Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Grzeskowiak, Julita K.; Tscheliessnig, Anne; Toh, Poh Choo; Chusainow, Janet; Lee, Yih Yean; Wong, Niki S.C.; Jungbauer, Alois

doi:10.1016/j.chroma.2009.04.014

Cited by 13 publications

(13 citation statements)

References 21 publications

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“…Within the last 10 years there have been efforts to replace Protein A chromatography with less-costly non-affinity methods, such as ion-exchange chromatography steps in series. One embodiment uses cation exchange (CEX), followed by anion exchange (AEX), and then hydrophobic interaction chromatography (HIC) [5,6] to significantly reduce purification costs. However, to achieve a high enough dynamic binding capacity for a single-cycle capture step using current resin beads, one needs to use low volumetric throughput (i.e., high residence time) [3], which limits productivity.…”

Section: Introductionmentioning

confidence: 99%

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

Chenette

Robinson

Hobley

et al. 2012

Journal of Membrane Science

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This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min.

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Section: Introductionmentioning

confidence: 99%

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

Chenette

Robinson

Hobley

et al. 2012

Journal of Membrane Science

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“…The adjustment can either enhance or reduce the binding of IgG 1 to the resin, depending if a separation in bind‐elute mode or flow‐through mode is desired. In a previous paper 17, we reported the modification of protein pattern and loss of IgG 1 during the conditioning step of the feedstock for CEX. The loss of product during the conditioning step indicated the requirement of optimization of this step.…”

Section: Resultsmentioning

confidence: 99%

“…For IgG 1 quantification, we used the CIM Protein A HLD disk monolithic column (BIA Separations, Klagenfurt, Austria). We applied the protocol evaluated by Tscheliessnig and Jungbauer 17 with minor modifications. HPLC analyses were carried out on an Agilent 1100 Series HPLC system (Waldbronn, Germany) at flow rate of 1 mL/min.…”

Section: Methodsmentioning

confidence: 99%

Two‐dimensional difference fluorescence gel electrophoresis to verify the scale‐up of a non‐affinity‐based downstream process for isolation of a therapeutic recombinant antibody

Grzeskowiak

Tscheliessnig

et al. 2010

Electrophoresis

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For therapeutic antibody production Protein A chromatography is often replaced by non-affinity-based purification sequences, which are considered as more economical. 2-D DIGE was applied for evaluation of scale-up of non-affinity based process of a humanized monoclonal antibody, anti-Rh(D) IgG(1), in comparison with other conventional analytical methods, like SDS-PAGE, Western blot, or SEC. Due to a high sensitivity of this technique (125 pg protein/spot) and high dynamic range of five orders of magnitude, low molecular weight impurities were detected in purified samples. Cation exchange chromatography was efficient capture step for IgG(1) purification in laboratory and pilot scale. The differences between samples after first purification step in laboratory and pilot scale were compensated with second purification step where almost the same protein pattern was observed. 2-D DIGE is a helpful tool for monitoring of purification effects and for scale-up verification of downstream processes.

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“…Life Sci. We characterized the optimized DNA precipitation method via common methods such as SEC as well as with two-dimensional difference gel electrophoresis (2D-DIGE), which was recently presented as suitable for monitoring downstream processes [20,21]. In the presence of zinc, antibodies may progressively form aggregates, the removal of which is a current challenge when producing biopharmaceuticals.…”

mentioning

confidence: 99%

“…In the present investigation, we redesigned and optimized DNA preparation with divalent cations to generate antibody solutions with high yield and low DNA content. We characterized the optimized DNA precipitation method via common methods such as SEC as well as with two-dimensional difference gel electrophoresis (2D-DIGE), which was recently presented as suitable for monitoring downstream processes [20,21].…”

mentioning

confidence: 99%

Separation of recombinant antibodies from DNA using divalent cations

Satzer

Tscheließnigg

Sommer

et al. 2014

Engineering in Life Sciences

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New downstream methods for the purification of antibodies are required to meet the demands of current and future antibody applications, e.g. for mass production as cancer therapeutic. The standard chromatographic methods suffer from high material costs and mass transfer limitations. In this study, we established and characterized a method for DNA precipitation for antibody purification using divalent cations, particularly CaCl2, using four different antibodies. By implementing high‐throughput screening using a factorial design plan, we determined that CaCl2 concentration and PO43− concentration were significant factors, while temperature and pH were not significant. We detected DNA precipitation as well as host‐cell protein (HCP) reduction. Two‐dimensional difference gel electrophoresis (2D‐DIGE) revealed that improved HCP removal does not occur via an unspecific random mechanism such as the enclosure of proteins in the precipitate. CaCl2 precipitation of DNA and HCP can be combined with nonchromatographic methods such as precipitation and protein A affinity chromatography. This additional purification method not only enhances DNA removal, but also the removal of HCP and antibody multimers, which will reduce immunogenicity and increase homogeneity of the resulting drug.

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Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Cited by 13 publications

References 21 publications

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

Two‐dimensional difference fluorescence gel electrophoresis to verify the scale‐up of a non‐affinity‐based downstream process for isolation of a therapeutic recombinant antibody

Separation of recombinant antibodies from DNA using divalent cations

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