Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants.
Monoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue. Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.
Hassan
Sally
S
Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
van Dolleweerd
Craig J
CJ
Ioakeimidis
Fotis
F
Keshavarz-Moore
Eli
E
Ma
Julian K-C
JK
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D013696:Temperature; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics*
D000911:Antibodies, Monoclonal; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins