Author: Maa BaboviPublisher:
ISBN:
Category :
Languages : en
Pages : 0
Book Description
Comprehensive Characterization of Therapeutic Proteins by Top-down Mass Spectrometry
Author: Maa BaboviCapillary Electrophoresis-Mass Spectrometry
Author: James Q. XiaPublisher: Springer
ISBN: 3319462407
Category : Science
Languages : en
Pages : 80
Book Description
This book covers the latest developments in capillary electrophoresis-mass spectrometry for the analysis of therapeutic proteins. The application of capillary electrophoresis-mass spectrometry (CE-MS) coupling technology in the analysis of recombinant therapeutic proteins is detailed thoroughly. Specific topics include recent developments in coupling capillary electrophoresis with mass spectrometry for the quality control of monoclonal antibody therapeutics, top-down analysis of monoclonal antibody using the CE-MS platform, and detection of host cell protein impurities. Comprehensive characterization of antibody-drug conjugates (ADCs) by coupling capillary electrophoresis with mass spectrometry is also covered. This is an ideal book for scientists in the life science and biopharmaceutical industry who are working on characterizing the PTMs of monoclonal antibodies, as well as graduate students and researchers in the separation science and biological mass spectrometry fields.
Characterization of Protein Therapeutics using Mass Spectrometry
Author: Guodong ChenPublisher: Springer Science & Business Media
ISBN: 1441978623
Category : Science
Languages : en
Pages : 408
Book Description
This book highlights current approaches and future trends in the use of mass spectrometry to characterize protein therapies. As one of the most frequently utilized analytical techniques in pharmaceutical research and development, mass spectrometry has been widely used in the characterization of protein therapeutics due to its analytical sensitivity, selectivity, and specificity. This book begins with an overview of mass spectrometry techniques as related to the analysis of protein therapeutics, structural identification strategies, quantitative approaches, followed by studies involving characterization of process related protein drug impurities/degradants, metabolites, higher order structures of protein therapeutics. Both general practitioners in pharmaceutical research and specialists in analytical sciences will benefit from this book that details step-by-step approaches and new strategies to solve challenging problems related to protein therapeutics research and development.
Advancing Intact Protein Analysis by Top-down Mass Spectrometry
Author: Bifan ChenPublisher:
ISBN:
Category :
Languages : en
Pages : 215
Book Description
The study of proteins is critical for understanding cellular functions at the molecular level. Top-down mass spectrometry (MS) has emerged as a premier tool for global and comprehensive analysis of proteoforms. The top-down approach retains intact mass information, providing a "bird's-eye" view of the proteome and allowing for identification of novel proteoforms, in-depth sequence characterization, and quantification of disease associated post-translational modifications (PTMs). However, many technical challenges still exist. The research described here involves analytical development in top-down MS, particularly in the areas of enrichment, separation, and characterization of samples ranging from standard proteins and complex lysates, to large therapeutic biomolecules. Chapter 1 provides an introduction and review of recent advances in different aspects of top-down proteomics. Chapters 2 and 3 are related to the study of intact phosphoproteins. Specifically, chapter 2 describes the use of functionalized nanoparticles for enrichment and the subsequent coupling of online liquid chromatography (LC)-MS for characterizing endogenous phosphoproteins from complex cell lysates. Chapter 3 investigates how phosphorylation moieties might influence the efficiency of electron capture dissociation (ECD). Chapters 4 and 5 focus on the development of hydrophobic interaction chromatography (HIC) that could be coupled online directly with MS and its applications to therapeutic molecules (monoclonal antibodies). Chapter 6 describes a middle-down approach to obtain multi-attribute of both cysteine and lysine conjugated antibody-drug conjugates, which overcomes some current challenges using HIC-MS and the top-down approach. Overall, these analytical developments expand the toolbox of the top-down approach and generally facilitate the analysis of intact proteins.
Analytical Characterization of Biotherapeutics
Author: Jennie R. LillPublisher: John Wiley & Sons
ISBN: 1119384400
Category : Science
Languages : en
Pages : 426
Book Description
The definitive guide to the myriad analytical techniques available to scientists involved in biotherapeutics research Analytical Characterization of Biotherapeutics covers all current and emerging analytical tools and techniques used for the characterization of therapeutic proteins and antigen reagents. From basic recombinant antigen and antibody characterization, to complex analyses for increasingly complex molecular designs, the book explores the history of the analysis techniques and offers valuable insights into the most important emerging analytical solutions. In addition, it frames critical questions warranting attention in the design and delivery of a therapeutic protein, exposes analytical challenges that may occur when characterizing these molecules, and presents a number of tested solutions. The first single-volume guide of its kind, Analytical Characterization of Biotherapeutics brings together contributions from scientists at the leading edge of biotherapeutics research and manufacturing. Key topics covered in-depth include the structural characterization of recombinant proteins and antibodies, antibody de novo sequencing, characterization of antibody drug conjugates, characterization of bi-specific or other hybrid molecules, characterization of manufacturing host-cell contaminant proteins, analytical tools for biologics molecular assessment, and more. Each chapter is written by a recognized expert or experts in their field who discuss current and cutting edge approaches to fully characterizing biotherapeutic proteins and antigen reagents Covers the full range of characterization strategies for large molecule based therapeutics Provides an up-to-date account of the latest approaches used for large molecule characterization Chapters cover the background needed to understand the challenges at hand, solutions to characterize these large molecules, and a summary of emerging options for analytical characterization Analytical Characterization of Biotherapeutics is an up-to-date resource for analytical scientists, biologists, and mass spectrometrists involved in the analysis of biomolecules, as well as scientists employed in the pharmaceuticals and biotechnology industries. Graduate students in biology and analytical science, and their instructors will find it to be fascinating and instructive supplementary reading.
Development and Application of Top-down and Middle-down Proteomics for Comprehensive Protein Characterization in the Muscle Proteome
Author: Yutong JinPublisher:
ISBN:
Category :
Languages : en
Pages : 0
Book Description
Mass spectrometry (MS)-based proteomics, including top-down, middle-down and bottom-up approaches, has become the method of choice for protein identification, quantification, and characterization. Top-down proteomics represents a superior approach for comprehensive protein characterization by analyzing intact proteins, providing an overview of all the proteoforms present in a sample and allowing for identification of various proteoforms and in-depth characterization of sequences and post-translational modifications (PTMs). However, the protein mass range that can be analyzed by top-down approach is normally limited to 100 kDa. Middle-down proteomics, analyzing polypeptides from proteolysis, is a complementary strategy to top-down proteomics for characterization of large proteins (>100 kDa) and complex PTMs. In this dissertation, I developed and applied top-down and middle-down proteomics to comprehensively characterize the sarcomeric proteins from striated muscle tissues. Sarcomeric proteins are expressed in different isoforms produced by homologous genes, in the presence of various proteoforms produced by a single gene via genetic variations, alternative RNA splicing, and PTMs. The various isoforms and PTMs of sarcomeric proteins are associated with muscle contractile properties and thus affects the entire muscle function. However, a comprehensive characterization of sarcomeric protein isoforms and PTMs is still challenging due to the high complexity of the muscle proteome and the limitations in techniques for protein separation and characterization. The first part of this dissertation focuses on characterizing sarcomeric proteins below 100 kDa using top-down proteomics. Sarcomeric proteins from multiple skeletal muscle tissues were characterized by liquid chromatography and mass spectrometry plus (LC-MS+) strategy which combines online LC-MS and offline tandem MS (MS/MS) analysis. I also developed a reversed-phase chromatography method using monolithic column for highly efficient separation of sarcomeric proteins and combined it with high-resolution MS for top-down proteomics. The second part of this dissertation describes the development of middle-down proteomics for characterization of large sarcomeric proteins (>100 kDa). I developed a MS-compatible size-exclusion chromatography (SEC) method to purify myosin heavy chain from cardiac muscle tissue and characterize this protein by refining a middle-down MS strategy. I also developed a middle-down proteomics strategy to study the phosphorylation status of a serine-arginine-rich protein RNA-binding motif 20. Finally, combining top-down and middle-down proteomics, I described a strategy for the comprehensive characterization of a monoclonal antibody. Collectively, this dissertation provides comprehensive analysis of the isoforms and PTMs of sarcomeric proteins that will help elucidate molecular event in muscle contraction and disease pathologies. Furthermore, this dissertation presents a variety of novel LC and MS methods for protein separation and characterization, enabling the further characterization of other proteomes with deep proteome coverage.
Characterization of Therapeutic Proteins by Capillary Electrophoresis (CE) Coupled to Mass Spectrometry (MS)
Author: Nassur SaidPublisher:
ISBN:
Category :
Languages : en
Pages : 0
Book Description
Monoclonal antibodies (mAbs) are highly complex glycoproteins having a lot of micro-heterogeneities which can influence their effectiveness. As a consequence, it is necessary to develop robust analytical methods, sensitive and specific to characterize them with high accuracy. The purpose of this thesis was to develop analytical methods allowing the multi-level characterization of monoclonal antibody (cetuximab), and antibody drug conjugates (brentuximab vedotin), using on-online or off-line capillary electrophoresis - mass spectrometry coupling. In the first section, a middle-up proteomic approach of cetuximab was carried out using Off-line CZE-UV/MALDI-MS coupling to separate and to characterize Fc/2 and F(ab)'2 charge variants. A top-down characterization of Fc/2 fragments was also employed. Then a new strategy off-line CZE-UV/nanoESI-MS was used to allow the characterization of this partially digest mAbs. Finally, an online coupling by CESI-MS was developed to allow the fast and accurate analysis of middle-up cetuximab. In a second part, the combination of intact, middle-up and bottom-up proteomic carried out on CZE-UV/nanoESI-MS and CESI coupling allowed the most exhaustive characterization of brentuximab vedotin. This methodology allowed the analyze of DAR, the identification of fragments drug conjugates, the simultaneous characterization of the complete structure of antibody, a significant number of post-translational modifications, all peptides drug conjugates and the identification of diagnostic ions.
Internal Fragments in Top-down and Middle-down Mass Spectrometry
Author: Benqian WeiPublisher:
ISBN:
Category :
Languages : en
Pages : 0
Book Description
The interrogation of protein structure, especially identifying and localizing post-translational modifications (PTMs) and sites of ligand/small molecule binding, is crucial for understanding protein function in biological systems. In particular, the rapid increase of the use and development of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) for human health have necessitated the advancement of efficient and accurate analytical methods. Top-down and middle-down mass spectrometry (TD- and MD-MS) have become prominent analytical tools for protein characterization. However, obtaining complete protein sequence coverage by TD-/MD-MS can be limiting, particularly for proteins greater than 30 kDa, e.g., mAbs and ADCs. My dissertation research explores the utility of internal fragments, which are largely ignored by the MS community as they are difficult to assign, from both fundamental and application perspectives, for more efficient and comprehensive protein sequence, structure, and PTM characterization. On the fundamental side, we demonstrated a relationship between internal fragments and conventional terminal fragments. A better understanding of the fundamental formation mechanism of internal fragments aids the development of sequencing algorithms to assign internal fragments more accurately and reliably. On the application side, we started by analyzing standard disulfide-intact proteins using TD-MS, in which assigning internal fragments helped achieve near complete sequence coverage, and obtain disulfide bond position and connectivity information. This encouraged us to apply TD-MS on proteins of therapeutic significance, i.e., mAbs and ADCs, which are extremely complex disulfide-intact proteins. Incorporating internal fragments analysis allowed us to achieve the highest sequence coverage to date on an intact mAb by TD-MS, and enabled the access of important PTM information and drug binding information on intact ADCs. We then expanded this application by applying MD-MS on reduced mAbs and ADCs. Analyzing internal fragments in MD-MS helped us achieve comprehensive characterization of mAbs and ADCs which is a significant improvement from TD-MS and is comparable to the results obtained from the routinely utilized bottom-up peptide mapping method. Lastly, we demonstrated that assigning internal fragments generated by collision-based fragmentation also helps deliver higher-order structure information of multi-subunit protein assemblies. In summary, this dissertation work contributes to advancing the technique and instrumentation surrounding TD- and MD-MS workflows to achieve better characterization of proteins, especially biotherapeutics, and identification of specific proteoforms.
Chapter Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry
Author: Laura HeikausPublisher:
ISBN:
Category :
Languages : en
Pages :
Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.
Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry
Author: Hartmut Schl√oterPublisher:
ISBN:
Category : Science
Languages : en
Pages : 0
Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.