Development of robust and stable cell lines that express high-quality products in large quantities is a critical first step in bioprocess development. As the “workhorse” expression host for manufacturing glycoprotein-based therapeutics — from antibodies to enzymes and beyond — Chinese hamster ovary (CHO) cells are the subject of most industry discussion regarding cell-line development and engineering. Most methods, technologies, and strategies apply regardless of the type of protein to be expressed or the type of cells expressing it. This featured report provides an update on the latest cell-line development techniques and workflows for working with CHO cells and other hosts —Pseudomonas fluorescens, in particular — along with some forward-looking discussion of evolving genetic engineering technologies, versatile analytical instrumentation, and innovative approaches for highly complex multimeric antibodies.

Introduction: Cell-Line Engineering and Development at BPI West
by Cheryl Scott
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. BPI’s senior technical editor reports here on the main themes and some key presentations from the track that addressed the latest developments in cell-line engineering and development. 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. 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 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.
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Genome Editing for Cell-Line Development
by Cheryl Scott, with Ben Haley
Industry researchers have gone beyond scratching the surface of what CRISPR/Cas9 and other methods can do for biotherapeutic development and production. In a March 2022 talk at BPI West in San Diego, Ben Haley of Genentech (Roche) presented inspiring examples that lit the way toward even more potential uses. In “End-to-End Platform Optimization for Loss-, Gain-, and Modification-of-Function Genetic Engineering,” he touted the importance of CRISPR/Cas9 technology as a powerful breakthrough with many applications. And he showed how the “knock-out” technique for loss-of-function screening has been the most widely used so far, with many researchers increasingly working with gain- and modification-of-function options as well. Haley illustrated the utility of functional genomics, especially for drug discovery and developability assessments. For example, modification-of-function can be used to elucidate protein structure–function relationships and enhance recombinant therapies through in situ mutagenesis and to evaluate how such drugs affect tumors. Lineage tracing is helping scientists parse out biological pathways, especially related to tumor evolution. And both gain-of-function and loss-of-function work are helping to identify new targets for protein, DNA/RNA, and even cell therapies. Here we discuss where these technologies have come from and where they could take the industry in the future.
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Cell-Line Development for Expressing IgM Antibodies
by Cheryl Scott, with Christina Pingchuan Tsai
Immunoglobulin G (IgG) antibodies remain dominant in the monoclonal antibody (MAb) drug-development pipeline. By contrast, immunoglobulin M (IgM) molecules are much larger and consequently more challenging for biomanufacturing and therapeutic application. Essentially multimers of five or six familiar Y-shaped IgG molecules, joined at their bases in a hand-like presentation, IgMs have 10 or 12 binding moieties and thus superior binding power (avidity) compared with IgG isomers. IGM Biosciences of Mountain View, CA, believes that IgMs could be more effective than IgGs at treating certain diseases —in particular serving as cancer immunotherapies. Many researchers in the past have been stymied by technical barriers to IgM manufacturing. But over the past 10 years, IGM scientists and engineers have made a series of breakthroughs that now make the company the only developer producing engineered IgM antibodies efficiently at g/L scale in a dedicated GMP-compliant facility. Here, Christina Tsai (senior scientist leading IGM’s cell-line development group) expands on the topics of her BPI West talk, “Cell-Line Characterization for IgM Molecules in Cell-Line Development Process.”
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Pseudomonas fluorescens: Cell-Line Development of a Commercially Proven Platform for Biopharmaceutical Manufacturing
by Russell Coleman, Elizabeth Orchard, and Yinghui Lee
Formerly known as Pfenex expression technology, the Pelican Expression Technology platform (PET) is a microbial expression system based on the Gram-negative bacterium Pseudomonas fluorescens. The platform was designed for rapid, robust, and cost-effective production of proteins, antibodies, and peptides for human therapeutics, vaccines, and other applications. PET expression enables rapid delivery of products through preclinical and clinical development and into commercialization. Four commercial products come from it so far. As authors from Ligand Pharmaceuticals describe here, the key first step in delivering a robust PET protein-production process is to identify an optimal combination of production strain and fermentation conditions. The group uses an innovative approach to strain screening that leverages the unique properties of its proprietary bacterial strain. Ligand’s automation-enabled, high-throughput parallel screening method can identify an optimized strain rapidly for further development. Taking advantage of the obligate aerobic nature of the PET microbe, the platform produces enough biomass at 96-well expression scale to generate a target protein in quantities required for analyses of quantity and quality. In a case study, these workflows enabled identification of a strain candidate for a neutralizing recombinant human Fab antibody fragment for continued product development within 12 weeks.
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Analytical Tools to Accelerate Cell-Line Development Workflows
by Brian Gazaille, with Lukas Klein and Dirk MĂĽller
Cell line developers screen thousands of clones to identify the most stable and productive candidates for biomanufacturing. Such assessments require considerable time and resources for preparation and execution of multiple cultivations and analytical assays. In March 2022, Lukas Klein and Dirk Müller (Sartorius) delivered a BPI Ask the Expert webinar about integrating their company’s Ambr 15 Cell Culture bioreactor and Octet label-free biolayer interferometry (BLI) technology to accelerate cell-line development. Case studies highlighted applications for media screening and platform optimization. Leveraging the bioreactor’s automation capability with the instrument’s high-throughput analytical capability significantly streamlines execution of such studies. Adding Modde software helps users further optimize microculture parameters.
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