Removal of Albumin Using Blue Sepharose® Chromatography Media

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016

Blue Sepharose® 6 Fast Flow or prepacked HiTrap™ Blue HP 1 ml and 5 ml columns (Figure 4.2) can be used to remove albumin either before or after other purification steps (see Table 4.1). The albumin binds in a nonspecific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand, Cibacron Blue F3G-A, coupled to Sepharose.

Fig 4.2. Prepacked with Blue Sepharose® High Performance, HiTrap™ Blue HP columns offer fast  and simple removal of albumin by affinity chromatography.


Use HiTrap™ Blue HP 1 ml or 5 ml columns to remove host albumin from mammalian  expression systems or when the sample is known to contain high levels of albumin that might mask the UV absorption of other protein peaks.

Do not use Blue Sepharose® chromatography media if the immunoglobulin or other target molecule has a hydrophobicity similar to that of albumin.


Table 4.1. Options for the removal of albumin by affinity chromatography using Blue Sepharose® chromatography media

  Capacity/ml medium1 Comments
HiTrap™ Blue HP HSA 20 mg Removal of albumin Prepacked 1 ml and 5 ml columns
Blue Sepharose® 6 Fast Flow  HSA >18 mg Supplied as a suspension ready for column packing

1 Protein binding capacity varies for different proteins


Figure 4.3. Effective removal of albumin from human plasma using a HiTrap™ Blue HP 1 ml column.


Figure 4.3 shows the removal of human serum albumin from plasma using HiTrap™ Blue HP 1 ml.

The protocol for removal of albumin using HiTrap™ Blue HP 1 ml and 5 ml columns is described below.


Buffer preparation

Binding buffer: 20 mM sodium phosphate, pH 7.0, or 50 mM potassium hydrogen phosphate (KH2PO4), pH 7.0

Elution buffer: 0.02 M sodium phosphate, 2 M sodium chloride, pH 7.0, or 0.05 M potassium hydrogen phosphate, 1.5 M potassium chloride, pH 7.0


Albumin removal

  1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied), laboratory pump, or chromatography system “drop to drop” to avoid introducing air into the system.
  2. Remove the snap-off end at the column outlet.
  3. Wash out the ethanol with 3 to 5 column volumes of distilled water.
  4. Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min (1 ml column) and 5 ml/min (5 ml column).
  5. Apply the sample using a syringe fitted to the Luer connector or by pumping it onto the column. For optimal results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml column) during sample application*.
  6. Wash with binding buffer (generally at least 5 to 10 column volumes) until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for washing.
  7. Elute with elution buffer using a one-step or linear gradient. For step elution, 5 column volumes is usually sufficient. For linear gradient elution, 10 to 20 column volumes are usually sufficient. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for elution.
  8. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding buffer. The column is now ready for a new purification.

* 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a 1 ml HiTrap™ column; 5 ml/min corresponds to approximately 120 drops/min when using a 5 ml HiTrap™ column.



Store in 20% ethanol at 2°C to 8°C.


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