Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG1)

Gillespie, Ron; Nguyen, Thao P.; Macneil, Sean; Jones, Laurie; Crampton, Shon; Vunnum, Suresh

doi:10.1016/j.chroma.2012.06.037

Cited by 65 publications

(25 citation statements)

References 41 publications

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“…The chromatographic behavior observed in our work for our glycosylated IgG2 is in many ways consistent with the observations of Gillespie et al [17] for an aglygosylated IgG1.…”

Section: Discussionsupporting

confidence: 80%

“…[17] showed that a two-peak elution behavior could also be observed with CEX resins, including Fractogel EMD SO3-, when an aglycosylated IgG1 antibody was loaded on the column and then eluted with a salt gradient. However, unlike the results of Voitl et al [16], in this case while the early eluting peak was comprised of monomer only, the late eluting peak was a mixture of monomer and aggregated species.…”

Section: Motivations and Backgroundmentioning

confidence: 99%

“…The first is to explore the elution behavior of a pure glycosylated antibody on the CEX resin Fractogel EMD SO3-in a direct follow-up to the study of Gillespie et al [17]. Glycosylation is expected to improve stability of the mAb 5.1 with acetic acid) and then loaded with 0.5 mL of 2 mg/mL mAb.…”

Section: Introductionmentioning

confidence: 99%

“…Gillespie et al [17] used whole-protein on-column HX-MS to differentiate between native and unfolded forms of their aglycosylated IgG1 antibody eluting from a Fractogel EMD SO3-CEX column. Their HX-MS results confirmed that species present in the late eluting peak, which comprised a mixture of monomeric and aggregated mAb, experienced more solvent exposure while bound to the column than the species the early eluting peak, suggesting that conformational changes were induced by interactions with the CEX resin.…”

Section: Introductionmentioning

confidence: 99%

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Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

Guo

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This work elucidates the molecular interactions that are responsible for protein unfolding and aggregation on the surface of certain cation exchanger resins. The chromatographic behavior of a monoclonal antibody (mAb) that exhibits a two-peak elution behavior is studied for a range of strong cation exchange resins and with varying load buffer pH and composition. The two-peak elution behavior is very pronounced for the tentacle and polymer-grafted resins, but is essentially absent for hydrophilic macroporous resins. Confocal Laser Scanning Microscopy (CLSM) shows that this behavior is related to the unique kinetics of protein binding in the tentacle-type resin Fractogel. Hydrogen-Deuterium Exchange Mass Spectrometry (HXMS) proves that the two-peak elution behavior is tied to conformational changes that occur when the mAb binds. Circular dichroism suggests that the propensity of different mAbs to form stabilizing intermolecular structures can be related to their chromatographic behaviors. Another mAb exhibiting a threepeak elution behavior on the CEX resin POROS XS was also investigated. Dynamic Light Scattering (DLS) shows that the third peak contains significant levels of aggregates formed in the column that only slowly revert to monomeric species after elution. Circular dichroism and HXMS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HXMS analysis indicate that the aggregates are generated by a destabilized, unfolded intermediate that is slowly formed on the resin. The two early eluting peaks observed regardless of hold time are shown to comprise exclusively monomeric species and form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics. This work has important practical implications in downstream processing for the industrial production of biopharmaceuticals as well as broad scientific value from a biomolecular perspective.ii Acknowledgements I would like to give my deepest gratitude to my academic advisor, Professor Giorgio Carta, for his guidance and support during my PhD study. I will never forget the many hours we spent together discussing my research results, drawing sketches of protein aggregation mechanisms, and fixing all sorts of experimental equipment.

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“…The chromatographic behavior observed in our work for our glycosylated IgG2 is in many ways consistent with the observations of Gillespie et al [17] for an aglygosylated IgG1.…”

Section: Discussionsupporting

confidence: 80%

Section: Motivations and Backgroundmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

Guo

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“…This may be due to the fact that dimerization is very slow at pH 5, and the equilibrium is shifted towards monomers at high salt concentrations [9], which are conditions in which the antibodies are eluted. The interaction of antibodies with the stationary phase promotes the formation of aggregates [23][24][25], but the mechanism is unknown. Figure 4 presents the simulated chromatogram of the CIEX step.…”

Section: Resultsmentioning

confidence: 99%

Model-Based Comparison of Antibody Dimerization in Continuous and Batch-Wise Downstream Processing

2015

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Monoclonal antibodies are generally produced using a generic platform approach in which several chromatographic separations assure high purity of the product. Dimerization can occur during the fermentation stage and may occur also during the downstream processing. We present here simulations in which a traditional platform approach that consist of protein A capture, followed by cation-exchange and anion-exchange chromatography for polishing is compared to a continuous platform in which dimer removal and virus inactivation are carried out on a size-exclusion column. A dimerization model that takes pH, salt concentration and the concentration of antibodies into account is combined with chromatographic models, to be able to predicted both the separation and the degree to which dimers are formed. Purification of a feed composition that contained 1% by weight of dimer and a total antibody concentration of 1 g/L was modeled using both approaches, and the amount of antibodies in the continuous platform was at least 4 times lower than in the traditional platform. The total processing time was also lower, as the cation-exchange polish could be omitted.

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Adsorption Equilibrium

2020

Protein Chromatography

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Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG1)

Cited by 65 publications

References 41 publications

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

Model-Based Comparison of Antibody Dimerization in Continuous and Batch-Wise Downstream Processing

Adsorption Equilibrium

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