0:01
My name is Christian and I'm managing director at Numaferm.
0:05
And we as company, yeah, we are working on peptides, proteins and something that we call pepteins.
0:12
It's a word that you don't need to know automatically.
0:17
So let's start with an introduction to this word.
0:50
So the let's start my presentation with the definition of pepteins, which we did.
0:56
Hopefully that's acceptable for you.
0:58
What we found in the last couple of years being initially solely peptide company that peptides are regarded as molecules containing not more than 40 amino acids based on FDA nomenclature, being still small molecule.
1:13
And what we as being biochemists and biologics also knew from former times that proteins being like the long variants of peptides, they are typically consisting of 150 or 200 amino acids being fairly large.
1:28
And we also observed that in between those two worlds of peptides and proteins, actually both worlds are very different in terms of behaviours of molecules, despite being built of amino acids between both worlds, there's a kind of gap on the landscape.
1:43
We call this gap pepteins having unique functionalities and very importantly are very tough to be produced because neither chemical synthesis is very strong and producing long chain peptides, but also recombinant technologies being available on the market, they're not so easy to be adjusted for short proteins, inside the family of pepteins are quite interesting.
2:10
Yeah, subfamilies, something like not really antibodies, but small antibodies named nano bodies or antibody fragment single chain antibodies.
2:19
That's something that's within the peptein family.
2:21
But also, for example, we have it here, vaccines being built off many epitopes or valence disulphide-rich candidates with very rigid structure are also part of this family.
2:36
And all of them are not so easy to be produced.
2:40
Our company has developed a new approach at the end.
2:43
So our company is about the technology that we have developed.
2:46
Let's therefore jump onto the technology first.
2:50
On the top level, what we have developed as a production platform, we call that Numatech.
2:55
And it's a platform to produce peptides and proteins.
2:59
Please allow me just to phrase and now pepteins to keep with one word independently of the sequence, the lengths or the functionality and the processes that we develop a really cool and various means.
3:10
And also the products that we release are very pure.
3:13
And if we now have a more detailed look onto this production platform, we will find 2 core technologies.
3:20
The first technology is called Numasec.
3:23
This is actually coming from my PhD times more than 10 years ago.
3:26
What I was able to achieve is that I really could, yeah, develop the first secretion approach for Gram-negative bacteria like E coli.
3:35
So we only need in this technology to fuse or target of course genetically to a so-called Sectag protein.
3:44
We co-express a transport complex sitting in the gram negative cell wall and that's insufficient to produce targets with the outside of E coli.
3:51
And I talk about normal B strains, K strains or whatever you like.
3:55
It really works fantastic.
3:56
And here's now the big surprise.
3:58
We don't use this technology anymore at our company.
4:00
It's a little bit frustrating for me having developed that, but that's the work, that's how it is.
4:05
And to keep it very brief, what's the reason why we don't apply it anymore?
4:08
It's really working.
4:09
Yeah, I can guarantee you.
4:11
The reason is it's still a recombinant approach.
4:14
What does it mean compared to chemical synthesis?
4:17
Being a peptide company those days, it was just impossible to meet time expectations from our clients being used to get the material very quickly by this great chemical synthesis.
4:29
And being a recombinant approach means it's slow because you need to adjust the process towards the target in terms of the environment, pH values, salts and cells.
4:39
So being a recommended approach, unfortunately it was not successful in competing with chemical synthesis.
4:44
I'm only explaining you this technology.
4:47
As I mentioned, we only use it in few projects because we learnt here and very important thing for us and this ‘Eureka moment’, I call it was that we have found that the secreted Sectag proteins here in the surrounding are highly soluble proteins.
5:04
In contrast, if you don't co-express the transport complex sitting here, of course these proteins keep in the cells, right?
5:11
But here they're not soluble at all.
5:13
They aggregate as so-called inclusion bodies.
5:15
It's aggregated protein stuff.
5:17
Normally you're regarded as waste.
5:20
We found out what's ongoing, why this happens because we were fascinated by this observation those days at the university.
5:26
And we found out that these Sectag proteins contain so-called GG repeats, calcium binding domains.
5:32
Since in the cells the calcium ion concentration is very low, no binding event happens, keeping these Sectag proteins unstructured aggregation prone.
5:42
But after the secretion process to the surrounding where the calcium ion concentration can be high, what happens is the calcium ions bind and this binding event converts the entire protein into a very stable soluble protein.
5:54
We have shown by the way also that the system works for enzymes, very large enzymes up to 9100 kilodaltons being secreted functional.
6:01
This means after the Sectag with its cargo on is outside of E coli, these fusion proteins reach the final functional form.
6:11
We have proven that in many examples and based on those observation, we were able to develop a technology that we apply nowadays, and we call that a biochemical production platform.
6:22
I will come to the point why I will guide you know through this technology step by step that you get an understanding on how it works.
6:30
First of all, what we do is we fuse the targets now to a so-called ‘Switchtag’ protein at the end.
6:39
It's similar to these Sectag protein.
6:41
It contains calcium binding domains and what we do now, and the first step is something that might be a surprise for people being used to recombinant systems.
6:48
What we do is we produce birational inclusion bodies, so aggregated nonfunctional proteins regarded normally as waste.
6:56
Before I come to the what's the problem with inclusion bodies?
6:59
I come to the advantages because it's accepted, I believe very much that inclusion bodies have distinct advantages being mentioned here like high tide expression, high purity, all this stuff mentioned here.
7:11
The big issue about them is how do you generate the functional protein out of these aggregated proteins scientifically?
7:20
We are in the science conference.
7:21
So I try to find the scientific explanation for this observation of the problem.
7:28
What you have is with these aggregated proteins at the end, unfolded proteins shown here.
7:33
And this is an energy landscape.
7:38
One second, this is the energy landscape, and you want to reach a native state during the following step.
7:43
But what you will find is that on this track you will have intermolecular interactions yielding aggregates, oligomers, and at the end precipitation.
7:54
Because of this inclusion body based processes, despite all these advantages that I mentioned, are at the end inefficient because you lose your stuff during the refolding step.
8:04
Luckily, quite some decades ago a smart guy found something where we have for what he receives the Nobel Prize.
8:13
This guy, Mr Anfinsen demonstrated with some with an enzyme, that the primary structure of a protein is sufficient to give the information for proper folding.
8:23
That's what he got this noble prize for.
8:25
This means, by the way, we have confirmed that in those secretion studies.
8:31
This means that if you have a nice environment for proper protein folding, you get your functional stuff.
8:36
Actually, what is nature doing using chaperones, preventing intermolecular interactions?
8:42
So based on these thinkings, we then thought let's try to do the following.
8:47
Let's try to get the advantages of the inclusion bodies into our hands by working on the refolding issue.
8:53
And an ideal technology would prevent intermolecular interactions during the refolding step.
9:00
And that's actually the technology that we have developed.
9:04
What we do nowadays is we fuse targets to the Switchtag protein and produce inclusion bodies.
9:09
We take them out and then we refold the unfolded proteins in the presence of calcium ions.
9:15
Learn from the secretion approach.
9:16
Remember what happens then is these calcium ions bind to the Switchtag proteins converting them quantitatively to highly soluble proteins.
9:25
And during our studies in the last years, we learned that the fuse target to the Switchtag proteins are now keeping in solution.
9:33
So the Switchtags inter prevents the intermolecular interactions, keeping the target and solution and giving it the time to fold properly along the thermodynamic landscape.
9:45
We have proven that this technology works nowadays initially with peptides, nowadays with for proteins up to 600 amino acids.
9:53
So we are operating this technology now for peptides, proteins and pepteins. Another big thing that we needed to develop.
10:02
So this really worked since couple of years, but what we learned being again a peptide company that our clients that we worked for that didn't accept a Switchtag protein being attached to the target, right?
10:14
Of course not.
10:15
But if you are familiar with recombinant systems, you will most likely know that cleaving off an affinity tag or a fusion partner is not so easy.
10:25
You need to work with proteases or whatsoever, but there's no protease platform around that you can always use to cleave off your target without amino acid keeping attached, so-called natively.
10:38
But this was a clear expectation from our peptide clients.
10:41
So we needed to work on a protease platform that can be always applied be applied to release our Switchtag protein from the target, what it's all about and we have worked on that with partners.
10:52
We have based our developments on the so-called TEV protease.
10:55
It's you know, it's a market available sequence specific protease recognising 7 amino acids shown here and it cleaves after the six amino acids in front of the so-called P1 prime position.
11:05
And the TEV protease accepts small amino acids at this position like serine, glycine or alanine, but it doesn't accept other amino acids or very inefficiently.
11:15
So the TEV protease actually it's quite nice thing if your target has an N-terminal consisting of a small amino acid, but it doesn't work for others.
11:24
Still based on our evaluation, this was the best protease being available on the market, but by far not good enough for our platform idea having everything in the platform.
11:35
So we work with different partners on that, and they came up indeed with a very nice solution for us.
11:40
They have to develop for us the so-called Numacut TEV protease and keep it to keep it very brief.
11:46
What they achieved for us is they have developed an enzyme that still recognise these 6 amino acids being highly specific, but please independently of the N terminus.
11:56
So this Numacut now enables us to use our Numaswitch technology to produce traces, targets independently of the secrets very easily.
12:06
We use this protease since two to three years and our partners always ask for more material, you know, for some internal studies and we shipped it always we said OK, great, the demand is on the market.
12:17
So we released this enzyme as our one and only product.
12:20
We don't have products, but this was we were somehow forced to do that.
12:24
So now this protease can also be sourced on our homepage and our distribution partners.
12:31
Yeah, taken together, what we do is we produce inclusion bodies.
12:34
Everyone can do that, but we can we refold them efficiently by our strategy, we can cleave off your targets tracelessly from by the Numacut protease.
12:43
Taken together, it's very easy now to produce pure targets and I come to the schedule behind that later on.
12:50
So this is an experimental, representative experimental data set.
12:55
What you can expect when we apply this number switch platform, first of all, we train our cells and then there are strategies around you now to precisely induce the production called expression.
13:08
So we throw something in and then the cells start to produce the inclusion bodies containing the Switchtag and the target.
13:14
We take it out, we have done the unfolded proteins, and we do the refolding.
13:18
You see here quantitative refolding.
13:20
Then we give the Numacut protease to it.
13:22
It release a target from the Switchtag protein.
13:25
And you see here if you are doing RP-HPLC analysis, how pure already the target is.
13:30
Here are the proteogenic impurities and you more or less only need to collect the target, making it very easy.
13:36
You also see here already is a high purity level.
13:38
We only need to separate these minor impurities from the target.
13:45
People tend to ask how Mr Anfinsen is great, but you know, I'm not really convinced it's refolding step is sufficient to generate functional proteins up to 600 amino acids.
13:57
Indeed it works and here are a few examples we can talk about.
14:00
So we have for example produced anti-microbial peptides with our system.
14:04
So that also works because the aggregated anti-microbial peptides are not functional inside E coli.
14:10
We take them out and then we switch it on to be functional.
14:13
The same holds true for growth factors.
14:15
Here's an example of hEGF, nanobodies, so camelid single chain antibodies, but also here for single chain FC proteins and we also apply it for fusion proteins like that.
14:27
And here are always in shown functional data.
14:29
Due to time considerations, I cannot go to details, but this was done by internal or external partners showing that's functional as reference material.
14:39
So even for the most complex target and our record internally is a target containing 6 disulphates, this technology works.
14:46
And why does it work?
14:48
Because I mentioned we give the target time to fold properly.
14:51
And if you think about disulphate containing targets, then we do a redox shuffling system and then also it works out to reach a low energy level.
15:00
Isomers, the correct, typically the correct isomer has the lowest energy level.
15:05
This is an overview about what you can expect from the Numaswitch technology.
15:09
So you can expect we handle short peptides, actually we handle also much shorter peptides going up to 600 amino acids being enzymes, binding proteins, APIs and whatsoever.
15:22
We have a very standardised upstreaming approach and midstreaming approach.
15:25
So fermentation and extraction of the including bodies and then the downstream we did developed with our team, you know, meeting your expectation in terms of scalability or whatsoever.
15:35
Typically we don't use HPLCS or affinity chromatographs due to the high purity of the material.
15:40
So it's easier, cheaper than to scale up, but sometimes we do.
15:43
It really depends on the project.
15:46
Production timelines are very short.
15:48
This is up to kilogramme batches.
15:50
Production titles are shown here.
15:51
So just to give you a few numbers for those who are interested, Being forced by chemical synthesis and we are still forced chemical synthesis is great.
16:02
Yeah.
16:02
So you can just use your amino acid and click it to your target.
16:05
It's, I mean, a big advantage.
16:07
We're not there, but we worked a lot on let's say expanding the natural.
16:11
So we have established protocols for state-of-the-art click chemistry to add fatty acids to targets.
16:17
We have developed protocols for C-terminal amidation and N-terminal acetylation.
16:21
And also we have implemented a strategy.
16:23
This was not invented by us.
16:25
It just implemented from very smart people where they that can incorporate non-natural amino acids already during the fermentation.
16:35
This is at the end what we offer.
16:37
Yeah, we are a company doing service for our partners and these time schedules here are very important for us to be successful.
16:45
In the peptide area, we do milligramme sampling.
16:48
So including a cell and development and milligramme sampling within a month.
16:51
We say that's the speed of chemical synthesis, but it's very quick for recombinant technology.
16:54
So I tend to say we bring the speed of chemical synthesis to the world of recommend people by our biochemical tool.
17:02
Also gramme scale samples are done with a couple of months and then GMP material can be supplied on the year nine months.
17:12
This is just an overview.
17:13
So we really do everything at Dusseldorf here.
17:15
Our labs are here in this building.
17:18
We are, yeah, we have everything for upstreaming, midstreaming, downstreaming, analytics.
17:22
We do almost everything in house up to a 100 gramme non GMP scale, everything exceeding we have a partner network to produce at large scale, small scale GMP whatsoever.
17:33
This is just a little bit feedback from partners we work with together.
17:37
So we have roughly 60% pharma clients, biotech or large pharma, but also 40% non-pharma clients from artery sector or cosmetic things like that.
17:49
And yeah, here now his error showing we are indeed, I didn't say it wrong.
17:54
We are in this building and yeah, our mission is really to help out.
17:58
Yeah, we want to apply our technology giving you your materials quickly pure at with a scalable approach and that's us.
18:09
Thanks for listening.
