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Table 1. Medium characteristics.
Mean particle size 90 μm
Particle size range 45–165 μm
Bead structure Highly cross-linked 4% agarose, spherical
Linear flowrate
Base matrix 150–250 cm/h, 0.1 MPa (1 bar), XK 50/60 column, bed height 25 cm
Swelling factor 4–5 ml drained medium/g
Coupling capacity 13–26 mg α-chymotrypsinogen/ml
pH stability*
long term 3–11
short term (CIP) 3–11
* Refers to stability of coupling between ligand abd base matrix. Ligands can be less stable.
Sepharose 4 Fast Flow matrix
Sepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its pre-
activated CNBr form, it offers much improved performance when compared
with the well established CNBr-activated Sepharose 4B. The Sepharose 4
Fast Flow matrix has higher rigidity and can thus be run at high flow rates
(see Table 1).
The higher mechanical strength of the cross-linked matrix makes it well-
suited for use in large columns. Scaling up a purification developed on CNBr-
activated Sepharose 4 Fast Flow is therefore simple and more predictable.
The coupled product is stable at low pH, which is often required for elution
from some immunoadsorbents.
For applications that require operation at high pH, note that the amide bond
formed when using the companion product NHS-activated Sepharose 4
Fast Flow is stable up to pH 13 for normal use.
2. Coupling
CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried in the
presence of additives. These additives need to be washed away at low
pH (pH-2–3) before coupling the desired ligand. The use of low pH (pH2–3)
preserves the activity of the reactive groups, which otherwise hydrolyse at
high pH.