{"id":6720,"date":"2023-10-02T11:56:00","date_gmt":"2023-10-02T11:56:00","guid":{"rendered":"https:\/\/www.waters.com\/blog\/?p=6720"},"modified":"2026-01-21T11:39:32","modified_gmt":"2026-01-21T11:39:32","slug":"polysorbate-80-exploring-its-impact-on-igg-stability","status":"publish","type":"post","link":"https:\/\/www.waters.com\/blog\/polysorbate-80-exploring-its-impact-on-igg-stability\/","title":{"rendered":"Polysorbate 80: Exploring its Impact on IgG Stability"},"content":{"rendered":"\n<p class=\"wp-block-paragraph\">As drug safety regulations intensify, manufacturers must ensure their formulations, like polysorbate 80, are free from visible particles and contain a limited number of subvisible particles larger than 10 \u00b5m (per FDA requirements). These particulates aren\u2019t just due to process instability but also the degradation of formulation excipients.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Mechanical stress during manufacturing can cause protein aggregation, negatively impacting product quality and safety. Aggregation is often the result of weak interactions between exposed hydrophobic patches of partially or fully unfolded proteins. A variety of conditions such as pH shifts, elevated temperatures, and additional mechanical stress can induce protein unfolding, thus promoting the formation of protein aggregates.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">As a result, developing robust formulations that limit protein unfolding and aggregation is crucial for ensuring protein drug product quality.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6720_2b8849-44 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6720_2b8849-44 .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-6720_2b8849-44\"><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-heading6720_05205b-a0, .wp-block-kadence-advancedheading.kt-adv-heading6720_05205b-a0[data-kb-block=\"kb-adv-heading6720_05205b-a0\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6720_05205b-a0 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6720_05205b-a0[data-kb-block=\"kb-adv-heading6720_05205b-a0\"] 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-heading6720_05205b-a0 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6720_05205b-a0[data-kb-block=\"kb-adv-heading6720_05205b-a0\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6720_05205b-a0 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6720_05205b-a0\">Polysorbates: A Key Player in Preventing Protein Aggregation<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">To prevent aggregation, polysorbates are frequently used. These nonionic surfactants primarily consist of sorbitan polyoxyethylene (POE) fatty acid esters. The protective function of polysorbates in protein formulations is well-studied, with two primary mechanisms identified: (1) polysorbates acting as a chaperone for aggregation-prone hydrophobic sites on protein surfaces, thus supporting the folded structure, and (2) polysorbates competing with proteins at interfaces, thereby reducing interface exposure.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Polysorbate 80 (POE sorbitan monooleate, PS80) is a popular surfactant in commercially available biopharmaceutical formulations due to its effectiveness in preventing protein adsorption and aggregation. However, there\u2019s a catch: the grade and quality of PS80, particularly during processes generating mechanical stress, can significantly influence protein stability, especially IgG.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">What\u2019s more, long-term storage of polysorbate at low temperatures can lead to free fatty acid particle formation, potentially compromising drug effectiveness. Traditional analysis methods like light obscuration (LO) and flow imaging (FI) struggle to identify degradation sources. Over time, they can become susceptible to enzymatic hydrolysis by host cell proteins (HCPs), resulting in visible and subvisible particle formation that could compromise a drug\u2019s effectiveness.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Conducting high-throughput, sensitive, and specific analysis of polysorbate particles has been difficult due to their complex chemistry and low concentrations formulation environments, making it a perennial needle in a haystack problem.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6720_e3699d-1e .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6720_e3699d-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-6720_e3699d-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-heading6720_537705-99, .wp-block-kadence-advancedheading.kt-adv-heading6720_537705-99[data-kb-block=\"kb-adv-heading6720_537705-99\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6720_537705-99 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6720_537705-99[data-kb-block=\"kb-adv-heading6720_537705-99\"] 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-heading6720_537705-99 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6720_537705-99[data-kb-block=\"kb-adv-heading6720_537705-99\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6720_537705-99 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6720_537705-99\">Backgrounded Membrane Imaging Used to Explore Functionality of Polysorbate 80<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">The role of PS80 on protein stability is a topic of ongoing debate. In addition, the impact of partially degraded PS on protein aggregation is not yet well understood. This has led researchers to conduct a study of the functionality of PS80.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">In the\u00a0<a href=\"https:\/\/jpharmsci.org\/article\/S0022-3549(19)30651-3\/fulltext\" target=\"_blank\" rel=\"noreferrer noopener\">study<\/a>, a monoclonal antibody (mAb), IgG, was subjected to three different mechanical stress conditions in the presence of multicompendial (MC) and Chinese pharmacopeia (ChP) grade PS80. IgG formulations were supplemented with partly hydrolyzed PS80 to investigate the effect of its degradants on protein stability.<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"934\" height=\"611\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-36.png\" alt=\"Figure 1. Aggregation over time of 5 mg\/mL IgG formulated with different amounts of MC-PS80 or ChP-PS80, stressed by shaking in 2R glass vials at RT, as analyzed by SEC, UV (OD350 nm), DLS and FIM. From \u201cWhat Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress,\u201d by A. Grabarek, 2019, 12, JPharmSci, volume number 109, page(s) 871-880. Copyright 2023 Elsevier Inc.\" class=\"wp-image-6723\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-36.png 934w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-36-300x196.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-36-768x502.png 768w\" sizes=\"(max-width: 934px) 100vw, 934px\" \/><figcaption class=\"wp-element-caption\">Figure 1. Aggregation over time of 5 mg\/mL IgG formulated with different amounts of MC-PS80 or ChP-PS80, stressed by shaking in 2R glass vials at RT, as analyzed by SEC, UV (OD350 nm), DLS and FIM. From \u201cWhat Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress,\u201d by A. Grabarek, 2019, 12, JPharmSci, volume number 109, page(s) 871-880. Copyright 2023 Elsevier Inc.<\/figcaption><\/figure>\n<\/div>\n\n\n<p class=\"wp-block-paragraph\">PS80\u2019s functionality was assessed by measuring protein aggregation and particle formation induced during mechanical stress by using several analytical methods. These included: size-exclusion chromatography (SEC), dynamic light scattering (DLS),\u00a0flow\u00a0imaging microscopy (FIM), and\u00a0backgrounded membrane imaging (BMI).<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Researchers used the <a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers.html\">Aura<\/a> System\u00a0to perform BMI for micrometer-sized particle characterization and quantification. Under laminar air flow conditions, 30 \u03bcL of the sample was transferred on a polycarbonate 96-well membrane filter plate (Waters) with a 0.4-\u03bcm pore size. A vacuum of 350 mbar was applied after filling six wells to allow a flow of liquid through the membrane and entrapment of particles. Subsequently, 90 \u03bcL of highly purified water was used to wash each well after sample transfer to remove any soluble material. Each sample was measured in triplicate.<sup>1<\/sup><a id=\"_msocom_1\"><\/a><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">The study found no significant differences in PS80 functionality between MC and ChP grades across the stress tests. However, as the degree of PS80 hydrolysis increased, higher counts of subvisible particles were measured after stress. Furthermore, higher levels of PS80 degradants at a constant PS80 concentration may destabilize the IgG. Researchers concluded that MC and ChP grade PS80 were equally protective, but PS80 degradants compromise IgG stability.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6720_ddcff0-7c .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6720_ddcff0-7c .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-6720_ddcff0-7c\"><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-heading6720_78bd4f-3f, .wp-block-kadence-advancedheading.kt-adv-heading6720_78bd4f-3f[data-kb-block=\"kb-adv-heading6720_78bd4f-3f\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6720_78bd4f-3f mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6720_78bd4f-3f[data-kb-block=\"kb-adv-heading6720_78bd4f-3f\"] 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-heading6720_78bd4f-3f img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6720_78bd4f-3f[data-kb-block=\"kb-adv-heading6720_78bd4f-3f\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6720_78bd4f-3f wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6720_78bd4f-3f\">High-Throughput Polysorbate Particle ID using Aura Assay<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Degradation of polysorbate results in three distinct fatty acids: lauric, myristic, and palmitic acid. These acids create particles with unique morphological features that can be pinpointed using Aura System\u2019s labeling and imaging capabilities.\u00a0Through\u00a0the application of BODIPY\u2122 FL C<sub>16 <\/sub>(Thermo Fisher Scientific), a high-affinity stain, researchers can accurately identify FFAs.<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"940\" height=\"325\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-37.png\" alt=\"BODIPY FL C16 staining 3 different free fatty acids.\" class=\"wp-image-6724\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-37.png 940w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-37-300x104.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-37-768x266.png 768w\" sizes=\"(max-width: 940px) 100vw, 940px\" \/><figcaption class=\"wp-element-caption\">BODIPY FL C<sub>16<\/sub>\u00a0staining 3 different free fatty acids.<\/figcaption><\/figure>\n<\/div>\n\n\n<p class=\"wp-block-paragraph\">Aura System\u2019s polysorbate degradation assay\u00a0streamlines the process of detecting degraded excipients in protein formulations with elevated concentrations. This method is adaptable, capable of managing a wide range of sample volumes from 5 \u00b5L to 10 mL, and can simultaneously analyze up to 96 samples for the existence of free acid particles. This efficiency significantly reduces both time and effort.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Importantly, the Aura System\u2019s polysorbate assay is sensitive enough to identify free fatty acids (FFAs) at concentrations relative to lauric acid exceeding\u00a09.38\u00a0\u00b5M (which translates to more than 2183 counts\/mL), and can quantify them at levels above 18.75 \u00b5M, or over 2976\u00a0counts\/mL.<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"778\" height=\"513\" src=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-38.png\" alt=\"LogFL2 counts (&gt;5 micron) by log (lauric) uM. In the presence of hIgG, Aura System offers ultra-sensitive detection and quantification limits for free fatty acids. \" class=\"wp-image-6725\" srcset=\"https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-38.png 778w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-38-300x198.png 300w, https:\/\/www.waters.com\/blog\/wp-content\/uploads\/image-38-768x506.png 768w\" sizes=\"(max-width: 778px) 100vw, 778px\" \/><figcaption class=\"wp-element-caption\">In the presence of hIgG, Aura System offers ultra-sensitive detection and quantification limits for free fatty acids.<\/figcaption><\/figure>\n<\/div>\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6720_4794cf-aa .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6720_4794cf-aa .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-6720_4794cf-aa\"><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-heading6720_2df790-b8, .wp-block-kadence-advancedheading.kt-adv-heading6720_2df790-b8[data-kb-block=\"kb-adv-heading6720_2df790-b8\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6720_2df790-b8 mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6720_2df790-b8[data-kb-block=\"kb-adv-heading6720_2df790-b8\"] 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-heading6720_2df790-b8 img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6720_2df790-b8[data-kb-block=\"kb-adv-heading6720_2df790-b8\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6720_2df790-b8 wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6720_2df790-b8\">Conclusion<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\"><a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/particle-analyzers\/aura-ptx.html\">Aura<\/a>\u00a0System simplifies the detection and distinction of degraded components in protein-based samples at any point in the drug production process. Requiring only 5 \u00b5L of sample, this method is both specific and sensitive, capable of analyzing 96 samples in a matter of hours, thereby outpacing alternative techniques.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">In addition, this advanced technology can identify and differentiate crucial degraded particles from polysorbate formulation based on their shape, appearance, and unique labeling with BODIPY FL C<sub>16<\/sub>, making it an indispensable tool.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-6720_c933ad-27 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-6720_c933ad-27 .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-6720_c933ad-27\"><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-heading6720_4dc9ea-4f, .wp-block-kadence-advancedheading.kt-adv-heading6720_4dc9ea-4f[data-kb-block=\"kb-adv-heading6720_4dc9ea-4f\"]{font-style:normal;}.wp-block-kadence-advancedheading.kt-adv-heading6720_4dc9ea-4f mark.kt-highlight, .wp-block-kadence-advancedheading.kt-adv-heading6720_4dc9ea-4f[data-kb-block=\"kb-adv-heading6720_4dc9ea-4f\"] 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-heading6720_4dc9ea-4f img.kb-inline-image, .wp-block-kadence-advancedheading.kt-adv-heading6720_4dc9ea-4f[data-kb-block=\"kb-adv-heading6720_4dc9ea-4f\"] img.kb-inline-image{width:150px;vertical-align:baseline;}<\/style>\n<h2 class=\"kt-adv-heading6720_4dc9ea-4f wp-block-kadence-advancedheading\" data-kb-block=\"kb-adv-heading6720_4dc9ea-4f\">References<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li><a href=\"https:\/\/jpharmsci.org\/article\/S0022-3549(19)30651-3\/fulltext\">https:\/\/jpharmsci.org\/article\/S0022-3549(19)30651-3\/fulltext<\/a><\/li>\n\n\n\n<li>Application Note: <a href=\"https:\/\/www.waters.com\/nextgen\/global\/library\/application-notes\/2025\/high-throughput-detection-of-degraded-polysorbate-in-biological-formulations-with-fmm.html\">High-Throughput Detection of Degraded Polysorbate in Biological Formulations with FMM<\/a> <\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>As drug safety regulations intensify, manufacturers must ensure their formulations, like polysorbate 80, are free from visible particles and contain a limited number of subvisible particles larger than 10 \u00b5m (per FDA requirements). These particulates aren\u2019t just due to process instability but also the degradation of formulation excipients. 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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. 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