4.2. Chromatography of Plasma on Q Sepharose FF

The flow rate was 15 mL min⁻¹ during plasma application and 25 mL min⁻¹ during other purification steps. The volume of the Q Sepharose FF column was 46 mL.

Equilibration, sample, and washings with 10 CV of Buffer A, according to Section 4.1 and Section 4.2, and 10 CV of Buffer B (25 mM sodium citrate, 200 mM NaCl and 5 mM CaCl₂, pH 6.0) (200A) were carried out. Next, 10 CV of 5 mM to 100 mM CaCl₂ (in Buffer B) linear gradient and 5 CV stepwise Buffer B + 100 mM CaCl₂ were undertaken. After a second wash with 5 CV of Buffer B (200B), elution with 4 CV of Buffer C (25 mM sodium citrate, 500 mM NaCl and 5 mM CaCl₂, pH 6.0) was performed.

Resin was regenerated by sequentially washing with 2 CV 1M NaOH (with 1 h pause), 5 CV 2M NaCl and 5 CV purified water and stored in 0.2 M NaOH.

Equilibration, sample, and washings with Buffers A and B were carried out as indicated in Section 4.1, Section 4.2 and Section 4.2.1 Protein elution with 5 CV Buffer B + 15 mM CaCl₂, and 5 CV Buffer B + 65 mM CaCl₂ were performed. Washing with Buffer B (200B) and elution with Buffer C were then carried out according to Section 4.2.1. Resin regeneration and storage were carried out as in Section 4.2.1.