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Advancing Structure Characterization of PS-80 by Charge-Reduced Mass Spectrometry and Software-Assisted Composition Analysis

Published:September 03, 2021DOI:https://doi.org/10.1016/j.xphs.2021.08.036

      Abstract

      The commercially available Polysorbate 80 (PS-80) is a highly heterogeneous product. It is a complex and structurally diverse mixture consisting of polymeric species containing polyoxyethylenes (POEs), fatty acid esters, with/or without a carbohydrate core. The core is primarily sorbitan, with some isosorbide and sorbitol. Depending on the sources of fatty acids and the degrees of esterification, multiple combinations of fatty acid esters are commonly observed. A number of POE intermediates, such as polyoxyethylene glycols, POE-sorbitans, POE-isosorbides, and an array of fatty acid esters from these intermediates remain in the raw material as well. The complex composition of PS-80 is difficult to control and poses a significant characterization challenge for its use in the pharmaceutical industry. Here, we present a novel solution for PS-80 characterization using ultra high-performance liquid chromatography coupled with charge-reduction high resolution mass spectrometry. Post column co-infusion of triethylamine focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation, resulting in a simpler ion map and enhanced measurement of PS-80 species. The data processing workflow is designed to programmatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. The 2-dimensional graphical representation of the data helps visualize these features. Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and shall be a useful tool to study formulation and stability of pharmaceutical preparations. The power of this approach was demonstrated by comparing the composition of PS-80 obtained from different vendors.

      Keywords

      Abbreviations:

      PS-80 (Polysorbate 80), HX (multicompendial grade PS-80), SR (super refined grade PS-80), CP (Chinese pharmacopeia grade PS-80), POE (polyoxyethylene), PEG (polyoxyethylene glycol), POE-S (POE-sorbitan), POE-IS (POE-isosorbide), EO (ethylene oxide), RP-LC (reverse phase liquid chromatography), ESI-HRMS (electrospray ionization-high resolution mass spectrometry), RP-UHPLC (reverse phase ultra-high-performance liquid chromatography), TEA (Triethylamine), 2D Ion Density Map (2-dimensional mass spectrometry ion density map)
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      References

        • Kanthe A.D.
        • Krause M.
        • Zheng S.
        • et al.
        Armoring the interface with surfactants to prevent the adsorption of monoclonal antibodies.
        ACS Appl Mater Interfaces. 2020; 12: 9977-9988
        • Grabarek A.D.
        • Bozic U.
        • Rousel J.
        • et al.
        What makes polysorbate functional? Impact of polysorbate 80 grade and quality on IGG stability during mechanical stress.
        J Pharm Sci. 2020; 109: 871-880
        • Biddlecombe J.G.
        • Smith G.
        • Uddin S.
        • et al.
        Factors influencing antibody stability at solid–liquid interfacesin a high shear environment.
        Biotechnol. 2009; 25: 1499-1507
        • Nayem J.
        • Zhang Z.
        • Tomlinson A.
        • Zarraga I.E.
        • Wagner N.J.
        • Liu Y.
        Micellar morphology of polysorbate 20 and 80 and their ester fractions in solution via small-angle neutron scattering.
        J Pharm Sci. 2020; 109: 1498-1508
        • Mahler H.-C.
        • Huber F.
        • Kishore R.S.K.
        • Reindl J.
        • Rückert P.
        • Müller R.
        Adsorption behavior of a surfactant and a monoclonal antibody to sterilizing-grade filters.
        J Pharm Sci. 2010; 99: 2620-2627
        • Chou D.K.
        • Krishnamurthy R.
        • Randolph T.W.
        • Carpenter J.F.
        • Manning M.C.
        Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation.
        J Pharm Sci. 2005; 94: 1368-1381
        • Braun A.C.
        • Ilko D.
        • Merget B.
        • et al.
        Predicting critical micelle concentration and micelle molecular weight of polysorbate 80 using compendial methods.
        Eur J Pharm Biopharm. 2015; 94: 559-568
        • Donbrow M.
        • Azaz E.
        • Pillersdorf A.
        Autoxidation of polysorbates.
        J Pharm Sci. 1978; 67: 1676-1681
        • Ha E.
        • Wang W.
        • Wang Y.J
        Peroxide formation in polysorbate 80 and protein stability.
        J Pharm Sci. 2002; 91: 2252-2264
        • Yao J.
        • Dokuru D.K.
        • Noestheden M.
        A quantitative kinetic study of polysorbate autoxidation: the role of unsaturated fatty acid ester substituents.
        Pharm Res. 2009; 26 (2303-2013)
        • Jones M.T.
        • Mahler H.-C.
        • Yadav S.
        • et al.
        Considerations for the use of polysorbates in biopharmaceuticals.
        Pharm Res. 2018; 35: 148
        • Bates T.R.
        • Nightingale C.H.
        • Dixon E.
        Kinetics of hydrolysis of polyoxyethylene (20) sorbitan fatty acid ester surfactants.
        J Pharm Pharmacol. 1973; 25: 470-477
        • Tomlinson A.
        • Demeule B.
        • Lin B.
        • Yadav S.
        Polysorbate 20 degradation in biopharmaceutical formulations: quantification of free fatty acids, characterization of particulates, and insights into the degradation mechanism.
        Mol Pharm. 2015; 12: 3805-3815
        • Doshi N.
        • Demeule B.
        • Yadav S.
        Understanding particle formation: solubility of free fatty acids as polysorbate 20 degradation byproducts in therapeutic monoclonal antibody formulations.
        Mol Pharm. 2015; 12: 3792-3804
        • Saggu M.
        • Liu J.
        • Patel A.
        Identification of subvisible particles in biopharmaceutical formulations using raman spectroscopy provides insight into polysorbate 20 degradation pathway.
        Pharm Res. 2015; 32: 2877-2888
        • Kishore R.S.K.
        • Kiese S.
        • Fischer S.
        • Pappenberger A.
        • Grauschopf U.
        • Mahler H.-C.
        The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics.
        Pharm Res. 2011; 28: 1194-1210
        • Wasylaschuk W.R.
        • Harmon P.A.
        • Wagner G.
        • Harman A.B.
        • Templeton A.C.
        • Xu H.
        • Reed R.A.
        Evaluation of hydroperoxides in common pharmaceutical excipients.
        J Pharm Sci. 2007; 96: 106-116
        • Ueyama E.
        • Tamura K.
        • Mizukawa K.
        • Kano K.
        Realistic prediction of solid pharmaceutical oxidation products by using a novel forced oxidation system.
        J Pharm Sci. 2014; 103: 1184-1193
      1. Polysorbate 80 for injection [9005–65-6].
        Chinese Pharmacopeia. 2015; 4
      2. C. USP. The United States Pharmacopeia. USP 40 NF 35; NF Monograph: Polysorbate 80. (2017)

        • C.O. Europe
        European pharmacopoeia (PhEur) European Medicines Agency.
        9th ed. 2017: 2267-2271
      3. Japanese Pharmacopeia 17th Edition, the MHLW Ministerial Notification No. 64. (2016)

        • Vaclaw C.
        • Merritt K.
        • Pringle V.
        • et al.
        Impact of polysorbate 80 grade on the interfacial properties and interfacial stress induced subvisible particle formation in monoclonal antibodies.
        J Pharm Sci. 2021; 110: 746-759
        • Penfield K.W.
        • Rumbelow S.
        Challenges in polysorbate characterization by mass spectrometry.
        Rapid Commun Mass Spectrom. 2020; 34: e8709
        • Cheng Y.
        • Hu M.
        • Zamiri C.
        • et al.
        Rapid high-sensitivity reversed–phase ultra high performance liquid chromatography mass spectrometry method for assessing polysorbate 20 degradation in protein therapeutics.
        J Pharm Sci. 2019; 108: 2880-2886
        • Yang K.
        • Hewarathna A.
        • Geerlof-Vidavsky I.
        • Rao V.A.
        • Gryniewicz-Ruzicka C.
        • Keire D.
        Screening of polysorbate-80 composition by high resolution mass spectrometry with rapid H/D exchange.
        Anal Chem. 2019; 91: 14649-14656
        • Hvattum E.
        • Yip W.L.
        • Grace D.
        • Dyrstad K.
        Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: specific determination of oxidation products of thermally oxidized polysorbate 80.
        J Pharm Biomed Anal. 2012; 62: 7-16
        • Ayorinde F.O.
        • Gelain S.V.
        • Johnson Jr J.H.
        • Wan L.W.
        Analysis of some commercial polysorbate formulations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
        Rapid Commun Mass Spectrom. 2000; 14: 2116-2124
        • Snelling J.R.
        • Scarff C.A.
        • Scrivens J.H.
        Characterization of complex polysorbate formulations by means of shape-selective mass spectrometry.
        Anal Chem. 2012; 84: 6521-6529
        • Tani T.H.
        • Moore J.M.
        • Patapoff T.W.
        Single step method for the accurate concentration determination of polysorbate 80.
        J. Chromatogr A. 1997; 786: 99-106
        • Li Y.
        • Hewitt D.
        • Lentz Y.K.
        • Ji J.A.
        • Zhang T.Y.
        • Zhang K.
        Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.
        Anal Chem. 2014; 86: 5150-5157
        • Katzenmeyer B.C.
        • Hague S.F.
        • Wesdemiotis C.
        Multidimensional mass spectrometry coupled with separation by polarity or shape for the characterization of sugar-based nonionic surfactants.
        Anal Chem. 2016; 88: 851-857
        • He Y.
        • Brown P.
        • Bailey Piatchek M.R.
        • Carroll J.A.
        • Jones M.T.
        On-line coupling of hydrophobic interaction column with reverse phase column -charged aerosol detector/mass spectrometer to characterize polysorbates in therapeutic protein formulations.
        J Chromatogr A. 2019; 1586: 72-81
        • Zhang R.
        • Wang Y.
        • Tan L.
        • Zhang H.Y.
        • Yang M.
        Analysis of polysorbate 80 and its related compounds by RP-HPLC with ELSD and MS detection.
        J Chromatogr Sci. 2012; 50: 598-607
        • Borisov O.V.
        • Ji J.A.
        • Wang Y.J.
        • Vega F.
        • Ling V.T.
        Toward understanding molecular heterogeneity of polysorbates by application of liquid chromatography-mass spectrometry with computer-aided data analysis.
        Anal Chem. 2011; 83: 3934-3942
        • Hurtado P.P.
        • Lam P.Y.
        • Kilgour D.
        • Bristow A.
        • McBride E.
        • O’Connor P.B.
        Use of high resolution mass spectrometry for analysis of polymeric excipients in drug delivery formulations.
        Anal Chem. 2012; 84: 8579-8586
        • Nohmi T.
        • Fenn J.B.
        Electrospray mass spectrometry of poly(ethy1ene glycols) with molecular weights up to five million.
        J Am Chem Soc. 1992; 114: 3241-3246
        • Stephenson J.L.
        • McLuckey S.A.
        Reactions of poly(ethylene glycol) cations with iodide and perfluorocarbon anions.
        J Am Soc Mass Spectrom. 1998; 9: 957-965
        • Lennon J.D.
        • Cole S.P.
        • Glish G.L.
        Ion/molecule reactions to chemically deconvolute the electrospray ionization mass spectra of synthetic polymers.
        Anal Chem. 2006; 78: 8472-8476
        • Huang L.
        • Gough P.C.
        • DeFelippis M.R.
        Characterization of poly(ethylene glycol) and pegylated products by LC/MS with postcolumn addition of amines.
        Anal Chem. 2009; 81: 567-577
        • Forstenlehner I.C.
        • Holzmann J.
        • Scheffler K.
        • Wieder W.
        • Toll H.
        • Huber C.G.
        A direct-infusion- and HPLC-ESI-Orbitrap-MS approach for the characterization of intact PEgylated proteins.
        Anal Chem. 2014; 86: 826-834
        • Larriba C.
        • de la Mora J.F.
        • Clemmer D.E.
        Electrospray ionization mechanisms for large polyethylene glycol chains studied through tandem ion mobility spectrometry.
        J Am Soc Mass Spectrom. 2014; 25: 1332-1345
        • Wang S.
        • Xing T.
        • Liu A.P.
        Simple approach for improved LC-MS analysis of protein biopharmaceuticals via modification of desolvation gas.
        Anal Chem. 2019; 91: 3156-3162
        • Bush D.R.
        • Zang L.
        • Belov A.M.
        • Ivanov A.R.
        • Karger B.L.
        High resolution CZE-MS quantitative characterization of intact biopharmaceutical proteins: proteoforms of interferon-β1.
        Anal Chem. 2016; 88: 1138-1146
        • Belov A.M.
        • Zang L.
        • Sebastiano R.
        • et al.
        Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry.
        Electrophoresis. 2018; 39: 2069-2082
        • Griffin W.C.
        Calculation of HLB values of non-ionic surfactants.
        J Soc Cosmet Chem. 1954; 5: 249-256
        • Pasquali R.C.
        • Taurozzi M.P.
        • Bregni C.
        Some considerations about the hydrophilic-lipophilic balance system.
        International J Pharm. 2008; 356: 44-51
        • Sun H.
        • Yang R.
        • Wang J.
        • et al.
        Component-based biocompatibility and safety evaluation of polysorbate 80.
        RSC Adv. 2017; 7: 15127-15138
        • Li X.
        • Chandra D.
        • Letarte S.
        • et al.
        The methodology has been demonstrated in a recent publication: profiling active enzymesfor polysorbate degradation in biotherapeutics by activity-based protein profiling.
        Anal Chem. 2021; 93: 8161-8169