how can we streamline influenza virus purification?,how can we streamline … · 2020. 7. 20. ·...
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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, haemophiliaand 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 ofbetter 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.
CURRENT VIRUS PRODUCTION PROCESSES
Figure 1Schematic representation of current and proposed virus production process
CONCLUSION: FLEXIBLE STREAMLINED VIRUS PRODUCTION
Mustang Q XT ion exchange chromatographyis a valuable alternative for the purification ofinfluenza virus, and by extension for otherviruses, from clarified cell culture feedstock
Allows for faster, simpler and economicalprocessing
EXPERIMENTAL STRATEGY
MEMBRANE CHROMATOGRAPHY
© 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
Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: [email protected] • Web: www.pall.com/biopharm
Feed solution:Influenza virus A/Puerto Rico/8/1934 (H1N1) cultured in HEK-293 suspension cells. Clarifiedusing a Pall Seitz® P series depth filter V100P
80
0.001
0.002
0.003
0.004
0.005
0.006
AU
Min9 10 11 12 13 14
1. Design of Experiment (DoE)• Critical parameters (loading and elution conditions)• Quality attributes (virus yield, HCP, DNA)
• AcroPrep™ Advance Filter Plates• Vacuum manifold
• Virus detection (HA assay)• HCP quantification (ELISA)• DNA (PicoGreenu assay)
3. Analytical Testing
2. Screening on 96-Well Plates
4. Result Analysis• Design space for optimum performances
5. Transfer to Mustang Q XT Acrodisc Device
Buffer
Sorbent bedFilter plate
7.0
Load pH
Elut
ion
pH
5.04.84.64.44.24.0
8.5
8.0
7.5
6.5
6.0
5.5
5.0
HCP (ppm)
150-200
200-250
250-300
300-350
Load
pH
Elution pH
Elut
ion
Cond
SELECTION OF BIND/ELUTE CONDITIONS USING HTS
Conditions for bind/elute Binding ElutionpH 6.8 5.8Conductivity (mS/cm) 6.2 55.6HA units per well 85.9 Non Applicable
• Comparing streamlined process in full single-use mode with standardre-use process
• 40 batches/year, 120 M doses/year
• DSP yield constant at 46%
• BioSolve Software Model
Annual CoGs reduced due to lower CAPEX and eliminationof 1 purification step
Additional 10-fold reduction in water consumption
PROCESS ECONOMICS
0 2 4 6
Capital
Materials
Consumables
Labour
Other
Annual CoGs
Million USDStandard
-14%
+28%
-11%
Streamlined
-63%
Excellent HCP (binding) and DNA (elution)clearance at elevated conductivity
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
pH
Con
duct
ivity
(mS/
cm)
Con
duct
ivity
(mS/
cm)
Virus binding yield (%)
Contour Plot of Virus Binding Yield
> – – – – – – – – – – <
90 95 95 99
99
1 1 15
15 30 30 40 40 50 50 60 60 70 70 80 80 90
HCP content in FT (%)
Contour Plot of HCP Content in FT
pH8 7 6 5 4
pH7 8 6 5 4
15.0
12.5
10.0
7.5
5.0
Con
duct
ivity
(mS/
cm)
15.0
12.5
10.0
7.5
5.0
Con
duct
ivity
(mS/
cm)
15.0
12.5
10.0
7.5
5.0
> – – – – – – – – <
1,200,000
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
DBC (HA units/mL)
Contour Plot of DBC
8 7 6 5 4
15.0
12.5
10.0
7.5
5.0
95 100
HCP content in FT (%)
1.12e+006 2e+006
DBC (HA units/mL)
99 100
Virus binding yield (%)
Sweet Spot
> – – – – – – – – <
99
1 1 50
50 80 80 90 90 92 92 94 94 96 96 98 98 99
pH7 8 6 5 4
Open pore structure with direct access to ion exchange binding sites allow higher flow rates
Higher binding capacities for largermolecules
Ready-to-use devices
Reduced hardware investment andvalidation cost
TRANSFER TO SCALABLE DEVICE
Virus DNA HCP DBC, Recovery, Clearance, Clearance, Productivity, Particle/mL % % % particle/hr/L9 x 1011 75.1 99.9 95 2 x 1014
Data obtained on Mustang Q XT Acrodisc Units with the conditions definedby HTS s tudy
AcroPrep™ Advance XT Acrodisc Filter Plates Units XT5 XT140 XT500014 µL membrane 0.86 mL 5 mL 140 mL 5000 mLper 1 mL well
Conditions selected on 96-well plate in the HTS provide excellent results for virus DBC and purity on XT Acrodisc unitwith Mustang Q membrane
Virus DBC in the upper 1011 range
Good recovery of ~75 % with excellent DNA and HCP (>95%) clearance and process productivity
Results from Minitab� analysis Binding ElutionVirus yield /recovery (%) Non Applicable 90.8HCP removal (%) >99.5 >99.8DNA removal (%) 0 >99.7