"Sartorius' in house cell line development service actually began as a small startup named CELLCA GmbH in two thousand and five and has improved over many years to become one of the leading technologies on the market. A Sartorius research fellow, Christophe Zier, will walk us through the evolution of Sartorius' cell line development technology. Christophe joined CELLINK in two thousand and seven where he established a leading platform technology for the generation of high expressing cell lines. Welcome, Christophe.

Yeah, thanks for the nice introduction. So I start with a little timeline here. So the company was founded as CELLINK in two thousand and five. In twenty fifteen, the company have been acquired by Sartorius.

And since beginning of twenty twenty, we have this great new building, which is a Cell Line Development Center of Excellence, where we do all our cell line development services and also the further development of the technology.

So, the company CELLINK was founded with the mission to develop a really a protein manufacturing technology, which is enabling the biopharmaceutical industry to produce biologics fast, cost efficient, in high quality and at scale. So at that time, back in two thousand and five, this was missing, in the market and we developed this really from scratch.

How did we do that? So we have set up a technology consisting of four components, which you see here. So we had to develop a host cell line, an expression plasmid, cell culture medium and also the process design. And of course, also a lot of procedures and know how to round it. So from the transfection over how to generate stable cell lines, how to cultivate the cells, how to single cell cloning and so on and so forth.

And in the following, I want to start now with the what did we do for the first version of the COD technology. So it took us about five years to come up with the first CLD one point zero version of this technology.

And this is what I want to show you in the next slides. I will focus mostly on these cell line and expression vector plasmid due to some time restrictions.

And I will deal with the other ones only to a lower extent.

So the beginning, the host cell line, so what was the starting point? We have started with a CHO DG44 cell line where we got the license from Chase in from University of Columbia. So this is a DHF artificial cell line and we started with an adherent growing cell line in serum containing medium.

And the first step was now to adapt it to industry needs. So what you can see here is the first eighty passages of these adherent cell line. So we started with a phase where we had some FCS in. We removed this step by step.

And then we had a long phase of where we did adaptation to serum free chemically defined medium in suspension.

What you can see here is that the viability looks already nice here, close to one hundred percent. However, we had a lot, a lot of fluctuations here in the cell growth. So it was for sure not consistent at that point.

And what did we do? We did a lot of more cultivation. So on top of these eighty, we cultivated for more than one hundred additional passages. So the whole adaptation process took more than one year in this cell culture medium. And what was the result? The result you see here.

So a very nice, very consistently growing host cell line with a high level of viability, extremely stable, and with a really nice doubling time. So what we did here is, so we worked on this promoter here, which is driving the DHFR and we went with already a relatively weak promoter, so the HSSVTK.

But we also inserted further changes, a few point mutations, some truncations.

And what we also did, and this was also quite successful in the end, we added a hairpin structure. So after the promoter, before the start codon, we inserted a hairpin to slow down the ribosomes in the translation process.

And we tested this in a simple readout. So just the different TK promoters here, which were driving reporter molecule, secreted alkaline phosphatase in that case. And what you can see very nicely, so here is the Y type situation, the original TK and we could really reduce very largely the expression level depending on the different modifications we did.

We also could achieve enhanced expression levels by implementing these elements.

Good. So this was now just some work on the plasmids. I only showed you maybe thirty percent of what we did due to this time restriction. But the result was, Okay, we had now a nice plasmid with a good topology with the right promoters for heavy chain, light chain, but also the selection mica containing chromosome elements.

And we also had to set up, of course, the cell line development workflow. So how do you now do your cell line development? And this is briefly shown here. So at that time we have chosen a so called mini pool approach, meaning that you seed a certain number of cells into ninety six well plates with either no MTX or very low level of MTX component is of course the cell culture medium.

And this is now extremely short. So behind this nice picture, there is five years of really intense development work with hundreds if not thousands of experiments and fifty, sixty, seventy different recipes which were tested and evaluated.

And the final result is what is now a day called fossil extractor, so a medium which is available at Sartorius.

And this is a chemically defined animal origin free medium, also protein and peptide free. And in fact, it's a kind of a medium family, right, because it comprises the cell culture medium which you take for your cell line development for cultivation expansion, but also production medium, which is used for fat batch processes as well as the respective feed media, which you need.

That already at that time, we could achieve extremely high titers, so up to nine grams per liter here already.

And we had an excellent scalability, as you can see, because the behavior of these clone in the different scales, in the different bioreactors is extremely comparable. So first of all, not a lot, I have to say, regarding development because once we had this technology and we saw this great performance of the technology, we had a phase of about five years, which I call here commercialization phase, right? So here the task was to take this technology, to get client projects, to apply it, to develop cell lines.

What was also important relatively early was to establish a quality system, so just which are usually used in the field are not really performing that high in our systems such as the EF1 alpha for example.

Then we also added a very interesting one which we came across, which is a mouse CMV promoter.

And you see that this mouse CMV is really nice. The promoter really outperforms what we had so far, and it is also not prone to any silencing.

Another aspect which is can really help to boost expression and which we addressed as the next step is the signal peptide.

So also here we tested a certain selection of signal peptides. What we used so far was here the standard was a serum albumin signal peptide. We tested a few more and you see already here that this what we what is designed as signal peptide nine showed a very nice performance, which was also confirmed with quite a number of products here. So in that case, four products. And we saw a different amount of increase compared to the standard, but for all products we saw an increase. So that was very good.

And yes, indeed, in whatever situation we tested it, this novel SP9 signal peptide outperformed the current standard.

And now, obviously, I mean, we tested the promoter, we test the signal peptide, now we combine this together.

So again, for molecules, in that case, antibodies, also Fc fusions, we compared the two point zero where we applied MTX. And what we did here now was that we combined CMV SP9 without MTX. And because this was also something we want to achieve to get rid of these MTX selection process, then we generated clones and evaluated them.

And here you see these four products. So you always have the CLD version with high MTX, with low MTX, and here the CMV plus SP9 without any MTX. And you see that it's a little different for the different products, but what we concluded was that with the novel CMB plus SP9, we either got even better results without MTX, which is important, or at least similar results to compare to the old version, CLD two point zero with MTX. And so this was now our new approach, our new expression plasmids combining this MCMB and the SP9.

Good. And having that now, we could heavily simplify the silane development process because we canceled everything which contains MTX, right? So all mini pools here was two point five five and ten were canceled.

We also, in that context, could cancel the whole MPP workflow. So we just went without MTX, only mini pools.

And what was also very nice to see is that we also could speed up the expansion process quite significantly.

Furthermore, we didn't have any medium exchange anymore. So from the very beginning, we went now with the CELLINK x2 medium.

So very much of simplification of the whole workflow and also further saving in time. So from DNA to RCP now in eighteen weeks instead of twenty. TEG technology in this bluish color with our SP9 signal peptide. And this was really helpful, as you can see. So the yellow bars, a combination of UTR BetaTec and our signal peptide is really the best combination you can find and gave much better results than the current standard here.

The same we did for the Fc fusions where we saw that the sigmoid peptide didn't play such a role, But also here, beta tag with or with our sigmoid peptide outperformed what we had before."