Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 197)

Section 2
Guidelines When Using Protein A Methods
2.1 Sample Preparation Information
Affi-Prep protein A cartridges are convenient, ready-to-use devices for the isolation and
purification of monoclonal and polyclonal antibodies. Protein A binds specifically and with
high affinity to the Fc portion of immunoglobulins, in particular IgG. Bio-Rad protein A cartridges
are designed for routine affinity purification of most IgGs from serum, ascites, and cell culture
supernatant. The binding capacity of protein A depends on the species, subtype, and source
of the particular IgG. In addition, the total capacity of protein A cartridges also depends on
several other factors, such as flow rate during sample application, sample concentration, the
temperature at which the purification is carried out, and the composition of the buffers used for
the separation. Profinia protein A methods are compatible with most commercially available
protein A cartridges and are optimized to provide fast, easy, and reproducible
chromatographic separations including integrated desalting and cartridge regeneration.
Protein A generally binds to IgG most effectively between pH 7–9, although the optimal
binding pH varies by species. Elution is generally achieved by decreasing the pH (2.5–4).
When possible, the sample should be adjusted to the composition of the binding/equilibration
buffer (see Table 2 for recommendations). If a concentrated source of IgG is used, such as
serum, then this is best accomplished by diluting the sample with binding buffer to a minimum
of a 1:5 ratio (or greater). Increased recoveries of concentrated samples can be achieved by
using sample load volumes of 5 ml or greater. In cases where the concentration of the sample
is low and the sample volume is large (for example, > 25 ml for a 1 ml cartridge, such as with
cell culture supernatants) it may not be practical to dilute the sample further with binding
buffer. When working with dilute samples, the pH of the sample should be checked and
adjusted to an optimum binding pH (~7–9) with an appropriate buffer, such as 1 M Tris pH 8
or similar, in order to maximize binding to the protein A cartridge. Immediately prior to load-
ing on the instrument, samples should be filtered through a 0.2 or 0.45 µm filter to prevent
clogging of the system filter and cartridge. This is especially important when loading large vol-
umes of serum or plasma.
2.2 Protein A Cartridge Selection
Profinia protein A methods are designed to use 1 ml or 5 ml protein A cartridges. Since the
total IgG content of serum differs between species, use a sample size such that the amount
of IgG in the sample does not exceed the maximum cartridge binding capacity. The
concentration of antibody in cell culture supernatant can vary considerably between
hybridoma clones (5–50 µg/ml). The approximate cartridge binding capacity for a mm Affi-
Prep protein A cartridge is approximately 15–20 mg of human IgG. If the anticipated
amount of IgG in the starting sample exceeds the capacity of a 1 ml cartridge, then it is rec-
ommended to either select the 5 ml cartridge purification option or decrease the sample
volume applied to a 1 ml cartridge. To minimize the possibility of cross contamination, it is
suggested that individual protein A and desalting cartridges are dedicated to the purification
of a unique sample or IgG species, as in the case of hybridoma cell culture supernatants.
Profinia
protein A methods include regeneration steps for the protein A cartridge to strip any
remaining bound immunoglobulins following elution. For methods that include integrated
desalting, using the recommended buffers in Table 2 will ensure that the Bio-Rad P6
Desalting cartridges are cleaned, stored, and ready for re-use at the completion of the
method.
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