Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 2. Nonionic micellar systems

Autor(es)

Rodrigues Teixeira-Pinto, Renata Garcia ^[1] ; Molino, Joao Vitor Dutra ^[2] ; Santos-Ebinuma, Valeria Carvalho ^[3] ; Pessoa, Jr., Adalberto ^[1] ; Valentini, Sandro Roberto ^[1] ; Brandao Pereira, Jorge Fernando ^[3] ; Lopes, Andre Moreni ^[3]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo FCF USP, Sch Pharmaceut Sci, Dept Biochem & Pharmaceut Technol, Sao Paulo - Brazil

^[2] Ronin Inst, Montclair, NJ - USA

^[3] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, Araraquara - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Separation and Purification Technology; v. 210, p. 69-79, FEB 8 2019.
• Citações Web of Science: 0

Resumo

Lipopolysaccharide endotoxins (LPS) are the most common contaminant pyrogenic compounds found in intracellular recombinant biomolecules purified from Gram-negative bacteria, such as Escherichia coli. Thus, the purification downstream processing should guarantee the effective removal of LPS from the final bioproduct, particularly, therapeutic biopharmaceuticals. Aqueous two-phase micellar systems (ATPMS) appear to be an excellent strategy to purify recombinant biopharmaceuticals from the cell lysate of E. coli, reducing high LPS concentrations. In order to demonstrate the effectiveness of ATPMS as a biopharmaceutical purification platform, the influence of inorganic salt electrolytes (NaCl, Li2SO4, KI, or KNO3) on the partitioning of green fluorescent protein (GFP) and LPS removal using ATPMS composed of n-decyl tetraethylene oxide (C10E4) was evaluated. The impact of different LPS concentrations on GFP partitioning was also studied. The addition of electrolytes (Le., NaCl or Li2SO4) to the C10E4-based ATPMS have reduced the phase forming temperatures to very mild conditions (ca. 17.00 and 13.00 degrees C, for NaCl and Li2SO4, respectively). The selective partitioning ability of the proposed ATPMS was further demonstrated, where a complete removal of the LPS from the micelle poor phase (REMLPS > 98%) and a preferential GFP recovery (RECGFP = 97%, K-GFP > 7) to the micelle-poor phase was obtained. The GFP partitioning was even enhanced by increasing LPS loading (10(4)-10(6) EU/mL), probably due to the formation of mixed micelles between LPS and C10E4. It is here demonstrated that a C10E4/buffer + salt-based ATPMS can be a useful and straightforward platform for the removal of endotoxin contaminants and the purification of recombinant biopharmaceuticals from E. coli cell lysates. (AU)

• Processo FAPESP: 07/51978-0 - Estudo da adicao de alguns sais inorganicos no sistema micelar de duas fases aquosas aplicado a remocao de lipopolissacarideo(lps).
• Beneficiário: Renata Garcia Rodrigues da Silva
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 14/19793-3 - Otimização e aumento de escala de processos com líquidos iônicos como nova ferramenta de purificação da proteína verde fluorescente recombinante produzida por Escherichia coli "GFPurIL"
• Beneficiário: Sandro Roberto Valentini
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 05/60159-7 - Remoção de endotoxina presente em meio fermentado contendo biomoléculas utilizando sistemas micelares de duas fases aquosas
• Beneficiário: André Moreni Lopes
• Modalidade de apoio: Bolsas no Brasil - Doutorado