CIMac™ ANALYTICAL COLUMNS FOR IN-PROCESS CONTROL OF

ADENOVIRUSES
L. Urbas¹, N. Brument², J. Ruščić³, D. Marc¹
1 BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia | 2 Atlantic Gene Therapy, INSERM U1089 (vector core), Nantes, France | 3 University of Zagreb, Faculty of Science, Department of Biology, Zagreb, Croatia

INTRODUCTION
Determining the concentration of viruses is a crucial step in any production process. The most commonly used methods for virus
quantification are either based on the infectivity of the virus (plaque assay, TCID50) determination of their genomic material (qPCR), or protein
content (SRID, ELISA) and are very cumbersome and time consuming. HPLC analytical methods represent a fast alternative to these assays
since they provide information on the virus content and purity in a matter of minutes. Due to the structural properties of the monolithic
supports, monolithic analytical columns offer a great advantage over particle based HPLC columns in terms of time and their ability to
separate large biomolecules,
like viruses, VLPs, pDNA.
In this poster the performance of the CIMac™ Adeno Analytical Column – a monolith based anion exchange column, designed for fast and
reproducible analyses of adenoviruses was evaluated. CIMac Adeno column can be used for designing a fast finger printing method that is
applicable for monitoring the DSP production process of adenoviruses. Once the basic analytical parameters like linearity and sensitivity are
determined using a purified adenoviral standard, the metod can be applied for quantitative determination of adenoviruses.

Monolith Chromatography RESULT

MONOLITH CHROMATOGRAPHY MONITORING YOUR ADENOVIRUS DOWNSTREAM PROCESS - A QUICK FINGERPRINTING METHOD QUANTITATION OF YOUR ADENO SAMPLES
• Unlimited load volume of lysate 40 Using a CIMac™ Adeno-0.1 Analytical Column and UV detection at 260 nm, the method was linear in the range
35 A280
• Binding sites inside the channel of 4.8E+08 VP to at least 1.9E+10 VP; LOD was 4.8E+08 VP. One order of magnitude lower sensitivity (4.8E+07

Absorbance [mAU]
30 A260
• No dead-end pores or void-volume 25 VP) can be achieved by using a fluorescence detector (fluorescence detection at λexc = 280 and λem = 335).
20
• No stagnant zones 15
10
• No diffusion limitations 5
1400000

• Performance-independent of flow rate 0 1200000

-5 0 3 6 9 12 15 18
• Large channels (2 µm) - optimal for binding large -10
1000000
Time [min]
molecules, such as viruses, VLPs and DNA 800000

Area
y = 6E - 05x
• Flow-independent resolution 40 600000 R² = 0,9996
35
• High flow rates (up to 10 CV/min) A280 400000
30

Absorbance [mAU]
• Purification within minutes 25
A260 200000

• Scale-up straightforward 20 0
15 0,00E+00 1,00E+10 2,00E+10
10
Virus Titer
5
0
60 120
-5 0 2 4 6 8 10 12 14 16 18
1,93E+10
-10
Time [min] 50 7,73E+09 100
1,93E+09
40 9,67E+08
40 80
35 A280 4,83E+08

Absorbance mAU)
Absorbance [mAU]
30 30
A260 gradient

Buffer B (%)
25 60
20 20
15 40
10 10
5
0 20
0
2 mL/min -5 0 3 6 9 12 15 18
0 1 2 3 4 5 6 7 8 9
-10
Time [min] -10 0
1 mL/min

0.5 mL/min -20 -20

Time (min)
Chromatographic conditions:
The CIMac™ Adeno-0.1 Analytical Columns were run with an Explorer 10 FPLC System (GE Healthcare) under control of the Chromatographic conditions:
Unicorn software. Sample: rAd5, Flow rate: 0.5 mL/min, sample volume: 50 µL, sample diluted in buffer A: 20 mM Tris, pH 8.0, The CIMac™ Adeno-0.1 Analytical Columns were run with an Agilent series 1100 (Agilent, Waldbronn,
method: a NaCl gradient from 0 to 1 M in Tris, pH 8 was applied to elute in 30 CV. Germany) HPLC system, sample: Ad5 wild type purified with CsCl (replication genes are deleted), buffer A: 20
mM Tris 5% glycerol pH 7.4, buffer B: 20 mM Tris 2 M NaCl 5% glycerol pH 7.4, flow rate: 1 mL/min.
CONCLUSIONS
The CIMac™ Adeno Analytical column can be used as a fast finger printing method, that gives you information about the production process of your Adenoviruses.

Using a CIMac™ Adeno-0.1 Analytical Column and UV detection at 260 nm, the method was linear in the range of 4.8E+08 VP to at least 1.9E+10 VP; LOD was 4.8E+08 VP.
Acknowledgments
Thanks to M. Pennors for her appreciated technical
contribution!
One order of magnitude lower sensitivity (4.8E+07 VP) can be achieved by using a fluorescence detector (fluorescence detection at λexc = 280 and λem = 335).

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