0:00
And it's a pleasure to introduce our first speaker of the session, who is Richard Easton, who is the Technical Director, structural analysis of the company, the BioPharmaSpec.
0:12
Now Richard obtained his PhD in glycoprotein structural characterization using mass spec from Imperial College of Science, Technology and Medicine.
0:24
And after a postdoctoral.
0:26
In the field of glycan elucidation, Richard worked at GSK, then Amoscan Limited, and now currently at BioPharmaSpec.
0:38
So Richard, thank you for your talk, the title of which is Application of Varied Mass spectrometry Techniques to synthetic Peptide Characterization.
0:49
Thank you.
0:51
Thank you to all of you for turning up.
0:54
Obviously I have the challenge straight after lunch of keeping everyone awake.
0:58
I don't know where the lights are going to be dimmed, but it gets even harder if that happens.
1:02
So please bear with me and no snores if you can help that.
1:12
OK, so first, just to give a little bit of background about who we are.
1:16
So BioPharmaSpec is a contract research organisation.
1:19
We focus very much on the structural analysis of proteins like proteins, peptides, oligonucleotides and conjugations thereof.
1:28
So things like ADCs, AOCs as well.
1:31
We have 5 labs around the world.
1:34
We have a lab in Jersey in the Channel Islands in the UK, so slightly unusual place but nice to be particularly in the summer.
1:41
We have a lab in America as well, just outside Philadelphia.
1:44
And as of earlier this year, we now have three other labs in Europe.
1:48
So we have a lab in Lithuania, one in Germany and one in Bergamo in Italy that have just come online.
1:56
So the company is about just over 10 years old now and was set up by Professor Howard Morris, who also set up the M-Scan group of companies that I worked for before BioPharmaSpec sort of evolved subsequent to M-Scan and a few of us moved out from M-Scan into to BioPharmaSpec.
2:15
But Professor Morris has a very significant history in terms of applying mass spectrometry to protein glycoprotein structural characterization.
2:26
So he developed a lot of the techniques that we use nowadays as real sort of cornerstone procedures for this kind of analysis.
2:33
So peptide mapping, disulfide bridge analysis, he was involved in elucidating a lot of the fragmentation pathways that peptides undergo when they, the breakup in mass spectrometers and also in conceptualising the Q-TOF geometry instruments that are now very much a cornerstone of the kind of work that a lot of protein chemists and mass spectrometrists will use.
2:57
So BioPharmaSpec, we do have a very strong focus on science, but also on problem solving.
3:01
And the company, the offering is greater than just the mass spectrometry.
3:06
So we fulfil all of the requirements from ICH Q6B for full structural characterization.
3:15
So what are synthetic peptides?
3:17
Well, fairly obviously synthetic peptides are those that are manufactured by a chemical process rather than a biological process.
3:24
If you look at the documentation, the FDA defines them as 40 amino acids or less to qualify as a peptide.
3:32
And just to put, I've just put a few examples on there.
3:34
So Teriparatide, Semaglutide, Liraglutide, you know, fairly common kind of things that we see as peptides. And both the EMA and the FDA, as you would expect, have produced guidelines for these kind of molecules.
3:47
So from a quality point of view, people know what the expectations are, how to assess these, how to handle them and what to look for in these kind of products.
3:58
So why would we produce things, excuse me, synthetically?
4:02
Well, biological systems are really the only choice for producing large molecules, so large proteins, but peptides small in size can be readily produced in a synthetic sense in large amounts.
4:16
And it does give you the freedom to play around in a sense to express your creativity in terms of what you want to do to produce these products.
4:27
So you're not just beholden to what the cell can do, but you can start to put your own chemistries in place.
4:33
So if you wish you can put in stereoisomers D amino acids are very often incorporated specifically in certain locations for their own functional reasons.
4:44
You can do other things as well, of course in there.
4:46
So acylation you might try or your own unique chemical modifications.
4:51
It's really down to what you're doing and what you're designing the drug to do.
4:55
And these can be done much more easily at the peptide level.
4:59
The idea, of course, is to put specific chemical modifications in that are going to be very efficacious in various different ways.
5:07
So it may be that you're looking to increase the half-life of the product, maybe protease resistance or just the activity of the product itself.
5:14
Maybe it's a targeting thing.
5:16
Whatever that particular design is, you can design it into your synthetic peptide, but then of course you have to go and do all the testing to make sure that you've done it right.
5:27
Like I say, the regulatory authorities have come up with their guidelines for what the expectations are for these class of molecules and you can see them both shown on the screen here.
5:38
So the EMA one is on the left and this really details in general terms what's expected for synthetic peptide analysis.
5:47
The FDA does a similar thing.
5:50
They don't just define what the expectations are analytically, but it's their guideline.
5:56
And it's also help you to decide whether you should go through an NDA or an ANDA type application.
6:02
And that's really based on the impurity profiles that you see within the sample.
6:05
But nonetheless, the fundamental principles of the guidelines are there to help you understand what is expected analytically.
6:15
So what are these expectations?
6:16
Well, I've just sort of summarised them very briefly on the screen here.
6:20
And as you can expect, as you would expect really in sort of conjunction with the idea of what's expected from Q6B, you're expected to look at the primary structure, the secondary and tertiary structure.
6:31
Even for peptides, there's consideration for tertiary structure in the molecule.
6:37
We have to look at the full sequence and generate full sequence for the peptide.
6:42
Any disulfide bridges that may be present also need to be assessed as well and orthogonal techniques should be considered.
6:51
Regulatory authorities.
6:52
They do like the idea of orthogonality, and we should consider that in the kind of analysis that we do.
6:57
So the data becomes a very strong self-supporting package in terms of the structural investigations that you've done.
7:06
Impurities play a big part in considerations for this class of product.
7:13
So both guidelines recognise that they know that there are risks associated with these impurities.
7:18
So we do have to look at these in great detail and emphasis is quite rightly placed on this within both sets of guidelines.
7:28
And impurities, of course, can be both product-related from the actual process of manufacturing the sample itself, as well as process related in terms of things that may be coming in through impurities or raw materials that are being used or whatever it is that is coming into contact with the sample, as it were.
7:46
So there's different classes of impurities, of course, and we need to consider all of those.
7:53
Mass spectrometry can help us with an awful lot of this.
7:56
Not completely.
7:57
So I want to give that impression that this is one technique that can do everything.
8:01
But mass spectrometry to a very large extent can help us with a lot of those areas.
8:07
So we can generate a huge amount of information from different forms of mass spectrometry.
8:13
Intact mass analysis is fairly easily performed by LCMS, as I'm sure many of you are aware.
8:21
This will give us a very accurate overall mass for the molecule.
8:24
So if we have produced it correctly, we know what we expect to see.
8:28
If we don't see that mass or if we see other components that are present in there, we're starting to learn something about the impurity profile as well, so we can generate some information from a fairly simple LCMS experiment.
8:41
Amino acid sequencing can be performed by MS as well.
8:44
Very often we can do this completely through the mass spectrometric analysis.
8:49
There's an expectation that we sequence this in absolute terms so we know precisely what the amino acids are.
8:54
Mass spectrometry can do this for us as well.
8:58
Disulphide bridges we can assess of course as well.
9:01
Another part, excuse me, of any necessary characterization should those bridge components be present in your sample.
9:10
Fragment ions can be generated in real time in mass spectrometers depending on the type of mass spectrometer that you're using.
9:16
And this again is a great support to the kind of assignments that we're able to generate.
9:22
You see a mass that you think may be related to a peptide or possibly is an anomalous mass fragmentation data that you've generated can help you identify what that particular peptide is, if it's out of a peptide mapping assessment.
9:35
And that will go some way to tell you what that particular modification that is on there is if it shouldn't be present.
9:40
So you've got this mechanism for assessing the data rather than just relying on a mass that maybe doesn't quite make sense.
9:46
There's underlying information that we can use to support that investigation.
9:52
The peptide mapping data itself can support the intact mass data.
9:56
Obviously everything should add up to the total at the end.
9:58
That's what you expect the mapping to do.
10:01
So that becomes a sort of an orthogonal test to the intact mass.
10:04
So we're starting to generate data that is working together as people like to see.
10:09
And the fact that we can generate these fragment times means that potentially depending if the fragment times are strong enough and clear enough, we can identify where these modifications are because that modification will fragment along with a particular mean wise it's attached on.
10:28
So we can locate these maybe anomalous masses or whatever it is that we're looking for as well.
10:35
So there's a lot of useful information that comes out of this in an intact sense, in a peptide sense, in a fragmentation ion generating sense as well.
10:46
So I'll talk a little bit about sequencing specifically in a mass spectrometric sense.
10:55
We already know that there's an expectation that the sequence is identified unambiguously.
10:59
And like I say, this can be performed either completely or virtually completely by mass spectrometry depending on the actual nature of the peptide.
11:08
And I've just shown here the most common kind of fragmentation pathway that we tend to classically see in a lot of the sort of, I hesitate to use the word standard, but more regular analysis that we perform in the sort of electric spray LCMS type sense.
11:25
So this is the kind of fragmentation that you may see in this that occurs in the source of an instrument or if you're carrying out particular MS-MS type analysis.
11:35
So in this case, we have protonation of the backbone nitrogen in the amide link and either a lone pair of electrons on the carbonyl oxygen moving to form a triple bond and the carbon nitrogen bond breaks to produce a so-called b ion.
11:53
And this would carry the N terminus of the peptide.
11:57
Or we can get rearrangement around this CO bond and produce the y ion.
12:04
And this is the C terminus of the peptide.
12:07
And of course, these are the two forms we see because they carry a charge.
12:12
So we need that charge to actually be able to move the things through the mass spectrometer.
12:15
So we see the b or the y ion, OK?
12:19
And we're seeing either the N terminus or the C terminus of the molecule, OK.
12:24
Now this can happen any one of these bonds, OK.
12:29
And because it happens at each bond, it's happening down the backbone of the molecule, which means we're losing amino acids each time.
12:37
So that means we can generate a series of fragment ions that we can actually work out because amino acids side chains have different masses, what the sequence of the molecule is, OK.
12:48
Now the b ions and the y ions are not generated equally.
12:52
So molecules tend to have a propensity to produce y ions much more frequently.
12:58
But you do see b ions as well.
12:59
And so you can see both types, they can support one another.
13:03
So this is just a quick example of some of the data, excuse me, that we can generate and this is just one particular peptide.
13:11
And you can see how if you just look at the red trace here.
13:15
And I've tried to match these up to this.
13:17
So we're looking at y ions on the red trace and you can see we've got a lot of clear strong signals here and we're fragmenting down the peptide backbone.
13:30
So these masses here are unique to the individual amino acids because majority, and I notice I use that word majority have unique masses.
13:40
So we can use those mass differences to sequence the peptide.
13:43
And because we're sequencing using the y ions, the peptide runs from the N terminus, EG, V, N and so on.
13:50
So that's the direction into C that we're reading.
13:56
But unfortunately not all amino acids have different side chain masses.
14:02
And the main culprits here are the isobaric amino acids leucine and isoleucine because they are simply isomers of one another.
14:10
So in this form of fragmentation, the mass loss is identical.
14:14
It's 113 Dalton.
14:15
So you don't know whether you've lost a leucine or an isoleucine.
14:18
So you have not unambiguously identified the sequence of your molecule.
14:24
Mass spectrometry can still come to the rescue, but in this case we have to use a different form of fragmentation, and this is an electron based fragmentation mechanism that we find on newer types of mass spectrometers are that are coming out now at BioPharmaSpec we're fortunate to have some of these.
14:43
So we have the ZenoTOF 7600, Waters Cyclic IMS and in this instance we're able to fragment slightly differently down the backbone, but also at the same time fragment side chains off and produce so-called w ions and the leucine and isoleucine fragmentations are different.
15:06
You can see the rearrangement here on the side chain.
15:09
So the fragment ions actually have different masses in this electron based fragmentation mechanism.
15:17
So when we look at the data that we generate from mass spectrometer wherever we have a leucine or a isoleucine z ion fragments lose either 43 for leucine or 29 for an isoleucine.
15:28
So we can use this sub fragmentation to categorically identify which of the two amino acids that we have.
15:37
So I'd like to also just talk briefly about impurity investigations.
15:40
As I've mentioned, they are a significant part of structural characterization for these kinds of molecules.
15:48
The EMA and FDA do talk a lot about this.
15:51
It's quite understandable when you think of the importance of the well, the nature of the chemical synthesis and the opportunity to produce impurities is high because every cycle you are not going to get 100% yield.
16:03
You are using various different reagents that will have their own contaminants that you're bringing into the process.
16:08
So you need to consider the various kinds of impurities that may be present.
16:12
And I've just listed some of the examples that they talk about stereoisomers is one, DL configurations.
16:17
You could have truncated species if the yield hasn't gone to 100% in each cycle.
16:22
Likewise, you could maybe have sort of extended chains, side chain reactions, oxidations, deamidations are the classical ones.
16:28
But other things may happen depending on your chemistry.
16:31
The molecule could aggregate and on top of that you could have process related impurities such residual solvents and heavy metals that have come in from the process itself.
16:39
So there's a lot of scope for issues in your molecule.
16:45
But again, or I should just say first of all, in terms of impurity investigations, any techniques that we use have to be specific and clear.
16:53
So the output allows us to clearly identify that impurity, and we need high sensitivity so we can get the best level of detection that we can.
17:01
And to a large extent mass spectrometry can help us with a lot of these.
17:08
The ones that are very heavily based around effects on the protein itself in terms of extensions, deletions and such like are also mass altering.
17:19
So standard LC-MS approaches are very good for that.
17:22
We can see these kind of things in an intact mass analysis.
17:25
Like I was saying, you know, that sort of generation of the intact data will show you minor components as well, stereo isomers.
17:34
We can also investigate through an LC-MS approach.
17:36
This is a multiple reaction monitoring system, and we'll talk about this shortly briefly.
17:42
Or we can use GC-MS.
17:44
We can also use GC-MS for residual solvents, or we can use another a form of inductively coupled plasma mass spectrometry to look for heavy metals as well.
17:53
So all sorts of different MS applications for investigations into impurities in the sample.
18:01
So stereoisomers, I'll just talk about very briefly in the interest of time.
18:06
Naturally found amino acids are in the L form, but the D form can be included and is included in various different peptides for various different reasons, and a few are just listed here.
18:19
Any D isomers that appear elsewhere are impurities.
18:22
They still need to be considered.
18:23
We need to sort of demonstrate effectively that they're not present as well as demonstrating any D isomers that are meant to be there are.
18:32
So we can do this, like I said, by GC-MS or LC-MS.
18:36
Fundamentally the principles are in terms of getting the sample to the instrument and the same in the sense that you have to break the sample down to individual amino acids.
18:44
They have to be specifically tagged and labelled such those that you don't lose the chirality of the amino acid.
18:51
And then you can use either a GC-MS based mechanism or an LC based mechanism to separate the individual D and L forms.
18:59
OK, and I've just shown them here.
19:01
This is an LC-MRM method for separation.
19:05
You can see how the L forms in red separate nicely from their equivalent D forms in blue by this.
19:13
So I will just go through the last slide just to tell you a little bit what LC-MRM is.
19:19
So this is basically a quadrupole method.
19:21
So we're still using LC-MS, but this is now on a quadrupole type instrument and this is a mechanism whereby the sample is ionised.
19:29
We use the first quadrupole in the system to select the component of interest.
19:34
The second quadrupole carries out fragmentation of that particular component and then the third quadrupole will select is set to select a fragment mass from that component.
19:45
So here so we can actually take fragment and detect particular masses for each amino acid.
19:53
And because we can set these quadrupoles to select various start and end masses, we can generate a lot of data.
20:01
For the individual amino acids through a sequence, they're not just set for one particular mass, but they can cycle through the different masses that they're meant to assess.
20:11
So with that, I will stop.
20:12
Thank you very much for your attention and for staying awake.
20:16
We're at booth 55 if you want to come and have a chat about anything.
20:20
Meanwhile, I'm happy to answer any questions.
