Cho-k1 Cell Culture Medium

[Annette Fazzari]

Asubclone of the parental CHO cell line, which was derived from the ovary of an adult Chinese hamster. Cells require proline due to the absence of the gene for proline synthesis, the block in the biosynthetic chain lies in the step converting glutamic acid to glutamine gamma serialdehyde. They undergo morphological changes in response to cholera toxin.

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Recombinant therapeutic proteins were introduced >20 years ago and now generate >$99 billion in annual revenue from a broad range of products, including monoclonal antibodies, growth factors, hormones, blood factors, interferons and enzymes1. For these biopharmaceuticals, CHO-derived cell lines are the preferred host expression systems because of their advantages in producing complex therapeutics and manufacturing adaptability. CHO cells can be genetically manipulated and grown either as adherent cells or in suspension. Methods for cell transfection, gene amplification and clone selection in CHO cells are well characterized and widely used. Furthermore, CHO cells have an established history of regulatory approval for recombinant protein expression. Most importantly, these cells perform human-compatible, post-translational modifications (e.g., glycosylation), thereby improving therapeutic efficacy, protein longevity and reducing safety concerns. Various cell-line engineering strategies have been developed for CHO cells to enhance post-translational modifications, such as antibody glycosylation and protein sialylation2. As a result, CHO cell lines now play a dominant role in bioprocessing research and the development of therapeutic biopharmaceuticals, delivering up to several grams per liter of these products in highly optimized production processes3.

The genome sequences of CHO cell lines represent useful tools that have been unavailable to the bioprocessing community. Thus, applying genome-scale techniques to generate hyperproductive cell lines has been restricted to using expressed sequence tags (ESTs) and the potential of the 'omic technologies has not been fully realized4. To address this, we present a public draft genome sequence and comprehensive annotation of the ancestral CHO-K1 cell line. We investigate the CHO-K1 genome and transcriptome for insights into protein glycosylation and viral susceptibility because these processes affect the yield and quality of therapeutic protein production.

We note that the genomes of cell lines derived from CHO-K1 over the past few decades may contain large-scale rearrangements and that even clonal populations are known to diverge into heterogeneous subpopulations5,6. Thus, we anticipate that further analyses and sequencing studies with other clonal populations and cell lines will be required. Nevertheless, the dissemination of this ancestral CHO genome sequence should be a valuable public resource.

To assign scaffolds to chromosomes, we isolated and amplified individual chromosomes from single molecules using a microfluidic device (Online Methods)9. Each chromosome preparation was amplified, barcoded and sequenced on an Illumina HiSeq(2000) (2 100 bp reads). The reads from each chromosome preparation were aligned to the assembled scaffolds and the frequency of paired-end reads aligning from each chromosome preparation was computed and normalized. Metrics derived from the normalized frequencies were used for assigning scaffolds to a particular chromosome preparation (Supplementary Notes). All of the longest scaffolds that represent 50% of the assembly (top N50 scaffolds) had chromosome reads mapping to them; 68% of the top N50 scaffolds could be unambiguously mapped to unique chromosome preparations (Table 1).

Different chromosomal counts have been reported for the CHO-K1 karyotype10, presumably due to its genomic instability. To find evidence of multiple or duplicate chromosomes across the 22 sample preparations, we used the frequency of the paired-end reads aligning from each chromosome preparation to compute the correlation between the N50 scaffolds (Supplementary Notes). Scaffolds that are from the same chromosome will be highly correlated owing to physical connection. Clustering of this correlation matrix revealed 21 large, discrete noninteracting blocks, which can be interpreted as the chromosomes containing the respective scaffolds (Fig. 1a and Supplementary Notes). Consistent with this result, classical karyotyping found 21 chromosomes in CHO-K1 (Fig. 1b and Online Methods).

(a) Chromosomal preparations from CHO-K1 were sequenced and the reads were aligned to the scaffolds. For each of the N50 scaffolds, a vector was used to represent the read alignments in the 22 preparations. Using this metric, a correlation matrix was generated between all the N50 scaffolds. Upon clustering the matrix, 21 clusters of highly correlated scaffolds emerged, suggesting that the scaffolds are associated with 21 chromosomes in CHO-K1. (b) Classical karyotyping of CHO-K1 reveals 21 chromosomes.

For each GOslim biological process category, the fraction of all GO terms in that category is shown for human, mouse, rat and CHO genomes. GOslim classes that are significantly enriched and show the highest and lowest coverage of human and mouse genes in the CHO genome are highlighted in red (*) and green (**), respectively. P value cutoff and coverage in human and mouse were used to determine significance.

The therapeutic proteins secreted by CHO cells often include post-translational modifications including N- or O-linked glycosylation. For some of these proteins, differential glycosylation can substantially affect functional activity and/or in vivo circulatory half-life18. Furthermore, such modifications can induce immune responses if they differ from native human glycans. Therefore a genome-scale assessment of CHO glycosylation is important in the understanding of CHO-derived glycoprotein quality.

Out of 300 human genes associated with glycan synthesis and degradation, only three genes (ALG13, CHST7 and CHST13) lack homologs in the CHO-K1 genome (Supplementary Table 13). As almost all glycosylation genes are found in CHO-K1, we expect that the expression and activities of these gene products are more important than their presence in the genome for determining the diversity of glycan structures on protein products in CHO. In RNA-Seq data for exponentially growing CHO-K1 cells, we detected about half of the predicted glycosylation genes (Fig. 3a). N-glycan transferases, mannosyltransferases, sugar-nucleotide synthesis genes and hyaluronoglucosaminidases were enriched for expression or completely expressed. These classes are critical for constructing the core parts of the glycan chains or dictating glycan localization. The significantly depleted classes (P N-acetylgalactosamine (GalNAc) transferases.

CHO cell lines often produce glycoforms similar to human glycans. However, CHO cells do not produce the bisecting GlcNAc branch, which is found on about 10% of human IgG glycoforms19. The CHO LEC10 cell line remedies this with a gain-of-function mutation that induces MGAT3 expression, coding for GnTIII/GlcNAcTIII, which adds the bisecting GlcNAc residue20. The fact that the LEC10 cell line gains this functionality suggests that the gene is present in the parent strain. Consistent with this, a homolog to this gene is found in the CHO-K1 genome but is not expressed (Fig. 3b,i).

Glycan sialylation can have an impact on the function, longevity and immunogenic effects of proteins. Sialic acids often are the terminal sugar on N-linked glycans. These sugars may increase the lifespan of glycoproteins in the circulatory system by covering the penultimate galactose, which otherwise would bind to the hepatocyte asialoglycoprotein receptor and subsequently be degraded24. The CHO-K1 genome has homologs to all six human ST3Gal enzymes, which form α(2,3) linkages of sialic acid to galactose. Moreover, these genes are expressed as well (Fig. 3b,iii). Although homologs also exist for the human ST6Gal genes, which catalyze α(2,6) linkages of sialic acid to galactose, the transcriptome data show no evidence for ST6Gal gene expression. This is consistent with the observation that CHO cells do not normally show ST6Gal activity19, whereas terminal α(2,3)-linked sialic acid residues are abundant.

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