Due to its two-step workflow, a nonaffinity-based purification process is well suited for the
purification of immunoglobulin M antibodies.
» by Payal Khandelwal, Global Product Manager, Protein Purification Business, Bio-Rad Laboratories, Inc.
Immunoglobulin M (IgM) antibodies are currently a hot target for in vitro diagnostics and therapy for vari- ous maladies, including cancer and
infectious diseases. The IgM backbone
is essentially formed by a crosslinkage
of five or six IgGs. However, IgMs are
structurally and biochemically much
more complex than IgGs. For one, IgMs
are more heavily glycosylated than IgGs.
They are very sensitive to pH conditions, making them highly susceptible to
degradation or precipitation. Therefore,
they are soluble in a narrow range of
conditions. This makes their downstream
IgM purification is made even more
complex by their large size, labile nature
and complex physicochemical properties.
The diffusion constant for IgMs is about
half of IgGs, which means IgMs need half
the flow rate to attain similar capacity and
separation performance on a resin. Hence,
the purification techniques used for IgGs
cannot be translated directly to IgMs. In
addition, affinity chromatography, which
is commonly used for IgG purification,
also cannot be successfully applied to pro-cess-scale IgM purification. The acidic conditions required for elution of targets from
an affinity resin are too harsh and adverse
for IgM stability.
Therefore, a nonaffinity-based strategy
can be explored for basic/neutral IgM
purification, utilizing an ion exchange
and a mixed-mode resin. Optimization of
the buffer pH and conductivity lead to a
protocol that could minimize both product- and process-related impurities and
produce a pure, stable fraction of IgM.
Purification of basic/neutral IgM
Purification workflows typically involve
multiple steps to ensure high purity of
the target. This application incorporated
capture and polish steps in the purification
workflow. A capture resin must ideally
Figure 1: SDS-PAGE analysis of the basic/neutral IgMs purified on Nuvia S and CHT-II-40. M: Protein marker, L: Load, FT: Flowthrough, EP: Eluted
protein, HC: Heavy chain, LC: Light chainp