Serum preparation and methods for the large-scale production of IgG monoclonal antibody.
In this study we demonstrated that fetal calf serum (FCS) depleted of IgG by protein G affinity chromatography (G-FCS) is superior to whole FCS or serum-free culture media as a culture supplement for the production of purified IgG monoclonal antibodies (MAb). One hundred ml FCS was applied to a 25 ml protein G Sepharose 4 Fast Flow Column, which was shaken gently for 2 days at 4 degrees C. The procedure was repeated using a protein G column for an additional day. G-FCS was used at a concentration of 5% in RPMI 1640 medium to grow the mouse myeloma cell line P3X63.Ag8.653, which secretes an IgG-1 mouse-human chimeric monoclonal antibody (TVE-1). Cell density, viability, doubling time, and antibody production were used as indices to compare the efficacy of this medium with that of whole FCS medium, AIM-V (Gibco, USA) and other serum-free media. The results demonstrate that cell growth and antibody production in -GFCS medium did not differ significantly from that in FCS medium, but were significantly better than in the serum-free media (p < 0.001). TVE-1 antibody in the spent tissue culture media was purified by 50% ammonium sulfate precipitation and Protein A affinity chromatography. An antibody that was more than 99% pure was obtained. Endotoxin analysis revealed that the IgG depletion process does not generate a significant level of endotoxin in FCS (< 0.06 EU/ml).(ABSTRACT TRUNCATED AT 250 WORDS)
Zeng
Q S
QS
John Wayne Cancer Institute, Santa Monica, CA 90404.
Takeyama
H
H
Kanda
S
S
Irie
R F
RF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001709:Biotechnology; D002417:Cattle; D002455:Cell Division; D002846:Chromatography, Affinity; D003470:Culture Media / Q000737:chemistry; D005069:Evaluation Studies as Topic; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007074:Immunoglobulin G