Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates.
Identifieur interne : 000371 ( Main/Exploration ); précédent : 000370; suivant : 000372Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates.
Auteurs : Hannah R. Doss [États-Unis] ; Mathura Raman [États-Unis] ; Ryan Knihtila [États-Unis] ; Naresh Chennamsetty [États-Unis] ; David Wang [États-Unis] ; Alan Shupe [États-Unis] ; Nesredin Mussa [États-Unis]Source :
- Journal of chromatography. A [ 1873-3778 ] ; 2019.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Antibodies, Monoclonal, Immunoglobulin G.
- chemical , isolation & purification : Antibodies, Monoclonal, Immunoglobulin G.
- methods : Chemistry, Pharmaceutical.
- Chromatography, Gel, Hydrophobic and Hydrophilic Interactions, Molecular Weight.
Abstract
When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.
DOI: 10.1016/j.chroma.2019.03.044
PubMed: 30954243
Affiliations:
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Doss</name><affiliation wicri:level="2"><nlm:affiliation>Biologics Manufacturing Sciences and Technology, Global Product Development and Supply, Bristol-Myers Squibb Company, Devens, MA, USA; Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA. 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A</title><idno type="eISSN">1873-3778</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antibodies, Monoclonal (chemistry)</term><term>Antibodies, Monoclonal (isolation & purification)</term><term>Chemistry, Pharmaceutical (methods)</term><term>Chromatography, Gel</term><term>Hydrophobic and Hydrophilic Interactions</term><term>Immunoglobulin G (chemistry)</term><term>Immunoglobulin G (isolation & purification)</term><term>Molecular Weight</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Anticorps monoclonaux ()</term><term>Anticorps monoclonaux (isolement et purification)</term><term>Chimie pharmaceutique ()</term><term>Chromatographie sur gel</term><term>Immunoglobuline G ()</term><term>Immunoglobuline G (isolement et purification)</term><term>Interactions hydrophobes et hydrophiles</term><term>Masse moléculaire</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Antibodies, Monoclonal</term><term>Immunoglobulin G</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Antibodies, Monoclonal</term><term>Immunoglobulin G</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Anticorps monoclonaux</term><term>Immunoglobuline G</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Chemistry, Pharmaceutical</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Chromatography, Gel</term><term>Hydrophobic and Hydrophilic Interactions</term><term>Molecular Weight</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Anticorps monoclonaux</term><term>Chimie pharmaceutique</term><term>Chromatographie sur gel</term><term>Immunoglobuline G</term><term>Interactions hydrophobes et hydrophiles</term><term>Masse moléculaire</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Massachusetts</li><li>New Jersey</li><li>État de New York</li></region></list><tree><country name="États-Unis"><region name="État de New York"><name sortKey="Doss, Hannah R" sort="Doss, Hannah R" uniqKey="Doss H" first="Hannah R" last="Doss">Hannah R. Doss</name></region><name sortKey="Chennamsetty, Naresh" sort="Chennamsetty, Naresh" uniqKey="Chennamsetty N" first="Naresh" last="Chennamsetty">Naresh Chennamsetty</name><name sortKey="Knihtila, Ryan" sort="Knihtila, Ryan" uniqKey="Knihtila R" first="Ryan" last="Knihtila">Ryan Knihtila</name><name sortKey="Mussa, Nesredin" sort="Mussa, Nesredin" uniqKey="Mussa N" first="Nesredin" last="Mussa">Nesredin Mussa</name><name sortKey="Raman, Mathura" sort="Raman, Mathura" uniqKey="Raman M" first="Mathura" last="Raman">Mathura Raman</name><name sortKey="Shupe, Alan" sort="Shupe, Alan" uniqKey="Shupe A" first="Alan" last="Shupe">Alan Shupe</name><name sortKey="Wang, David" sort="Wang, David" uniqKey="Wang D" first="David" last="Wang">David Wang</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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