Purification or Removal of Coagulation Factors

VIISelect, VIIISelect, IXSelect, Heparin Sepharose High Performance, Heparin Sepharose 6 Fast Flow, Capto Heparin

Blood coagulation factors form an extremely important group of proteins for research, medical and clinical applications.

Different coagulation factors involved in bleeding disorders are selectively purified using VIISelect, VIIISelect, and IXSelect. Hemophilia type A is caused by a deficiency or defect in factor VIII (FVIII) while hemophilia type B is known as factor IX (FIX) deficiency. Factor VII (FVII) is used for hemophilia patients with FVIII or FIX deficiencies who have developed inhibitors against the replacement coagulation factors.

In addition, coagulation factors obtained from plasma or expressed as recombinant proteins in various cell types can be purified by Heparin Sepharose and Capto Heparin chromatography media. For details about Heparin Sepharose High Performance, Heparin Sepharose 6 Fast Flow and Capto Heparin, see Purification or removal of DNA-binding proteins in this chapter. The protocols are also applicable for coagulation factors.

Chromatography media characteristics

The characteristics of VIISelect, VIIISelect, and IXSelect chromatography media for purification of coagulation factors are shown in Table 3.9.

Table 3.9. Characteristics of VIISelect, VIIISelect, and IXSelect chromatography media

Product Ligand Composition pH stability1 Average particle size (µm)
VIISelect Recombinant protein (Mr 14 080) produced in Saccharomyces cerevisiae. Ligand coupled to Capto. Short term: 2 to 12 Long term: 3 to 10 75
VIIISelect Recombinant protein (Mr 13 000) produced in S. cerevisiae. Ligand coupled to Capto. Short term: 2 to 12 Long term: 3 to 10 75
IXSelect Single-chain antibody fragment (Mr 13 151) directed against FIX and produced in Saccharomyces cerevisiae. Ligand coupled to Capto. Short term: 2 to 12 Long term: 3 to 10 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

A wide range of chromatography media packs and prepacked columns for purification of coagulation factors is available (Table 3.10).

Table 3.10. Chromatography media and prepacked columns for purification of coagulation factors

Product Binding capacity Maximum operating flow Comments
VIISelect FVII, ~ 8 mg/ml medium At least 600 cm/h in a 1 m diameter column with 20 cm bed height1 Supplied as suspension ready for column packing.
VIIISelect Typically 20 000 IU/ml medium Up to 300 cm/h at 30 cm bed height1 Supplied as suspension ready for column packing.
IXSelect FIX, ~ 6 mg/ml medium At least 600 cm/h in a 1 m diameter column, with 20 cm bed height1 Supplied as suspension ready for column packing.
HiTrap IXSelect, 1 ml FIX, ~ 6 mg/column 4 ml/min Prepacked 1 ml column.
HiTrap IXSelect, 5 ml FIX, ~ 30 mg/column 20 ml/min Prepacked 5 ml column.
HiScreen IXSelect FIX, ~ 28 mg/column 4.6 ml/min Prepacked 4.7 ml column.

1 20°C using buffers with the same viscosity as water at < 0.3 MPa (3 bar, 43.5 psi).

Purification of FVII

A commercially available drug approved for infusion therapy was spiked in human plasma, and FVII was purified (Fig 3.7A). Gel electrophoresis was run on SDS-PAGE gradient gels, 8% to 16%, under nonreducing conditions. Figure 3.7B shows the high purity of FVII in the eluted fractions obtained using VIISelect.

Purification of FVII

Fig 3.7. (A) UV280 absorbance curve for purification of FVII from spiked plasma using VIISelect for the initial capture step. (B) SDS-PAGE of the FVII drug before and after purification on VIISelect. The gel was stained with Deep Purple total protein stain and scanned in a Typhoon™ scanner.

Capture of FIX

Capture of FIX from a Chinese hamster ovary (CHO) cell lysate was performed using IXSelect chromatography medium (Fig 3.8A). Fractions from the FIX capture step were analyzed by SDS-PAGE using a FIX reference preparation as standard (Fig 3.8B). The identity of the target protein was confirmed by Western blot analysis (Fig 3.8C).

Capture of FIX

Fig 3.8. (A) FIX capture using IXSelect. (B) SDS-PAGE analysis of fractions from FIX capture step. (C) Western  blot analysis of fractions from FIX capture step. Data from customer evaluation.

Purification of antithrombin III from bovine plasma

Figure 3.9 shows the result from purification of antithrombin III from bovine plasma using HiTrap Heparin HP. The antithrombin III eluted in the second peak.

Purification of antithrombin III from bovine plasma on HiTrap Heparin HP, 1 ml

Fig 3.9. Purification of antithrombin III from bovine plasma on HiTrap Heparin HP, 1 ml.

Storage

Store at 4°C to 8°C in 20% ethanol.

Performing a separation

VIISelect

Binding buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5
Elution buffer: 50 mM Tris, 1.5 M NaCl, 50% (v/v) propylene glycol, pH 7.5
Regeneration buffer: 100 mM glycine
  1. Equilibrate with 6 to 10 CV of binding buffer.
  2. Load the sample.
  3. Wash with 10 to 12 CV of binding buffer.
  4. Elute with 6 to 12 CV elution buffer.
  5. Regenerate the column with regeneration buffer.

VIIISelect

Binding buffer: 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, 0.02% Tween 80, pH 7.0
Wash buffer 1: 20 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, 0.02% Tween 80, pH 6.5
Wash buffer 2: 20 mM histidine, 20 mM calcium chloride, 1 M sodium chloride, 0.02% Tween 80, pH 6.5
Elution buffer: 20 mM histidine, 20 mM calcium chloride, 1.5 M sodium chloride, 0.02% Tween 80 dissolved in
50% ethylene glycol at pH 6.5
  1. Equilibrate with 10 CV of binding buffer.
  2. Load the sample in loading buffer.
  3. Wash with 5 CV of wash buffer 1.
  4. Wash with 5 CV of wash buffer 2.
  5. Elute with 5 to 10 CV of elution buffer.

IXSelect

Binding buffer: 20 mM Tris-HCl, 150 mM NaCl, pH 7.4
Wash buffer: 20 mM Tris-HCl, 500 mM NaCl, 0.01% Tween 80, pH 7.4
Elution buffer: 20 mM Tris-HCl, 2 M MgCl2, pH 7.4
Regeneration buffer: 100 mM glycine, 100 mM NaCl, pH 2.0
  1. Equilibrate with 6 to 10 CV binding buffer.
  2. Load the sample.
  3. Wash with 10 CV of binding buffer.
  4. Wash with 10 CV washing buffer.
  5. Wash with 3 CV of binding buffer.
  6. Elute with 5 to 10 CV elution buffer.
  7. Regenerate the column with regeneration buffer.

Cleaning

The following are suggestions for solutions to be used during cleaning. Prolonged exposure to pH < 2.0 and pH > 12.0 should be avoided. The required cleaning is strongly dependent on the sample used, number of runs, conditions of the chromatography media etc. and has to be designed for each application.

  • PAB (120 mM phosphoric acid, 167 mM acetic acid, 2.2% benzyl alcohol). Store in dark.
  • 10 mM sodium hydroxide
  • 100 mM citric acid

Materials