U.S. government researchers have made a significant step forward in developing a fully automated process for cell-line development. The National Institute of Allergy and Infectious Diseases (NIAID), part of the NIH, hopes the new improvements are a move towards reducing cell-line development timescales to help tackle future pandemics.
“The novel piece of this work was incorporating the best instruments into our existing pipeline while compensating for the limitations we had,” explains Nadia Amharref, PhD, staff scientist in the vaccine production program lab at NIAID.
Amharref and the team had already developed a cell-line development workflow for anti-malarial monoclonal antibodies (mAbs) before beginning their new work. They began by constructing an expression vector with the gene of interest. This was followed by transfection into CHO cells, the development of pools of stably transfected cells, and then single cell cloning to find the best mAb producers.
The stable pools were grown in semi-solid media and a ClonePIX® instrument was used for automated screening of the highest producers. These clones were subsequently grown in shake flasks, and in a high-throughput bioreactor.
Stringent regulations for cell-line development
According to Amharref, “This platform allowed us to generate good average titers with a good average vector using our in-house expression vector.” However, the team found that the highest producing cells displayed inconsistent growth in a bioreactor. They also generated variable yields.
In addition, although ClonePIX allowed them to calculate that only one cell had been picked for further development, it could not be proved sufficiently for regulatory approval as regulations for cell-line development become more stringent, she says.
To get around this issue, the team incorporated a VIPS™ (Verified In-Situ Plate seeding) instrument into their workflow. This images the droplet deposited into 96-well plates for further growth to ensure it contains a single cell. The team also adopted a Cell Metric imager to study the contents of each well.
Finally, to speed up the cell-line selection process and avoid high producers being outgrown by fast-growing, low-producing cells, they adopted a mini-pool strategy—transfecting only a few cells at a time. Challenges included finding instruments that could fit into their lab’s existing footprint. They also found that, when they incorporated the VIPS, they needed a custom media to turn a single cell into a viable monoclonal colony.
The team now plans to take their work forward by acquiring the latest versions of Cell Metric and VIPS, as well as incorporating artificial intelligence for single-cell recognition.
“We’re still optimizing our media to improve timelines. There are commercially available supplements but, for our own cell lines, we want customized for the best results,” she points out.
Finally, they hope to incorporate quality assays at an early stage into the process. Amharref will be speaking about the work at the Bioprocessing Summit in Boston later this month.
