Detergents are commonly used in hydrophobic cell membrane protein purification and viral removal/inactivation. These detergents
must eventually be removed during the purification process. If a detergent has low critical micelle concentration (CMC), it
can be difficult to remove from solution using techniques such as dialysis or gel filtration. Therefore, a detergent with
a low CMC like Triton X-100 requires different removal techniques. Triton X-100 is a non-ionic detergent with an approximate
molecular weight 625 and CMC value of 0.24 mM. The structure of Triton X-100 is shown in Figure 1.
Figure 2: Dynamic binding capacity of Amberchrom CG161 for cephalosporin C and BSA.
Amberchrom CG161 was chosen as a chromatographic solution for the removal of Triton X-100. It is a polystyrene divinylbenzene
polymer used for the purification of peptides and antibiotics. This resin has been used in commercial purification processes
for several decades and is an excellent choice for detergent removal because of its unique pore structure and hydrophobic
nature. As shown in Figure 2, Amberchrom CG161 has a high capacity for small molecules such as cephalosporin C (479 MW), and
virtually no capacity for larger proteins such as BSA (67000 Da). Therefore, detergents should effectively be removed from
protein feedstocks using Amberchrom CG161.
Results and Conclusions
Table 1: Capacity comparison for Triton X-100.
For detergent capacity experiments, Amberchrom CG161 was compared with another commercially available resin which is sold
specifically for detergent removal, SDR HyperD (Pall Life Sciences, East Hills, New York, USA). Amberchrom CG161M and SDR
HyperD were packed into separate glass 1.0 cm ID × 7.0 cm L columns and the columns were used to determine the dynamic binding
capacities for Triton X-100. A phosphate buffered solution containing 5 mg/mL of Triton X-100 was passed through the column
at a flow-rate of 2 mL/min (152 cm/hr) and the effluent was collected. After complete saturation of the column (200 mL of
effluent collected), the experiment was terminated. The 1% breakthrough capacity and total capacity were then calculated.
Conditions: Column: 1cm ID × 7cm L Flow-rate: 2 mL/minute Sample: 5 mg/mL Triton X-100 Mobile phase: Sodium phosphate buffer, pH 7.4 Detection: UV @ 280nm
As can be seen in Table 1, Amberchrom CG161M demonstrated higher 1% and total capacity values for Triton X-100 as compared
to SDR HyperD. Amberchrom CG161 chromatographic resin is an effective tool for the removal of detergents from biological samples.
It provides high capacity for non-ionic detergents such as Triton X-100.
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