Purification of recombinant human B-domain-deleted factor VIII using anti-factor VIII monoclonal antibody selected by the surface plasmon resonance biosensor.
The surface plasmon resonance (SPR) biosensor measures the real-time kinetics of noncovalent interaction between a receptor and its ligand. Monoclonal antibodies (mAbs) against recombinant factor VIII (rFVIII) were screened from 127 mAb candidates using the SPR biosensor for the purpose of affinity purification of rFVIII. Each mAb showed a different association and dissociation capacity for rFVIII at each buffer condition. One mAb, F8-38, was selected for immunopurification of rFVIII. To characterize the selected mAb F8-38, the immunopurification results on the anti-FVIII mAb F8-38 affinity gel and the anti-von Willebrand factor (vWF) mAb affinity gel were studied. Immunopurification by the anti-vWF affinity gel showed a lower binding capacity of rFVIII and resulted in low purification efficiency. On the other hand, immunopurification by the anti-FVIII affinity gel exhibited a 3.5-fold binding capacity and a 2-fold purification efficiency compared to those of the anti-vWF affinity gel. The amounts of proteins and DNAs derived from host cells and mouse IgGs derived from the affinity matrix in the affinity eluate were similar to those of the anti-vWF affinity gel. In conclusion, the SPR method of immunopurification is a useful technology in the screening of mAbs aimed at the development of an affinity purification procedure, and the mAb F8-38 was selected using this technology on the basis of the purification procedure of rFVIII.
Oh
H K
HK
Central Research Center, Korea Green Cross Corporation, 227-3 Kugal-Ri, Kiheung-Eup, Yongin City, Kyunggi-Do 449-900, Korea.
Lee
J M
JM
Byun
T H
TH
Park
S Y
SY
Kim
Y H
YH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006825:Hybridomas; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry
D000911:Antibodies, Monoclonal; C451034:B-domain-deleted factor VIII; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin