Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 195)

Table 1. Protocol steps for Profinia 1 ml protein A plus 10 ml desalting method with a
single sample
1.3 Required Equipment and Materials
Bio-Rad Affi-Prep protein A 1 ml and 5 ml Bio-Scale Mini cartridges are designed to be
used with the pre-programmed protein A methods on the Profinia instrument. In addition, a
number of other pre-packed protein A cartridges available in 1 ml and 5 ml sizes from other
chromatography suppliers have been tested and can be used with the pre-programmed
protein A methods. When using cartridges from other suppliers, it may be necessary to
obtain the correct fittings to adapt the cartridges to the luer-style fittings of the Profinia
instrument cartridge connection ports. Protein A methods that include integrated desalting
will also require the use of Bio-Rad Bio-Gel P6 desalting cartridges, which are available in
10 ml and 50 ml sizes, to match the corresponding cartridge sizes required in the
pre-programmed protein A methods. Complete Profinia buffer and purification kits specific
for protein A methods are not available; however, Table 2 lists the most common recommended
buffers and reagents used for protein A purifications. All buffers and solutions can be made
or purchased as concentrates as described in Table 2. Prepared buffers should use high
quality, chromatography grade reagents and should be pre-filtered through a 0.2 or 0.45 µm
filter to remove particulates prior to use with the system. Although the buffer concentrations
are preset in the protein A methods, buffer concentrations of 1x, 2x, 3x, 4x, and 5x can be
selected and saved as an alternative protein A method in the Program Method mode on
the Profinia instrument.
When using the Profinia protein A plus integrated desalting methods, the Profinia desalting
purification kit or individually packaged Profinia buffers can be used to supply most of the
reagents recommended for the protocol. If the Profinia desalting purification kit is used, a
protein A elution/regeneration buffer, such as those recommended in Table 2, will need to
be prepared separately (see Table 2 for buffer compositions). Protocols for preparation of
recommended protein A elution/regeneration buffers are included in Section 5 of this
appendix.
Method Steps Buffer Name
Buffer
Port
Inlet Buffer
Conc. Fraction Port
Outlet
Column
Volume Flow Rate
[ml/min]
Protein A
Cart.
Desalting
Cart.
System Priming All Buffer and Sample Ports
B0,1,3,4,5,6,7,8
, S1 - Waste (W) - 20 Offline Offline
Equilibrate 1 Water B0 - W 2 4 Offline Online
Equilibrate 2 Desalting Buffer B4 5x W 3 4 Offline Online
Water Wash Water B0 - W - 20 Offline Offline
Equilibrate 1 Water B0 - W 2 2 Online Offline
Equilibrate 2
Protein A Equilibration/Wash
Buffer B1 5x W 10 2 Online Offline
Load S1 Sample B9 1x W->1A 2-50 ml 1 Online Offline
Wash-1 C1
Protein A Equilibration/Wash
Buffer B1 5x 1A->1B 12 1 Online Offline
Elute 1 C1
Protein A Elution/Regeneration
Buffer B3 5x 1B->W Up to 10 1 Online Offline
Elute 2 C1
Protein A Elution/Regeneration
Buffer B3 5x W 3 1 Online Online
Elute 1 C2 Desalting Buffer B4 5x W Up to 1 2 Offline Online
Elute 2 C2 Desalting Buffer B4 5x 1D 0.4 2 Offline Online
Elute 3 C2 Desalting Buffer B4 5x W 1 2 Offline Online
Clean 1 C2 Clean 1 Buffer B5 2x W 1 4 Offline Online
Clean 2 C2 Clean 2 Buffer B6 4x W 2 4 Offline Online
Prime B3
Protein A Elution/Regeneration
Buffer B3 5x W - 20 Offline Offline
Clean 1 C1
Protein A Elution/Regeneration
Buffer B3 5x W 5 2 Online Offline
Clean 2 C1
Protein A Equilibration/Wash
Buffer B1 5x W 5 2 Online Offline
Water Wash Water B0 - W - 20 Offline Offline
Store C1 20% EtOH B8 1x W 2 Online Offline
Water Wash Water B0 - W - 15 Offline Offline
Store C2 Profinia Storage Buffer B7 2x W 2 4 Offline Online
Water Wash Water B0 - W 20 Offline Offline
Clean Pump Seals Water B0 - W - 20 Offline Offline
System Line Cleaning Water
B0,
B9, B10 -
System and
Pump Seals - 20 Offline Offline
20% EtOH Wash 20% EtOH B8 1x W - 15 Offline Offline
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