Aggregation of Antibody-Drug Conjugates: The Light ...... Aggregation of Antibody-Drug Conjugates:...
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www.crystal.csiro.au Aggregation of Antibody - Drug Conjugates: The Light Scattering Toolbox for Screening and Characterization Chris Broomell and Daniel Some , Wyatt Technology Corporation, 6300 Hollister Ave, Santa Barbara, CA 93117 Size Distributions by FFF-MALS scattered w I Mc Aggregation Analyses of Stressed and Unstressed ADCs by FFF-MALS Abstract Antibody-Drug Conjugates (ADCs) show much promise as effective therapeutics for cancer and other diseases. However, they often exhibit an increased aggregation propensity compared to their unmodified counterparts due to non-specific interactions arising from attached drug and linker moieties. Light scattering offers multiple techniques for addressing the challenges of formulation screening, and characterizing both aggregates and propensity for aggregation. We present these tools in the context of ADCs and the dependence of aggregation profiles on linker chemistry. © Wyatt Technology Corporation 2014 – All Rights Reserved Linker-Induced Instability Studied by DLS High-Throughput Dynamic Light Scattering Dynamic light scattering (DLS) analyzes Brownian motion via the intensity fluctuations of light scattered by macromolecules and nanoparticles in solution. DLS determines directly diffusion coefficients D t . Particle radius R h is calculated from the Stokes-Einstein equation: www.synaffix.com Identical mAb and drug, different linkers T onset = 63.9°C T onset = 48.6°C Protein stability is typically studied by temperature of melting (T M ) or onset of aggregation. ADC 2 unfolds and aggregates at a lower temperature, and is therefore less stable than ADC 1 . These data suggest distinct aggregation pathways. Typical Fractogram – Unstressed Sample Typical Fractogram – Stressed Sample Peak1 Peak 2 Peak 3 % M w % M w % Unstressed 0.75 148.4 97.75 231.5 1.5 Stressed 1.40 149.6 95.1 297.1 3.5 M w is determined by the ratio of light scattering signals to UV. Mass fraction is determined by integrating UV peaks. FFF- MALS is highly sensitive to small quantities of aggregates due to the dependence of scattered intensity on M w . 149.4 kDa (monomer) Higher-order species present in stressed sample ~339 kDa (average) * Small shoulder suggestive of fragmentation. This is present to some extent in all samples. * Peak 2 (Monomer) Peak 1 (Fragment) Peak 3 (Oligomer) Average M w and Mass Fraction (n=2) M w = 149.2 kDa from MALS Multi-angle, static light scattering (MALS) analyzes molar mass M w from first principles, via the average intensity of scattered light I and concentration c: ADC 1 ADC 2 ADC 2 ADC 1 DLS determines size distributions without fractionation, providing polydispersity estimates. High-throughput screening via the DynaPro® Plate Reader II is a powerful tool for characterizing multiple aspects of biotherapeutic stability: • Degree and type of aggregation • Thermal conformational stability (T M , T onset ) • Chemical conformational stability • Colloidal stability ℎ = 6 This analysis is independent of questionable reference standards and column calibration. In an Asymmetric-Flow FFF separation channel, macromolecules and nanoparticles are gently pushed against a semipermeable membrane by crossflow. Smaller particles diffuse back up towards the center of the channel. Laminar channel flow induces a parabolic flow velocity profile, causing smaller particles to elute earlier. ADC drug and linkers molecules are often hydrophobic, making them incompatible with fractionation via size-exclusion chromatography (SEC). Field flow fractionation (FFF) separates macromolecules and nanoparticles by size without a stationary phase, eliminating most of the non-ideal surface interactions prevalent in SEC. Coupling an Eclipse™ FFF device to a Wyatt DAWN® HELEOS® II MALS detector creates a versatile system for accurate and robust characterization of molar mass and size distributions from 1 nm to 1000 nm. SEC-MALS FFF-MALS CG-MALS DLS MP-PALS Molar Mass Size Charge Conjugation Interactions Conclusions High-throughput screening via the DLS Plate Reader is essential for assessing stability and aggregation of ADCs, and the impacts of linker chemistry on the onset of aggregation. FFF-MALS yields high-resolution characterization of aggregates, leading to a better understanding of the aggregation process. In a separate study, MALS-UV-RI analysis determined drug-antibody ratio (DAR). The light scattering toolkit contributes to complete characterization of ADCs, leading to an efficient and rapid development timeline. Temp (°C) 7.0 Radius (nm) 600 Time (min) 1.0x10 9 1.0x10 8 1.0x10 7 1.0x10 6 1.0x10 5 1.0x10 4 1000 100 10 1.0 0.1 0 Molar Mass 10.0 12.0 14.0 16.0 18.0 20.0 22.0 1.0x10 9 1.0x10 8 1.0x10 7 1.0x10 6 1.0x10 5 1.0x10 4 1000 100 10 1.0 0.1 0 Time (min) 10.0 15.0 20.0 25.0 1.0 0.5 0.0 Relative Scale Time (min) Molar Mass 10.0 12.0 14.0 16.0 18.0 20.0 22.0 Radius (nm) Temp (°C) % Intensity Radius (nm) 20 10 0 0.1 1.0 10.0 100.0 0 20.0 40.0 60.0 400 200 6.0 5.0 20.0 40.0 60.0
Transcript of Aggregation of Antibody-Drug Conjugates: The Light ...... Aggregation of Antibody-Drug Conjugates:...
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www.crystal.csiro.au
Aggregation of Antibody-Drug Conjugates: The Light
Scattering Toolbox for Screening and Characterization
Chris Broomell and Daniel Some, Wyatt Technology Corporation, 6300 Hollister Ave, Santa Barbara, CA 93117
Size Distributions by FFF-MALS
scattered wI M c
Aggregation Analyses of Stressed and Unstressed ADCs by FFF-MALS
Abstract
Antibody-Drug Conjugates (ADCs) show much promise as effective therapeutics for cancer
and other diseases. However, they often exhibit an increased aggregation propensity
compared to their unmodified counterparts due to non-specific interactions arising from
attached drug and linker moieties. Light scattering offers multiple techniques for
addressing the challenges of formulation screening, and characterizing both aggregates and
propensity for aggregation. We present these tools in the context of ADCs and the
dependence of aggregation profiles on linker chemistry.
© Wyatt Technology Corporation 2014 – All Rights Reserved
Linker-Induced Instability Studied by DLSHigh-Throughput Dynamic Light Scattering
Dynamic light scattering (DLS) analyzes Brownian motion via the
intensity fluctuations of light scattered by macromolecules and nanoparticles in solution.
DLS determines directly diffusion coefficients Dt. Particle radius Rh is calculated from the
Stokes-Einstein equation:
www.synaffix.com
Identical mAb and drug, different linkers
Tonset = 63.9°C Tonset = 48.6°C
Protein stability is typically studied by temperature of melting (TM)
or onset of aggregation. ADC2 unfolds and aggregates at a lower
temperature, and is therefore less stable than ADC1.
These data suggest distinct aggregation pathways.
Typical Fractogram –Unstressed Sample
Typical Fractogram –Stressed Sample
Peak1 Peak 2 Peak 3
% Mw % Mw %
Unstressed 0.75 148.4 97.75 231.5 1.5
Stressed 1.40 149.6 95.1 297.1 3.5
Mw is determined by the ratio of light
scattering signals to UV. Mass fraction is
determined by integrating UV peaks. FFF-
MALS is highly sensitive to small quantities
of aggregates due to the dependence of
scattered intensity on Mw.
149.4 kDa (monomer)
Higher-order species present instressed sample
~339 kDa (average)
* Small shoulder suggestive of fragmentation.This is present to some extent in all samples.
*
Peak 2 (Monomer)
Peak 1 (Fragment)
Peak 3 (Oligomer)
Average Mw and Mass Fraction (n=2)
Mw = 149.2 kDafrom MALS
Multi-angle, static light scattering (MALS) analyzes
molar mass Mw from first principles, via the average
intensity of scattered light I and concentration c:
ADC1
ADC2
ADC2ADC1
DLS determines size distributions without
fractionation, providing polydispersity estimates.
High-throughput screening via the DynaPro® Plate
Reader II is a powerful tool for characterizing multiple
aspects of biotherapeutic stability:
• Degree and type of aggregation
• Thermal conformational stability (TM, Tonset)
• Chemical conformational stability
• Colloidal stability
𝑅ℎ =𝑘𝑇
6𝜋𝜂𝐷𝑡
This analysis is independent of questionable
reference standards and column calibration.
In an Asymmetric-Flow FFF separation channel, macromolecules and nanoparticles are gently pushed against a semipermeable membrane by crossflow. Smaller particles diffuse back up towards the center of the channel. Laminar channel flow induces a parabolic flow velocity profile, causing smaller particles to elute earlier.
ADC drug and linkers molecules are often hydrophobic, making them incompatible with
fractionation via size-exclusion chromatography (SEC). Field flow fractionation (FFF)
separates macromolecules and nanoparticles by size without a stationary phase,
eliminating most of the non-ideal surface interactions prevalent in SEC.
Coupling an Eclipse™ FFF device to a Wyatt DAWN® HELEOS® II MALS detector creates a
versatile system for accurate and robust characterization of molar mass and size
distributions from 1 nm to 1000 nm.
SEC-MALS FFF-MALS CG-MALS DLS MP-PALS
Molar Mass Size Charge Conjugation Interactions
Conclusions
High-throughput screening via the DLS Plate Reader is essential for assessing stability and
aggregation of ADCs, and the impacts of linker chemistry on the onset of aggregation.
FFF-MALS yields high-resolution characterization of aggregates, leading to a better
understanding of the aggregation process.
In a separate study, MALS-UV-RI analysis determined drug-antibody ratio (DAR).
The light scattering toolkit contributes to complete characterization of ADCs, leading to
an efficient and rapid development timeline.
Temp (°C)
7.0
Rad
ius
(nm
)
600
Time (min)
1.0x109
1.0x108
1.0x107
1.0x106
1.0x105
1.0x104
1000
100
10
1.0
0.1
0
Mo
lar
Mas
s
10.0 12.0 14.0 16.0 18.0 20.0 22.0
1.0x109
1.0x108
1.0x107
1.0x106
1.0x105
1.0x104
1000
100
10
1.0
0.1
0
Time (min)
10.0 15.0 20.0 25.0
1.0
0.5
0.0
Re
lati
ve S
cale
Time (min)
Mo
lar
Mas
s
10.0 12.0 14.0 16.0 18.0 20.0 22.0
Rad
ius
(nm
)
Temp (°C)
% In
ten
sity
Radius (nm)
20
10
0
0.1 1.0 10.0 100.0
0
20.0 40.0 60.0
400
200
6.0
5.020.0 40.0 60.0
