Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 194)

Appendix I
Profinia Protein A and G Method
1.1 Background Information
The Profinia protein purification system is an automated, user-friendly instrument for the
purification of affinity-tagged proteins and antibodies. The system has been designed to
use standard chromatography buffers, resins, and reagents with easy-to-use pre-programmed
purification methods stored on the instrument. There are five main types of applications
currently used in the Profinia system: native IMAC, denaturing IMAC, GST, desalting, and
protein A and G. The Protein A and G method will be referred to as the Protein A method
throughout this section. This section provides information on the use of protein A methods
in the Bio-Rad Methods and Program Methods modes of the Profinia instrument.
1.2 Description of Method
The Profinia protein A method is designed for the affinity purification of antibodies (typically
IgG) from serum, ascites, or hybridoma cell culture supernatant using protein A (or in some
cases, protein G) chromatography media. The instrument has two main types of protein A
methods that can be used for the purification of IgG: protein A affinity, and protein A plus
integrated desalting. Both methods are designed to use either 1 ml or 5 ml pre-packed
protein A cartridges and 10 ml or 50 ml P6 desalting cartridges (respectively), when using
the protein A plus integrated desalting methods. Cartridges for protein A applications are
available from Bio-Rad as 1 ml and 5 ml Bio-Scale Mini Affi-Prep protein A cartridges that
are designed for direct connection to the Profinia instrument. Each ml of Bio-Rad Affi-Prep
protein A resin typically yields 5–15 mg of purified IgG, depending on the species, sample
type, and antibody subclass. The Profinia protein A methods allow the selection of one or
two samples per run, a choice of standard or low flow-rates for sample loading (1 ml
cartridges, 1 or 0.5 ml/min; 5 ml cartridges, 5 or 2.5 ml/min) or extended cartridge washing
for each sample, similar to the options available for the IMAC and GST pre-programmed
methods. Following system priming and cartridge equilibration, the protein A method is
programmed to load sample from the S1 and/or S2 ports onto the cartridge. The available
pre-programmed Bio-Rad protein A methods are designed for loading volumes between
2–50 ml per sample. For larger sample volumes, the protein A methods can be customized
to load volumes (in ml or L) ranging between 0–999. Refer to Section 11 for system
requirements when using large sample or buffer volumes on the Profinia instrument. Table 1
summarizes the method steps for a typical Bio-Rad Profinia 1 ml protein A plus 10 ml
desalting method with 1 sample. The steps outlined in Table 1 are similar for the 5 ml
protein A methods; however, flow-rates are increased proportionally to accommodate large
volumes of the 5 ml protein A and 50 ml desalting cartridges. In the case of the protein A
affinity only methods (without integrated desalting), the steps associated with the desalting
cartridge are absent, and elution of the protein A cartridge is collected directly into 1D (or
2D) fraction without desalting.
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