Toward product attribute control: developments from genome sequencing
Graphical abstract
Introduction
Chinese hamster ovary (CHO) cells have emerged as the key platform host for the production of therapeutic proteins. As a result, there is community-wide interest in ways to improve and control CHO cell characteristics as a host for heterologous protein expression. In particular, there has been effort invested on enhancing productivity via cell line development, genetic engineering, metabolic engineering, and bioprocess engineering [1, 2] and there is now a growing interest in understanding how to engineer CHO cells and their growth to help define product quality attributes. Well-defined product attribute control will ultimately arise from a detailed understanding of the underlying cell biology which can provide a relationship between genotype (sequence, annotation, and gene expression levels) and quality attributes of the product (e.g. glycosylation). However, unlike other important genomes, the CHO genome has only recently been sequenced [3]. This development has been enabled by new technologies which have significantly reduced the cost required to sequence mammalian genomes. For example, it had cost approximately $300 million to sequence the human genome in 2001 [4].
The advent of so-called second-generation sequencing (SGS) technology enabled relatively low-cost, high-throughput sequencing [4, 5]. While there are different SGS technologies, most are characterized by massively parallel sequencing (millions of concurrent reactions) and generation of DNA libraries without vector-based cloning steps (for a detailed review, see [6]). The rates of data generation, compilation, and processing have advanced rapidly with developments in sequencer technologies, computing power, and assembly algorithms [5] which heralds the possibility of linking genome sequence to host cell characteristics including the range of possible product quality attributes. Looking forward, emerging technologies will provide insights not only on the relationship between genome and host cell, but also on epigenetic changes and their impact on cell behavior.
In this review, we summarize recent developments in CHO (and Chinese hamster) genomics studies and the emerging understanding of distinct characteristics of the CHO (and Chinese hamster) genome. We will also discuss future applications of CHO genomics toward bioprocess engineering but limit our discussion to the genome.
Section snippets
Genome sequencing of CHO cells
Traditionally (before SGS), CHO DNA sequencing efforts were limited to a small set of genes (less than 700 sequences found in the GenBank database in 2004) individually isolated and sequenced by the traditional Sanger method [7]. In 2005, Wlaschin et al. sequenced expressed sequence tags (ESTs) from a cDNA library of the CHO DXB11 cell line to create a CHO specific cDNA microarray [7]. The sequencing of 4219 ESTs yielded 2602 unique sequence assemblies, of which 76% were successfully annotated
Chromosomal rearrangements and genomic instability
One feature of CHO cells is the significant number of chromosomal rearrangements among different cell lines — rearrangements that can occur over time or during cell line development within the same cell line [12, 13••, 16] and contribute to issues with cell line (in)stability. While sequence variations, such as SNVs and small insertions/deletions, may affect gene expression (by changing the sequences of mRNAs and proteins), larger structural variations can impact gene expression by varying gene
Future applications
Moving forward, there is the immediate need by the community to establish a community-wide reference genome that can serve as the basis for future studies of the relationship between the genome and cell line properties. Once a baseline reference genome is established it is important for the community to define genes and the regulation of genes important for product attribute control. For example, genome-scale reconstruction models can help define cell line properties and other mathematical
Conclusion
The availability of the CHO and Chinese hamster genomes is now opening the door to functional gene annotation, comparative genomics, and gene expression analysis of many production cell lines which will ultimately permit one to define product quality attributes of interest. However, there is a need for further refinement of existing reference genomes to facilitate a better understanding of the relationship between the genome and cell line performance.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors would like to thank the National Science Foundation for financial support and Leila Choe for important comments.
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