Virus Concentration by Ultrafiltration

• Virus Isolation, Propagation, and Characterization
• General Methods for Viral Propagation
• Virus Concentration for High Titer Virus Stocks
• Virus Concentration with Amicon^® Ultra Centrifugal Ultrafiltration Devices
• Conclusions
• Centrifugal Ultrafiltration Devices

Virus Isolation, Propagation, and Characterization

Figure 1. Ultrafiltration for concentration of virus. Fluid passes through the membrane filter, while large viral particles are retained.

Virus isolation and propagation methods are critical in research, vaccine production, and diagnostic workflows. Basic research labs use viral stocks to study viral biology and pathogenesis. Vaccine production requires small-scale and large-scale preparation of virus. And although most diagnostic laboratories now use non-culture approaches for viral disease diagnosis, virus isolation in cell culture remains a useful approach when viable isolates are needed, when viable and nonviable virus must be differentiated, and in cases where culture-based methods are able to provide results faster than non-culture methods.

Viruses can be propagated in cell culture to generate virus stocks. Cultured cells are inoculated with viral stock from seed virus, a commercial source, or infected tissue. After incubation, infected cells are lysed to harvest viral particles, or released virus is harvested directly from cell supernatants. The virus stock is then titered, aliquoted, and stored at −80 °C for later use.

Isolation of virus from clinical samples also exploits cell culture for viral propagation. Clinical samples are prepared by sample homogenization and extraction into a liquid medium, which is then centrifuged and used to inoculate cell cultures.

In order to achieve high titer virus stocks, it is necessary to concentrate purified virus particles. Amicon^® Ultra and Centricon^® Plus centrifugal ultrafiltration devices with regenerated cellulose membranes can be used to concentrate virions and virus solutions (Figure 1). This protocol describes the use of Amicon^® and Centricon^® centrifugal ultrafiltration devices for the concentration of three common types of viruses: lentivirus, adenovirus and adeno-associated virus (AAV).

General Method for Viral Propagation

1. Packaging cells: Host cells were plated to achieve 80–85% confluence.
2. Transfection: Cells were infected with viable virus particles.
3. Harvesting virus: Depending on the virus, either cell supernatant or pelleted cells, or both, were used to purify the recombinant virions. Four rounds of freeze/thaw cycles were applied to release virus from cells. Cell debris was then separated from cell lysate by centrifugation.
   
4. Virus purification: Virus was purified from cell lysate by density gradient ultracentrifugation or column/membrane chromatography.
5. Purified virus solutions were concentrated and buffer exchanged into an appropriate storage solution using either passive dialysis, diafiltration, or centrifugal ultrafiltration with Amicon^® Ultra and Centricon^® Plus 70 devices.

Virus Concentration for High Titer Virus Stocks

To produce high titer viral stocks, the purified virus can be concentrated using centrifugal ultrafiltration. The correct choice of device, membrane material, molecular weight limit or cut-off, centrifugation speed, centrifugation time and buffer composition are critical for high recovery of infective viral particles. Amicon^® Ultra centrifugal ultrafiltration units have specified nominal molecular weight limits (NMWL) defined by their filter membrane. Amicon^® Ultra centrifugal ultrafiltration devices with a 50 kDa NMWL Ultracel^® were used to concentrate adenovirus and adeno-associated virus (AAV) solutions. For lentivirus concentration, devices with both 50 kDa and 100 kDa NMWL membranes were used.

Factors to consider when selecting an ultrafiltration method and membrane nominal molecular weight size:

1. Viral particle size: Size can be estimated from published sources, or by measurement techniques such as microscopy, laser diffraction, and dynamic light scattering.
2. Size of key separation targets in solution: The size of proteins, antibodies, drugs, chemicals, and other particles that need to be separated will affect membrane size selection.
3. Sample volume: Processing volumes ranging from ≤0.5 mL to 70 mL are compatible with centrifugal ultrafiltration (cUF) devices.

To retain viral particles, the molecular weight cut-off of the filter membrane needs to be smaller than the particle (~2 times smaller than the molecular weight of the viral particle), but large enough to allow smaller components to filter through
(Table 1).

Table 1. Selection of ultrafiltration membrane NMWL based on viral particle size.

Ultrafiltration Membrane NMWL (kDa)	Nominal Pore Size (nm)	Recommended Ultrafiltration Nominal Retaining Size Range
		Diameter (nm)		Molecular Weight (kDa)
		Minimum	Maximum	Minimum	Maximum
3	0.3	1	1.5	6	20
10	1	3	9	20	90
30	3	9	15	60	180
50	5	15	30	100	300
100	10	30	90	200	900

Virus Concentration with Amicon^® Ultra Centrifugal Ultrafiltration Devices

Table 2 shows results for concentration of adenovirus, lentivirus, and AAV from both crude cell lysate and virus purified by chromatography. Viral particles were effectively concentrated to high titers with high recoveries using Amicon^® Ultra centrifugal ultrafiltration devices.

Table 2. Concentration of virus using ultrafiltration devices. We do not recommend concentrating the virus to higher than 1013 particles/mL due to potential virus aggregation.

Starting sample	Device	Starting virus titer	Starting volume (mL)	Concentration factor	Spin time at 1500 x g (min)	Recovery (%)
Adenovirus in crude cell lysate	Amicon® Ultra-4 50 kDa	1 x 108	4	27X	20	>100
Adenovirus purified, in 1M salt buffer	Amicon® Ultra-4 50 kDa	1 x 108	4	45X	10	85
AAV in crude cell lysate	Amicon® Ultra-4 50 kDa	2 x 107	4	20X	35	50
AAV in crude cell lysate	Amicon® Ultra-15 50 kDa	2 x 107	10	25X	45	76
AAV diluted in 100 mM Tris, 250 mM NaCl buffer	Amicon® Ultra-4 50 kDa	5 x 106	4	20X	20	100
Lentivirus in cell culture supernatant	Amicon® Ultra-4 100 kDa	9 x 104	4	100X	30	>100
Lentivirus in cell culture supernatant	Amicon® Ultra-4 50 kDa	9 x 104	4	100X	30	93
Lentivirus in cell culture supernatant	Centricon Plus-70 100 kDa	8 x 103	65	80X	30	81

It is important to remember that most of the viruses cannot be efficiently concentrated and stored in low salt buffers. Centrifugal concentration of the virus in < 250 mM salt solutions leads to virus aggregation and decreased infectivity.

Concentrated viral stock can be filter-sterilized through 0.22 µm filters. Ultrafree^®-MC GV filter units can be used for volumes of < 0.5 mL.

Conclusions

We developed a method to validate ultrafiltration using Amicon^® Ultra and Centricon^® Plus centrifugal devices as a fast and cost-effective means for achieving high titers of viruses. This method can be used to concentrate viruses from cell culture supernatants, cell lysates, and other sources. After concentration, viral stock can be filter-sterilized using Ultrafree^® filter units. For highest recovery of infective viral particles, it is critical to select the proper ultrafiltration device, membrane filter, molecular weight cut off (MWCO), centrifugation speed, and centrifugation time.

Centrifugal Ultrafiltration Devices

Device Nominal Molecular Weight Limit, NMWL (kDa)	Amicon® Ultra centrifugal ultrafiltration devices				Centricon® Plus 70 centrifugal devices
	0.5 mL	2 mL	4 mL	15 mL	15 – 70 mL
3	UFC5003		UFC8003	UFC9003	UFC7003
10	UFC5010	UFC2010	UFC8010	UFC9010	UFC7010
10 (IVD*)			UFC4010D*	UFC9010D*
30	UFC5030	UFC2030	UFC8030	UFC9030	UFC7030
50	UFC5050	UFC2050	UFC8050	UFC9050
100	UFC5100	UFC2100	UFC8100	UFC9100	UFC7100

* registered for IVD use