Bio-Rad Bio-Scale™ Mini Affi-Prep® Protein A Cartridges - Manual (Page 3)

Section 1
Introduction
Protein A chromatography is one of the most reliable and specific methods for immuno-
globulin purification. The Affi-Prep protein A support is a macroporous affinity chromatography
matrix developed to take advantage of the specificity of protein A for medium to high pressure
chromatographic applications. This product is particularly useful for the analytical and
preparative requirements of the biotechnology industry, where there is a need for scale-up
purification of monoclonal and polyclonal antibodies. The Affi-Prep protein A matrix allows
high linear flow rates and exhibits excellent mechanical strength. These key features allow the
direct transfer of experimental conditions from the initial analytical methods development stage
to the final production scale purification of product. When the Affi-Prep protein A matrix is used
in conjunction with the optimized Affi-Prep protein A MAPS II buffers, high efficiency binding
of a wide range of immunoglobulins, including all isotypes of murine IgG, is achieved.
Advantages of the Affi-Prep protein A support include:
• Binds a wide range of monoclonal and polyclonal antibodies, including murine IgG
1
• Macroporous polymeric support is non-compressible under normal operating conditions
• Linear flow rates up to 2,000 cm/hr are achievable
• High flow rates at all column diameters for easy scale-up
• Compatible with most commonly used chromatographic buffers
• Stable pH range from 2–10
• Multiple chromatographic cycles without loss of capacity
• Ligand leakage is extremely low
• Easy to use
1.1 Principle
Protein A, from Staphylococcus aureus, has the property of binding with high specificity to
the Fc region of immunoglobulins from most mammalian species. 1 When coupled to a suitable
hydrophilic support, protein A can be used to purify IgG, to selectively remove IgG prior to
analysis of other immunoglobulin classes, or to adsorb immune complexes to purify antigens.
2
The affinity of IgG for protein A is not the same for all species or isotypes. For this reason
MAPS II buffers have been developed to optimize the binding and recovery of many
immunoglobulins, including murine IgG
1
.
1.2 Comparison of IgG Recoveries
Protein A preparations have been used extensively to purify both polyclonal and monoclonal
IgG and IgG subclasses from a variety of mammalian species. 2 Currently, protein A affinity
chromatography is being widely used to purify monoclonal antibodies from murine ascites fluid
or culture medium supernatants.
3 However, the usefulness of protein A purification in this
application has been limited because, using published methods, most IgG
1
immunoglobulins
have low affinity for protein A. This results in poor IgG
1
retention on protein A affinity
supports. 4-7 Since many murine IgG monoclonal antibodies belong to the IgG
1
subclass, poor
IgG
1
retention represents a significant problem in their purification.
Figure 1 shows the results achieved with the Affi-Prep protein A MAPS method, using
MAPS II buffers, for the purification of several murine monoclonal antibodies including IgG
1
,
IgG
2a
, and IgG
2b
subclasses.
1
LIT79B 9/1/98 8:12 AM Page 1