David Horn Thermo Fisher Scientific San Jose, CA Orbitrap mass spectrometry, a high-confidence screening tool in biopharmaceutical product development David Horn Thermo Fisher Scientific San Jose, CA Today 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

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software Confident antibody analysis using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions Here’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.

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software High resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions

MAb 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, 10000 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.

Structure of an antibody Mass ~150 kDa 2 light chains (~20 kDa each) 2 heavy chains (~50 kDa each) 16 disulfide bonds Glycan heterogeneity Various degradation pathways: Deamidation Oxidation Disulfide scrambling Sequence truncation Deglycosylation Aggregation Here 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.

The Challenge in Monoclonal Antibody Analytics Today’s focus MAb Development Workflow MAb Analytics Target Identification and Validation MAb Generation Drug Discovery Cell Line Development Clone Screening and Selection Preclinical Development Cell Culture and Purification Process Development Clinical Development Formulation Lot Release Testing Stability Studies Pre-Commercialization Product Improvements Patent Extensions Biobetters Post-Commercialization Product Titer Purity/Impurities Product ID - Intact mass - Sequence coverage Product Quality - Charge var. - Aggregates - Fragments - Modifications DMPK/Metabolite Glycans Trends that increase the number of MAb samples requiring analysis: Increasing #s of MAb candidates in pipeline Advances in automation in cell culture & recovery dev., formulation screening QbD guidelines requiring enhanced MAb quality monitoring There 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 pipeline Advances in automation, upstream, for example, in cell culture & recovery development, or downstream, for example in formulation screening Implementation 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

Thermo Scientific + Dionex: Combining Best-in-Class Technologies Best-in class consumables for protein science High resolution protein separation columns Bio-HPLC & UHPLC+ Platforms for Protein & MAb Characterization & QA/QC Unique ion-chromatography solutions Leading HR/AM mass spectrometers Leading chromatography & MS data systems Thermo 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 Focus 7 7

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software Confident antibody analysis using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions Here’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.

A 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.

The Orbitrap Mass Analyzer φ The first Orbitrap was introduced in 2005 The Orbitrap is a Fourier transform mass spectrometer Ions oscillate at a frequency proportional to m/z Image current detection produces a “transient” that is converted to a mass spectrum via a Fourier transform. High resolution, mass accuracy, and throughput As 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.

Q Exactive and Orbitrap Elite – What’s New? Quadrupole mass selection HCD MS/MS Advanced signal processing and electronics Improved ion optics High sensitivity and throughput! Orbitrap Elite New compact high field Orbitrap Velos Pro Ion Trap Advanced signal processing and electornics Improved vacuum Ultra high resolution and mass accuracy!

Advanced signal processing produces higher resolution for isotopically-resolved intact proteins 48+ charge state of yeast enolase (46.6 kDa) is baseline resolved Orbitrap Elite LTQ Orbitrap Velos Advanced signal processing during the detection actually leads to a 2x improvement in resolving power for the same detection time Advanced signal provides higher resolving power. Higher resolution = improved mass accuracy = higher confidence. 12

Advanced Signal Processing Produces Higher Resolution for Unresolved Intact Proteins Q Exactive @ 12500 resolution 2700 2720 2740 2760 2780 2800 2820 2840 2842.63 m/z Glycoforms Orbitrap Elite @ 15000 resolution 2700 2720 2740 2760 2780 2800 2820 2840 m/z The 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

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software High resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions

Q Exactive MS - High Performance for Intact Proteins HCD Cell MS/MS Quadrupole Mass Filter m/z 50-4000 0.4-10 amu wide Precursor selection SIM scan Orbitrap Mass Analyzer Resolution 140K Mass accuracy better than 2 ppm Advanced signal processing Ion Source Improved sensitivity High transmission across full m/z range – important for high m/z peaks from intact proteins Bent flatapole Efficient transfer is important High resolution, hardware improvements, and advanced signal processing all contribute to improve intact protein characterization

Intact MAb on a Q Exactive 54+ 55+ 53+ 2680 2700 2720 2740 2760 2780 2800 2820 m/z 2745.77 2695.89 2797.57 2742.78 2794.54 2748.78 2800.63 2698.82 2701.77 2751.77 2803.64 2791.73 2740.08 2704.65 2754.64 2806.69 2770.30 2737.33 High S/N Well resolved glycoforms across full m/z range Clean baseline Smooth distribution of charge states The 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. 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 m/z

ReSpectTM 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 resolved As 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.

Detected Charge States for G0F/G1F glycoform Measured m/z Calculated Mass Delta Mass Da Delta Mass (ppm) 45 3294.733 148217.65 0.75 5.04 46 3223.126 148217.45 0.54 3.66 47 3154.570 148217.43 0.52 3.52 48 3088.869 148217.35 0.44 2.97 49 3025.845 148217.06 0.15 1.03 50 2965.348 148217.04 0.14 0.93 51 2907.220 148216.86 -0.05 -0.31 52 2851.332 -0.04 -0.30 53 2797.548 148216.65 -0.26 -1.73 54 2745.762 148216.74 -0.17 -1.12 55 2695.858 148216.81 -0.09 -0.64 56 2647.737 148216.88 -0.02 57 2601.301 148216.73 -0.18 -1.19 58 2556.469 148216.79 -0.11 -0.76 59 2513.159 148216.98 0.07 0.48 Our 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 (148216.906) The calculated average mass is very consistent for all charge states

Mass Measurement Accuracy for 52+ charge state of IgG G0F/G1F Δ= 7 ppm 2835 2840 2845 2850 2855 2860 2865 2870 m/z G1F/G1F or G0F/G2F Δ = 10 ppm G0F/G0F Δ= 0 ppm G0/G0F Δ= 12 ppm G1F/G2F Δ = 12 ppm Actual 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

Mass 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 gradients Two different Q Exactive instruments were used Data were acquired on 3 different days Deconvoluted mass spectra were produced using Protein Deconvolution 1.0 software Reproducibility in mass and relative abundance were determined for the 5 major glycoforms of IgG

Q Exactive produces reproducible mass measurement for IgG glycoforms IgG Glycoform Mass Measurement Accuracy (ppm) RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F 1 -10.5 0.7 -13.8 -18.0 2 -3.2 -4.3 -6.9 3.2 N/A 3 -11.6 -1.1 -8.8 -11.2 -12.0 4 5.1 -5.0 -2.6 5.6 5 -14.3 3.0 -5.4 -5.9 6 -8.6 -2.2 -12.2 -12.5 -12.9 7 -6.6 -12.3 -14.8 -10.1 Far left column shows the 7 different RAW files The 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 -12.3 (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 measurements Mass measurement is highly reproducible, even across instruments

IgG glycoform relative abundances (%) Q Exactive produces reproducible relative abundances for IgG glycoforms IgG glycoform relative abundances (%) RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F 1 12.9 74.1 100.0 67.0 23.4 2 12.3 76.0 71.4 29.8 3 12.0 72.8 66.2 22.0 4 12.2 75.0 23.6 5 12.7 75.7 63.6 21.6 6 13.2 75.4 64.8 21.0 7 76.6 64.7 CV 3.4% 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 runs Run 2 used a different gradient and thus there was an overlapping species that did not occur in the other runs.

What does low ppm mass measurement provide? IgG, 150 kDa ppm error Mass error (Da) Detectable Modificcation 5 0.75 Da 1 disulfide bond 10 1.5 Da 2 or more disulfide bonds 15 2.25 Da 3 or more Disulfide bonds 20 3 Da 3 Disulfide bonds 30 4.5 Da 5 Disulfide bonds 40 6 Da 6 Disulfide bonds 50 7.5 Da 8 Disulfide bonds What 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.s At <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.

Q Exactive Summary The Q Exactive produces very high mass accuracy for intact antibodies These masses and abundances can be very reproducibly measured This indicates that the Q Exactive will be excellent for high throughput confirmation of biopharmaceuticals

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation of OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software High resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions

Protein Deconvolution 1.0 Workflow software for intact protein mass determination Supports all Orbitrap mass spectrometers Includes 2 deconvolution algorithms: Xtract for isotopically resolved proteins ReSpect for isotopically unresolved proteins (e.g. IgG) Target release date: Early November For 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 biopharmaceuticals Keep 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.

Protein Deconvolution 1.0 – A Workflow Design Create/Select Method Select Algorithm Load File Here 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..

Protein Deconvolution Method Parameters Estimated Target Mass Once 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.

Protein Deconvolution – Chromatogram Tab Select chromatogram The 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

Protein Deconvolution - ReSpect Deconvolution Fast Data Processing: ~1-2 s Deconvoluted Spectrum After 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

Protein Deconvolution - Report Comprehensive, Exportable Report Finally, 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.

Outline Challenges in the characterization of biopharmaceuticals Introduction to the new generation in OrbitrapTM mass spectrometers Confident intact antibody analysis using the Q ExactiveTM Hybrid Mass Spectrometer Introducing Protein Deconvolution 1.0 software High resolution analysis of antibody subunits using the Orbitrap EliteTM Hybrid Mass Spectrometer Summary and conclusions

Orbitrap Elite for High Performance Protein Characterization Electron Transfer Dissociation Compact high-field Orbitrap analyzer Advanced Signal Processing 240,000 resolution, low ppm mass accuracy Velos Pro Ion Trap Selectable m/z range CID, MSn HCD Cell High sensitivity ion optics This is a schematic of the Orbitrap Elite This is a hybrid instrument, with a Velos Pro Ion Trap connected to an Orbitrap New higher field Orbitrap produces up to 240,000 resolution My 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 capabilities HCD cell for high resolution, high mass accuracy MS/MS fragmentation ETD (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 techniques New high field Orbitrap provides higher resolution and mass accuracy Electron transfer dissociation especially important for glycoproteins

Orbitrap Elite: Compact High-Field Analyzer All other Orbitraps Orbitrap Elite 12 mm 20 mm 30 mm 10 mm Compact Orbitrap unique to Orbitrap Elite Reduced Orbitrap size results in 2x improvement in resolution Compact Orbitrap + advanced signal processing = 4x resolution improvement in over previous generations of Orbitraps 240,000 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.

Isotopically Unresolved vs. Isotopically Resolved 1379.70 Orbitrap Elite: IgG light chain 17+ Resolution = 15,000 Isotopically unresolved (first beat only) 1377.19 1378.84 1378.02 1381.61 1380.70 1381.87 1377.0 1377.5 1378.0 1378.5 1379.0 1379.5 1380.0 1380.5 1381.0 1381.5 1382.0 1382.5 m/z 1379.74 Orbitrap Elite: IgG light chain 17+ Resolution = 240,000 Isotopically resolved (2 or more beats) 1379.63 1379.80 1379.57 1379.92 1379.98 1379.51 Distance between peaks = ~1 Da/(charge state) Brief comparison of isotopically-resolved and unresolved proteins This 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 Da Beyond 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 complexity 1379.45 1380.04 1380.10 1379.39 1378.74 1380.22 1377.10 1378.57 1379.27 1380.33 1377.39 1381.62 1377.80 1378.16 1380.63 1381.33 1381.86 1382.21 1377.0 1377.5 1378.0 1378.5 1379.0 1379.5 m/z 1380.0 1380.5 1381.0 1381.5 1382.0 1382.5 Isotopic resolution provides improved mass accuracy and higher peak capacity

Isotopically-Resolved IgG Heavy Chain (~50 kDa) 54+ of IgG heavy chain Acquired at 240,000 resolution on Orbitrap Elite Baseline resolved! 932.8726 932.8544 932.9092 932.8905 Δm/z = 0.018 IgG light chain is about 50 kDa, well within the means for the Orbitrap Elite to produce isotopic resolution In this case, the IgG was resolved in an LC timescale. Notice that the peak separation is 0.018 m/z units. With isotopic resolution, we have low ppm mass accuracy. 932.2 932.4 932.6 932.8 933.0 933.2 933.4 933.6 m/z Isotopic resolution for the heavy chain on an LC timescale

What does ppm mass measurement mean? Heavy Chain, 50 kDa ppm error Mass error (Da) Detectable Modifications 5 0.25 Da Deamidation, Disulfide reduction 10 0.5 Da Deamidation, disulfide reduction 15 0.75 Da Disulfide reduction 20 1 Da 30 1.5 Da --- 40 2 Da 50 2.5 Da We 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 chain The reduction of a single disulfide bond on a 50 kDa protein. Any amino acid substitution (except for Q->K)

Accurate MW Determination of reduced IgG light chain 1200 1400 1600 1800 2000 2200 2400 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 1303.0917 z=18 1465.9155 z=16 1234.6137 z=19 1563.5760 z=15 1675.1174 z=14 1172.8326 z=20 1803.8945 z=13 1954.1323 z=12 2131.7785 z=11 1117.1264 z=21 IgG light chain 18+ charge state 240,000 resolution 1302.6 1303.0 1303.4 1303.8 m/z Xtract deconvolution We 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 = 23424.4845 Target mass = 23428.416 4 Dalton Mass Deviation  2 S-S? How do we confirm this? Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University

“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/MS Peptide mass fingerprinting has some disadvantages, including introduction of artifacts into the sample and there is no guarantee of 100% sequence coverage An 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 method High resolution mass spectrometry is a must References: Kelleher et al, “Top Down versus Bottom Up Protein Characterization by Tandem High Resolution Mass Spectrometry”, J. Am. Chem. Soc., 1999, 21, pp. 806-812 Bondarenko 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, pp. 1415-24. We have a couple of options: Digest the protein trying to keep the disulfides intact Peptide map, looking for all combinations of linked peptides with cysteines and hope their mass is unique Perform MS/MS and find some software for cross-link ID Or Fragment the intact light chain itself using “top down” mass spectrometry I 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.

Top-Down Analysis of mAb Light Chain – Electron Transfer Dissociation (ETD) Xtract produces >200 mass values ProSightPCTM search results in unambiguous identification of the IgG light chain (E-value = 1.4e-16) Fragments detected between the two internal cysteines: 1670 1672 1674 1676 1678 1680 m/z 1680.45 1802.8888 z=13 1954.5505 z=12 1545.0427 z=7 2132.2379 z=11 This 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… 2345.6667 z=10 1416.4512 z=8 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z

Top-Down Analysis of mAb Light Chain – HCD Xtract produces >500 mass values ProSightPCTM 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. 1697.00 1686.85 1696.57 1711.01 1687.28 1675.26 1694.00 1715.44 1665.84 1699.43 1715.86 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. 1666.27 1678.13 1686.28 1663.84 1672.83 1656.83 1691.85 1704.85 1679.84 1702.86 1656.33 1708.58 1661.55 1670.84 1682.84 1655 1660 1665 1670 1675 1680 1685 1690 1695 1700 1705 1710 1715 m/z

Top 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 data The 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 coverage Combined ETD and HCD results produced fragments at 53 backbone cleavage sites, 13 more than HCD or ETD alone No fragments are identified between the disulfide-bound cysteines due to cyclization

Summary – Orbitrap Elite The Orbitrap Elite is well suited for both intact protein confirmation as well as top down protein characterization Top 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

Summary and Conclusions Orbitrap-based systems are excellent for biopharmaceutical characterization The Q Exactive and Orbitrap Elite are the best Orbitrap systems yet for intact biopharmaceutical analysis Protein Deconvolution 1.0 produces highly accurate confident intact protein masses and abundances ProSightPC 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

Thermo Scientific BioPharma Capabilities Sample Analysis Sample Preparation Data Interpretation Unregulated Regulated Research Discovery DMPK QC/QA The most complete portfolio of BioPharma solutions

Acknowledgements Marketing Software R&D Hardware R&D Andreas Hühmer Thomas Moehring Markus Kellmann Yi Zhang Zhiqi Hao Seema Sharma Rosa Viner Vlad Zabrouskov Amy Zumwalt Shannon Eliuk Reiko Kiyonami Justin Blethrow Julian Saba Scott Peterman Software R&D Steve Chaput Doug Miller Tom McClure Grace Li Paul Gazis Helen Tran Shijun Li Barbara Gibson Hardware R&D Alexander Makarov Jae Schwartz Martin Zeller

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