Improving Cell Lines
SAFC Biosciences (www.sial.com) has an active research project to find new value-added component and media formulations for the biopharma industry,” says Kevin Kayser, Ph.D., R&D manager, cell line engineering. The project evaluates the effects of specific genes on recombinant protein production in CHO cell lines and uses that to improve their growth and productivity characteristics as well as to engineer new cell lines for therapeutic protein production.
This biomarker discovery project for recombinant protein engineering optimizes media and also examines apoptosis as a way to increase cell density and extend cell life in particular cell lines. For this, SAFC does its own DNA sequencing and also accesses DNA sequence information through membership in the CHO Consortium.
“We place the sequences on microarrays and look at the difference between phenotypes,” in terms of cell productivity, working mainly with mAbs, says Dr. Kayser. That information is used to find a receptor that expresses in productive cells only. Then, he says, the question is “whether we can add a media component to affect the receptor and increase productivity.
“Because so many factors control the phenotype, we use siRNA to suppress gene expression” in order to ensure that the gene presumed to cause the increased productivity actually does. “We’ve built the architecture for evaluating the genes in the CHO transcriptome to determine their effects, including assays and a discovery platform. Now we are getting interesting phenotypes from our cell lines and assaying the differences” between productive and non-productive genes, Dr. Kayser adds.
Some of the findings are still proprietary, but broadly include some of the biological modulators of recombinant proteins such as secretion, assembly, and translation processes.
One of the benefits is the increased knowledge of CHO cell lines. “The industry moved quickly into CHO cell lines and didn’t do some of the basic biology work,” Dr. Kayser says. Consequently, cell line optimization has traditionally been a statistical endeavor. “This approach steps back to gain a greater understanding of the basic biology of CHO cells to help us engineer better media for our customers.” The goal, he says, is to bring rational design to bioprocess cell-line optimization.
Selexis (www.selexis.com) is interested in accessibility of the gene of interest within a transfected cell. The company says its work resulted in an expression technology that has increased recombinant protein expression in more than 30 mammalian cell lines by up to 20-fold. The technology, called MARtech™ (for matrix attachment region), “controls the promoter accessibility of the expression cassette,” helping the chromatin unwind so the protein vectors carrying the genes can insert themselves at a single, productive integration site without chromosomal breaks, according to Cori Gorman, Ph.D., head of technology development. “Therefore, you’re guaranteed the gene will be transcribed and the protein will be produced. MARtech is so stable you can remove drug selection and have production for a long time,” she says. Selexis’ experiments have remained productive for more than nine months with drug selection, Dr. Gorman notes.
“The benefit of having an extremely stable producing cell line is that you don’t have to search for productive clones,” and the elimination of the drug selection eases scale-up. Selexis found that it could screen fewer than 30 cells to determine that the gene expression and distribution didn’t change. Other methods required screening hundreds or thousands of cells, Dr. Gorman says.
The next step, she adds, is to optimize each step of cell line development and to develop smaller and more robust vectors.