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Application of aqueous two‐phase micellar system to improve extraction of adenoviral particles from cell lysate
Author(s) -
Molino João Vitor Dutra,
Lopes André Moreni,
Viana Marques Daniela de Araújo,
Mazzola Priscila Gava,
da Silva Joas Lucas,
Hirata Mario Hiroyuki,
Hirata Rosário Dominguez Crespo,
Gatti Maria Silvia Viccari,
Pessoa Adalberto
Publication year - 2017
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1627
Subject(s) - lysis , chromatography , aqueous solution , aqueous two phase system , adenoviridae , cell , chemistry , incubation , cell culture , hek 293 cells , microbiology and biotechnology , biology , biochemistry , genetic enhancement , gene , genetics
Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two‐phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK‐293) cells to a concentration of 1.4 × 10 10 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X‐114 in a 2 3 full factorial design with center points. The systems were formed with Triton X‐114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X‐114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 10 10 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3‐fold, purifying it by 2.2‐fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two‐phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.