Vaccine Product Characterization by AF4-MALS: Particle size distribution and aggregation
Martijn Schenning Analytical Development | 18Mar16
Melinda, Goddess of Healing Melinda’s artwork reflects her journey living with HIV.
Melinda, Goddess of Healing Melinda’s artwork reflects her journey living with HIV.
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Content
Janssen vaccines Objective Feasibility study Optimization Method performance Conclusions
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Janssen vaccines – AdVac platform
Selected genes responsible for protective immune response
Virus or parasite
Production on PER.C6® cells
Commercial scale volumes
of vaccine
Vaccine Created after
intensive purification
AdVac ® vector
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Janssen vaccines – AdVac platform
Selected genes responsible for protective immune response
Virus or parasite
Production on PER.C6® cells
Commercial scale volumes
of vaccine
Vaccine Created after
intensive purification
AdVac ® vector
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Structure • 90 – 100 nm diameter • Total mol. weight: 150 MDa • Non-enveloped, Icosahedral (20 planes) • Core: double stranded DNA • Molecular weight of proteins: ~7 – 200 kDa
At Janssen • Subtypes Ad26 and Ad35 • Transgene inserts:
Ebola HPV (human papillomavirus) HIV (human immunodeficiency virus) RSV (respiratory syncytial virus)
Janssen vaccines – Adenovirus
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Objective – Adenovirus aggregation
• Aggregation – What: Biological phenomenon where viruses/proteins accumulate – Why: Could induce or reduce immune response and have
toxic/adverse effects – How: Currently no method available in Janssen IDV that
quantitatively determines the percentage of aggregated adenovirus
• Guidelines – No clear guidance in Pharmacopoeia for virus aggregates. – Vaccine/adenovirus product should be characterized to assure safety,
efficacy and potency of our product.
Transmission electron microscopy (TEM) pictures of virus like particle
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Objective – Technique selection
Objective: Development of a FFF-MALS method for the quantification of adenovirus aggregation in drug substance and drug product Method requirements: Sufficient precision, working range, matrix compatible, and robust Reportable result: Percentage of aggregated adenovirus particles
DCS
SEC
DLS
AUC
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Outlet Inlet Top View Side View
Frit Ultra-Filtration Membrane
Asymmetric-flow field flow fractionation
Wyatt technology Europe
Objective – Field flow fractionation
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Stage 1: Injection/Relaxation Inlet Exit Injection
Wyatt technology Europe
Objective – Field flow fractionation
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Inlet Outlet
Stage 2: Elution Injection
Objective – Field flow fractionation
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Objective – Multi angle light scattering
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Angular Dependence of Light Scattering
Detector at 0° scattered light in phase
Detector at θ, scattered light out-of-phase
( ) ( ) 2
scattered θθ PdcdnMcI
∝
Intramolecular interference leads to a reduction in scattering intensity as the scattering angle increases.
anisotropic scattering
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Objective – Why AF4-MALS?
Broad size range (1 nm – 100 µm) (adenovirus ±80 nm)
Minimal shearing (aggregation study)
Combined with several detectors (RI, UV, and MALS)
Non-destructive separation
Direct injection (minimal sample preparation)
Many parameters to be optimized
Some proteins stick to all the available membranes
Dilution may dissociate reversible aggregates
Open system requires a MLII lab for virus work
Complex and ‘new’
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Feasibility study – Spacer and membrane Spacer Membrane
250S
RC 10 kDa
PES 30 kDa
CTA 10 kDa
RC 30 kDa
350W RC 10 kDa
PES 10 kDa
PES = Polyethersulfone RC = Regenerated Cellulose CTA = Cellulose Triacetate
0 20 40 60 80 100
Recovery
CV 8%
CV 2%
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Adenovirus FFF separation + UV260 & MALS detection
time (min)0.0 10.0 20.0 30.0 40.0 50.0
0.0
0.5
1.0 LS UVmonomer
aggregates
Optimization – Setting the cut-off point
time (min)0.0 10.0 20.0 30.0 40.0 50.0
0.0
20.0
40.0
60.0
80.0
100.0
LS UV
Smal
l par
ticle
s = p
rote
in
Artif
act
Adenovirus FFF separation + UV260 & MALS detection
Aden
oviru
s
RMS
radi
us (n
m)
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Calculate mean RMS radius
ASTRA provides std dev on µ
Set upper radius limit at 3x std dev above mean radius
Fluctuations in std dev
Set upper radius limit as mean +5% (>3x std dev)
Optimization – Setting the cut-off point
But is this a fair cut-off?
time (min)0.0 10.0 20.0 30.0 40.0 50.0
0.0
20.0
40.0
60.0
80.0
100.0
LS UV
Adenovirus FFF separation + UV260 & MALS detection
RMS
radi
us (n
m)
Setting the cut-off point between monomeric and aggregated particles
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Justification of point of divergence
RMS conformation and Rh/Rg plots reveal a change in particle shape past the point of divergence (upper radius cut-off point) indicative of aggregated adenovirus particles
Cut-off is fair
Optimization – Setting the cut-off point
RMS conformation plot
Ad26.mos2.gag-pol DS[L608-043]
Molar Mass (g/mol)
71.0x10
rms
radi
us (n
m)
20.0
30.0
40.0
50.0
60.0
70.080.090.0
100.0
conformation plot slope0.85±0.00
Monomer
RMS conformation plot
RMS
radi
us (n
m)
Day 2 - SSC 3(003)[L950-086]
40.0 60.0 80.0 100.0 120.0 0.0
50.0
100.0
150.0
Mon
omer
RMS radius vs. Rh(Q) Plot
Hydrodynamic Radius (nm)
RMS
radi
us (n
m)
RMS vs Rh plot
time (min)0.0 10.0 20.0 30.0 40.0 50.0
0.0
20.0
40.0
60.0
80.0
100.0
LS UV
Mon
omer
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.
Small particles past the upper limit?
Optimization – Quantification by UV 260 nm
Direct read out = 21.5%
Cumulative rms radius
Cum
ulat
ive
WEI
GH
T ra
dius
RMS radius (nm)
25.59 91.43 + 25.59 x 100% = 21.9%
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Method performance – Linearity and working range
Linearity
Output of the method is “percentage of aggregated adenovirus” lack of standards hinders true linearity testing
R² = 0,9989
-
50,00
100,00
150,00
200,00
250,00
300,00
350,00
400,00
2,00E+09 2,20E+10 4,20E+10 6,20E+10 8,20E+10
virus particles
Linear sample recovery
Virus particles
Working range
0%
10%
20%
30%
40%
50%
60%
70%
80%
2,00E+09 2,20E+10 4,20E+10 6,20E+10 8,20E+10 1,02E+11
Aver
aged
agg
rega
te p
erce
ntag
e
virus particles
Aggregate percentage per injection
Aggr
egat
ed a
deno
viru
s (%
)
Virus particles
Percentage of aggregated adenovirus is constant over the full working range: 1.20x1010 and 9.60x1010 viral particles per injection
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Repeatability Intermediate precision
Coefficient of variation 5 – 7% Coefficient of variation 7 – 13%
Method performance – Precision Ag
greg
ated
ade
novi
rus (
%)
Aggr
egat
ed a
deno
viru
s (%
)
Day Virus particles
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Cum
ulat
ive
wei
ght r
adiu
s
Cumulative rms radius
RMS radius (nm)
Cum
ulat
ive
wei
ght r
adiu
s
Cumulative rms radius
RMS radius (nm)
Method performance – Stability indicating potential
Stress conditions Expected result
Thermal stress Denaturation of proteins
Mild oxidation Oxidation of proteins, lipids, nucleic acid
pH Protein deamidation
Freeze-thaw cycles Denaturation of protein
Red no stress Blue thermal 45 min Pink thermal 120 min LGreen oxidative stress Brown-ish pH stress D-Green Freeze-thaw
Vector 1
Vector 2
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• Accuracy could not be determined (lack of ref standard), but method exhibits high precision (low run-to-run and day-to-day variation) and robustness
• AF4 is a separation technique with nearly no artifacts/shear force which allows for studying adenovirus particles in their most native/inherent form
• AF4 is capable of “separating” the monomeric and multimeric form of adenovirus particle
• The upper limit cut-off point is set in a fair/justifiable way
• Quantification by UV 260 nm / cumulate weight fraction is the best method to determine the percentage of aggregated adenovirus particles
Conclusions
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Acknowledgement
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Pascal
Annemiek Bryan Bojana Tatev
Lars Johan Jonathan
Miranda
Ewoud Bennie
Thank you
Melinda, Goddess of Healing Melinda’s artwork reflects her journey living with HIV.