1:32
I was asked to do this talk little bit late notice and I had a look at the market for oligos and one of the things I found was there was quite a few different numbers out there.
1:44
But I think the main take home that I found interesting was this exponential growth that was being predicted for oligos and essentially regardless of the value that you like, it was expected to double by 2027.
2:00
I also noticed to date there was around 11 FDA approved oligonucleotide therapeutics currently out there on the market.
2:07
And I thought there’s quite a way to go.
2:12
But I think one of the main things in the take homes that I kept reading through a lot of this market research that I was doing was the notion of the strict standards that are currently there for manufacturing and for the drugs itself.
2:28
And I thought, OK, well, this is where maybe Waters comes into play.
2:33
And me, myself.
2:34
So I work in the European demo lab.
2:36
I spend my days managing the pharma and biopharma team, but also speaking to customers every day who come to us with a problem.
2:44
And I'd say kind of our top hot topics that we get asked for is how can we identify, characterise, look at impurity, look at sequencing of oligos.
2:55
That's started to now creep in as well, the notion of oligo mapping for even bigger simRNAs when we need to digest them and start looking maybe down more the peptide mapping route and purity today for this talk, because that's an awful lot and I'm splitting it with my colleague.
3:14
I'm going to concentrate on mapping
3:17
Sequencing is what I'm going to look at today.
3:23
I'm also going to concentrate because in my team we tend to concentrate more down the high-res mass-spec end.
3:30
So I'm going to concentrate today primarily on the XEVO G3 QToF.
3:36
I don't know if anyone's heard about the Bioaccord system.
3:38
It's been marketed quite heavily by Waters at the moment as our easy-to-use kind of QC monitoring platform.
3:46
I think what's quite nice is with the launch of the G3, you'll get that higher-res, but we're bringing in all of those easy to use functionalities.
3:55
So we've tried to listen, I think to the customers.
3:58
So where traditionally high-res mass spec calibration.
4:01
I mean, I've worked in mass spec for 20 years now and back then, you know, you'd spend a day calibrating a mass-spec.
4:09
You know, you're talking now press “go” and get a coffee and come back.
4:15
Another thing I'm probably going to mention quite a lot is something called Waters Connect, which is the new software platform.
4:24
I always see it a little bit.
4:25
If you think of for anybody with an iPhone in the hand and the iOS and you've got your apps for Android users, I don't know the equivalent, I'm sorry.
4:35
Again, I'm going to talk about CONFIRM Sequence mainly today, which is where we do all of our oligo sequencing.
4:42
But if anybody's interested in any of these other applications, feel free to come and seek me out today.
4:48
It's not the only application that we have for oligos.
4:55
So CONFIRM Sequence.
4:56
This is what our new software looks like.
4:59
This has been designed specifically to sequence oligonucleotides.
5:04
It's got very nice libraries here.
5:06
You can make any weird and wonderful oligonucleotide you want.
5:11
Any monomers, you can add them into your library.
5:16
You can draw them if you want as well.
5:18
If you think a little bit like ChemDraw, if that's the way you wanted to do it, you can insert a MO file.
5:23
So it's pretty user friendly in any way you want.
5:26
So you can build these up, put them into your software, put your data in, press go, and what it should do is give you an output of a dot map for your oligo, for your sequence.
5:39
Once you've built your sequence, now it does this on deconvoluted data.
5:45
You don't have to trust the dot map.
5:46
If this was live right now, I'd be able to click over each one of these dots and you'd be able to bring up your raw spectra as well so you can check it.
5:56
You haven't got to just go I'm going to rely on this if you wanted to.
6:00
Some people like to dig into the raw data and other people don't.
6:06
It works on the McLuckie fragmentation rules for anybody who's interested in that as well.
6:16
So what I wanted to do as well is not just stand here and give a bit of a marketing speech on kind of software and oh, isn't this all great?
6:24
I wanted to show some real data as well.
6:26
You know, we're all scientists in this room.
6:29
So what I'm going to talk about today is 3 samples.
6:33
Gem 91 is something we use as a bit of a standard across waters.
6:37
It's 25-mer phosphonothioate oligonucleotide.
6:42
I'm going to mention a 40 MER, which is something we brought in as well.
6:46
It's just unmodified 40 nucleotide strand of DNA.
6:50
And I'm going to talk about a couple of isomers as well.
6:58
I think I've already mentioned that I'm going to primarily talk about the XEVO G3 today.
7:04
This was launched at ASMS last summer.
7:08
Essentially, it's got, we've got increased sensitivity, increased dynamic range and increased robustness.
7:14
Now I've been running in my lab, the G3 now I think for eight weeks on oligo benchmarking.
7:20
And I think I can stand here in honesty and say that it's just ran 8 weeks solid.
7:27
We haven't really done anything with it.
7:29
So I was glad that we've done that benchmarking so I could stand here today and be quite honest with everybody in the room.
7:38
This is how the calibration looks now.
7:40
So for anyone, whoever used mass links, when you just have to go and click ions and click pages and it take forever, you can now just click on and off whatever calibration you want, press go, it'll go away, do its detector setup and do all those things for you.
7:55
So it really is now pretty easy to use.
8:00
This is our Gem 91 data.
8:03
This is our dot map.
8:04
This is a 25-mer, the phosphonothioated, and we built the sequence doesn't take very long at all in the software and hit Go and we got 100% coverage.
8:18
All this data is actually done with HFIP:TEA Fast 6-minute gradient system.
8:27
I think it says at the bottom.
8:28
And this is actually Mse data.
8:30
So for anybody who doesn't know Mse, you've got MS, MSMS which has your precursor ion selection and MSE, which fragments everything that's in your sample.
8:42
So it's incredibly quick because there's no optimisation of that precursor ion.
8:47
And then the collision energies, you can actually run a collision ramp.
8:51
So it just gives you a very quick look see at your fragmentation.
8:56
But for a 25mer and 6 minutes method, minimal development, we can get 100% coverage no problem here on this oligo.
9:09
We also looked at MSMS because I know some people, certainly the customers I speak to want MSMS, not just Mse.
9:17
There's a bit of a debate on is that the standard at the moment?
9:21
I don't know.
9:21
You know, it depends who I speak to.
9:24
So we've put that in as well.
9:25
You can see here at the bottom the other dot maps.
9:28
So for each precursor that we've taken in, you can take in different charge states, so essentially different precursor lines and then you can start stepping them up so that you get increased coverage as you go.
9:42
But you can see that again, we get perfectly fine 100% coverage for that 25mer.
9:49
We then wanted to push the system a little bit more and went, well, let's go bigger again, this is an intact mass.
9:56
This is purely for sequencing and we pushed it a little bit further and a little bit annoyingly, you'll see there's smack bang in the middle, which tends to be the case for larger oligos.
10:06
You start to struggle to sequence there around the middle.
10:11
I'm not happy with this and currently this is running and I'd hope that by this morning one of my team might have gone.
10:17
We've got it.
10:19
But what we're doing at the moment, because we're running this in HVIP, we're currently running this in a different mobile phase to try and push down to that lower charge data so that we can increase that coverage.
10:30
And the date is coming off as we speak.
10:32
So if someone finds me at about 1:00 this afternoon, we might have them too in the middle.
10:39
I just wanted to mention as well low-level impurities.
10:42
I know there's no kind of standard out there out at the moment.
10:46
There's FDA guidance for these low-level impurities.
10:49
We often go to research to look, but I think it was quite nice to see that we were down in the 0.08% impurity in terms of full length products.
10:58
So the sensitivity really is there for this system.
11:06
Time permitting, as you probably heard my, you know, originally my PhD was ion mobility mass spectrometry and was all very originally all about the hardware.
11:17
And we threw some of our samples down a couple of the IMS devices.
11:24
So that's the SYNAPT in the white and the cyclic IMS there on the right in the black.
11:29
I don't want to go too deep into this, but essentially what you get is that third dimension, that orthogonal separation of size and shape with ion mobility.
11:37
So you have your separation in your LC by polarity, size and shape and then your mass detection.
11:45
What it allows us to do is to plot these, what we would call these drift plots, where you have drift time, which is a function of the molecule size and shape and mass to charge.
11:56
Now where this becomes quite nice for sequencing is it allows you to essentially take almost snippets of the drift time mass to charge, send that down to your mass detection and avoid everything else, all the other ions around it.
12:16
And what that does is it reduces that noise level.
12:19
So you can bring up your oligo signal essentially.
12:24
And when we did this, you can see this naggy oligo with the thymine there in the middle that we were struggling to sequence.
12:31
Once we start using this technique, we start hitting that 100% again, which is why we're confident that by shifting that charge state, we will hit the same.
12:44
The other thing on the cyclic. So the cyclic IMS is a little bit more special again.
12:47
So the synapt is a drift tube device.
12:49
The cyclic as suggested, is round and you can send your irons round and round as much as you want, ergo increasing the resolution of the ion mobility.
13:00
And you'll see that these were two sets, a mixed set of isomers there.
13:05
It's just a 7mer that we looked at with a base switched, an AU base that was switched round.
13:14
And you can see that when we send it through the instrument once you get one nice peak and normally, you'd look at that down a normal mass back and go it's one compound.
13:24
As we start stretching out how much we can separate and we start increasing that resolution on size and shape, we start seeing a split peak.
13:33
So you see around pass 10, you can just about start seeing an edge of a peak.
13:38
When we get to around 45 passes around that IMS device, we can see two distinct peaks.
13:44
So it's letting us say yeah, we have isomers there.
13:47
I've spoken to some customers who say they don't want to see the diastereomers, stick them all back in one pass.
13:54
And I have others who are interested in them.
13:57
It's up to you, right?
13:58
I mean, I think that's the nice thing in Waters the instruments are there depending on your needs really.
14:05
And lastly, I just wanted to show you can see the mass specs there when we've pulled out.
14:12
So in the software, you can actually drag as you would an LC peak drag underneath and pull out your extracted ion and that's where you can then start pulling that out.
14:23
But again, this date, this would be unseen under any of the normal conditions without the cyclic.
14:29
I think I'm just about on time.
14:34
I just wanted to say thank you.
14:37
This is my team, the European Biopharma and pharma team.
14:40
So really this is their work.
14:42
So it's a privilege for me to stand here with it.
