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Thank you for joining my talk.
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I'm Ebi from GeneFrontier and in this talk I'm going to give you a flavour about what you can do with cell-free technologies and maybe cell-free technology sounds niche to you, but it's getting popular quickly nowadays with the new application including AI machine learning approach.
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I'm so excited about that.
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So I hope I can share that excitement with you through my talk.
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So let's get started from who we are.
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We are GeneFrontier based in Tokyo area.
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And since 2010, we are committing to the cell-free technology, the unique cell-free technologies.
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And our mission is to change the world and inspire the world with our unique cell-free technologies that is PUREfrex.
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And PUREfrex is the approach in the cell-free.
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And cell-free is the approach to break the cell for the breaking through the limitation of the living cell system.
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But our approach is one more step further.
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That means we only use necessary molecules for individual transcription and translation or IBTT like this.
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And we prepare each component separately and purify them and then rebuild in a tube like this.
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So I would say it's a pure system based technology.
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So if you are familiar with pure system technology, it was published in Nature Biotech 2001 and it was embedded by Professor Ueda in Tokyo University them and we have been collaborating with him for long time and we upgraded the pure system technology and launched as PUREfrex like this.
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It has various advantages.
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I would say the rebuilt type cell-free system just adding DNA here, totally molecular based system moves on like this.
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So I would say it's integrated enzymatic reaction for making peptide or protein.
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Just in a few hours you can get your product.
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As you can imagine, it's easy to add or remove something from this system for your application.
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For example, you can precisely control the translation factors or chaperones, or even you can add the detergent or other chemicals into the system.
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And you can precisely control the physical condition like a temperature or redox state.
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And redox state is very important factor for making the protein that including the disulfide bridges like a antibody or the peptides.
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With that you can simply imagine the easy incorporation of non-natural amino acid through arbour separation.
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For example, this is example of the fluorescence labels, non-natural amino acid incorporated into the products like this, very easy to go and you can simply add the nano disc for the solubilization of the membrane protein like this.
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And in this case we use the product from the Cube Biotech German based company and it works quite well.
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And also you can combine the detergent as I mentioned, and we validated various type of detergent like this.
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And in most cases you can go very higher concentration of the detergent and it's compatible with our IVDG system in PFX.
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So it's very helpful not only for the membrane protein, but also for the difficult to express protein.
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And we are improving the yield through the version up with good reproducibility like this.
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So that is because we precisely control at molecular bases, OK.
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And so I would say it's a versatile and robust platform for making protein or peptide.
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And we expecting the huge potential as a new platform in biotechnology field and we are improving the PUREfrex as through the collaboration with many academia like this.
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And for the productivity, you can express mainly we are using molecular machinery from E coli.
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But for the expression product, there's no bias for eukaryotic protein as well.
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So you can express the eukaryotic protein as well as prokaryotic protein and small one like peptide size to, large one like beta galactosidase or IgG which is coming to the latest slide.
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And it's easy to purify the product.
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Simple purification leads to this purity and as I mentioned it, increasing the productivity like this.
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And in case of the GFP, you can see the colour of GFP like this and if you can reach to the one milligramme per ML yield in this case.
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And we are expanding our capability for manufacturing of the PUREfrex itself and we can distribute from micro litre scale to even little scale as well for one bunch.
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And actually a some pharmas are already using our kit in little scale in their facility already.
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And for the productivity that yield, I would emphasise one key success factor here that is the design of the DNA template.
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So we again when we are using the molecular machinery from E coli, so the codon usage should be applied to E coli basis.
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But just using measure codon is not good enough.
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This is example of the heavy chain from the Herceptin and at the end terminus of the product, the construct you if you can use, you should use AT rich codon not major codon that leads to the huge increase in the yield like this.
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Not only the case, we applied this rule to various type of protein like this.
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So PD-1, PD-L1 and GMCSF is a remarkable case like this.
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Just using Ath cotton for the N terminus, you increase the productivity like this.
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This is a very minor modification that, but it could a could have huge impact in the yield.
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So please keep in mind and we are looking into that mechanism in our facility and we compare the GC percent or 80% in and the yield as well as the stability of the messenger RNA.
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And those findings we incorporated into a publication here and we just published last year.
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And also we incorporated as the knowledge of this kind of things into our manual, which can be downloaded freely from our website.
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And also we established that our own software for the optimization of the DNA construct for PUREfrex expression and we are free to consultation.
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We are free to consult the DNA construct for you.
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So if you can send us, I mean as it sequence for your product, we are happy to optimise the DNA sequence and get back to you for free.
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And if you like, we are happy to share that software itself for free, although it works in Macintosh for now.
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But anyway, if you have interest, please contact us later.
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And with that, you can improve the timeline in the expression from days to hours compared to the traditional methods.
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Starting from the DNA, you can get your protein just in a few hours.
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And you can use not only plasmid but also the linear template like a PCR product.
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And in most cases, you don't need to purify the product because our system doesn't have any other factor except IBTD.
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So that has good fit to the AI machine learning approach in the expression process.
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And if you'd like to purify the product, you can simply utilise the tag system like a purification and the cleavage for protease like this.
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So we are using that kind of system in a facility as well.
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So and it works very well.
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And from the collaboration with academia, this is the work done by a researcher from Tokyo University.
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They try to establish the sophisticated validation platform for VHH and its target.
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And they incorporated another new type of DNA synthesis method like this.
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And together with PUREfrex, they established the validation platform just in two days.
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They can get the comprehensive data just in two days like this and as the details was published in this paper quite recently.
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So if you have interest, please check it out.
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And this, those are the example of what you can express with PUREfrex and especially the antibody or antibody related molecules.
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Of course, you can express the SCSB, SCFB or Fab and in those examples, we use the linear template as a PCR product and like this.
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And in this case, we express the Herceptin derived molecules and we reached to .5 milligramme per ml in this in those cases.
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And we expressed, we succeeded in the expression of the Blinatumomab, the bispecific molecules.
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And I heard that Blinatumomab was difficult to express in E coli.
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But we succeeded in the expression with slight modification or optimization of the condition with PUREfrex.
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And of course, we can express the full size IgG as well.
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And in this case you can get IgG just in two days starting from the DNA template and function is the same as commercially available half safety in this case just difference is it our product from PUREfrex not glycosylated because it's self resistant.
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But it's good enough for the early validation study I believe.
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And this is the example from the third party a Bonum Therapeutics, the US based biotech.
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And they are developing the unique type of immuno-cytokine like this.
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And they made the different type of mutant of their immuno-cytokine and they synthesised their mutant with PUREfrex and validated in the cell based assay.
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And they moved on to the cell based assay even without purification because PUREfrex has low LPS.
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So you can think about the seamless functional assay in cell based assay as well.
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And from the other example except antibody related molecules, this was reported by Novo Nordisk.
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They succeeded in the expression of the functional insulin and mutant of the insulin with PUREfrex with precise control of the disulfide with a precisely control of the redox state like this.
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And I'm a very appreciate their great effort for this validation study.
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And the table here is a little bit busy table, but this was reported by it's a research group from Stanford University.
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And they tried to validate the expression of the 20 different types of therapeutic molecules from the hormone or cytokine including GLP one.
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And they compared the different type of platform for the expression including PUREfrex, our product here and the other type of crude Cellulite type self-system.
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And interesting point is only PUREfrex our products shows the success in all the expression of those molecules and the other system failed in some of them in the expression.
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So we believe this table shows the versatility of PUREfrex as expression platform.
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And more than that, you can express the enzyme even though they have the multiple disulfide bridges like this in TPA case this has a disulfide bridges and it's non-serial type disulfide bridges.
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But with precise control of the redox state, you can express the functional enzyme as well.
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And from the third party a Roche Diagnostics succeeded in the expression of the Fab mutant with PUREfrex and they executed the high throughput validation for bio core analysis with PUREfrex even without purification of their product.
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And the another example is Japan based biotech iBody.
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They have unique technology for making antibody starting from the single B cell cloning and they use pair flex for the expression of the each clone in Fab format and it works quite well.
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And another example is also the Epsilon molecular engineering which is another biotech company in Japan.
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They also have unique platform for the screening of cyclic peptide and VHH based on the cDNA display and MGS and PUREfrex is used for their cDNA display and the high support expression for functional screening afterwards for PHH.
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And the other example is from Fujifilm.
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They just studied the CRO business for the screening of cyclic peptide including non-natural amino acid and they have unique mRNA display and PUREfrex is used for this process.
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And also they use PUREfrex for the high script expression for functional screening of their cyclic peptide afterwards.
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And many researchers are publishing the papers using PUREfrex and those are some examples.
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And again, a Stanford research group publish the paper about the sophisticated validation platform for the comprehensive analysis for the interaction between peptide and its binding molecules like this.
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And PUREfrex is used here.
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And going down, another research group is using PUREfrex for the high throughput expression of antimicrobial peptide and they executed a functional screening afterwards without purification in this case.
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And Roche Pharma reported the VHH screening with ribosome display with PUREfrex like this.
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And academia reported the ribosome display with PUREfrex for the epitope mapping in this case.
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And like that a PUREfrex is used for in visual display technology as well and it's working well.
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And the other report is here for mRNA display and cDNA display here.
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And in this case, the mRNA display is a researcher compared some product similar to our product, but they mentioned our product is the best for mRNA display because of the low amount of contamination such as RNA.
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So we are happy for that.
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And this is a research from the Nature and they are reporting the senior cDNA display with PUREfrex and that is getting for is a bunch of folding data and that is for aiming for the next generation of AlphaFold.
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And we are happy to contribute that kind of exciting research through our reactions and by ourselves.
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We are essentially working on the ribosome display and you can express the scFv or BHH or cyclic peptide and even Fab you can display the ribosome display with PUREfrex and like this.
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So you don't need to convert the Fab into SCP or single chain.
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So we have IP around here for sophisticated ribosome display system.
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And we have been licencing to the pharma including suit drop like this.
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And lastly, I want to emphasise again about PUREfrex, the advantage of the PUREfrex.
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PUREfrex has good fit to the AI machine learning approach with great controllability and reproducibility at molecular bases and with precise control of the platform and optimised platform can give you a great advantage in your R&D.
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That is a take home message from you, from me to you.
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And if you have interest or any new idea, I'm happy to talk with you.
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And we have a booth here.
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So if you have any, if you come up with a new idea, please stop by our booth.
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Thank you for your attention.
