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Evaluation of a microfluidic electrophoresis device coupled to an Orbitrap mass spectrometer for the characterization

Evaluation of a microfluidic electrophoresis device coupled to an Orbitrap mass spectrometer for the characterization
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Evaluation of a microfluidic electrophoresis device coupled to an Orbitrap mass spectrometer for the characterization of biotherapeutics proteins. Stephane Houel1, Terry Zhang1, Erin Redman2, Scott Mellors2, Aaron Bailey1, Chris Petty2, Aran Paulus1, Kai Zhou1, Jonathan Josephs1., 1Thermo Fisher Scientific, San Jose, CA, USA, 95134; 2908 Devices, Boston, MA, USA ABSTRACT Purpose: To evaluate the performance of a chip-based electrophoresis device coupled to an Orbitrap mass spectrometer for intact and sub-unit mass analyses and peptide mapping. Results: Using the ZipChip device, the lysine variants of the NIST mAb were successfully identified at the intact and sub-unit level. For the glycoforms G0F/G1F and G1F/G1F of trastuzumab emtansine, the average DAR values were respectively 3.46 and 3.47, which are in accordance with previously published data. Using the ZipChip device, a sequence coverage in excess of 97% was observed for the light and heavy chains after a 10 min peptide mapping experiment. The discovery and development of biotherapeutics continues to accelerate. The complexity of these agents and the increasing requirements to characterize them for both safety and efficacy places a large burden on the analytical scientists tasked with these challenging demands. Intact and sub-unit mass analysis as well as peptide mapping are widely used to get insights on biotherapeutics. Often these assays are LC-MS based, but the development of a new microfluidic capillary electrophoresis device could offer a fast and sensitive orthogonal mode of separation. Here we evaluate the performance of a chip-based electrophoresis device coupled to an Orbitrap mass spectrometer for some of the major workflows used to characterize biotherapeutics Sample Preparation The analyzed samples were the NIST mAb and trastuzumab emtansine. For intact mass analysis, the sample was simply diluted in water prior to analysis. For sub-unit mass analysis, the sample was first diluted in Tris HCl 0.1M and digested with the IdeS protease. Subsequently, the sample was denatured with Guanidine 8M and reduced with DTT. The final step is a buffer exchange with the dilution buffer provided by 908 Devices in the peptide kit. For peptide mapping, NIST mAb was denatured in guanidine HCl and Tris followed by reduction and alkylation with DTT/IAA. The alkylation was quenched with DDT following buffer exchange in 50 mM Tris using BioSpin™ 6 columns (Bio-Rad Laboratories). Trypsinization was performed at 37 oC for 30 min and quenched by lowering the pH with formic acid. Finally, the sample was diluted with the peptide dilution buffer provided in the 908 devices peptide kit. Methods Microfluidic electrophoresis The ZipChip™ HR chip from 908 Devices (Boston, MA) was used for all experiments. For intact and subunit mass analysis and peptide mapping, field strengths of 500V, 220V and 400V were respectively used. Mass Spectrometry Mass spectrometer: A Thermo Scientific™ Q Exactive™ HF MS operated in high mass range (HMR) mode was used for the intact mass analysis and in standard mode operation for peptide mapping. A Thermo Scientific™ Q Exactive™ Plus in protein mode was used for the sub-unit mass analysis. Data Analysis For all of the experiments, Thermo Scientific™ BioPharma Finder™ 2.0 software was used. Figure 1. a) Data were collected using the ZipChip device coupled to a Q Exactive Plus or Q Exactive HF instrument and processed with BioPharma Finder softwar. b)The ZipChip HR is a twenty two centimeter etched channel with a neutral hydrophilic coating with an integrated nanoelectrospray emitter RESULTS A) Intact Mass Analysis 1) NIST mAb Figure 2. a) Electropherogram of NIST mAb (0.1 ug/uL). b) Spectra of the NIST mAb and the lysine variants. c) Zoom in the region of the charge +30 ions. Table 2. Deconvoluted masses and intensities of the NIST mAb for the glycoforms G0F/G1F and G1f/G1F at different concentrations. Injection Volume = 0.65 nL Table 1. Deconvoluted masses of the NIST mAb and lysine variants major glycoforms.

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