To make biotherapeutic antibodies and recombinant proteins, scientists turn to cell lines. In most circumstances, the top picks include human embryonic kidney cells, such as HEK293, or Chinese hamster cells (CHO). Often, a team of researchers uses one method during development and another in production, but that demands extra care to ensure that the product is exactly the same as the one tested. That can be tricky.
Knowing more about the biochemical machinery of a cell line and how to control it promises advances in making biotherapeutic antibodies and recombinant proteins.
As Daniel Ivansson, staff research engineer at GE Healthcare Life Sciences, notes, “Engineering host cell lines improves the starting phenotypes and reduces screening needed in cell-line development.”
Transfection—incorporating a desired protein-generating DNA construct into a cell line—offers various opportunities to improve the production process. For instance, putting the transfected gene where you want it is helpful in production. Ivansson says that “site-directed integration of recombinant antibody genes reduces variation and improves capacity of cell-line development.” In addition, Ivansson notes that synthetic promoters can be used to fine-tune the expression of a gene.
In short, the more a scientist knows about a cell line’s genetic system, the better it can be controlled. Understanding more about “the gene construct used to encode and drive expression of a candidate antibody can have a major impact on both the yield and the quality,” Ivansson explains, “and much can be gained by tailoring the sequence for each given protein candidate.”
Down-under decisions
At the University of Sydney, Veysel Kayser, associate professor on the faculty of pharmacy, and his team use HEK and CHO cell lines to express biotherapeutic antibodies with, as Kayser explains, “an aim to develop biosimilar and biobetter monoclonal antibodies.”
Recently, Kayser and his colleagues described a method to use polyethylenimine to stably transfect HEK293 cells and quickly select the stable clones [1]. Kayser and his colleagues wrote: “The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. … This method can be transferable to the development of an expression system for the production of biosimilar antibodies.”
As for what he looks for in a cell line, Kayser says it must make enough product, and do so as easily as possible. So he wants a cell line to supply “high-yield protein expression and easy handling with standard protocols.”
Collecting the clones
Some companies also are making tools that help scientists screen for desired clones. “Traditionally, cell-line development has been considered a numbers game, but more automation is now being employed,” says Simon Keen, group leader, cell line development, at Abzena.
Robotics can be used to move containers, and software can control many of the tasks. In addition, Keen notes that software can assist in the “decision-making process, using strictly defined criteria to select which clones to progress, allowing fully automated cell-line development.”
Keen and his colleagues use many of these automation tools when screening for clones. For example, they “moved over to screening in shaking, fed-batch cultures at a much earlier stage using deep-well, multiwell plates and [TPP] TubeSpin bioreactors,” Keen says. “This gives a better indication of which clones will be the most productive in a fed-batch production system at an earlier stage.” In the last step of screening, the scientists at Abzena use an ambr 15 cell-culture platform, which is a single-use product from Sartorius Stedim Biotech.
Keen concludes, “The time scales are kept short by the inclusion of a robust platform process which can be quickly and reliably scaled up, and the use of single-use bioreactors ensures that manufacturing campaigns can be completed quickly, and turnaround times are kept to a minimum.” With this approach, Abzena has developed cell lines that produce 1 gram to 3 grams per liter.
Stable transfection of a cell can take time and quite a bit of expertise; transient transduction is easier and faster.
As the names suggest, though, the former keeps making the protein for a long time, and the latter only makes it for a short time, typically days to a few weeks.
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Using the MaxCyte STX Scalable Transfection System, Krista Steger, consultant at MaxCyte, and her colleagues showed that it’s possible to transiently transfect CHO cells and produce higher levels of antibodies than in the past [2]. This enables researchers to progress to later stages in development before migration to stable cell-line antibody production is needed.
Beyond simplicity, other factors can encourage the kind of cell line selected. “HEK and CHO proteins have different glycosylation patterns,” Steger explains, “and if all of the early work is done in HEK cells, then the CHO proteins may be less efficacious, because [they are] glycosylated differently.”
Reducing steps with recombinases
To sidestep cloning, some companies develop stable cell lines that include site-specific recombinases. The recombinases “allow genes to be targeted to known transcriptional hot spots in the genome, cutting out the need to screen clonal isolates,” says Michael Weiner, vice president of molecular sciences at Abcam.
Abcam’s pMINERVA uses recombinase enzymes in E. coli, and it creates complete antibodies (IgGs) rather than fragments. This technology, says Weiner, reduces “the time it takes to convert antibody fragments to IgGs from a week to overnight [3].”
Weiner points out that the recombinant antibodies provide very consistent products, because they are defined by a DNA and protein sequence. “This means they have excellent batch-to-batch consistency and provide specific and reproducible results,” he explains.
Extent of stability
So far, for long-term experiments, scientists have been putting their time and efforts into creating a stable cell line rather than a transient one. “But now you can delay that, because transiently transfected CHO cells can make enough protein to get you pretty far down the pipeline,” Steger explains.
How far you can go depends on the specific case—not to mention your own cell-line philosophy—but you might go as far as early toxicology studies. “It’s getting fuzzier and fuzzier, when you need to make the transition,” says Steger. “Some people in pharma say that they use transient transfection for early toxicology studies but switch over to a stable cell line for later tox studies—to be sure that they’re working with what they’ll use for production.”
Eventually, computational tools will help scientists experiment with ways to engineer the host genome and transfected gene for the best outcome.
So far, says Ivansson, “knowledge to enable in silico selection of a gene-construct design lags behind.” With ongoing advances in computer hardware and software, plus the benefits of big data, modeling and simulation should soon play a big part in using cell lines to build tomorrow’s biotherapeutic antibodies and recombinant proteins. When that happens, scientists will really crank up the output!
References
[1] Elgundi, Z, et al., “Laboratory scale production and purification of a therapeutic antibody,” J Vis Exp, 119:e55153, 2017. [PMID: 28190027]
[2] Steger, K, et al., “CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines,” J Biomol Screen, 20:545-551, 2015. [PMID: 25520372]
[3] Batonick, M, et al., “pMINERVA: A donor-acceptor system for the in vivo recombineering of scFv into IgG molecules,” J Immunol Methods, 431:22-30, 2016. [PMID: 26851519]
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