0:00
So Thomas has more than 30 years of experience in the field of analytical chemistry from various positions in the pharmaceutical and agrochemical industry as well as the instrument manufacturing lab solution providers arena.
0:11
In his current position, Thomas is responsible for the BD in the field of pharma at Agilent Technologies.
0:18
And may I ask you to introduce yourself, please?
0:24
I'm a market specialist, but I cover the genetic part, the molecular biologist part.
0:30
And this is why we’ve split up this talk because we have to really cover a broad range of topics.
0:36
Thank you.
0:37
So I'm very curious to know why we should really work with you.
0:42
Really.
0:43
Thank you.
0:55
I've been at Agilent for 23 years.
0:59
And when Agilent started off being formed out of Hewlett Packard, it started off with the Bioanalyzer.
1:05
You may be well aware of and on a quality control.
1:08
This is a long time ago, however, as you've just seen the presentation a second ago, that industry is right on the spot of the heart of molecular biology and regulation and everything related to genetics and molecular biology.
1:25
So let's start here.
1:27
Our talk is named Agilent solution for nucleic acid-based vaccine development and production.
1:32
And as outlined here, you start typically with a sequence of analytical steps in that industry starting off from sample QC, down to formulation and final product.
1:50
If you deal with an RNA molecule and it's running out of itself, OK.
1:59
And this is what makes up the QC steps in a typical vaccine development pipeline here. From nucleic acid isolation sequencing down to in vitro transcription, RNA quality checks, Poly(A) analysis, formulation and final product.
2:28
In that industry, RNA became the key focus.
2:42
So it started off in 1961. mRNA got synthesised for the first time in the lab in vitro 1984.
2:53
In 2005, the key invention happened that nucleoside modification was identified and the RNA is shown to be non immunogenic.
3:07
And 2008 and 2010, BioNtech and Moderna started off with the first showcases to inject RNA and to start with cancer therapies.
3:27
In 2021, as you’re well aware, FDA approved the first vaccines.
3:33
And since then I think we witnessed an explosion in that field.
3:37
Basically I think it's like now everybody knows Lego.
3:43
But in these days that industry is really starting a Lego play and having a huge promise to deliver all aspects of molecular biology, all aspects of tissue function and all aspects of cell regulations.
4:01
Because now we have the Lego box to intervene to have strategies to have therapies to really be effective, whether it's an infectious disease or a cancer therapy. So we can apply these toolboxes.
4:20
In a typical IVT RNA preparation workflow there are various steps of QC here, starting from the DNA material, which is used as a template for the production process.
4:31
The QC step of the IVT RNA product itself, the further polishing and further modification of that RNA.
4:40
One step is the addition of the Poly(A) tail.
4:44
Some other steps might have a look at the capping and of course the QC step, the final product and looking for RNA stability and potential degradation.
4:59
This is a process which has manufacturers.
5:04
You will know that company (Pfizer) is setting the stage for that industry and you can nicely see how over the process of several weeks. Even between manufacturing sites, various QC steps are kicking in.
5:21
At the same time.
5:21
You will notice there is one prominent instrument here which is the so-called fragment analyzer.
5:27
It's not the Bioanalyzer, it's not based on microfluidic.
5:30
This instrument is based on capillary electrophoresis and it's well used on these various QC steps from initial QC quality testing to final checks.
5:46
This is what the vaccine looks like in a final QC step.
5:53
This is basically what everybody or most of us got injected with in the last two years.
6:01
So this is what the RNA looks like in an electropherogram, which is nicely illustrating the point that you have one specific size that you are away from impurities.
6:24
4.1 kb RNA to be to be effective in the tissue.
6:32
The instrument of choice here is the fragment analyzer.
6:35
This is what it looks like.
6:36
It's a model system.
6:39
Basically you have three different bench top models according to capacity and having a 16 or 96 capillary set up.
6:50
It has various kits for the assays, but then you're going to go on DNA, on RNA analysis
7:03
And per application, these kits are separated by different sizing range so that you can choose the appropriate chemistry.
7:12
Kits are qualitative or quantitative, and it's just that you have up to six different array formats to configure the instrument.
7:23
This is the core of the instrument.
7:27
It's a capillary system.
7:30
So you have multiple capillaries in an optical array being analysed so that you can have a full automated system even running out of 96 well plates.
7:46
Well, it's decreased any platform preparation time because the chemistry is well established and is easy to set up.
7:57
The methods are available and easy and it's very cost effective.
8:06
And now I'm going to hand over to Thomas.
8:17
So what Goetz said is let's say that the hard work.
8:21
So you find your candidates, you do all your science, the magic to end up with a with a product that's something that you think is efficient or shows the effect you want to see.
8:37
But now the magic happens to put it in a formulation that makes your vaccine available for the system.
8:47
So against the disease, the flu, whatever your vaccine is for.
8:52
And as Goetz said and as we saw before, there are a lot of applications on the market now. One of the ways which I find really smart is to use the so-called LNP's, the lipid nanoparticles to make your available for the human body in the end.
9:18
And of course, this is a process, it sounds easy, but it's very complicated to find the right mixture of the building blocks of the lipid nanoparticle and then of course get the vaccine to travel in this in the body and then do the effects that one wants to see.
9:42
So that's a real process here.
9:45
And we are looking at this part of the process now in the next couple of minutes.
9:51
So if you want to understand the building blocks behind the lipid nanoparticle, it's typically that we have our RNA in this case which is negatively charged.
10:04
We have our cationic lipids; we have some helper lipids.
10:08
They aggregate around the core.
10:12
The outer shell consists of PEC lipids formed as an outer layer, which makes it very comfortable for the body to get the drug inside.
10:23
If you would administer the drug as it stands, maybe we have side effects, we have effects that our body doesn't like them, but those are little shuttle services for our vaccine that hide what we are really going to administer.
10:42
And that's a production process which must be highly reproducible.
10:48
And to measure if on one hand, let's say upstream, if our recipe makes sense of which percentage of which of the building blocks we need.
11:03
We have a nice method here, which is an LC based method, liquid chromatography utilising the Agilent 1290 Infinity II Bio LC.
11:13
That's a long name, I know.
11:15
And we use evaporative light scattering detection, which is ELSD very simple set up here, a gradient with a flow rate of 0.4 mil per minute.
11:29
And again, upstream, you don't know exactly what to expect.
11:34
You need a lot of different columns, a lot of different massive development facilities.
11:41
So we have here set up, an MCT with a column selection well with six columns in it.
11:49
And in the method we can switch or toggle between the different columns for method development.
11:57
Of course, once we developed our method, we don't need those six.
12:00
We have one column then and we look at why that is important later.
12:07
So here are the different building blocks as you can see here, those different lipids.
12:20
You play around with a mixture, and you change the cholesterol percentage or the neutral lipid percent.
12:29
And as you can see here, that's a chromatogram out of this ELSD detector where we have all our four building blocks and of course the abundance we can make out the relative percentage.
12:43
And again in method development we do different percentages.
12:49
So every single thing will go up and down.
12:52
And once we settled for a recipe that forms a nice uniform lipid nanoparticle for the administration later, then of course this method can be used as a QC method later.
13:06
So then we fix this method, we validate this method and as you can see, nice sharp peaks, very nice resolution here.
13:15
So that would be one part of the story with also one more check mark.
13:23
We need linearity because later on we can of course not always expect the same concentration of our building block or of our LNPs.
13:38
That's why we need a certain level of reproducibility and linearity hand in hand if you wish.
13:48
So you can see from 4.86 nanomole all the way up to 2.2 picomole.
13:55
We are relatively linear here for all our different ingredients.
14:02
And then the second part is our actual vaccine that also has to be monitored.
14:09
And here that would be another way of doing a final batch release or QC or whatever your downstream part is.
14:20
For this we can use Bio-MDS which is also light scattering, in this case dual angle light scattering.
14:28
We can even use an isocratic method here with size exclusion chromatography.
14:35
Different columns available with different pore size.
14:38
Depending on the size of our vaccine, we can use different pore size columns.
14:45
Really the columns we are using are called AdvanceBio.
14:52
So whenever you read the term AdvanceBio, you would know that this column is for biological molecules.
14:58
So it's optimised for the big ones.
15:02
But that's just to give you an indication which columns we were using.
15:07
That is a standard BSA and its fragments to kind of see if the instrument performs well.
15:16
We do it at the 90° angle here, the light scattering experiment and we also have as a reference a UV of 260 nanometers here.
15:28
Those are the two signals.
15:30
And the real interesting bit is that this is the actual BioNTech Pfizer vaccine.
15:37
So we saw in the example before the different molecules of the LNP and this is now the drug itself, the vaccine itself.
15:49
And for QC purposes you can with one injection see the loaded LNP, one fine distinct signal.
15:58
And of course, there would be criteria for the for the height or the area of this peak, which would give you the actual QC result.
16:08
We have two nice application notes around that.
16:12
If you want to see more details, you can see as a reference here the actual application note numbers or you can take a picture if you wish.
16:24
And just to mention one very nice thing that we think in our portfolio of consumables we have a broad range of different columns: Reversed Phase, Anion Exchange, HILIC, Size Exclusion, and so on and so forth.
16:46
So wherever you are in your process, we might have the right column for you.
16:52
And why do I mention that?
16:53
Well, number one is of course you need a column anyway.
16:56
So you should use one that is capable to show resolution as I showed in my example here.
17:06
The other thing is we even have some bio inert columns which nicely fit our bio inert LC system.
17:13
So it's all for the sticky bio molecules that give you some headache.
17:18
We can help you to overcome that.
17:21
And what I really specifically like is that we can scale up our experiments.
17:28
So if you are upstream in the R&D mode, you work in an analytical scale, you use one column filling, you use one, let's say reverse phase, we call it PLRP-S for example, as you can see here in a 4.6 millimetre diameter.
17:44
But we have the same column fill or the same stationary phase also all the way up to 25 millimetres or even bigger.
17:56
So preparative scale you can have the same that you used in R&D and do a tech transfer all the way to your QC lab without changing the column.
18:08
So that's what we think comes in handy.
18:13
And as you can see here, if you have an oligo separation 15 mer, 20 mer and 25 mer all the way up to sometimes even 100 mer, you get very nice resolution with the AdvanceBio oligonucleotide column.
18:28
And as I said, it can be either analytical scale or preparative scale.
18:34
We have the hardware, we have the columns, we have reagents, consumables, all you might possibly need here for your experiments.
18:45
And this is finally showing you that, for example, starting from 4.6 all the way to 100 millimetre inner diameter.
18:57
As you can see from different flow rates. Here we offer the full range from analytical to preparative.
19:06
And if you have any more questions or if you need more information on what we were discussing.
19:12
And of course, many other workflows in the world of RNA and vaccines and oligos and all the nice stuff.
19:19
Come and see us at booth 8 please.
