plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a plurality of CpG sites of a CHO cell, wherein the CpG sites of the CHO cell are selected from at least Tables 5a - 5f.

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