Discover the Best Decoration Bill Format for Animal Science

Decoration bill format for Animal science

hello we're glad you've joined us for this live webinar rethinking the timeline required for Quality by Design studies in protein process development I am Brenda Kelly Kim labyrinth I'll be moderating today's session today's educational web seminar presented by lab routes leading scientific social networking website and provider of virtual events and webinars advancing scientific collaboration and learning it's sponsored by perkin-elmer PerkinElmer instrumentation helps to advance biotherapeutics research by automating small-scale protein purification and protein characterization to learn more please visit us at .piofl.com I'd to do so simply type them into the Q&A box which will open when you click the green Q&A button at the lower left of the presentation window questions will be answered after the presentation to enlarge the slide window please click the screen icon in the lower right hand corner of slide window if you experience any technical problems seeing or hearing the presentation just click on the support button found at the top right of the presentation window or report your problem by clicking on the green Q&A button at the lower left I will now present today's speakers Lou fabry is a senior member of the CSL research staff heading up the protein chemistry purification group he leads the high-throughput purification of protein and macromolecular assessment of proteins for research based studies in CSL limited Lou will be followed by bill clarity a bioprocess scientist with Merrimack pharmaceuticals within the process development group where he is charged developing methods and characterizing molecules as well as supporting process to development and manufacturing prior to this role you work for eight years first at wyatts and Pfizer contributing to analytical development efforts across their pipeline bill is a WPI grad the substantial experience and assay development across multiple research areas and validation GMP environment I'll now turn it over to lose to begin to the presentation hi it's a loose I agree here from yourself and my talk today is that based on protein purification and the high throughput protein production in the research based industry settings so by way of introduction what I would like to speak about is just a little bit about their cell and who we are given that we're here in Australia and then specifically about what the research group does and then from there sort of going to what we do within the protein purification group now one of the things I'll talk about is the fact that we produce quite a lot of proteins within the research group and one of the key tools for us to be able to do that is to be able to achieve the throughput and we did this array of multi-dimensional chromatography traditionally with these technologies such as the FDA Express but more recently what we've been able to do is miniaturize purification process and utilized liquid handling to see such as for Janice to produce the purification that we need in a Hydra commode now once we've been able to do that one of the things we need to do make sure we can take full of is producing utilizing the janitor of multi-dimensional chromatography and ultimately I'll just make the concluding remarks about have a successful we've been in producing a purification system utilizing liquid handling system magenta so psl is a global specialty biopharmaceutical company and we last year had 25 billion dollars in sales in plasma products and predominantly these days apprentice such as argument and uniform with the manager we are now opposes second Rogers up flu vaccine companies in the world and CFL employed 16,000 employees will arrive in as many is going to come treat and some of those are expensive it's that others are actually significant manufacturing practices are that it's a special group last year CSL thanks 460 million dollars in R&D investment alone so there's quite a huge focus on our own game and develop a new product if in CSL more recently we've introduced to recombinant protein based therapeutics a Delvian and that Scylla and these are both recombinant protein based therapeutics providing treatment in both hemophilia A and hematuria both a day well more long term some of the types of proteins of CSL is also trying to develop as well and additional other coagulation therapy recombinant proteins are recombinant antibodies and there's quite a lot of work in our pipeline to develop seeing as potential need therapeutics for quite a few different therapies so in terms of one of the purposes of the research group we predominantly try to discover league protein candidate and then move those through the discovery quad lines into developing them as a drug candidate and then consequently are dealing moving these things through clinical trials and into into being a pharmaceutical products in such a competitive market being able to do this fast efficiently and effectively is really importantly important key in terms of developing therapeutics and within research arranged to identify key protein very early on look at the ability of having food can be manufactured and focused on generating these elite proteins typically we're also working with very small amounts of protein and we need to do this so that we can produce a large quantity of protein once selected the protein other growth lead optimization and ultimately solar cell line development within the research group before moving on to product development and then ultimately on to manufacturing so ideally what we try to do within the research group he's actually not only that I've defined as the efficacy of the lead candidate but also powerful transition through the formulation and the purification steps that will be coming coming on board in the different processes so we produce quite a lot of proteins and using this is essentially the basis of my talk you is looking at the numbers of proteins that we produce for Leo anybody's watching and how we are able to handle that sort of the mouth so typically for example last year we produced and have those two products we use approximately about 1,500 proteins annually and this could be as little as an overgrown quantities of protein all the way through to gram protein requirements for things like preclinical tops any animal studies that we do as well as we're also the esso development we own stages of the discovery process we also generate material for reference shock as well as feeding to equally poor cell line development so ultimately for us two types of selection processes that we have the key consideration for appointing you what the affinity of the protein might be and identifying that in terms of a selection process is critical and also then looking at the efficacy of the mountain material from the proof of concept study in animal studies now these two things are the primary selection two products area for pelicula equality candidate but what we've introduced as really as has many other industry company is the concept of manufacturability and so I dealing what that means for us can we produce a clean product as other research space can we look at how reproducible encoding to do is it consistently producing material with our ideal modifications or ideally no modifications and in terms of is it stable at a molecular level so do we see occupation for example do we do look at any other post translational modifications and if we do have these things and we will consider them and part of the selection criteria equally looking at whether protein aggregates is critical as well as whether we can formulate the material in button that would be typical during the downstream processes so how do we achieve this throughput that's right one of the key proteins that we work with Suzanne so keep a physically workish give multi-dimensional chromatography given that we do essentially purification with the group utilizing multi-dimensional chromatography is important from being able to achieve the throughput that we require the multi-dimensional chromatography of course is the separation of molecules through multiple steps and not multiple stationary phases sequentially to increase the separation performance of the purification and for us automated multidimensional chromatography made utilizing multidimensional chromatography but in doing that with an instrument independent of the user so typically what happens is that the aleutian is one protein during the first gradually fade we'll be looking into a hobby route that ultimately be introduced and in turn into the second column and the loop itself is an interface between the two different places chromatography now in terms of doing this and traditionally as I said before we eat a live human such as yet to experience the crisis chromatography that I'm talking about I think like a first step of affinity purification and then subsequently salty North products we journey to an appropriate butter for us each of the individual instrument is capable of running for proteins and any one stage and at a time this particular class we have six of these particular units in our group and that meant for example the we were able to produce 24 handfuls per day assuming that you would join your energy and decently however if you do inside excluding what that meant was actually for protein six proteins today which was equivalent to the sea creature months that we have so ideally this room could be is 70 proteins clinic or 100 samples all the way through or middle is about 20 proteins too late so obviously one needs to look at other processes for being able to maximize their troop however one of the good things about this instrument that we also identified was that there's a fairly good correlation between what we would expect as a theoretical recovery of each of in terms of what we see so them that the amount loaded so for example for as little as two milligrams of protein the overall observed Republic is around eighty five percent on two stages of chromatography and when you're using as less as little as portaak milligram this level goes down to about happiness on cutting classes and recovery typically the with this instrument for physics we below quantified milligrams the just the law of diminishing returns would mainly you learn for mostly protein so ideally looking for an additional instrument is critical and also looking at being able to do more than just 20 to 100 samples Kuwait was pretty pretty important one of the things that we looked into with utilizing the janet as we instrument of choice and so here what I've done in slides put up the extra method for purification you can see what we do here is we produce transient salon media and then we apply that to an affinity protein and purification in the math lecture and then by a series of washes we then allude that material and then from the Aleutian it goes on to our defaulting or projects cruises petition itself goes undergo school cleaning in place before a new sample is introduced onto the column and hence the process continues with the Janus we can do the same thing but obviously in a much smaller scale and utilizing columns such as the at home we were able to have a 96-well plate array and the introduction of a new shovel arm on the Janus allows us to move between racks and doing washes and then subsequent solutions so ideally we can utilize it as they apologize type extraction actually I generate a purification the fractions then of course they're losing to a 96-well plate then can be taken on for subsequent purification so that could be for example defaulting or it could be projects cruising on a different instrument one of the things we click is very early on with looking at what the dynamic binding capacity of the column on oxygen policy at all columns would be on the Janus as compared to reactor and then we slide what you can see sort of the resident I'm then technically beginner and then also to reactor you can see there's a significant overlap and correlation from the three areas that they overlap for the 25 minutes second residence time and people is a very close agreement in a dynamic binding capacity and this is because all other parameters are capable of being kept constant so things like the respective load and the linear flow velocity means tenses one screen equal why we Janice isn't shown at the higher concentration is course of the fact that it would be impractical practical to load as much longer residence time for the for the year for the genesis so looking at the Janus platform essentially we do affinity purification and then going on to for exclusion or dissolving the robo columns themselves coming out of that hole are shown your bottom lip and try to describe and you can see at this point and some of these some of the shower drain into the dry bones and one of the the critical features of this colony that it hasn't only rather only at the top of the column that causes the seal on on the very spend arms as they go into the the needles as they go into the column and their corner feel that as a pneumatic drive of the sample going through pushes the liquid through the column so essentially operates like Collinwood on a hplc and actually is able to accurately deliver volleys and on the right-hand side we see how big these conv and typically we have something like about a hundred microliters of reading within these columns and the beauty is the the size of these columns he said with a hundred microliters and a dynamic on capacity is typically about 30 milligrams and milk that means for a hundred lucky literacy you could probably put as much as the battery in your grams of protein and that's recoverable if if you are able to put it within the low groaning required from Jana and then subsequent to the actual purification the fate of the elution coming off East enemy column events either default image and the bottom part of the slide and what we utilize is a 96-well defaulting array plate column consumes of 96 columns with one military sausage or than 92 and it's a company called BNP that these could be purchased from and the maximum loading onto these columns of three hundred microliters but ideally the elution completely coming off the road back on and then be applied directly onto these columns and dissolved in in once said if so the question is required then you can also just packing material onto to something like an agilent system with an appropriate content so that you can then fractionated directly off that and we utilize Palkia calling for the autosampler as well as for the for the fractionation device to make sure that to deal with the throughput that we require and typically what this means is that we can get through at least 200 sample on a size exclusion and as many is about 500 samples per week if we actually need to go talk to my Lords out of that triple subsequent to the defaulting for the quality exclusion there are many other instruments that we utilize on the jet are coming off the Janus to also deal with the throughput of providing the energy in our own you have to do sighs inclusion that we have capabilities of looking at multi angle right scattering sds-page on the GX to as well as protein conversation quantitation on the on the job then some examples with our on the caliper gx2 instrument and you can see this is 45 sample of different antibody runs in a single run which for us is providing quite a lot of information on the purity and the quality of material that's coming off our Jana fisherman likewise the drop sense is a tree microliter nano UV that is capable of generating pruning quantitation in an unfixed well a race on that and the beauty and instantly because the place I was quite small it has a linearity up to about 30 absorption units so therefore a lot of samples this means is no dilution required which allows you to be a lot more accurately no UV quantitation determination and finally in terms of the other analytics that we can do you describe exclusion analysis releases that quite a few proteins run on on overlaid onto the ranch exclusion to showing that we can run it means about 500 proteins weekly Artemis column but having done men we recently identified that we can lead to Janet for multi-agent Martin should very multi-dimensional chromatography and one of the one of the things that the guys in my group spent doing and the last year or so was to really optimize secure vacation so we can use it at the purification platform and punishment and group is quite instrumental in doing this for many of the other members in the group as well as also Michael a broken person Alma who originally came up with the script that we use for the purification and increases being recently published in the Journal of chromatography for those who would be interested in having a look at this paper so again the purification is that agenus utilizing agenus with a transient cellulite condition may yield typically about the former load and this can be as little as 50 micrograms or a half a milligram all the way through without three milligrams of protein it's applied to the map collect on the road open 100 microliters there's a wash as well as evolution process and then the subsequent our desalting or if I between as well as been for cleaning in places and when we look at the test protein such as looking for grand haven to clear human unicorn or polyclonal in globulin to derive you can see that for the amount loaded on the left-hand panel verse and cream and recovered which is the open circles there's a fairly good correlation with the material coming of the probe protein a so we're getting quite a neat recovery and we're seeing the final yield which is the clothes con drops down a little bit of course because the the way the trapping occurs on the default and calm but for Carlos in less than 2 million times retrieving as much as bad eighty-two percent repetitive yield or recovery coming off at con compared to that cp8 and for higher loads and you can see this is because that we're starting to see material coming out or we're going to reach the capacity they column and material coming out in your breakthrough p Please Please by the open triangle one of the other things that we tested on these columns very early on with what the what the potential cross-contamination was other in the JC our contamination take the Jacqueline where contamination or subsequent run contamination and what you can see we exclude the saturated concrete formula grants the material then subsequent rounds with PDF essentially don't identify any carrier protein and so this is really important when you're considering utilizing these things as a platform / typically what we have identified that becomes computer Farquaad in a linear fashion up to that you're my heart milligrams of protein and obviously to make more and get more recovered however the recovery won't be as good as at the lower concentrations in tempe to finish the cupboard we would look at real life samples was applied as much as 200 antibodies on to the Janus me and what you can see there is that there is a direct relationship between Ian downloaded the amount recovered and foremost protein sewing things they tend to fall in these patterns and when you look at the speed the speed which is quite easy to produce a hundred samples in an eight-hour appearing and that this material then can go on to defaulting and so typically one need much is about point 25 milligrams of protein in a milk for that for milk of material or one milligram of material can be loaded onto the cone balloon to be education and this correlates well can you turn to the relationship with an amplitude versus are recovered material on the Janice and so looking at the Janus is an example of 400 antibodies to fight on the actor and [Music] and basically on a two-step protein a going on consulting or whether the throughput is a lot slower one can see this feeling better after a good pass linear relationship are in the low probably of course of the actors that be through putting the rock for now I'll just jump one month slides killer and what we also looked at is that some of the proteins in terms of the 200 that we loaded onto the these columns not we're not recovered in levels that we would have expected but they're sort of run very very far from ideal and when we look at the samples that are darkened in this case 1327 and the quasi on supplies including it's interesting to notice for example whether you really call recovery from illegal material really bad behaviour ideal proteins and so ideally what this suggesting that this is one can even use expression versus recovery as though sort of a predictor of power around will perform and if you couple that with the size region then one can see whether these antibodies would be in the being used to take on any further so one of the key reasons that the purification on the janet has also become very capable is that they have been advancing in transient expression that allows for us to be able to reach the quantities required during the load process so for example from 250 different on a coil antibody expression the average expression level is about a half grant / Mel so on a formula gram sorry I'm a formula right this is equivalent on average to about 2 milligrams loaded which is a really good concentration to be loading onto these rubber columns and when we look at how many proteins are less than the point 5 milligrams which ideally would need to be minimum amount loaded on calm we only see about four points four percent of these proteins samples at that threshold so ideally what that means is about 80 three-point-six percent of these proteins would be loaded at greater than one milligram an early 4.4% of these would not be applied to the image analysis for the expiation really good system and dovetails very well with the chance for purification and this is about contrast to our previous expression systems such as the Freestyle any human embryonic kidney set one where for example on 400 clone on average we with being about point our 50 micrograms the milk flow ting so ideally what that means is only about point seven five things we find could be processed by this method and essentially 99% of them could not utilize the magenta so having an expression system is capable of producing grades and point 25 milligrams to know what prank is really critical to having the right study material concentration to be able to load onto these column so in conclusion or some of the concluding remarks even the CFL group produces about 1500 points annually to support the research group the Janus is used for a large number of these purifications so that we can do see antonis purification many proteins on the aerobic on and be able to put these inappropriate buses for other implications or violently assaulting ography affinity revenues that we use I think got a protein a protein g we neglect and the concentration would have to be greater than 125 milligrams per ml and typically formula load can be loaded onto the column up to four mil we've seen columns at last and the final note that I've gosselin saying that the scene Rover columns can it clean and recycle of runs on the Unruh Jenna but typically we were more comfortable with using these columns at about 15 cycles and then introducing a an analysis of the columns before we then do additional run on them and this is because they're there has been observed deterioration the only and sometimes these columns and fail after about 15 cycles so any using for 15 cycles won't quite get quite as quite a few runs batted in columns and and by testing the month additionally you can get quite a few and identify the ones that are going to be a problem um and you send through additional cycles as well so the last thing I have to do to thank the guys in my group Peter and the other guys in the group as well as well as within molecular biology food can be a charge and I think we did quite a lot of the expression work control chamber and and Georgina came in with inquest normal between people and repair leon help to actually get a liquid handling system as a purification system in Okinawa with my collector and Jeremy Lambert and just thank you everyone for being here and listen kid all right good morning everybody my name is Bill flirty and i'll be doing me the second portion of the stock kettle my presentation is the assessment of targeting arm pegylation in a complex with its own therapeutic we're going to examine using the caliper GX 2 c-e SDS system as a tool to positively identify the pegylated conjugate that functions as a targeting arm for our encapsulated chemotherapy lipids own drug product this product is known as mmm 302 it's currently in Phase two it specifically targets human epidermal growth factor receptor 20 it's known as r2 and our hope is that it provides patients that have are even treated with other drug regimens a new and effective treatment the bulk of my discussion will be focused on the scfv protein portion of the molecule that talk to specifically targets or two and how we can access the chronicle assess the product quality of this protein 12 the start of B bulk drug substance a quick note about Merrimack who we are and what we do we are a company located in cambridge massachusetts all of our pipeline projects are going after oncology targets or a group of scientists and engineers dedicated to helping the world treat and ultimately cure and eradicate cancer your systems biology model that looks to define and treat the cause of disease so we do a lot of molecular modeling to find specific targets for our drugs so the GX why we're using it and what it is so we have the GX 2 system the blue boxes as it's known colloquium around the company why do we use it it's very easy to use for even for unfamiliar at this you can provide a plate for testing simply having to dilute samples it's high throughput um easy to run day of experiments and provide real-time data is necessary it's like running up to a chels at once much faster to completion and much easier to analyze the data as well as the consistency being very very very consistent good chip-to-chip reproducibility and very low variability so the molecule mmm 302 it's a me natal episomal construct that we use here at Merrimack it's made of three primary portions the top portion is the protein to incorporate the protein into the Olympus ohm we conjugate it with a peg molecule and it is a number of those are incorporated on to the final drug substance which is the encapsulated chemotherapy nano episome drug product so the important portion and the portion that we're going to specifically look at today is ensuring that we have the tag linker on our protein so that it can be inserted correctly into the lipid zone so our purification process for this molecule is obviously much more complicated than a standard mad so here's an oversimplified version of the purification it's all the way through first column second column and you fgf2 we defined your initial protein purification the scfv is the protein it's then prepared for conjugation conjugated with a peg molecule it goes through another set of purification steps and is incorporated into the liposome so so we want to devise a strategy where we can have one simple method to help identify and quanti the status of our protein throughout the purification conjugation and live is owned corporation it's three individual steps across a significant significantly longer purification and lipid on construction train then would be done typically for a map so here is the an electropherogram directly from the GX I'll walk through this this has both the scfv protein portion and the kanji apportioned shown same same electropherograms so on the y-axis you have your fluorescence and on your ex you have the sides the scfv is approximately twenty-seven twenty-eight kilo dalton protein it exists as both the monomer and I American see both species there d sav conjugate is shown in brown and red in this trace the two samples here are distinct lack of kanji that we've produced we know that there are some differences in a tag elation pattern the conjugates I want to want to show if the GX can identify a mono pegylated to a single tag edition conjugate compared to an over pegylated in ecology of material so the TKE at 29 and 30 kilotons is the model pegylated conjugate ticket 36 is an over pegleg kanji it and the peak eps 41 is also an older pegylated kanji so the GX is able to detect three distinct species is protein congee at mixture within three kilo dalton chokes the static confirms we've suspected from sds-page separations the gels are partially able to resolve the species but smearing and low concentration coupled with lack of quantitation on the gel in provide with the most definitive data to allow us to demonstrate an understanding the conjugation state and the status of material to move forward and fulfill incorporation so this is a significant increase in sensitivity we can positively identify the conjugate and multiple conjugate species and we have an ability to understand the overt I galatian rate of our conjugate which can change from lock to lock here is another electropherogram from the GX this is a football full view so we want to look again at what this tells us so it actually tells us a great deal of information we can see the status of the single chain the scfv as both a monomer in a dimer we can see the mono pegylated conjugate and they over calculated conjugate we can see that there's no fraction of the protein that exists and we're seeing a definitive size separation across a very small change in size this is the same samples under a reducing condition to prove that we aren't just seeing Ashley artifacts we ran under reducing conditions as well the only changes that we are that we see here is a reduction of our anticipated reduction of a reducible dimer of the single chain fvfv so we can further confirm that the kanji species that we are seeing are in fact the conjugates that we are claiming to be so now that we have an understanding of our ability to see the conjugate can we quantitate the species that are there looking at the pegylated conjugated various constant diluted concentrations we're able to understand we're able to see if we can quantitate the conjugates so our conjugate linearity from our starting concentration down below our anticipated concentration is it correlates fairly well we know the final concentration of our protein in North Zone drug product and we were able to quantitate well below that one our biggest hurdle with the kanji at standard curve is the percent of the old regulated material present in each lot in detail we know the amount will vary slightly the curve here is using total area all conjugate species observe the total protein concentration should not be impacted by presence of separately distinction apology gate species but a correlation should be done on different Lots conjugate with high and low percent over conjugated material to understand the one-to-one correlation this may prove to be a fully quantitative methods for poking total protein and illiberal incorporation simple it's very promising and offers a significant increase in quantitative understanding over our typical sds-page methods now that I've developed a method to observe intentionally quantitate the contrary material find a way to abstract it from the liposome is imperative so we did some initial detergent experiments to dissociate the conjugate formalism itself here are some substandard detergent treatments all compatible with the GX platform we have Triton bridge 35 and a few others all of these proves to be I have too much background noise or not sufficiently able to separate the conjugate from the liposome so new ideas need to be explored to understand oh if we could simply see the conjugate once they have been incorporated into the old Azam drug product here's is a pretty messy electropherogram this is power a look at our all about drug components and how they obscure our ability to see our actual protein and how our take home message here is the conjugate species that we want to see is scared by all of the other components of the drug product what can we do to understand to remove these other components and see our protein of interest so the typical GX method only calls for a dilution so if we explore the technique up front that allow us to clear Olympic components in the chemotherapy reagent that don't allow the quantitative protein will we be able then to see our protein of interest here are two initial look at purifying away the rigid and additional material so our first was using a molecular lake off-spin filter to remove small lipid components and the chemotherapy and concentrate a conjugate protein it would give us a great signal and declare acceleration contamination as it worked unfortunately it was only able to concentrate the chemotherapy and I was not able to recover the protein of interest a second method that we attempted was an ethanol precipitation and subsequent chillin at minus 80 Celsius so we had a significant bolus of ethanol to separate this intercepted two sample spin it healthy protein and described supernatant hopefully only reconstituting the protein in this prep we froze the sample to encourage the lipids to break apart and provide additional stress to remove contaminants or lipid portions of our proteins and leaving our conjugate intact invisible this preparation recovers protein we still see significant signal from other components I notice during this portion that they lifted the pellet process was not as optimal as we would like to see so it's possible that we carried through some of the lipid components and we're not looking too early at our conjugate material here we see the final prep that we undertook the the ethanol precipitation and compared in fats in red and we're comparing that against our initial no prep lipids ohm drug product loaded directly onto the plate so what we had wanted to see was removal of all the intermediate signal between what's here is about eight and thirty kill adult so what we were able to do is resolve and clear the drug components that were obscuring your view we do still see a significant peak at about five seconds flat kill Dalton's doxorubicin that is coming through with our final drug product but it is not obscuring our ability to see our protein so this is the final condition that we move forward with fully able to resolve our conjugate species away from the liposome and extract it from the lipid zone here we have the reference conjugate reference material that we'd used for initial quantitation just experiment compared with a conjugate that is precipitated from an actual representative product what you can see in in blue is the conjugate reference and what you can see in red is the material we were able to abstract from the middle of his own so what this tells us is that we are able to pull that conjugate out and recover it quite well the conjugate reference here is diluted to any specific concentration that concentration is approximately what we would expect to see coming out of the lip is owned and we are within five percent we totally protein recovery by total area of our anticipated concentration here we have a look at repeatability these are two separate precipitation drug product precipitations done on two consecutive days and run on the same chip so what we're able to see is that we are able to completely resolve this protein on multiple days on multiple preps and get very very good very consistent recovery from day to day an experiment to experiment so we want to make sure that we are are seeing what we what we think we're seeing so we devised reverse-phase hplc method to separate out the conjugate the mono pegylated conjugate as well as the over pegylated console conjugate and here we have the mono peg conjugate the single tag away to conjugate and they over pegylated ecology it so we confirm the presence of both in the same material using an orthogonal method and took this method and put it on the front end of a mass spec which allowed us to here's a some mass spectra data that shows specifically the presence of both model and over pegylated conjugate species using that regard phase hplc UV method what this tells us is that everything that we saw on the GX is confirmed and we can use the GX as a in process daily measurement tool as opposed to having to run math data each time we have a sample which significantly increases our throughput and allows us to provide real-time answers to the group's so conclusions the GX will be proposed as a commercial law release methods for evaluating size heterogeneity in the ecology we will be able to report the unconjugated single chain protein the mono pegylated conjugate protein and the over pegylated conjugate protein which is a significant increase in our ability to see these species over the traditional sds-page methods the protein precipitation and condition optimization enable analysis of conjugate southsider today directly from the nan ilysm they are able to extract to be conjugate from the final drug product and quantitate and fully observe the protein has reported out the data implies that the method is repeatable in robust as a relevant want a shin approach again we can report multiple species and absolute quantitation should be achieved with some optimization the experiments are ongoing that would be the ultimate goal would be to fully absolutely quantitate all the different species that were pulling out of the drug product now with that I just want to say thank you to my team at merrimack also analytics Department and the nano episomal development team that I work with closely during this process oh thank you thank you both Lou and bill for bringing us that informative presentation a quick reminder for our audience on how to make your question simply type them in the Q&A box found by clicking on the green to a button at the lower left of the presentation window we'll get to as many questions as our time permits and we do have our first question an attendee asks i'm looking into equipment for high-throughput HEK expressions what's shaking speed do you use in your incubators I've heard that you need a very high shaking speed so I said I that's more question I could you um ah get off yep we are we're only doing for a lot of this small scale expressions very small volume and and from what I understand the fairly standard method that actually probably doesn't all those speeds are high they're not excessively hard I do know that they utilize tinkle rodas and yeah and the actual and flasks that they use of the five mil 24 10 mil tube that they use being sort of specifically designed to create very deterioration and I'd be happy to put you in contact with the molecular expression guys could be happy to share from the day oh I'm pretty sure we've actually published some of that any one of our early papers where we've taken material and and looked at generating a platform for a hard to put expression and so so yeah so so I'm sorry I can answer that means a lot of detail but yeah that that's I don't think the speaker physically hi okay we have another question you change cell strain to improve expression level did you also look at the vector I'm pretty sure that the victims they used were the the manufacturing victims for the for that particular XP so long for the salon interest in a selected Australian for high-throughput expression and it just enables that expression to go ahead having said that when we actually generate larger quantities and material we do codon optimization as well as vector optimization for for the purification so that we can get the yields up but typically look for example like in that slide where i showed in 250 anybody's that's a good path purification taking CDRs out of anybody's and then just going and putting it into our frame I you know our format platform for expression okay thank you another question for you Luis can you elaborate a bit on the upstream expression process I'm not entirely sure what they would like us to elaborate on to be honest that question has a bit more detail please okay I'm sure our attendee can type in some more detail in the meantime we'll move on to another question from another attendee I saw mostly method development details presented rather than qbd approaches what was the connection to cutie is this directed more toward my presentation I believe perhaps all right yeah this did have more than that the development aspect to it however I would say that the throughput of GX lends itself to the ability to run far more samples in our detergent application and all of our standard care development was it increases the time was significantly lesson through using the lab ship as opposed to typical sds-page style methods so it's not a true quality by design approach but the throughput and the data received enabled a lot more lessons learned in the facial area time ok and I'll see another college go ahead I just a follow on from that bring your I suppose in respect to my talk and the early phase of discovery or research identifying issue we part of how we can assign this or the quality of tributes and the design parameters that you acquire in qbd so having some understanding of what our proteins behave at that early stage it's really very you know the initial part of that a QED approach and and I think that's where we would have a role to play in that of course it's not to be deeper say that it's sort of starting to think about what we need to do when we come to that study I see okay we do have the next question for bill I'm surprised that the peg peaks are not broad is the peg of a very defined side yes the peg molecule is a uniform in size and it is relatively small so we are able to separate those rocks if it's about three KD difference on each for each peg added this is not peg 2000 or any of the large pegs but we do know the size of this peg and it's and we're able to see even one versus two on the system okay great we have another question what's the approximate sensitivity of purity what percent scfv / pegylated species can you see we were able to see down to approximately one percent total protein in the various different different species which is a significant improvement over the SDS page and the older bioanalyzer methods well we wouldn't be able to detect these species at such a low percentage okay thank you I'm afraid that is all the time we have now for questions today I want to thank our audience for bringing such a great bunch of information for questions to our presenters bill or lose you have any final comments for the audience no just anger you to participate year and follow me if anyone's got any additional questions beyond this tool from happy to answer them to a later stage great i activate my echo those sentiments thank you very much thank you both for being here I also do want to thank our sponsor perkin-elmer from making this presentation possible today if we didn't get your question we will follow up via email this webcast can be viewed on demand through December 2016 lab roots will alert you via email when it's available for replay we encourage you to share that email with your colleagues who may have missed today's live event until next time thank you for participating today and goodbye