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Projeto de investigação
DOCTOR OF PHILOSOPHY IN BIO-ENGINEERING SYSTEMS
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Magnetic purification of antibodies
Publication . Dhadge, Vijaykumar Laxman; Roque, Ana; Barros, Raquel
This work aimed at the development of magnetic nanoparticles for antibody purification and at the evaluation of their performance in Magnetic fishing and in a newly developed hybrid technology Magnetic Aqueous Two Phase Systems. Magnetic materials were produced by coprecipitation and solvothermal approaches. Natural polymers such as dextran, extracellular polysaccharide and gum Arabic were employed for coating of iron oxide magnetic supports. Polymer coated magnetic supports were then modified with synthetic antibody specific ligands,namely boronic acid, a triazine ligand (named 22/8) and an Ugi ligand (named A2C7I1). To optimize the efficacy of magnetic nanoparticles for antibody magnetic fishing, various solutions of pure and crude antibody solutions along with BSA as a non-specific binding protein were tested. The selectivity of magnetic nanoparticle for antibody, IgG, was found effective with boronic acid and ligand 22/8. Magnetic supports were then studied for their performance in high gradient magnetic separator for effective separation capability as well as higher volume handling capability.
The magnetic materials were also supplemented to aqueous two phase systems, devising a new purification technology. For this purpose, magnetic particles modified with boronic acid were more effective. This alternative strategy reduced the time of operation,maximized separation capability (yield and purity), while reducing the amount of salt required.
Boronic acid coated magnetic particles bound 170 ± 10 mg hIgG/g MP and eluted 160 ± 5 mg hIgG/g MP, while binding only 15 ± 5 mg BSA/g MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 105 M-1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed/g MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions.
Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification
Publication . Santana, Sara D. F.; Dhadge, Vijaykumar L; Roque, Ana Cecília Afonso; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); DQ - Departamento de Química; ACS - American Chemical Society
Dextran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 10⁴ M⁻¹. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
OE
Número da atribuição
SFRH/BD/72650/2010