Editing Adenovirus Purification using Q Membrane (section)

== Flow Rate Affect on Capacity  ==
<div>To determine whether the flow rate of Mustang membrane would 
be a significant barrier in using this technology for viral purification, 3.7 x 10<sup>11</sup> adenoviral plaque forming units from a CsCl purified stock were loaded at flow rates of 20 and 40 column volumes per minute. These flow rates are 200 and 400 times faster than traditional resin-based chromatography, which would not be able to capture adenovirus at these speeds. Biological plaque forming units (pfu) were assayed by agar overlay of infected cells in 60 mm<sup>2</sup> (Table 1). 

'''Table 1'''<br> Adenovirus Particle Counts and Plaque Forming Units (pfu) 

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! 1.1 
! Adenovirus Particles 
! &nbsp;% Recovery&nbsp;&nbsp;&nbsp;&nbsp;
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| Loading capacity&nbsp;&nbsp;&nbsp;&nbsp; 
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| Start 
| 1.0 x 10<sup>13</sup> 
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| Final 
| 8.0 x 10<sup>12</sup> 
| 80
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! 1.2 
! Adenovirus pfu&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 
! &nbsp;% Recovery&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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| Flow rate capacity&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 
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| Start 
| 3.7 x 10<sup>11</sup> 
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| At 20 cv/min 
| 2.4 x 10<sup>11</sup> 
| 65
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| At 40 cv/min 
| 1.7 x 10<sup>11</sup> 
| 46
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! 1.3 
! Adenovirus pfu/mL 
! Adenovirus pfu
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| Cell lysate recovery 
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| Fraction A6 
| 1.6 x 10<sup>10</sup> 
| 5.0 x 10<sup>10</sup>
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| Fraction A7 
| 2.4 x 10<sup>11</sup> 
| 4.8 x 10<sup>11</sup>
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| Fraction B9 
| 1.0 x 10<sup>9</sup> 
| 2.0 x 10<sup>9</sup>
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From this information, it appears that speed does affect binding 

capacity but only when the speed is already 400 times faster than other resin-based chromatographic protocols. 

<br> '''''Adenovirus PFU Recovery from Cell Lysate<br>'''''Adenovirus-containing supernatant from centrifuged 

and filtered cell lysate was adjusted to a final concentration of 0.4M NaCl and loaded at 2 mL per minute onto the 180 µL Pall Mustang Q Acrodisc filter unit. Elution of adenovirus was achieved with a linear gradient from 0.4M to 0.6M NaCl (Figure 4). Fractions were buffer exchanged with a standard HEPES buffer and assayed for biological plaque forming units (Table 1.3). 

<br> '''Figure 4''' 

&lt;img src="http://www.pall.com/images/laboratory/pg171Fig4.jpg"&gt; 
</div>


CsCl Purification Method Versus Mustang Q Purification
</div><div style="padding-top: 10px; margin-bottom: 10px;"><div>Adenovirus preparations which started with the same cellular 
lysate material filtered to 0.2 µm were then purified using either Mustang Q membrane or ultra-centrifugation CsCl gradient. Side-by-side analysis such as particle count recovery, infectious units, cytotoxicity, and purity on SDS-PAGE were performed. 

Both methods gave comparably high titers and transgene expression, and the total amount of virus recovered from each method was equivalent (Table 2). One clear advantage of the Mustang Q membrane process was that purified virus could be obtained within a single day, compared to the multi-day process needed for the conventional method. However, the Mustang Q membrane chromatography purified material contained higher total protein and thus a lower final specific activity. Regardless, the Mustang Q membrane chromatography purified virus demonstrated far less cytotoxicity in primary neurons then normally experienced with CsCl purified virus. 

'''Table 2<br>'''Biological Assay Data on Adenovirus Serotype 5 Activity in Crude Prep, Mustang Q Purified Adenovirus and CsCl Purified Adenovirus 

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! Crude Prep 
! Mustang Q<br> 
Purification Method 

! CsCl Purification<br> 
Method 

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| Total volume 
| 3.5 mL<br> 
(remaining volume) 

| 3 mL<br> 
(remaining volume) 

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| <br> 
| <br> 
| <br>
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| Number of infected<br> 
cells for each method 

| 2.5E+08 of<br> 
293S cells 

| 2.5E+08 of<br> 
293S cells 

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| <br> 
| <br> 
| <br>
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| Total infectious<br> 
particles (TCID50%) 

| 1.15E+10 
| 1.98E+10
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| <br> 
| <br> 
| <br>
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| Infectious particles/mL<br> 
(TCID50%/mL) 

| 3.3E+09<br> 
(28.7% recovery) 

| 6.6E+09<br> 
(33.3% recovery) 

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| <br> 
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| OD (OPU/mL) 
| 3.65E+12 
| 3.85E+11
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| <br> 
| <br> 
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| Total OD (OPU) 
| 1.27E+13 
| 1.15E+12
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| <br> 
| <br> 
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| Total protein mg (BCA) 
| 82.31 
| 14.38
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| <br> 
| <br> 
| <br>
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| Protein mg/mL (BCA) 
| 23.51 
| 4.79
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