Virus Purification

Quick Purification Strategy

Adenovirus Purification
Adenoviruses are commonly used as gene transfer vectors in gene therapy and clinical trials. However, their large size,
complexity, and acid labile nature lead to challenges in their downstream purification. The following purification strategy
provides an easy two-step protocol to purify adenoviruses.

Initial resin
screening

UNOsphere S
Nuvia S
(cation exchange) (cation exchange)™

™

Nuvia cPrime(mixed mode)

UNOsphere Q(anion exchange)

Nuvia Q(anion exchange)—++++—
++++—
++++™

™

™

™

Results
Virus in flowthroughVirus in eluate

Conclusion

+++++

+++
++

Poorly suited for bothcapture and polish steps

Considered for direct mass capture

Virus binding to Nuvia cPrimerequires low salt and pH
conditions. Therefore it cannot
be used for the polish step afteranion exchange, where theresulting feedstream would
likely have high ionic strengthand high pH.

Nuvia Q can adsorb virus at
higher NaCl concentrations
relative to UNOsphere Q.
Nuvia Q selected forthe polish stepNuvia cPrime selected forthe mass capture stepResults in ~500 mM NaCl eluate

ResultsHarvest

Nuclease-treated harvest

Nuvia cPrime

HCP, ng/1010 particles

ND

3,022

582

HC dsDNA, ng/1010 particles

3,144

30

ND

<0.02

Total virus (x1011 particles)

30.6

31.8

18.4

16.4

HCP, host cell protein. HC dsDNA, host cell dsDNA. ND, not determined.

Refer to experimental details on page 2.

Nuvia Q

Adenovirus Purification Strategy Details
Capture Conditions
Step1

BufferColumn Volume

2345

6

7

Flow Rate,cm/hr
250

Equilibration

25 mM histidine,pH 6.0 (buffer A)

10120

150Hexon

10075Sample Loading

Culture supernatantdiluted 1:3
with buffer A

48120PentonCore (V)5037Hexon (VI)Core (VII)

25WashBuffer A

5120

201510Elution

75 mM Tris, 525 mM
NaCl, pH 8.5

3120
SDS-PAGE of intermediates and the final product. Lane 1, MW marker; lane 2, Nuvia
cPrime load; lane 3, Nuvia cPrime flowthrough; lane 4, Nuvia cPrime elution/Nuvia Q load;lane 5, Nuvia Q flowthrough; lane 6, Nuvia Q pre-elution; lane 7, Nuvia Q product.

Polish Conditions
StepBuffer

Equilibration

75 mM, pH 8.0(buffer A)

Column Volume

Flow Rate,
cm/hr

10120

Visit bio-rad.com/web/ProcessResins to get technical details
about the resins used in this workflow.
Visit bio-rad.com/web/ProcessApplications to see otherapplications in which these resins can be used.

Nuvia cPrime eluate
diluted 1:1 withbuffer A

2120

Wash 175 mM, 250 mM
NaCl, pH 8.0

2120

Wash 215 mM Tris, 440 mM
NaCl, pH 8.5

3120

Elution

75 mM Tris, 1 M
NaCl, pH 7.55120

Sample Loading

ResourcesDownload bulletin 6713 to get details about this workflow.

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Bulletin 6880 Ver A

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