Protein Solutions for Biotherapeutics

Protein solutions eBook Biopharma 2 3 Enabling every step in your protein therapeutics journey Early development phases identify potential molecules to develop as drug candidates. Includes the search for pipeline candidates. Requires: High-throughput capabilities and versatile, advanced tools. In process development, an in-depth understanding of process effect on biomolecules allows for quicker progression through the development pipeline. Requires: Robust and reproducible analyses to monitor molecular attribute changes with speed and confidence. At this stage, the analytical focus shifts to gaining a deep product understanding. Requires: Advanced tools to answer in-depth molecular questions with full confidence. Early discovery and research Product characterization Process and analytical development From discovery to commercialization, Thermo Fisher Scientific provides expertise and a superior array of scalable tools, services, and support, designed to deliver high-quality results and accelerate your productivity and innovation. Small changes in manufacturing conditions can have a significant impact on product quality. To ensure the release of safe and effective products, advanced analytical methods are implemented to closely monitor CQAs for each batch. Requires: Reproducible, robust, GMP compliance-ready methods. Quality control and manufacturing Protein therapeutics are a type of biotherapeutics derived from genetically engineered human proteins. They offer several advantages over small-molecule drugs, allowing for more precise targeting of complex functions. Compared to chemical drug production, the production process for protein-based therapeutics is intricate and variable, with a higher risk of product degradation and contamination. The final product is fragile and can be affected by factors like temperature, storage duration, denaturants, solvents, oxygen, and pH changes. As a result, protein biologics require strict controls in manufacturing, transport and storage. An array of analytical approaches and workflows facilitate comprehensive characterization, allowing for full product understanding which can save significant time and development costs. Throughout process development and manufacturing, these analytical tools are required for reliable monitoring of product microheterogeneity ensuring drug safety and efficacy. This eBook will provide a comprehensive explanation of the different workflows specifically designed for protein analysis, highlighting the benefits that each workflow offers. Protein therapeutics: At a glance 4 5 Learn more about intact protein analysis Thermo Scientific™ Orbitrap Exploris™ 240 MS Excellent sensitivity, allows the use of much less sample for high quality intact MS analysis Thermo Scientific™ Orbitrap Exploris™ MX MS Built for usage in the routine environment Complemented by compliance-ready software Thermo Scientific™ Chromeleon™ Biotherapeutic protein Value-added workflow benefits • Direct infusion techniques or separation allow intact protein analysis • Screen, identify, and characterize intact proteins with higher productivity and confidence using the Intact Protein workflow in Thermo Scientific™ BioPharma Finder™ software • BioPharma mass extension available for Orbitrap Exploris 240 and 480 • Quickly confirm intact protein molecular weight and determine glycoform heterogeneity of biopharmaceuticals Thermo Scientific™ Orbitrap Exploris™ 480 MS Intuitive instrument control for easy execution of analysis Easy-to-use software for data processing Intact protein analysis Efficient intact protein analysis for rapid assessment enabling confident identification There is a need for a more efficient protein analysis approach that saves time on sample preparation and offers simple data analysis. The ideal solution provides high throughput, information-rich data, and fast assessment of post-translational modification levels. Webinar - Workflows for robust and sensitive separation of mAbs, intact proteins at subunit and peptide level using capillary chromatography by LC-MS Thermo Scientific™ Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer CID and HCD, optionally with UVPD, ETD, PCTR, can be used in any MS stage, followed by mass analysis in either the ion trap or ultrahigh resolution Orbitrap mass analyze Early discovery and research Development QC Use the wheel to navigate to your area of interest. Protein therapeutics workflows Intact protein analysis Native intact analysis Aggregate analysis Subunit analysis Glycan analysis Higher order structure analysis Charge variant analysis Peptide mapping Multi-attribute method Host cell protein analysis 6 7 Learn more about intact protein analysis Intact protein analysis under near-native conditions Analysis of intact proteins and protein-complexes, in their true biological states Analyzing complex therapeutic proteins like antibody-drug conjugates (ADCs) is a significant challenge. The Thermo Scientific Orbitrap Exploris mass spectrometers, coupled with size exclusion or ion exchange chromatography and compatible buffers, enable routine intact protein analysis under native conditions. Webinar - NIBRT: Ion exchange chromatography hyphenated to mass spectrometry for fast and robust characterization of mAb variants under native conditions Thermo Scientific™ Orbitrap Exploris™ 480 MS Intuitive instrument control for easy execution of analysis Easy-to-use software for data processing Thermo Scientific™ Orbitrap Exploris™ 240 MS Excellent sensitivity, allowing the use of much less sample for high quality native MS analysis Thermo Scientific™ Orbitrap Exploris™ MX MS Built for usage in the routine environment. Complemented by compliance-ready software Thermo Scientific™ Chromeleon™ Biotherapeutic protein Early discovery and research Thermo Scientific™ Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer Advanced MS capabilities and optional mass range of up to 16’000 m/z allow scientists to take advantage of versatile fragmentation techniques and advanced ion reaction techniques like proton transfer charge reduction Development Thermo Scientific™ Q Exactive™ UHMR MS The first UHMR MS to combine substantially increased sensitivity and mass resolution at high m/z, MS2, and psuedo-MS3 capabilities in a single platform QC Value-added workflow benefits • Use Thermo Scientific MAbPac SEC-1 Size Exclusion Columns for high-resolution separation of monoclonal antibody (mAb) analysis, including monomers and aggregates • BioPharma option to extend mass range available for the Orbitrap Exploris 240 and 480 MS systems • Excellent mass spectrometric data, intuitive instrument control and easy-to-use software for data processing • Q Exactive™ UHMR Hybrid Quadrupole Orbitrap™ Mass Spectrometer offers precise mass determination, peak interference mitigation, and the ability to study heterogeneous protein assemblies Learn more about aggregate analysis Thermo Scientific™ Vanquish™ Duo UHPLC System The Vanquish Duo UHPLC system provides two independent flow paths connected to two individual detectors. This unique system lets you run two analyses in parallel, either identical or not, on a single system to double throughput, accelerate method development, and improve sample characterization Aggregated proteins Value-added workflow benefits • High resolution separation of fragments, monomers and aggregates • BioPharma option to extend mass range available for the Orbitrap Exploris 240 MS • Thermo Scientific™ Vanquish™ Duo UHPLC system for Dual LC provides simple and rapid high-throughput analysis of aggregates • Compliance-ready analytical setup using Thermo Scientific™ Chromeleon™ software for the monitoring stage Aggregate analysis High resolution separation of monomers and aggregate peaks Video - Application Note Video - A universal Chromatography Method for Aggregate Analysis of Monoclonal Antibodies The consistent quality of biologic products must be ensured by monitoring protein aggregates throughout the production process, as aggregates can have serious negative effects. Failure to monitor protein aggregates can result in incorrect drug dosage, decreased solubility, and activity loss. Thermo Scientific™ Vanquish™ UHPLC System & Thermo Scientific™ Orbitrap Exploris 240 MS Aggregate analysis by HRAM MS facilitates the analysis of the proteins in their native form, without the need for sample preparation, while providing information on aggregation and fragmentation Characterization Routine monitoring 8 9 Learn more about subunit analysis Subunits Value-added workflow benefits • Subunits are readily separated by reversed-phase (RP) UHPLC using Thermo Scientific MAbPac RP columns • Deconvolution of subunit mass spectrometry (MS) spectra is easy with powerful Thermo Scientific BioPharma Finder software • Thermo Scientific™ Pierce Fab Preparation Kit uses immobilized pepsin protease to digest human or mouse IgG antibodies to make separate Fab and Fc antibody fragments Subunit analysis Easy deconvolution by harnessing the power of Thermo Scientific BioPharma Finder Software Monoclonal antibody (mAb) subunit analysis involves the antibody fragmentation through digestion and reduction of IgG antibody molecules. The cysteine protease produced by S.pyogenes, known as IdeS, is highly specific. IdeS digestion followed by reduction generates three subunits that are readily separated by reversed-phase (RP) UHPLC using Thermo Scientific MAbPac RP columns. Deconvolution of subunit mass spectrometry (MS) spectra is easy with powerful Thermo Scientific BioPharma Finder software. Webinar - Workflows for robust and sensitive separation of mAbs, intact proteins at subunit and peptide level using capillary chromatography by LC-MS Thermo Scientific™ Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer Quantify more samples at lower concentrations using faster acquisition, while achieving greater coverage using a revolutionary new hardware design featuring dual ion routing multipoles Vanquish UHPLC System & Orbitrap Exploris 240 MS The extreme resolving power of the Thermo Scientific Orbitrap mass analyzer ensures isotopic resolution of monoclonal antibody subunits and facilitates middledown sequencing. Thermo Scientific™ Vanquish UHPLC System Take advantage of MAbPac SEC-1 columns which offer superior, reproducible separation of monomers, aggregates, and fragments resulting from proteolysis Characterization Routine monitoring Learn more about higher order structure analysis Biotherapeutic protein Value-added workflow benefits • Sample preparation and labelling is made easy by our partners, Trajan Scientific and Medical. Trajan’s LEAP HDX sampler system enables automated labelling and digestion • The Vanquish Neo UHPLC system combines an unrivaled degree of innovation to deliver 24/7 reproducible separations of complex mixtures at maximum performance for a variety of high-sensitivity LC-MS workflows Higher order structure analysis A robust method for the analysis of protein conformation, conformation dynamics, and protein-protein interactions Full structural characterization is critical where local conformational changes can impact safety and efficacy. Hydrogen deuterium exchange (HDX) mass spectrometry (MS) is a powerful analytical approach for studying the dynamics of higher order structure of protein-based therapeutics. We offer a unique total solution for your HDX workflow. Webinar - Dr Patrick Griffin - Hijacking Molecular Plasticity to Fine Tune Nuclear Receptor Signaling: Chemical Biology and Precision Therapeutics Thermo Scientific™ Orbitrap Exploris™ 480 MS Intuitive instrument control for easy execution of analysis Easy-to-use software for data processing Thermo Scientific™ Orbitrap Exploris™ 240 MS Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer fast tracks your path to highconfidence data and identification Thermo Scientific™ Neo™ Nano LC & Thermo Scientific™ Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer Delivering the ultimate flexibility to expand experimental scope, and with built-in intelligence, Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer ensures the highest data quality for HDX-MS experiments • Thermo Scientific BioPharma Finder software supports all HDX-MS data analysis, including peptide identification, PTM analysis and HDX-unique protection factor plots at the single residue level Early discovery and research Development 10 11 Learn more about charge variant analysis Biotherapeutic proteins Value-added workflow benefits • Simplified and fast ion exchange analysis (IEX) of charge variants using a pH gradient • Obtain fast and highly robust, reproducible HPLC gradients using Thermo Scientific™ CX-1 pH gradient buffer kits. The CX-1 buffers save time in method development, facilitate method transfer to QA/QC • The Orbitrap Exploris 240 MS offers high sensitivity, allowing for the detection and quantification of low abundance charge variants • Vanquish UHPLC systems offer dependable, flexible, and productive separations Thermo Scientific™ Vanquish™ UHPLC System & Orbitrap Exploris 240 MS Speed and accuracy are key criteria for laboratories performing charge variant profiling with an out of the box sample capacity of 216 samples. The Vanquish UHPLC system and the Orbitrap Exploris 240 MS offers the speed and accuracy required for charge variant profiling Charge variant analysis Full solutions from mobile phase preparation to purpose-built software Webinar - Taking Charged Variant Analysis to the next level Heterogeneity of monoclonal antibodies must be monitored and revealed by charge sensitive techniques. Two workflows specific to your needs: 1. Separation by charge followed by mass spectrometry (CVA-MS) allows for characterization of post-translational modifications (PTMs) at the intact protein level. 2. Salt gradient cation-exchange chromatography can be used to monitor mAb charge variants. Vanquish™ UHPLC System Obtain new benchmarks in accuracy, precision and sensitivity in charge variant analysis with the Thermo Scientific™ Vanquish™ UHPLC systems. Providing biocompatibility with a state-of-the-art quaternary or binary high-pressure solvent blending, these ultra-high performance liquid chromatography systems share all Vanquish values, such as a design focused on uptime, robustness and reliability Characterization Routine monitoring Learn more about peptide mapping Value-added workflow benefits • Automation options and kit-based protein digestion for the reproducible results • High throughput peptide mapping with Vanquish Duo UHPLC system • Complete product characterization: Sequence coverage, PTMs, Disulfide bonds and Sequence variants Accurate peptide mapping is crucial for determining sequence coverage, identifying and quantitating post-translational modifications, determining disulphide bonds, identifying sequence variants, and performing de novo sequencing of proteins. Failure to accurately perform peptide mapping can result in incomplete or incorrect protein characterization, compromising the safety and efficacy of biologic products. Webinar - Rapid Automated Peptide Mapping Vanquish Tandem LC System & Orbitrap Exploris 240 MS The Vanquish Duo allows for tandem LC-MS. With this approach, a greater number of sample injections can be performed in the same timeframe, enhancing throughput and significantly reducing MS idle time Vanquish Tandem LC System & Orbitrap Exploris 480 MS Accurate mass and resolving power provide the most effective way to accurately and confidently identify peptides. Confident and complete sequence coverage with versatile Thermo Scientific™ BioPharma Finder™ software Thermo Scientific™ KingFisher™ Duo Prime Purification System Thermo Scientific™ SMART Digest™ Kit provides fast and simple protein digestion with high reproducibility and sensitivity, in a format that’s compatible with automation Peptides Peptide mapping High throughput peptide mapping with Vanquish Duo UHPLC system Characterization 12 13 Learn more about Multi-Attribute Method Value-added workflow benefits • Seamless and direct method transfer from lab-to-lab • End-to-end, compliance-ready platform for deployment from development to QC • Enterprise software platform for connectivity at global scale leveraging the power of enterprise Chromeleon and Ardia Platform software. • Confident and consistent attribute identification and monitoring with high resolution and accurate mass data Webinar - Pfizer: Evaluation of the Orbitrap Exploris MX Mass Detector Multi-Attribute Method A platform from discovery and development to QC The Multi-Attribute Method (MAM) is a peptide mapping-based method used to quantify multiple potential CQAs simultaneously. Thermo Scientific™ MAM 2.0 is a powerful high resolution accurate mass-based workflow that enables comprehensive characterization and monitoring of quality attributes from research to quality control (QC). MAM is in alignment with the QbD approach to the development of biopharmaceuticals, which is advocated by regulatory agencies and is being adopted by biopharmaceutical companies. Vanquish Flex or Horizon UHPLC System & Thermo Scientific™ Orbitrap Exploris™ MX enables confident monitoring of critical quality attributes Thermo Scientific™ Vanquish™ Flex or Horizon UHPLC System & Orbitrap Exploris 240 enables comprehensive characterization of product quality attributes Research & Development Attribute Characterization | Product Quality Attribute Monitoring Manufacturing & QC Critical Quality Attribute Monitoring | New Peak Detection Peptides with or without PTMs Thermo Scientific™ eWorkflow™ Characterization Routine monitoring Digestion Learn more about host cell protein analysis Value-added workflow benefits • Ready-to-use single informatics workflow for peptide mapping and HCP data processing and visualization • HRAM MS data combined with Thermo Scientific™ Proteome Discoverer™ software provides confident HCP identifications The analytical solution for Host Cell Protein (HCP) monitoring must be both robust and fast, and capable of identifying and quantifying multiple HCPs in a single chromatographic separation. To meet the growing demand for increased process understanding, scientists are turning to reliable UHPLC systems coupled with HRAM mass spectrometers for accurate and reliable HCP monitoring throughout the purification process. Video - Optimized sample preparation for low ppm detection of Host Cell Proteins (HCPs) in biotherapeutics with Orbitrap-based MS detection HCPs • Increased sensitivity enabled by micro-flow rate separation allows detection of low abundant HCPs KingFisher Duo Prime Purification System Non-denaturing protein digestion with SMART Digest kit reduces intra-sample dynamic range and increases host cell protein (HCP) identifications Vanquish™ High-throughput LC systems & Orbitrap Exploris 480 MS Detection of HCPs ranging in concentrations from <0.5 ppm to 200 ppm Identify and quantify multiple HCPs in a single chromatographic separation, allowing for reliable HCP monitoring throughout the purification process Vanquish™ Neo UHPLC System & Thermo Scientific™ Orbitrap™ Astral™ MS The Orbitrap Astral Mass Spectrometer helps overcome the challenges of insufficient throughput, low abundant HCP ID and provides sub ppm quantification Host cell protein analysis Fast analysis achieved by harnessing the power of Orbitrap technology Characterization 14 15 Complete characterization of monoclonal antibodies under native and denaturing conditions This document provides a comprehensive overview of the use of the Thermo Scientific Orbitrap Exploris 480 mass spectrometer equipped with BioPharma Option for the full characterization of antibody samples. The primary objective is to provide optimal LC-MS conditions to achieve conclusive information on the molecular weight and proteoform heterogeneity at the intact mAb, subunit, and peptide levels. A key challenge in analyzing therapeutic monoclonal antibodies (mAbs) is the macro-heterogeneity deriving from various N-linked glycan species and structural heterogeneity deriving from various endogenous modifications. These modifications contribute to micro-heterogenous proteoform mixtures of covalently assembled molecules, which vary in size, charge, and hydrophobicity and could impact mAb chemical properties.The solution to this challenge is the application of different workflows for characterizing biopharmaceuticals on the new LC-MS platform, including intact mass analysis under native and denaturing conditions, subunit analysis complemented with Middle-Down analysis, and peptide mapping. The Orbitrap Exploris 480 mass spectrometer equipped with the BioPharma Option is used to achieve these analyses with high sensitivity, scan speed, and resolution to achieve results with the highest confidence. Learn more about glycan analysis Value-added workflow benefits • Flexible solutions for all analytical strategies • Fit for purpose sample preparation options • HRAM MS for outstanding intact protein or glycopeptide analysis • Easy-to use fit for purpose software capabilities from discovery to control Glycan analysis Comprehensive toolbox for glycan analysis Analysis of glycans can be complex as no single method will provide all information, but there are many different approaches for glycan analysis. Thermo Fisher Scientific offer full solutions to analyze glycans utilizing UHPLC, UHPLC-MS and IC for analysis of glycoforms. Webinar - Advancing carbohydrate analysis - Glycans Thermo Scientific™ Orbitrap™ Ascend Tribrid™ Biopharma mass spectrometer Quantify more samples at lower concentrations using faster acquisition, while achieving greater coverage using a revolutionary new hardware design featuring dual ion routing multipoles OR Glycans Vanquish UHPLC System & Orbitrap Exploris Platform or Thermo Scientific™ ICS-6000 ™ Ion Chromatography system Monitor released N- & O-glycan without labelling Flexibility: Ion chromatography with pulsed amperometric detection (HPAEPAD) OR LC with charged aerosol detection (CAD) Characterization Routine monitoring HCD Intact native Intact denatured Subunits Peptide mapping Top/Middle-Down Application Note - View the PDF Application note spotlight 16 17 Seamless LC-MS method transfer in a biopharmaceutical development laboratory Symphogen, a Denmark-based biopharmaceutical company, employs an LC-MS system, made up of a Thermo Scientific Vanquish Horizon Duo UHPLC and Thermo Scientific Q Exactive Plus hybrid quadrupoleOrbitrap mass spectrometer, for the development and lead selection studies of complex therapeutic proteins and protein mixtures. Recently, Symphogen aimed to augment their laboratory capabilities by introducing a new generation benchtop Orbitrap-based high-resolution accurate mass (HRAM) mass spectrometer, the Thermo Scientific Orbitrap Exploris 480 mass spectrometer. • The primary challenge was to ensure the transfer of the execution of the native mass analysis measurements hyphenated to size exclusion chromatography (SEC-MS) from the Q Exactive Plus MS to the new Orbitrap Exploris 480 MS. The new MS platform had to deliver consistent high-quality data, comparable to the existing Q Exactive Plus MS system, across hundreds of samples in each lead selection study. • The solution involved a systematic evaluation and optimization of method parameters on the Orbitrap Exploris 480 MS to generate high-quality data comparable to the Q Exactive Plus MS platform. Figure 6 shows the assessment of robustness through the investigation of glycoform level determination reproducibility. The results demonstrate the new platform’s robustness, with remarkably low variability and %CV values, thus ensuring a seamless method transfer between the two platforms. Application note spotlight 4 × 96-well plates Vanquish Duo UHPLC System for tandem LC or LC-MS Q Exactive Plus MS with BioPharma Option Instrument control and data processing in Chromeleon CDS 5000 5200 5400 5600 5800 6000 6200 6400 6600 0 10 20 30 40 50 60 70 80 90 100Relativ e Abundance G0F/G0F G0F/G1F G0F/G2F 147500 148000 148500 Orbitrap Exploris 480 MS with BioPharma Option • Instrument control and data processing for ID testing in Chromeleon CDS • For method optimization, including assigment and quantitation of glycoforms/proteoforms data processing with Protein Metrics Byos software 5000 5200 5400 5600 5800 6000 6200 6400 6600 0 10 20 30 40 50 60 70 80 90 100Relativ e Abundance G0F/G0F G0F/G1F G0F/G2F 147500 148000 148500 A) B) Vanquish Duo UHPLC System for tandem LC or LC-MS 4 × 96-well plates Application Note - View the PDF Confident peptide mapping and disulfide bond analysis of an IgG2 monoclonal antibody The document discusses an application brief related to peptide mapping and disulfide bond analysis of an IgG2 monoclonal antibody, denosumab, using the Thermo Scientific Orbitrap Exploris 240 mass spectrometer. The primary goal was to demonstrate the utility and capability of the mass spectrometer for the routine characterization of biotherapeutics through LC-MS peptide mapping. This procedure allows for the full sequence coverage, high confidence in low level posttranslational modifications (PTMs) identification, and ease in interpreting complex data • The major challenge in the biopharmaceutical industry is ensuring the quality, efficacy, and safety of biotherapeutic proteins, such as monoclonal antibodies, due to variations in production processes and the intrinsic complexity of these proteins. There are many critical quality attributes (CQAs) that must be monitored, including the detection of inter- and intrachain disulfide bonds and low-level posttranslational modifications (PTMs). Additionally, the industry requires instruments and analytical methods that can be transferred and adopted routinely across organizations. • The Orbitrap Exploris 240 mass spectrometer provides operational simplicity and flexibility, enabling a wide range of characterization assays to be performed with high confidence. It was used to perform peptide mapping analysis to confirm 100% sequence coverage of denosumab, enabling the detection of low-level PTMs and the location of disulfide bonds. Application note spotlight 1:C23/1:C89 2:C368/2:C426 1:C135/1:C195 2:C149/2:C205 2:C22/2:C96 1:C215/2:C136 2:C262/2:C322 2:C224, C225, C228, C231/2:C224, C225, C228, C231 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Minutes 10 20 30 40 50 60 70 80 90 100 Relative Abundance S-S bond type Position Δ ppm RT Intrachain LC1 ATLSCR / LEPEDFAVFYCQQYGSSPR 1:C23/1:C89 -1.2 25.95 LC2 SGTASVVCLLNNFYPR / HKVYACEVTHQGLSSPVTK 1:C135/1:C195 -2.1 24.15 HC1 LSCAASGFTFSSYAMSWVR / AEDTAVYYCAK 2:C22/2:C96 -2.7 28.95 HC2 STSESTAALGCLVK / DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK 2:C149/2:C205 -0.9 32.54 HC3 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK / CK 2:C262/2:C322 -0.6 26.76 HC4 NQVSLTCLVK / WQQGNVFSCSVMHEALHNHYTQK 2:C368/2:C426 0.4 22.87 Interchain LC-HC GEC / GPSVFPLAPCSR 1:C215/2:C136 -2.5 21.86 Hinge KCCVECPPCPAPPVAGPSVFLFPPKPK / KCCVECPPCPAPPVAGPSVFLFPPKPK 2:C224, C225, C228, C231/ 2:C224, C225, C228, C231 -0.02 28.05 Peptide sequence Application Note - View the PDF 18 19 IdeS-cleaved mAb subunit analysis with LC-HRAM-MS: a quick and accurate comparison of biosimilar and originator biotherapeutics The document discusses the application of a middle-up approach for the characterization of biotherapeutics, using the Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. This technique is crucial for the fast and reliable characterization of monoclonal antibody variants and modifications, which is of paramount importance in the biopharmaceutical industry. • The key challenge in the biopharmaceutical industry is to establish the quality and safety of biosimilar therapeutics, which are drugs with minimal variations from their originator. These variations can occur due to multiple factors, including expression system, growing conditions, purification steps, or final formulations. Therefore, it is crucial to monitor and quantify these variations to correlate them to any potentially different in vivo activity, such as different clearance time or other interactions within the patient. • The solution to this challenge is the use of a middleup approach, which involves the use of liquid chromatography hyphenated with high-resolution, accurate-mass spectrometry (LC-HRAM-MS) for the comparison of monoclonal antibody drug substances and their respective biosimilars. This approach minimizes sample handling and artifacts and provides quicker or complementary information. As illustrated in Figure 2, the LC-MS analysis of IdeS digested bevacizumab biosimilar provided high quality data, which allowed for a confident identification and sequence verification of light chain and Fd region and a rapid analysis of Fc region variants, including glycoform and N-terminal lysine loss. Relative Intensity 0 10 23942.8376 23428.5729 + LVTNS ∆ppm: 1.6 20 30 40 50 60 70 80 90 100 Mass 23000 23200 23400 23600 23800 24000 24200 Application Note - View the PDF Proton transfer charge reduction (PTCR) This document provides a comprehensive overview of the experiments conducted for the analysis of monoclonal antibody (mAb) subunits under denaturing conditions. The experiments utilize Liquid Chromatography (LC) and Mass Spectrometry (MS) analysis with various reagents, columns, solvents, and flow rates. The document details the LC conditions, MS conditions, data analysis, results, and discussion. • The primary challenge in this context is the characterization of the primary sequence of biotherapeutics, specifically monoclonal antibodies (mAbs). Traditional methods such as Bottom-Up Mass Spectrometry (BU MS) and Top-Down Mass Spectrometry (TD MS) have limitations in terms of sequence coverage and the complexity of MS2 spectra, especially for larger mAb subunits. • The solution proposed in this document is the application of Proton Transfer Charge Reduction (PTCR) in conjunction with Electron Transfer Dissociation (ETD) for middle-down analysis of mAbs. This approach simplifies fragment ion spectra, leading to improved sequence coverage and increased confidence in product ion matching. …CPPCPAPELLG GPSVF… Reduction (TCEP/DTT) F(ab’)2 Fc ~ 50 kDa ~ 100 kDa IdeS ~ 25 kDa ~ 25 kDa 2x Fd’ ~ 25 kDa 2x Fc/2 2x Lc Mannose Fucose N-acetylglucosamine A B Time (min) 6.0 8.0 10.0 12.0 Fc/2 Lc Fd’ 800 1000 1200 m 1400 1600 1800 /z 1147.6 m/z 1148.6 20+ Monoisotopic mass (theoretical) Monoisotopic mass (experimental) ΔM (ppm) 22,928.1799 22,928.09975 +3.5 ppm Application Note - View the PDF Application note spotlight Application note spotlight Resources BioPharma learning center Peptide mapping Glycan analysis Host cell protein analysis Subunit analysis Native intact mass analysis Protein aggregate analysis Charged variant analysis Multi-attribute method Higher order structure analysis Protein homepage Find the column for your application Biopharma webinar portal, searchable by application © 2024, 2025 Thermo Fisher Scientific Inc. 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