Ask the Expert: A Robust, Stable Platform for Biologics Development

Sean Liour (vice president for project management at GenScript ProBio) delivered an Ask the Expert presentation on 18 March 2020 to explore the advantages of his company’s platform for cell-line development. Liour explained that successful biologic development hinges on robust host cell lines, capable expression vectors, and discriminating clone-screening systems. Overviewing relevant technologies and capabilities, Liour illustrated how his company’s ProCLD platform helps sponsors navigate cell-line development.

Liour’s Presentation
Researchers must weigh their options carefully when selecting a host cell line because that decision will facilitate or hinder future operations, ultimately influencing cost of goods (CoG) and product quality. Hosts should be selected to facilitate process development and good manufacturing practice (GMP) production. For instance, high titers are essential, as is a short timeline from transfection to clone selection. But successful hosts perform consistently well over time: Choosing a stable cell line can minimize operational changes during process development. Ensuring that a host can adapt to suspension and serum-free growth conditions similarly can ease the transition to GMP production. Identifying a host with a high but well-tuned secretory capacity can streamline downstream process development by minimizing product-related impurities from the beginning. Other early considerations should include a host’s protein-folding capability and glycosylation machinery.

Vector engineering can improve expression levels, increase stability, and streamline clone screening. But researchers must take care to maintain expression reduction in prolonged cultures and to ensure high stability without adding selection pressure. Preventing integration-dependent repression and negative-position effects helps to overcome vector-engineering challenges, as can using promoters, enhancers, and other expression-augmenting elements to optimize vector composition. Although both strategies are common, it is also helpful to use chromatin open elements, insulators, and CpG islands to overcome gene silencing as well as to use Cre-loxP and clustered regularly interspaced short palindromic repeats (CRISPR) for site-specific integration of genes and selection markers.

Several cell-screening technologies are available, so scientists should explore the advantages and drawbacks of each system. Limiting dilution (LD) is common because it is inexpensive, uses simple equipment, and does not damage cells. But it has low seeding efficiency, requires considerable time, and is susceptible to contamination. Regulatory agencies also require two LD rounds to establish monoclonality, and that can slow down operations when speed is critical. Investigating other techniques can accelerate timelines and increase test accuracy.

GenScript’s ProCLD platform incorporates all the above criteria. Using CHOK1 cells, which are stable and robust enough for manufacture, and which have a well-documented parentage, the company initiates cell-line development with three to five months of pool screening. It tests three to five vectors during that process. From more than 200 pools, six are analyzed for titer in 10 96-well plates using a Solentim VIPS (verified in-situ plate seeding) benchtop system. VIPS technology enables fast, gentle, and automated single-cell seeding. Its companion device, the Cell Metric whole-well imager, offers high-resolution scanning of clones and can provide visual confirmation of monoclonality. After Cell Metric analysis, 30 clones move on to fed-batch evaluation, after which six remain. Those enable construction of a primary cell bank
(PCB) for stability assessment and finally selection of a top clone.

The ProCLD platform enables selection of robust, stable clones for monoclonal antibody (MAb) production. Top clones generally yield 3–8 g/L of MAb, although 10 g/L is common. Such clones also perform well in 60- and 90-generation studies. Combined with the platform’s speed — the period from DNA synthesis to selection of six top clones takes 14 weeks, and selection of a top clone takes four more — the ProCLD process can help researchers move confidently from cell-line development to process development.

Questions and Answers
What are the differences among primary, working, and master cell banks? Primary banks comprise cells produced from a company’s top clones, and such material can be used to perform stability studies that ground selection of a single clone. Working and master cell banks derive from that single clone. The former are used in GMP production; the latter are amassed for drug-substance characterization testing.

What are key advantages of the ProCLD platform? It has a proven track record. It continually yields cells with high titers and stable production qualities. The platform’s speed and its ability to establish monoclonality are also clear benefits to researchers.

More Online
The full presentation of this webcast can be found on the BioProcess International website at the link below.

Watch the full webcast now.