April 15, 2017 (Vol. 37, No. 8)
For Real Gains, Try Engineering Better Cell Lines
Cell-line development has been an ongoing priority in the life sciences. With cell- and tissue-based assays rapidly replacing animal studies, researchers are increasingly reliant on cells that can demonstrate greater biological relevance. Similarly, therapeutic biotechnology is constantly seeking to improve production cells’ performance with respect to productivity and product quality.
Given that both research and industrial concerns are demanding better cells, it should come as no surprise that that cell-line development has healthy growth prospects. According to market research firm Global Market Insights, cell-line development should become a $7.5 billion market by 2023, up from $2.8 billion in 2015, reflecting an annual growth rate of 13%.
Such figures mean opportunity to market participants, who are already positioning themselves in the competitive cell-line development landscape. For example, in February, GE Healthcare entered a three-year collaboration with the Austrian Centre of Industrial Biotechnology for identifying new techniques for optimizing CHO cell-line performance. The collaboration’s initial emphasis is on CHO cell-line growth and expression.
In its original announcement of the deal, GE Healthcare noted that CHO cells had not traditionally been the object of optimization due to limitations of existing optimization technologies and a demanding regulatory climate.
The Austrian Centre of Industrial Biotechnology and GE Healthcare are introducing a cell-line engineering research collaboration to bring increased productivity to biomanufacturers. The goal of the three-year partnership is to explore and identify new tools and methods to modify and optimize CHO cell-line performance.
Improving CHO Cells
“The recent productivity gains are mainly due to optimization of media, feed, and processes plus improved expression vector and selection workflows,” comments Daniel Ivansson, staff research engineer, GE Healthcare. He adds that GE Healthcare intends to bring about future productivity gains through the “engineering and design of the cell itself.” The company is emphasizing the development of CHO starter cells with enhanced phenotypes.
Current optimization methods involve empirical, trial-and-error methods for selecting clones from suboptimal starter cell lines. Modern gene-editing and analysis tools have changed the picture to the point where more detailed characterization of cell performance is possible with improved starting cells. Transcriptomics, in particular, provides insights into the regulation of cellular performance.
“Among the tools we will employ are high-throughput formats for assessing product secretion and cellular growth of desired phenotypes,” Ivansson details. “We will also use RNA-Seq, global epigenetic analysis, genome sequencing, and metabolomics to characterize phenotypes. We will also use proteomics as needed.”
Difficult Proteins
Early in 2017, Selexis announced that it was applying its SUREtechnology Platform™ to cell lines for three Sanofi biotherapeutics: a “naked” antibody, a bispecific antibody, and a complex recombinant vaccine.
Selexis CEO Igor Fisch, Ph.D., explains how one platform cell-improvement technology can serve the production of such disparate products. He begins by noting that generating clinically and commercially viable therapeutic proteins involves well-known transfection, transcription, translation, and transport of products via the cell’s secretion pathways, whereby it is folded, modified, and secreted into the media.
“However, parameters for maximal secretion vary significantly between proteins,” he points out. “Transcriptional, metabolic, folding, post-translational modifications, and transport requirements can be quite different, even among monoclonal antibodies.”
By employing appropriate epigenetic elements, Selexis obtains high transcriptional levels of mRNA, thus bypassing the need for strong selection processes based on drugs such as methotrexate (MTX) or l-methionine sulfoximine (MSX).
Next, applying whole-genome sequencing to the company’s proprietary suspension-adapted CHO-K1 cell line with unique bioinformatics analysis tools, Selexis can address potential secretion issues for bispecifics, large multimeric proteins, or vaccines.
“Using these techniques,” Dr. Fisch explains, “we can engineer CHO-M to overcome expression issues by generating CHO-M libraries engineered in defined pathways to allow proper folding, glycosylation, and transport to the membrane for secretion.” Through this approach, Selexis has recently demonstrated a 30-fold increase in expression for a difficult-to-express vaccine protein.
Selexis also uses whole-genome sequencing for clonal assessment of cell lines, including the integrity of the transgene copies at all locations in the genome and the site of integration of the transgene in the host cell genome. This approach ensures that no bias exists in the clonality assessment, compared to mRNA or partially digested gDNA sequencing.
Selexis realized several years ago that novel therapeutic proteins would entail complex manufacturing. At the time, the company identified bottlenecks that affected secretion, folding, and productivity, particularly for difficult-to-express proteins. This was the justification for developing the SURE CHOM-Mplus Libraries.
Selexis was the first company, in collaboration with the University of Lausanne and the Swiss Institute of Bioinformatics, to sequence and annotate its CHO-K1 line. It used these data to improve the cell’s production characteristics across the transcription, translation, and secretion continuum.
Selexis works with a variety of cell lines, including CHO, BHK, HEK-293, B cell, and C2C12 cell lines.
Most recently, Selexis launched a novel metabolic selection method based on the co-expression of a vitamin B5 transporter. The method exploits the dependence on vitamin B5 evidenced by mammalian cells, which use the essential nutrient to sustain efficient energy production.
“This method yields smaller polyclonal cell populations and produces higher yields of the recombinant proteins,” asserts Dr. Fisch. And it can be used to identify and isolate cell lines that grow even when they produce recombinant proteins that are toxic for CHO-M. Cell lines that grow under such circumstances do so because of positive metabolic selection.
Selexis reports that it has used its SUREtechnology Platform to generate nearly 80 clonal research cell banks (RCBs) that have reached clinical and commercial manufacturing. The platform has made it possible to generate stable and high-performing manufacturing cell lines in approximately three months from the time of transfection, with productivity levels reaching 1–7 g/L for monoclonal antibodies. The company also asserts that its SUREdevelopment Process improves productivity and jump-starts the scale-up process for CMOs.
CHO Power to the People
All commercially available manufacturing-worthy CHO cell lines currently require some form of licensing. Terms vary, depending on the provider and the degree to which the cells have been manipulated.
Wild-type CHO lines adapted to suspension growth in animal component-free media are available for a relatively low initial fee, notes Jamie Freeman, Ph.D., product manager at Horizon Discovery, who adds that it is also his understanding that each time a product is filed under an IND, the provider receives an additional fee. Cells that have been manipulated to be null for the glutamine synthetase (GS) gene—which allows selection of high-expressing cells in media lacking glutamine (now pretty much industry-standard)—entail significant initial cost plus annual payments that rise with achieving product milestones.
“Therefore, almost all commercially available cell lines incur significant downstream costs,” explains Dr. Freeman. “Moreover, license agreements restrict users in modifications they can make to the cells through gene editing or vector manipulation, and in media optimization as well.”
Horizon Discovery addressed these issues through the release of its GS-knockout CHO-K1 cell line for a one-time fee with no downstream costs, and by encouraging further innovation in CHO cell-line development. “We are doing this by releasing the sequenced genome of our host cell line, which will be a significant resource,” says Dr. Freeman. Horizon Discovery has also provided its cells gratis to aid in the advancement of basic research.
“While we provide the cells with an expression vector and a recommended culturing protocol, we do not oblige their use,” informs Dr. Freeman. “Customers are free to explore different options that fit their specific processes.”
Horizon Discovery generates CHO-K1 GS Null cells using its proprietary gene-editing platform. The editing tool, rAAV, uses homologous recombination to create precise edits, from point mutations to whole exon knockouts. The company licenses rAAV as well as the rights to use it to engineer cells for bioproduction with no “reach through,” which ensures use unfettered by intellectual property issues.
Horizon Discovery uses the CRISPR/Cas9 gene-editing system to generate proof-of-concept edits in its cells. Once the company identifies targets with the greatest impact on desirable CHO cell-line improvements (for example, productivity or product quality), it uses rAAV to re-engineer the cells to provide a manufacturing-ready cell line.
Horizon Discovery is also applying these technologies to modify a range of pathways in parallel, to continually improve the biomanufacturing capabilities of its cells. “We’re redefining the gold standard in cell-line technology,” Dr. Freeman tells GEN. “We appreciate the complexity of biological systems, and we understand the need to modulate several regulatory pathways in parallel to achieve the phenotype we are aiming for.”
Examples include cells that remain 100% viable at harvest, thus improving product quality (or the cells could be cultured longer, resulting in higher titres but at the expense of degradation of product generated earlier in the batch0. “If this modification is paired with an edit such as knocking out secreted proteases or sialidases,” advises Dr. Freeman, “the resulting phenotype would be a synergy of the two, leading to increased titre with no compromise on quality.”
Horizon Discovery believes its minimally IP-encumbered CHO cell lines will help lower manufacturing costs by increasing clone productivity, decreasing variability in product consistency, and improving bioproduction efficiency. And as a business entity, Horizon Discovery recognizes the economic implications.
“We are adopting a disruptive commercial approach to established technology,” Dr. Freeman admits, “but future engineered lines will be truly disruptive technology.
“We are attempting to improve the CHO cell factory beyond a pure selection system by rewiring the metabolism of the cell to maximize protein expression and secretion, achieve consistent product quality, and reduce development time,” Dr Freeman continues. “Application of our CRISPR/Cas9 screening expertise to engineer product-specific solutions, particularly for difficult-to-manufacture proteins, will open commercially viable pathways for this class of formerly inaccessible targets.”
Analysis and Characterization
Engineered cell lines undergo constant evaluation during their development. Where genomics defines the success of transfection or gene editing, proteomic analysis uncovers a cell’s actual activity. For production cells, the obvious measure of success is productivity, but other quality- and productivity-related proteomic indicators can signal viability, growth characteristics, or product quality.
A proteomics platform has been developed by Biognosys. The platform, known as Hyper Reaction Monitoring (HRM-MS™), quantifies up to 9,000 proteins per sample, thus enabling monitoring of altered protein expression in genetically modified cells.
HRM is a label-free proteomics workflow based on data-independent acquisition (DIA). It uses high-resolution LC-MS/MS, most notably the Thermo Fisher Scientific Orbitrap platforms (QExactive or Orbitrap Fusion series) or the Sciex TripleTOF series. It seeks to answer two questions related to protein profiling of engineered cells. First, what happens to the proteome (for discovery proteomics)? Second, what happens to proteins of interest (targeted proteomics)?
“Discovery of unbiased proteomics aims to understand global proteomic dynamics in a cell, tissue, or organism,” states Stephan van Sint Fiet, Ph.D., chief commercial officer at Biognosys, “and is applied in biomarker research, drug and target discovery, pathway modeling, mechanism-of-action studies, and many other areas.”
HRM allows investigation of a cell line’s global expression pattern, and how expression changes during cell-line development and during the production of biotherapeutics. Global protein abundance is an important piece of data.
“However, one should keep in mind that most proteins rely on post-translational modifications (PTMs) like N-glycosylation for their stability and proper solubility,” clarifies Dr. van Sint Fiets. “PTMs containing peptides can be enriched and quantified on a global scale using HRM-MS.”
Protein folding is another quality attribute of interest. “To investigate folding,” says Dr. van Sint Fiets, “we have recently acquired an exclusive license that combines HRM-MS with technology that enables the global investigation of protein conformational changes in native cell lysates, with no need to purify the protein of interest.
Hyper Reaction Monitoring (HRM), a label-free discovery proteomics workflow developed by Biognosys, allows investigation of a cell line’s global expression pattern. HRM was used to carry out single-shot proteome profiling of HeLa, Jurkat, and HEK-293 cell lines and generate this heatmap, which identifies and quantifies thousands of proteins across the cell lines. Distinctive differences in proteome expression can be observed.
The Final Word?
Horizon Discovery’s Dr. Freeman notes that in the 30 years that therapeutic proteins have been produced in CHO cells, the only significant parental cell-line improvement with broad commercial uptake has been the single-gene GS knockout. Given Horizon Discovery’s expertise in this area, this represents a significant opportunity for further development of CHO lines through genome engineering, which has not yet been extensively utilized to optimize performance. But not all experts are sold on the idea that genetic manipulation can achieve tangible benefits in CHO cells.
Florian Wurm, Ph.D., emeritus professor at the Swiss Federal Institute of Technology and founder and CSO of the protein expression services company ExcellGene, has long maintained that improvements in upstream titers have resulted almost entirely from improved understanding of culture media and feeds. The problem with the CHO cell, as a substantial body of research demonstrates, is its inherent genetic instability and heterogeneity.
All cell lines undergo genetic changes over time that result in significant phenotypic differences. The CHO cell, moreover, undergoes more subtle genomic drifting whose effects may not readily be anticipated.
“Cell engineering by genomic interaction is a very tough issue in CHO, a cell host that is constantly changing,” comments Dr. Wurm. “This is why I am critical of the overstated expectations from genomic sequencing and its applications. At ExcellGene, we routinely generate 5–10 g/L using entirely unmodified CHO cells.”
