hello everybody my name is chantha giulian and welcome to this presentation i work at novo nordisk pharma tech i am the product manager for our recombinant insulin for use in cell culture media during this presentation i will introduce you to a doe statistical approach for media optimization and i will present be presenting two case studies as well where insulin was used in media so i will begin with a short introduction [Music] also a summary of the um the function of insulin in cell culture then i will present a case study on using the doe approach for cho cell media and i will finally present two case studies using cho cells and hec293 cells so a few words about who we are at nordisk farmer tech we are located in denmark and we are 100 percent owned by novo nordisk which is the global leading manufacturer of therapeutic insulin at novo nordisk pharma tech we are a supplier of ingredients to the pharmaceutical and bio bio pharmaceutical manufacturers worldwide including recombinant insulin so what is insulin cellular functionality in vivo insulin is a major metabolism regulating hormone it is secreted by beta cells of the eyelids of langerhans in the pancreas the major function of insulin is to maintain low blood glucose levels in vitro insulin functions as a growth factor insulin is actually a member of a family of growth factors including the insulin-like growth factors igf-1 and igf-2 insulin regulates cell growth and differentiation cell survival and cellular glucose uptake by modulating transcription dna synthesis replication and stimulating protein translocation different intracellular signaling pathways are initiated by insulin binding to its receptor such as the activation of pi3 kinase akt signaling triggering glucose homeostasis with glucose transporter glut4 translocation and fusion with the plasma membrane and the activation of res erg pathway with activation of transcription dna synthesis and sidewalls now the cell culture media hindu industry has advanced over the years from serum based media to animal derived component free media a continuing trend is to shift towards defined systems to support manufacturing quality requirements of cell therapy gene therapy tissue processing vaccines and recombinant protein today the most extended practice in the culture of mammalian cells is the use of commercially available chemically defined cd media these media are optimized to support sub growth and recombinant protein production and are usually very complex however this their composition is usually proprietary and unknown for the user when used for a specific cell line cd media may not support all its specific requirements and therefore additional supplementation with other compounds at optimal concentrations may provide substantial improvement in cell performance there are different approaches to media optimization the case study presented here was conducted in a partnership with dr frances codia at the university of barcelona and we used the chosen line cho is still the preferred mammalian cell host for production of recombinant protein including including maps media supplementation is generally addressed by means of a one factor at a time or classical design of experiment approach but these techniques may not be efficient in initial screening phases as they are time consuming and costly in this study a doe statistical approach was used to achieve the best performance in increasing both self-world and map production through supplementation with animal-free recombinant compounds eight components as you can see on the slides on the slide were selected according to their capacities to improve cell culture conditions in a chosen culture system a placard berman design any box banking design were used and synergies between compounds could be detected with a reduced number of experiments by using this method compared to more conventional conventional design and this led to a fast and efficient screening and concentration optimization phase the first part of the work consisted of a folded over ppd so plackett-burman design and was used to detect possible synergies between different compounds in the screening phase pbds are developed to eliminate factors having no or negligible effects on over the responses this means that only main effects are considered without detection of factor interactions leading to potential incorrect decisions so this would be the traditional pbd which is shown on graph a on the other hand a folded over matrix pbd here in this example on graph b we have a 20 run 24 run folded over ppd improves the detection of factor interactions in the screening phase of a full doe approach growth kinetics were followed every 24 hours during 8 days that allowed to determine the maximum viable cell concentration for each condition tested the 8 compounds were screened at 2 levels a low level meaning no additive and a high level that was defined based on toxicity experiments the classical approach showed transfer in was the only factor affecting cell growth that would be on graph a while the model containing interactions on graph b showed insulin and two significant two-way interactions between transfer and insulin and transferring into an 80. therefore recumbent transferrin and insulin and tween 80 were chosen for subsequent concentration optimization using the bucks bank and design to optimize the concentrations so in this phase in the bucks banking design phase the range of insulin concentration was reduced since no improvement in cell growth was observed beyond two milligrams per liter of insulin 3d plots were constructed for visual analysis of the response illustrating different combinations of insulin transferrin in between 80. the highest level of cell concentration were obtained with a high level of transfer in or with the highest level of insulin and a moderate level of transfer in that is seen in graphs d2f and as the transfer and concentration increased the optimum optimum condition moved from higher to lower insulin concentrations and that is seen in graphs a to c the optimum with transferrin was achieved with a very high level at 57 milligrams per liter and this was considered difficult to be adopted in practical terms due to the high cost of transferrin and therefore 21.3 milligram per liter of transferrin 2 milligram per liter of insulin and 1.8 times of tween 80 was selected as being the optimum condition and under this condition the predicted maximal maximum viable cell concentration was nine plus minus one million cells per milliliter and for the full data and statistic analysis you can refer to a published paper called a statistical approach to improve compound screening in cell culture media that was published in engineering in life science in 2018 by professor gaudia's group now following the doe approach optimal conditions were validated against negative control cd media without supplementation and cell growth and cell productivity were measured the triangle in light blue the line with the triangle in light blue shows that cell growth and the cd optimized media versus the dark blue line which is the negative control and the live blue circle line shows that my map titus in the cd optimized media was better than the one with the pink triangles which was which was the negative control and under these optimized conditions the viable cell concentration reached their predicted nine million cells per milliliter in a map tighter of 120 milligrams per liter to summarize the sequential use of this modified placard berman in combination with the box banking design led to a 1.5 fold increase in cell growth and a two-fold increase in a map titer in suspension batch culture as a follow-up to this study the effect of insulin media supplementation was tested in three types of commercially available cho cd media that are called here cd1 cd2 and cd3 and that was to compare cell growth and cell productivity in a suspension batch joe cell culture system the black diamonds show the negative control cd media without insulin and the green and light blue lines represent respectively one milliliter one milligram per liter and five milligram per liter of insulin supplementation and already after 48 hours culture a significant improvement in cell growth was achieved with the addition of insulin and in all three cd media differences in several peak at around 100 120 hours were observed before a viable cell density drop and this is due to the saturated culture conditions the same cd chill media were tested for map productivity with and without insulin supplementation and here we achieve up to 66 percent increase of target protein production with one milligram per liter of insulin supplementation a second case study was generated in collaboration with the national research council in canada conducted the in the lab of dr aziza mensur the aim of this project was to test the effect of insulin supplementation in hec293 cell growth and on virus production an in-house suspension cell line was utilized to in the study uh the in-house serum free 3f61 cells have previously successfully successfully been used for the production of influenza virus virus-like particles lentiviral vectors adenovirus and adeno-associated virus here they were tested in a commercially available cdhac293 media called cd1 and an in-house cd hack 293 media a that is called cd2 here with and without incidence implementation insulin additions were performed every 72 hours at two different concentrations 10 and 20 milligram per liter of insulin the dark blue line here represents the negative control with no insulin and in cd1 the cell density remains below 1.5 million cells per liter with addition of insulin on the other hand viable cell density reached about 6 million cells per milliliter and there were no significant difference between the two concentrations of insulin in the second set of experiment it was performed on cd2 with this media this media was developed specifically for this cell line so with or without insulin a cell density of nearly 6 million cells per liter per milliliter is achieved this level can be reached about three days earlier with the addition of insulin thereby accelerating the process the effect of insulin supplementation on cd media was also tested on influenza virus productivity infection was performed with a multiplicity of infection in moi of 0.01 that means one bar viral particle for every 100 cells h1n1 puerto rico 8 34 zero type was selected and three different concentrations of insulin ranging ranging from five milligram per liter to 25 milligram per liter were tested insulin was added at the time of infection together with the addition of trypsin the viral titer was measured by immunoblot assay against h.a epitope in this case insulin supplementation led up to a 1.6 full increase of viral production compared to the control cd media lastly should you need recombinant human insulin animal-free insulin for your cell culture media the recombinant insulin that we offer is manufactured by our mother company nuvo nordisk and it is manufactured according to the highest highest available standards on the market gmp standards and it is analyzed in compliance with the european pharmacope and the us from eccopay and we also offer a complete documentation package you are welcome to contact us for more information [Music] or of course to visit our home page at nominal disclaimertech.com if you want to get more information thank you very much for your attention today and please do not hesitate to write with your questions we will be happy to respond thank you