A comprehensive CE/ESI-MS solution for BioPharma applications

Transcription

1 A comprehensive CE/ESI-MS solution for BioPharma applications Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometers with ZipChip system

2 Fast CE Separation Nanospray Sensitivity HRAM Spectrometry A unique and powerful combination of CE/ESI and MS technology designed for BioPharmaceutical applications Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometer platforms coupled with the 98 Devices ZipChip system can characterize a wide range of bio-analytes via unique microfluidic capillary electrophoresis (CE) separation and high resolution accurate mass (HRAM) spectrometry identification. This exceptionally fast, sensitive, and accurate CE/ESI-MS workflow complements LC-MS technology to address analytical challenges in BioPharma. The unique ZipChip system and powerful Q Exactive MS combine with Thermo Scientific BioPharma Finder software and ZipChip consumables to offer a comprehensive CE/ESI-MS solution for intact antibodies and antibody drug conjugates (ADC), antibody subunits, and peptide analyses. 2

3 Electropherograms of intact trastuzumab corresponding to spectra a,b,c shown below a b c a Full MS spectra 1. ppm G/GF ppm GF/GF ppm GF/G1F ppm (G1F/G1F) or (GF/G2F) ppm G1F/G2F Deconvoluted spectrum z= Native 1 ng of sample injected Intact mab Analysis in HMR mode The ZipChip system and Q Exactive MS platforms can quickly analyze intact mabs in native, partially denatured, and fully denatured conditions to support characterization of biotherapeutics under a diverse range of conditions. The CE/ESI-MS analysis can be completed within 3 minutes Confidently achieve high resolution accurate mass spectra in intact native, partially denatured, and fully denatured states on the Thermo Scientific Q Exactive Plus, HF, HF-X hybrid quadrupole-orbitrap MS with the BioPharma option Sample consumption can be as low as picograms to nanograms Major glycoforms are identified by BioPharma Finder software b z= Fully denatured 16 pg of sample injected c z= Partially denatured 16 pg of sample injected ZipChip interface z= BioPharma Finder software MS data were acquired on the Q Exactive HF-X mass spectrometer equipped with the BioPharma option 3

4 Intact mab Analysis in HMR mode The ZipChip system and Q Exactive MS platforms are unique and powerful in their ability to separate and identify different intact antibody charge variants in native and denaturing conditions. Baseline separation of intact mab charge variants resulting from different levels of Lysclipping can be achieved within three minutes by the ZipChip system High resolution accurate mass spectra of all lysine variants are confidently detected on Q Exactive Plus/HF/HF-X MS with the BioPharma option Three major glycoforms from each of the three lysine variants are identified by BioPharma Finder software with mass accuracies better than 1 ppm Lys Lys 3.5 Lys z=26 z= z=25 z= Deconvoluted MS spectrum Full MS spectra zoom zoom zoom CE/ESI-MS electropherogram of intact infliximab: separation of lysine-variants RT 2.6 min RT 3.5 min RT 4.5 min z= [GF/GF] 2 Lys Lys [GF/G1F] 2 Lys Lys [(GF/G2F)/(G1F) 2 ] 2 Lys ng of sample injected Lys 1 Lys Lys Sliding window deconvolution with Respect algorithm Theoretical and experimental masses of infliximab glycoforms of lysine variants Lys-Variant Glycoform Theor. Average Exp. Average Δ (ppm) 2 Lys GF/GF Lys GF/G1F Lys (G1F)2 or (GF/G2F) Lys GF/GF Lys GF/G1F Lys (G1F)2 or (GF/G2F) Lys GF/GF Lys GF/G1F Lys G1F)2 or (GF/G2F) MS data were acquired on the Q Exactive HF-X mass spectrometer equipped with the BioPharma option 4

5 Antibody-Drug Conjugate Analysis in HMR mode All the different forms of Trastuzumab Emtansine can be analyzed extremely fast with extremely low sample consumption Heterogenetic ADCs can be successfully characterized within 1 minute by utilizing a ZipChip system and Q Exactive Orbitrap MS workflow with BioPharma Finder software. DM1 No sample pre-treatment is needed DM1 Powerful sliding Nwindow capability enabled by N NH2 N N H H NH 2 BioPharma Finder for highly accurate H H software allows average masses The calculated average DAR values are consistent with previous published data Trastuzumab Emtasine Trastuzumab Emtasine Trastuzumab Emtasine Trastuzumab lysine-linked ADC Emtasine lysine-linked ADC Average Drug-to-Antibody (DAR) Ratio DAR DAR DAR DAR DAR DAR Relative GF/G1F Accuracy Abundance DAR Form (ppm) DAR DAR DAR DAR DAR DAR DAR DAR DAR DAR7 55 DAR DAR6 ADC 3 ng of sample injected MS data were acquired on the Q Exactive HF mass spectrometer equipped with the BioPharma option 5

6 Fd regions LCs mab Sub-unit Analysis in Protein Mode Fast, sensitive, and accurate antibody subunit analysis can be accomplished by the ZipChip system and Q Exactive Plus or HF/HF-X MS platforms. Separation of infliximab mab subunits can be achieved in 3 minutes by the ZipChip system The sliding window method combined with Thermo Scientific Xtract deconvolution algorithm in BioPharma Finder software enables monoisotopic mass determination of each subunit The subunits of the infliximab antibody mab and the lysine variant have different migration times and were identified at less than with 5 ppm after processing with BioPharma Finder software Fc/2 regions F(ab ) 2 region IdeS digest IdeS digested infliximab 12 k 2.11 Fc / (Fc/2) -Lys 2.26 F( ab ) 2 15 k z=48 z= z=44 z= z=45 z=41 z= Deconvolution with Respect F( ab ) Fc / (Fc/2) -Lys IdeS digest plus reduction Fc/2 regions IdeS digested + reduced infliximab (Fc/2) -Lys Fc /2 12 k LC Fd Fc / (Fc/2) -Lys LC Fd Deconvolution with Xtract Deconvolution with Xtract (Fc/2 GF) -Lys Fc/2 GF (Fc/2 G1F) -Lys Fc/2 G1F LC (Fc/2 GF) -Lys Fd (Fc/2 G1F) -Lys Fc/2 GF Fc/2 G1F 25 IdeS digest plus reduction IdeS digest Subunit Monoisotopic Theo. Experimental Δ (ppm) Light chain Fd Fc/2 GF Fc/2 G1F (Fc/2 GF) -Lys (Fc/2 G1F) -Lys F(ab ) (av.) Fc/2 GF (2 SS bonds) Fc/2 G1F (2 SS bonds) (Fc/2 GF) -Lys (2 SS bonds) (Fc/2 G1F) -Lys (2 SS bonds) MS data was acquired on the Q Exactive HF-X mass spectrometer equipped with BioPharma option

7 Peptide Mapping Analysis in standard mode The combination of ZipChip sample separation, Q Exactive produced HRAM MS and MS/MS spectra, and BioPharma Finder software enables fast and accurate peptide identification. Peptides can be separated and analyzed in a 1-minute CE-MS/MS experiment 98% sequence coverage based on MS/MS data for the light chain and heavy chain can be confidently achieved The plug-and-play ZipChip system delivers stable nanospray and nanospray level sensitivity with only 1 nanogram of sample injected for analysis Flag Protein NIST mab light chain NIST mab heavy chain Unidentified RT (min) Proteins Number of MS Peaks MS Peak Area Sequence Coverage Abundance (mol) NSIT mab light chain % 1.% 41.67% NSIT mab heavy chain % 97.6% 56.35% Unidentified % MS data was acquired on the Q Exactive HF mass spectrometer equipped with BioPharma option 7

8 Ordering information Product Spectrometer Cat. No. Q Exactive Plus MS 7263 BioPharma Option for Q Exactive Plus Q Exactive HF MS BioPharma Option for Q Exactive HF 7266 Q Exactive HF-X MS BioPharma Option for Q Exactive HF-X 7266 BioPharma Finder software OPTON-3592 CE/ESI ZipChip Interface (Autosampler version) ZipChip Interface (Manual version) ZipChip Autosampler Consumables ZipChip HS ZipChip HR ZipChip Metabolites Assay Kit ZipChip Peptides Assay Kit ZipChip Intact Protein Assay Kit Find out more at thermofisher.coipchip For Research Use Only. Not for use in diagnostic procedures. The ZipChip Interface is a Class 1 Laser Product (complies with 21 CFR 14.1 and except for deviations pursuant to laser notice No. 5). 217 Thermo Fisher Scientific Inc. All rights reserved. ZipChip is a registered trademark of 98 Devices. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. BR64967-EN 617M