In this on-demand webinar, Dr. Devika Kalsi and Dr. Xin Liu illustrate how they can reduce cloning times from 25 to 10 weeks
Dr. Devika Kalsi, FUJIFILM Diosynth Biotechnologies and Dr. Xin Liu, Sphere Fluidics
Speed of access to the clinic is a priority when developing a biopharmaceutical product. Therefore, reducing GMP manufacturing time remains a challenge for CHO cell line development workflows. However, the goal of a rapid development process must be balanced with high quality requirements and maintaining regulatory compliance. A production cell line used for the manufacture of biopharmaceuticals must be derived from a single-celled progenitor. As a result, traditional cell line development workflows often contain two cycles of cloning a single cell, resulting in a lengthy process exceeding six months.
In this SelectScience® expert webinar, now available on-demand, learn how Dr. Devika Kalsi, FUJIFILM Diosynth Biotechnologies and Dr. Xin Liu, Sphere Fluidics, combine single cell deposition, imaging and screening capability of the productivity of Sphere Fluidics. Cyto-Mine® technology with plate imaging method to create a new workflow for generating high quality clonal cell lines with high probability and assurance of monoclonality in a single cloning run.
Read on for highlights from the Q&A discussion and register now to watch the webinar on demand.
What is the minimum expression enhancement that this system can reliably select?
XL: There is a known phenomenon when binding and it’s called the hook effect. Essentially, if your target molecule crosses a certain threshold, you start to see small subunit (SSU) drawdown. That’s why you see these two high-concentration populations starting to overlap. But what we see is that the incubation window and isodynamic range are good enough to allow us to reliably detect a solution from two cells or in other cell types, for example, the primary B cells.
What we see is very diverse among end users. So I think in terms of productivity, enrichment is highly dependent on their host cell lines, their track record of optimizing their platform including media including downstream, upstream and various processes .
Apollo-directed evolution: Have the cells simply been grown under the desired stress conditions and rely on selection for spontaneous mutations or epigenetic changes to yield the desired trait?
DK: We wanted to be able to handle all the lanes at once. So, yes, we wanted to know which cell lines or which host population would be best able to withstand stresses and out-compete maladaptive variants. This is essentially the method we adopted to improve our whole cell line.
Are there other tests you can use for other types of antibody formats that you can use to select for high producing cells?
XL: Yes, in general we offer a standard test kit called Cyto-Cellect™, which has been optimized to specifically detect human IgG or human Fab fragments. And as I said, the benefit of using FRET is that the user can potentially customize the assay for their specific molecule of interest. All you have to do is find a pair of recombinant probes capable of boosting the target on the protein of interest.
Do you use advanced analytics during screening to interrogate direct product quality attributes such as glycan profile and other post-translational modifications? Does this take into account your selection criteria in addition to the title?
DK: Within Apollo X we assess the quality of the product at the ambr 15 stage. And we use analytics to look at glycan profiles and things like high and low molecular weight species and check the purity of the product we get and compare it to the standard for that molecule. Then we take that into account when we select 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 about 50% clonal growth in a 96 well plate. So you’re looking at about 14 days from seeding that cell in that plate to then having an outgrowth, to having enough cells to then move on to your next step. After 14 days we have about 50% clonal growth and then we are able to pull the cells out and kind of blow them up and then go into this amber 15 screen.
How many Cyto-Mine runs do you run in a CLD project?
DK: In a CLD project, we would perform five Cyto-Mine runs. In this setting, each Cyto-Mine cycle would have a transfection pool which is placed into the machine to then generate cell lines derived from that transfection pool. So you would have a diverse set of cell lines at the end, as they come from five different transfection events.
What is the maximum number of picogoutlets you can assess in a Cyto-Mine analysis?
XL: In a Cyto-Mine analysis, the user has the ability to choose the number of droplets to generate for the downstream process, ranging from approximately one milli to two millidroplets.
In Apollo X, how many cell lines are evaluated at each stage, i.e. in Cyto-Mine and then it’s ambr 15, then in bioreactors?
DK: The Cyto-Mine level when we have this 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 continue, the next screen is amber 15, where we’ll take up to 48 cell lines in the microbioreactors, and then evaluate the growth, product titer, and product quality over the fed-batch of 14 days to process. In the end, when we move on to bioreactors, it is a maximum of three sectors that we would advance. Or if the customer has decided that they only want this one cell line, we will take this cell line forward and do more experiments on it.
What technique do you use for the initial valuation of titles before they go to amber?
DK: For the 96-well plate screen, we use the Octet platform to assess titer support.
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