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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation Figure 4. Spectral library creation in Empower. Pre-characterized glycan names were input in the Component Tab (left) to create the library. The collected information can be added to the library via the dropdown menu (right). INTRODUCTION Glycosylation is one of the most common and complex post-translational modifications on monoclonal antibodies. Due to its direct correlation to the efficacy of antibody drugs, glycosylation has been recognized as a critical quality attribute (CQA) of drug product. To ensure the accuracy and consistency of glycosylation profiles, the suitable characterization and monitoring methods are critical for the product quality throughout the product lifecycle. To this end, methods that can increase the confidence in CQA assessment are highly desired for product quality control. As an orthogonal technique, MS detection has been increasingly applied in regulatory environment as an orthogonal detection technique for its high sensitivity and specificity of measuring product quality attributes. The ACQUITY QDa mass detector requires minimal knowledge to operate and can be readily adapted to the existing LC-UV or LC/FLR workflow, making it easy to fit in QC environment. In this study we present a LC-FLR/MS workflow using MS enabled Spectral Library matching algorithm for improved productivity and confidence of analysis of glycan profiles. IMPROVING ROUTINE ANALYSIS OF GLYCAN PROFILES IN A QC ENVIRONMENT USING SPECTRAL LIBRARIES Brooke Koshel; Ximo Zhang; Robert Birdsall; Joe Fredette; Min Du; Ying Qing Yu Scientific Operations, Waters Corporation, Milford, MA METHODS LC conditions QDa condition References 1. Exploiting Rapifluor-MS labeling to Monitor Diverse N-Glycan Structures via Fluorescence and Mass Detection. Waters application note, 720005353EN 2. Rapidly Monitoring Released N-Glycan Profiles During Process Development Using RapiFluor-MS and the ACQUITY QDa Detector . Waters application note, 720005438EN RESULTS AND DISCUSSION CONCLUSION Spectral Library provides a tool for glycan identification and routine analysis Added MS detection increases confidence in results compared to optical-based assays Empower enabled automated data processing and reporting Readily investigate out of spec results LC system ACQUITY UPLC H-Class Bio Detector ACQUITY FLR detector (λ excitation =265 nm, λ emission =425 nm) ACQUITY QDa MS detector (performance model) LC column ACQUITY Glycan BEH Amide column, 1.7 μm, 130 Å, 2.1 × 150 mm Column temp. 60 °C Mobile phase A H 2 O with 50 mM Ammonium Formate Mobile phase B ACN Informatics Empower 3 Mass range 350-1250 m/z Mode ESI Positive Capillary voltage 1.5 kV Cone voltage 10 V Probe temp 500 °C Sample rate 2 points/sec Figure 2, The ACQUITY ® QDa Mass Detector. The QDa detector is a single quadrupole mass detector that can be easily incorporated into an existing LC stack. Time (min) Flow (mL/min) %A %B Initial 0.400 25.0 75.0 35.00 0.400 46.0 54.0 36.50 0.200 100.0 0.0 39.50 0.200 100.0 0.0 43.10 0.200 25.0 75.0 47.60 0.400 25.0 75.0 55.00 0.400 25.0 75.0 Gradient table Figure 1, Improved workflow for routine analysis using MS-enabled Spectral Library. With the collected LC-MS data from reference standard, orthogonal information such as name, retention time, and base peak mass, are stored in the Spectral Library and used to match against the samples. Once built, the Spectral Library is universal to all projects in Empower to ensure the consistency of spectral matching. The Spectral Library can be used for routine analysis of glycan profiles to increase confidence in data analysis, provide orthogonal mass spectra confirmation, and support investigations into assay failures. Figure 7, Spectral Library matching results of the detected five peaks. The results table shows retention time, base peak mass, and matched spectral name of detected peaks. A custom field “Match_Result” was used to compare the glycan identification based on retention time and MS spectra. Results can be exported as report via Empower in an automated fashion. Workflow for Spectral Library Building and Matching Orthogonal MS information to support investigations Start Figure 5. MS Library Search tab. Parameters can be adjusted for the best matching. Figure 6. Spectral matching for glycan sample. Using the RFMS glycan standard as reference, Peak 1-5 are the detected glycans from an intact mAb sample using a defined processing method in Empower. Spectral matching was automatically performed with the Empower processing method. The retention time filter can be used to address challenging glycan spectra such as isomers for improved matching ability. A custom field used as indication of matching results MS spectra of Glycan isomers Glycan isomers Extract MS spectra from auto-integration Input glycan names in Empower processing method Retention time filter Helps identify isomers Spectral Library matching results MS spectra Released glycan from Trastuzumab Failure of matching due to the additional m/z peak from co-eluted Man5 Open investigation and re-develop method for higher resolution Figure 8. Investigations supported by orthogonal information. Using released glycan from Trastuzumab as a sample, a 35 min gradient partially resolved the Man5 and A2G1a peak, causing the additional peak (774 m/z) in the MS spectrum of A2G1a, which leads to a mismatch. The orthogonal MS data allows for efficient investigations to identify method failure points and recommend the appropriate course of action. Collect LC-MS data of reference standard Matching Results Data acquisition of glycan samples Enter glycan name and create library Adjust noise threshold Set library searching parameter Figure 3. LC/MS data acquisition. The QDa was used as an in- line detector to provide MS information. Retention time and base mass of each peak were obtained via automated integration in Empower. A threshold of relative intensity can be set up to reduce the background noise in Spectral Library. Report of glycan profiles XIC XIC 1) Acquire LC/MS Reference Data 2) Build Spectral Library 3) Run Samples 4) Report
