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Speed to clinic is a priority during the development of a biopharmaceutical. Consequently, reducing time to GMP manufacture remains a challenge for CHO cell line development workflows. However, the goal of a fast development process must be balanced with requirements for high quality and the maintenance of regulatory compliance. A production cell line used for the manufacture of biopharmaceuticals should be derived from a single-cell progenitor. Accordingly, traditional cell line development workflows often contain two rounds of single-cell cloning, resulting in a lengthy process exceeding six months.

In this expert SelectScience® webinar, now available on-demand, learn how Dr. Devika Kalsi, FUJIFILM Diosynth Biotechnologies and Dr. Xin Liu, Sphere Fluidics, combine the single-cell deposition, imaging, and productivity screening capability of the Sphere Fluidics’ Cyto-Mine® technology with a plate imaging method to create a novel workflow for the generation of high-quality clonal cell lines with high probability and assurance of monoclonality in a single round of cloning.

Read on for highlights from the Q&A discussion and register now to watch the webinar on-demand

What is the minimum expression improvement that this system can reliably select?

XL: There is a known phenomenon when you do the binding and it's called the hook effect. Essentially, if your target molecule goes above a certain threshold, you start to see the draw of the small subunit (SSU) . That's why you see those two high-concentration populations start to overlap. But what we see is that the incubation window and the isodynamic range are good enough to allow us to reliably detect solution from either two cells or in other cell types, for example, primary B cells.

What we see is highly diversified among the end-users. So, I think in terms of productivity, enrichment is highly dependent on their host cell lines, their track record of optimization of their platform, including the media, including the downstream, upstream, and various processes.

Apollo-directed evolution: Were cells simply grown under the desired stressed conditions and relying on the selection of spontaneous mutations or epigenetic changes to give the desired trait?

DK: We wanted to be able to manipulate all pathways in one shot. So, yes, we wanted to find out which cell lines or which of our host population would be most able to withstand the stresses and be able to outcompete the unsuitable variants. That was essentially the method we took to improve our whole-cell line.

Are there any other assays you can use for other types of antibody formats that you can use to select high-producer cells?

XL: Yes, in general, we provide an off-the-shelf assay kit called Cyto-Cellect™, that has been optimized to specifically detect human IgG or human Fab fragments. And as I said, the advantage of using FRET is potentially the user can customize the assay for their specific molecule of interest. All you need to do is find a pair of recombinant probes that can boost the target on the protein of interest.

Are you employing any advanced analytics during screening to interrogate direct product quality attributes such as glycan profile and other Post-translational modifications? Does this factor into your selection criteria in addition to titer?

DK: Within Apollo X, we do evaluate the product quality at the ambr 15 stage. And we use analytics to look at the glycan profiles and things like high-molecular and low-molecular-weight species and check the purity of the product that we're getting and compare it against the standard for that molecule. Then we take that into account when we're selecting at the end of the cell line development process.

What is the outgrowth of single-cell cloning in 96-well plates?

DK: For Apollo X, we get around 50% clonal outgrowth in a 96-well plate. So, you're looking at around 14 days from the seeding of that cell in that plate to then have an outgrowth, to have enough cells to then take forward into your next stage. By that 14-day standpoint, we have around 50% clonal outgrowth, and then we're able to take the cells out and, kind of, bulk them up to then go into that ambr 15 screen.

How many Cyto-Mine runs do you perform in a one CLD project?

DK: In one CLD project, we would carry out five Cyto-Mine runs. Within that, each Cyto-Mine run would have one transfecting pool that is put into the machine to then generate cell lines derived from that transfecting pool. So, you'd have a diverse set of cell lines by the end of it, because they're coming from five different transfection events.

What is the max number of picodroplets you can evaluate in one Cyto-Mine run?

XL: In one Cyto-Mine run, the user has the option to choose the number of droplets to be generated for the downstream process, ranging from about one milli up to two millidroplets.

In Apollo X, how many cell lines are evaluated at each stage, i.e., at Cyto-Mine and then it's ambr 15, and then in bioreactors?

DK: The Cyto-Mine level when we've got that outgrowth in the 96-well plate, we would evaluate up to 300 cell lines, and we would evaluate them for titer. And then we'll move on, the next screen is the ambr 15, where we'll take up to 48 cell lines into the microbioreactors, to then assess for growth, product titer, and product quality over the 14-day fed-batch process. By the end, when we're going to bioreactors, it’s a maximum of three lines that we would take forward. Or if the customer has decided that they just want this one cell line, we'll take the one cell line forward and do more experimentation on it.

What technique do you use for the initial evaluation of titers before it goes to ambr?

DK: For the 96-well plate screen, we use the Octet platform to assess for titer support.

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