Fast-Trap Adenovirus/Lentivirus Purification and Concentration Kit

Frequently Asked Questions

• These kits have been optimized and tested using a VSV-G pseudotype Lentivirus, Adenovirus serotype 5 and AAV serotype 2. Other similar viruses may work, but have not been evaluated for performance. We have heard from customers that the AAV kit will work with AAV serotypes 1 and 7 as well.

• For Adenovirus, the equivalent of between 2-20 T-150cm² flasks generally yields sufficient amounts of virus for use with this kit. However, the titer of the total number of viral particles should be used as the basis for the amount of virus used with the Fast-Trap kit (e.g. depending on cells, specific virus, processing conditions, etc. the amount of virus generated per T-flask will vary considerably. Therefore, some customers may reach capacity with 10 flasks while others may reach capacity with 20.
  
  For Lentivirus, the equivalent of between 2-8 T-75 flasks generally yields sufficient amounts of virus for use with this kit. Again, the titer of the total number of viral particles should be used as the basis for the amount of virus used with the Fast-Trap kit.
  
  For AAV, the kit can handle virus produced from up to 1025 cm² cell culture surface area. Typically this limit is equivalent to 13 T-75 flasks but again, the titer of the total number of viral particles should be used as the basis for the amount of virus used with the Fast-Trap kit.

• Yes. It is possible to load sample in sequential steps as long as capacity of the device is not exceeded. Also, make sure to change out the 50ml collection tube with an empty tube to ensure the filtrate volume does not exceed 50ml.

• No. However, optimal results are achieved when using a nuclease pre-treatment step prior to purification. Faster flow rates have been observed with samples treated with Benzonase.

• As virus binds to the membrane there is less space available in the pores of the membrane thereby decreasing the rate at which liquid can flow through the membrane. A significant decrease in flow rate may be observed if too much virus is loaded onto the device. Purification of virus titers above recommended capacity is best performed by splitting the challenge between multiple purification devices.

• Ensure the vacuum pressure is at least 15” Hg for the device to function properly. Also, make sure the Fast-Trap purification device and 50ml conical tubes are tight and securely fastened.

• A crude virus stock with too much cellular debris will clog the clarification unit. This can be prevented by performing the post-harvest centrifugation step (after freeze/thaw for Adenovirus) until no debris is visible in the supernatant. If a clarification unit clogs, the crude virus sample can undergo an additional centrifugation step followed by clarification with another Steriflip-HV unit.

• Yes. Diluting the starting virus sample in the 10X binding buffer allows for optimal binding of the maximum amount of virus particles to the membrane.

• A second 3ml elution typically results in an extra 1-5% recovery.

• No. Sterilization of final concentrated sample may be performed using a Millex-GP Filter Unit.

• The purified virus eluted from the Fast-Trap device is in a high salt buffer. Exchanging purified virus into the optimal buffer for storage and/or downstream processing is recommended. An example of one storage buffer is: 20mM Tris-HCl pH 8.0, 250mM NaCl, 5% sorbitol, and 0.001% PF68 (pluronic acid)

• Titer of starting samples exceeded maximum capacity of Fast-Trap device (Adenovirus capacity <1x10¹³ total particles/1x10¹¹ infectious particles, AAV capacity is 1x10¹² total viral particles, and Lentivirus capacity < 2x10⁸ infectious particles)
  
  Reducing the sample volume below 500µl for Adenovirus or 200µl for Lentivirus in the Amicon Ultra during buffer exchange/concentration may cause the virus to aggregate resulting in decreased recovery of infectious virus particles.
  
  Also, ensure that during the elution procedure 5 drops of elution buffer have filtered through the membrane prior to the 5 minute incubation. This allows maximum interaction between the elution buffer and the virus adsorbed onto the membrane. Failure to follow this procedure may result in decreased virus recovery.
  
  Purification of crude virus containing sucrose/glycerol (sometimes used by customers to stabilize Lentiviral samples) can result in low virus recovery.

• Yes. Millipore provides an optional funnel attachment (Catalogue Number: SC50FL025) for the top of the Fast-Trap purification device that allows equilibration, sample, wash, and elution buffers to be added directly to the purification device.

• The Adenovirus kit was developed using Adenovirus Serotype 5, which is the most commonly used Adenovirus for virus purification. We do not know whether or not the kit will work the same with other serotypes of Adenovirus. It may, but it has not been tested on other serotypes.
  
  The Lentivirus kit was developed using the VSV-G Serotype of Lentivirus, which is the most commonly used Lentivirus type for virus purification. We do not know whether or not the kit will work the same with other pseudotypes of Lentivirus.
  
  Tha AAV kit was developed using AAV serotype 2. We have heard from customers that it works with AAV serotypes 1 and 7 but we have not tested this ourselves.

• Flow rate is expected to decrease when using high virus titer. Do not exceed device capacity of approximately 1x10¹³ total virus particles for the Adenovirus kit, 1x10¹² total viral particles for AAV and 2x10⁸ infectious particles for the Lentivirus kit.

• The elution buffer contains high (>1M) salt/Tris-HCl (pH 8), and should be buffer exchanged (w/2 spin protocol) with the Amicon Ultra before using for their application or addition of glyerol for long-term storage. If it is not buffer exchanged, the tube could form salt crystals and might not freeze properly. Also, if kept in the elution buffer, their downstream applications (i.e. with cells) might be adversely impacted.

	1. Examples of "exchange" buffer could be:
		a. 20 mM Tris-HCl, pH 8/200 to 250 mM NaCl/10 mM MgCl2. b. 20 mM Tris-HCl, pH 8/250 mM NaCl/10 mM MgCl2/5% Sorbitol c. If recovery is low, add a biological surfactant such as 0.001% PF68 (10% Pluronic acid, Sigma #P5556) to "a" or "b" above.
	2. Customer can also try the buffer that they typically use for an "exchange" buffer.

• The Fast-Trap Lentivirus was compared side-by-side with the System Biosciences PEG-it precipitation: (Catalogue Number LV810A-1):

• Both products had similar recovery (~57% of input 2.2x10⁷ infectous viral particles).
• However, the purity (using gel electrophoresis and Western Blotting) was very poor with the PEG-it when compared to the Fast-Trap
• Although there is a lot of hands-off time with the PEG-it, it took over 12.5 hours to complete, vs. < 2 hours with the Fast-Trap.

• New membranes were developed for these kits. For Lentivirus and Adenovirus the membrane is an anion exchanger. For Adeno-Associated Virus the membrane is a cation exchanger.

• The AU cut offs by virus are the ones recommended in the virus purification tech brief for the specific virus type.