Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments.
Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.
Zimmerman
Timo
T
MorphoSys AG, Lena-Christ-Str. 48, D-82152 Martinsried, Germany.
Petit Frère
Corinne
C
Satzger
Marion
M
Raba
Monika
M
Weisbach
Manuela
M
Döhn
Katrin
K
Popp
Andreas
A
Donzeau
Mariel
M
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001666:Binding Sites, Antibody; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D042783:Endothelial Cells / Q000502:physiology; D004731:Endotoxins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D008670:Metals; D008958:Models, Molecular; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry
D000911:Antibodies, Monoclonal; D003902:Detergents; D004731:Endotoxins; D007128:Immunoglobulin Fragments; D008670:Metals; D018799:Intercellular Adhesion Molecule-1; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40