hello and welcome to this presentation my name is chantal giulian i work for novorodisk pharma tech i am global product manager responsible for our recombinant insulin for using cell culture media today i will be discussing if recombinant insulin should be a factor to consider for viral production i will be beginning with a short introduction about novo nordisk pharma tech and who we are then i will briefly discuss viral systems their status today then i will move further on to our hypothesis on the use of insulin for viral production then i will be presenting two case studies one on adeno-associated viruses and one on lentivirus and i will have a brief conclusion afterwards novo nordisk pharma tech is a fully owned doctor company of novo nordisk novo nordisk is the danish global leading manufacturer of insulin for therapeutic use the vonardis pharma tech is also located in denmark where we have our headquarters and just above 200 employees we supply recombinant insulin to cell medium manufacturers and biofarm manufacturers worldwide we have local sales offices in singapore and in the u.s and in the us we also have a local distribution center for our insulin viral systems today i think we can agree that for the past 10 to 15 years we have seen a tremendous development in this area and today viral systems are commonly used in both r d and therapeutic applications where they receive more and more acceptance by patients and practitioners and where we find some of the most effective cancer therapies available today so they are used for delivering transgenes in cell and gene therapies and in vaccine production there are numerous clinical trials ongoing worldwide at the moment and this is both for new product approvals and also for new applications for existing products there are currently 22 gene therapy products approved by the usfda and 11 in europe by the mea and in the us only close to 50 new therapies are in significant late clinical trial stages so this creates this is an area in expansion and it creates increasing needs for production scale up optimizing processes and increasing yields now whether you are a medium manufacturer or a biofarm manufacturer many today turn to animal component free or chemically defined cell culture media for safety reasons these media can be complemented by adding metabolites such as interleukins for example or growth factors such as recombinant insulin this is what we want to take a closer look at today the effect of insulin on viral production if we look at already published data [Music] several have reported significant improvements when using insulin in cell culture media for viral production if we look at the left-hand side these are results reported by the national research council of canada where they used hector93 cells for influenza he protein production they used the chemically defined media where they added 10 milligrams incident per liter of media and they observed a four-fold increase in viable cell growth and a 60 increase in viral titer if we look at the right hand side these are results reported by the university of barcelona where they also use hector93 cells here for producing hiv one gag virus like particles or vlps they also used a chemically defined medium this time adding 20 milligrams of insulin per liter of media here they observed the an almost twofold increase in vlp production and a proportional improvement in cell growth therefore we had very good reasons to believe that recombinant insulin would have a positive effect on the production tighter for lentivirus and avs using hectonana tree cells we commissioned two studies at quasal biotechnology they are a cell line and cell media manufacturer and supplier we wanted to look at the effects of insulin concentration and addition time on cell growth and viral expression the variables that we have been looking at you can find them in the table at the bottom of the slide so we look at four different concentrations of insulin either 0 5 10 or 20 milligrams per liter of cell culture medium and adding times was either at cell inoculation two hours before transfection or four hours after transfection we use a four plasmid packaging system for lentivirus and a three plasmid packaging system for aav where we included three serotypes av two five and eight we use suspension adapted hec 293 cells and we used 125 mil shake flasks with a 30 ml working volume we use quacell's own chemically defined transfection medium for packaging and growth and we supplemented with feed 03 medium two hours before transfection and our transfection region was pei here we have illustrated the production process for antivirus and avs so on the left hand side for lentivirus after seeding and transfection after 48 hours we after transfection we harvested the lv particles from the supernatant and we infected ht1080 cells with the supernatant and 72 hours later we detected the target gene gfp by fax and for aav after seeding and transfecting we harvested the av particles and 72 hours later the viral dna was extracted and the itr target gene was detected by real-time pcr method if we look at the results for aav first so we observed no significant effect of insulin concentration or addition time on cell viability or density and this goes for all three serotypes on the other hand if we look at viral titer we have observed very significant improvements using insulin on the viral titer if we start on the left hand side for aav2 so the highest increase in tighter was 56 percent compared to the control when using 10 milligrams insulin per liter medium and the optimal addition time was inoculation if we look in the middle for av5 here we observe so the highest improvement in tighter when using insulin was 68 higher tighter compared to the control hereby using five milligrams insulin per liter of medium and the optimal addition time was at inoculation or two hours before transfection and again on the right hand side for av8 so generally a positive effect of insulin on the viral tighter here the highest increase was 51 percent compared to the control here using five milligrams per liter of media the optimal addition time here was two hours before transfection using the ue software for multi-factor analysis we could conclude here that adding recombinant insulin had a significant positive effect on the titer of both av 2 5 and 8 although it had no obvious effect on cell density or viability and that the insulin adding time had an effect on virus tighter therefore our recommendation would be adding 5 to 10 milligrams insulin per liter of medium at the time of inoculation or two hours before transfection can improve aav tighter in serum free suspension systems now if we look at the results for lentivirus again we did not see a significant effect of insulin concentration or adding times on cell viability however here we saw an effect on cell density over time so the highest improvement we have served here was a 40 increase in cell density compared to the control when using five milligrams insulin per liter of medium and the most optimal results were obtained here when adding insulin four hours after transfection compared to the time of inoculation or two hours before transfection now looking at the the viral titer so all the way through here we can see a positive effect on the titer when using recombinant insulin as compared to the control the most significant improvement we observed here was when using 10 milligrams insulin per liter of media where we saw a 120 percent increase in viral tighter compared to the control and here the most optimal adding time was two hours before transfection so again using the oe software for multi-factor analysis we can conclude that adding recombinant insulin had a significant effect positive effect on cell density and virus tighter for lentivirus although no obvious effect on cell viability and also insulin adding time had an effect on cell density and virus tighter although no obvious effect on cell viability therefore here our recommendation would be that adding 10 milligrams of recombinant insulin per liter of medium two hours before transfection can improve cell density and tighter in serum free suspension systems now looking back at the results we obtained for these two studies when we initiated the studies we were expecting to see significant improvements both in cell growth viability density and viral titer although it is obvious that or the results were more obvious on the viral character and we did not necessarily obtain improvements on the other parameters and we have come across a few other studies where others also have observed an improvement in vile tighter when using recombinant insulin but not necessarily any significant effect on other parameters then we can speculate as to why we observe results like this so we know that insulin acts through different intracellular pathways in the cell and that it acts through cell differentiation it acts on cell differentiation and glucose uptake pathways we know that insulin has it counteracts apoptosis cell apoptosis and it promotes mitogenic pathways and it can interact on cellular signaling pathways that can be directly used or exploited by viruses so we believe that this could be an explanation for these results although this would require further investigation in conclusion again we can say that using recombinant insulin for lentivirus and these three zero types of aavs two five and eight had a very significant positive effect on tighter and in one case also on cell density and this is similar to already published results on the same topic now of course whether you are preparing your own cell culture media or you are purchasing a commercially available subculture media these media would require optimization and especially under scale-up processes depending on the cell line that you're using or the the virus that you're using various type or serotypes or the end product that you're looking for uh different levels of insulin would be needed uh depending on your process and on the media that you are using and of course if we want to look into further optimization of the use of insulin in cell culture media we could look at refining concentrations for example in the interval between 5 and 10 milligrams per liter and we could also look at further optimizing and refine refining adding times we are interested in using or testing recombinant insulin for your cell culture media cell-based processes you can contact us we offer the highest quality recombinant insulin on the market for use in cell culture media our insulin is manufactured in gmp facilities by our mother company novo nordisk we offer a full documentation package and services and our product is of course in full compliance with the european pharmacope and the u.s pharmacope you are welcome to contact us through our webpage or our linkedin profile if you have any questions or wish to know more about this presentation or our products i would like to thank you very much for your attention today and i also want to extend special thanks to dr henry yu at quasal biotechnology and also several colleagues at nugent's pharma tech that have helped prepare this presentation thank you very much