Fc-region variants with modified fcrn- and protein a-binding properties
a region variant and protein a-binding technology, applied in the field of igg fcregions, can solve the problems of reduced clearance, difficult production of bispecific antibodies in one expression cell line, poor yield of 12.5%, etc., and achieve the effect of avoiding systemic side effects
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example 1
Expression and Purification
[0599]Transient Transfections in HEK293-F System
[0600]The monospecific and bispecific antibodies are generated by transient transfection with the respective vectors (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) are transfected with a mix of the respective expression vectors and 293fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells are seeded at a density of 1*106 cells / mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after the cells are transfected at a cell density of approx. 1.5*106 cells / mL with approx. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total vector DNA (1 μg / mL) encoding the heavy or mo...
example 2
[0606]Coupling to Streptavidin Sepharose:
[0607]One gram streptavidin Sepharose (GE Healthcare) is added to the biotinylated and dialyzed receptor and incubated for two hours with shaking. The receptor derivatized Sepharose is filled in a 1 mL XK column (GE Healthcare).
[0608]Chromatography Using the FcRn Affinity Column:
[0609]Conditions:
[0610]column dimensions: 50 mm×5 mm
[0611]bed height: 5 cm
[0612]loading: 50 μg sample
[0613]equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5
[0614]elution buffer: 20 mM Tris / HCl, with 150 mM NaCl, adjusted to pH 8.8
[0615]elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer, 10 CV elution buffer
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