{"id":6673,"date":"2024-08-09T14:11:00","date_gmt":"2024-08-09T14:11:00","guid":{"rendered":"https:\/\/www.waters.com\/blog\/?p=6673"},"modified":"2026-01-20T14:18:07","modified_gmt":"2026-01-20T14:18:07","slug":"complete-guide-to-protein-identification-and-characterization","status":"publish","type":"post","link":"https:\/\/www.waters.com\/blog\/complete-guide-to-protein-identification-and-characterization\/","title":{"rendered":"Complete Guide to Protein Identification and Characterization"},"content":{"rendered":"\n<p>Protein identification and characterization is one of the most important components of expressing and manufacturing recombinant proteins for biologics. It involves a wide variety of analytical tools and techniques because of the very complexity of proteins themselves, starting with 21 amino acids arranged in&nbsp;a nearly infinite&nbsp;number of ways, and then folded into three-dimensional structures.\u202f&nbsp;<\/p>\n\n\n\n<p>Understanding a protein\u2019s structure is key to understanding its function, and the creation of biological therapies, including antibodies, recombinant proteins, vaccines and other molecules, depends upon clear and&nbsp;accurate&nbsp;characterization.&nbsp;<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"903\" height=\"903\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13.png\" alt=\"Protein structure\" class=\"wp-image-6674\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13.png 903w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13-300x300.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13-150x150.png 150w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13-768x768.png 768w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13-650x650.png 650w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-13-350x350.png 350w\" sizes=\"(max-width: 903px) 100vw, 903px\" \/><\/figure>\n<\/div>\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_1145ab-89 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_1145ab-89 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_1145ab-89\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81, .wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81[data-kb-block=\"kb-adv-heading6673_7f2e5f-81\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81[data-kb-block=\"kb-adv-heading6673_7f2e5f-81\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_7f2e5f-81[data-kb-block=\"kb-adv-heading6673_7f2e5f-81\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6673_7f2e5f-81 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_7f2e5f-81\">Protein Identification Methods&nbsp;<\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Mass spectrometry (MS) \u2013 <\/strong>Whole proteins are first ionized, and then enter a mass&nbsp;analyzer, or&nbsp;identified&nbsp;at peptide level. The spectrometer&nbsp;identifies&nbsp;protein structure by mass fingerprinting&nbsp;or tandem mass spectrometry. Peptide masses are compared to online databases to make the closest protein matches.&nbsp;<\/li>\n\n\n\n<li><strong>Edman degradation\u202f\u2013 <\/strong>Purifies proteins by removing one residue at a time from the amino end of a peptide, using phenyl isothiocyanate. The process does not damage the protein and separates one residue at a&nbsp;time, but&nbsp;is not as useful for larger proteins.\u202f\u202f&nbsp;<\/li>\n\n\n\n<li><strong>Peptide mass fingerprinting\u202f\u2013 <\/strong>This high-throughput method involves&nbsp;endoproteases&nbsp;that cleaves unknown protein into smaller peptides. The mass of these peptides can be measured using MS, and resulting lists of \u201cpeptide peaks\u201d compared to protein databases.\u202f&nbsp;<\/li>\n\n\n\n<li><strong>Database searching and bioinformatics tools <\/strong>\u2013 Data from MS and fingerprinting is compared using computer programs and databases to find the closest match to your sample.\u202f&nbsp;<\/li>\n\n\n\n<li><strong>Immunoassays\u202f\u2013<\/strong> Identifies proteins by their interactions with specific antibodies. These tests include ELISA (enzyme-linked immunosorbent assays),&nbsp;western blotting (separation of proteins by molecular weight, electrophoresis transfer to a membrane, and probing with antibodies), and immunoprecipitation (precipitating a protein antigen out of solution using an antibody that specifically binds to that protein).&nbsp;<\/li>\n\n\n\n<li><strong>Size exclusion chromatography&nbsp;(SEC)\u202f\u2013 <\/strong>Using beads of specific dimensions packed in a column, these methods separate proteins according to size. When samples&nbsp;enter into&nbsp;the column, different molecules will have different elution rates.\u202f&nbsp;<\/li>\n\n\n\n<li><strong>Affinity chromatography\u202f\u2013<\/strong> This technique also uses a column and cellulose beads. A substrate (or sometimes a coenzyme) is bound covalently to the beads at the top of the column. Those proteins that have a binding site for the immobilized substrate will bind, while all other proteins will be eluted.&nbsp;<\/li>\n<\/ul>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_54938a-82 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_54938a-82 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_54938a-82\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae, .wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae[data-kb-block=\"kb-adv-heading6673_fd0630-ae\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae[data-kb-block=\"kb-adv-heading6673_fd0630-ae\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_fd0630-ae[data-kb-block=\"kb-adv-heading6673_fd0630-ae\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_fd0630-ae wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_fd0630-ae\">Protein Characterization Techniques<\/h3>\n\n\n\n<p>Protein characterization elucidates the primary sequence, higher-order structure, post-translational modifications, interactions, and biological activities of proteins. Protein characterization involves a wide range of analytical tools and techniques. There cannot be a \u201cone-size fits all\u201d technology to&nbsp;determine&nbsp;the chemical makeup,&nbsp;structure&nbsp;and function of large molecules with different aggregation states, charges, size, and&nbsp;three-dimensional&nbsp;configuration. But characterizing proteins in your samples, especially potential therapeutics, is vitally important to&nbsp;determine&nbsp;the safety,&nbsp;efficacy&nbsp;and purity of your protein-based therapies.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_be51a0-50 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_be51a0-50 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_be51a0-50\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24, .wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24[data-kb-block=\"kb-adv-heading6673_be355c-24\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24[data-kb-block=\"kb-adv-heading6673_be355c-24\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_be355c-24[data-kb-block=\"kb-adv-heading6673_be355c-24\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_be355c-24 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_be355c-24\">Protein Analysis Equipment&nbsp;<\/h3>\n\n\n\n<p>Protein analysis involves tools that detect,&nbsp;purify&nbsp;and&nbsp;identify&nbsp;proteins, and begin to characterize structure and function. Equipment can include traditional methods like electrophoresis, separating proteins by size and charge, western blotting, marking targets with antibodies, and mass spectrometry, measuring mass-to-charge ratios. More modern methods include&nbsp;dynamic&nbsp;light scattering (DLS).\u202f&nbsp;<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Protein separation (electrophoresis)\u202f<\/strong>places proteins in a gel&nbsp;and&nbsp;observes&nbsp;their mobility in the presence of an electric field. Proteins can be separated by solubility, size,&nbsp;charge&nbsp;and binding. The most common is SDS-PAGE&nbsp;(short for sodium dodecyl&nbsp;sulfate&nbsp;polyacrylamide gel electrophoresis), which separates proteins based on molecular weight.\u202f&nbsp;<\/li>\n\n\n\n<li><strong>Western blotting<\/strong>\u202fidentifies&nbsp;proteins extracted from cells, and separates them by size, transfers them to a solid support (the blot), and uses primary and secondary antibodies to target proteins thus providing identification of proteins based on biological associations.\u202f&nbsp;<\/li>\n\n\n\n<li><strong><a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/light-scattering-instruments-and-systems.html\">Light scattering<\/a><\/strong>\u202fis a more modern\u00a0approach, and\u00a0is more sensitive with larger molecules. For\u00a0instance, DLS\u00a0can sensitively detect small quantities of polypeptide aggregates in preparations. The<a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers.html\">\u00a0Aura\u00a0Systems\u00a0family <\/a>accurately measures particle size distribution, based on backgrounded membrane imaging (BMI) and fluorescence membrane microscopy (FMM). These instruments provide valuable information about the size distribution of particles in a sample, helping researchers and manufacturers understand the characteristics of their products.\u00a0<\/li>\n<\/ul>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_5e9b2d-7b .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_5e9b2d-7b .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_5e9b2d-7b\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61, .wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61[data-kb-block=\"kb-adv-heading6673_527dd6-61\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61[data-kb-block=\"kb-adv-heading6673_527dd6-61\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_527dd6-61[data-kb-block=\"kb-adv-heading6673_527dd6-61\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_527dd6-61 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_527dd6-61\">Biologics Formulation Instruments&nbsp;<\/h3>\n\n\n\n<p>During manufacturing of therapeutic molecules,&nbsp;it\u2019s&nbsp;important to have the right instrumentation to&nbsp;monitor&nbsp;protein conformation, predict thermal stability, and measure aggregate formation.\u202f&nbsp;<\/p>\n\n\n\n<p><strong>Differential Scanning Calorimetry (DSC)<\/strong>\u202fcharacterizes the thermal stability of proteins by measuring enthalpy (\u0394H) and temperature (Tm) of&nbsp;thermally-induced&nbsp;structural transitions of molecules.&nbsp;DLS&nbsp;can&nbsp;analyze&nbsp;particle mobility and charge (Zeta potential) using the technique of Electrophoretic Light Scattering (ELS), and the molecular weight of particles in solution using Static Light Scattering (SLS). Other systems, like&nbsp;<a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers\/aura-ptx.html\">Aura&nbsp;PTx&nbsp;System<\/a>, use\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/applications\/biopharma-and-pharma\/protein-therapies\/particle-counting-and-identification.html\" target=\"_blank\" rel=\"noreferrer noopener\">BMI<\/a>\u202fwith two channels of\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/applications\/biopharma-and-pharma\/protein-therapies\/particle-counting-and-identification.html\" target=\"_blank\" rel=\"noreferrer noopener\">FMM<\/a>\u202fto detect and&nbsp;identify&nbsp;subvisible particles that can form aggregates and threaten the stability of biologicals. Additionally, FMM enables characterization of formulation excipients as well such as polysorbate.&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_8b6956-f6 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_8b6956-f6 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_8b6956-f6\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94, .wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94[data-kb-block=\"kb-adv-heading6673_e98c63-94\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94[data-kb-block=\"kb-adv-heading6673_e98c63-94\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_e98c63-94[data-kb-block=\"kb-adv-heading6673_e98c63-94\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_e98c63-94 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_e98c63-94\">Protein Characterization Tools&nbsp;<\/h3>\n\n\n\n<p><strong>Chromatography<\/strong><em>:<\/em>\u202fHigh&nbsp;performance liquid chromatography (HPLC),&nbsp;SEC, ion-exchange chromatography (IEC), and affinity chromatography separate proteins based on size, charge, hydrophobicity, or specific interactions, enabling purity analysis and separation of protein isoforms or variants.&nbsp;<\/p>\n\n\n\n<p><strong>Electrophoresis<\/strong><em>:<\/em>\u202fSDS-PAGE, isoelectric focusing (IEF), and capillary electrophoresis (CE) separate proteins based on size, charge, or isoelectric point,&nbsp;facilitating&nbsp;purity analysis, subunit composition, and post-translational modifications.&nbsp;<\/p>\n\n\n\n<p><strong>MS<\/strong><em>:<\/em>\u202fLiquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption\/ionization mass spectrometry (MALDI-MS)&nbsp;identify&nbsp;and quantify proteins, peptides, and post-translational modifications, elucidating primary sequence, structural variations, and interactions.&nbsp;<\/p>\n\n\n\n<p><strong>Spectroscopy<\/strong><em>:<\/em>\u202fUltraviolet-visible (UV-Vis) spectroscopy, fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy allow protein structural analysis as well as analysis of folding, conformational changes, and ligand binding.&nbsp;<\/p>\n\n\n\n<p><a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers.html\" target=\"_blank\" rel=\"noreferrer noopener\">The Aura&nbsp;Systems&nbsp;family<\/a>\u202f(in particular the&nbsp;Aura&nbsp;PTx&nbsp;System&nbsp;for proteins) uses a combination of&nbsp;BMI, a microscopy method,&nbsp;and fluorescence membrane microscopy, which uses fluorescent dyes or antibodies for faster,&nbsp;reliable characterization all on one instrument. With Aura&nbsp;Systems, researchers can understand the stability and purity of the proteins being made.&nbsp;<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"1024\" height=\"682\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-14.png\" alt=\"Aura PTx System to speed up protein formulation development and screening\" class=\"wp-image-6677\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-14.png 1024w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-14-300x200.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-14-768x512.png 768w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/figure>\n<\/div>\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_b17636-a5 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_b17636-a5 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_b17636-a5\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85, .wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85[data-kb-block=\"kb-adv-heading6673_3d818f-85\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85[data-kb-block=\"kb-adv-heading6673_3d818f-85\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_3d818f-85[data-kb-block=\"kb-adv-heading6673_3d818f-85\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_3d818f-85 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_3d818f-85\">Protein Characterization Assays&nbsp;<\/h3>\n\n\n\n<p><strong>Biophysical assays:<\/strong>\u202fDLS and\u00a0DSC\u00a0characterize protein size, shape, stability, and interactions with ligands or other molecules.\u00a0<\/p>\n\n\n\n<p><strong>Immunological assays<\/strong>:&nbsp;ELISA, Western blotting, immunoprecipitation, and flow cytometry detect and quantify proteins, epitopes, or specific protein-protein interactions,&nbsp;facilitating&nbsp;immunoassays, biomarker detection, and protein-protein interaction studies.&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_99b9b1-28 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_99b9b1-28 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_99b9b1-28\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7, .wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7[data-kb-block=\"kb-adv-heading6673_98da90-c7\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7[data-kb-block=\"kb-adv-heading6673_98da90-c7\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_98da90-c7[data-kb-block=\"kb-adv-heading6673_98da90-c7\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_98da90-c7 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_98da90-c7\">Protein Formulation&nbsp;<\/h3>\n\n\n\n<p>The process of assembling the primary structure of proteins (amino acids), followed by secondary, and tertiary (three-dimensional) structures into final pharmaceutical protein products requires steps that ensure stability, structural&nbsp;integrity&nbsp;and function. The protein must be stabilized so it tolerates manufacturing processes and&nbsp;remains&nbsp;stable and active during transportation, storage, and administration. Formulation development aims to ensure the stability, efficacy, safety, and manufacturability of biopharmaceutical products by selecting&nbsp;appropriate excipients, buffers, pH, and dosage forms.&nbsp;<\/p>\n\n\n\n<p>Factors that influence stability and efficacy include aggregate formation, which&nbsp;impacts&nbsp;protein structure (and therefore function). It is important to distinguish aggregated active pharmaceutical ingredients (API) from other particle types for understanding the root cause of instability.\u202f\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers\/aura-ptx.html\" target=\"_blank\" rel=\"noreferrer noopener\">The Aura&nbsp;PTx&nbsp;System\u2019s\u202f<\/a>\u202f96-well aggregate and particle imaging system can rapidly size, count, and characterize particles and&nbsp;identify&nbsp;them as proteins, non-proteins, excipients, or other types of molecules. This system is much more efficient and&nbsp;accurate&nbsp;than traditional flow cell systems used to distinguish protein size and count.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_e98a96-1e .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_e98a96-1e .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_e98a96-1e\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80, .wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80[data-kb-block=\"kb-adv-heading6673_c98da5-80\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80[data-kb-block=\"kb-adv-heading6673_c98da5-80\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_c98da5-80[data-kb-block=\"kb-adv-heading6673_c98da5-80\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_c98da5-80 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_c98da5-80\">Protein Aggregation Analysis&nbsp;<\/h3>\n\n\n\n<p>Aggregates in protein samples are usually segments of broken polypeptide chains which \u201ccome along for the ride\u201d when manufacturing protein therapies. They can, however, significantly reduce target protein stability and reduce the efficacy of a therapeutic agent. In addition, exposure to air, solid, light or changing temperatures (often parts of the pharmaceutical development process) can produce protein particles.&nbsp;<\/p>\n\n\n\n<p>Successful protein therapies require product quality measurements in the late discovery\/early development stages that detect,\u00a0count\u00a0and characterize formulation excipients, ensuring protein stability. But traditional methods require hours of sorting through images and\u00a0relatively large\u00a0volumes, and adopting complex machine learning libraries, and often overlook key sample aggregates and degraded compounds.<a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers\/aura-ptx.html\">\u202fAura PTx System<\/a>, with its combination of BMI\u00a0and two\u00a0FMM\u00a0channels, quickly provides count, size and morphology, and differentiates cellular, protein or extrinsic aggregates.\u00a0<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_06afeb-5d .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_06afeb-5d .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_06afeb-5d\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20, .wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20[data-kb-block=\"kb-adv-heading6673_dd14e8-20\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20[data-kb-block=\"kb-adv-heading6673_dd14e8-20\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_dd14e8-20[data-kb-block=\"kb-adv-heading6673_dd14e8-20\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_dd14e8-20 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_dd14e8-20\">Molecular Weight Characterization&nbsp;<\/h3>\n\n\n\n<p>Techniques for characterizing proteins by molecular weight include SDS-PAGE, MALDI-TOF MS, and\u00a0SEC or SEC-MALS. SDS-PAGE is the most common\u00a0technique and\u00a0separates proteins by molecular weight (and not by charge or folding).\u00a0It\u2019s\u00a0often used in biochemistry, forensics,\u00a0genetics,\u00a0and molecular biology. MALDI-TOF is a form of mass spectrometry that\u00a0determines\u00a0the ratio of mass to charge, which\u00a0determines\u00a0masses and elemental composition of proteins. Once MALDI\u00a0was developed, this technique became popular to study proteins.\u00a0SEC, also known as gel filtration or\u00a0HPLC, is a powerful technique for aggregate and fragment analysis in the research, development, and manufacturing of biotherapeutic proteins, such as insulin and monoclonal antibodies.\u202f\u00a0<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_0e4743-b0 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_0e4743-b0 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_0e4743-b0\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70, .wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70[data-kb-block=\"kb-adv-heading6673_49b292-70\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70[data-kb-block=\"kb-adv-heading6673_49b292-70\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_49b292-70[data-kb-block=\"kb-adv-heading6673_49b292-70\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_49b292-70 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_49b292-70\">HPLC Purity Analysis&nbsp;<\/h3>\n\n\n\n<p>HPLC, as mentioned above, is used for protein purity studies. It allows for highly selective separation of sample material. HPLC separates molecules based on their size by filtration through a gel. The gel is made of beads&nbsp;containing&nbsp;pores of a specific size distribution, charge, and\/or affinity. Separation occurs when molecules of&nbsp;different sizes, charges or affinities are included or excluded from the pores within the matrix. Larger analytes will elute first, while the smaller molecules interact more with the stationary phase and will elute later.&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_c2ba72-fd .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_c2ba72-fd .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_c2ba72-fd\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26, .wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26[data-kb-block=\"kb-adv-heading6673_5d7a5c-26\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26[data-kb-block=\"kb-adv-heading6673_5d7a5c-26\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_5d7a5c-26[data-kb-block=\"kb-adv-heading6673_5d7a5c-26\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_5d7a5c-26 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_5d7a5c-26\">Protein Charge Variants Analysis&nbsp;<\/h3>\n\n\n\n<p>Large biotherapeutic proteins can go through enzymatic post-translational modifications during manufacturing, such as glycosylation and lysine truncation. In addition, chemical modifications can occur during purification and storage. Because of this, FDA and other regulators require that these proteins go through charge variants analysis. This analysis has traditionally been performed using ion exchange chromatography with salt gradients, while newer techniques rely on pH gradients instead of salt. Isoelectric focusing using&nbsp;CE&nbsp;has also been used as a fast global method, but it still has longer run times and is prone to human error. This often requires a very pure sample as it cannot handle contaminated or heterogeneous samples well.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_120efe-b1 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_120efe-b1 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_120efe-b1\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8, .wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8[data-kb-block=\"kb-adv-heading6673_dfc609-c8\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8[data-kb-block=\"kb-adv-heading6673_dfc609-c8\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_dfc609-c8[data-kb-block=\"kb-adv-heading6673_dfc609-c8\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_dfc609-c8 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_dfc609-c8\">Aggregation and Fragment Content Analysis&nbsp;<\/h3>\n\n\n\n<p>Aggregates and fragments are common impurities in biopharmaceuticals that have an impact on product efficacy, safety, and stability.\u00a0SEC\u00a0can be coupled to electrospray ionization\u00a0MS\u00a0for the characterization of size variants of therapeutic monoclonal antibodies (mAbs). Quadrupole time-of-flight (Q-TOF) MS can also be\u00a0optimized\u00a0for more sensitivity. In addition, analytical ultracentrifugation can aid in separating fragments.\u00a0<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_cded4a-f3 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_cded4a-f3 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_cded4a-f3\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78, .wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78[data-kb-block=\"kb-adv-heading6673_cc3fe3-78\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78[data-kb-block=\"kb-adv-heading6673_cc3fe3-78\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_cc3fe3-78[data-kb-block=\"kb-adv-heading6673_cc3fe3-78\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_cc3fe3-78 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_cc3fe3-78\">Identifying&nbsp;and Quantifying Host Cell Protein Residues&nbsp;<\/h3>\n\n\n\n<p>Host cell proteins are impurities produced by the host organism during biotherapeutic manufacturing. Purification usually removes most of these residues from the final product, but residual proteins that&nbsp;remain&nbsp;with the manufactured antibody therapy, even at low levels, can be immunogenic. FDA and other regulators today dictate that&nbsp;practically no&nbsp;protein impurities be present in a final biotherapeutic product.&nbsp;MS&nbsp;techniques are useful for identification and quantitation of HCPs lingering in recombinant therapeutic products, and an improvement over ELISA and other analytical methods. BMI and FMM microscopy analytical techniques in the&nbsp;Aura&nbsp;PTx&nbsp;System can also be&nbsp;very&nbsp;effective&nbsp;at detecting proteins and&nbsp;aggregates.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_4f7518-c9 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_4f7518-c9 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_4f7518-c9\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45, .wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45[data-kb-block=\"kb-adv-heading6673_26a4d4-45\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45[data-kb-block=\"kb-adv-heading6673_26a4d4-45\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_26a4d4-45[data-kb-block=\"kb-adv-heading6673_26a4d4-45\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_26a4d4-45 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_26a4d4-45\">Sequence Coverage\/Peptide Mapping&nbsp;<\/h3>\n\n\n\n<p>Determining&nbsp;the exact,&nbsp;accurate&nbsp;sequence of your therapeutic protein is critical to successful characterization. This method works by digesting proteins down to their fundamental&nbsp;peptides and&nbsp;ensures a full sequence of the biotherapeutic molecule. Techniques for sequence coverage and peptide mapping include HPLC, and HPLC coupled with MS. MS techniques can provide high accuracy and selective data, especially with&nbsp;very complex&nbsp;biomolecules like&nbsp;mAbs.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_892253-55 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_892253-55 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_892253-55\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h3 class=\"wp-block-heading has--font-size\">N-terminal and C-terminal Sequence Confirmation&nbsp;<\/h3>\n\n\n\n<p>Amino (N-terminal) sequence analysis&nbsp;identifies&nbsp;the order of amino acids of your protein, starting at the N-terminal. N-terminal sequences significantly&nbsp;impact&nbsp;half-life, subcellular localization, and post-translational modifications of proteins. Similarly, C-terminal (acid terminal) sequence analysis&nbsp;identifies&nbsp;amino acid orders at the C-terminal end of the protein. This can provide information on protein folding, secondary structure, and the arrangement of functional domains. Two principal methods for conducting this sequence confirmation are&nbsp;MS&nbsp;and Edman Degradation.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_0ca1f6-58 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_0ca1f6-58 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_0ca1f6-58\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de, .wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de[data-kb-block=\"kb-adv-heading6673_d8e582-de\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de[data-kb-block=\"kb-adv-heading6673_d8e582-de\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_d8e582-de[data-kb-block=\"kb-adv-heading6673_d8e582-de\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_d8e582-de wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_d8e582-de\"><strong>Post-translational Modification Analysis<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Post-translational modifications (PTMs) are variants introduced in proteins after they are expressed. Epigenetics studies have shown that PTMs can be innocuous or can dramatically alter the function of proteins, and thus it is necessary to detect PTM effects in any biotherapeutic product. Common PTMs can range from the&nbsp;very simple&nbsp;to the complex, and include phosphorylation, glycosylation, ubiquitination,&nbsp;nitrosylation, methylation, acetylation, lipidation and proteolysis. They are most often enzymatic (caused by enzyme actions). Techniques for&nbsp;identifying&nbsp;and&nbsp;analyzing&nbsp;PTMs can vary by the type of PTM, and include SDS-PAGE, Western blotting, chromatin immunoprecipitation (ChIP), and other techniques.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_3ee901-3c .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_3ee901-3c .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_3ee901-3c\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c, .wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c[data-kb-block=\"kb-adv-heading6673_6b81a9-1c\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c[data-kb-block=\"kb-adv-heading6673_6b81a9-1c\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_6b81a9-1c[data-kb-block=\"kb-adv-heading6673_6b81a9-1c\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_6b81a9-1c wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_6b81a9-1c\"><strong>Disulfide Bond Analysis<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Disulfide bonds are covalent bindings between the&nbsp;sulfur&nbsp;atoms found on residues of the amino acid cysteine. They are the only covalent link found between&nbsp;polypeptides&nbsp;and&nbsp;stabilize protein structure. Reducing agents like tris (2-carboxyethyl) phosphine hydrochloride (TCEP), beta-mercaptoethanol&nbsp;(BME), and dithiothreitol (DTT) can disrupt disulfide bonds and therefore\u202fprotein stability.\u202fDisulfide bond analysis is usually conducted by MS and HPLC.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_22fd1f-84 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_22fd1f-84 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_22fd1f-84\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd, .wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd[data-kb-block=\"kb-adv-heading6673_f13c93-cd\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd[data-kb-block=\"kb-adv-heading6673_f13c93-cd\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_f13c93-cd[data-kb-block=\"kb-adv-heading6673_f13c93-cd\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_f13c93-cd wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_f13c93-cd\"><strong>Glycosylation Modification Analysis<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Protein glycosylation is an important PTM, and can change protein folding, conformation, distribution,&nbsp;stability&nbsp;and activity. Glycosylation involves the addition of a variety of sugars to proteins, which then control cell attachment to the extracellular matrix and protein-ligand interactions in the cell. Glycoproteins are detected,&nbsp;purified&nbsp;and&nbsp;analyzed&nbsp;by, in order, glycan staining and visualization, glycan crosslinking to agarose or magnetic resin for&nbsp;labeling&nbsp;or purification, or proteomic analysis&nbsp;by MS.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_f65959-d6 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_f65959-d6 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_f65959-d6\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd, .wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd[data-kb-block=\"kb-adv-heading6673_079817-dd\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd[data-kb-block=\"kb-adv-heading6673_079817-dd\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_079817-dd[data-kb-block=\"kb-adv-heading6673_079817-dd\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h3 class=\"kt-adv-heading6673_079817-dd wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_079817-dd\"><strong>Analysis of Glycosylation Sites and&nbsp;Glycoforms<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Glycosylation will vary according to where in the cell the sugar molecule binds, and according to the structure and chemical binding site of the sugar molecule. These oligosaccharides can affect protein-protein interactions by either allowing or blocking proteins from binding to cognate interaction domains. Since they are hydrophilic, they can also alter protein solubility. Techniques for analysis include glycan staining, enrichment and analysis using lectins, and mass spectrometry.&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_7b6d73-bf .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_7b6d73-bf .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_7b6d73-bf\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Analysis of Sialic Acid<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Sialic acid is the most prevalent sugar found on mammalian cell surfaces. They are essential to cell adhesion and immune modulation, and they bind selectins and lectins. Sialic acid and monosaccharide analysis is required by ICH Q6B guidelines for biopharmaceutical production. Typically, sialic acid is quantified by fluorescence&nbsp;labeling&nbsp;and analysis via HPLC with fluorescence detection.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_593449-d2 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_593449-d2 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_593449-d2\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n<style>.wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15, .wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15[data-kb-block=\"kb-adv-heading6673_67559d-15\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15[data-kb-block=\"kb-adv-heading6673_67559d-15\"] mark.kt-highlight{font-style:normal;color:#f76a0c;-webkit-box-decoration-break:clone;box-decoration-break:clone;padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6673_67559d-15[data-kb-block=\"kb-adv-heading6673_67559d-15\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6673_67559d-15 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6673_67559d-15\">Overview: Protein Characterization Technology&nbsp;<\/h2>\n\n\n\n<p>Proteins are&nbsp;arguably the&nbsp;most complex molecules in living cells and tissues, and their use as biopharmaceuticals requires integrating various technologies for comprehensive analysis to ensure their safety and efficacy.\u202f&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>MS&nbsp;Characterization<\/strong>&nbsp;<\/h3>\n\n\n\n<p>As described previously,&nbsp;MS&nbsp;is an invaluable tool to characterize and&nbsp;analyze&nbsp;proteins&nbsp;as well&nbsp;as fragments, aggregates and PTMs that can&nbsp;impact&nbsp;protein development and stability. MS techniques are used to&nbsp;determine&nbsp;structure, function, folding and interactions,&nbsp;identify&nbsp;proteins&nbsp;from its mass of peptide fragments, and to accurately count proteins in a sample. The development of high-throughput and quantitative MS proteomics within the last 20 years has expanded the scope of MS.&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Characterization by CD&nbsp;Spectrometry<\/strong>&nbsp;<\/h3>\n\n\n\n<p>CD&nbsp;measures the differential absorption of left- and right-handed circularly polarized light, by optically active compounds at different wavelengths. The incident light on the sample switches between LCP and RCP light. As the incident light switches direction of polarization, the absorption changes and the differentiated molar absorptivity can be calculated. CD can study secondary structure changes and set the stage for a conformational analysis of proteins. CD can also be used to examine protein\/nucleic acid stability (folding,&nbsp;unfolding&nbsp;and refolding) in&nbsp;the presence of pH, denaturants,&nbsp;temperature&nbsp;and other factors.\u202f&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>FMM\/SIMI\/BMI<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Forming the basis of&nbsp;protein and protein fragment analysis&nbsp;conducted using Aura Systems,\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers\/aura-ptx.html\" target=\"_blank\" rel=\"noreferrer noopener\">Aura PTx<\/a>&nbsp;System\u202fis the first and only system that combines\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/applications\/biopharma-and-pharma\/protein-therapies\/particle-counting-and-identification.html\" target=\"_blank\" rel=\"noreferrer noopener\">BMI)<\/a>\u202fwith two\u202f<a href=\"https:\/\/www.waters.com\/nextgen\/global\/applications\/biopharma-and-pharma\/protein-therapies\/particle-counting-and-identification.html\" target=\"_blank\" rel=\"noreferrer noopener\">FMM)<\/a>\u202fchannels to quickly provide count, size, ID, imaging and morphology with differentiated cellular and protein aggregates, excipients such as degraded polysorbate, and\/or extrinsic particle characterization.&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>DLS<\/strong>&nbsp;<\/h3>\n\n\n\n<p>DLS is used to measure particles from 0.2 nm to 10 \u00b5m. These instruments work using Brownian motion, in which lighter particles move faster than heavier (and therefore larger) ones. A laser illuminates the\u00a0particles,\u00a0and the scattered light is\u00a0analyzed. Fluctuations in intensity\u00a0determine\u00a0size distribution in a sample.\u00a0<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Characterization Through X-Ray Crystallography<\/strong>&nbsp;<\/h3>\n\n\n\n<p>X-ray crystallography is based on the capture of x-ray images as they travel through a crystallized form of a target molecule (in this case, a protein). It is&nbsp;an accurate&nbsp;way to&nbsp;determine&nbsp;the three-dimensional structure of a protein. To use this technique, the crystallographer obtains protein crystals, records the diffraction pattern formed by x-rays passed through the crystals, and then interprets the data using computer software. The result is an atomic-resolution model of a protein.&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Nuclear Magnetic Resonance Spectroscopy Characterization<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Nuclear magnetic resonance (NMR) occurs when nuclei in an unmoving magnetic field is disturbed by an oscillating magnetic field; the nuclei generate an electromagnetic signal, whose frequency depends on the magnetic field applied. With x-ray crystallography and cryogenic-electron microscopy, NMR is one of three techniques that is used to elucidate the structure of proteins. Unlike nuclear magnetic imaging, protein NMR uses algorithms to create three-dimensional models of the sample of interest. Protein NMR is conducted on thoroughly purified samples.&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Cryo-Electron Microscopy Characterization<\/strong>&nbsp;<\/h3>\n\n\n\n<p>Another technique that can&nbsp;determine&nbsp;the structure of a protein, cryo-EM is an emerging and promising technology to obtain high-resolution membrane protein structures, which are not possible with other techniques like NMR or X-ray crystallography. Cryo-EM consists of several applications like single particle analysis (SPA), cryo-electron tomography (cryoET), and micro electron diffraction (MicroED). It has been revolutionary in structural biology, infectious disease research, and drug discovery.&nbsp;<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"903\" height=\"598\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-16.png\" alt=\"FDA Center for Food Safety Applied Nutrition CFSAN 6540 8754867859\" class=\"wp-image-6679\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-16.png 903w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-16-300x199.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-16-768x509.png 768w\" sizes=\"(max-width: 903px) 100vw, 903px\" \/><\/figure>\n<\/div>\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_fe254e-43 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_fe254e-43 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_fe254e-43\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Advancing Protein Characterization&nbsp;<\/h2>\n\n\n\n<p>Because of the complex nature (not to mention importance) of proteins,&nbsp;it\u2019s&nbsp;necessary to develop integrative approaches that combine multiple techniques for comprehensive analysis and protein characterization. Future trends in characterization appear to lie in higher throughputs and better atomic-level resolution, as well as the ability to&nbsp;quickly and reliably determine non-proteins and protein fragments and aggregates, particularly as more protein-based biotherapeutics are developed and enter the marketplace.\u202f&nbsp;<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6673_32a76b-be .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6673_32a76b-be .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-6673_32a76b-be\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">References<\/h2>\n\n\n\n<p><a href=\"https:\/\/www.usp.org\/biologics\/proteins\">https:\/\/www.usp.org\/biologics\/proteins<\/a><\/p>\n\n\n\n<p><a href=\"https:\/\/www.biopharminternational.com\/view\/usp-standards-to-support-the-characterization-of-mabs-applications-and-general-chapter-129\">https:\/\/www.biopharminternational.com\/view\/usp-standards-to-support-the-characterization-of-mabs-applications-and-general-chapter-129<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Protein identification and characterization is one of the most important components of expressing and manufacturing recombinant proteins for biologics. It involves a wide variety of analytical tools and techniques because of the very complexity of proteins themselves, starting with 21 amino acids arranged in&nbsp;a nearly infinite&nbsp;number of ways, and then folded into three-dimensional structures.\u202f&nbsp; Understanding&#8230;<\/p>\n","protected":false},"author":60,"featured_media":6679,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_seopress_robots_primary_cat":"none","_seopress_titles_title":"%%post_title%% %%sep%% %%sitetitle%%","_seopress_titles_desc":"Learn all about protein identification and characterization, an important component of expressing and manufacturing recombinant proteins for biologics.","_seopress_robots_index":"","_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"_kad_post_classname":"","footnotes":""},"categories":[228,1],"tags":[171,51,52,722,727,726],"class_list":["post-6673","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-biopharmaceutical","category-general","tag-biologics","tag-biopharma","tag-biopharmaceutical","tag-particle-analysis","tag-protein-characterization","tag-protein-identification"],"acf":[],"taxonomy_info":{"category":[{"value":228,"label":"Biopharmaceutical"},{"value":1,"label":"General"}],"post_tag":[{"value":171,"label":"biologics"},{"value":51,"label":"biopharma"},{"value":52,"label":"biopharmaceutical"},{"value":722,"label":"particle analysis"},{"value":727,"label":"protein characterization"},{"value":726,"label":"protein identification"}]},"featured_image_src_large":["https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-16.png",903,598,false],"author_info":{"display_name":"Waters Staff","author_link":"https:\/\/www.waters.com\/blog\/author\/dmason\/"},"comment_info":"","category_info":[{"term_id":228,"name":"Biopharmaceutical","slug":"biopharmaceutical","term_group":0,"term_taxonomy_id":228,"taxonomy":"category","description":"We help ensure the medicines people take every day are reliably safe and effective, from the common aspirin to the most complex biotherapy. Tests performed using our liquid chromatography-mass spectrometry (LC-MS) instruments ensure the efficacy and safety of these complex, protein-based drugs well before they reach the patient.","parent":25,"count":42,"filter":"raw","term_order":"0","cat_ID":228,"category_count":42,"category_description":"We help ensure the medicines people take every day are reliably safe and effective, from the common aspirin to the most complex biotherapy. Tests performed using our liquid chromatography-mass spectrometry (LC-MS) instruments ensure the efficacy and safety of these complex, protein-based drugs well before they reach the patient.","cat_name":"Biopharmaceutical","category_nicename":"biopharmaceutical","category_parent":25},{"term_id":1,"name":"General","slug":"general","term_group":0,"term_taxonomy_id":1,"taxonomy":"category","description":"","parent":0,"count":52,"filter":"raw","term_order":"0","cat_ID":1,"category_count":52,"category_description":"","cat_name":"General","category_nicename":"general","category_parent":0}],"tag_info":[{"term_id":171,"name":"biologics","slug":"biologics","term_group":0,"term_taxonomy_id":171,"taxonomy":"post_tag","description":"","parent":0,"count":17,"filter":"raw","term_order":"0"},{"term_id":51,"name":"biopharma","slug":"biopharma","term_group":0,"term_taxonomy_id":51,"taxonomy":"post_tag","description":"","parent":0,"count":50,"filter":"raw","term_order":"0"},{"term_id":52,"name":"biopharmaceutical","slug":"biopharmaceutical","term_group":0,"term_taxonomy_id":52,"taxonomy":"post_tag","description":"","parent":0,"count":58,"filter":"raw","term_order":"0"},{"term_id":722,"name":"particle analysis","slug":"particle-analysis","term_group":0,"term_taxonomy_id":722,"taxonomy":"post_tag","description":"","parent":0,"count":19,"filter":"raw","term_order":"0"},{"term_id":727,"name":"protein characterization","slug":"protein-characterization","term_group":0,"term_taxonomy_id":727,"taxonomy":"post_tag","description":"","parent":0,"count":1,"filter":"raw","term_order":"0"},{"term_id":726,"name":"protein identification","slug":"protein-identification","term_group":0,"term_taxonomy_id":726,"taxonomy":"post_tag","description":"","parent":0,"count":1,"filter":"raw","term_order":"0"}],"_links":{"self":[{"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/posts\/6673","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/users\/60"}],"replies":[{"embeddable":true,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/comments?post=6673"}],"version-history":[{"count":0,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/posts\/6673\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/media\/6679"}],"wp:attachment":[{"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/media?parent=6673"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/categories?post=6673"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.waters.com\/blog\/wp-json\/wp\/v2\/tags?post=6673"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}