Production of humanized antibody against human high-affinity IgE receptor in a serum-free culture of CHO cells, and purification of the Fab fragments.
We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.
Takai
T
T
Bioscience Research & Development Laboratory, Asahi Breweries, Ltd., Kitasoma-gun, Ibaraki, Japan.
Takahashi
K
K
Akagawa-Chihara
M
M
Fukada
M
M
Yuuki
T
T
Shibuya
I
I
Okumura
K
K
Ra
C
C
Yokota
T
T
Okumura
Y
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D016466:CHO Cells; D002469:Cell Separation; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000302:isolation & purification*; D010206:Papain / Q000737:chemistry; D017455:Receptors, IgE / Q000276:immunology*
D000906:Antibodies; D016895:Culture Media, Serum-Free; D007140:Immunoglobulin Fab Fragments; D017455:Receptors, IgE; D007073:Immunoglobulin E; D010206:Papain