Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines.

Cell line development for biomanufacturing processes: recent advances and an outlook.

At the core of a biomanufacturing process for recombinant proteins is the production cell line. It influences the productivity and product quality. Its characteristics also dictate process development, as the process is optimized to complement the producing cell to achieve the target productivity and quality. Advances in the past decade, from vector design to cell line screening, have greatly expanded our capability to attain producing cell lines with certain desired traits. Increasing availability of genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development. These developments are poised to help biosimilar manufacturing, which requires targeting pre-defined product quality attributes, e.g., glycoform, to match the innovator's range. This review summarizes recent advances and discusses future possibilities in this area.

Global insights into the Chinese hamster and CHO cell transcriptomes.

Transcriptomics is increasingly being used on Chinese hamster ovary (CHO) cells to unveil physiological insights related to their performance during production processes. The rich transcriptome data can be exploited to provide impetus for systems investigation such as modeling the central carbon metabolism or glycosylation pathways, or even building genome-scale models. To harness the power of transcriptome assays, we assembled and annotated a set of RNA-Seq data from multiple CHO cell lines and Chinese hamster tissues, and constructed a DNA microarray. The identity of genes involved in major functional pathways and their transcript levels generated in this study will serve as a reference for future studies employing kinetic models. In particular, the data on glycolysis and glycosylation pathways indicate that the variability of gene expression level among different cell lines and tissues may contribute to their differences in metabolism and glycosylation patterns. Thereby, these insights can potentially lead to opportunities for cell engineering. This repertoire of transcriptome data also enables the identification of potential sequence variants in cell lines and allows tracing of cell lineages. Overall the study is an illustration of the potential benefit of RNA-Seq that is yet to be exploited.

Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells.

Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

Exploring the transcriptome space of a recombinant BHK cell line through next generation sequencing.

Baby Hamster Kidney (BHK) cell lines are used in the production of veterinary vaccines and recombinant proteins. To facilitate transcriptome analysis of BHK cell lines, we embarked on an effort to sequence, assemble, and annotate transcript sequences from a recombinant BHK cell line and Syrian hamster liver and brain. RNA-seq data were supplemented with 6,170 Sanger ESTs from parental and recombinant BHK lines to generate 221,583 contigs. Annotation by homology to other species, primarily mouse, yielded more than 15,000 unique Ensembl mouse gene IDs with high coverage of KEGG canonical pathways. High coverage of enzymes and isoforms was seen for cell metabolism and N-glycosylation pathways, areas of highest interest for biopharmaceutical production. With the high sequencing depth in RNA-seq data, we set out to identify single-nucleotide variants in the transcripts. A majority of the high-confidence variants detected in both hamster tissue libraries occurred at a frequency of 50%, indicating their origin as heterozygous germline variants. In contrast, the cell line libraries' variants showed a wide range of occurrence frequency, indicating the presence of a heterogeneous population in cultured cells. The extremely high coverage of transcripts of highly abundant genes in RNA-seq enabled us to identify low-frequency variants. Experimental verification through Sanger sequencing confirmed the presence of two variants in the cDNA of a highly expressed gene in the BHK cell line. Furthermore, we detected seven potential missense mutations in the genes of the growth signaling pathways that may have arisen during the cell line derivation process. The development and characterization of a BHK reference transcriptome will facilitate future efforts to understand, monitor, and manipulate BHK cells. Our study on sequencing variants is crucial for improved understanding of the errors inherent in high-throughput sequencing and to increase the accuracy of variant calling in BHK or other systems.

Conserved microRNAs in Chinese hamster ovary cell lines.

MicroRNAs (miRNAs), a class of short (20-24 nt) non-coding RNAs that direct post-transcriptional repression of messenger RNAs, increasingly have been shown to play a key role in regulating cellular physiology. We investigated the prevalence of miRNAs in Chinese hamster ovary (CHO) cells by high-throughput sequencing. Six cDNA libraries of small RNAs from four CHO cell lines were constructed and sequenced by Illumina sequencing. Three hundred fifty distinct miRNA and miRNA* sequences were identified through homology with other species, including mouse, rat, and human. While the majority of the identified miRNAs appear to be expressed ubiquitously, many miRNAs were found to have a wide range of expression levels between cell lines. The identification of these miRNAs will facilitate investigations of their contribution to the hyperproductivity trait.

Global assessment of protein turnover in recombinant antibody producing myeloma cells.

The global turnover rates of cellular proteins and the secretion rate of a recombinant immunoglobulin G (IgG) in a myeloma cell line, NS0, were determined using SILAC proteomic analysis. After complete labeling of cellular proteins with (13)C(6), (15)N(4)-arginine, cells were transferred to unlabeled medium and the decay of the labeled arginine in proteins was monitored during exponential cell growth. After PAGE separation and mass-spectrometric identification of proteins, those detected with high confidence over at least three time points were used for the determination of turnover rates. Among the 224 proteins quantified with a protein half-life, about 15% have a degradation rate constant lower than one-tenth of specific growth rate. For most proteins, the turnover rate is insignificant in its overall dynamics. Only 6.3% of proteins have a half-life shorter than the cell doubling time. For IgG secretion, both heavy and light chain molecules follow the same kinetic behavior with a half-life estimated to be 2h. The label decay curve appears to show a second region with very slow kinetics, raising the possibility of two populations of IgG molecules with different secretion characteristics.

Reaching the depth of the Chinese hamster ovary cell transcriptome.

The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to highly variable. This observation suggests that more challenges are yet to be resolved before direct sequencing can be used as a true quantitative measure of transcript level and for differential gene expression analysis. With the depth that high-throughput sequencing methods can reach, one can expect that the entire transcriptome of this industrially important organism will be decoded in the near future.

Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment.

Recombinant Chinese hamster ovary (CHO) cells selected for high productivity are capable of secreting immunoglobulin G (IgG) molecules at a level that rivals plasma cells in vivo. Following butyrate treatment at 33 degrees C, further increases in productivity are observed. To better understand the mechanisms by which this increased productivity is incurred, the transcriptional response of an antibody-producing cell line undergoing these treatments was investigated using oligo-DNA microarrays. Using distance calculations, more than 900 genes were identified as kinetically differentially expressed between the butyrate-treated 33 degrees C culture and the untreated culture. Furthermore, transcript levels of the heavy and light chain IgG genes increased following treatment. Using stable isotope labeling (SILAC), the secretion rate of IgG was investigated by tracking the decay of the isotope label upon switching to unlabeled medium. Both treated and untreated cultures exhibited very similar IgG secretion kinetics. In contrast, the intracellular IgG content was found to be elevated following treatment. This result suggests that increased productivity under treatment is attributable to elevated cellular secretory capacity, rather than shorter holding times in the secretory pathway. This hypothesis is further supported by the results of gene set enrichment analysis (GSEA), which revealed that elements of the secretory pathway, including Golgi apparatus, cytoskeleton protein binding and small GTPase-mediated signal transduction are enriched and thus may play a role in the increased recombinant protein production observed under butyrate treatment at 33 degrees C.

Second International Conference on Accelerating Biopharmaceutical Development: March 9-12, 2009, Coronado, CA, USA.

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme "Delivering cost-effective, robust processes and methods quickly and efficiently." The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.

Developing genomic platforms for Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used in recombinant protein production, yet despite their importance in bioprocessing, few genomic resources have been developed for this cell line. Over the past several years, we have made considerable progress in the development of genomic tools for CHO. Using Sanger-based sequencing technology, we have accrued a sequence repertoire of more than 68,000 expressed sequence tags (ESTs), representing more than 28,000 unique CHO transcripts. Using closely related species, we have functionally annotated this sequence set and have currently achieved significant representation in a number of functional classes, including some closely tied to recombinant protein production. This sequence repository has been used to design custom CHO Affymetrix arrays for transcriptome analysis. Illumina Solexa deep sequencing technology was also applied to study the CHO cell transcriptome and survey the identity and expression of small RNAs. These applications demonstrate the utility of genomic tools, and illustrate the applicability of emerging next-generation sequencing technologies.