Affinity Chromatography Step
The initial step in the purification train is a standard Protein A affinity chromatography separation carried out by loading
the filtered culture medium from the bioreactor onto a column pre-equilibrated with phosphate-buffered saline (PBS). The medium
is loaded overnight at room temperature under conditions that allow a contact time of approximately 30 s. The column is then
washed through with an acetate saline buffer at pH 5.0, and the antibody is eluted with an acetate saline buffer at pH 3.0.
The eluted product is adjusted to pH 6.0 and diluted to a final conductivity of 8.0 mSi/cm before loading onto the Q/S dual
adsorber. As shown by the elution profile in Figure 1, it is possible to recover up to 90% of the product in this step at
a >90% purity.
Figure 1. Teleukin elution profile after the affinity chromatography step, based on absorbance at 280 nm
The Polishing Step: A Q/S Dual Adsorber System
The polishing step is carried out using two Sartobind ion exchange adsorbers, a 7-mL Sartobind Q for the AEX step, and a 70-mL
Sartobind S for the CEX step. These are arranged in series on an Amersham Akta Pilot (GE Healthcare, Uppsala, Sweden) as shown
in Figure 2. However, for washing and elution, the two adsorbers can be operated independently, as shown in Figure 3. The
two operational modes are selected by the operator using the Unicorn 4.12 management software (GE Healthcare).
Figure 2. The Q/S dual adsorber system operating in series during the polishing step
Before loading, the system is set to the "in series" mode and the adsorbers are washed through with 1 M NaCl and then sanitized
in place with 1 M NaOH. After this treatment, the system is washed with 50 mM NaCl in pH 6.0 acetate/EDTA buffer (F2 buffer
A). After washing and sanitizing, the eluate from the affinity purification can be loaded onto the Sartobind Q adsorber at
a rate of up to 150 mL/min, a process that can take up to 40 min for a maximum 6 L of feed (or up to 500 mg of antibody),
although greater amounts can be processed by splitting into multiple batches. In the "in series" mode, the flow-through containing
the antibody elutes from the Sartobind Q step directly into the Sartobind S adsorber, wherein the target antibody binds to
the sorbent. The system is then washed with 10 volumes of loading buffer before the operator switches it to the independent
mode, allowing the Sartobind S adsorber to be washed with another 15 volumes of buffer at a flow rate of 350 mL/min before
Figure 3. The Q/S dual adsorption system operating in independent mode to facilitate elution from the Sartobind S adsorber.
It is also possible to operate the Sartobind Q adsorber independently (not shown).
Salt gradient elution is carried out in independent mode at 350 mL/min using a continuous gradient from F2 buffer A (50 mM
NaCl, conductivity 8.0–8.5 mS/cm) to F2 buffer B (like buffer A but 1 mM NaCl, conductivity 86.0–87.0 mS/cm), which goes from
0% to 100% F2 buffer B in 20 volumes. This step takes from 40 to 60 min and has a yield of up to 95%. A typical elution profile
is shown in Figure 4.
Figure 4. Teleukin elution profile after the affinity chromatography step, based on absorbance at 280 nm