Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation

Farsang, Evelin; Murisier, Amarande; Horváth, Krisztián; Beck, Alain; Kormány, Róbert; Guillarme, Davy; Fekete, Szabolcs

doi:10.1016/j.jpba.2019.02.024

Cited by 30 publications

(15 citation statements)

References 24 publications

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“…Each of these buffers will also control the pH at the column surface. Other approaches have used mixtures of buffer types, charged buffers, and zwitterionic buffers, such as citric acid, succinic acid, Bis-Tris propane, piperazine, triethanolamine, and CAPSO. ,,, The problem with such heterogeneous mixtures lies in the varying abilities of each component to buffer at the surface of the column and to be replaced from the charged sites on the column during the gradient and equilibration. A negatively charged component, such as succinic acid, will be repelled from the surface of a cation exchange resin.…”

Section: Resultsmentioning

confidence: 99%

“…Recently the intentional increase in salt concentration during the pH gradient has been reported to aid in elution and give an additional dimension of separation. , The name given to this technique was “salt-mediated pH gradient elution” . In this instance, the recommended pH gradient used MES with a p K a value of 6.1 and DAP (1,3-diamino-2-propanol) with a p K a value of 9.6 to control the pH.…”

Section: Resultsmentioning

confidence: 99%

“…A mixture of pI protein standards covering a pI from 7 to 10 was injected (L 1–3 , lentil lectins: pI 7.6–8.2; T, trypsinogen: pI 8.4–8.7; R, ribonuclease A: pI 9.6, and C, cytochrome C: pI 10–10.5). (A) Salt-mediated pH gradient, using a MES/DAB system . (B) Zwitterionic CX-1 pH gradient buffer system.…”

Section: Resultsmentioning

confidence: 99%

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New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization

et al. 2020

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Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization

et al. 2020

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“…IEX chromatography can be run in three different elution modes: (1) salt gradient, (2) pH gradient, and (3) the combination of the two methods, which is often referred as salt‐mediated pH gradient [39]. The latter elution mode merges the benefits of the salt‐ and pH gradients, namely that proteins will elute more or less in the order of their p I and will elute in sharp peaks thanks to peak focusing effect of the increasing ionic strength [74]. Because of the nonvolatile nature of the salts (NaCl, KCl) and buffers (MES, phosphate) commonly used in IEX mobile phases, IEX is inherently not compatible with MS detection.…”

Section: Analytical Methods For Fc‐fusion Proteins Characterizationmentioning

confidence: 99%

Therapeutic Fc‐fusion proteins: Current analytical strategies

Duivelshof

Murisier

Camperi

et al. 2020

J of Separation Science

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Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of iso-

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“…7 As a conventional and nondenaturing technique, ionexchange chromatography (IEX) has been widely used to separate and isolate protein charge variants during protein purification and for subsequent characterization. [22][23][24] Upon the separation of charge variants by IEX, current strategies to determine the effects of modifications on specific charge variant peak involve isolating the peak of interest followed by various mass spectrometric analyses, such as intact mass analysis, peptide mapping, and glycan analysis. 10,25 In addition to being limited by time and resources, this two-step approach may overlook the minor species that do not exhibit distinctive UV peaks and introduce artifacts because of the lengthy sample preparation processes.…”

Section: Introductionmentioning

confidence: 99%

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Shi

Xiao

Dillon

et al. 2020

mAbs

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2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

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Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation

Cited by 30 publications

References 24 publications

New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization

New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization

Therapeutic Fc‐fusion proteins: Current analytical strategies

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

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