3.3. Virus Purification via Heparin-Affinity and Sucrose Gradient Ultracentrifugation

The protocol described herein outlines the production of HBV particles from HBV- or recombinant HBV-expressing cell lines (e.g., HepAD38, HepG2.2.15). An HBV genotype D high-producing cell line (HepG2-pB-HBV1.3) is available upon request.

Step 1: Proliferate producer cells until they reach confluence on 5 collagen-coated T175 flasks.

CRITICAL Cultivate all HepG2-based cell lines on collagen-coated plates and flasks. It is sufficient to collagen-coat the flasks by rinsing them with a H₂O/0.02% Collagen R solution for 5 min followed by aspiration of the remaining Collagen solution.

Step 2: Collagen coat a 10-layer Corning HYPERflask with 550 mL of H₂O/0.01%-Collagen R solution and incubate overnight at room temperature. Take care that upper and lower surfaces are equally well coated. Collect H₂O/0.01%-Collagen R solution and store at 4 °C for future HYPERflask collagen-coatings. Solution can be reused approx. 5 times.

Step 3: Pre-warm 550 mL of cultivation medium to 37 °C and add 90% of the cells trypsinized off the 5 collagen-coated T175 flasks into the pre-warmed medium bottle. Cultivate the remaining 10% cells again in the 5 collagen-coated T175 flasks for step 6.

CRITICAL Dispense the cells homogeneously by pipetting them against the wall of the T175 flask.

Step 4: Pour 550 mL of the warm cell-suspension into the HYPERflask.

TROUBLESHOOTING

CRITICAL Remove all remaining air by applying pressure to the center of the HYPERflask, enabling homogenous cell spreading and guaranteeing space for medium extension during 37 °C incubation to avoid a pressure burst of the HYPERflask.

Step 5: Culture the cells in the HYPERflask for 4 days at 37 °C in a 5% CO₂ atmosphere to achieve confluence on the upper surfaces of the flask.

Step 6: Pre-warm 550 mL of cultivation medium to 37 °C, trypsinize the cells regrown in the 5 collagen-coated T175 flasks (step 3) and suspend cells in the pre-warmed medium. Discard medium from the HYPERflask and add 550 mL of cell-suspension to the HYPERflask, flip the flask and cultivate it upside down overnight to allow attachment and growth of cells on the lower surfaces of the flask.

CRITICAL Dispense the cells homogeneously by pipetting them against the wall of the T175 flask.

CRITICAL Avoid drying out of cells in the HYPERflask by minimizing time between medium exchange.

Step 7: Cultivate HYPERflask at 37 °C in a 5% CO₂ atmosphere and harvest the supernatant in 4 day intervals.

CRITICAL Discard the first medium collected after each cell plating cycle because it contains a high number of dead and non-attached cells.

TROUBLESHOOTING

CRITICAL As it is known that there is a maturation of HBV particles (t_1/2 = 4.7 h) [36] and secretion of HBV particles takes place over time, do not harvest supernatant earlier than 2 days after each medium exchange. Depending on the cell density, cells will start detaching and dying after 5–6 days without medium exchange, leading to an optimal exchange interval of 4 days. To facilitate a weekly routine, we recommend exchanging medium in alternating 3 day/4 day intervals.

CRITICAL Store cell culture supernatants at 4 °C overnight prior to heparin-affinity chromatography in order to precipitate serum lipoproteins and cell debris.

CRITICAL Consider: Titer of HBV in the supernatants should be higher than 10⁷ GE/mL to achieve adequate final titers.

PAUSE POINT Generation of HBV-rich supernatant is now completed. Store supernatants at 4 °C but consider that HBV infectivity drops with increasing storage time. For best results, process supernatants after overnight incubation at 4 °C. To facilitate a weekly routine, we recommend purification of two pooled supernatants from the same HYPERflask every 7 days.

Step 8: Filter cold, HBV-containing supernatant through a 0.45 µm sterile filter to remove precipitated proteins and remaining cell debris.

TROUBLESHOOTING

CRITICAL For best filtration results and saving of filter devices, we recommend pre-filter inlays.

Step 9: Assemble the purification apparatus without connecting the heparin columns and wash the system with 50 mL of 70% ethanol followed by 50 mL 1× PBS solution (Figure 7a,b).

TROUBLESHOOTING

CRITICAL Ensure that the tubing is completely filled with PBS and all remaining air bubbles are removed. Failure to do so will lead to damage of the heparin columns.

Set-up of the heparin-affinity chromatography apparatus: (a) Schematic and (b) Pictured set-up for loading of heparin columns. Heparin columns are assembled in parallel connection for loading; (c) Schematic and (d) Pictured set-up for elution of heparin columns. Heparin columns are assembled in serial connection for elution.

Step 10: Connect 2 heparin columns (HiTrap heparin 5 mL) to the stopcocks in a parallel connection (Figure 7a,b) for every 550 mL of supernatant. To facilitate a weekly routine, we recommend using 4 heparin columns for the weekly volume of 1100 mL.

TROUBLESHOOTING

Step 11: Store filtered supernatant on ice and perfuse it through the heparin columns at a flow rate of approx. 10 mL/min per column (e.g., 40 mL/min for 4 heparin columns).

TROUBLESHOOTING

CRITICAL Higher loading speed may lower the lifetime of the columns.

Step 12: After a complete perfusion of the supernatant, remove the columns and flush the system with elution buffer.

CRITICAL It is important that the columns do not run dry. Ensure that no air is left in the system before you reattach the columns.

Optional To increase HBV stock purity, but at costs of lower final concentration, columns can be washed with 10 mL of 1× PBS per column.

Step 13: For elution, assemble columns using a serial connection (Figure 7c,d) and elute with 2 mL/min elution buffer. If you use more than 1 column, discard for each additional column 4 mL of the eluate due to the dead volume and collect the next 17 mL (e.g., using 4 columns, discard 12 mL and collect the following 17 mL (Figure 4e)).

TROUBLESHOOTING

CRITICAL Due to variations in dead volume, do not discard any eluate if you elute from a single column.

CRITICAL Do not exceed the 2 mL/min elution speed in serial connection to avoid damaging the columns and tubing.

CRITICAL Do not exceed more than 4 columns per elution since they can obstruct during serial elution due to high protein aggregation.

CRITICAL Do not store the eluent on ice since HBV particles can precipitate due to high salt concentration. Keep eluent at room temperature until step 14.

PAUSE POINT Purifying and concentrating HBV via heparin-affinity chromatography is now completed and eluent is ready to be further purified, concentrated and buffer exchanged via sucrose gradient ultracentrifugation. We recommend to not exceed storage of eluent for more than 2 h.

Step 14: Carefully overlay sucrose solutions and collected eluate in a SW32Ti ultracentrifugation tube by the following instructions: (from bottom to top) 3 mL sucrose 60%; 7 mL sucrose 25%; 9 mL sucrose 15% and 17 mL eluate (Figure 5a).

Step 15: Centrifuge the gradient at 175,000× g (32,000 rpm) at 10 °C for 3.5 h to perform a separation (Figure 5b).

TROUBLESHOOTING

CRITICAL Use fast acceleration and brake.

Step 16: Fractionate the gradient in 2 mL fractions using a fraction recovery system. The second fraction from the bottom of the gradient is the HBV-rich fraction (Figure 5b).

TROUBLESHOOTING

CRITICAL If you do not have access to a fraction recovery system, you may carefully insert a long blunt cannula into the tube and aspirate 2 mL from the bottom. Discard this fraction and aspirate another 2 mL from the bottom to obtain the HBV-rich fraction.

Step 17: Clean Heparin HiTrap columns by applying 20 mL of 10× PBS solution per column at 5 mL/min in parallel connection. Flush them afterwards with heparin column storage buffer and store them air-tight at 4 °C.

Step 18: Clean tubings, stop-cocks and fraction recovery system with 70% ethanol to inactivate potentially remaining HBV and store at room temperature.

Optional Divide HBV stock into smaller aliquots prior to freezing. For in vitro infection experiments, we recommend diluting the sucrose stock 1:1 with fetal bovine serum to lower viscosity and to stabilize HBV particles by covering them with lipoproteins.

PAUSE POINT Generation of HBV-rich stock is now completed. Stocks should have titers up to 100-times higher than the supernatant (typically higher than 10¹⁰ GE/mL) and can be stored for more than three years at −80 °C without a notable loss in infectivity. However, we recommend to avoid multiple freeze-thaw cycles and storage in non-frozen stages due to the loss of infectivity (Figure 6b,c).

Overall → Problem: HBV is not infectious → Possible reasons: HBV has been precipitated or denatured → Solution: Check all used buffers. For correct osmolarity, it is crucial to prepare sucrose gradient solutions in 1× PBS. Due to high-salt concentration during elution, it is important to use elution buffer at room temperature and not to store eluate on ice. Thoroughly wash out ethanol-containing buffer with 1× PBS prior to usage of the purification apparatus.

Step 6 → Problem: Supernatant is cloudy (note: supernatant can be slightly cloudy due to detached cells without affecting the purification process) → Possible reasons: Dead detached cells or bacterial contamination → Solution: Cultivate a 25 mL supernatant-sample in a collagen-coated T175 flask for 4 days and examine it under the microscope.

→ In the case of bacterial contamination (obvious cloudiness and visible bacteria at 40× magnification): start over again from Step 1 and ensure to thoroughly clean spilled medium in the screw thread of the lid after each medium exchange.

→ In the case of dead cells: Check HBV titer (HBsAg or GE/mL) of the supernatant at the next medium exchanges. If titer is further decreasing start over at Step 1. If titer is stable, continue with the protocol and try to avoid to keep too much air in the flask after each medium exchange. Carefully pat out the bubbles in front of the air trap of the HYPERflask. Consider cells are growing on both sides of the layers such that remaining bubbles will lead to punctual drying-out and death of some cells. Additionally, try to minimize time during medium exchange to avoid overall drying-out of the cells.

→ In the case of living cells: Cells probably did not attach adequately to the HYPERflask or have been detached. Check HBV titer in the supernatant of the next medium exchange. If titer has been decreased further start over at Step 1, check collagen solution and increase collagen incubation time in the HYPERflask for up to 2 days. If titer is stable or has been increased, pay attention to a more gentle medium exchange and avoid shaking or friction of the flask.

Step 6 → Problem: HBV titer is less than 10⁶ GE/mL → Possible reasons: Cells are not dense enough (consider that the bottom layer of the HYPERflask can be examined under the microscope) or do not produce sufficient amounts of HBV → Solution: Keep cells in culture and determine HBV titer at each medium exchange. If titer is not increasing over time until reaching a plateau due to producer cell confluence, use a lower passage of the cell line, escalate antibiotic selection or perform a single-cell selection of a high-producer cell clone or replace the producer cell line (note: a high-producing cell line (HepG2-pB-HBV1.3) is available on request). Check for contamination with other cells, bacteria or mycoplasma.

Step 10 → Problem: Supernatant is running through parallel heparin columns at different flow-rates (note: slight differences in flow-rate are due to manufacturing and are unproblematic since it will balance during the purification process)→ Possible reasons: One or more columns are blocked or have been used too many times → Solution: Wash blocked column with 50 mL of 10× PBS. If problems remain, exchange the blocked column.

Step 10 → Problem: Blocking of heparin columns during loading → Possible reasons: Remaining cell debris or proteins plugging the column → Solution: Cooling of the supernatant overnight at 4°C to allow clotting of cell debris and proteins prior to filtering (Step7). Carefully wash blocked column with 50 mL of 10× PBS. If problems remain, exchange it with a new heparin column.

Step 12 → Problem: Blocking of heparin columns during elution → Possible reasons: HBV concentration is too high and virus is precipitating inside the columns → Solution: Reduce the volume of supernatant applied to each column during loading or elute each column individually. Carefully wash blocked column with 50 mL of 10× PBS. If problems remain, exchange with new heparin columns.

Step 12 → Problem: HBV is not present in the eluate → Possible reasons: HBV does not bind to the columns or HBV does not elute → Solution: Check HBV titer pre- and post-column. If HBV did not bind to the columns and titers do not differ, exchange the columns. If titers differ, check elution buffer.

Step 1–7: Cultivation of producer cells and collection of HBV particle-rich supernatant: 14 days → Production of supernatant every 3 to 4 days for more than 12 months.

Step 8–13: Purification and concentration of HBV particles via heparin-affinity chromatography: 90 min

Step 14–18: Buffer exchange, concentration and purification of HBV particles via sucrose gradient ultracentrifugation: 4 h