Overview of characterization & Quantitation by LS: from ......CONFIDENTIAL © Wyatt Technology Corporation –All Rights Reserved 20CQA 1: Particle concentration chromatograms time

CONFIDENTIAL © Wyatt Technology Corporation – All Rights Reserved 1

Overview of characterization & Quantitation by LS:from Proteins to AAVs to BioNPs

November, 2019


Topics

Analyze proteins by SEC-MALS

Typical protein applications

Protein conjugate analysis

Quantify AAV CQAs by SEC-MALS

Particle concentrations of total, full, and empty AAVs

Capsid content (full to empty ratio)

Percentage of aggregation

Characterize large AAV aggregates and other bioNPsby FFF


Topics





Capsid particle concentration

Capsid content (Full to Total ratio)




SEC-MALS-UV-DRI

Well validated and used in QC for other biologics (proteins, PEGylated proteins, polysaccharides, protein-polysaccharide conjugates)

CFR21-Part11 compliant software, IQOQ Robust instruments with outstanding technical support teams


Define Peaks

LS dRI

time (min)

19.5 20.0 20.5 21.0

Rela

tive S

cale

0.0

0.5

1.0

SEC-MALS-UV/RI MethodSEC provides separation and the molar mass is measured by online MALS and concentration detectors.

LS

Concentration[mg/mL]

Mi


SEC-MALS(DLS)-UV-DRI


SEC-MALS-UV-RI: Protein conjugates

Protein MW

Carbohydrate MW

UV trace, Mammalian Cell

Carbohydrate MW

UV trace, Insect Cell

Glycosylated Proteins


Full and Empty AAV (protein-DNA)

MWs for the protein capsid and encapsulated DNA can be reliably determined for full or empty AAVs.

Protein Conjugate Data vs. time

time (min)

12.5 13.0 13.5 14.0 14.5 15.0

Mo

lar

Mass (

g/m

ol)

1.0x105

1.0x106

1.0x107

AAV9_Full_30uL_2

LS

Total Protein Modifier

Capsid: 3.7 MDa

DNA: 1.2 MDa

Total: 4.9 MDa

Protein Conjugate Data vs. time

time (min)

13.0 13.5 14.0 14.5 15.0

Mo

lar

Mass (

g/m

ol)

1.0x105

1.0x106

1.0x107

AAV9_EMPTY_30uL_1

LS

Total Protein Modifier

Capsid: 3.7 MDa

Total: 3.7 MDa

DNA: <0.03 MDa


Topics

Protein analysis by SEC-MALS









What is hot now in the biopharmaceutical field


Gene therapyUse vector (or transporter) to deliver DNA or RNA to cells.

To learn more about gene therapy, visit

https://learn.genetics.utah.edu/content/genetherapy/

DOI: 10.1126/science.aan4672


Gene therapy

http://sparktx.com

Particle Size &

Concentration

Payload

Content

Aggregation

Degree

Needs to quantify

50years

2300trials

2AAV-based

More background


Adeno-associated virus (AAV) as the vector

AAV vector is an ideal gene delivery vehicle

— Absence of pathogenicity

— Low immunogenicity

— Infectious to most cells

— Predictable targeting site

— Icosahedral, 12 serotypes, ~ 25 nm in diameter

— Capsid MW: 3-4 Mda, filled ssDNA: 1.55 Mda (4.7Kb)


Quality attributes in viral vector manufacturing

Naso, Michael F., Brian Tomkowicz, William L. Perry, and William R. Strohl. "Adeno-associated virus (AAV) as a vector for gene therapy." BioDrugs 31, no. 4 (2017): 317-334.


Analytical tools to quantify AAV vectors

– Real-time polymerase chain reaction (qPCR)

– Optical and electron microscopy (TEM, cryoEM)

– Analytical ultracentrifugation (Sedimentation)

– Light scattering coupled with separation (SEC-MALS, FFF-MALS)

Adeno-associated virus (AAV) as the vector

• Particle concentration• Capsid content (Cp/Vg)• Aggregation


CQA 1: Particle concentrationchromatograms

time (min)

5.0 10.0 15.0 20.0

Rela

tive S

cale

0.0

0.5

1.0

PC_AAV9_EMPTY PC_FULL PC_Full_Empty=1-2 PC_Full_Empty=1-3 PC_Full-Empty = 1-5 PC_Full-Empty = 1-10

LS

F:EMeasured AAV particle

concentration [N/mL]

0 to 1 (E) 9.773E+13

1 to 0 (F) 3.870E+13

1 to 1 6.799E+13

1 to 2 7.405E+13

1 to 3 8.437E+13

1 to 5 8.526E+13

1 to 10 9.022E+13

• Particle concentration from eluted mass calculation. Only assumption is 100% mass recovery.

• Consistent correlation with other quantitative methods.

AAV samples provided by Virovek Inc.


CQA 2: Capsid Content (Cp/Vg or total/full)

Cp: capsid particle concentration, empty + full AAV

Vg: viral genome particle concentration, full AAV alone

• Excellent agreement with theoretical value on capsid content. • Excellent correlation with AUC data.


CQA 3: Quantify Aggregation

Results Fitting

time (min)

10.0 12.0 14.0 16.0

Nu

mb

er

Den

sit

y (

part

icle

s/m

L)

1.0x107

1.0x108

1.0x109

1.0x1010

1.0x1011

1.0x1012

1.0x1013

Number Density 1 LS

• Total particle concentration is calculated for each data slice. • Percentage of monomer and aggregates can be calculated and used to quantify

AAV aggregation.


AAV Critical Quality Attributes (cQA) Measured by SEC-MALS

SEC-MALS could measure the following cQAs:

• Particle concentration

• Capsid content (Cp/Vg)

• Aggregation (FFF-MALS will be needed for measuring large aggregates)

Advantages of the SEC-MALS method

• Easy, fast (30 minutes or less), nondestructive, and able to measure all three cQAs in one single run. Additional parameters (Rg, Rh, impurities) could also be measured.

• Be able to analyze samples without a priori knowledge about the sample or calibration.

• Robust, repeatable, consistent, and calibration measurement once the method is optimized.

• With the potential to be implemented in manufacturing and QC.

© Wyatt Technology Corporation – All Rights Reserved 24

• SEC-MALS

– SEC may be able to resolve monomer and oligomer

– SEC is not be the right tool to quantify large aggregates

• Eclipse AF4-MALS

– AF4 provides better separation of confirmation of aggregate %

– HMW aggregates visible by AF4-MALS may be removed by SEC column

chromatograms

volume (mL)

5.0 10.0 15.0

Rela

tive S

cale

0.0

0.5

1.0

AAV_filled_SEC-MALS-DLS_proconj

UV

WTC-050S5

fragmentoligomersLarge aggregates?

chromatograms

time (min)

10.0 15.0 20.0 25.0 30.0

Rela

tive S

cale

0.0

0.5

1.0

AAV Sample 2_FFF AAV Sample 1_FFF

UV

Large aggregates

fragment oligomers

AAV Aggregates by AF4-MALS


Topics










Wyatt FFF Platform for other delivery vehicles

AAV aggregates Other viral vectors (lentivirus, adenovirus, …) Liposomes/LNPs Exosomes


0

50

100

150

200

10 20 30 40 50 60 70

Geom

etric

R

adiu

s (n

m)

T ime (min)

Geometric Radius vs. Time DukeB_011st order

Isolation: high resolution fractionation

• The fractions can be collected for subsequent assays.


geometric radius vs. volume

CsCl_Fx05R45C10_25uL(02)[CGI2] CsCl_Fx05R45C10_25uL(01)[CGI2]

volume (mL)

15.0 20.0 25.0 30.0

geo

met

ric

rad

ius

(nm

)

0.0

50.0

100.0LS

Identification: sizing by MALS and DLS

Adenovirus

• Small amount of aggregates will not be detected by batch DLS.• Rg and Rh can be measured by MALS and online DLS, respectively.

http://upload.wikimedia.org/wikipedia/commons/0/0b/Adenovirus.jpg




Quantitation: particle sizing and counting

Parameter AFM/TEM FFF-MALS % Difference

Total Particle Count 2.9 X 1010 2.8 X 1010 2

Average Radius [nm] 43.0 45.0 5

Z. Wei, et al., "Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity", Journal of Virological Methods, 144(1-2), 122-132, (2007)

T. Bousse,et al., "Quantitation of influenza virus using field-flow fractionation and multi-angle light scattering for quantifying influenza A particles", Journal of Virological Methods, 193(2), 589-596, (2013)

geometric radius vs. volume

CsCl_Fx05R45C10_25uL(02)[CGI2] CsCl_Fx05R45C10_25uL(01)[CGI2]

volume (mL)

15.0 20.0 25.0 30.0

geo

met

ric

rad

ius

(nm

)

0.0

50.0

100.0LS


Biophysical characterization: shape & payloadBurchard-Stockmayer Plot

cellulose rod spherical NP

time (min)

15.0 20.0 25.0 30.0 35.0 40.0

RM

S R

ad

ius/R

h(Q

)

0.0

0.5

1.0

1.5

LS

Spherical NP as reference

Cellulose Nanocrystal

• Particle size can be measured by MALS (Rg) or DLS (Rh).

• Rg/Rh and apparent particle weight can determine payload/cargo content.


Molar Mass vs. time

online (from Filled Liposome 1) online (from Empty Liposome 2)

time (min)

14.0 16.0 18.0 20.0 22.0 24.0

Mo

lar

Mass

(g

/mo

l)

71.0x10

81.0x10

91.0x10 dRI

Empty liposome

Filled liposome

Genetic payload from apparent particle weight


Lentiviral Vector by AF4-MALS

Particle weight, concentration and size can be measured by MALS and DLS.

Define Peaks

time (min)

8.0 10.0 12.0 14.0 16.0 18.0

Rela

tive S

cale

0.0

0.5

1.0

LS UV dRI

1 1

Molar Mass vs. volume

volume (mL)

10.0 12.0 14.0 16.0 18.0 20.0

Mo

lar

Mass (

g/m

ol)

1.0x107

1.0x108

1.0x109

1_MW

LS

Hydrodynamic Radius (Q) vs. volume

volume (mL)

14.0 16.0 18.0

Hyd

rod

yn

am

ic R

ad

ius

(Q)

(nm

)

100.00

1_ND

LS

radius vs. volume

volume (mL)

14.0 15.0 16.0 17.0 18.0

rad

ius (

nm

)

100.00

1_ND

LS

1.0x1010(mL)


Adenoviral Vector by AF4-MALS

• Freeze-thaw– Isolate and quantify virus size with

Eclipse AF4-MALS

– Sensitive and robust aggregation assessment

• Fresh vs. aged– Elution time and peak shape are not

representative of size distribution

– Eclipse fractionation with DAWN MALS detector determines absolute size

• Buffer effects– Measure differences in size

distribution between buffers

– Quantify number of particles: 1.7 ×1010 for each case

Shortly after

thawing

12 hr. later3.5 hr.

later Salt

added

Aged, 10µLFresh,

25µL

Tris buffer

Formulation buffer


Liposome & Lipid Nanoparticle (LNP)

http://www.arbutusbio.com/our-science/lnp-delivery-platform.php

https://www.cordenpharma.com/all-news-press/newsletter-

spotlight/lnp-options-for-sirna-mrna-or-crispr/


LNP: DLS vs. FFF-MALS

Subtle differences by DLS vs. detailed fingerprints by FFF-MALS


LNP: DLS vs. FFF-MALS

Subtle/less reproducible differences by DLS vs. detailed/consistent fingerprints by FFF-MALS


Quantify encapsulated mRNA inside LNP

Encapsulated mRNA can be calculated either from the deduction of the free mRNA peak or apparent MW differences.

Molar Mass vs. time

time (min)

20.0 25.0 30.0

Mo

lar

Mass (

g/m

ol)

1.0x108

1.0x109

Fill1 Empty1_UV260

UVEmptyFilled


Wyatt solutions for AAV and other BioNPs

SEC - MALS Particle concentration

Capsid content

Aggregation

FFF - MALS Large aggregates

Other viral vectors,

exosome, liposomes,

LNP

DLS PlateReader

Size distribution

Stability screening

Particle concentrations

Thank you!

Documents

Overview of characterization & Quantitation by LS: from ......CONFIDENTIAL © Wyatt Technology Corporation –All Rights Reserved 20CQA 1: Particle concentration chromatograms time