TY - JOUR
T1 - Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography
AU - Franco-Medrano, Diana Ivonne
AU - Guerrero-Germán, Patricia
AU - Montesinos-Cisneros, Rosa María
AU - Ortega-López, Jaime
AU - Tejeda-Mansir, Armando
N1 - Publisher Copyright:
© 2016, Springer-Verlag Berlin Heidelberg.
PY - 2017/3
Y1 - 2017/3
N2 - The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.
AB - The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.
KW - Leishmaniasis
KW - Perfusion chromatography
KW - Plasmid DNA
KW - Vaccines
UR - http://www.scopus.com/inward/record.url?scp=85000968637&partnerID=8YFLogxK
U2 - 10.1007/s00449-016-1714-6
DO - 10.1007/s00449-016-1714-6
M3 - Artículo
SN - 1615-7591
VL - 40
SP - 463
EP - 471
JO - Bioprocess and Biosystems Engineering
JF - Bioprocess and Biosystems Engineering
IS - 3
ER -