IgG4 (cB72.3) was prepared, isolated, and concentrated to 10 mg mL–1 as described previously.20 (link),26 (link),27 (link) MabSelect SuRe resin (Cytiva, UK) was analyzed
in this study. Used MabSelect SuRe samples, harvested from the inlet
and the outlet of an AXIChrom 981 mL column after 25 cycles of purification,
were donated by GSK Biopharm Process Research as described previously.27 (link) A MediaScout ResiQuot (ATOLL, Weingarten, Germany)
was used to pack and equilibrate 20.8 μL (Vresin) of resin into the wells of a 96-well Supor filter
plate with a pore size of 0.45 μm. Purified IgG4 was diluted
with phosphate buffer (pH 7.4, 50 mM PBS, 150 mM NaCl) to prepare
a range of concentrations (CO) of 1, 2,
3, 4, 5, 6, 7, and 9 mg mL–1. Aliquots (200 μL, Vsample) of IgG4 solution at each concentration
were individually added to packed resin samples in filter plates which
were then mixed for 45 min at 1000 rpm at ambient temperature. The
filter plate was centrifuged at 493 g for 2 min to
remove the excess unbound IgG4 (Ceq) into
individual wells of a 96-well plate. The amount of excess unbound
IgG4 which flowed through at each loaded concentration was determined
on a nanodrop lite at OD280 nm with E1% = 13.7. The concentration of unbound mAb in the flow through
was used to determine mAb binding capacity (Q) of
the resin using the mass transfer equation38 (link) given below (eq 1 ).
in this study. Used MabSelect SuRe samples, harvested from the inlet
and the outlet of an AXIChrom 981 mL column after 25 cycles of purification,
were donated by GSK Biopharm Process Research as described previously.27 (link) A MediaScout ResiQuot (ATOLL, Weingarten, Germany)
was used to pack and equilibrate 20.8 μL (Vresin) of resin into the wells of a 96-well Supor filter
plate with a pore size of 0.45 μm. Purified IgG4 was diluted
with phosphate buffer (pH 7.4, 50 mM PBS, 150 mM NaCl) to prepare
a range of concentrations (CO) of 1, 2,
3, 4, 5, 6, 7, and 9 mg mL–1. Aliquots (200 μL, Vsample) of IgG4 solution at each concentration
were individually added to packed resin samples in filter plates which
were then mixed for 45 min at 1000 rpm at ambient temperature. The
filter plate was centrifuged at 493 g for 2 min to
remove the excess unbound IgG4 (Ceq) into
individual wells of a 96-well plate. The amount of excess unbound
IgG4 which flowed through at each loaded concentration was determined
on a nanodrop lite at OD280 nm with E1% = 13.7. The concentration of unbound mAb in the flow through
was used to determine mAb binding capacity (Q) of
the resin using the mass transfer equation38 (link) given below (
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