Dr. Mark Garner: What I'd like to talk about today are some technologies which we have been working on to characterize biologics; that is, biotherapeutics. This is really an exciting area in the pharmaceutical industry and an area of a lot of growth where there's some novel therapeutic entities coming online that will need to be characterized and also will need quantitative assays, so the kinds of assays one typically does by LC/MS for either protein or peptide-based biotherapeutics.

Intact protein analysis is really a workhorse assay done all day, every day, looking at intact molecular weight, looking for degraded forms, unstable forms, oxidized, deamidated; that's really a workhorse assay.

Peptide mapping is increasingly important, both LC/MS and LC/MS/MS, to verify the identity of the product, to look for sequence variance, things like that.

And of course, post-translational modifications, both biological modifications like glycan analysis--which, of course, is one of the main determinants of immunogenicity. It tells you about the biological activities of core fucosylation. It's so important for many monoclonal antibodies--but also non-biological modifications, so looking at stability; do I see oxidation happening on my methionines; am I seeing degradation of the protein. So, one needs to look for both post-translational modifications from biological processes and just chemical modifications which happen.

And also quantitation is very important, quantitation both of intact proteins or quantitation, as we'll show, of digested proteins by looking at the peptides.

The main system we'll be talking about today is the AB SCIEX TripleTOF 5600 system, which we launched at ASMS 2010. It's both high mass accuracy, high resolution, but it has the speed and sensitivity and the quantitative capability of a triple quadrupole. Combined with the high resolution, it really brings together what you need in a mass spectrometer system, sensitivity, speed, resolution, and mass accuracy, but also the dynamic range that you need to do quant, and you're getting that all at the same time.

The way that we do that is by combining the front end from our 5500 platform triple quadrupole and QTRAP mass spectrometers with a new high resolution, very fast time-of-flight mass analyzer, and that combination really gives you the best of both worlds.

So, just to talk about the applications of that, maybe I'll start out by talking about characterization of a monoclonal antibody using the TripleTOF system. So, typical IgG therapeutic antibodies are typically IgG type 1 or IgG type 4, both large proteins typically around 150,000 molecular weight. They're symmetrical homodimers with both a heavy and light chain; a very typical IgG structure.

So, what is it that one would like to do to be able to characterize an antibody? Obviously, you'd like to be able to get an intact mass to show that it has the correct structure, that the structure has integrity so there's not too much degradation, confirm sequence by peptide mapping either by MS or MS/MS, look at the carbohydrates to ensure that you have the correct glycosylation, look for non-biological modifications, map the disulfide bonds, and so what one really wants to be able to do are these kinds of assays.

And as any large molecule, you know that there's a lot of heterogeneity possible in an IgG, either at the termini, at glycosylation sites, is their Fab glycosylation, residual heavy or light chain aggregates and so forth. And one thing I would mention, which is becoming an area of increasing interest, of course, are sequence variants where you have a low level, 1 percent or can even be less, of an abnormal sequence which can be due either to the presence of a mutated gene in the cell culture strain that's being used to produce it, it can be due to aberrant tRNA charging when the wrong amino acid gets interpreted. But, being able to find and quantitate sequence variants is becoming more and more important.

So, typically what one would like to be able to do is to look--on the left hand of this slide--to look at the intact antibody, but also look at the so-called level 2 where we reduce and look at half antibodies, look at light chain separate from heavy chain, do a digestion, and then start looking at peptides, and then finally start looking at the glycan groups on the protein and characterizing those.

First, I'll talk about intact molecular weight of antibodies. So, these were the conditions we used to look at a particular monoclonal antibody against actin. And this shows you what the data looked like. It's an intact IgG going from the top at 10 microgram on column down to the bottom one microgram on column. You see you get really good sensitivity and a good resolution. If we expand it, the box on the right, you see that actually each of the peaks in the series has a certain amount of heterogeneity to it, which is not entirely surprising in a monoclonal antibody, but it does indicate that we have the sensitivity to be able to see those, we don't know at this point, degraded forms, alternately modified forms, whatever, but we do see heterogeneity in the prep.

So, you take this data, and you do a deconvolution. So, where typically, of course, you have this charge series from a single species where for each peak you have an accurate measure of M over Z, but you don't know M and you don't know Z, so we have to do a deconvolution to determine that. And if we do the deconvolution, the charge series shown at the top and the deconvolved mass graph on the bottom shows a series of molecules separated by exactly the mass of the hexose. So, it looks like we have an intact antibody where we have several species which have had one hexose unit cleaved off of the glycan; at least the data at this point are consistent with that. So, we have the mass accuracy to be able to pick out that small difference at high confidence from the reconstructed data.

We can also do that--in this case, it's on the reduced light chain, so we have the--this is looking at the--the top here is the charge series of the light chain of that same antibody. Then, when we do mass reconstruction, look at the reconstructed mass graph on the bottom of this, we see a small peak, 17 Daltons different from the main light chain peak, which is very consistent with it being a pyroglutamic acid conversion of the end terminus, a very common end terminal modification. But, again, we have the mass accuracy, we have the sensitivity that we pick up that modification very easily using the TripleTOF.

Our conclusion here is that using the TripleTOF system we have the sensitivity and the mass accuracy that allows us to do very high resolution characterization of these antibodies, that we can look at heterogeneity at the glycosylation level, we can look at heterogeneity at the termini and be able to make assignments of those based on the determined mass.

In addition to doing intact mass, of course, one wants and needs to be able to do peptide mapping, both to--as a further identification of the protein and to confirm the sequence. And I won't be going into that today, but this just shows you some of the high resolution MS/MS spectra that one can get on the TripleTOF system. So, the top panel, the blue, is the total ion chromatogram, the pink is the TIC of an MS/MS, and then on the bottom panel we show the high resolution MS/MS based on that. And given that high resolution MS/MS data, we can search using ProteinPilot to get a complete sequence.

This shows you a screenshot of that triptych digest of the monoclonal antibody. This is a screenshot of ProteinPilot showing that you can pull up from that data really good sequence coverage and really high confidence identity. So, the ability to do both peptide mapping and intact protein analysis here at a high resolution, I think, is really important.

So, the other thing that we mentioned in the beginning is the quantitative ability of the TripleTOF system. Typically, one thinks of time-of-flight mass spectrometers as high resolution, but not particularly quantitative. But, for the TripleTOF 5600 it does quantitation very well.

And so, if you think about it, doing an MRM, which is, of course, the gold standard for quantitation, it's an MS/MS-type experiment where I only look at one of the second-generation product ions from the main ion. However, in a TOF system I see all of those ions, so I can quantitate based on any individual fragment ion or I can look at multiple fragment ions and use any one of those or a sum of those to do quantitation. And we call that workflow high resolution MRM because I'm looking at a product ion as I would do in an MRM assay, but in the time of flight I see a large number of product ions, I see essentially all of them, so I can quantitate on any of them or I can start summing them and doing the quantitation that way. So, we call this the MRMHR workflow.

I'm collecting all of the data anyway, so by looking at the sum of those XICs--and if you look at the top graph, it shows you the individual--in this case, I'm looking at Y7, Y6 and Y8 ions. If I sum those, I get a much better CV than I do for any one individually. So, that ability to collect data on multiple product ions, sum it up, it gives me better counting statistics, and I can then get really good quantitation.

This just shows--done for angiotensin looking at, in this case, Y2, B5 and Y6, the ions in the insect on the lower right, which are circled. If I do a summation using those three ions, I get really good linearity over five orders, and I can quantitate based on that. And in this case, the quantitation is done using MultiQuant software. So, I get very high resolution quantitation using MultiQuant software MRMHR on the TripleTOF 5600 to really complete that package of being able to do qualitative analysis, like intact protein analysis, like peptide mapping, but now I can also do quant and ask how much of what I want is there.

Just to summarize, we think that the TripleTOF 5600 has a lot of advantages for analysis of biologics. The high mass accuracy, high resolution for qualitative characterization, the speed to be able to do really good MS/MS and get good peptide mapping results, and then being able to do quantitative assays as well. And one thing I didn't talk about today is based on the MRMHR assay from the TripleTOF, it's easy then to transfer those assays to the QTRAP system for routine high throughput quantitation.

And I think with that, I will close.