A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole- Orbitrap LC-MS/MS Zhiqi Hao, 1 Yi Zhang, 1 David Horn, 1 Seema Sharma, 1 Shiaw-Lin Wu, 2 Irene Ae-Ning Lin, 3 Yi-Hsuan Pan, 3 Ya-Fen Yang, 3 and Andreas F. R. Huhmer 1 1 Thermo Fisher Scientific, San Jose, CA, USA; 2 Barnett Institute, Northeastern University, Boston, MA, USA; 3 CGMP BPPF, Development Center for Biotechnology, Taipei, Taiwan
Overview Purpose: A LC/MS-based workflow solution was developed for robust, accurate and comprehensive intact monoclonal antibody (mab) characterization. Methods: Q Exactive quadrupole-orbitrap mass spectrometers were used for intact mass measurement and top-down sequencing. Full MS spectra of intact or reduced mab were analyzed using Protein Deconvolution 1. that utilizes the ReSpect TM algorithm for molecular mass determination. The top-down msx HCD spectra were analyzed using ProSight PC 2.. Results: A mass error of less than 1 ppm was routinely achieved for intact mab mass measurement. Using an on-line high resolution top-down MSMS approach, over % of the fragmentation site was achieved for intact light chain that covers % sequence. Results from this study indicates that both precise mass measurement and extensive, high confident sequence information can be obtained for intact mab using this workflow solution that combines high resolution MS, fast chromatography, high throughput msx HCD and accurate data analysis. Introduction Monoclonal antibodies (mabs) are increasingly developed and utilized for the diagnostic and therapeutic treatment of diseases including cancer. Due to the heterogeneity of mab products, thorough characterization is necessary for their reproducible as well as safe production. Among the analytical tools used for the analysis of therapeutic mab, mass spectrometry has become more and more important in providing valuable information on various protein properties, such as intact mass, amino acid sequence, post-translational modification including glycosylation form distribution, minor impurities due to sample processing and handling and high order structure, etc. In this study, a high resolution LC-MS based workflow solution was developed for robust, accurate and comprehensive intact mab characterization. The fast chromatography, the superior resolution and mass accuracy provided by the Q Exactive Orbitrap TM MS, and accurate data analysis of this workflow provides high-confident screening tool to accelerate biopharmaceutical product development cycles. Methods Samples: Four intact mabs were used in this study. To reduce intact mab, the sample was incubated for one hour at C in 6 M guanidine-hcl containing mm DDT. HPLC: Thermo Scientific ProSwift RP-1R monolithic column (1 x mm) was used for desalting and separation of light and heavy chain. LC solvents are.1% formic acid in H 2 O (Solvent A) and.1% formic acid in acetonitrile (Solvent B). Column was heated to 8 C during analysis. Flow rate was µl/min. After injection of µg mab, a 1 min gradient was used to elute mabs from the column (.min, 2%B; 1.min, 3%B; 3.min, %B; 4.min, 98%B; 7.min, 98% B; 7.1min, 2%B; 1.min, 2%B). Mass Spectrometry: Q Exactive Orbitrap instruments (Figure 1) were used for this study. Intact and reduced mabs were analyzed by ESI-MS for intact molecular mass. Top-down MSMS was performed using high energy collision dissociation with a unique spectrum multiplexing feature (msx HCD). In this data acquisition mode, fragment ions produced from several individual HCD events, each on a precursor of a different charge state of the reduced mab, were detected together in the Orbitrap mass analyzer. The spray voltage was 4kV. Sheath gas flow rate was set at 1. Auxiliary gas flow rate was set at. Capillary temperature was 27 C. S-lens level was set at. In-source CID was set at 4 ev. Resolution was 17, or 1, for full MS and 1, for top-down MSMS. The AGC target was set at 1E6 for full scan and 2E for MSMS. Maximum IT was set at 2 ms. 2 A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS/MS
Data Processing: Full MS spectra of intact or reduced mabs were analyzed using Protein Deconvolution 1. (Figure 2) that utilizes the ReSpect algorithm for molecular mass determination. Mass spectra for deconvolution were produced by averaging spectra across the most abundant portion of the elution profile for the mab. The averaged spectra were subsequently deconvoluted using an input range of 2 to, an output mass range of 1 to Da, a target mass of 1 Da, and minimum of at least 8 consecutive charge states from the input spectrum to produce a deconvoluted peak. To identify glycoforms, the masses were compared to the expected masses with the various combinations of commonly found glycoforms. The top-down msx HCD spectra were analyzed using ProSightPC TM 2. under the single protein mode with a fragment ion tolerance of ppm. Figure 1. Schematics of Q Exactive Mass Spectrometer HCD/C-Trap Combo Cell Spectrum Multiplexing Quadrupole Mass Filter (Precursor Ion Selection) Bent Flatapole S-lens (3-xSensitivity) Orbitrap with Enhance Resolution Capillary Key Features of The Q Exactive instrument The incorporation of S-lens at the source dramatically enhanced its sensitivity. Quadrupole mass filter enables precursor selection for data-dependent MS2 and selected ion monitoring. Advanced signal processing increased resolution by two folds, which results in a maximum resolution of 1, and a maximum scan speed of 12Hz at a resolution of 17,. Spectrum multiplexing and parallel ion injection/orbitrap detection significantly improved duty cycle. Figure 2: Protein Deconvolution 1. A screenshot of the software with the source and deconvoluted spectrum and a list of the components for that protein at the bottom of the page. In this application, the user has a choice from two algorithms, Xtract or ReSpect, depending on whether or not the target protein is isotopically resolved. 1) Select data file 2) Choose appropriate deconvolution parameters 3) Create an averaged spectrum from a chromatographic peak 4) Perform deconvolution ) Print or save report Thermo Scientific Poster Note PO6329-1_e 2/12S 3
RT:. - 1.2 9 8 8 7 6 4 3 3 2 2 1 1.72 1.26 1.82 2.49 3.3 3.81.41.93.96 6. 6.9 6.26.79 9.61 9.92 1.6 12.24 13.66 14 1 2 3 4 6 7 8 9 1 11 12 13 14 Time (min) mabs were analyzed using he ReSpect algorithm for volution were produced by f the elution profile for the voluted using an input to Da, a target tive charge states from the To identify glycoforms, the the various combinations of pectra were analyzed using fragment ion tolerance of Results Figure 3: LC-MS Result of mab A B 6.8 6.83 7.8 21182_AntiHer2_ug_4 #497-9 RT:.78-6.21 AV: 113 NL: 1.8E7 T: FTMS + p ESI sid=4. Full ms [.-.] 274.7679 9 8 8 7 6 4 3 3 2 2 1 1 2281.311 2213.1896 2149.749 29.86 1977.362 233.62 2647.713 21.378 26.489 213.186 243.8234 281.336 27.2147 296.331 32.84 Total Ion Chromatogram Average Spectrum of Intact mab Charge Envelope 388.8689 314.911 3223.169 3294.7339 3369.6496 33.61 36.42 18 2 22 2 2 28 3 32 3 3 After each of the mab datasets were analyzed using software, the masses were compared to the masses amino acid sequence with the various combinations o found on mabs. One such result is shown below in Fi Figure : Deconvoluted spectrum for a composition and mass errors - G -.7 ppm GF+G. ppm G+GF 2xMan G+G oluted spectrum and a list ge. In this application, the t, depending on whether or ) Select data file ) Choose appropriate deconvolution parameters ) Create an averaged spectrum from a chromatographic peak ) Perform deconvolution ) Print or save report C Relative Abundance 21182_AntiHer2_ug_3 #499- RT:.83-6.22 AV: 12 NL: 1.6E7 T: FTMS + p ESI sid=4. Full ms [.-.] 274.7682 2797. Figure 4: Consistency of instrument performance 8 3 2 1 8 3 2 1 9 8 8 7 6 4 3 3 2 2 1 1 2742.87 2748.771 271.7736 274.63 27 27 278 28 282 28 28 Q Exactive 1 2647.727 2692.9366 269.87 2644.8711 2698.8269 26.637 2647.7466 2644.871 26.6611 GF+G1F 2794.3 GF+GF G+G1F GF+G2F 28.62 283.6411 G1F+G2F 286.649 2692.9218 2848.2716 269.8679 281.342 2698.8414 284.443 287.893 Five micrograms of mab were desalted and eluted from a ProSwift RP-1R monolithic column using a 1min gradient and analyzed using ESI-MS on the Q-Exactive. The was mab eluted over one minute as shown in (A). The average spectrum over the elution time shows a nicely distributed complete charge envelope of the mab (B). A zoom-in view of each charge state reveals five major glycosylation forms that are baseline separated (C). Q Exactive 2 262 263 26 26 26 26 268 26 2 271 2 Mass difference of major components is < 6 ppm between instruments To measure the mass accuracy and reproducibility of m Exactive in conjunction with ReSpect, the mab sample times using two different instruments over three differen mass accuracy are shown in Table 1 and the results for various glycoforms are shown in Table 2. Table 1: ppm mass deviations from expe for the most abundant glycoforms ppm mass mea RAW file Q Exacve G+GF GF+GF GF+G 1 1-1..7 2 1-3.2-4.3 3 1-11.6-1.1 4 1.1 -. 2-14.3 3. 6 2-8.6-2.2 7 2-14.3-6.6 The average ppm error for all 34 measurements was 6. deviation of 6.4 ppm. This indicates that the Q Exactive for confirmation of protein primary structure. Table 2. Relative abundance for the mo glycoforms Relave abu RAW file Q Exacve G+GF GF+GF GF+G1 1 1 12.9 74.1 1 2 1 12.3 76. 1 3 1 12. 72.8 1 4 1 12.2 7. 1 2 12.7 7.7 1 6 2 13.2 7.4 1 7 2 12.9 76.6 1 For the top glycoforms, the relative intensity reproduc percent. 4 A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS/MS
After each of the mab datasets were analyzed using the Protein Deconvolution software, the masses were compared to the masses expected for the known amino acid sequence with the various combinations of glycoforms commonly found on mabs. One such result is shown below in Figure. Figure : Deconvoluted spectrum for a mab with known composition and mass errors -7 ppm GF+G1F -.7 ppm -8. ppm GF+GF GF+G2F (or 2G1F). ppm G+GF 2xMan G+G -.9 ppm G1F+G2F G2F+G2F G1F+G2F+SA To measure the mass accuracy and reproducibility of mab samples on the Q Exactive in conjunction with ReSpect, the mab sample was analyzed several times using two different instruments over three different days. The results for ppm mass accuracy are shown in Table 1 and the results for relative abundance of the various glycoforms are shown in Table 2. Table 1: ppm mass deviations from expected target masses for the most abundant glycoforms ppm mass measurement errors RAW file Q Exacve G+GF GF+GF GF+G1F GF+G2F G1F+G2F 1 1-1..7-1. - 13.8-18. 2 1-3.2-4.3-6.9 3.2 N/A 3 1-11.6-1.1-8.8-11.2-12. 4 1.1 -. - 2.6.1.6 2-14.3 3. - 6.9 -.4 -.9 6 2-8.6-2.2-12.2-12. - 12.9 7 2-14.3-6.6-12.3-14.8-1.1 The average ppm error for all 34 measurements was 6.9 ppm with a standard deviation of 6.4 ppm. This indicates that the Q Exactive is a very powerful platform for confirmation of protein primary structure. Table 2. Relative abundance for the most abundant glycoforms Relave abundances RAW file Q Exacve G+GF GF+GF GF+G1F GF+G2F G1F+G2F 1 1 12.9 74.1. 67. 23.4 2 1 12.3 76.. 71.4 29.8 3 1 12. 72.8. 66.2 22. 4 1 12.2 7.. 67. 23.6 2 12.7 7.7. 63.6 21.6 6 2 13.2 7.4. 64.8 21. 7 2 12.9 76.6. 64.7 21.6 For the top glycoforms, the relative intensity reproducibility is within a few percent. Thermo Scientific Poster Note PO6329-1_e 2/12S
RT:. -. 36. 9 8 _3 8 7 6 4 3 3 2.7 2.61 1 41.91 1 42.73 32.8 33.67 2.76.4 7.86 9. 11.4 1. 18.38 2.22 24.66 28.78 1 1 2 2 3 3 4 Time (min) NL: 9.26E8 TIC MS 211119_ Reduced_1 k_dionex To obtain amino acid sequence, on-line, top-down MS/MS was applied to the reduced mab samples using msx HCD. Besides the improved throughput from spectrum multiplexing, the advanced signal processing provides improved resolution and higher Orbitrap scan speeds, which is critical for on-line protein top-down sequencing. As a result, high resolution, information rich spectra were generated on the one minute LC elution time for reduced mab samples. For the light chain, over % sequence coverage was achieved, including the N-terminal variable region, with a mass error of less than ppm for fragment ions. The result is shown below in Figure 6. Figure 6: On-line top-down sequencing of light chain Total Ion Chromatogram 37.7 38.72 3.39 41.7 43.77 4. 211119_Reduced_tHCD1light_Dionex #112-1 RT: 36.-36.71 AV: 29 NL: 1.12E T: FTMS + p ESI Full msx ms2 148.14@hcd1. 198.16@hcd1. 113.3@hcd1. 1213.@hcd1. 128.87@hcd1. 136.23@hcd1. [... 1176.187 z=9 9 8 8 7 6 On-line top-down HCD spectrum of light chain 18.18 437.27 4 3 3 2 2 1 1 324.1919 24.384 123.6177 1 996.4786 1322.89 z=8 9.281 739.3983 13.297 839.4744 216.98 8.22 1446.393 z=3 296.1969 686.2 177.448 2 8 12 1 Analysis using ProSight PC % sequence coverage < ppm fragment mass error ProSightPC E-value=.71e-69 Conclusion ProSwift RP-1R monolithic column provides robust and efficient separation of mabs. Q Exactive MS produces accurate and reproducible mass analysis for intact mab analysis. Q Exactive on-line top-down analysis generates extensive sequence information for reduced mab, offering a fast way to confirm sequence identity. Protein deconvolution suite enables fast and accurate calculation of the intact mass of mabs. ProSight PC offers confident sequence assignment for high resolution top-down spectrum generated by Q Exactive instrument. Both precise mass measurement and extensive, high confident sequence information can be obtained for intact mab using this workflow solution that combines high resolution MS, fast chromatography, high throughput msx HCD and accurate data analysis. ReSpect is a trademark of Positive Probability, Ltd. ProSightPC is a trademark of Proteinaceous, Inc. All other trademarks are the 6 A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS/MS
www.thermoscientific.com/dionex Thermo Scientific Dionex products are designed, developed, and manufactured under an ISO 1 Quality System. 212 Thermo Fisher Scientific Inc. All rights reserved. ReSpect is a trademark of Positive Probability, Ltd. ProSightPC is a trademark of Proteinaceous, Inc. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. U.S./Canada (847) 29 7 Brazil () 11 3731 1 Austria (43) 1 616 1 2 Benelux (31) 2 683 9768 (32) 3 33 42 94 Denmark (4) 36 36 France (33) 1 39 3 1 1 Germany (49) 6126 991 Ireland (33) 1 644 64 Italy (39) 2 1 62 1267 Sweden (46) 8 473 338 Switzerland (41) 62 2 9966 United Kingdom (44) 1276 691722 Australia (61) 2 942 233 China (82) 2428 3282 India (91) 22 2764 273 Japan (81) 6 688 1213 Korea (82) 2 263 28 Singapore (6) 6289 11 Taiwan (886) 2 871 66 PO6329-1