it's very it's a very big pleasure to be here today so my name is Sarah busamano and I'm a global product manager and I'm an artist Pharma Tech and today's talk is gonna focus on insulin and it's used as a supplement in animal-free cell culture media to improve cell proliferation and cell productivity so before I start I would like to give you a brief introduction about Northeast pharmatech so I guess that some of you in the in the audience know number Nordisk which is our mother company which is the world leader manufacturer of insulin for diabetes we are not this Farm attack and we started our business in 1949 with the name we were called theft chemicals we were producing quads which are antimicrobial agents and Affinity resins then we got acquired by Nova Nordisk and we got fully integrated into um now an artist product supply of raw material for different biomanufacturing processes in another Northeast so we were supplying Affinity resin specialty enzymes at the same time we were selling rods to the external market then we also started selling and distributing novenority skin slim but for non-therapeutic use so as ingredients in cell culture media and then we changed name to known artist pharmatech So based on many years of experience in the supply chain of normal disk we gained extensive experience in supplying raw material to biomanufacturing processes so our product portfolio covers the entire biomanufacturing process both upstream and downstream processes we have insulin to support cell growth cell proliferation we have process enzymes for protein modifications but we are also expanding our uh portfolio our enzymes portfolio with cell culture enzymes we are soon to launch um some cultural enzymes for cell dissociation and special form also stem cells I would like to invite you to our booth number three to talk with us about our new enzymes we have Affinity resins and also here we are expanding our portfolio with customized resin solutions for the purification of a broader range of molecules and then we have our reports which are the antibicrobial agents that are used as a for in the formulation of different type of drugs um yes but today we are going to talk about insulin in cell culture media so insulin is a very interesting protein has a very fascinating history because insulin as a first isolated in 1921 from dogs pancreas and then only one year later has been injected for the first time in diabetic patients one year later it was produced in such an amount that was able to cover to supply the old North America um and this was insulin that was extracted from cattle and Peaks I already here in the night in the 20s no one artist was playing an important role in a commercializing um insulin for diabetes then 50 years later insulin porcine insulin was produced for the first time in Nikolai so insulin was the very first therapeutic protein to be produced as a recombinant protein and a few years later human insulin was produced recombinant and the funny part is that insulin started as a this therapeutic protein that became the first therapeutic protein produces recombinant that being that has been incorporated into the biomanufacturing of other uh therapeutic proteins like monoclonal antibodies because insulin is a very unique ingredient in cell culture media in serum freestyle cultural media formulations so traditionally we are all very aware that cells to be maintained in culture need to be supplemented with nutrients and traditionally these nutrients derive from federal government serum um which is a rich mixture of components um you have vitamins proteins lipids growth factors you name it so it's a rich mixture of components the really supplement cell growth and it's such a powerful tool that we're still talking about it and we are still using it in our cell cultures 70 years after it was first discovered however there are some limitations coming with the use of fatal bovine serum first of all is um is a mixture of ingredients so it's not well defined so it's not well defined the amount of its components and it's subjected to a lot to lot variabilities you can imagine deriving from animals uh derive some animals so this means there is also a high risk of high animal derived contaminations and is also um linked to Big ethical concerns because of the way it's manufactured from and the way is extracted from fetuses so there is this trend in the biopharma industry to move away from um from serum and to develop serum free media so it's widely widely used in free media that's are still free of course but still need to be supplemented with some nutrients in order to make the cells leaving cell culture and these nutrients can be can derived from hydrolysis for example or from Individual proteins that are supplemented to the media again idealize it it's a mixture of components so it's not well defined and again in this trend moving away from animal derived and going towards more defined culture media formulation the latest to be identified the latest media to be um discovered is a chemically defined media which is the most the most widely used chemical defined media and biomanufacturing where every single ingredient is well identified well characterized and measured so this ensures high repressibility of the um of the process but in this uh trip towards a more and more defined media formulation we are we are losing such culture media performance because the cells really need to be supplemented with um with some metabolites in order to live and be maintained in cell culture and there are a broad range of metabolites that can be supplemented it really depends on the cell types on the cell application on the production of the cells and so on but there is a rather Universal ingredient in the serum free and chemically defined culture media and this is the insulin that has been found to be um similarly in almost all cell types that been tested so how is insulin working at the cellular level to stimulate cell proliferation so insulin is binding to the insulin receptor which is a tyrosine kinase receptor but after binding gets phosphorylated intracellularly and activates metabolic pathways including glucose intake and this is these are the pathways that are beneficial for for diabetes this happens at the physiological levels of insulin but when insulin is added at higher concentrations which is the case when you supplement oops sorry when a supplement insulin into the cell culture insulin is also binding to the insulin-like growth factor one receptor so ihf1 receptors this is also tyrosyncinase receptor belonging to the same family of the insulin receptor and after binding again autophospholates and activate intracellular Pathways for cell growth differentiation and cell survival so in a way insulin is acting on two different levels to support the graph both acting on metabolic pathways and on cell growth and survival pathways of course there are other members of the insulin family of the growth factor insulin family that binds to the igf-1 receptor and this is hf1 um that can bind to its own receptor activating the same Pathways however the there is a limitation in idf-1 that can be bound by igf-1 binding proteins and when this proteins bind to hf1 they prevent the its binding to the receptor therefore it cannot activate the downstream Pathways while insulin is not subjected to The Binding uh to this binding protein so it can always activate both metabolic and cell survival pathways so for many reasons insulin has been widely adopted in different biomanufacturing processes in the biopharma industry so it's an ingredient in this culture media that can be adapted to different scales of production uh currently it's been widely used for different biomanufacturing for different therapeutic products like monochronous antibodies but also for the production of viral vectors for cell engine therapy and also for the production of viruses and virus virus particles for the preparation of vaccines um it is really important when you use of course an ingredient in biomanufacturing to have the highest purity of the highest quality and we our insulin is actually the highest uh Puritans in that you can actually find because it's the same insulin that is administered to diabetic patients so it's highly pure and it's constantly um exceeding the pharmacopoeia requirements both in Europe and in the US and as I said the beginning is animal free because it's producing yeast so it's um the bright side also is that our insulin is already approved by different regulatory agencies for the for different applications and um it's because it's produced by the world leader manufacturer of insulin can rely on many different um manufactured sites around the globe and this ensure the security of the supply chains also in this difficult times like the ones we are living now and it's produced in multi-term capacities as well okay so let's move a little bit into the science now so we have I'm providing here a few evidence of how insulin is actually supporting cell growth cell proliferation and serum free uh cell culture media so I'm going to show you just a few examples uh there are more of course and there will be more so now this first example is um we supplemented insulin into a chemically defined media for the growth of Choke A1 cells producing monoclonal antibodies so in this experiment um the cells were grown in um yeah of course in the bezel media without insulin or in the presence of different concentrations of insulin we tested two five and then milligrams yes okay so we tested different concentration of insulin and there was supplemented on the at the Baseline in the Basel media so we observed that and then we followed sorry the third proliferation over time we observed that on day nine there was a peak in cell proliferation also in cells they were not supplemented with insulin so without insulin but this peak was even higher um in the cells that were supplemented with insulin two micro milligram per liter so among all the concentrations of insulin that we tested in this experiment the lowest concentration actually worked better than the highest and we observed that this increase in cell proliferation was not um I was not a consequence of a change in cell viability indeed the cells um were not dying more without insulin they were just not proliferating as much as in the presence of insulin and then we looked at the production of these cells that were producing a monoclonal antibody and we looked at the production of the IGG on day 9 day 11 day 13 for all the three um concentration of insulin that we tested and in almost all cases we observe an increase in the production of monoclonal antibodies in the process of insulin with the peak observed on day 13 when insulin was supplemented at concentration of two milligram per liter and this increase correspond to a 50 percent increase in the monoclonal antibody yields where without insulin we reached a foreground per liter of monoclonal antibody with insulin we reached almost six gram per liter of monoclonal antibody in the culture then we move to the second example in this example we looked at the effect of insulin on the proliferation and productivity of act 293 cells they were adapted to grow in suspension and these cells were producing uh were used for the production of influenza virus so in this study we have the cells act 293 were adapted to grow and suspension in he now's chemically defined media and were then supplemented with different concentrations of insulin here we tested 10 and 20 milligram per liter so an insulin was supplemented every two every 72 hours so without insulin you can clearly see that the chemically defined media that we are using in this experiment was not able to fully support the cell growth and circular Refrigeration so the cells were struggling to survive while with insulin of course we observed this four-fold increase in self-proliferation already a 10 milligram and even higher 20 milligram per liter of insulin and in a way we can say that insulin is alleviating the growth limitations that are observed in the chemically defined media that does not contain serum does not contain proteins but um so this is why the cells could not could not grow as beautifully so but then when the x293 cells were transfected with virus to produce H1N1 influenza virus and were supplemented with insulin we did not observe any change in cell ploreiferation rate of the cells after insulin supplementation even when we supplemented very high concentrations I mean even 10 hundreds um microgram per milliliter of insulin so there was no effect after um infection with the influenza virus but these cells were producing High um height title of high amounts of H1N1 influenza virus in fact we observed that after supplementation of insulin already at very low concentration of insulin far from the 100 milligram plated of the previous slide so already a five milligram per liter there is a 60 percent increase in the production of the H1N1 influenza virus and but this increase in the virus yield was not uh was not due to an increase in the cellular Refrigeration and then in this uh last study here we again looked at 293 cells proliferation and productivity but producing adenovirus and Antivirus again insulin was supplemented in the chemically defined media for the growth of this x293 cells they were adapted to Growing suspension so in this study we have um suspension adaptive 293 cells growing in flasks they were respected with the three plasma systems for the production of three different stereotypes of adenovirus we looked at the nanovirus 2 5 and 8. and the cells were we also looked at the cells transfected with a four plasmic system for the lentivirus and cells were constructed with the Pai Pei transfection system for transient transfection so these cells were also supplemented with different concentrations of insulin there was added a three different addition times so we looked at addition at the cell inoculation or two hours before inoculation or four hours after and then 72 hours after in infection we looked at a cell proliferation of the cells and the virus virus virus production for adenovirus and Antivirus so for adenovirus for all three stereotypes that we looked at we observed a general increase in the viral title after supplementation of insulin and all the different concentrations of insulin that we tested we tested 5 10 and 20 microgram per millimeter of insulin in cell culture and among the three um the three concentrations that we tested the 10 microgram per milliliter in all cases yeah as you can see with the asterisks here so the 10 microgram milliliter was the concentration of insulin that brought to the highest viral title for adenovirus especially when um and this increase corresponded to a 1.5 fold increase for all the stereotypes that we looked and the highest increase was observed when when insulin was supplemented two hours before infection also in this case like in the previous case study we did not see any change in the cellular Refrigeration rate when the cells were transfected even presence of insulin meaning that the cells are acting on different cellular mechanisms to activate viral production and then lastly with uh with Lindsay virus we also observed a general increase following insulin supplementation with the highest increase corresponding to a two-fold increase in a lentivirus title observed when insulin was supplemented at 20 microgram milliliter again two hours before inoculation in this case we also observed a change an increase in the self-proliferation rate in as a consequence of insulin supplementation Okay so in conclusion so we saw different different applications different cell types uh different media but um we can say that in all cases that insulin is actually acting to intensify the production of the either monoclonal antibodies or viral vectors as in as shown here so insulin really works to intensify the production across different types modalities and workflows as well and it is very very important to really fine tune you might be aware you're aware of it that fine tune different um supplements in the third culture because you can really work magic to to improve your work um your biomanufacturing process if you think that a two-fold increase but doesn't mean corresponds that potentially you could run two times less volumes to have the same amount of um viruses or proteins that you're running of course cost analysis needs to be run and but you can you can really save um time and you can have benefits on your process economy by really fine-tuning the right supplements like insulin and here as I said we have a few examples of possible applications insulin is already widely used in biomanufacturing so we have already broad portfolio of applications but we are always eager to learn more we always want to test insulin more so yeah we're open uh to learn more from you and I would like to invite you to our booth number three today and tomorrow and to discuss any synergies or possibilities to investigate more yes thank you [Applause] thank you very much Sarah so we opened the board for questions um yes please choose the microphone if possible for the viral Vector applications um for like influenza or lentiviruses did you when you were looking at the titers did you check physical titers by like Eliza on the surface proteins or did you do that via infection like actual functional particles it was we looked at the for the influenza virus we looked at the HR h a hemogloating expression by I am by philosophytometry So Sorry by with antibody so it was like a physical testing not application testing testing and then antiviruses uh then we looked at the again for the lentivirus we looked at the gfp expression because it was yeah with the facts by facts and for the denovirus we measured by PCR thank you yes since uh thank you for a very nice presentation uh you really showed that instrument has an influence on the production levels both in child cells and 293 cells what strikes me a little bit is that the influence in the crow on 293 cells is much higher than onto our cells when you look to the uh culture without insulin so can you say something about maybe a different mechanism between two cell lines the influence of insulin on the cell how does insulin enter the cell is there a difference okay yes we really is a valid question because we really observe big changes in how insulin acts on different cell lines different cell types of course and there are also changes on in the same cell line but for different applications also on the type of virus that is produced by the same cell line so if you also see how in the x293 cells that are producing adenovirus or that are producing antivirus they behave differently so the ones producing and nanovirus are not growing after infection with the insulin but the other ones are growing with insulin after infection so there are insulin is acting not only on the on this metabolic and cell growth Pathways but is activating also some intracellular Pathways that are the ones that are used by the virus as well and this is phosphorylation of akt MPI 3K so we have evidence showing that after insulin you have a peak in the phosphorylation of ekt which is actually this same pathway that is also used by for example influenza virus too so in a way insulin is prolonging the time that can be used by the virus to pack to do the packaging and to be to pop up from outside from the cell so in a way so there are other mechanisms that are activated by the instrument and then you have tested where there are is there an active insulin receptor on child cells sorry again these are an active insulin receptor to to enter the cells into insulin onto your cells there can be yes they can be and but there's also what is also known is that the insulin receptors at very high concentrations can be internalized so in a way we think that insulin is acting more on the igf-1 receptor for the cell proliferation because insulin receptor can internalize it can still be um let's say it can go again onto the cell surface but it's not very clear to which concentrations but um yeah thank you very much yeah thank you thank you very much for this very nice talk it was very nice to see the difference between the hack and the show cells like but I was just wondering so for the production of maps in a show have you observed any impact on the product quality um no we did not but I will say that the product quality is pretty high because we it's widely monoclonal antibodies already in the market are using insulin so I wouldn't say that there is an effect on product quality if not it's a good one yeah yeah okay thank you thank you okay we have a question we have time for one final question yes please okay thanks for our presentation uh one question around uh the different case study you had uh presented here where we found a different concentration of insulin affects the cell floor Refrigeration correct so uh because we saw that sometime microgram per milliliter then sometime 10 sometime 20. so is there any thought process that uh about how means we have to do the development study always or is there any standardization uh we are thinking of yeah so the one thing is that optimization is the is the crucial word because um it's very important to optimize if we cannot say 10 Micron per milliliter works for all cell types it's very it really varies as you say as you see it varies from in the same line for different applications so imagine across different cell lines and also within the same size within the same application we also observe differences in a concentration when insulin is added in the bezel Media or it was added in the feeding media for example in fed batch cultures so there is big changes but I think it's really important the optimization really pays back because if you can really optimize and fine-tune it's I think you can get the best because it doesn't necessarily have to be highest concentration of insulin most likely you need to use the lowest concentration to have the most optimal result so um yeah so thank you very much thank you Sarah thank you