Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 198)

For additional cleaning-in-place options for protein A cartridges, refer to the Utilities section
of the manual for information on the affinity cartridge cleaning-in-place method and the
information provided from the manufacturer of the protein A cartridge used.
2.3 Protein A Method Parameters and Instruction for Use
The default protein A method in the Profinia protein A purification system requires the use
of buffers as concentrates which are then proportioned to a 1x formulation by the instrument
using the preprogrammed methods. The formulations provided in Table 2 list the most
commonly recommended buffers for protein A purifications, as well as cleaning and storage
solutions designed for optimal regeneration and cartridge re-use.
Optimal binding and elution conditions for IgG are both sample type and antibody dependent.
In general, binding/equilibration/wash buffers for protein A purifications should be between
pH 7–9. The recommended buffer for binding/equilibration/washing is the Profinia desalting
buffer (pH 7.4). Two commonly recommended elution buffers for Protein A separations are
solutions of sodium citrate (pH 2.7–4) or glycine (pH 2.5–3). For methods that include
integrated desalting, elution with a sodium citrate buffer at pH 3 is recommended (see
Section 5 of the appendix). In addition, the width of the elution peak is influenced by the
concentration of the elution buffer; higher buffer concentrations produce narrower elution
peaks. In cases where milder elution conditions are required, use of protein A affinity only
methods is recommended to ensure that the entire IgG elution peak is collected.
When preparing for a protein A purification, bottles should be clearly labeled with the buffer
name and the buffer position number (1–8) for insertion in the correct location on the
instrument. The position number on the bottle must match the position number on the
instrument to properly run the preprogrammed methods. Follow the on-screen guide on the
instrument for the correct set-up of a protein A method and refer to the Quick Start Guide in
Section 4 of the instrument manual for detailed instructions. The sample load should be in
the range of 2–50 ml; however, a minimum load volume of 5–10 ml is recommended to
avoid sample loss due to small volumes. The recommended A
280
for 1 mg/ml solution of
IgG is 1.4. A typical run profile for 1 ml of human serum diluted 1:5 in 1x PBS (Profinia
desalting buffer) and using the recommended buffer formulations in Table 2 is shown in
Figure 1.
Note: For Protein G purifications, it may be necessary to reduce the affinity peak diversion
volume from 3 ml (for a 1 ml protein A and G plus desalting method) to 2.5–2.7 ml in
Program Method Mode in order to optimize recovery of the antibody.
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