Protein A chromatography for antibody purification

Hober, Sophia; Nord, Karin; Linhult, Martin

doi:10.1016/j.jchromb.2006.09.030

Cited by 469 publications

(348 citation statements)

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“…The selective purification steps usually comprise the mAbs adsorption to a protein A resin, followed by two further chromatography steps which remove host cell proteins (HCP), DNA, aggregates, any leached protein A and provide an adequate level of overall viral removal [3,4]. Although chromatographic separations have been the workhorse of most purification processes, several limitations have been pointed out, such as (i) batch operation, (ii) low capacity, (iii) complex scale-up, (iv) time-consuming and high pressure packing processing, (v) slow intraparticle diffusion, (vi) low chemical and proteolytic stability and consequent contamination of the final product, and (vii) the high cost of the resins [5][6][7][8][9]. The replacement of some of these chromatographic steps by non-chromatographic alternatives, with high capacity and throughput, has been then suggested [7,[10][11][12].…”

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confidence: 99%

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Rosa

Azevedo

Sommerfeld

et al. 2013

Biotechnology Journal

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An aqueous two‐phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back‐extraction, and washing) was set up and validated in a pump mixer‐settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155‐fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22‐fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.

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confidence: 99%

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Rosa

Azevedo

Sommerfeld

et al. 2013

Biotechnology Journal

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“…Staphylococcal Protein A (SPA) is a cell wall-associated protein domain exposed on the surface of the gram-positive bacterium Staphylococcus aureus (Hober et al 2007). SPA has significance to biotechnology and bioscience because of its specific binding to the Fc portion of immunoglobulin G (IgG) from many mammals (Jungbauer and Hahn 2004).…”

Section: Introductionmentioning

confidence: 99%

Recombinant Protein A Immobilized on Cross-linked Cellulose Microspheres for Immunoglobulin G Adsorption from Human Plasma

Cao¹,

Zhu²,

Chen³

et al. 2016

BioResources

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Cross-linked cellulose microspheres (CL-CMs) were successfully prepared using an inverse crosslinking suspension method from a cellulose solution with sodium hydroxide/urea aqueous solution as a solvent and epichlorohydrin as the crosslinker. The effects of epichlorohydrin content on the appearance and dispersity, average pore volume, moisture content, and wet real density of CL-CMs were studied. The microspheres presented a good spherical shape and porous surface structure. After activation with NaIO4, the recombinant protein A was immobilized onto the surface of CL-CMs to form an immunoadsorbent. Adsorbents containing various amounts of protein A were applied to adsorb immunoglobulin G (IgG) from human plasma. The maximum IgG adsorption capacities with static adsorption and dynamic adsorption were 23 and 13 mg, respectively, per gram of CL-CMs carrying 6.8 mg of recombinant protein A. Therefore, CL-CMs immobilized with recombinant protein A have great potential for application in the field of blood purification.

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“…[23][24][25] Solid supports for affinity purification media must be carefully designed to achieve an efficient purification process. 26 Porous materials with large surface areas and pore diameters larger than that of the target protein (430 nm for IgG), enable high binding capacity and fast adsorption kinetics. 27 The supports must be hydrophilic (ideally a hydrophilic gel such as agarose) to reduce non-specific adsorption of proteins on the surface.…”

Section: Introductionmentioning

confidence: 99%

Preparation of nanogel-immobilized porous gel beads for affinity separation of proteins: fusion of nano and micro gel materials

Hoshino

Arata

Yonamine

et al. 2014

Polym J

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We describe the preparation and evaluation of nanogel-immobilized porous gel beads (GB) for application as a protein purification medium. Nanogel particles (NP) that bind with the Fc fragment of immunoglobulin G (IgG) were immobilized on the pore surface of macroporous hard GB containing quaternary ammonium cations on the surface via multipoint electrostatic interactions. The amount of NPs that were irreversibly immobilized in 1 ml of GB slurry was determined to be~30 mg using fluorescent-labeled NPs. Images obtained via scanning electron microscopy established that the NPs were uniformly immobilized on the surface of the pores without blocking the macropores. The model target protein (IgG) was reversibly captured by the NPimmobilized GBs through NP-IgG interactions. NP-immobilized GBs have potential applications as novel affinity purification media for proteins, combining inexpensive and stable ligands with high-performance supports.

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Protein A chromatography for antibody purification

Cited by 469 publications

References 50 publications

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Recombinant Protein A Immobilized on Cross-linked Cellulose Microspheres for Immunoglobulin G Adsorption from Human Plasma

Preparation of nanogel-immobilized porous gel beads for affinity separation of proteins: fusion of nano and micro gel materials

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