0:46
Thank you very much.
0:47
That awkward moment when you realise your bio was a bit too long.
0:52
I get the dubious honour of being in between all of you and lunch, but more importantly and between myself and lunch, so I'll try and be efficient.
1:01
We are Genovis, if you don't know who we are, we're the guys with the colourful stand and the funny characters.
1:06
I see a lot of familiar faces here, so plenty of you do know who we are.
1:10
I've moved on the slide, but we provide enzymes and solutions for mainly characterization of antibodies or antibody-based formats, but we are expanding into other kind of formats as well.
1:22
As we've heard through a lot of talks particularly at this conference and others we know that the field is moving in wild and crazy directions in terms of molecules and we need to be able to keep up to support you guys.
1:34
So if you do know us, you'll be aware that our first product or most famous product is FabRICATOR for below hinge of IgG.
1:44
A lot of our early products were focused on IgG digestion and specific IgG deglycosylation with and endoglycosidases specific for the FC.
1:54
But we are expanding, we've got now enzymes for IGA digestion, IGN digestion.
1:59
We've got a wide profile for a wide profile of products for glycan characterisation as well as some really interesting kits and technologies for antibody conjugation for site specific ADC preparation for example, and also to produce homogeneous glycoform antibodies for structure function kind of relationship assessment.
2:20
So we are growing, we are expanding.
2:21
But for this talk I will focus on antibody digestion tools for specifically hinge mutated antibodies.
2:31
So just a bit of a go back what we do, we focus on mid level analysis and for mass spectrometry that mass is really useful.
2:41
It's really easy, little to no sample prep and it can be very quick and good for high throughput characterisation.
2:48
But your destiny is in the hands of the complexity of your molecule.
2:53
This is just an example of three samples from a clone screening study.
2:57
It's very simple, some of them, but some of them can be really complex glycosylation, complex modification profiles.
3:04
The information that you can get from this can be quite limited.
3:08
So rather than digesting down to the peptide level, we can digest at the middle level, digest at the hinge of the antibody to do mid-level characterization.
3:16
In this case here we digest below the hinge reduce to get the FD prime light chain, single chain FC, beautiful size subunits for high resolution mass spectrometry, we can do short chromatography runs.
3:28
I mean, this is 10 minutes, but we can get this down to 3 minutes.
3:32
I think we've shown in previous posters and actually you get the information that you can get from an intact mass, but you can get a lot more information about potential modifications and you can start to localise those modifications as well onto the different subunits.
3:46
So that's why that's where we sit.
3:49
This is probably the most basic slide you'll see about Fc mutated antibodies at the entire conference.
3:56
But the point is that these are becoming more and more prevalent.
4:00
We are fully aware that the hinge mutations to modulate FC effector function, either to silence or enhance to kind of either promote activation of different effective functions or kind of suppress them, but also improve safety profiles or to alter the half-life.
4:22
Which is great because it means we've got a lot more tools in the toolbox to be able to target different conditions and diseases.
4:31
But for a company that specialises in very specific enzymes digesting antibody hinges, it's not particularly useful for us.
4:40
This is just a snapshot of some of the different kind of mutations that are prevalent.
4:45
I'm going to focus for this on the silencing mutations, particularly those guys within the hinge.
4:50
We could fill slides of the different kind of mutations that are currently being used and developed.
4:55
There's been some really nice talks and discussions about these.
4:59
Well, the key thing is that we need to be able to support you guys if you are working with these kind of molecules.
5:06
FabRICATOR is amazing.
5:07
It doesn't really work with these kind of molecules.
5:12
The specificity of the enzyme is really good until you start fiddling down with the hinge.
5:16
And that's one of the reasons why we like to try and keep up with this.
5:21
And this is the our latest offering that we have to support the digestion of mutated antibodies.
5:27
So this is FabRICATOR xtra to digest antibodies that do contain those mutated hinges.
5:32
We've tested it on a few different types of mutated antibodies and these is for people who have said to us, we really like to use FabRICATOR, but we can't use FabRICATOR because it doesn't work.
5:43
So this allows those kinds of customers to be able to do some of the similar type of work that they would want to do for below hinge digestion.
5:51
And this is kind of shows that we've got a LALA and a FALA.
5:54
So the IDT four kind of homologue we're focused on here.
5:58
We've got the heavy chain which doesn't really get digested very well with the FabRICATOR, but it gets digested down into the light into the FD prime and the single chain Fc.
6:09
Interestingly, for this enzyme, we do actually get 2 sites of glycol.
6:12
There 2 sites of digestion, which is quite different for some of our enzymes.
6:16
And actually the primary site of digestion will change depending on the mutation that you do have in your hinge.
6:22
And actually the ratio between the primary and the second digestion site does actually change as well.
6:27
For characterization purposes.
6:29
This is generally fine, may or may not be fine.
6:31
But actually it's really interesting that this enzyme kind of exists.
6:36
You know, we've got our enzymes are from natural sources.
6:39
We produce them recombinantly in the majority of cases and to actually find an enzyme that is capable of digesting the non-native antibodies is actually quite fascinating.
6:50
And actually for us it's a bit of a step outside of our usual.
6:55
Normally I have enzymes.
6:56
If you've used them before, typically you can follow the instructions exactly as we say.
7:01
They will work very efficiently without any need for optimization.
7:05
The beauty of something like a FabRICATOR is that it's a platform method compared to say a Papain, for example, you don't get over digestion, you don't get under digestion.
7:14
You don't need to do optimization of conditions between different samples.
7:18
Whereas for this you probably have to, depending on the type of mutation that you have, you may need to add more enzyme, leave it a bit longer, tweak some of the conditions slightly to get a bit of an efficient digestion.
7:29
And that's kind of what we show here.
7:30
We have a LAGA mutated hinge where we've had to add twice the amount of enzyme and leave it for twice as long.
7:36
And this was quite a challenging one to digest.
7:38
This is an FES digestion which required an overnight digestion and three times the amount of enzyme.
7:44
So put more enzyme in, leave it longer.
7:47
It's just, it's not complex enzymology.
7:49
But actually for this, it's something that we do recommend is required and actually talking to us about it as well.
7:56
Simple, easy.
7:58
It's not easy because as we know, everything moves, everything you know develops and actually when you have mutated hinges to silence the FC, are they always as silent as you think they are?
8:13
And in a lot of cases, they actually probably weren't.
8:16
So there's all whole different type of molecules with additional mutations, particularly in the CH2 region LALAPG, D265S, D265A, for example.
8:26
And one of them is this really challenging molecule that we could digest here.
8:31
The problem we've found with the FabRICATOR xtra is some of these mutations actually prevent the enzyme from digesting efficiently.
8:40
Even though these are what, 100 amino acids away from the digestion site, they actually do cause an impact on the enzyme.
8:47
So it's not as simple as this will digest everything, which is a really big shame for us unfortunately.
8:53
But actually it really means that we can talk to the people who are going to use this and they can support us to understand how this enzyme works.
9:02
So we like a challenge, so we try and address a challenge.
9:05
We've hopefully tried to start addressing some of the challenges below hinge.
9:08
But below hinge digestion isn't always necessary.
9:11
Sometimes people have said they really need it, but actually above hinge digestion can be just as good depending on what you want to get over your experiments.
9:20
And as well as producing new enzymes, actually finding alternative uses or kind of showing different capability of our existing enzymes is another strategy as well.
9:30
So we have three guys here, FabDELLO, GingisKHAN, and FabALACTICA, all clear above the hinge.
9:35
And these are less susceptible because of how they interact with the antibody.
9:41
These are less susceptible to some of those particularly those CH2 mutations.
9:46
And what we found is we can actually digest a lot of the kind of non digestible substrates that we've had with FabRICATOR using these enzymes that we already had in our portfolio.
9:58
And just to show this as an example, we have a LALAPG mutated IgG 1 here we have a light chain, heavy chain and we can digest it.
10:05
We have a little bit of undigested heavy chain, but compared to what we see with FabRICATOR or the FabRICATOR xtra, we get a nice digestion above the hinge.
10:13
So you can characterise your antibody.
10:16
This is purely for visual purposes.
10:19
And actually Nasri beautifully described this in the previous talk and it's the range of different bispecifics, so moving on to bispecific or multispecific type molecules now is just huge.
10:34
And this is from 2017, so it's even bigger now.
10:37
There are so many different formats that are available with so many different bits clogged onto different parts of the antibody.
10:45
I kind of see it like mobile phones.
10:46
They started a certain size and then everyone tried to really get as small as possible and have an active subunit as small as they could.
10:52
And now they're bigger again with more functionality, with more features, and actually again, for us at Genovis, really difficult.
11:02
And one of the things that people need to do with bispecifics, this is a simple example of a bispecific is chain pairing characterization.
11:10
So we can show in the next couple of slides how we can use our products to do chain pairing characterization to ensure the correct assembly of the bispecific.
11:20
There are lots of technologies now available to try and prevent chain mispairing, of course, but it can still happen.
11:27
And potentially you need to be able to show that you haven't got it in your product.
11:32
Of course this is the product we want could have potentially heterodynamic FC species where you've got the correct FC but the incorrectly assembled light chain or similarly you could have the correctly or incorrectly assembled light chain, but you might have homodynamic FC species as well.
11:48
So the ability to be able to detect these different incorrect forms is important.
11:55
So we've shown this previously with FabDELLO compared to an intact mass which you can see maybe the half antibodies are actually really nicely able to be separated in the INTACT level, which makes sense.
12:07
And some of the different mispaired forms can be separated chromatographically.
12:12
But within this you may get inefficient separation of some of the incorrectly formed chain pairs.
12:20
Also, it all depends on the difference in mass between the correct and the incorrect forms.
12:26
If there's a particularly small mass difference between the two, you may not be able to see this particularly well.
12:31
The intact level, they may not separate as well chromatographically.
12:34
So the intact mass may not be as efficient as a subunit approach.
12:39
Because by digesting in this case above the hinge, we can see the correct Fabs that we want as well as the mispaired ones.
12:44
We get really nice separation of them and we can actually start to really see what we've got, what we shouldn't have and how much of each one we have.
12:53
To take that to the mutated antibodies as well.
12:59
This is one of the key applications that a lot of our users were wanting to do.
13:05
They wanted to do chain pairing of mutated bispecifics below the hinge.
13:09
They wanted to use FabRICATOR.
13:11
They couldn't use FabRICATOR because the difference of doing this below the hinge as opposed to above the hinge is that you can look at both the heavy and light chain pairing or mis paired variants at the same time.
13:22
Within the same Fab 2 fragment.
13:23
You can see the light chain variants as well as to make sure that the two heavy chains are connected in the right way as opposed to just seeing the Fabs.
13:30
You don't know if those Fabs are necessarily connected correctly to each other.
13:35
In this case, we've shown where the mispaired or some of the mispaired would be.
13:40
And this actually highlights a further point and a further challenge for us, which I'll touch on a little bit more later.
13:45
This was a beautifully clean sample that we managed to get.
13:47
So we had to say if you did have some mispairs, this is where they would be.
13:52
But this shows again an advantage of using a below hinge approach.
13:57
And in this case, we have our digestion of FALA, FALA mutation, which is nice and easy digested with FabRICATOR xtra.
14:04
But again, to show we can do this above hinge, this is an LFLE hinge with AD265A mutation and mutation is really difficult to digest with the FabRICATOR extra We show that using another one of our existing products, the FabALACTICA, we can digest above the hinge to separate out the different parts of the bispecific to assess the chain pairing.
14:27
And this is actually one of my final slides.
14:29
And because we're trying to keep up with the market, we're trying to keep up with what people are actually doing.
14:39
We're quite limited.
14:40
We're very limited in the molecules that we can buy.
14:43
We can buy molecules from the clinic over in Sweden.
14:47
What we can buy was in early-stage development 10 years ago.
14:51
The molecules in early stage development now may or may not become products.
14:56
At what point in the future for us to be able to really support what is being done now, we actually do help because we need to be able to work with customers, with customers like you to be able to help us with access to molecules.
15:16
I know it's difficult.
15:17
I know it's really hard because of confidentiality and because of you want to make sure that your molecules are kept secret.
15:24
But help us help you know, if you want to be able to digest certain types of molecules, we can work with us.
15:32
And if anyone wants to discuss the possibility of working together, we do have some collaborations with some customers and we actually do provide them with product and help on particular kind of samples that really helps us.
15:45
This is such a vast area, particularly with the mutated hinges.
15:49
We want to make sure that as many people as possible are able to get the results that they need.
15:54
And even in this case here, you know, we're showing the different products that we have and the molecules that we've tested and the success we've had.
16:02
Just because we see something here, it doesn't mean that every single version of that particular mutation or molecule will have the same level of success.
16:12
So it's kind of a step into the unknown for us.
16:16
We're used to things working.
16:17
10 years ago, 95% of antibodies would be able to be digested in FabRICATOR nice and easy for us.
16:24
But it's much more of a challenge now.
16:26
That's great.
16:26
We love a challenge, but actually it's kind of a plea.
16:30
If anyone's got any crazy funky samples, they want to see if they can help us and we even work with people who are looking to develop enzymes. We have done that in the past as well.
16:45
So this shows that yes, we've launched a new product specifically for hinge-mutated antibodies.
16:52
There are some examples where it doesn't work particularly well.
16:56
We know that, but there are examples of existing products we have where actually it does.
17:00
So the best approach for you is the approach that gives you the result that you want.
17:06
So we can do efficient middle level digestion of hinge mutated antibody formats, maybe not all the time, hopefully most of the time.
17:14
And I know there are some people here who have had success.
17:16
I know there are some people here who have not had so much success.
17:19
But the best thing that you can do is talk to us.
17:21
If you have something that you've tried and hasn't worked particularly well, let us know and we can certainly try and see what we can do going forward.
17:29
Ultimately, the best tool is the best tool that's for you.
17:32
Whatever you want to get out of your analysis, whether it's below hinge or above hinge, we hopefully try and find a way to support you in your work.
17:41
So thank you.
17:42
There is still time to come and see us.
17:43
I've got my wonderful colleagues, Hannah and Maria here.
17:46
We're on Booth 4.
17:47
Still time to come and speak to us.
17:49
And I will take questions if there is time.
