Toward product attribute control: developments from genome sequencing

doi:10.1016/j.copbio.2014.05.001

Current Opinion in Biotechnology

Volume 30, December 2014, Pages 40-44

https://doi.org/10.1016/j.copbio.2014.05.001 Get rights and content

Highlights

•
Genomes can enable product attribute control in the future.
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Genomes of Chinese hamster and CHO cells have been sequenced and assembled.
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Chromosomal/genomic instability is a major challenge in CHO engineering.
•
A community-wide reference genome is important to have.

Chinese hamster ovary (CHO) cells are important hosts for the production of therapeutic proteins. Recent genome sequencing studies provide an initial baseline of information useful for understanding cell line performance in terms of product quality attributes. However, the lack of a well-established reference genome together with concerns about genome stability have not yet permitted the community to define the detailed relationship between the genome and cell line performance. Emerging efforts to define a new reference genome, together with new data on genome stability, herald an era where cell line's with defined genomes can be combined with defined process parameters to yield product quality attribute control.

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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Foreseeing the increasing complexity in pharmaceutical products and processes, FDA published a guideline encouraging the biopharma industry to use process analytical technology (PAT) as “a framework for innovative pharmaceutical development, manufacturing, and quality assurance” as early as 2004.11 It calls for shifting the testing, monitoring and control from the end-of-process to in-process, in order to gain deeper understanding of the process and the product as well as their potential correlations.12–15 As a result, a number of PAT tools have been developed and widely applied to real-time monitoring of bioreactor cell culture processes, many of which are in the form of sensors or spectroscopic probes, such as pH, temperature, dissolved oxygen (DO), near infrared (NIR), mid infrared (MIR) and Raman.16–20
In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.
Simultaneous Monitoring and Comparison of Multiple Product Quality Attributes for Cell Culture Processes at Different Scales Using a LC/MS/MS Based Multi-Attribute Method
2020, Journal of Pharmaceutical Sciences
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As outlined by the FDA,18,19 QbD principles enable biopharmaceutical companies to better understand their products and hence design processes that will ensure consistent product quality instead of solely relying on final product testing.20,21 One of the key emphasis of the QbD approach is to shift the testing, monitoring and controlling from the end-of-process to in-process testing to gain deeper understanding of both the process and the product while exploring their correlations.22–25 While the CQAs are the important elements within the QbD framework, the critical process parameters’ (CPPs) are the levers that allow adjustment and control of the process to yield consistent products with the desired CQAs profile.26–29
In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.
The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells
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Therefore improvement of CHO-based bioprocess performance and product characteristics of biopharmaceutics, e.g. by molecular engineering approaches, is of great interest to academia and industry (Baik and Lee, 2014). An essential basis for this purpose is the CHO K1 genomic sequence and transcriptome data that became recently available and will enable a deeper understanding of genotype–phenotype relationships in terms of product quality attributes (Baik and Lee, 2014; Lewis et al., 2013; Rupp et al., 2014). A missing link between these cellular levels in CHO cells, however, is the yet unknown CHO cell epigenome.
Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype–phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.
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What is missing is how to efficiently use this flood of data for bioprocess optimization, manipulating pathways associated with high productivity and/or desired PTMs. Links between process conditions, intracellular dynamics and CQAs are starting to be established, and process or cell engineering strategies implemented to maximize product yields and maintain consistent quality [4•,43•,46,51,52]. To fill the gaps in process knowledge, hybrid modelling frameworks, combining first principles with non-parametric models such as multivariate data analysis can be explored [53,54].
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A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level
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View full text

Toward product attribute control: developments from genome sequencing

Highlights

Graphical abstract

Introduction

Section snippets

Genome sequencing of CHO cells

Chromosomal rearrangements and genomic instability

Future applications

Conclusion

References and recommended reading

Acknowledgements

Biotechnol Adv

Nature

Production of recombinant protein therapeutics in cultivated mammalian cells

Nat Biotechnol

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

Nat Biotechnol

The race for the $1000 genome

Science

A decade's perspective on DNA sequencing technology

Nature

Next-generation DNA sequencing methods

Annu Rev Genomics Hum Genet

EST sequencing for gene discovery in Chinese hamster ovary cells

Biotechnol Bioeng

The complete nucleotide sequence of Chinese hamster (Cricetulus griseus) mitochondrial DNA

DNA Seq

Genomic sequencing and analysis of a Chinese hamster ovary cell line using Illumina sequencing technology

BMC Genomics

Chinese hamster genome sequenced from sorted chromosomes

Nat Biotechnol

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

Nat Biotechnol

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines

Biochem Biophys Res Commun

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Biotechnol Bioeng

Chinese hamster genome database: an online resource for the CHO community at www.CHOgenome.org

Biotechnol Bioeng

The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-band, C-band, and autoradiographic analyses

Chromosoma

Relative impact of nucleotide and copy number variation on gene expression phenotypes

Science

Fusion genes and rearranged genes as a linear function of chromosome aberrations in cancer

Nat Genet

Cell-by-cell dissection of gene expression and chromosomal interactions reveals consequences of nuclear reorganization

PLoS Biol