 Over the last 30 years, protein-based medicines have emerged as therapeutically and commercially important drugs. However, as these biopharmaceuticals are proteins, they are only marginally stable and so their structure can be perturbed by small changes in the environment encountered during manufacture. So our initial study which we published earlier last year, we were interested in how extensional flows and shear flows come down into proteins. So what happens with extensional flows? This could occur in filtration for example, where you have a flow moving slowly and then all of a sudden there's a contraction in the flow and the fluid is forced to rapidly accelerate. So using this model here, this is a GCSE which we stressed in our previous study, you could be trundling along slowly and then at the contraction point, as the flow accelerates, the protein may stretch out exposing aggregation prone regions and then in our capillary here, we have a high shear region, so we have a shear flow where we have layers of fluid travelling at different speeds which may influence the aggregation. Now rather than a mock-up of the device light we have here, our real device consists of two syringes connected together with a capillary, we shuffle our protein of interest between the two syringes and then perform a range of analytical techniques afterwards, one of which was a pelleting assay where we spin the sample down and then quantify the amount of protein left in solution afterwards. As you can see on the screen, we took an array of different proteins in our previous study of various topologies and pharmaceutical interest from our collaborators, Medinium. Now we stressed our proteins for twenty to a hundred passes, but what we could see is they have markedly different aggregation behaviour, but this was all done at the same speed. So for BSA what we found is that when we stress it for a hundred passes, a range of different speeds, there's a plunger velocity and equivalent strain rate where you start to see aggregation in a region where we didn't get any. So what we thought for these biopharmaceutical proteins which are much more susceptible to the effects of extensional flow is that perhaps this speed threshold may occur earlier and that the subsequent aggregation landscape may be more complicated. So in this study we really put our different proteins, so this is for the BSA and the two antibodies under the extensional flow and this is also included in the CL course. So basically these are the representation of the 3D plot or 3D surface, the number of passes plunger velocity versus the protein aggregation. So you can see like BSA requires some sort of the threshold to unfold and followed by the aggregation. However the two different proteins or the two different maps they are really sensitive to the extensional flow and they can easily differentiate based on their 3D surfaces as well. So if you see the STT for example which is more resistant to this one so it's covered most of the blue area which correspond to nearly 20 to 40%. However the WFL which is more aggregation which is mostly covered the red of the area and despite these two proteins differ only by six residue. So as Amit has shown you these two proteins that only differ by six residue show remarkably different behavior under different fluid strains and stresses. This led us to think about assessing the ability of this device in formulations studies and this graph here shows that the most aggregation-prone protein here WFL showing black bars is essentially resistant to any changes in aggregation propensity. However the more manufacturable protein STT shows that histidine buffer this protein forms less well in histidine buffer but performs very well in phosphate buffer as this graph shows the percentage of protein in the pellets. And interestingly another protein called MAB1 shows completely inverse behavior. So together this study shows that extension flow can be used to assess different candidate proteins for their aggregation behavior early on in development. It can also be used as a formulation tool to allow production of difficult to produce proteins and it can also be used to investigate whether bioprocess parameters can be changed to allow the production of difficult to produce proteins.