Editing Adenovirus Purification using Q Membrane (section)

== Introduction  ==
<div>Adenovirus vectors (rAd) are widely used in gene therapy to 
deliver genes of interest. It is therefore essential to develop a purification process that clears the adenovirus of undesired contaminants while purifying high titre stock adenovirus. The current standard method of purification uses a CsCl density gradient, which is formed during centrifugation. The limitations of this procedure include the speed of the process, the cost of trying to scale up to pharmaceutically relevant levels and the potential carryover of high molecular weight proteins. 

A chromatographic method, which includes a matrix that has a high dynamic binding capacity for the rAd and is able to withstand high flow rates to render faster purification, will enable researchers to purify concentrated quantities of rAd and scale the process from bench to production. Mustang Q membrane is such a&nbsp; chromatography&nbsp; matrix and has the additional benefits of being available in a sterilizable, disposable format so that cleaning validation protocols and all costs associated with that process are eliminated. Other benefits of Mustang Q chromatography include maintaining resolution at faster flow rates than traditional ion exchange resins, eliminating column packing, and easily adapting to bench-scale work. All of these factors will speed up the drug development process. 
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Materials and Methods 
<div style="padding-top: 10px; margin-bottom: 10px;"><div>'''''Adenovirus Preparation'''<br>''Harvest infected cells by pelleting and resuspending the pellet in PBS. Lyse host cells by three freeze-thaw cycles and separate the cell debris by spinning down at 20,000 x g for 60 minutes at 4 ºC. The crude cell lysate can then be filtered through a 25 mm Acrodisc® syringe filter with 1.2 µm Supor® membrane followed by a 25 mm Acrodisc syringe filter with 0.2 µm Supor membrane. This two-step filtration process may require more then just a single Acrodisc filter depending on the effectiveness of the pelleting procedure. At this stage, the filtered rAd containing lysate should be clear. 
Incubate rAd at room temperature for 30 minutes with either 100 units of Benzonase or 100U DNAase/RNAse cocktail per mL of cell lysate. Filter with 0.2 µm filtration (25 mm Acrodisc PF syringe filter with Supor membrane, PN 4187) to ensure that the lysate is free of particulate matter. Adjust suspension to a final concentration of 0.3 M NaCl. This crude lysate sample can be applied directly onto a Mustang membrane for isolation of high titre rAd. The Mustang Q membrane column (PN MSTG25Q6 or coin) should be preconditioned with 0.3 M NaCl in 25 mM HEPES, pH 7.4 (Figure 2) for a quick purification process. 

'''''Binding Capacity Determination''<br>'''To determine binding capacity of Mustang Q membrane for rAd, aCsCl purified adenovirus preparation (1.24 E4 VP/mL) was loaded onto an Acrodisc unit with Mustang E membrane (0.03 mL bed volume). Optical density was determined at 280 nm for real time monitoring of breakthrough (Figure 1). 

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'''Figure 1<br>'''Determination of Dynamic Binding Capacity of Mustang Q Membranes for Purified rAd 

&lt;img src="[http://www.pall.com/images/laboratory/pg169Fig1.jpg http://www.pall.com/images/laboratory/pg169Fig1.jpg] /&gt; 

The binding capacity for adenovirus in crude lysate was determined 

(Figure 2) by isolating the adenovirus on an Acrodisc device, quantifying the material isolated (data not shown) then reloading the virus (1.8E12 VP/mL) onto an Acrodisc unit with Mustang membrane and monitoring for the breakthrough point. The curve in Figure 2 indicates that the dynamic binding capacity of Mustang membrane for crude lysate purified virus at 10% breakthrough is 4.9 x 10<sup>13</sup> VP/mL of membrane. This shows that the membrane is capable of binding significant amounts of virus for both concentrating purified viral samples or purifying virus out of crude lysate, which could save time and money, and decrease the exposure of a researcher to hazardous chemicals by eliminating the use of CsCl. 

<br> '''Figure 2<br>'''Determination of Dynamic Binding Capacity of Mustang Q Membranes for rAd from Lysate 

&lt;img src="http://www.pall.com/images/laboratory/pg170Fig2.jpg"&gt; 

'''''Elution of Adenovirus from Mustang Q Membrane'''''<br> 

To determine the elution conditions for rAd from Mustang membrane, an ÄKTA Explorer* 10 LC system was setup in a 4 ºC refrigerator and ~3<sup>11</sup> infective adenovirus particles in 0.2M NaCl were loaded onto an 0.18 mL bed volume (BV) Mustang Q column. The viral particles were eluted at 20 column volumes (CV)/min (3.5 mL/min) using buffer A (0.2M NaCl in 20mM HEPES pH 7.5, 1mM MgCl<sub>2</sub>) and buffer B (1M NaCl in 20mM HEPES pH 7.5, 1mM MgCl<sub>2</sub>). The gradient was 0 to 40% B in 0.5 CV, then held at 40% B for 20 CV and a final elution gradient of 40 to 100% B in 10 CV 0.5 mL fractions was collected. 

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An aliquot of each fraction was run on an SDS-PAGE gel to determine which fractions contained adenovirus. Sucrose was added to a final concentration of 8% to each fraction containing adenovirus. These fractions were pooled for further processing. This pooled sample was then concentrated and desalted on a Microsep™ centrifugal device with 300K Omega™ membrane (PN OD300C41 or OD300C46). 

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The viral peak eluted at 30 mS/cm in the 0 to 40% B gradient (Figure 3). The cellular DNA eluted in the 40 to 100% B gradient (based on the OD<sub>280</sub>/OD<sub>260</sub> intensities). These two peaks were completely resolved when the viral peak was washed with 20 CV of 40% B. This extended hold at 40% B was necessary to ensure the viralpeak reached baseline. 

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'''Figure 3''' <br> 

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&lt;img src="http://www.pall.com/images/laboratory/pg170Fig3.jpg"&gt;<br> &nbsp; 
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