Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant

Protocolo

1. Preparation of the Chromatography System

1. Prepare the chromatography system by sequentially rinsing the sample and buffer lines each with sterile water, 1 N sodium hydroxide, water, and wash buffer (20 mM sodium phosphate buffer containing 150 mM sodium chloride, pH 7.0) at a linear flow rate of 50 mL/min.
2. Prepare a heparin 50 µm column (10 × 10 cm, 7.9 mL of column volume (CV)) by sequentially running column cleaning buffer (5 CV sterile 20 mM sodium phosphate buffer containing 2 M sodium chloride, pH 7.0) and 5 CV wash buffer at a linear flow rate of 7 mL/min.

2. Purification of Baculovirus Vector

1. Set up the chromatography system as follows:
   1. Use the sample loading inlet port for loading the baculovirus supernatant.
   2. Use the buffer inlet port A1 for loading the wash buffer. Prepare a bottle with at least 500 mL of wash buffer and place the wash buffer line into the bottle.
   3. Use the buffer inlet port A2 for loading the elution buffer (20 mM sodium phosphate with 1.5 M sodium chloride, pH 8.0). Prepare a bottle with at least 500 mL of elution buffer and place the elution buffer line into the bottle.
   4. Insert several 50 mL conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluted baculovirus.
2. Equilibrate the heparin column with five 7.9 mL column volumes (CVs) of wash buffer (40 mL) at a linear flow rate of 7.0 mL/min.
3. Load 250 mL of baculovirus supernatant onto the heparin column using the sample pump (inlet sample port) of the chromatography system at a linear flow rate of 2.0 mL/min.
4. Run 10 CVs (80 mL) of wash buffer through heparin column at a linear flow rate of 2.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) has returned to baseline and becomes stable.
5. Elute the baculovirus particles from the 7.9 mL heparin column with 5 CVs (40 mL) of elution buffer at a linear flow rate of 4.0 mL/min. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.
6. Post-elution, immediately dilute the eluted baculovirus supernatant 10-fold using 20 mM sodium phosphate buffer in water to prevent inactivation of baculovirus particles from osmotic shock during subsequent centrifugation.

Materiais

Name	Company	Catalog Number	Comments
Akta Avant 150	GE Healthcare	28976337	Chromatography system
POROS Heparin 50 µm Column	ThermoFisher Scientific	4333414	Heparin column
Wash buffer	In-house	Non-catalog item	20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride
Elution buffer	In-house	Non-catalog item	20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride
50 ml Conical tube	ThermoFisher Scientific	14-959-49A	For collection of Baculovirus supernatant
Column cleaning buffer	In-house	Non-catalog item	20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride
Sterile water	In-house	Non-catalog item	For Akta Avant cleaning
Sodium hydroxide	Sigma-Aldrich	1.09137	For Akta Avant cleaning