Purification or Removal of Albumin

Blue Sepharose High Performance, Blue Sepharose 6 Fast Flow, Capto Blue, Capto Blue (high sub)

Albumin binds to Cibacron Blue F3G-A, a synthetic polycyclic dye that acts as an aromatic anionic ligand binding the albumin via electrostatic and/or hydrophobic interactions. Similar interactions are seen with coagulation factors, lipoproteins and interferon. Cibacron Blue

F3G-A is linked to Sepharose to create Blue Sepharose AC media (Figure 3.1).

Figure 3.1. Partial structure of Blue Sepharose Fast Flow and Blue Sepharose High Performance.

Capto Blue and Capto Blue (high sub) have a more rigid agarose base matrix compared with Blue Sepharose 6 Fast Flow, which results in improved pressure/flow properties, optimized pore structure, and high chemical stability to support cleaning-in-place (CIP) procedures.

Use Blue Sepharose or Capto Blue to remove host albumin from mammalian expression systems, or when the sample is known to contain high levels of albumin that can mask  the visualization of other protein peaks seen by UV absorption.

Advice on the selection of techniques for the removal of albumin during antibody purification is given in the handbook Affinity Chromatography, Vol. 1: Antibodies, 18103746 from Cytiva.

Cibacron Blue F3G-A also shows certain structural similarities to naturally occurring  molecules, such as the cofactor NAD+, that enable it to bind strongly and specifically to  a wide range of proteins including kinases, dehydrogenases, and most other enzymes  requiring adenylyl-containing cofactors.

Chromatography media characteristics

Characteristics of Blue Sepharose and Capto Blue chromatography media are summarized  in Table 3.1.

Characteristics of Blue Sepharose and Capto Blue chromatography media

Table 3.1. Characteristics of Blue Sepharose and Capto Blue chromatography media

Ligand density	Composition	Binding capacity	Average particle size (µm)
Blue Sepharose High Performance	4 mg/ml	Cibacron Blue F3G-A coupled to Sepharose High Performance using the triazine method.	Short term: 3 to 13 Long term: 4 to 12	34
Blue Sepharose 6  Fast Flow	6.7 to 7.9 µmol/ml	Cibacron Blue F3G-A coupled to Sepharose 6 Fast Flow using the triazine method.	Short term: 3 to 13 Long term: 4 to 12	90
Capto Blue	13 µmol/ml	Cibacron Blue F3G-A coupled to Capto	Short term: 3 to 13 Long term: 2 to 13.5	75
Capto Blue (high sub)	18 µmol/ml	Cibacron Blue F3G-A coupled to Capto.	Short term: 3 to 13 Long term: 2 to 13.5	75

¹ Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Purification options

Blue Sepharose and Capto Blue are available in chromatography media packs for packing  into empty columns. The media are also available in prepacked columns for convenience. Purification options for the media and prepacked columns are shown in Table 3.2.

Purification options for Blue Sepharose and Capto Blue chromatography media and prepacked columns

Table 3.2. Purification options for Blue Sepharose and Capto Blue chromatography media and prepacked columns

Binding capacity	Maximum operating flow	Comments
Blue Sepharose 6 Fast Flow1	Human serum albumin (HSA), 18 mg/ml medium	> 750 cm/h2	Supplied as a suspension ready for column packing.
HiTrap® Blue HP, 1 ml	HSA, 20 mg/column	4 ml/min	Prepacked 1 ml column.
HiTrap® Blue HP, 5 ml	HSA, 100 mg/column	20 ml/min	Prepacked 5 ml column.
HiScreen Blue FF	HSA, 85 mg/column	4.6 ml/min	Prepacked 4.7 ml column.
Capto Blue	HSA, 24 mg/ml medium	At least 600 cm/h1	Supplied as suspension ready for packing.
HiScreen Capto Blue	HSA, 118 mg/column	4.6 ml/min	Prepacked 4.7 ml column
Capto Blue (high sub)	HSA, 30 mg/ml medium	At least 600 cm/h1	Supplied as suspension ready for packing.

¹ In a 1 m column with 20 cm bed height at 20 °C using process buffers with the same viscosity as water.

² Appendix 4 to convert flow velocity (cm/h) to volumetric flow rate (ml/min) and vice versa. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Purification examples

Figure 3.2 shows the use of HiTrap^® Blue HP for purification of increasing amounts of human serum albumin. The process is easily scaled up by connecting several 1 ml or 5 ml HiTrap^® columns in series.

Figure 3.3 shows the use of Blue Sepharose 6 Fast Flow for the separation of HSA from interferon β.

Figure 3.2. Scaling up on HiTrap^® Blue HP gives predictable separations and quantitatively reproducible yields.

Figure 3.3. Purification of human serum albumin and interferon b on Blue Sepharose 6 Fast Flow

In these examples elution is achieved by increasing the ionic strength of the buffer or changing the polarity of the buffer. Changing the pH of the buffer can also work, but the correct cofactor is preferable for the elution of specifically bound proteins.

Performing a Separation

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

Elution buffer: 50 mM KH₂PO₄, 1.5 M KCl, pH 7.0 or 20 mM sodium phosphate, 2 M NaCl, pH 7.0

1. Equilibrate the column with 5 CV of binding buffer.
2. Adjust the sample to starting conditions and apply to the column, using a syringe or a pump.
3. Wash with 10 CV of binding buffer or until no material appears in the eluent (monitored by absorption at A280 nm).
4. Elute with 5 CV of elution buffer (step elution) or with 0% to 100% elution buffer in binding buffer (gradient elution).

Cleaning

Wash with 5 CV of high pH (100 mM Tris-HCl, 500 mM NaCl, pH 8.5) followed by low pH (100 mM sodium acetate, 500 mM NaCl, pH 4.5). Repeat four to five times. Re-equilibrate immediately with binding buffer.

Remove precipitated proteins with 4 CV of 100 mM NaOH at a low flow rate, followed by washing with 3 to 4 CV of 70% ethanol or 2 M potassium thiocyanate. Alternatively, wash with 2 CV of 6 M guanidine hydrochloride. Re-equilibrate immediately with binding buffer.

Remove strongly hydrophobic proteins, lipoproteins and lipids by washing with 3 to 4 CV of up to 70% ethanol or 30% isopropanol. Alternatively, wash with 2 CV of detergent in a basic or acidic solution, e.g. 0.1% nonionic detergent in 1 M acetic acid at a low flow rate, followed by 5 CV of 70% ethanol to remove residual detergent. Re-equilibrate immediately in binding buffer.

Chemical stability

Stable in all commonly used aqueous buffers, 70% ethanol, 8 M urea, and 6 M guanidine hydrochloride.