An epigenetic clock for CHO cells

Abstract

The present invention relates to an in vitro method for predicting the chronological age of a test Chinese Hamster Ovary (CHO) cell or population of test CHO cells, the method comprising the steps of: (a) determining the test methylation level of a set of specific CpG sites from genomic material obtained from the test CHO cell or from the population of test CHO cells from a CHO cell line using a DNA-methylation bead-based array; (b) comparing the test methylation level determined from step (a) with a reference methylation level of the same set of specific CpG sites from an age-correlated reference sample, thereby establishing the epigenetic age and predicting the chronological age of the test CHO cell or the population of test CHO cells wherein the set of specific CpG sites comprises at least 10%, preferably 30%, more preferably 50% of the CpG sites indicated in Table 1.

Description

[0001] 202400021 Foreign Filing 1

[0002] AN EPIGENETIC CLOCK FOR CHO CELLS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for establishing an epigenetic clock for Chinese Hamster Ovary (CHO) cells that can then be used to determine the epigenetic age of a CHO cell and to estimate the chronological age the CHO cell.

[0005] BACKGROUND OF THE INVENTION

[0006] Chinese Hamster Ovary (CHO) cells are known to be the workhorses for the industrial production of recombinant therapeutic proteins since 1987 and are hence widely used for biologies production. About 70% of all recombinant biopharmaceutical proteins and all monoclonal antibodies approved since 2016 are being manufactured in CHO cells. Several advantages of utilizing CHO for biologies production include tolerance to genetic manipulations, ease of adaptation to manufacturing process scales, rapid growth rates, and ability to perform human-compatible post-translational modifications. As the biomanufacturing process advances towards continuous processes that lead to consistency and production of stable proteins, longer culture durations and older cell ages are involved. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. Accordingly, it is important to know exactly how old a CHO cell is before the cell is used for large scale production of desired proteins. Currently, the age of a CHO cell from a population of CHO cells is estimated based on the accurate recording of the lab technicians working on the cells and the age provided by the laboratories from which the cells are purchased. This may not necessarily be correct and is subject to many levels of human error.

[0007] Accordingly, there is still a need in the art for a robust analytical tool to estimate the chronological age of CHO cells as accurately as possible to ensure efficient production of recombinant proteins in the CHO cells and to reduce the chances of downstream production of undesired proteins or proteins of poor quality.

[0008] DESCRIPTION OF THE INVENTION

[0009] The present invention attempts to solve the problems above by providing a robust method for establishing the epigenetic age of test CHO cells that may be used to predict the chronological age of the test CHO cells with improved specificity, accuracy and precision. In particular, the method for establishing the epigenetic age of test CHO cells involves firstly establishing an epigenetic clock for CHO cells and then using the clock to predict the chronological age of the CHO cells.

[0010] Age-correlated DNA methylation changes at discrete sets of CpGs in the human genome have been identified and used to predict age (Horvath, S. (2013). These “epigenetic clocks” specific for CHO cells can estimate the DNA methylation age in specific CHO cells. Epigenetic / biological age is highly correlated with chronological age.

[0011] These age-correlated biomarkers are particularly useful tools for large scale protein production as they facilitate determining the age of monitoring CHO cells and provide objective quality assurance. CHO cells 202400021 Foreign Filing 2 present a unique challenge for performance biomarker development, as they combine considerable economic importance with a relatively short lifespan.

[0012] The epigenetic clock for CHO cells is established by determining the methylation levels of a specific set of CpG sites from CHO cells at different known time points. Each known time point is then connected to the methylation levels of the specific set of CpG sites to obtain an age-correlated reference sample. This age- correlated reference sample is then used to determine the chronological age of test CHO cells and / or populations thereof.

[0013] According to a first aspect of the present invention, there is provided an in vitro method for predicting the chronological age of a test Chinese Hamster Ovary (CHO) cell or population of test CHO cells, the method comprising the steps of:

[0014] (a) determining the test methylation level of a set of specific CpG sites from genomic material obtained from the test CHO cell or from the population of test CHO cells using a DNA- methylation bead-based array;

[0015] (b) comparing the test methylation level determined from step (a) with a reference methylation level of the same set of specific CpG sites from an age-correlated reference sample, thereby establishing the epigenetic age and predicting the chronological age of the test CHO cell or the population of test CHO cells, wherein the set of specific CpG sites comprises at least 10%, preferably 30%, more preferably 50% of the CpG sites indicated in Table 1.

[0016] The present invention provides a new epigenetic clock for CHO cells with a substantially improved generalization capability and robustness. In particular, the better design and selection of CpG sites used in the method according to any aspect of the present invention have risen from potentially evaluating the whole genome of the CHO cell with an optimized workflow, possibly including the normalization process to circumvent bias and errors from the methods used in the state of the art, enabling the selection from over ~30 million methylation sites to the list of CpG sites provided in the method according to any aspect of the present invention. The final design of the CpG sites for clocks in the method according to any aspect of the present invention is through a selection matrix with more than ~ 200 parameters to result in the development of a robust clock. More in particular, the method of selection of CpG sites used in the method according to any aspect of the present invention is more accurate compared to that used in the state of the art age clocks where the CpG sites are predetermined CpG sites (restricted to promotor regions) unlike those of the method according to any aspect of the present invention where the CpG sites are with functional elements of the genome and therefore reflect the whole age-related biological processes.

[0017] Epigenetics technologies thus provide a solution for predicting the chronological age of CHO cells that indirectly helps in quantitative and qualitative analysis of protein production. In particular, the set of specific CpG sites for determining methylation levels may comprise environmental specific CpG sites or dynamic CpG sites i.e., sites which seem to have a crucial role in several environmental conditions; CpG sites in the viral promoters (CMV and SV40 promoters) and / or CpG sites from regulatory regions of 202400021 Foreign Filing 3 candidate genes from pathways which are significant in certain important biological processes for the CHO cell (e.g. metabolic linked genes, protein production linked genes, cell growth / division linked genes, and methylation linked genes). More in particular, the set of specific CpG sites from wherein the test methylation levels and reference methylation levels are determined are from the CHO cell genome.

[0018] The term ‘CHO cell genome’ herein refers to the genomic DNA of the CHO cell that excludes the DNA of a virus, particularly CMV and SV40, that are used to introduce foreign DNA to the cell. In particular, the CHO cell genome may denote the cell with a genome make-up that is in a form as seen naturally in the wild. The term may also include genes which have been added to the CHO genome by genetic modification (i.e. with regard to improved production of protein etc.) but not necessarily or not genes and promoters of viruses that have been used to introduce the genes into the CHO genome. The term “CHO cell genome” therefore may exclude virus genes and promoters and / or may include endogenous or homologous genes of the CHO cell and / or genetically modified endogenous or homologous genes of the CHO cell and / or intergenic genes, DNA found between the genes of the CHO cell.

[0019] The CHO (test) cell refers to any CHO cell from any CHO cell line and refers to immortal Chinese Hamster Ovary cell line (CHO) derived from Cricetulus griseus. In particular, the CHO cell line may be selected from the group consisting of CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO- DXB11 (ATCC), ExpiCHO-S™ cells (Thermo Fisher Scientific), Freestyle™ CHO-S™ cells (Thermo Fisher Scientific), CHO 1 -15 [subscript 500] (ATCC) and Agarabi CHO (ATCC).

[0020] As used herein, the term ‘population of CHO cells’ refers to more than one CHO cell or a group of CHO cells from a single CHO cell line. The CHO cell line comprises cells from a single parental clone In particular, the majority of the cells are in the same stage of the cell cycle such that determining the epigenetic age of one or a few of the CHO cells from the population of the CHO cells is indicative or representative of the whole population of CHO cells.

[0021] As used herein, the term ‘parental clone’ refers to a cell line derived from host cells (a CHO cell line) in which a transgene has been integrated into the genome. The term ‘subclone’ as used herein in relation to a parental clone refers to a clonal cell line derived from parental clone having the same genotype but a different phenotype due to epigenetic changes.

[0022] The term “chronological age” refers to the calendar time that has passed from day 0 of the CHO cell.

[0023] The epigenetic age depends on the biological state or condition of a CHO cell or of a population of CHO cells and takes into account the circumstances of the cell culture (such as stress, nutrition, etc.). The terms “epigenetic age”, “methylation age”, and “biological age” have identical meanings and are used interchangeably in the context of the present application.

[0024] In step (a) according to any aspect of the present invention, CpG sites within the genomic DNA of the CHO cells were identified and the methylation level of those CpG sites is determined. Accordingly, 202400021 Foreign Filing 4 method step (a) involves a DNA methylation profiling process, preferably bisulfite sequencing. Therein, cytosine residues in the genomic DNA are transformed to uracil, while 5- methylcytosine residues in the genomic DNA are not transformed to uracil.

[0025] Whole genome bisulfite sequencing is a genome-wide analysis of DNA methylation based on the sodium bisulfite conversion of genomic DNA, which is then sequenced on a next-generation sequencing platform. The sequences are then re-aligned to the reference genome to determine methylation states of the CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil.

[0026] In particular, methylation levels are measured using a DNA-methylation bead-based array. In particular, a DNA methylation-based array provides a convenient platform for simultaneous analysis of large numbers of CpG sites.

[0027] Arrays allow for a high-throughput and robust method to determine semi-quantitative / quantitative DNA- methylation information through a small sample of extracted DNA of interest. These custom designed arrays may use Illumina iScan and Infinium platform technology or an equivalent thereof, which allows on each chip for example 100,000 different bead types that covalently bind DNA methylation probes. Each probe represents one CpG Methylation site at the end of the probe sequence. DNA samples undergo bisulfite conversion, amplification, fragmentation, precipitation and resuspension steps before hybridization on an array chip. Once on the chip the DNA hybridizes to the beads for each CpG site so that methylation changes at each site can be detected specifically through single nucleotide extension. This is especially advantageous as the array based method is simple and the results of the array are accurate and reproducible. Further, compared to traditional sequencing which can take weeks to generate data, the array technology has a much shorter turn-around time. The volume and complexity of data generated is lesser compared to sequencing making it computationally less intensive. This allows for quicker computation to achieve interpretable results from experimental groups. Overall microarray technology is roughly 10x faster and 10x cheaper than traditional sequencing while still quantifiable for the methylation level at specific CpG sites. The term “array” as used herein refers to an intentionally created collection of probe molecules which can be prepared either synthetically or biosynthetically. The probe molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports. In particular, an array provides a convenient platform for simultaneous analysis of large numbers of CpG sites, for example, 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.

[0028] In particular, the array comprises a plurality of different probe molecules that can be attached to a substrate or otherwise spatially distinguished in an array. Examples of arrays that may be used according to any aspect of the present invention include slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays and the like. In one example, array technology used according to 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. 202400021 Foreign Filing 5

[0029] In particular, the array according to any aspect of the present invention may be an array of arrays, also referred to as a composite array, having a plurality of individual arrays that is configured to allow processing of multiple samples simultaneously. Examples of composite arrays and the technology behind them are disclosed at least in US 6,429,027 and US 2002 / 0102578. A substrate of a composite array may include a plurality of individual array locations, each having a plurality of probes, and each physically separated from other assay locations on the same substrate such that a fluid contacting one array location is prevented from contacting another array location. Each array location can have a plurality of different probe molecules that are directly attached to the substrate or that are attached to the substrate via rigid particles in wells (also referred to herein as beads in wells). In one example, an array substrate can be a fibre optical bundle or array of bundles as described in US6,023,540, US6,200,737 and / or US6,327,410. An optical fibre bundle or array of bundles can have probes attached directly to the fibres or via beads. A skilled person would be able to easily determine which substrate will be most suitable for the array according to any aspect of the present invention. W02004110246 further discloses other substrates and methods of attaching beads to the substrates that may be used in the array according to any aspect of the present invention.

[0030] In one example, a surface of the substrate may have physical alterations to enable the attachment of probes or produce array locations. For example, the surface of a substrate can be modified to contain chemically modified sites that are useful for attaching, either-covalently or non-covalently, probe molecules or particles having attached probe molecules. Probes may be attached using any of a variety of methods known in the art including, an ink-jet printing method, a spotting technique, a photolithographic synthesis method, or printing method utilizing a mask. W02004110246 discloses these techniques in more detail. In one example, the array according to any aspect of the present invention may 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.). An array of beads useful according to any aspect of the present invention can also be in a fluid format such as a fluid stream of a flow cytometer or similar device. Commercially available fluid formats for distinguishing beads include, for example, those used in XMAP(TM) technologies from Luminex or MPSS(TM) methods from Lynx Therapeutics. The term “solid support”, “support”, and “substrate” as used herein 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 synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like.

[0031] The array or microarray according to any aspect of the present invention may be a very high density array, for example, those having from about 10,000,000 probes / cm2to about 2,000,000,000 probes / cm2or from about 100,000,000 probes / cm2to about 1 ,000,000,000 probes / cm2. High density arrays are especially useful according to any aspect of the present invention for including the multitude of CpG sites on the array. The array according to any aspect of the present invention may be used to analyse or evaluate such pluralities of loci simultaneously or sequentially as desired. In one example, a plurality of 202400021 Foreign Filing 6 different probe molecules can be attached to a substrate or otherwise spatially distinguished in an array. Each probe is typically specific for a particular locus and can be used to distinguish methylation state of the locus.

[0032] The term “probe molecules” or ‘probes’ as used interchangeably herein refers to a surface immobilized molecule that can be recognized by a particular target. Probes used in the array can be specific for the methylated allele of a CpG site, the non-methylated allele of the CpG site or both or for the methylated allele of a non-CpG site, the non-methylated allele of the non-CpG site or both.

[0033] The term “target” as used herein refers to a molecule that has an affinity for a given probe molecule. Targets may be naturally occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed according to any aspect of the present invention are methylated and nonmethylated CpG sites. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended.

[0034] The term “complementary” as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, 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 said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Perfectly complementary refers to 100% complementarity over the length of a sequence. For example, a 25- base probe is perfectly complementary to a target when all 25 bases of the probe are complementary to a contiguous 25 base sequence of the target with no mismatches between the probe and the target over the length of the probe.

[0035] To quantify the methylation level, various established protocols may be used to calculate the beta value of methylation, which equals the fraction of methylated cytosines in a specific location.

[0036] The term “CpG site”, “clock CpG” or “CpG location” as used in the context of the present invention refers to a CpG position that is potentially methylated. Methylation typically occurs in a CpG containing nucleic acid. The CpG containing nucleic acid may be present in, e.g. a CpG island, a CpG doublet, a promoter, an intron, or an exon of a gene or in an intergenic region. For instance, the potential methylation sites may encompass the promoter / enhancer regions of the indicated genes.

[0037] The “set of specific CpG sites in the genomic CHO cell refers to the CpG locations showing the best correlations with age. In particular, the set of specific CpG sites comprises at least one of the CpG sites indicated in Table 1. More in particular, the set of specific CpG sites comprises at least 10% of the CpG 202400021 Foreign Filing 7 sites indicated in Table 1. The set of specific CpG sites comprises at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% of the CpG sites indicated in Table 1 . In one example, the set of specific CpG sites consists of all the CpG sites indicated in Table 1 of Example 1 .

[0038] The age-correlated reference sample serves as a control and represents an average methylation level at a pre-determined and specific chronological age. Aging in CHO cell culture is determined by the number of days the cells have been in culture since the day they were thawed from their cryopreserved state. To achieve this, the cells are periodically passaged every 3-4 days when they reach their highest cell density. The aging process is initiated on day 0 when the cells are thawed and continues up to day 60, during which approximately 16 passages are performed. In particular, the genomic DNA is obtained from samples of CHO cells in a cell line / population of cells at specific time points within the chronological lifespan of a CHO cell and DNA methylation measured of specific CpG sites at these specific time points. In particular, the lifespan of the CHO cell is up to 2 months. As an example, the sample DNA may be stratified into five age (0 d, 15 d, 30 d, 45 d and 60 d) groups. More in particular, the sample DNA may be stratified into 12 age (3 d, 7 d, 10 d, 14 d, 17 d, 21 d, 24 d, 28 d, 31 d, 35 d, 38 d, 42 d) groups. Elastic net is then carried out on the samples. Details regarding suitable sample materials will be provided below. Ideally, the sample material covers the entire life cycle of the CHO cell under investigation.

[0039] The method for predicting the chronological age of CHO cells according to any aspect of the present invention may be used for testing a single individual CHO cell or for testing a CHO cell population, such as a CHO cell line, CHO clone (i.e. CHO cells from a single parental clone). The biological sample material deriving from the CHO cell or from the population to be tested may be the genomic DNA from one cell or a group of cells.

[0040] The sample size (i.e. the number of cells from which the DNA is taken at each time point) has to be determined in view of the actual cell density, i.e. with the actual number of cells belonging to the population to be tested.

[0041] Step (b) may be performed with a mathematical algorithm and in particular with a statistical prediction method.

[0042] The selection of the CpGs, which define the clock, i.e. the set of specific CpG sites of step (a), may be done with a penalized regression. In this case, the evaluation of a newly sequenced test sample is done by evaluating the methylation values applying the existing regression function of the clock. In accordance therewith, a trained regression function is preferably applied in step (b).

[0043] According to a further aspect of the present invention, there is provided a computer-implemented method of establishing an epigenetic clock for CHO cell, the method comprising:

[0044] (a) identifying and determining the methylation levels of specific CpG sites within the genomic DNA obtained from a plurality of CHO cells and representing specific time points within the chronological lifespan of the CHO cells; and 202400021 Foreign Filing 8

[0045] (b) correlating the CpG methylation levels of the CpG sites obtained in step (a) with chronological age using a penalized regression model.

[0046] The plurality of CHO cells in step (a) have representative ages ranging between 0 to 60 days, 1 to 60, 1 to 55 or 1 to 50. In particular, the representative ages range between 1 to 60 days. In particular, in step (a) the plurality of CHO cells refer to cells from a single CHO cell line or population of CHO cells from a parent clone.

[0047] In particular, the penalized regression model is elastic net linear regression model. Elastic net is used to select CpGs, differentially methylated regions (DMRs), lowly methylated regions (LMRs) and CpGs of a subset of that are most predictive of age of the CHO cell. These selected specific CpGs according to any aspect of the present invention can then be used to develop a model for predicting cell chronological age based on DNA methylation values. More in particular, the Elastic net algorithm is a machine learning method that combines traditional Lasso and ridge regression techniques, placing emphasis on model sparsity while appropriately balancing the contributions of correlated variables. It is particularly useful in the method according to any aspect of the present invention for constructing linear models where the number of variables is substantially greater than the number of samples.

[0048] In particular, the specific CpG sites within the genomic DNA of the CHO cells according to any aspect of the present invention, are distributed in the CHO genome .

[0049] The methylation level of the CpG sites according to any aspect of the present invention is determined using a DNA-methylation bead-based array.

[0050] According to another aspect of the present invention, there is provided a computer program loading into a memory of a computer, implementing the method according to any aspect of the present invention.

[0051] According to a further aspect of the present invention, there is provide a tangible, computer-readable medium comprising a computer-readable code that, when executed by a computer, causes the computer to perform operations comprising:

[0052] (a) receiving information corresponding to the methylation levels of specific CpG sites obtained from a plurality of CHO cells and representing specific time points within the chronological lifespan of the CHO cells; and

[0053] (b) correlating the CpG methylation levels of the CpG sites of step (a) with chronological age using a penalized regression model.

[0054] According to yet another aspect of the present invention, there is provided a use of a DNA-methylation bead-based array for determining the chronological age of a test CHO cell or a population of test CHO cells from a CHO cell line. 202400021 Foreign Filing 9

[0055] According to a further aspect of the present invention, there is provided a bead-based DNA methylation array comprising at least: a plurality of distinct locations, each location having at least one probe molecule comprising a nucleic acid sequence complementary to a plurality of CpG sites of a CHO cell, wherein the beadbased DNA methylation array is used in the method according to any aspect of the present invention.

[0056] Unless stated otherwise, all percentages (%) given are percentages by mass.

[0057] The examples adduced hereinafter describe the present invention by way of example, without any intention that the invention, the scope of application of which is apparent from the entirety of the description and the claims, be restricted to the embodiments specified in the examples.

[0058] BRIEF DESCRIPTION OF FIGURES

[0059] Figure 1 is a PCA plot for all CpG sites in the CHO cell of Example 1 .

[0060] Figure 2 is a PCA plot for Differentially Methylated Positions (DMPs) of Example 1 .

[0061] EXAMPLES

[0062] Example 1 :

[0063] Determination of CHO age in shaker flask cultures

[0064] CHO cell culture

[0065] Her83 CHO (A*Star BTI) cells were cultured in EX-CELL Advanced CHO Fed-batch medium (Sigma- Aldrich, cat. no. 14366C) supplemented with 6 mM L-glutamine. For the aging experiment, the cells were maintained in three shaker flasks (as biological replicates) for 42 days (12 passages). Passaging was carried out twice a week when viability was >90% and cell density was >2E+06 cells / ml. The cell pellets were collected during each passage to isolate genomic DNA.

[0066] DNA Extraction

[0067] DNA was extracted using the PureLink Genomic DNA Isolation Minikit kit (Invitrogen), including RNAase treatment following the manufacturer's instructions. DNA quantity was measured by PicoGreen assay and DNA quality is assessed via NanoDrop (Thermo Scientific) to ensure the A260 / 280 ratio is < 1 .8. A small amount of sample was then also analysed using automated electrophoresis on TapeStation (Agilent) to ensure each sample contained high molecular weight DNA.

[0068] Bisulfite Conversion and BeadChip Analysis

[0069] The genomic DNA samples were then subjected to bisulfite conversion using the EZ DNA Methylation- Gold™ Kit (Zymo Research). The methylation levels were then quantified using Evonik’s customized methylation BeadChip kits (Illumina) which can analyze over 60,000 methylation sites quantitatively across the genome at single-nucleotide resolution. After bisulfite conversion, samples were processed 202400021 Foreign Filing 10 through a three-day workflow including sample amplification, fragmentation, precipitation, hybridization to BeadChip and X-stain according to Infinium HD Methylation Assay (Illumina, Document # 15019519 v07), before being imaged on the iScan (Illumina) where intensity files for the computation of beta values were generated.

[0070] Data processing

[0071] The customized chip array data processing was performed in R version 4.1 .2 using sesame version 1 .14.2. DNA methylation level for each site was calculated as methylation p-value. Beta values were defined as methylated signal / (methylated signal + unmethylated signal). The SeSAMe pipeline (Zhou et al. 2018) was used to generate normalized p-values and for quality control. The pipeline first infers Infinium 1 channel, followed by dye Bias Correction, the Low intensity- based detection calling and making (based on p-value) was done with pOOBAH. Background subtraction based on normalexponential deconvolution using out-of-band probes noob (Triche et al. 2013) and optionally with extra bleed-through subtraction were also implemented. After obtaining the beta values, control probes were filtered out of the data frame. CpG sites with NA beta values were also removed from the data. This resulted in 26585 CpG sites remaining and the Principal Component Analysis (PCA) plot for these sites were plotted using the prcomp followed by autoplot functions. The resulting PCA plot is shown in Figure 1.

[0072] To obtain Differentially Methylated Positions (DMPs), beta values for the samples were extracted using the same method as above. Differential Methylation Positions (DMPs) among all days of culture samples were extracted using the dml function in sesame. DMPs in this analysis is defined as sites with FDR < 0.05 (BH method). This resulted in 7719 CpG sites (Table 1) and the PCA plot for these sites were plotted using the prcomp followed by autoplot functions. The resulting PCA plot is shown in Figure 2.

[0073] Table 1 : List of 7719 DMPs between different days in culture samples:

[0074] 202400021 Foreign Filing 11 202400021 Foreign Filing 12 202400021 Foreign Filing 13 202400021 Foreign Filing 14 202400021 Foreign Filing 15 202400021 Foreign Filing 16 202400021 Foreign Filing 17 202400021 Foreign Filing 18 202400021 Foreign Filing 19 202400021 Foreign Filing 20 202400021 Foreign Filing 21 202400021 Foreign Filing 22

Claims

202400021 Foreign Filing 23CLAIMS1 . An in vitro method for predicting the chronological age of a test Chinese Hamster Ovary (CHO) cell or population of test CHO cells, the method comprising the steps of:(a) determining the test methylation level of a set of specific CpG sites from genomic material obtained from the test CHO cell or from the population of test CHO cells from a CHO cell line using a DNA-methylation bead-based array;(b) comparing the test methylation level determined from step (a) with a reference methylation level of the same set of specific CpG sites from an age-correlated reference sample, thereby establishing the epigenetic age and predicting the chronological age of the test CHO cell or the population of test CHO cells wherein the set of specific CpG sites comprises at least 10%, preferably 30%, more preferably 50% of the CpG sites indicated in Table 1.

2. The method according to any one of the preceding claims, wherein the specific CpG sites from the test and reference methylation levels are from the CHO cell genome.

3. The method according to any one of the preceding claims, wherein the test CHO cell and / or the population of test CHO cells have a life span of up to 60 days.

4. A method of establishing an epigenetic clock for CHO cell, the method comprising :(a) identifying and determining the methylation levels of specific CpG sites within the genomic DNA obtained from a plurality of CHO cells and representing specific time points within the chronological lifespan of the CHO cells; and(b) correlating the CpG methylation levels of the CpG sites obtained in step (a) with chronological age using a penalized regression model.

5. The method according to claim 4, wherein the plurality of CHO cells in step (a) have representative ages ranging between 1 to 60 days.

6. The method according to either claim 4 or 5, wherein the specific CpG sites within the genomic DNA of the CHO cells are distributed in the CHO genome.

7. The method according to any one of the claims 4 to 6, wherein the methylation level of the CpG sites is determined using a DNA-methylation bead-based array.

8. A computer program loading into a memory of a computer, implementing the method of any one of claims 4 to 7.

9. A tangible, computer-readable medium comprising a computer-readable code that, when executed by a computer, causes the computer to perform operations comprising:202400021 Foreign Filing 24(a) receiving information corresponding to the methylation levels of specific CpG sites obtained from a plurality of CHO cells and representing specific time points within the chronological lifespan of the CHO cells; and(b) correlating the CpG methylation levels of the CpG sites of step (a) with chronological age using a penalized regression model.

10. A bead-based DNA methylation array comprising at least: a plurality of distinct locations, each location having at least one probe molecule comprising a nucleic acid sequence complementary to a plurality of CpG sites of a CHO cell, wherein the bead-based DNA methylation array is used in the method according to any one of the claims 1 to 3.11 . Use of a DNA-methylation bead-based array according to claim 10, for determining the chronological age of a test CHO cell or a population of test CHO cells from a CHO cell line.

12. Use of the method according to any one of the claims 1 to 3, for determining the chronological age of a test CHO cell or a population of test CHO cells from a CHO cell line.

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