Introduction: Cell-Line Engineering and Development at BPI West

Cheryl Scott

May 24, 2022

14 Min Read


Critical to the manufacturing of biotherapeutics is development of robust and stable cell lines that express high-quality products in large quantities. Chinese hamster ovary (CHO) cells are the “workhorse” expression host for manufacturing glycoprotein-based therapeutics — from antibodies to enzymes and hormones and beyond — making them the subject of most industry discussion regarding both cell-line development (CLD) and engineering. Most methods, technologies, and strategies apply regardless of the type of protein to be expressed or the type of cells expressing it.

On 15–17 March 2022, Informa Connect Life Sciences presented the main BioProcess International US West Conference and Exhibition program live in San Diego, CA. That was followed the next week with online-only roundtable discussions on key topics from the in-person sessions. Here I report on the main themes and some key presentations from the track that addressed the latest developments in CLD. What stood out most to me was how the industry is finding increasingly nuanced approaches toward meeting its goals of process optimization and intensification, speed and efficiency in development, and balancing clonality and product quality.

Tuesday, 15 March 2022
Optimization and Intensification: Bringing new biologics to market requires a “fail-fast” approach incorporating flexibility and risk management that enables companies to move the most promising molecules into (and through) clinical testing as soon as possible. Many modern product candidates now target rare-disease and other niche markets, which intensifies cost pressures on their development and manufacturing. Complex biologics also demand stringent process controls to ensure good yields of consistently high-quality products. Traditional fed-batch platforms designed for production of “blockbuster” biologics often struggle to meet the requirements of modern development. So the industry is moving toward intensified process methods, and CLD groups find themselves faced with the challenge of optimizing cell lines for such production modes.

In “Rapid High-Throughput CHOZN Stable Cell Line Development Platform,” Whitney Liu (senior scientist at Bristol Myers Squibb, BMS) described a high-throughput transposon-based CLD system to produce stable pools of CHO cells — modified by a zinc-finger–nuclease method — that could express proteins to required quality at high titers. The new platform, she said, enables BMS to design and screen as many vectors and vector combinations as required to help select lead cell lines for high productivity and quality. It also shortens expression timelines by a month, enabling progress from DNA transfection to large-scale bulk pool production in seven to 10 weeks. The company has applied this technology to monoclonal antibody (MAb) projects and to even more complex biotherapeutic molecules.

Liu also reported on work in progress to adapt the FectoPRO-mediated transfection process from Polyplus Transfection to a transient expression method for rapid production of high-titer and high-quality test materials. Based on high-throughput chemical transfection, this platform also uses ATUM’s Leap-In transposases with CHOZN cells in a 24- or 96-well format. For this work, the CLD group has integrated the following:

• high-throughput imaging and cell counting to monitor stable-pool recovery
• an Octet assay (Sartorius), a Zephyr G3 SPE workstation (Perkin Elmer), and analytical size-exclusion chromatography (SEC) for titer evaluation and quality assessment
• Solentim’s verified in situ plate seeding (VIPS) and Ambr 15 bioreactors from Sartorius for single-cell cloning and bioprocess development.

In “Cell Line Development and Plasmid Optimization to Improve AAV Titers,” Ping Liu (associate director of CLD at Regenxbio) introduced her company’s efforts to adapt human embryonic kidney (HEK293) cells from adherent to suspension cultures. The CLD group also has improved adenoassociated-virus (AAV) productivity of those cell lines through multiple cloning efforts. “To improve AAV titers, we made a sequential modification of our helper plasmid,” Liu said. “By combining the new cell lines and new helper plasmids, we improved our overall transient yield >20-fold while maintaining the product quality.”

In “End-to-End Platform Optimization for Gain-, Loss-, and Modification-of-Function Genetic Engineering,” Benjamin Haley (senior principal scientist at Genentech/Roche) described optimized platforms for gene editing and functional genomics of different cell types. He said that although rapid evolution of gene-editing tools has made it possible to suppress, activate, and mutate genomes precisely “at unprecedented scale,” the full potential for such technologies has yet to be realized. See page 6 for my discussion with Haley on this topic.

Wednesday, 16 March 2022
Speed and Efficiency: Talks on Wednesday focused on improving speed and efficiency in both cell-line engineering and development.

In “Use of Stable CHO Pools to Reduce Time to Clinic,” Yves Durocher (head of mammalian cell expression at the National Research Council of Canada’s Human Health Therapeutics Research Center) presented results from his laboratory’s efforts to use stable CHO cell pools to produce SARS-CoV-2 trimeric spike proteins as a vaccine candidate. Here, speed was the most important parameter, and Durocher explained that “the current pandemic has contributed to a broader acceptance from the regulatory authorities for using stable CHO pools to accelerate clinical evaluation of potentially life-saving drugs.”

As Whitney Liu had done the previous day, he showed that clonality is not necessarily the key to product quality and consistency. Along with transient gene expression (TGE), the stable-pool approach is an alternative to beginning CLD with a focus on clonality. Stable pools have demonstrated good productivity (with some pools matching the expression titers of stable clones), robustness, and product quality (again, equivalent to that of CHO clones).

Durocher quoted an opinion paper from 2018 (1): “The greatly reduced time and expense associated with stable pools and TGE, combined with the improved understanding of the heterogeneity of CHO clone-derived populations, strongly supports the careful consideration of these alternative methods for achieving fast, economical, and safe evaluation of biotherapeutics in [toxicology] and early phase clinical trials.” He also pointed to a recently published report from Merck authors who used a transposase-enzyme method to generate a CHO pool that could produce neutralizing antibodies to the SARS-CoV-2 spike protein at high levels (2). That team took a mere 4.5 months to progress from a DNA sequence to 2,000-L scale production of phase 1 clinical materials at 1.3 g/L. A team from WuXi Biologics published its own similar success in 2021, but with a three-month timeline (3). And a Chinese national laboratory team just published similar results this year — also demonstrating equivalence of results with those of single-cloned CHO cells (4).

Pointing out that the viral spike protein is “notably difficult” to produce, Durocher then described his laboratory’s inducible-expression method for doing so at nearly 1 g/L levels and showed how the pool then could be used to produce clones for further development. “Stable CHO pool platforms are likely to become more and more integrated to support manufacturing for early clinical studies in post-pandemic times,” he concluded.

In “Technology and Data Strategies for Fast, Quality-Driven Cell Line Selection,” Paul Rousseau (engineer III at Biogen) showed how his company leveraged historical data and high-throughput techniques to develop a CLD platform that works for all cell lines and therapeutic candidates. “Clinical projects aim to generate the highest-titer clonal cell lines meeting product quality specifications on the fastest possible timelines,” he said, echoing Durocher. “This process is demanding for well-behaved MAbs but further complicated when considering multiple or novel protein candidates.”

In “Development of Biotherapeutic Cell Lines for Continuous Manufacturing,” Ying Huang (senior principal scientist in biologics process research and development at Merck) discussed CLD process optimization to support a perfusion-culture platform. Such process-intensive technologies increasingly are used to improve biomanufacturing outputs. That puts even more emphasis on cell-specific productivity and robustness in development of cell lines for such high-intensity processes. Merck’s “clone by design” process made use of the stable-pool approach Durocher had described earlier along with mock perfusion, mRNA assays, and Ambr 250 cell culture (Sartorius). Those integrated assays helped the team identify top-producing clones that work well for continuous manufacturing, reaching ~2 g/L/day productivity at the 2-L perfusion bioreactor scale.

The second day ended with a keynote presentation: “Monoclonality and Sequence Variants of Production Cell Lines: Have We Reached a Sweet Spot?” In it, Sampath Kumar (senior director and head of cell line development in biologics process development at Takeda Pharmaceuticals) showed how CLD groups must address the inevitability of sequence variation in an environment so focused on monoclonality of cell lines. Next-generation sequencing (NGS) and peptide mapping are valuable tools for eliminating sequence variants, but the industry faces a dilemma regarding investments in doing so and the need to balance what is ideal against what is necessary.

Since 2014, regulators have emphasized clonality in CLD, with pushback from the industry even as technologies were adopted to meet those expectations. In 2016, well-respected experts from major companies pointed out that clonality is “only one of many factors affecting product quality” and suggested that consistency and quality of drug substance should be the main emphasis (5). “Referring to a production cell line as a ‘clone’ or to the ‘clonality’ of a cell bank is misleading,” they said, because of the inherent genetic variation that occurs when cells divide. Those concerns were echoed a year later by noted Swiss scientist Florian Wurm (6). Studies showed that clonally derived cell lines inevitably become heterogeneous in culture and that clonal derivation does not ensure either expression stability or consistency of performance. “A single cell takes more than 100 generations to reach production scale,” Kumar said. “Therefore, the industry needs to demonstrate product quality and process consistency.”

Regulators have responded to those concerns, and Kumar cited a 2019 publication in which authors from the US Food and Drug Administration (FDA) agreed that product quality should be paramount (7). But they also showed how documented clonality remains important. Kumar said that a consensus is emerging in which the industry adopts methods to validate clonality while accepting that clonally derived cell lines quickly turn heterogeneous — leading to a semantic change from “monoclonal” to “clonally derived” cell lines. Although clonal derivation is expected, similar emphasis must be placed on product quality and process consistency. The emergence of sequence-variant analysis methods is helping companies monitor and document the impacts of genetic variation over time, especially with the high-throughput capabilities of NGS. Kumar highlighted NGS and peptide mapping as complementary and orthogonal technologies.

Thursday, 17 March 2022
Genetics and Characterization: The third day’s keynote presentation, “Repairing the Repairers: Stabilizing the CHO Genome By Editing DNA Repair,” took up where Kumar had left off. Nathan Lewis (associate professor in pediatrics at the University of California, San Diego) reminded attendees that the CHO genome is known for its instability. But he explained that the sources of that instability can be addressed. “We have discovered a subset of DNA-damage repair genes that harbor mutations, and by remedying these mutations, DNA damage repair is improved, and expression stability can be extended.” Single-nucleotide polymorphism (SNP) reversal and overexpression of repaired genes can be used to restore necessary functions and produce more stable clones.

The final keynote presentation of the CLD track, “Conditional Gene Expression Systems (in CHO Cells) Using Precise, Customizable Multi-Input Biomarker Sensing,” expanded on Lewis’s ideas. Ron Weiss (professor at the Synthetic Biology Laboratory of the Massachusetts Institute of Technology) showed how researchers are pushing the envelope of genetic engineering through assembly and delivery of genetic circuits. With his information-technology background, Weiss is a noted pioneer in synthetic biology, which he described as a way to program cells “like we program computers.” He illustrated progress in increasingly sophisticated systems for live-cell sensing, artificial neuronal networks, and cell lines for site-specific, scalable expression with precisely controlled posttranslational modifications.

In “Lessons Learned from a Multiyear CHO -Omics Initiative,” Hooman Hefzi (scientist II at Biogen) reported on a platform his company created by integrating years of collected data. “The plasticity that has made CHO cells so useful for generating life-saving therapeutics also has made it difficult to tease apart actionable information from targeted, small-scale ‘omics’ efforts” he said. The “-omics initiative” at Biogen was meant to determine how modern genomics, proteomics, metabolomics, and related technologies could be integrated into routine process development activities.

For example, Hefzi showed how RNA sequencing (transcriptomics) could enable comparisons across programs, clones, and culture time points to discover process trends and track targeted metabolites. Ultimately, these efforts have increased the amount of meaningful information available to cell-line and process developers, enabling them to apply that understanding to solve problems and improve cell lines. He concluded by expressing his hope for implementing “actionable -omics” earlier in clone assessment for better results in the future.

In “Improving the Efficacy and Productivity of Recombinant Biologics Produced in CHO Cells By Genome Editing,” Song Zhiwei (senior principal investigator in bioprocessing technology at Singapore’s Agency for Science, Technology, and Research) showed how his laboratory inactivated the glutamine synthetase (GS) gene in CHO cells through genome editing. The researchers evaluated a congenital GS mutation that causes glutamine deficiency in humans as an attenuated selection marker for CHO CLD, creating a panel of new GS mutants as potential selection markers. Using the resulting selection system, the team generated CHO cell lines to produce afucosylated versions of three different MAbs.

In “Cell Line Characterization for IgM Molecules in Cell Line Development Process,” Christina Tsai (senior scientist at IGM Biosciences) pointed to the need for productivity and product-quality assessments in cell-line characterization during CLD for biologics production. She illustrated the special concerns that very large IgM antibody molecules bring to the game. See page 12 for an in-depth discussion.

In “Developability: Early Preparation for a Successful Product,” Sarah Auclair (scientist at Sanofi) highlighted the importance of developability assessments in evaluating and “derisking” potential biotherapeutics very early on in their campaigns. “We seek to understand [a protein’s] physical and chemical stability as well as to determine its compatibility with an established manufacturing process and proposed administration method.” Although incompatibilities can be mitigated for otherwise valuable product candidates, she cautioned that doing so brings timeline delays and ultimately increases cost of goods (CoG).

With case studies, she illustrated Sanofi’s strategy to test manufacturability and device compatibility early on. In one example, early assessment of a product’s sensitivity to reducing conditions helped the CLD team determine whether bioreactor modifications would be required. In another, a viral inactivation platform was tested by subjecting a product to a stepwise acid-sensitivity assay, enabling the team to provide guidance on its pH limits in cell culture.

Tuesday, 22 March 2022
The next week, a live, online-only interactive panel discussion followed the main BPI West program. Panelists were speakers from the week before — Sampath Kumar of Takeda Pharmaceuticals, Whitney Liu of BMS, moderator Nathan Lewis of UCSD, and Christina Tsai of IGM Biosciences — with the addition of Valentina Ciccarone (director of gene expression at MacroGenics, Inc.). They addressed rapid development of high-quality clones and finding a balance between speed and optimization.

Kumar pointed out that cloning and clone screening take up most of the time required for CLD, even with the help of new technologies and instrumentation. Ciccarone highlighted the value of screening not only for expression titer but also for product quality. Automated, microplate-scale suspension-culture systems are showing good promise for that work. “There is no shortcut,” said Tsai, emphasizing the importance of identifying CQAs as early as possible.

Kumar pointed out the complexity of enzymes and blood factors when it comes to measuring and monitoring product quality: “We can’t simply go by mass, but [rather] by activity.” Lewis agreed, noting a case in which glycosylation was critical to both activity and half-life of a protein in development. Ciccarone explained that even 1-mL scaled-down culture volumes could be enough to produce material for such analyses. Liu highlighted the value of NGS for clonality and cell-line analysis. And site-specific gene integration was touted as one of many up-and-coming technologies that are likely to aid in future CLD.

“We like how unstable CHO cells are,” Lewis remarked, because it makes them adaptable to different conditions and requirements. The challenge comes with locking in desirable features throughout CLD and seed-train development. That’s where -omics and DNA damage-repair methods ultimately should come into use.

BPI West will return to the San Diego Convention Center during the week of 27 February through 3 March 2023. I hope to see you there.

1 Stuible M, et al. Beyond Preclinical Research: Production of CHO-Derived Biotherapeutics for Toxicology and Early Phase Trials By Transient Gene Expression or Stable Pools. Curr. Opin. Chem. Eng. 22, 2018: 145–151;

2 Agostinetto R, et al. Rapid CGMP Manufacturing of COVID-19 Monoclonal Antibody Using Stable CHO Cell Pools. Biotechnol. Bioeng. 119(2) 2021: 663–666;

3 Zhang Z, et al. Reshaping Cell Line Development and CMC Strategy for Fast Responses to Pandemic Outbreak. Biotechnol. Prog. 37(5) 2021: e3186;

4 Xu G, et al. Quality Comparability Assessment of a SARS-CoV-2 Neutralizing Antibody Across Transient, Mini-Pool-Derived and Single-Clone CHO Cells. mAbs 14(1) 2022:

5 Frye C, et al. Industry View on the Relative Importance of “Clonality” of Biopharmaceutical-Producing Cell Lines. Biologicals 44, 2016: 117–122;

6 Wurm FM, Wurm MJ. Cloning of CHO Cells, Productivity, and Genetic Stability — A Discussion. Processes 5(2) 2017: 20;

7 Welch JT, Arden NS. Considering “Clonality”: A Regulatory Perspective on the Importance of Clonal Derivation of Mammalian Cell Banks in Biopharmaceutical
Development. Biologicals 62, 2019: 16–21;

Cheryl Scott is cofounder and senior technical editor of BioProcess
International, part of Informa Connect; 1-212-600-3429; cheryl.[email protected].

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