Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography

Müller, Egbert; Vajda, Judith

doi:10.1016/j.jchromb.2016.01.036

Cited by 49 publications

(30 citation statements)

References 31 publications

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“…The isotherms show highly favorable adsorption, as expected for Protein A affinity chromatography . Both Langmuir fits present a rectangular shape that levels between 0.2 mg mL −1 and 0.4 mg mL −1 equilibrium concentration, depending on the resin.…”

Section: Resultsmentioning

confidence: 55%

“…The isotherms show highly favorable adsorption, as expected for Protein A affinity chromatography. [21,23] Both Langmuir fits present a rectangular shape that levels between 0.2 mg mL −1 and 0.4 mg mL −1 equilibrium concentration, depending on the resin. The isotherms are highly favorable, with TP PA presenting a higher equilibrium binding capacity (q m = 96 mg mL −1 packed bed) and higher affinity (with and K D = 0.0042 mg mL −1 ) than MSS (q m = 79 mg mL −1 packed bed and K D = 0.0079 mg mL −1 ).…”

Section: Langmuir Adsorption Isothermsmentioning

confidence: 99%

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Antibody Binding Heterogeneity of Protein A Resins

Silva

Plewka

Tscheließnig

et al. 2019

Biotechnology Journal

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Protein A affinity chromatography is a core unit operation in antibody manufacturing. Nevertheless, there is not enough understanding of in‐column antibody adsorption in the Protein A capture step. This work aims to investigate in situ the establishment of an antibody (trastuzumab) layer during Protein A chromatography both in terms of energetic contributions and uptake kinetics. Flow microcalorimetry is employed as a technique with an in situ operating detector, which provides an understanding of the thermodynamics of the adsorption process. In addition, the antibody uptake rate is also investigated in order to establish a correlation between its diffusion on the stationary phase and the associated thermodynamics. Two resins with different particle size, intraparticle porosity, and a Protein A ligand structure are studied: the synthetically engineered B‐domain tetrameric MabSelect SuRe and the synthetically engineered C‐domain hexameric TOYOPEARL AF‐rProtein A HC. The uptake rate follows a pore diffusion model at low equilibrium time, showing a slower diffusivity after a certain time because of the heterogeneous binding nature of these two resins. In addition, the microcalorimetric studies show that adsorption enthalpy is highly favourable at low isotherm concentrations and evolves toward an equilibrium with increasing surface concentration. These data suggest that the relationship between adsorption enthalpy and the establishment of the antibody layer in the Protein A chain is consistent with heterogeneous adsorption.

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Section: Resultsmentioning

confidence: 55%

Section: Langmuir Adsorption Isothermsmentioning

confidence: 99%

Antibody Binding Heterogeneity of Protein A Resins

Silva

Plewka

Tscheließnig

et al. 2019

Biotechnology Journal

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“…These differences have shown impacts on mAb binding capacities or elution behavior (Ghose et al, 2005;Müller & Vajda, 2016). In the mAbfree mock load runs, using EQ-intermediate-wash process, similar LRVs were obtained for each virus on Eshmuno A® or MSS, all of which were over 2.4 logs (Table 2).…”

Section: Resin Typementioning

confidence: 81%

“…Virus clearance differences appear between resins in the presence of mAbs (Table 2) suggesting that the different protein A ligands alter the virus-mAb interaction. Conformational changes are known to occur on both the mAbs and protein A on binding (Bolton, Selvitelli, Iliescu, & Cecchini, 2015;Müller & Vajda, 2016;Tashiro & Montelione, 1995). It is likely that different mAbs with the same FC region that dominates binding would have a similar minor effect on any protein A conformation changes so that the differences seen between different mAb products would not occur if they led to protein A-virus binding.…”

Section: Different Protein a Resins Show Low Levels Of Comparable Virusmentioning

confidence: 99%

Characterizing and enhancing virus removal by protein A chromatography

Pan¹,

Becerra-Arteaga

Tran³

et al. 2019

Biotech & Bioengineering

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Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal. K E Y W O R D S Chinese Hamster Ovary cells, Eshmuno ® A, high throughput screening (HTS), protein A chromatography, virus removal Biotechnology and Bioengineering. 2019;116:846-856. wileyonlinelibrary.com/journal/bit 846 | Abbreviations: CHO, Chinese Hamster Ovary; RVLP, retrovirus-like particles; HTS, high throughput screening; XMuLV, xenotropic murine leukemia virus; MMV, murine minute virus;RT-QPCR, real-time quantitative PCR; LRV, log reduction value. *Pan and Becerra-Arteaga have contributed equally to this study.

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“…Many of these commercially available resins are made up from several repeated binding units, which in many cases have been shown to significantly increase the DBC of the ligand. 20 Here, we examined whether multimerization of the Z Ca domain can lead to a higher binding capacity of the Z Ca resin in an effort to optimize the domain for use as an affinity ligand in current biomanufacturing processes. The variants that were tested included a monomer, dimer, trimer and tetramer.…”

Section: Introductionmentioning

confidence: 99%

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

Scheffel

Kanje

Borin

et al. 2019

mAbs

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As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, ZCa, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of ZCa to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, ZCaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with ZCaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

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Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography

Cited by 49 publications

References 31 publications

Antibody Binding Heterogeneity of Protein A Resins

Antibody Binding Heterogeneity of Protein A Resins

Characterizing and enhancing virus removal by protein A chromatography

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

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