Presentation on theme: "David Horn Thermo Fisher Scientific San Jose, CA"— Presentation transcript:
1David Horn Thermo Fisher Scientific San Jose, CA Orbitrap mass spectrometry, a high-confidence screening tool in biopharmaceutical product developmentDavid HornThermo Fisher ScientificSan Jose, CAToday we will describing the application of the latest generation of Orbitrap mass spectrometers for biopharmaceutical analysis.Orbitrap systems are the highest resolution instruments in wide use on the market and are the dominant instrument in the field of proteomics.Until recently, the Orbitrap systems have been heavily used for “bottom-up” protein identification and this is a highly valuable tool for biopharmaceutical analysis. However, less is known about the capabilities of the Orbitrap for intact protein mass determination.I hope that I will prove to you that this mass analyzer is actually very well suited for biopharmaceuticals.1
2Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareConfident antibody analysis using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusionsHere’s the basic outline of today’s presentation:We’ll briefly cover a few of the difficulties in the analysis of biopharmaceuticals, focusing on IgG antibodies. However, the concepts described in the presentation today can be applied to any intact recombinant (or otherwise) protein.We will focus on two instruments: The Q Exactive and the Orbitrap Elite.We will take a slight detour in the discussion of instrumentation to talk about new software packages for intact protein mass determination.Finally, we will follow up with a summary.
3Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareHigh resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusions
4MAb Characterization and QA/QC is Challenging …unlike Aspirin This present some new analytical challenges...MAb(150,000 Da)Aspirin(180 Da)Recombinant proteins are extremely complex molecules.Aspirin = “typical small molecule drug” has a molecular weight of roughly 180 Da with the chemical formula C9H8O4.In comparison, an antibody has a roughly 7000 C, H, 1700 N, 2000 O, 40 C. >20,000 atoms overall.There are many different ways things can go wrong, including secondary and tertiary structure.Thus, confidence in the structure of the target antibody is very difficult to achieve.
5Structure of an antibody Mass ~150 kDa2 light chains (~20 kDa each)2 heavy chains (~50 kDa each)16 disulfide bondsGlycan heterogeneityVarious degradation pathways:DeamidationOxidationDisulfide scramblingSequence truncationDeglycosylationAggregationHere is a summary of some of those analytical challenges:Disulfide bonds hold the antibody together, but improperly linked disulfide bonds will lead to conformational changes and loss of drug efficacy.There is a conserved glycosylation site on the CH2 domain of the antibody, but the glycosylation is heterogeneous. Any change in the glycosylation population can lead to changes in efficacy.Also, there are a number of natural degradation pathways for proteins, including deamidation, oxidation, digestion, and aggregation that can occur over time.This required a wide variety of analyses, but the more of these that can be detected in a single assay the better.All of these modifications involve a mass change and thus mass spectrometry is a natural fit to the characterization of such molecules.
6The Challenge in Monoclonal Antibody Analytics Today’s focusMAb Development WorkflowMAb AnalyticsTarget Identification and ValidationMAb GenerationDrug DiscoveryCell Line DevelopmentClone Screening and SelectionPreclinical DevelopmentCell Culture and Purification Process DevelopmentClinical DevelopmentFormulationLot Release TestingStability StudiesPre-CommercializationProduct ImprovementsPatent ExtensionsBiobettersPost-CommercializationProduct TiterPurity/ImpuritiesProduct ID- Intact mass- SequencecoverageProduct Quality- Charge var.- Aggregates- Fragments- ModificationsDMPK/MetaboliteGlycansTrends that increase the number of MAb samples requiring analysis:Increasing #s of MAb candidates in pipelineAdvances in automation in cell culture & recovery dev., formulation screeningQbD guidelines requiring enhanced MAb quality monitoringThere is a growing challenge that many analytical groups in BioPharma/biotech faces today, which is, an increase in sample #s that need to be analyzed, submitted by the various different functions throughout the monoclonal antibody development process.There are several trends that contribute to this increase in samples requiring analysis:An increase in #s of monoclonal antibody candidates entering the pipelineAdvances in automation, upstream, for example, in cell culture & recovery development, or downstream, for example in formulation screeningImplementation of stricter QbD or quality-by-design guidelines that require enhanced sample quality monitoring at every step of the monoclonal antibody development and production process.The unfortunate reality is that the increase in workload is not always matched with an increase in lab resources and labor. The analytical groups often have no choice but to find ways to do more with less. The key goals here are to increase the speed of analysis, throughput & lab productivity.This throughput enhancement can be achieved in a number of ways (including LC), but we will show that mass spectrometry is one of these ways.For today’s webinar, we will focus on workflows at the upstream end of mAb development, specifically on the product ID and to some degree characterization of glycans.Goal of analytical labs: speed, throughput and productivity
7Thermo Scientific + Dionex: Combining Best-in-Class Technologies Best-in class consumables for protein scienceHigh resolution protein separation columnsBio-HPLC & UHPLC+Platforms for Protein & MAbCharacterization & QA/QCUnique ion-chromatography solutionsLeading HR/AM mass spectrometersLeadingchromatography & MS data systemsThermo now has a very broad portfolio of products for the analysis of recombinant biopharmaceuticals.We can provide consumables, columns, chromatography systems, mass spectrometers, and software comprising the entire workflow for protein characterization.Dionex is a leader in multidimensional separations for charge variant analysis and Thermo has best in class mass spectrometers.We will not focus on the chromatography aspect of protein separations today, but instead we will highlight how the new generation of Orbitraps are especially well suited for such analysis.Today’s Focus77
8Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareConfident antibody analysis using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusionsHere’s the basic outline of today’s presentation:We’ll briefly cover a few of the difficulties in the analysis of biopharmaceuticals, focusing on IgG antibodies. However, the concepts described in the presentation today can be applied to any intact recombinant (or otherwise) protein.We will focus on two instruments: The Q Exactive and the Orbitrap Elite.We will take a slight detour in the discussion of instrumentation to talk about new software packages for intact protein mass determination.Finally, we will follow up with a summary.
9A new season in life of Orbitrap mass spectrometry 2011 is the next season in the life of the Orbitrap product line.The seed was planted in 2005 with the release of the first Orbitrap and now we are several generations in the product line with a whole host of improvements..The Q Exactive and Orbitrap Elite especially demonstrate marked improvement for analysis of intact protein over previous generations of Orbitrap systems and this will be the focus of today’s presentation.
10The Orbitrap Mass Analyzer φThe first Orbitrap was introduced in 2005The Orbitrap is a Fourier transform mass spectrometerIons oscillate at a frequency proportional to m/zImage current detection produces a “transient” that is converted to a mass spectrum via a Fourier transform.High resolution, mass accuracy, and throughputAs most of you know the Orbitrap was invented by Alexander Makarov.Ions are injected, oscillate in various dimensions, where the axial motion is harmonic motion and is directly related to m/z.The induced currents from the ions oscillations along the z axis are detected and a Fourier Transform is done to produce the spectrum.Now improving the mass analysis performance in actually all about frequency, and being able to tell the difference in frequencies of motion for two neighboring masses. In general, you must get the frequency as high as possible.
11Q Exactive and Orbitrap Elite – What’s New? Quadrupole mass selectionHCD MS/MSAdvanced signal processing and electronicsImproved ion opticsHigh sensitivity and throughput!Orbitrap EliteNew compact high field OrbitrapVelos Pro Ion TrapAdvanced signal processing and electornicsImproved vacuumUltra high resolution and mass accuracy!
12Advanced signal processing produces higher resolution for isotopically-resolved intact proteins 48+ charge state of yeast enolase (46.6 kDa) is baseline resolvedOrbitrap EliteLTQ Orbitrap VelosAdvanced signal processing during the detection actually leads to a 2x improvement in resolving power for the same detection timeAdvanced signal provides higher resolving power.Higher resolution = improved mass accuracy = higher confidence.12
13Advanced Signal Processing Produces Higher Resolution for Unresolved Intact Proteins Q resolution27002720274027602780280028202840m/zGlycoformsOrbitrap resolution27002720274027602780280028202840m/zThe early detection of the transient results in much improved peak shape as well as S/N.In the IgG spectrum shown above, the glycoforms are very clearly resolved in the RAW data for both the Q Exactive and the Orbitrap Elite.Hardware and software improvements produce higher resolution peaks for IgG glycoforms
14Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareHigh resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusions
15Q Exactive MS - High Performance for Intact Proteins HCD CellMS/MSQuadrupole Mass Filterm/zamu widePrecursor selectionSIM scanOrbitrap Mass AnalyzerResolution 140K Mass accuracy better than 2 ppmAdvanced signal processingIon SourceImproved sensitivityHigh transmission across full m/z range – important for high m/z peaks from intact proteinsBent flatapoleEfficient transfer is importantHigh resolution, hardware improvements, and advanced signal processing all contribute to improve intact protein characterization
16Intact MAb on a Q Exactive 54+55+53+26802700272027402760278028002820m/zHigh S/NWell resolved glycoforms across full m/z rangeClean baselineSmooth distribution of charge statesThe Q Exactive can also be used to measure the mass of large proteins, such as intact monoclonal antibody. This is the average spectrum of 5ug antibody over 1min LC peak. A zoom-in view shows that the five major glycoforms are baseline separated, even at 17.5k resolution. The spectrum was deconvoluted using our protein deconvolution software, revealed 5 major glycoforms and a few minor forms as well. The measured values are only 7ppm away from expected values. The same antibody were also analyzed on separate days and different instruments. For the five major glycoforms, the average mass deviation is ~7ppm and the relative abundance varies by a few percents.180020002200240026002800300032003400360038004000m/z
17ReSpectTM Deconvolution of Q Exactive IgG Data G0F+G1F(-6 ppm)G0F+G1F(0 ppm)G0F+G2F (or 2 G1F)(-9 ppm)G0+G0(12 ppm)G2F+G2F(12 ppm)G1F+G2F(-12 ppm)G1F(9 ppm)G0F(9 ppm)G0+G0F(6 ppm)2xMan5(-9 ppm)10 glycoforms identified, all within ~12 ppm of theoretical average mass (+/-2 Da mass accuracy)2 deglycosylated forms detected with high mass accuracy at low relative abundance (both at ~2%)Deconvoluted spectrum produced by ReSpect is free of artifacts and the various forms of the protein are well resolvedAs we normally do for electrospray mass spectra of large intact antibodies, we use an algorithm called “Deconvolution”.For those of you who are not familiar with a deconvolution algorithm, it simply transforms a mass spectrum (which has mass/charge units) into a “Mass” spectrum.All the charge states for a given component, which there could be multiple dozen, are collapsed into a single peak at the mass value of that protein in the spectrum.We then match the masses from the deconvoluted spectrum to the calculated masses for our recombinant Mab and the various expected glycoforms and determine the mass measurement errors for each form.In this case, we identified 10 different glycoforms in the deconvoluted spectrum (which is expected for this sample) and we were surprised to see that they arll matched within about 12 ppm, which corresponds to less than 2 Da mass deviation.High mass accuracy even realized for low abundance components.ReSpect deconvolution algorithm used – will describe this in more detail a bit later in the webinar.
18Detected Charge States for G0F/G1F glycoform Measured m/zCalculated MassDelta Mass DaDelta Mass (ppm)450.755.04460.543.66470.523.52480.442.97490.151.03500.140.9351-0.05-0.3152-0.04-0.3053-0.26-1.7354-0.17-1.1255-0.09-0.6456-0.0257-0.18-1.1958-0.11-0.76590.070.48Our Protein Deconvolution software allows us to look at the different charge state peaks in the raw data and calculate the deconvoluted mass based on that charge state alone.We were surprised to see for this specific dataset that the calculated mass was surprisingly consistent for all the detected charge states for this experiment.This indicates that the deconvoluted mass is actually very accurate, since we are able to produce such accurate mass values for each of the individual charge states.The deconvoluted mass for each individual charge state are as accurate as the mass in the deconvoluted spectrum ( )The calculated average mass is very consistent for all charge states
19Mass Measurement Accuracy for 52+ charge state of IgG G0F/G1FΔ= 7 ppm28352840284528502855286028652870m/zG1F/G1F or G0F/G2FΔ = 10 ppmG0F/G0FΔ= 0 ppmG0/G0FΔ= 12 ppmG1F/G2FΔ = 12 ppmActual raw data!Chose a single charge state in the data. What we are showing is the actual raw spectrum zoomed across a 40 m/z-wide region in the data.This actually looks like a deconvoluted spectrum by itself.We took the m/z values of each glycoform, calculated the masses given that we know the charge state, and the ppm mass deviations from theoretical are shown here.As shown in the table in the previous slide, the mass measurement accuracy just taking this single charge state into account is nearly as good as the deconvoluted spectrum itself.High mass measurement accuracy can be obtained from a single charge state of an intact antibody
20Mass and Abundance Reproducibility of IgG data on a Q Exactive For the same IgG sample as previously shown, 7 different LC/MS runs with various 10 minute LC gradientsTwo different Q Exactive instruments were usedData were acquired on 3 different daysDeconvoluted mass spectra were produced using Protein Deconvolution 1.0 softwareReproducibility in mass and relative abundance were determined for the 5 major glycoforms of IgG
21Q Exactive produces reproducible mass measurement for IgG glycoforms IgG Glycoform Mass Measurement Accuracy (ppm)RAW fileQ ExactiveG0+G0FG0F+G0FG0F+G1FG0F+G2FG1F+G2F1-10.50.7-13.8-18.02-3.2-4.3-6.93.2N/A3-11.6-1.1-8.8-11.2-12.045.1-5.0-2.65.65-14.33.0-5.4-5.96-8.6-2.2-12.2-12.5-12.97-6.6-12.3-14.8-10.1Far left column shows the 7 different RAW filesThe second column shows the Q Exactive that was used for the analysis.The rest of the columns show the mass measurement deviation in ppm from the theoretical average masses for each of those glycoforms.G0F+G0F column – the mass deviations range from -6.6 to +3.0 (~10 ppm spread).The G0F+G1F column range from -2.6 to (10 ppm)On average, the ppm error is -7 ppm, which corresponds to roughly 1 Da lower than the expected mass.This is interesting – it turns out that the theoretical average mass varies dependent on carbon source and the values that we used may not match the isotope ratios for the bioreactor for which the protein was manufactured.Thus, the 6.4 ppm spread is a more realistic indication of the relative mass accuracy and this corresponds to roughly +/- 1 Da.-6.9 +/- 6.4 ppm mass tolerance across all measurementsMass measurement is highly reproducible, even across instruments
22IgG glycoform relative abundances (%) Q Exactive produces reproducible relative abundances for IgG glycoformsIgG glycoform relative abundances (%)RAW fileQ ExactiveG0+G0FG0F+G0FG0F+G1FG0F+G2FG1F+G2F112.974.1100.067.023.4212.376.071.429.8312.072.866.222.0412.275.023.6512.775.763.621.6613.275.464.821.0776.664.7CV3.4%1.6%N.A.3.9%14% (4.9%)When we look at the 7 different deconvoluted spectra and calculate relative abundances for each glycoform compared to the most abundant G0F+G1F form, these relative abundances are also remarkably consistent.There is one outlier (#2) that used a significantly different LC setup and thus there were some overlapping species that led to different results. However, 3 of these files used exactly the same gradient and the relative abundances were very close (5-7).Relative abundances are highly reproducible across runsRun 2 used a different gradient and thus there was an overlapping species that did not occur in the other runs.
23What does low ppm mass measurement provide? IgG, 150 kDappm errorMass error (Da)Detectable Modificcation50.75 Da1 disulfide bond101.5 Da2 or more disulfide bonds152.25 Da3 or more Disulfide bonds203 Da3 Disulfide bonds304.5 Da5 Disulfide bonds406 Da6 Disulfide bonds507.5 Da8 Disulfide bondsWhat does it mean if we can rely on 10 ppm mass accuracy on an intact antibody?A disulfide reduction (or oxidation ) leads to a 2 Da mass shift. At 10 ppm, we can probably detect when a few disulfide bonds have been reduced.I have an example that I’m not showing on the Orbitrap Elite where this was the case and we could confidently state on the intact level that this was happening.sAt <10 ppm mass tolerance, a mass spectrometer can detect:The reduction of two disulfide bonds on a 150 kDa protein.High confidence that there are few to no modifications to the sequence.
24Q Exactive SummaryThe Q Exactive produces very high mass accuracy for intact antibodiesThese masses and abundances can be very reproducibly measuredThis indicates that the Q Exactive will be excellent for high throughput confirmation of biopharmaceuticals
25Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareHigh resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusions
26Protein Deconvolution 1.0 Workflow software for intact protein mass determinationSupports all Orbitrap mass spectrometersIncludes 2 deconvolution algorithms:Xtract for isotopically resolved proteinsReSpect for isotopically unresolved proteins (e.g. IgG)Target release date: Early NovemberFor more information – create an account at the Thermo Proteomics Software Portal (http://portal.thermo-brims.com)ReSpect is a trademark of Positive Probability, Ltd.We have a bit of a “problem” that the instruments are so high in resolution, we can’t always use a standard charge state deconvolution algorithm.Fortunately, we already have an algorithm called Xtract for deisotoping and deconvolution of resolved isotope data.As I already mentioned, we are using an algorithm called “ReSpect” that we licensed from a componay called Positive Probability, Ltd.As I’ll briefly mention in a few slides, ReSpect not only produces accurate results, but it is unusually fast for a deconvolution algorithm.Borrowed an interface from small molecule metabolite screening application and we are considering this as a “screening” application for biopharmaceuticalsKeep track of updates on the Thermo Proteomics portal for Protein Deconvolution and all the proteomics software – please request an account and you can access tons of information about our various software products.
27Protein Deconvolution 1.0 – A Workflow Design Create/SelectMethodSelect AlgorithmLoad FileHere is a screenshot of the starting page for the software.User has a choice between Xtract and ReSpect.Then the user chooses the raw data file to be analyzed.Finally, the user then selects a method, which contains the various parameters for data processing..
28Protein Deconvolution Method Parameters Estimated Target MassOnce the user selects their data file and wants to create a new method, the software proceeds to the Parameters screen.This tab displays the various parameters required for ReSpect or Xtract deconvolution.This pane shows the parameters for ReSpect.This is split into commonly changed parameters and advanced parameters.The most important parameter for the user is to select a “Target Mass”. This target mass is used in conjunction with the instrument resolution to produce an appropriate theoretical model for an intact protein peak to be used with ReSpect. This is a rather complex calculation and we take the pain of this away from the user.More accurate the peak model, the better the results are going to be. The data shown for Q Exactive had this peak model set up for best deconvolution results.Instrument Resolution(Detected Automatically)The instrument resolution and the user-supplied protein target mass are used to calculate an accurate peak model for ReSpect deconvolution.
29Protein Deconvolution – Chromatogram Tab Select chromatogramThe next step is to produce an averaged spectrum that will ultimately be used for deconvolution.A user can choose between types of chromatograms to produce one that has the highest S/N on the chromatographic peak of the target protein.Then an average spectrum can be created by selecting the region in the chromatogram.That averaged spectrum will then be carried over to the next tab.Averaged spectrum created for deconvolution
30Protein Deconvolution - ReSpect Deconvolution Fast Data Processing: ~1-2 sDeconvoluted SpectrumAfter a user chooses to process the data, the deconvoluted spectrum in generated.ReSpect deconvolution is especially fast and can deconvolve a wide mass range in only a few seconds. Maximum entropy approaches can take minutes for wide mass ranges.When a user selects a mass from the deconvolution in the result below, the associated peak in the deconvoluted spectrum and the charge states for that mass are highlighted with the blue lines.If click on the plus on the left side of the listed mass, the information about the various charge states is shown, just like the example we showed for the Q Exactive.We also show the delta mass relative to the most abundant component on the far right side – can barely read the +162, +324 for the difference in the hexose modification.The peak list below can also be exported to Excel for offline processing if desired.Deconvolution Results
31Protein Deconvolution - Report Comprehensive, Exportable ReportFinally, the user can create a report with all the information from the preceeding screens, including the deconvoluted spectra, the source chromatogram, sample information, deconvoluted peak lists, and the parameters used to generate the results.These report can be saved as a PDF or printed.Version 1.0 out in November. We are aiming for very quick turnaround times for future versions so that features can be deployed as quickly as possible. You won’t wait 1 year for improvements.
32Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation in OrbitrapTM mass spectrometersConfident intact antibody analysis using the Q ExactiveTM Hybrid Mass SpectrometerIntroducing Protein Deconvolution 1.0 softwareHigh resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass SpectrometerSummary and conclusions
33Orbitrap Elite for High Performance Protein Characterization Electron Transfer DissociationCompact high-field Orbitrap analyzerAdvanced Signal Processing240,000 resolution, low ppm mass accuracyVelos Pro Ion TrapSelectable m/z rangeCID, MSnHCD CellHigh sensitivity ion opticsThis is a schematic of the Orbitrap EliteThis is a hybrid instrument, with a Velos Pro Ion Trap connected to an OrbitrapNew higher field Orbitrap produces up to 240,000 resolutionMy background is in FT-ICR mass spectrometry and with this instrument’s release I don’t miss it anymore.Velos Pro Ion Trap provides, CID fragmentation, ion selection, and MSn capabilitiesHCD cell for high resolution, high mass accuracy MS/MS fragmentationETD (a.k.a. electron transfer dissocation) is especially useful for characterization of glycoproteins. Collion-based fragmentation leads only to loss of glycans and no backbone fragmentation, while ETD leaves the glycans intact and only fragments the backbone.Multiple dissociation techniques provide several mechanisms for protein sequence characterization by top-down techniquesNew high field Orbitrap provides higher resolution and mass accuracyElectron transfer dissociation especially important for glycoproteins
34Orbitrap Elite: Compact High-Field Analyzer All other OrbitrapsOrbitrap Elite12 mm20 mm30 mm10 mmCompact Orbitrap unique to Orbitrap EliteReduced Orbitrap size results in 2x improvement in resolutionCompact Orbitrap + advanced signal processing = 4x resolution improvement in over previous generations of Orbitraps240,000 maximum resolution, enabling routine isotopic resolution for proteins up to 66 kDa.There are many variants of the Orbitrap analyzer. Here I would like to discuss in detail just one variant- so-called compact orbitrap. Actually, it is not that different from the standard trap- it is just 1.5 times smaller while central electrode is relatively thicker.However, this scaling down means that the entrance aperture reduced more than twice in cross-section and therefore we could expect a similar loss of sensitivity. We simply could not afford to lose on sensitivity, so a miniature lens system was developed to focus ions into a much smaller spot.Smaller size means also higher tolerance requirements which makes it harder to manufacture.Smaller geometrical size of the trap has some positive implications for image current detection. Indeed, capacitance of the trap drops proportionally to the size. Together with new transistors in the preamplifier, this allows to reduce total capacitance of the detection circuit and thus increase sensitivity.It turned out that the space charge shift in such system is smaller than in the standard trap at the same target.BSA – 67 kdA relatively routine to resolve.
35Isotopically Unresolved vs. Isotopically Resolved Orbitrap Elite:IgG light chain 17+Resolution = 15,000Isotopically unresolved(first beat only)1377.01377.51378.01378.51379.01379.51380.01380.51381.01381.51382.01382.5m/zOrbitrap Elite:IgG light chain 17+Resolution = 240,000Isotopically resolved(2 or more beats)Distance between peaks = ~1 Da/(charge state)Brief comparison of isotopically-resolved and unresolved proteinsThis is the IgG light chain (~24 kDa) run at two different resolution settings on the Orbitrap Elite.What we’ll notice on the bottom is that the broad peak above is converted to a number of much sharper peaks, each spaced by 1 DaBeyond the scope of the presentation to explain why this is, but suffice it to say that the narrower peaks provide significantly better mass accuracy and can handle spectra with much higher complexity1377.01377.51378.01378.51379.01379.5m/z1380.01380.51381.01381.51382.01382.5Isotopic resolution provides improved mass accuracy and higher peak capacity
36Isotopically-Resolved IgG Heavy Chain (~50 kDa) 54+ of IgG heavy chainAcquired at 240,000 resolution on Orbitrap EliteBaseline resolved!Δm/z = 0.018IgG light chain is about 50 kDa, well within the means for the Orbitrap Elite to produce isotopic resolutionIn this case, the IgG was resolved in an LC timescale.Notice that the peak separation is m/z units.With isotopic resolution, we have low ppm mass accuracy.932.2932.4932.6932.8933.0933.2933.4933.6m/zIsotopic resolution for the heavy chain on an LC timescale
37What does ppm mass measurement mean? Heavy Chain, 50 kDappm errorMass error (Da)Detectable Modifications50.25 DaDeamidation, Disulfide reduction100.5 DaDeamidation, disulfide reduction150.75 DaDisulfide reduction201 Da301.5 Da---402 Da502.5 DaWe easily have sub 5 ppm mass accuracy on the heavy chain.We can handle any modification that produces a mass difference greater than 1 Da.Almost any change will be detected on the intact protein level without requirement for digestion.At 5 ppm mass tolerance for a 50 kDa protein, we can detect:Deamidation of the IgG heavy chainThe reduction of a single disulfide bond on a 50 kDa protein.Any amino acid substitution (except for Q->K)
38Accurate MW Determination of reduced IgG light chain 1200140016001800200022002400m/z5101520253035404550556065707580859095100Relative Abundancez=18z=16z=19z=15z=14z=20z=13z=12z=11z=21IgG light chain18+ charge state240,000 resolution1302.61303.01303.41303.8m/zXtract deconvolutionWe used the same IgG sample as with the Q Exactive, but we instead wanted to analyze the antibody light and heavy chain.We did reduce the protein, but we did not actually fully reduce all the disulfide bonds in the process.We ran the data by LC/MS on the Orbitrap Elite and found a very well resolved isotopic cluster for the light chain as shown here.However, the mass was 4 Da lower than predicted by the target amino acid sequence.This suspiciously seems like 2 disulfides, especially since the light chain has two internal disulfides.How do we confirm this?Measured mass =Target mass =4 Dalton Mass Deviation 2 S-S?How do we confirm this?Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University
39“Top Down” vs. “Bottom Up” protein analysis Proteins are usually digested with a proteolytic enzyme and analyzed using peptide mass fingerprinting or data dependent MS/MSPeptide mass fingerprinting has some disadvantages, including introduction of artifacts into the sample and there is no guarantee of 100% sequence coverageAn alternative strategy is to use a “top down” methodology, where the intact protein is isolated and fragmented in the mass spectrometer using either a data-dependent or targeted acquisition methodHigh resolution mass spectrometry is a mustReferences:Kelleher et al, “Top Down versus Bottom Up Protein Characterization by Tandem High Resolution Mass Spectrometry”, J. Am. Chem. Soc., 1999, 21, ppBondarenko et al, “Mass Measurement and Top-Down HPLC/MS Analysis of Intact Monoclonal Antibodies on a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectrometer”, J. Am. Soc. Mass Spectrom., 2009, 20, ppWe have a couple of options:Digest the protein trying to keep the disulfides intactPeptide map, looking for all combinations of linked peptides with cysteines and hope their mass is uniquePerform MS/MS and find some software for cross-link IDOrFragment the intact light chain itself using “top down” mass spectrometryI came out of the McLafferty and I actually overlapped with Neil Kelleher when this workflow was being devised.Top down protein ID involves fragmentation of the entire protein rather than digestion of the protein and fragmentation of peptides.“Bottom up” protein digestion is not guaranteed to produce 100% sequence coverage and requires a separate experiment produce the peptide ID’s.I don’t have enough time to give a complete overview of top down, so I provided a couple of references.
40Top-Down Analysis of mAb Light Chain – Electron Transfer Dissociation (ETD) Xtract produces >200 mass valuesProSightPCTM search results in unambiguous identification of the IgG light chain (E-value = 1.4e-16)Fragments detected between the two internal cysteines:167016721674167616781680m/zz=13z=12z=7z=11This is an electron transfer dissocation spectrum of one of the charge states of the IgG light chain.It is a very complex spectrum as we can see here.The zoomed region of about 12 m/z has at least 10 different isotopic clusters.These data are analyzed using the Xtract algorithm to produce >200 unique masses, but there are considerably more isotopic clusters in the raw data because fragments will exist in multiple charge states.Then we used the ProSightPC software to unambiguously with an E-value of better than 1e-16 identify the IgG light that we thought we were analyzing.ProSightPC is top-down protein ID software created by Neil Kelleher’s group and sold by Thermo.Please visit the Thermo Proteomics Software Portal for more information.The sequence map we are showing here visualizes the MS/MS fragments that match the sequence at those sights.What we see is that all the fragments for ETD show up in the middle of the sequence between two internal cysteines.That’s interesting…z=10z=810001200140016001800200022002400260028003000m/z
41Top-Down Analysis of mAb Light Chain – HCD Xtract produces >500 mass valuesProSightPCTM search results in unambiguous identification of the IgG light chain (E-value = 1.4e-26)Fragments are detected between the two internal cysteines and at the termini up to the first cysteine.Perform the same type of analysis on the HCD data.In this case, Xtract produces more than 500 masses, which is very highly complex.ProSightPC search again produces an even more unambiguous identification with an E-value of 1e-26.The fragments also localize to the center of the sequence, but also significant sequence coverage of the N- and C-termini are found up to the first and second to last cysteine.1655166016651670167516801685169016951700170517101715m/z
42Top Down Fragment Map for IgG light chain identifies and localizes disulfide bonds Here is the combined top-down fragment map for the HCD and ETD light chain dataThe known disulfide bonds for the light chain are shown over the sequence.The disulfide bonds cyclize that portion of the sequence, preventing fragmentation from occurring.Another important point is that there are 21 sites where an N-terminal fragment and a C-terminal fragment were found. These “complementary” fragments are used to confirm the entire sequence and thus we have 100% sequence coverage without digestion.Also, it is important to note the complementarity of ETD and HCD and that more sequence coverage will be realized when using more than one collisional activation method.21 pairs of complementary fragments confirm 100% sequence coverageCombined ETD and HCD results produced fragments at 53 backbone cleavage sites, 13 more than HCD or ETD aloneNo fragments are identified between the disulfide-bound cysteines due to cyclization
43Summary – Orbitrap Elite The Orbitrap Elite is well suited for both intact protein confirmation as well as top down protein characterizationTop down protein characterization is an alternative to bottom-up peptide MS/MS for identification and confirmation of expected and unexpected changes to the target protein
44Summary and Conclusions Orbitrap-based systems are excellent for biopharmaceutical characterizationThe Q Exactive and Orbitrap Elite are the best Orbitrap systems yet for intact biopharmaceutical analysisProtein Deconvolution 1.0 produces highly accurate confident intact protein masses and abundancesProSightPC is applicable to biopharmaceutical applications (not just top down proteomics)High confidence results allow scientists in biopharmaceutical labs to increase sample throughput by bypassing more time-consuming experiments
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