Production and Purification of Baculovirus for Gene Therapy Application

Summary

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Abstract

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

Introduction

Baculovirus is primarily used for the production of recombinant proteins and vaccines in lepidopteran Spodoptera fugiperda (Sf)9 insect cells by using recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)^1,2,3,4. More recently, it is emerging as a promising vector for gene therapy application⁵. It is known to have a broad host and tissue tropism, infects both quiescent and proliferating cells, is non-pathogenic, and does not integrate into the host chromosome^4,5,6. Moreover, baculovirus can be produced in serum-free suspension culture which is scalable and allows for closed system processing for future clinical production¹.

The purity of baculovirus particles is important for achieving effective transduction while minimizing cytotoxicity^7,8,9. Baculovirus can be concentrated by ultracentrifugation or tangential flow filtration (TFF) with limited impact on its infectivity. However, these procedures not only concentrate virus particles but also cellular debris and proteins from Sf9 culture, which can be toxic in vitro (personal observation) and may induce inflammation or an immune response when used in vivo. To avoid this, especially when using highly concentrated virus stocks, infectious baculovirus needs to be purified and separated from contaminating particles.

Several methods have been reported for the purification and concentration of baculovirus vectors^10,11,12. Of the available approaches, heparin affinity chromatography allows for a single-step high level of purification with the low concentration of contaminating proteins¹². The method is based on the identification of heparan sulfate as the receptor for baculovirus^13,14. After loading Sf9 cell supernatant onto the column and binding of baculovirus, the column can be washed with physiologic (isotonic) buffer to remove unbound or loosely bound contaminating particles. Since the binding to heparin is reversible, baculovirus particles can be eluted with a high salt buffer, which is diluted immediately to physiologic (isotonic) salt concentration to prevent inactivation by osmotic shock¹². Moreover, the production of baculovirus, as well as capture on and elution from the chromatography column, can be performed using a closed-system process which is compatible with current good manufacturing practices (cGMP).

Here, we provide a detailed protocol for the manufacture, purification, and concentration of infectious baculovirus using affinity chromatography and centrifugation. Briefly, we produce baculovirus by transfection of Sf9 cells with a baculovirus plasmid DNA in adherent culture and further expand the infectious baculovirus in serum-free suspension culture. We purify baculovirus using heparin affinity chromatography and use ultracentrifugation as the final step to highly concentrate the vector for gene therapy application.

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Protocol

See Figure 1 for an illustration summarizing the protocol.

1. Purification of Baculovirus Plasmid DNA

1. Grow bacterial culture containing baculoviral plasmid DNA (bacmid)¹⁴ in 200 mL of LB broth with antibiotics; kanamycin (50 µg/mL), tetracycline (10 µg/mL), and gentamycin (7 µg/mL), for 16 h at 37 °C on an orbital shaker setting at 300 rpm.
2. Purify the bacmid DNA from the bacterial culture using standard alkaline lysis protocol¹⁵, and dissolve the bacmid in TE buffer.

2. Production of Baculovirus

1. Culture Sf9 cells in an orbital shaker incubator at 135 rpm and 28 °C in a polycarbonate Erlenmeyer flask containing serum-free insect culture medium^1,2,3.
   Note: If Sf9 cells are thawed from frozen stock, allow at least two weeks for the cells to recover and enter the exponential phase of growth.
2. Count the Sf9 cells harvested at exponential phase of growth with a hemocytometer after staining with Trypan blue. Seed 1 x 10⁶ viable Sf9 cells per well in a 6-well tissue culture-treated plate in 2 mL of medium. Allow the cell to attach for 1 h at 28 °C in an incubator.
3. Replace the growth medium with 1 mL of serum-free unsupplemented Grace's insect culture medium.
4. Transfect the bacmid DNA into the Sf9 cells using insect cell transfection reagent.
   1. Mix 8 µL insect cell transfection reagent with 100 µL of Grace's Insect medium.
   2. Dilute 2 µg of bacmid DNA into 100 µL of unsupplemented insect medium, and mix gently by vortexing.
   3. Combine the diluted DNA with the diluted insect cell transfection reagent, and mix gently. Incubate for 15 to 30 min at room temperature.
   4. Add the DNA-lipid mixture dropwise onto the cells. Incubate at 28 °C in an incubator for approximately 5 h.
5. Remove the transfection mixture from the cells and wash the cells with 2 mL of PBS.
6. Add 2 mL of insect culture medium and continue to culture at 28 °C in an incubator without changing the media.
   Note: If the bacmid contains a fluorescent protein, its expression can be detected in cells 48 h post-transfection. Baculovirus is produced by transfected Sf9 cells and secreted into the culture medium which subsequently infects the untransfected cells. The entire cell population becomes infected with baculovirus in 5 days and shows signs of viral infection, also called cytopathic effect. These include increased cell diameter, vacuoles/granules in the cytoplasm, cessation of cell growth and dead or lysed cells are present in cell culture. However, if the transfection or infection efficiency is not optimal, the culture may not show an obvious cytopathic effect. The cytopathic effect can be visualized with an inverted phase microscope at 200 - 400X magnification. Baculovirus infected cell can be detected by staining with anti-gp64 antibody¹¹.
7. Harvest baculovirus supernatant 5 days after transfection and label as the P₁ virus stock.
8. Store the baculovirus supernatant, which is light sensitive, in the dark with 0.5% bovine serum albumin (BSA) in PBS. Store at 4 °C for the short-term (up to 90 days) or at -80 °C for the long-term.
9. Seed 6 × 10⁶ Sf9 cells in a 10-cm tissue culture-treated plate in 10 mL of insect culture medium. Add 1 mL of initial baculovirus supernatant (P₁) to the cells.
10. Harvest baculovirus supernatant (P₂) at 5^th day post-infection.
11. Seed 50 mL of Sf9 cells (3 x 10⁶ cells/mL) in suspension culture in insect culture medium in a 250-mL polycarbonate Erlenmeyer flask and place the flask in an orbital shaker incubator. Add 2 mL of P₂ stock to the cells and shake at 135 rpm while maintaining a constant temperature of 28 °C.
    Note: Three or four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high-titer baculovirus production.
12. Sequentially amplify the baculovirus supernatants until the desired volume is obtained (P₃, P₄, ….) by increasing 10 to 50-fold volume of cell culture in each subsequent infection.
    Note: P₃ is the 3^rd round of infection in which 500 mL to 1 L of Sf9 cell culture can be infected with 10 to 20 mL of P₂ baculovirus supernatant; P₄ is the 4^th round of infection in which 5 to 10 L of Sf9 cell culture can be infected with 100 to 200 mL of P₃ baculovirus supernatant, and so on. Typically, Sf9 cells are seeded at 3 × 10⁶ cells/mL in serum-free insect culture media.
13. Centrifuge the baculovirus supernatant at 1,000 x g for 30 min at 4 °C to remove cellular debris and treat the baculovirus supernatant with 50 units/mL nuclease (250 units/µL of stock) for 2 h at 37 °C to digest the genomic DNA and RNA. Filter the supernatants through a 0.45 μm filtration unit.

3. 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.

4. 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.
7. Store 100 µL of each of the fractions, including the column flow-through collected during loading of the baculovirus supernatant, the wash buffer, and the eluate. Infect these baculovirus fractions to HT1080 to evaluate the purification process (See section 6).
8. Clean the column with column cleaning buffer and wash buffer. Finally, rinse the column with 5 CV sterile 20% ethanol in water at a linear flow rate of 7.0 mL/min and store at 4 °C.
9. Clean the chromatography system with water, 1 N sodium hydroxide, again with water, and 20% ethanol in water at a linear flow rate of 50.0 mL/min and store the system in 20% ethanol.

5. Concentration of Baculovirus

1. Pre-sterilize centrifuge tubes using an autoclave. Add an equal volume of baculovirus supernatant to each ultracentrifuge tubes in a tissue culture hood. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.
2. Place each of the ultracentrifuge tubes in opposing bucket of the SW 32 Ti rotor.
3. Run the ultracentrifuge at 80,000 × g for 90 min at 4 °C.
4. Open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet.
5. Resuspend the pellet of each tube by vigorously pipetting up and down with 200 µL PBS containing 0.5% (w/vol) BSA.
6. Transfer the concentrated baculovirus to cryovials (50 to 100 µL per vial) and store at -80 °C.

6. Titration of Baculovirus

1. The day before infection, count HT1080 cells using a hemocytometer after staining cells with Trypan blue. Seed 1 x 10⁵ HT1080 cells per well in a 6-well plate in 2 mL of Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). Seed several additional wells to be able to determine the exact number of cells present at the time of infection. Culture the cells in an incubator at 37 °C and 5% CO₂.
   Note: HT1080 cells are used for titration as they express a higher level of heparan sulfate as compared to the other cell lines¹⁶. Since HT1080 cells are adherent, titration is simpler and less time consuming as compared to using the traditional Sf9-based titration.
2. On the day of infection, determine the number of cells per well by counting cells from wells. Infect the cells by adding the diluted original baculovirus which had been set aside prior to column purification, and with samples from the column flow-through, wash, and elution fractions. For each sample, determine the dilution and volume empirically based on infectious baculovirus particles, and infect each well in triplicate.
3. Two days after infection, analyze the cells for the expression of the gene of interest. Cells can be analyzed using a flow cytometer if they express a fluorescent protein (e.g. GFP)¹⁷.
4. Calculate the titers (infectious unit per mL) based on the number of cells present at the time of infection, the dilution factor, and percentages of fluorescent protein in cells using the formula: (Number of cells during infection × percentage of fluorescent protein positive cells × dilution factor)/volume of baculovirus in mL.
   Note: For example, if total number of cells per well at the time of infection is 1.2 × 10⁵, the percentage of fluorescent protein is 5%, the dilution factor is 100, and the volume of diluted baculovirus added is 10 µL, the titer is 6 × 10⁷ infectious units (IU)/mL.

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Results

The protocol presented is in a flow diagram (Figure 1). Steps include the transient transfection of Sf9 cells with bacmid DNA to produce baculovirus in adherent culture in a plate, the subsequent amplification in serum-free suspension culture, nuclease treatment and clarification by centrifugation and filtration, and the purification using the heparin affinity chromatography followed by concentration with ultracentrifugation.

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Discussion

The protocol presented here describes the production of baculovirus in Sf9 cells in suspension culture and purification of baculovirus using a heparin affinity chromatography. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus. The protocol provided here shows a significantly improved recovery of baculovirus particles as compared to recoveries achieved by others⁹.

Due to broad host range and tissue tropism, se...

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Materials

Name	Company	Catalog Number	Comments
Akta Avant 150	GE Healthcare	28976337	Chromatography system
POROS Heparin 50 µm Column	ThermoFisher Scientific	4333414	Heparin column
Ultracentrifuge	Beckman-Coulter	Non-catalog item	Concentrates virus at high-speed
Polyallomer Ultracentrifuge tube	Beckman-Coulter	326823	Concentrates virus at high-speed
MaxQ 8000 orbital shaker incubator	ThermoFisher Scientific	Non-catalog item	Shaker for suspension culture
250 mL Erlenmeyer flasks	ThermoFisher Scientific	238071	Flask for suspension culture
1 L Erlenmeyer flasks	ThermoFisher Scientific	238072	Flask for suspension culture
Microscope (Olympus CKX41)	Olympus	Non-catalog item	Cell monitoring and counting
Table top centrifuge	ThermoFisher Scientific	75253839/433607	For clarification of Baculovirus supernatant
50 ml Conical tube	ThermoFisher Scientific	14-959-49A	For collection of Baculovirus supernatant
6-well plate	ThermoFisher Scientific	07-200-80	Tissue culture treated plate
10-cm plate	ThermoFisher Scientific	08-772E	Tissue culture treated plate
Stericup-HV, 0.45 µm, PVDF	EMD-Millipore	SCHVU05RE	Filtration unit
Kanamycin	ThermoFisher Scientific	15160-054
Tetracycline	Sigma-Aldrich	T7660
Gentamycin	ThermoFisher Scientific	15750-060
Bac-to-Bac Vector Kit	ThermoFisher Scientific	10360-014	Baculovirus expression system
DH10B-T1R  Competent cell	ThermoFisher Scientific	12331-013	Competent cell for bacmid
TE buffer	In-house	Non-catalog item	10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
Plasmid Maxiprep kit	ThermoFisher Scientific	K2100-06	For bacmid purification
Sf9 Cells	ThermoFisher Scientific	11496-015	Insect cells
Grace’s Insect Cell Culture Medium	ThermoFisher Scientific	11605-094	Transfection medium
PBS	ThermoFisher Scientific	20012227	Washing cells, diluting samples
HyClone SFX-Insect cell media	GE Healthcare	SH30278.02	Serum-free insect cell growth medium
Benzonase Nuclease	Sigma-Aldrich	E1014	Enzyme to degrade DNA and RNA
Baculovirus plasmid (bacmid) DNA	In-house	Non-catalog item	Bacmid for Baculovirus Production
Cellfectin II	ThermoFisher Scientific	10362	Transfection reagent for insect cells
Bovine serum albumin (BSA)	Sigma-Aldrich	A4737	Stabilizes Baculovirus
Cryovial	Thomas Scientific	1222C24	For storage of Baculovirus
HT1080 cell line	ATCC	CCL-121	Fibroblast cell line
DMEM	Sigma-Aldrich	D6429	Growth media for cell lines
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
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
Ethanol	Sigma-Aldrich	E7073	For Akta Avant cleaning