Journal ArticleDOI
Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin.
Hee Ho Park,Hyungsu Jeon,Hyung Jin Cha,Ji S. Bang,Mihyun Choi,Daekyung Sung,Won Il Choi,Jin Hyung Lee,Jae Sung Woo,Sangyong Jon,Sunghyun Kim +10 more
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TLDR
The ZZ-CSQ platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.Abstract:
Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.read more
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Current research approaches in downstream processing of pharmaceutically relevant proteins.
TL;DR: In this paper , a short review gathers essential research over the past three years that addresses novel solutions to overcome this bottleneck, including promising studies in the fields of chromatography, aqueous two-phase systems, precipitation, crystallization, magnetic separation and filtration for the purification of pharmaceutically relevant proteins.
Journal ArticleDOI
Magnetic composite membrane roll column for rapid and high efficiency separation of antibodies
TL;DR: In this article , a magnetic composite membrane roll column based on the "membrane sandwiched magnetism" strategy was proposed in order to overcome the drawbacks of conventional Protein A based methods, which consisted of high-density vinylimidazole (VIM) polymer brush modified hydrophilic membrane and Fe3O4 magnetic nanoparticles (MNPs, 5 mg).
Journal ArticleDOI
Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle
TL;DR: In this paper , the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) was optimized via precipitation with calcium chloride (CaCl 2 ) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na 2 ).
References
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Journal ArticleDOI
Development of therapeutic antibodies for the treatment of diseases
TL;DR: The preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation are outlined.
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Purification of recombinant proteins by fusion with thermally-responsive polypeptides
Dan E. Meyer,Ashutosh Chilkoti +1 more
TL;DR: The ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.
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Protein A chromatography for antibody purification
TL;DR: A minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications.
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The Increasingly Human and Profitable Monoclonal Antibody Market.
TL;DR: The monoclonal antibody (mAb) market has changed rapidly in the past 5 years: it has doubled in size, becoming dominated by fully human molecules, launched bispecific molecules, and faced competition from biosimilars.
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Calsequestrin and the calcium release channel of skeletal and cardiac muscle
TL;DR: Calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions, as indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca 2+ entry.