Protein characterization by mass spectrometry chapter 7 br266. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3d structures. 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. Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Method development for improved ricin and botulinum neurotoxin analysis abstract the illicit usage of toxic substances is increasing rapidly around the world, creating a need for comprehensive chemical methods for detecting and quantitating toxic agents.
This platform performance was demonstrated on several levels of analysis. To investigate the applicability of the directms mass spectrometry approach to. Protein identification and characterization through peptide mass spectrometry. Biological mass spectrometry ms offers a variety of approaches to study structure and behavior of complex protein drugs and has already become a default tool for characterizing the covalent structure of protein therapeutics, including sequence and posttranslational modifications. Presents practical applications of mass spectrometry for protein analysis and covers their impact on accelerating drug discovery and development covers both qualitative and quantitative aspects of mass spectrometry protein analysis in drug discovery principles, instrumentation, technologies topics include ms of peptides, proteins, and adcs, instrumentation in protein analysis, nanospray. One of the earliest applications of mass spectrometry in the development of protein pharmaceuticals was the confirmation of the amino acid sequence of recombinant proteins. Comprehensive characterisation of monoclonal antibodies. Mass spectrometrybased characterization of acidic glycans.
Current methods for the characterization of posttranslational. Currently there are over 50 investigators which utilize the facility for protein identification, quantitation, and characterization. Mass spectrometry ms has long been a major tool supporting development of each of these process areas with comprehensive information that few other techniques can match. Complete characterization of biotherapeutic proteins using. Characterization of protein impurities and sitespecific. Characterization of elisa antibodyantigen interaction using. However, mass spectrometry is now being actively tested as an alternative, which is capable of providing information on the conformational integrity of protein therapeutics during their production.
Characterization of protein complexes using chemical crosslinking coupled electrospray mass spectrometry timothy d. For the analysis of peptides, cems is an attractive complementary method to lcms, due. Protein structural characterization is an important aspect of biotherapeutic discovery and pharmaceutical development, primarily for the elucidation of structurefunction relationships, investigations of mechanisms of action, and determining proteinprotein interactions such as antibodyantigen binding interactions epitope mapping. Mass spectrometry based characterization of acidic glycans on protein therapeutics author links open overlay panel paul a. Fl was on the topic of characterization of protein therapeutics by mass. Mass spectrometrybased methods in characterization of the. Comprehensive characterization of the nglycosylation status of cd44s by use of multiple mass spectrometry based techniques. One of the largest groups of biotherapeutics is monoclonal antibodies mabs possessing various posttranslational modifications and potential degradation hotspots. Pdf mass spectrometric characterization in protein therapeutics discovery. Comparison of originator and biosimilar therapeutic. Protein analysis and characterization services, in line with the ich q6b guidance, including protein structure analysis, physicochemical properties, biological activity, immunochemical properties and purity and impurities determination. Capillary electrophoresismass spectrometry therapeutic. Highthroughput antibody screening from complex matrices.
Characterization of mabs using rp separations and high. Characterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. With the increase of the number of approved protein therapeutics in the market, comprehensive and reproducible characterization of these new generation drugs is crucial for the biopharmaceutical industry and regulatory agencies. Qualitative and quantitative host cell protein analysis using. Identification and characterization of proteins by mass. Books characterization of protein therapeutics using mass. Rapid level3 characterization of therapeutic antibodies by.
First book to focus on characterization by mass spectrometry of protein drugs. In characterization of protein therapeutics using mass spectrometry, expert contributors from academia and industry highlight current approaches and future trends. Qualitative and quantitative host cell protein analysis using mass spectrometry. Although liquid chromatography mass spectrometry lcms can be regarded as the gold standard for recombinant protein characterization, cems is an emerging technology in this. Monoclonal antibodies mabs are powerful therapeutics, and their characterization has drawn considerable attention and urgency. A continuously increasing number of therapeutic proteins are being released into the market, including. This book covers the latest developments in capillary electrophoresismass spectrometry for the analysis of therapeutic proteins.
Tools for the analysis and characterization of therapeutic. A combination of ionexchange chromatography ixc with online detection by native electrospray ionization mass spectrometry esi ms allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. Other readers will always be interested in your opinion of the books youve read. Characterization of the structure and interactions of p450 bm3 using hybrid mass spectrometry approaches laura n. Accelerating protein biotherapeutics from lab to patient. Characterization of protein therapeutics by mass spectrometry. Characterization of monoclonal antibodies using capillary. Jan 21, 2014 83 kaltashov ia, bobst ce, abzalimov rr, wang g, baykal b, wang s.
This chapter describes the current development of orbitrap mass spectrometry ms methods for the characterization of protein modifications to support the discovery and development of protein therapeutics in the pharmaceutical industry. Aug 05, 2016 pdf download characterization of protein therapeutics using mass spectrometry download full. Mass spectrometry in the development of protein biologics. Munro 1 the manchester institute of biotechnology, school of natural sciences, department of chemistry.
Mass spectrometry for the biophysical characterization of. Small and large peptides in the range of 365 amino acids have been separated, identified with 100. Summary this chapter focuses on the common molecular variants and degradation pathways of protein therapeutics generated under normal. Request pdf characterization of protein therapeutics by mass spectrometry. Native mass spectrometry ms is an increasingly important technique for the characterisation of recombinant proteinbased biopharmaceutical products such as monoclonal antibodies mabs, offering several benefits over ms workflows that involve protein denaturation steps. Apr 15, 2017 mass spectrometric data are being used extensively to support regulatory filings for protein and antibody therapeutics. Characterization of protein impurities and sitespecific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry. Books characterization of protein therapeutics using mass spectrometry free download. Higher order structure of protein therapeutics is an important quality attribute, which dictates both potency and safety. Whether youve loved the book or not, if you give your honest and detailed thoughts then people will find new books that are right for them. Illustrates the complete process for improving protein sequence coverage in the peptide mapping of monoclonal antibody by combining lcms with cems workflow.
Most crucial issues in the analysis of protein complex through ms include. Protein analysis using mass spectrometry lee, mike s. Mass spectrometry ms has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical. Qualitative and quantitative host cell protein analysis. Disulfide bond formation is an important posttranslational modification that helps to stabilize protein conformation. Sapkota medical research council protein phosphorylation and ubiquitylation unit, university of dundee, dow st. Protein identification and characterization through peptide. Rapid characterization of secreted recombinant proteins by. While modern experimental biophysics offers an impressive arsenal of stateoftheart tools that can be used for the characterization of higher order structure, many of them are poorly suited for the characterization of biopharmaceutical products. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. 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 impuritiesdegradants, metabolites, higher order structures of protein therapeutics.
Introduction mass spectrometry ms has become a vital tool in characterization of protein therapeutics and monoclonal antibodies because it can provide details of amino acid sequence and posttranslational modi. This book highlights current approaches and future trends in the use of mass spectrometry to characterize protein therapies. Mass spectrometric data are being used extensively to support regulatory filings for protein and antibody therapeutics. Recent advances in hydrogendeuteriumexchange mass spectrometry hdxms have provided a means to assess higher. Higher order structure characterization of protein. Jan 01, 2011 read characterization of protein therapeutics by mass spectrometry. Performance metrics for evaluating system suitability in. Above and beyond protein therapeutics, msbased methods for characterization of higher order structure are likely to accelerate the development of. With the need for high accuracy, sensitivity, and selectivity, the rapidly improving ms. Jun 11, 2009 characterization of protein impurities and sitespecific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry. Direct mass spectrometry characterization of recombinant proteins in yeast. Unambiguous characterization of analytes from complex matrices with high content information in a labelfree format continues to expand the application of mass spectrometry ms in drug discovery 1. Protein characterization by mass spectrometry mass spectrometry ms has become a powerful tool in proteomics for proteomewide analysis and characterization of proteins from a variety of organisms and cell types. Journal of the american society for mass spectrometry.
Overview of relevant techniques for the characterization of therapeutic proteins. Mass spectrometry ms has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical industry. This core is housed within the musc mass spectrometry facility which provides expertise, services, education, and training to enhance biomedical research endeavors through mass spectrometrybased proteomics. Recent advances in mass spectrometry provide tools for protein identification, protein characterization, relative and absolute. Characterization of protein therapeutics using mass spectrometry. Advances and challenges in analytical characterization of biotechnology products. Characterization of the structure and interactions of p450.
Recent advances in hydrogendeuteriumexchange mass spectrometry hdxms have provided a means to assess higherorder structure of protein. To investigate the applicability of the directms mass spectrometry approach to eukaryotic expression systems, we. The analytical characterization of biologics is intended to provide an understanding of the structures and properties of molecular variants, which can be generated during cell culture, protein purification, storage, and in vivo after administration. Brown convention center same location as the asms annual conference.
Characterization of mabs using rp separations and high resolution accurate mass hram mass spectrometry. Protein identification and characterization through. The application of capillary electrophoresismass spectrometry cems coupling technology in the analysis of recombinant therapeutic proteins is detailed thoroughly. Characterization of impurities and degradants in protein therapeutics by mass spectrometry li tao, michael ackerman, wei wu, peiran liu, and reb russell. Protein samples then need to be introduced to a mass spectrometer in buffer conditions. Tymiak describes recent developments and future trends in the characterization of protein therapeutics using. A tandem mass spectrometer further breaks the peptides down into fragment ions and measures the mass of each piece. Recent developments and future directions mass spectrometry. Unlike smallmolecular drugs 150600 da that have rigid structures, mabs 150 kda are engineered proteins that undergo complicated folding and can exist in a number of lowenergy structures, posing a challenge for traditional methods in structural biology. Mass spectrometer electrically accelerates the fragmented ions. Highresolution ms for structural characterization of protein therapeutics. Using mass spectrometry for protein complex analysis.
Rather than reading a good book with a cup of tea in the afternoon, instead they are facing with some infectious virus inside their laptop. Feb 26, 2019 mass spectrometry ms is considered to be a powerful method for quickly and efficiently identifying protein samples. Broadens readers understanding of protein glycosylation profiling using capillary electrophoresismass spectrometry and pk studies of biologics analysis using cems technologies. Characterization of elisa antibodyantigen interaction.
Comprehensive analysis of protein modifications by topdown. Protein analysis and characterization to meet the challenges of drug development can be complex. Advances and challenges in analytical characterization of. Protein sequencing and identification with mass spectrometry. Mass spectrometric analysis of protein species of biologics. Characterization of impurities and degradants using mass. Read characterization of protein therapeutics by mass spectrometry. Current methods for the characterization of posttranslational modifications in therapeutic proteins using orbitrap mass spectrometry. The book discusses mass spectrometry techniques as related to the analysis of protein therapeutics, structural identification strategies and quantitative approaches. Rapid level3 characterization of therapeutic antibodies. This paper provides a comprehensive characterization example of a representative mab trastuzumab herceptin as a continuation of our previous collaborative work, illustrating the benefits of cesi coupled with highresolution mass spectrometry. Characterization of protein therapeutics using mass. Tools for the analysis and characterization of therapeutic protein species marceline manka fuh, pascal steffen, hartmut schluter mass spectrometric proteomics, institute for clinical chemistry and laboratory medicine, university medical center hamburgeppendorf, hamburg, germany abstract. Characterization of protein complexes using chemical cross.