59
AFC
Protein a Affinity
chromatography
TOYOPEARL AF-rProtein A-650F
The standard TOYOPEARL AF-rProtein A-650F resin binds
human and mouse immunoglobulin G, IgM, and Fab frag-
ments. Typical static IgG binding capacity is > 45 mg/ml
resin and typical dynamic IgG binding capacity at 10 %
breakthrough is > 30 mg/mL resin at 2 minutes residence
time (1 mg/mL protein load). Fast mass transfer kinetics
support high binding capacities at high flow rates. IgG
breakthrough curves (Figure 7) at various linear veloci-
ties demonstrate the superior kinetic performance of
TOYOPEARL AF-rProtein A-650F.
The structure of the recombinant ligand and its multipoint
attachment to the base matrix enhances the stability of
TOYOPEARL AF-rProtein A-650F in 0.1 - 0.5 M NaOH. The
dynamic binding capacity remains high after repeated CIP
cycles. After more than 150 CIP cycles with 0.1 MNaOH at 16
min contact time per cycle more than 90 % of initial dynamic
binding capacity was retained (Figure 8). When performing
cleaning-in-place with 0.5 M NaOH the resin maintains
about 80 % of IgG binding capacity after 50 cycles.
TOYOPEARL AF-rProtein A-650F is also stable in ethanol,
6 M urea, 6 M guanidinium chloride, and 1 % phosphoric
acid, respectively. Static binding capacity of the resin is
not impaired when heated for 30 minutes to temperatures
of up to 90 °C. Figure 9 shows the thermal stability of the
resin. It can be stored at room temperature at production
site. Recommended conditions for long term storage are a
storage solution of 20 % ethanol and temperature of 4 - 8 °C.
figure 7
Breakthrough curves for h-IgG loading (polyclonal, 10 g/L)
Typical DBC at 10 % breakthrough: 30,5 g/L @ 100 cm/h (3 min residence
time) - 24 g/L @ 200 cm/h (1.5 min residence time); Column: 5 mm ID x 5 cm L;
Mobile phase: 20 mmol/L sodium phosphate buffer pH 7.2 containing
150 mmol/L NaCl; Sample conc.: 10 g/L; Residence time: 1.5, 2.0, 2.5, 3.0 min
Dynamic Binding Capacity
figure 8
Column: 5 mm ID x 5 cm L
10 column volumes binding buffer pH 7.4
5 column volumes elution buffer pH 3.0
3 column volumes binding buffer containing 0.1 mol/L NaOH,
16 min contact time
3 column volumes binding buffer pH 7.4
Cleaning-in-place Study with 0.1 M NaOH
figure 9
SBC (g/L-gel)
0
10
20
30
40
50
60
Temperature (
°C)
Ref.
40
50
60
70
80
90 100 Autoclave
Te perature stability
Resin: TOYOPEARL AF-rProtein A-650F; Mobile phase: deionized H
2
O; Auto-
clave settings: 120 °C, 1.2 bar, 15 min; Heating time: 30 min;
TOYOPEARL AF-rProtein is stable at 35 °C for least 3 years (data not shown)
TEMPERATURE STABILITY
