Most research labs can make cells that produce antibodies. The trick is making those antibodies in sufficient quantity and quality to use throughout clinical trials and for manufacturing scale-up. Speakers at the recent Informa Life Sciences’ “BioProcessing” conference in Paris and the “Maximizing Expression Levels through Total Process Optimization” conference in La Jolla later this month outlined their approaches to developing and optimizing bioprocess cell lines.
The options, according to Florence Wu, Ph.D., director of process development, PD-Direct, at Invitrogen (www.invitrogen.com), are to optimize the vector, host cell, media or bioreactor conditions, or to consider new phenotypic traits.
Invitrogen examined resistance to high osmolality—the concentration of particles in solution—and how this trait can be acquired. Manipulating the media’s osmolality offers a way of extending the productive life of cells. The standard practice of providing concentrated nutrient feeds to increase the productivity of fed-batch processes increases osmolality to the point at which cells are no longer productive or even viable.
“Our data indicates genetic selection plays a role in attaining tolerance to high osmolality, but it doesn’t exclude adaptation,” Dr. Wu says. This project examined results when osmolality levels were shifted from iso-osmotic levels of about 290 mOsm/kg (milli-osmols per kilogram) to hyper-osmotic concentrations of 450 mOsm/kg. “Grow rate declines, and viability, too,” she says.
Invitrogen used Revolution™, a new technology that introduces point mutations into cells to heighten the genetic diversity of the cell population, resulting in a highly heterogeneous pool of cells from which researchers selected interesting phenotypes, Dr. Wu says. Results on osmolality were based on the popular DG44 CHO cell line, “but previous studies for selecting for high-titer clones have been conducted on hybridoma and NS0 cells,” reports Dr. Wu. This, coupled with other advances, allows us to get a better handle on engineering new host cell lines that will provide a creative solution to our customers to improve current production levels, she says.
Although the extent of that role has not been quantified, this early knowledge “allows Invitrogen to offer a creative solution for customers,” concludes Dr. Wu.
Building a Productive Cell Line
ProBioGen (www.probiogen.de) is also engineering cells, developing a proprietary vector for CHO cell lines that increases yield by a factor of three. Volker Sandig, Ph.D., vp of cell and vector biology, says much of the key work involves stabilizing the promoter within the vector. “Our vector contains multiple elements, including two promoters working in cooperation and two markup genes.” This helps researchers locate the cells containing the gene of interest.
“Typically,” he says, “you create a vector—a DNA construct with a plasmid that carries a promoter with all the information a CHO cell needs to make its product.” The usual promoters come from long DNA viruses, like CMV or herpes, “but the cell has a way to shut off the introduced genes.”
Most strategies to protect the gene from inactivation are patented and charge royalties, making them less attractive for labs on tight budgets. ProBioGen says its approach is different. “We don’t charge royalties,” Dr. Sandig says. Instead, clients buy the cell line, and ProBioGen makes the product to the client’s specifications. “They come with a gene and want antibodies with certain receptors. They know the genetic information and tell us the amino acid composition.” The gene is then synthesized and optimized to increase productivity and quality of the final protein, says Dr. Sandig.
Cell-line development projects usually take about six months and are focused on evaluating multiple clones to determine growth rates, behavior in culture, productivity, and product quality. ProBioGen’s approach allows researchers to use only one clone, thus eliminating the comparison steps and speeding the process.
ProBioGen inserts a recombinase into the DNA that inserts at specific receptors built into the vector, allowing exact positioning of the vector, which then receives the gene of interest through gene exchange. The gene then replicated at that site in each clone. The result is a highly productive cell line.
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.
Creating Cell Lines Faster
Leinco Technologies (www.leinco.com) developed an automated high-throughput transient transfection production system to express, purify, and test multiple human mAbs for early discovery research. Traditionally, “You may spend years to get a producing cell line. Our system produces it within about five weeks,” according to Brian Villa, business development manager.
Each step of the process—bulk DNA preparation, mammalian transgene expression, transfection into CHO or HEK cells, and purification—takes about one week, Villa says. The speed is because of the efficiency of the transfection system, says Villa.
“Our system works with a variety of plasmids,” Villa says, and has low endotoxin levels of less than 0.06 endotoxin units per milligram.
“We’re always trying to achieve higher efficiency and productivity of cell lines and thereby increase capacity,” he says. Leinco achieves that by screening multiple antibodies simultaneously and by automating such downstream steps as antibody purification. Scale-up is also of interest. Although much of this type of work is done in HEK cells, Leinco focuses on CHO cells because they are frequently used to produce the final product, Villa explains. The technology is working at the 10-mg scale, performing four transfections per day.
Vivalis (www.vivalis.com) is developing a cell line based on chickens and ducks to replace the embryonated chicken eggs and primary chicken embryo fibroblasts that are traditionally used in vaccine production. This EBx™ cell line uses embryonic stem cells because of their genetic stability, diploid karyotypes, indefinite cell proliferation, and strong expression of ES-specific markers and also because of their production characteristics such as proliferation at high cell densities and growth in serum-free media.
Vivalis says the cell line can be engineered easily to produce recombinant human proteins and adherent cells from which proteins can be produced in stirred-tank bioreactors. The line also has a glycosylation profile quite similar to that of humans, so the proteins produced in this cell line may be close to their natural glycosylation. A biological master file was prepared for filing with the FDA this spring, according to Majid Mehtali, Ph.D., vp of R&D.