How Can We Streamline Influenza Virus Purification?
Aleksandar Cvetkovic1, René Gantier1 and Annelies Onraedt2
1Pall Life Sciences, Westborough, MA, USA; 2Pall International Sàrl, Fribourg, Switzerland
INTRODUCTION
Apart from their current application in viral vaccines, nanoparticle-based entities such as viruses and non-infectious virus-like particles are holding great promise in a myriad of clinical targets, including cancer, cystic fibrosis, Alzheimer’s, Perkins’s, haemophilia
and HIV/AIDs. Virus production processes (Figure 1) have been evolving on the upstream side, moving from chicken eggs in more reliable and controlled cell lines. The challenge for virus production processes is now downstream on the development of
better virus purification processes to meet ever increasing regulatory expectations in regards to contaminant removal, including host cell DNA and host cell protein (HCP) while mitigating high cost pressures.
Scope of the work was to develop a streamlined virus manufacturing process, by using Mustang® Q ion exchange membrane chromatography. The selection of bind/elute conditions was done by high-throughput screening (HTS) using 96-well plates.
Selected conditions were then transferred to a Mustang Q XT Acrodisc® device.
MEMBRANE CHROMATOGRAPHY
CURRENT VIRUS PRODUCTION PROCESSES
Open pore structure with direct
access to ion exchange binding
sites allow higher flow rates
Figure 1
Schematic representation of current and proposed virus production process
Higher binding capacities for larger
molecules
Ready-to-use devices
Reduced hardware investment and
validation cost
TRANSFER TO SCALABLE DEVICE
EXPERIMENTAL STRATEGY
DBC,
Particle/mL
9 x 1011
Elution pH
Feed solution:
Influenza virus A/Puerto Rico/8/1934 (H1N1)
cultured in HEK-293 suspension cells. Clarified
using a Pall Seitz® P series depth filter V100P
Elution Cond
1. Design of Experiment (DoE)
• Critical parameters (loading and elution conditions)
• Quality attributes (virus yield, HCP, DNA)
ad
Lo
Virus
Recovery,
%
75.1
DNA
Clearance,
%
99.9
HCP
Clearance,
%
95
Productivity,
particle/hr/L
2 x 1014
Data obtained on Mustang Q XT Acrodisc Units with the conditions defined
by HTS study
pH
Buffer
Sorbent bed
Filter plate
2. Screening on 96-Well Plates
• AcroPrep™ Advance Filter Plates
• Vacuum manifold
AcroPrep™ Advance XT Acrodisc
Filter Plates
Units
14 µL membrane
0.86 mL
per 1 mL well
XT5
5 mL
XT140
140 mL
XT5000
5000 mL
Conditions selected on 96-well plate in the HTS provide excellent results for virus DBC and purity on XT Acrodisc unit
with Mustang Q membrane
Virus DBC in the upper 1011 range
Good recovery of ~75 % with excellent DNA and HCP (>95%) clearance and process productivity
3. Analytical Testing
• Virus detection (HA assay)
• HCP quantification (ELISA)
• DNA (PicoGreenu assay)
AU
0.006
0.005
0.004
PROCESS ECONOMICS
0.003
0.002
0.001
0
8
9
10
Elution pH
4. Result Analysis
• Design space for optimum performances
11
12
13
14
HCP (ppm)
8.5
8.0
Min
250-300
7.5
• Comparing streamlined process in full single-use mode with standard
re-use process
200-250
7.0
6.5
150-200
6.0
5.5
5.0
-14%
Annual CoGs
300-350
4.0
4.2
4.4
4.6
Load pH
4.8
5.0
Other
• 40 batches/year, 120 M doses/year
-63%
Labour
5. Transfer to Mustang Q XT Acrodisc Device
• DSP yield constant at 46%
Consumables
• BioSolve Software Model
+28%
Annual CoGs reduced due to lower CAPEX and elimination
of 1 purification step
Materials
SELECTION OF BIND/ELUTE CONDITIONS USING HTS
Contour Plot of HCP Content in FT
HCP content
in FT (%)
12.5
10.0
7.5
5.0
4
5
6
pH
7
8
< 1
1 – 15
15 – 30
30 – 40
40 – 50
50 – 60
60 – 70
70 – 80
80 – 90
90 – 95
95 – 99
> 99
DBC (HA units/mL)
15.0
Conductivity (mS/cm)
15.0
Conductivity (mS/cm)
Contour Plot of DBC
< 1,000,000
1,000,000 – 1,030,000
1,030,000 – 1,060,000
1,060,000 – 1,080,000
1,080,000 – 1,100,000
1,100,000 – 1,120,000
1,120,000 – 1,140,000
1,140,000 – 1,170,000
1,170,000 – 1,200,000
> 1,200,000
12.5
10.0
7.5
5.0
4
1
50
80
90
92
94
96
98
10.0
7.5
5.0
4
5
6
pH
7
Conditions for bind/elute
pH
Conductivity (mS/cm)
HA units per well
pH
7
8
< 1
– 50
– 80
– 90
– 92
– 94
– 96
– 98
– 99
> 99
15.0
Conductivity (mS/cm)
Conductivity (mS/cm)
Virus binding
yield (%)
12.5
6
DBC in range of 1011 virus particles
per mL of membrane
DBC and virus recovery controlled
by virus and DNA content in load:
Improvement of elution yield could
jeopardize DNA clearance
Sweet Spot
Contour Plot of Virus Binding Yield
15.0
5
Capital
Excellent HCP (binding) and DNA (elution)
clearance at elevated conductivity
HCP content in FT (%)
95
100
12.5
1.12e+006
2e+006
Virus binding yield (%)
99
100
7.5
Binding
6.8
6.2
85.9
2
Standard
Streamlined
4
6
Million USD
CONCLUSION: FLEXIBLE STREAMLINED VIRUS PRODUCTION
Mustang Q XT ion exchange chromatography
is a valuable alternative for the purification of
influenza virus, and by extension for other
viruses, from clarified cell culture feedstock
5.0
4
8
0
Allows for faster, simpler and economical
processing
DBC (HA units/mL)
10.0
Additional 10-fold reduction in water consumption
-11%
5
6
pH
7
Elution
5.8
55.6
Non Applicable
8
Results from Minitab◆ analysis
Virus yield /recovery (%)
HCP removal (%)
DNA removal (%)
Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: biopharm@pall.com • Web: www.pall.com/biopharm
Binding
Non Applicable
>99.5
0
Elution
90.8
>99.8
>99.7
© 2014, Pall Corporation. Pall,
, Acrodisc, AcroPrep, Mustang and Seitz are trademarks of Pall Corporation. ® indicates a trademark registered in the USA.
◆ Minitab is a trademark of Minitab, Inc. PicroGreen is a trademark of Invitrogen. 7/14, GN14.9442