Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Spectrometer

Transcription

1 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Spectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej Bromirski Thermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

2 Overview Purpose: Improve the performance of bench-top Orbitrap mass spectrometers for large molecules and complex samples. Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector. Results: Measurements of the intact Humira antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 6 minute gradient sample runs from HeLa cells. Introduction This work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples. Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required. When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications. Large Molecules Image Current Detection Because image current detection is an interference detection method used in Fourier- Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to.5 seconds, the complete detection of the very first beat is crucial. Transient used Experimental Setup 5 µg Humira monoclonal antibody (m Thermo Scientific BioBasic- column µm particle size 5 min run time with linear gradient f acid at a 5 μl/min flow rate Resulted in a sample elution time Evaluation of signal intensity To evaluate the signal intensity containe transient portion is used while varying th the first five milliseconds contain a signi better signal transmission for the improv FIGURE. Transient sketch of a decaying beat pattern resulting from a FTMS. 5 ms 795. Technical Improvements To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms. To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse. Having these changes in effect, the transient dwell time reduced to <.5 milliseconds, as shown in Figure ms ms ms ms 6 8 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

3 FIGURE. Typical transients: Former design (Exactive ), Improved design (Q Exactive, Exactive Plus) Transient used FIGURE. Humira (8kD spectr ProMass Deconvolution Transient used Experimental Setup 5 µg Humira monoclonal antibody (ma (8 kda, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic- column of mm i.d. and mm length packed with 5 µm particle size 5 min run time with linear gradient from to 8% of acetonitrile with.% formic acid at a 5 μl/min flow rate Resulted in a sample elution time of ~5 min Evaluation of signal intensity To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design. FIGURE. Varied detect delay for 8 ms transient (development mode, xµscans, ms fixed inject time). The first five milliseconds show a significant signal contribution ms 5 ms ms ms ms RT: AV: T: N= RT: AV: T: N= RT: AV: T: N= RT: AV: T: N= RT: AV: T: N=68.9 FIGURE 5. Single experiment HCD 8 matching fragment humira_igg_std_hcd_pressure_aif humira_igg_std_hcd_pressure_aif humira_igg_aif #95- RT: #95- AV: 9 RT: NL: E AV: T:.@hcd8. [9.-.] R R= R= Heavy chain Light chain 6 R= R= R=876 Thermo Scientific Poster Note PN6595_E 6/S

4 .Inc.) loaded on a length packed with 5 ile with.% formic ient, a very short. It is visible that signal resulting in a - RT: AV: T: -5 RT: AV: T: Measurements The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 75 at to use the shortest available transient length; this will cover the entire first transient beat. This spectrum, shown in Figure, was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure. The mass accuracy stays below ppm and the different glycoforms are represented. Figure 5 shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond kda. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 8 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5. FIGURE. Humira (8kD spectrum, Deconvoluted spectrum using ProMass Deconvolution Humira_FullMS # RT:.8 AV: NL:.9E5 T: [.-.] FIGURE 5. Single experiment HCD fragmentation spectrum of Humira mab. 8 matching fragments identified by ProSight PC. humira_igg_std_hcd_pressure_aif 8 6 /7/ :6: PM humira_igg_std_hcd_pressure_aif humira_igg_aif #95- RT: #95- AV: 9 RT: NL: E AV: 9 NL:.8E T:.@hcd8. [9.-.] R=76 z= R= R= R= R= R= R=65 z= R=559 z=7 8.9 R=55 z= R= R= R= Complex samples Automatic gain control (AGC) To fully utilize the analytical performanc system, the number of ions injected to t measurement of the ion current is eithe records a very short transient, or by usi short section of the previous analytical s transient acquisition is used to calculate In some rare cases the number of ions resolution and the lower signal respons especially true for multiply charged ions To demonstrate this effect the AGC imp off and the maximum inject time was minute gradient LC chromatogram of a proteins and including the column wash retention times between 6 and 7 minu spectrum from this section shows multip the short AGC-prescan and therefore w spectrum shows the average of three m proteins become visible showing ions th of the AGC-prescan leading to further u the inject time for the analytical scan wi A valid previous workaround was to red inject time carefully to a dedicated level FIGURE 6. Chromatogram and mass gradient. During the colum wash the and peaks below the noise threshold range. RT: HeLa_LysC_noEM_e6_e5_mz5 fm5hcd_ 5 5 #86 RT: AV: N T: FTMS + p NSI Full ms [5.-.] T 9.9 R= R= R= R=7 z= R= R=65 HeLa_LysC_noEM_e6_e5_mz5 fm5hcd_ # RT: T: FTMS + p NSI Full ms [5.-.] R= R= R=97 z= R=775 R=99 R= FIGURE 7. Exactive Plus layout with analytical scan is acquired in the Orb 6-7 RT: AV: T: 8-9 RT: AV: T: 5 - RT: AV: T: Heavy chain Light chain humira_igg_std_hcd_pressure_aif #95- RT: AV: 9 NL:.5E T: humira_igg_aif FTMS + p ESI Full #95- ms.@hcd8. RT: [9.-.] AV: 9 NL:.8E R= R= R= R= R=66 R=86 5. R= Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

5 ctrum using of Humira mab. C = R= R=68 Complex samples Automatic gain control (AGC) To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 6 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 6 and 7 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class. FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 6 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range. RT: NL:.8E9 5.9 Base Peak F: ms MS 5. HeLa_LysC_noEM _e6_e5_mz5_ _fm5hcd_ HeLa_LysC_noEM_e6_e5_mz5 fm5hcd_ 5 5 #86 RT: AV: NL:.6E T: FTMS + p NSI Full ms [5.-.] Time (min) 5. R= z=5 R= R=696 R=6 R= z= R= R= R= 8.59 R= z= R=8 97. R=7 R=66 R= R=65 HeLa_LysC_noEM_e6_e5_mz5 fm5hcd_ # RT: AV: 9 NL:.7E T: FTMS + p NSI Full ms [5.-.] R=7 R=67 z= R=88 R= R=6 R=697 R=67 R= R= R=99 R=966 R=59 R=69 z= FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap. Measurement Results Using the CTCD, the HeLa run is repea To emphasize the effect by getting close AGC target was set to e6 for this expe during the column wash stage. The spe can be used for further confirmation. FIGURE 8. With the CTCD active, the overfilling. The mass spectrum conta RT: T IF #95- RT: AV: 9 T: [9.-.] AV: 9 NL:.8E R= R= R=56.86 R= R=77 5. R= Thermo Scientific Poster Note PN6595_E 6/S 5

6 ity of the Orbitrap trolled. The -prescan, which which uses the first rent from this short ext analytical scan. cause of the lower uisition. This is e noise threshold. ow was switched ure 6 shows a 6 artially digested nd of the run at ccurate. A single on t be resolved in le). The second rtially digested the short acquisition urrent. In this case, filling of the C-Trap. set the maximum e run with a 6 min. th, multiply charged ding retention time NL:.8E9 Base Peak F: ms MS HeLa_LysC_noEM _e6_e5_mz5 fm5hcd_ C-Trap Charge Detection (CTCD) To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling. Measurement Results Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation. FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest. RT: hela_lysc_wem_e6_e5_mz5 fm5hcd_ #9668 RT: 6.5 AV: NL:.7E6 Time (min) T: FTMS + p NSI Full ms [5.-.] R=57 R= R= R=7 8 z=5 R= z= R= R= R= R= R=99 R=87 R=7 z= R= NL:.5E9 Base Peak F: ms MS hela_lysc_wem_e 6_e5_mz5 fm5hcd_ R=96 R=8 R= R=6 R=67 R= ctive while the Conclusion The described hardware changes improve the measurement performance for large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than ppm for the deconvoluted spectrum. Using HCD fragmentation 8 matching fragments could be identified from a single experiment. The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run. Acknowledgements We would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC. The research funding of the 7th European Framework Program is appreciated (Health- F-8-68/PROSPECTS). Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

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