Cell Line Development for Biologics

Cell line development is the process by which the cellular machinery is co-opted to manufacture therapeutic biologics or other proteins of interest. You can use different expression systems for cell line development: bacterial, plant-based, yeast, mammalian. Most commonly used for the production of the complex protein is Chinese hamster ovary (CHO), cells grown in suspension cultures for eventual use in bioreactors at the manufacturing stage.


From producing cutting-edge vaccines and cancer-fighting drugs to developing revolutionary gene therapies, the use of cell lines to produce therapeutic products is changing all facets of the biopharmaceutical industry.

Cell line development is the process by which living cells are used to manufacture therapeutic biologics or other proteins of interest. Cell line development starts at small-scale suspension cultures, eventually transitioning to industrial-scale bioreactors at the manufacturing stage.

Cell line development is no simple task. Putting a lifesaving biologic, such as a targeted antibody, on the market requires years of drug discovery research, development, clinical trials and scaling up for commercialization. Establishing cell lines is a long process, with numerous steps and challenges (Figure 1).

Figure 1. Cell line development process. Automated steps described here are indicated in red. 

Figure 1. An overview of cell line development workflow steps. Automated steps indicated in red. 

 

Vector Construction

The vector construction step of cell line development requires identifying a gene sequence for a therapeutic product and assembling that sequence into a plasmid. (For more detail on vector assembly, see our Synthetic Biology materials). Plasmids are transfected into cell lines or delivered via a retroviral vector. Thousands of cells undergo the transfection process, increasing the possibility for chromosomal integrations and potential downstream production hits.

Stable Transfection and Selection

Once cells have been transfected with the construct of interest, screening for protein production is required. Single cells must then be isolated, grown in culture, and assayed for division time and product yield. Once these initial production screens have been accomplished, the top candidates are expanded and maintained to test for expression stability:

  • Single Cell Culture: By picking and culturing individual cells, researchers can identify the cells that have integrated the antibody’s DNA into their chromosomes. These cells produce the antibody of interest.
  • Selection: The host cells grow in 96-well plates, being monitored for both robust growth and high productivity. Cells are selected based on the titer of antibody they produce. Selection markers are then used to derive stable cell lines expressing the desired antibodies for further characterization. Eventually the top hits are trialed in different media conditions to optimize expression or other product attributes (e.g. glycosylation) before scaling up to production levels.

Single cells are seeded into individual wells, and high-resolution imaging of microplates is used to verify monoclonality. As with the early-stage iteration of this step, this process aims to minimize heterogeneity of the antibody by ensuring that the colony is grown from a single cell. Because the screening process takes months to complete, there are more concerns than a typical short-term cell culture workflow. Maintaining sterility is essential during various manipulations and throughout the weeks of growth from a single cell. The throughput required to identify a handful of optimal clones requires thousands of candidate wells, making data management and tracking challenging.

Single-Cell Depositing and Monoclonality Verification

Late-stage cell line development requires winnowing away lower-producing cell lines from a large pool of candidates. Cellular imaging is employed to verify that the cell line originated from a single cell. Afterward, viable daughter cells are sequenced to screen for concerning mutations.

Protein products go through an initial screen for critical quality attributes, such as structure, activity and stability, ensuring products are high-quality and as expected. As clones move through development and are seeded into lower density plates during hit picking, all previously captured data on these cell lines must be appended to new cell lines to maintain regulatory compliance. We can help overcome many challenges in this critical process by automating the establishment and screening of clones for cell line development.

Expansion and Characterization

During cell line expansion, researchers attempt to maintain optimal nutrient and physiological parameters for cells while transferring lines to a production environment. Parameters such as cell count, cell viability, metabolites, pH, dissolved oxygen and carbon dioxide concentrations are continuously monitored throughout this process. 

Researchers tweak media conditions so the cells can produce high yields of high-quality antibodies. Techniques such as glycosylation analysis, titer testing and genome sequencing are used as final characterization parameters of cell lines and as quality control checks.

Master Cell Banking

Cell lines that efficiently produce high-quality antibodies are stored in a master cell bank, where they’re securely cryopreserved for future use in process development and therapeutic protein production.

cell line engineering, cell line characterization, and process development steps in cell line development 

 

Products and methods described are not intended for use in diagnostic procedures.