Adsorption equilibrium and kinetics of monomer–dimer monoclonal antibody mixtures on a cation exchange resin

Reck, Jason M.; Pabst, Timothy M.; Hunter, Alan K.; Wang, Xiangyang; Carta, Giorgio

doi:10.1016/j.chroma.2015.05.007

Cited by 36 publications

(21 citation statements)

References 51 publications

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“…The identity of the purified species was confirmed by dynamic light scattering (DLS) with a Wyatt Dynapro Nanostar instrument from Waters Corporation (Milford, MA) instrument which gave hydrodynamic radii of 5.5 ± 0.1, 7.2 ± 0.1, and 16.3 ± 0.4 nm for monomer, dimer, and multimeric species, respectively. The monomer and dimer radii are consistent with those obtained experimentally by Reck et al for a different mAb. The large radius determined for the multimer suggests that this species is comprised of several mAb monomer units as reported by Ahere et al A rough estimate of the number of monomer units, n , present in these aggregates, can be obtained by assuming that they retain a globular shape.…”

Section: Methodssupporting

confidence: 91%

“…Longer times were not investigated experimentally. However, based on prior work by Reck et al on the kinetics of competitive binding of monomer‐dimer mAb mixtures in porous adsorbents, we expect that, given the large size of the pores in the POROS resin, the more weakly bound BSA would eventually diffuse out of the particles and ultimately be convected out of the column in a manner comparable to the behavior observed by Reck et al for mAb monomer‐dimer mixtures on a porous CEX resin. In any case, these results indicate that the presence of BSA, used here as representative of small HCP's, can interfere with binding of other species, including mAb multimers.…”

Section: Clsm Imagingmentioning

confidence: 72%

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Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Zhang

Iskra

Daniels

et al. 2017

Biotechnology Progress

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Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425-434, 2017.

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Section: Methodssupporting

confidence: 91%

Section: Clsm Imagingmentioning

confidence: 72%

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Zhang

Iskra

Daniels

et al. 2017

Biotechnology Progress

Self Cite

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“…In particular, it is evident that the overall predictive accuracy is limited by the model's own simplifying assumptions which are not always met in practice. For example, our previous work showed for two single component systems, lysozyme and a monoclonal antibody (mAb) on two different cation exchange resins, the SMA model could not quantitatively predict column elution profiles over a wide range of protein loads and salt gradients in part because of the inability of the SMA model to describe isotherms that exhibit an inflection point as a function of protein concentration . Furthermore, previous work has also shown the SMA model could not accurately describe the low selectivity between a monomer and dimer antibody on a cation exchange resin at low salt concentrations .…”

Section: Introductionmentioning

confidence: 99%

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads

Creasy

Reck

Pabst³

et al. 2018

Biotechnol. J.

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A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method shows good agreement with experimental elution curves for protein loads up to 40 mg mL column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components.

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“…For complex separations, in particular, it would be advantageous to optimize the chromatography operation by bead‐level monitoring while simultaneously analyzing the behavior of multiple target molecules. Despite the progress made in miniaturizing the different components of liquid chromatography and a significant amount of work on the investigation of intrabead analyte diffusion using confocal laser scanning microscopy, this type of multiplexed study and quantitative analysis associated with multiple target compounds has so far not been demonstrated in the context of rapid screening of operating conditions.…”

Section: Introductionmentioning

confidence: 99%

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules

Pinto

Soares

Aires-Barros

et al. 2019

Biotechnology Journal

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The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiolreactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.

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Adsorption equilibrium and kinetics of monomer–dimer monoclonal antibody mixtures on a cation exchange resin

Cited by 36 publications

References 51 publications

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules

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