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Introduced our first speaker of the second session, who's Andrew Kennedy, and he's a part of the European Assail team at AmbioPharm based in Glasgow.
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And after completing his PhD in Organic Chemistry at the University of Glasgow under the supervision of Professor Graham Cook, he then went on to specialise in peptide chemistry, process development and manufacturing with a major peptide CDMO.
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Currently, Doctor Kennedy focuses on providing peptide manufacturing solution to organisations in the pharmaceutical and biotech industry.
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So welcome Andrew, and we're looking forward to your talk on sustainable peptide synthesis.
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A novel cyclover-amine tag for efficient liquid phase synthesis of therapeutic peptides.
0:55
Thanks John.
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I'll make the titles a bit shorter next time.
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Thanks very much everybody for coming.
1:02
So I'll try and make it through.
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I'll be talking constantly for three days.
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So hopefully my voice is going to hold out for the next 15 minutes.
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So with peptide synthesis and more specifically solid phase peptide manufacturer, we've got a big problem.
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And basically, we use too much solvent, too much reagents, and usually those solvent reagents are toxic, which leads to a large amount of hazardous waste, which essentially means that peptides become more costly to produce and the way it's more costly to get rid of essentially.
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So hopefully, we're going to explore a possible solution to this today in this presentation.
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So my name is Andrew Kennedy.
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And for the last 20 years I've pretty much been working on GMP manufacture of peptides and development of novel methods of peptide synthesis.
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But basically for before preparing for this talk today, I had to think about why I got into the industry in the first place.
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So not to get too personal, but back in Inverness when I was 17, an aunt of mine was diagnosed with terminal breast cancer.
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And basically she was about the same age as I am now.
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And that got me thinking about drug discovery chemistry I want to do at university.
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So off to university I went and fast forward 26 years and I have to double check that 26 years I've been lucky enough to work with a number of organisations, research groups and customers and clients who normally develop cancer therapeutics, but also therapeutics for other diseases essentially.
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So basically what we're going to do today is kind of have a look at a potential sustainable peptide synthesis for basically a novel tag for liquid phase peptide synthesis.
2:47
So that was a bit about me, but basically to give you a bit of an introduction to AmbioPharm, it's only two slides.
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So we're basically a comprehensive CDMO peptide manufacturer.
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We're basically our partner for your clinical programme from preclinical manufacturing all the way through to GMP manufacturing and all the processes, sorry, all the ancillary support in between.
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So anything from non-GMP grade peptides for research lead development on to pharmacology tox studies et cetera through process development.
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So developing the process for that peptide target and that can be a variety of solid phase synthesis, liquid phase synthesis, et cetera and all the analytical method supports which is quite important.
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So that was method validation, method development, right through to GMP manufacturing, through phase 1, 2, 3 clinical trials and also to commercial scale as well.
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We also offer QC and regulatory support, so CMC writing, TMF writing, et cetera.
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So basically a one-stop-shop for your peptide clinical programme.
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So where are we?
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We're based in the US.
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This is at the North Augusta site in South Carolina.
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So we've got about 200 employees at that site.
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We have our main manufacturing site, Shanghai with about 300 employees and that's where we carry out most of the downstream and upstream processing for peptides with a mixture of solid phase, liquid phase and hybrid approaches.
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And we can produce around about just over 1000 kilos per year on different batch sizes.
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And we've just announced back in November that we broke ground in our expansion of that site in Shanghai, which will increase the capacity even further.
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So back to the case study, we're looking at going to concentrate on three different areas here.
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So first of all, reducing the environmental impact for sustainable peptide synthesis.
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So here we want to reduce the amount of toxic solvents, reduce the amount of hazardous reagents and also reduced amount of waste.
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Essentially what's going to is scaling up production.
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So it's all very well producing methods that are green chemistry are a bit more efficient, but if they can't be scaled up.
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They're pretty much next to useless for commercial manufacturing.
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So we want a scalable process and keep that process efficiency from bench to scale up in manufacturing and also keep the cost down as well.
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So basically, you know, there's a variety of different green solvents and green methods available, but if they're too expensive, we can't use them in industry.
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What we'll do here is basically optimise some techniques.
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So in solid phase synthesis, we usually concentrate on synthesis, cleavage, purification.
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I've seen a few talks this week, especially Fernando's yesterday about different options for TFA for the cleavage process, Dominic's talk this morning about purification using PEC, and then I'll talk about some options for synthesis here to make it a bit more greener.
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So the potential solution is to combine the benefits of solid phase synthesis and liquid phase synthesis.
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And basically we come up with TAG assisted liquid phase peptide synthesis.
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So what is TAG assisted LPPS?
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And if you're like me and spend most of your working life standing in front of solid phase synthesisers, you're probably not too familiar with it.
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But basically I'll try and give you a quick explanation.
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So a tag is basically attached to the growing peptide chain and the tag will be soluble in certain solutions.
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So for instance, here we've got THF or a mixture of THF and DMF.
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And this allows for a complete reaction in solution.
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So pretty close to a classical organic chemistry reaction, 1.1 and 1.2 equivalents, et cetera.
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So in order to kind of combine the purification benefits of solid phase, you can switch the solvent to either methanol, CN nitrile and then the tag becomes insoluble in solution, basically precipitating out and you can isolate that intermediate and then wash away excess reagents or side products.
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So again, it's combined to the benefits of solution phase synthesis.
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So the low equivalents, the high yield of a traditional chemical reaction with the separation efficiency of solid phase synthesis to be able to wash away those reagents and side products.
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So this reduces the amount of excess reagents used and solvents used and that leads to a sustainable synthesis of therapeutic biomolecules, which can be used for not just peptides but also oligos as well.
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So I've tried to summarise Albericio's paper with Sharma, basically describing the current landscape of liquid phase tag technologies.
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Won't go through them all, but basically we've got the benefits and the kind of weaknesses of each.
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And the kind of general theme is here that the benefits are, you know, you usually use them on small peptides, you can reduce the amount of solvents you use greener solvents, but the common theme for the weaknesses is pretty much as only applicable to short peptides at the moment.
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So we're going to concentrate here on the Cyclover tag by BioTides.
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And this is an example that we used in our process development lab.
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So the Cyclover tag is this is what it looks like, this is the structure.
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Where does the name come from?
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So the ‘Cy’ comes from the cyanuric core, which is essentially a triazine, and then a ‘Clover’, which comes in either 3 or 4 leaves, if you're lucky, which represents the four alkyl chains and pretty much looking like a surfactant type molecule here.
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And going back to our three principles, so reducing the amount of DMF that's used to reduce the amount of toxic solvents.
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We're also soluble in a single solvent.
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Usually you can't use mixtures, but you can solubilize in single solvent like THF.
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I'm basically reducing that range in consumption, so reducing down to 1.1, 1.2 equivalents, et cetera.
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For scale up.
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It's robust to acid and oxidation, sufficient for large scale production and efficient for somewhat longer peptides.
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So not so much to kind of get into 30-40mers, but compared to the other liquid phase tags, it seems to basically kind of manage better with extending that peptide chain.
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So optimising different stages, we've got efficient precipitation of products once we change the solvent leaving accessory agents and side products and solution and then allows for improved purification since you've got rid of a lot of those side products before entering that stage.
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So to give a high level view of the synthesis steps involved.
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So basically, we've got 4 steps dissolving, reacting, precipitating and filtering.
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So we start off by dissolving the starting material in THF or a mixture of THF/DMF 9:1, adding the amino acid addition and then also deprotecting that point.
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And that then couples to the extending the peptide chain and sorry, after deprotection then precipitate out that extra coupled extended peptide chain and then filter your product out for collection and then repeat that process over and over.
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So to give you an example, we've compared conventional solid phase synthesis to this liquid phase example.
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So I hate this when we can't say I can't mention sequences, but this is an 8mer peptide a reaction conditions for solid phase were basically two equivalents HBTU, DIPEA coupling with HOPt additive that could be swapped out for Oxyma, of course.
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But even here we've got 2 equivalents in solid phase.
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So even now we're kind of, we're kind of reducing the number of equivalents instead of using, you know, 5 or 10, which might be used in traditional solid phase synthesis methods.
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Here's some results, apologies for the integrations, but we've got a main purity of about 75%. Solvents used again DMF/DCM.
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So we're still using toxic solvents coupling time of about 3 hours per coupling. Quantity of solvents used, about 1/2 kilogrammes per millimole.
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And that still seems to be like quite a lot to be honest.
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The average yield and step is between 80 and 95%.
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But actually the 80% is really the C terminal loading.
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The rest are about 90 to 95% per coupling, a final year of 45% and accrued purity of 75%.
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So we look at the cyclover reaction steps.
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What we're going to do here is basically couple each amino acids and then trap any unreacted amino acid that's been added to that solution, deprotect the coupled amino acids to deprecate the Fmoc, and then precipitate out and isolate your product and again repeat that over and over again.
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So we compared two different synthesis methods.
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We were HBTU and DIEA on the left and then EDC on the right, and they pretty much performed kind of consistently.
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EDC was slightly better per yield, but you can see how like a small difference in each kind of coupling efficiency can affect the final yield overall.
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So EDC was slightly better than HBTU.
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A couple of tips for optimization here.
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You can also add what we call a sacrificial propylamine to the solution, which can trap success unreactive amino acids, and you can remove those from the solution.
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And this enables one pot sequential couplings and the protections without double insertions.
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So basically we can also reduce the amount of solvent used by instead of washing after every coupling, basically use precipitations at every other step to reduce that solvent usage even further.
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So our example here, 8mer peptides, this is the reaction conditions THF/DMF 9:1.
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We had 1.2 equivalents with amino acid precipitation in acetonitrile and say coupling with EDC and an additive.
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Here's the results.
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So a small increase in purity about 10%.
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We look at the table at the bottom, we basically got the solvents used, coupling times been reduced from three hours to two hours.
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Quantity of solvents reduced from 1.5 kilogrammes per millimole to 1 kilogramme per millimole.
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Average yield percent, it's still quite high, 90 to 95%.
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Final yield a bit higher than before, up to about 60% and the crude.
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Again around about 85%.
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So what this allows for is less consumption of amino acids, so a reduction of about 40% compared to solid phase.
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Lower solvent usage, so a reduction of about 30% compared to solid phase.
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And also being able to allow you to use greener solvents like THF or methyl THF, a higher crude purity, as we said for about increase of about 10%.
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So that's about it.
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It's kind of short and sweet today.
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So there's a summary of the two methods compared as I said, improved purity liquid phase, we've got about a 10% increase in this example.
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There's options to improve that even further by using those kind of optimization steps and optimising each precipitation even further.
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Yields can be improved by again, improving the precipitation conditions as well.
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So we'll like to use less solvents, greener solvents, and again, 30% reduction in consumption suitable for scale up.
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And basically we can efficiently scale up production while maintaining that high quality yield.
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And it doesn't require any specialised equipment.
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So yeah, so it can be implemented as long as you can carry out solid phase synthesis, you can basically use this method as well.
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So that's about it.
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And so thanks to the AmbioPharm team, a lot of this work was carried out by Dewey Sutton.
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Unfortunately, we didn't have a picture, but if you Google ‘Excited Chemist’, this chart pops up.
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So thanks very much, Dewey.
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And then also to our VP of Global R&D, Guoqing Zhang, who's heavily involved in a lot of our strategies for solid phase and liquid phase and hybrid synthesis.
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So myself and Dominic are here at the booth.
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If you haven't seen us already, come down and visit or you can scan the QR code, enter in some project details and we'll get back to you.
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Thanks so much for your time and I'll help you answer any questions.
