Hydrophobic Membrane Adsorbers for Large-Scale Downstream Processing - This alternative to column chromatography is suitable for flow-through as well as bind-and-elute purification operations. - BioPh
A Phenyl Membrane Adsorber for Large-Scale Purification
Table 1. Dynamic binding capacities (DBC) at 10% breakthrough for Sartobind phenyl membrane adsorber (3 mL membrane volume)
challenged with three different model proteins. All proteins were prepared as 1 g/L solutions in 50 mM phosphate buffer at
pH 7.0 with varying amounts of ammonium sulfate, and loading was done at 10 mL/min (18 s residence time).
The Sartobind phenyl hydrophobic membrane adsorber was recently introduced to the market.18,19 It is assembled into 30-layer radial flow process capsules with an 8-mm bed height. The HIC membrane adsorber resulted from
the combination of a newly designed macroporous membrane structure and covalently attached hydrophobic phenyl ligand. This
membrane structure was designed for high flow rates and binding capacities. Furthermore, the hydrophilic stabilized regenerated
cellulose membrane support possesses excellent mechanical and chemical robustness and demonstrates minimal unspecific interaction
with proteins, even at high concentrations of a lyotropic salt.19 The functionalized membrane exhibits increasing binding capacity in increasing ammonium sulfate concentrations, as expected
in HIC. Table 1 describes the binding capacity at 10% breakthrough for Sartobind phenyl challenged with three different model
proteins.
Figure 1. Flow-through mode operation with Sartobind phenyl membrane adsorber to remove high molecular weight (HMW) aggregates
for the purification of an E. coli expressed protein. The figure shows the percentage of aggregates bound onto the membrane
at increasing salt conditions.
In a recent paper, the separation properties of the HIC membrane were evaluated and compared to those of a traditional HIC
resin, phenyl Sepharose FF (low sub) from GE Healthcare (Chalfont St. Giles, UK).19 Using a protein mixture of cytochrome C, trypsinogen, and a polyclonal IgG, Sartobind phenyl demonstrated better resolution
than the HIC resin under the experimental conditions used for the evaluation. In addition to the bind-and-elute operation,
the authors demonstrated the application of Sartobind phenyl in flow-through mode for aggregate removal in a purification
process for a recombinant protein. The protein was loaded with varying amounts of ammonium sulfate such that the aggregates
bound while the monomer did not. After loading, the membrane was washed with deionized water to elute the bound aggregates.
The aggregate removal increased with increased ammonium sulfate concentration, as shown in Figure 1. Protein yield was >95%,
regardless of the salt conditions used in the loading buffer.
Nathalie Frau, PhD, is a senior scientist in purification process development, biotechnology division, Sartorius Stedim North America.
Articles by Nathalie Frau, PhD
