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.

2

Content

Janssen vaccines Objective Feasibility study Optimization Method performance Conclusions

3

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

4

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

5

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

6

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

7

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

8

Outlet Inlet Top View Side View

Frit Ultra-Filtration Membrane

Asymmetric-flow field flow fractionation

Wyatt technology Europe

Objective – Field flow fractionation

9

Stage 1: Injection/Relaxation Inlet Exit Injection

Wyatt technology Europe

Objective – Field flow fractionation

10

Inlet Outlet

Stage 2: Elution Injection

Objective – Field flow fractionation

11

Objective – Multi angle light scattering

12

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

13

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’

14

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%

15

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)

16

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

17

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

18

.

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%

19

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

20

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

21

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

22

• 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

23

Acknowledgement

23

Pascal

Annemiek Bryan Bojana Tatev

Lars Johan Jonathan

Miranda

Ewoud Bennie

mschenni@its.jnj.com

Thank you

Melinda, Goddess of Healing Melinda’s artwork reflects her journey living with HIV.