HOS Characterization, Stability, and
Comparability of ADCs
– Biophysical Analyses
Yin Luo*, Sharon Polleck, Lucy Liu
Analytical R&D, Biotherapeutic Pharmaceutical Sciences
Pfizer, Andover, MA
CASSS HOS 13-April-2015
What is an ADC Covalent conjugate of cytotoxic drug (payload) with mAb against
selected antigen on the surface of target cells
What are the advantages of ADC − increase efficacy
− decrease toxicity (lower dosing of toxic drug)
Why ADCs have these advantages ‒ enhanced drug specificity – mAb binding to target antigens
‒ superior Mode of Action – drug cytotoxicity within target cells
Linker and Payload
mAb
Antibody Drug Conjugate (ADC)
2
Target
cell
Cell death
mAb binding ADC internalization Cytotoxicity
Target
cell
mAb binding
Cell death
ADCC
CDC
Cytotoxicity
mAb cytotoxicity MOA ADC cytotoxicity MOA
Fc effector
functions
DNA / scaffold
destruction
Common antibody-drug conjugation strategies
3
Conjugation
strategy Typical locations
Typical DAR*
distribution
Semi site
specific
Cysteine (inter-chain S-S partial
reduction)
IgG1 IgG4
Lysine
Site
specific
Cysteine (engineered)
Glutamine tag (transglutaminase)
*Drug Antibody Ratio
4
Heterogeneity of inter-chain cysteine conjugation
Interchain S-S
partial reduction
Thioether
conjugation
Possible locations DAR
2
4
6
8
mAb
Challenges of ADC as biotherapeutics
CMC – complex manufacturing
– insufficient conjugations
– lot to lot reproducibility
– challenging analytics
Potency – uncontrolled payload loading
– maleimide exchange with HSA
– PK liability
– variable potency
Toxicity – linker instability resulting in premature drug release
– aggregation
– first pass metabolism in liver
5
Boswell, CA, R et al. Bioconjugate Chemistry 2011, 22, 1994 - 2004
Junutula, R et al. Nat Biotechol. 2008, 26, 925 – 932.
Junutula, R et al. Clinical Caner Res. 2010, 16, 4769 – 4778.
Analytical challenges
Heterogeneity (multiple payload
conjugation)
Payload hydrophobicity
Linker/payload stability
Aggregation propensity of ADC
Impact of conjugation on HOS
Relation of HOS to ADC function
Unconjugated species
etc
Foci of this
presentation
HOS: higher order structure
Protein higher order structure (HOS) and biophysical
methods for (low-resolution) characterizations
Far-UV CD Near-UV CD SEC-Multi Angle Light Scattering
(SEC-MALS)
FTIR Intrinsic fluorescence Analytical Ultracentrifugation (AUC)
Differential scanning
calorimetry (DSC) Field Flow Fractionation (FFF) - MALS
Dynamic light scattering (DLS)
Modified from
Figure 3-23
Lehninger “Principles
of Biochemistry”
5th Edition
HOS
Common
methods Common
methods
Case studies – Molecular information
Lysine conjugation 6 mAb3 (IgG4)
ADC3
Linker/Payload Conjugation
Partial reduction/
cysteine conjugation
Partial reduction/
cysteine conjugation
Average
DAR*
4
4 mAb1 (IgG1)
mAb ADC
mAb2 (IgG1)
ADC1
ADC2
MW ~1660
*Drug Antibody Ratio
Case studies –
Aggregation propensity of ADC in comparison
with mAb by SEC-MALS and AUC
8
Pfizer Confidential │ 9
Aggregation in mAb1 and ADC1 by SEC-MALS
0.E+00
1.E-05
2.E-05
3.E-05
4.E-05
5.E-05
6.E-05
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
5 6 7 8 9 10 11 12 13 14 15
Dif
fere
nti
al R
efra
ctiv
e In
dex
Mo
lar
Ma
ss (g
/mo
l)
Time (Minutes)
0.E+00
1.E-05
2.E-05
3.E-05
4.E-05
5.E-05
6.E-05
7.E-05
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
5 7 9 11 13 15
Dif
fere
nti
al R
efra
ctiv
e In
dex
Mo
lar
Ma
ss (g
/mo
l)
Time (Minutes)
mAb1 ADC1
Weight average
molar mass
Weight average
molar mass
SEC-MALS reported dimer as the main component of the HMMS of both mAb1
and ADC1 in similar quantities. ADC3 & mAb3 show similar profiles
Sample Monomer Dimer
Weight average
molar mass % Weight average
molar mass %
mAb1 148 99.0 295 1.0
ADC1 152 98.7 318 1.3
Aggregation in mAb1 and ADC1 by AUC
Pfizer Confidential │ 10
0
0.5
1
1.5
2
2.5
4 6 8 10 12 14 16 18 20
-0.02
0
0.02
0.04
0.06
0.08
0.1
4 6 8 10 12 14 16 18 20
C (
S2
0,w
)
S(20,w)
C (
S2
0,w
)
S(20,w)
HMMS
Monomer
0
0.5
1
1.5
2
2.5
4 6 8 10 12 14 16 18 20
-0.02
0
0.02
0.04
0.06
0.08
0.1
4 6 8 10 12 14 16 18 20
S(20,w)
C (
S2
0,w
)
C (
S2
0,w
)
S(20,w)
Monomer
HMMS
The mAb and ADC were run
in the SE-HPLC mobile phase
mAb1 ADC1
Sample Monomer (%) Dimer (%)
mAb1 >99
Aggregation in mAb2 and ADC2 by SEC-MALS
mAb
Sample Monomer “Other” Dimer
Weight average
molar mass % Weight average
molar mass % Weight average
molar mass %
mAb2 147 99.6 NA NA 311 0.3
ADC2 156 96.2 234 1.7 328 2.0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
8 10 12 14 16 18 20 22 24
UV
Ab
sora
nce
@ 2
80
nm
Mo
lar
Ma
ss (D
a)
Time (Minutes)
Dimer 328 kDa
Monomer 156 kDa
“Other” 234 kDa
ADC2 DAR = 4.2
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
8 10 12 14 16 18 20 22 24
UV
Ab
sorb
an
ce @
28
0 n
m
Mo
lar
Ma
ss (D
a)
Time (Minutes)
SEC-MALS reported dimer and “other” species with mass between monomer and dimer
Pfizer Confidential │ 12
mAb2
0.0
1.0
2.0
3.0
4.0
5.0
6.0
4 5 6 7 8 9 10 11 12 13 14 15
C (
s)
S
0.00
0.05
0.10
0.15
0.20
0.25
4 5 6 7 8 9 10 11 12 13 14
C (
s)
S
Monomer
Dimer
ADC2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4 5 6 7 8 9 10 11 12 13 14 15
C (
s)
S
0.0
0.1
0.2
0.3
0.4
0.5
4 5 6 7 8 9 10 11 12 13 14 15
C (
s)
S
Monomer
Dimer
“Other”
The mAb and ADC were run in the SE-HPLC mobile phase
Aggregation in mAb2 and ADC2 by AUC
AUC confirmed low levels of dimer and “other” species, but did not detect larger aggregates
Sample Monomer (%) “Other” (%) Dimer (%)
mAb2 98.6 NA 1.4
ADC2 92.1 5.1 2.8
Aggregation in ADC by SEC-MALS
Exemplary ADC early development materials
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000
275000
300000
325000
350000
375000
400000
425000
450000
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
11 12 13 14 15 16 17 18 19 20 21
Mol
ar M
ass
(g/m
ol)
Abso
rban
ce (A
U)
Time (Minutes)
UV
Molar Mass
13
8 k
15
0 k
17
7 k
29
0 k
35
4 k
SEC-MALS can provide average molar mass for SE-HPLC fractions to
characterize heterogeneous materials and help process development
Summary of aggregation characterization for ADC
SEC-MALS is useful to characterize/confirm the resolution of a SE-
HPLC method via the in-line measurement of average molar mass for
any given fraction (assay and process development support)
AUC is useful to characterize the size distribution of a sample using a
matrix-free separation mode, but results depend on fitting parameters
The two methods provide size information by different principles; each
has pros and cons, no “gold standard”
The case studies shown in this work indicate
– The multiple-site payload conjugations did not significantly increase
the aggregation propensity of the ADCs compared to the mAbs
– Low level new species smaller than dimer was observed, no new
species larger than dimer was above the detection limit
– The heterogeneity in conjugation products may be controlled by
process improvements
14
Case studies –
Characterization of ADC higher order structure
(HOS) using CD, fluorescence and DSC
15
Far-UV CD: ADC1 vs mAb1, ADC2 vs mAb2
16
De
(M-1
cm
-1)
Wavelength (nm)
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
195 205 215 225 235 245 255
hu6M024
PF-06647020
mAb2
ADC2
ADC1 vs mAb1 ADC2 vs mAb2
The multi-site payload conjugations did not alter the secondary structure of the mAbs
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
190 200 210 220 230 240 250
Δε
(M-1
cm-1
)
Wavelength (nm)
PF-06263507PF-06263507
huA1
mAb1
ADC1
17
Near-UV CD: ADC1 vs mAb1, ADC2 vs mAb2
-100
-80
-60
-40
-20
0
20
250 260 270 280 290 300 310 320 330 340 350De
(M
-1cm
-1)
Wavelength (nm)
mAb2
ADC2
ADC1 vs mAb1 ADC2 vs mAb2
The multi-site payload conjugations did
not alter the tertiary packing of the mAb1
in ADC1
Are the spectral differences indicative of
structural change in mAb2 by the payload
conjugation?
-40
-30
-20
-10
0
10
20
250 270 290 310 330 350
Δε
(M-1
cm-1
)
Wavelength (nm)
PF-06263507
huA1
mAb1
ADC1
18
Near-UV CD: Payload absorption in ADC2
-100
-80
-60
-40
-20
0
20
250 260 270 280 290 300 310 320 330 340 350
De
(M
-1cm
-1)
Wavelength (nm)
-95
-75
-55
-35
-15
5
250 260 270 280 290 300 310 320 330 340 350
De
(M
-1cm
-1)
Wavelength (nm)
mAb2+payload
ADC2
ADC2 vs (mAb2 + linker/payload)
The multi-site payload conjugations did not alter the tertiary structure of the mAbs
Linker/payload may also absorb, needs to be taken into account
ADC2 vs mAb2
mAb2
ADC2
Wavelength (nm)
250 275 300 325 350
-40
-30
-20
-10
0
10
20
Inotuzumab
F75626
20 °C
De
(M-1
cm-1
)
A
Near-UV CD: ADC3 vs mAb3
19
Wavelength (nm)
250 275 300 325 350
-200
-100
0
100
200
300 Inotuzumabozogamicin A
Inotuzumab
ozogamicin B
Inotuzumab
ozogamicin C
Inotuzumab
F75626
20 °CDe
(M-1
cm-1
)
B
mAb3 ADC3 vs mAb3
ADC3 (DAR=7.3)
ADC3 (DAR=6.6)
ADC3 (DAR=4.6)
mAb3
Linker/payload absorbance can dominate the spectrum, rendering the method
not suitable for structural characterization for the mAb in the ADC
Alternative method: Fluorescence spectroscopy
20
Fluorescence: ADC3 vs mAb3 Payload 3 CD and UV absorbance
Wavelength (nm)
350 400 450 500 550
Flu
ores
cen
ce I
nte
nsi
ty0
200
400
600
800
1000
inotuzumab
F75626
inotuzumab
ozogamicin
2007B0088
inotuzumab
ozogamicin
H87906
20 °C
A
mAb3
ADC3
mAb3 + payload 3
The linker/payload absorbance can also
affect fluorescence emission spectra
Giorgio, at al. (2005) Bioorganic
& Medicinal Chemistry 13:5072
excitation
wavelengths
Fluorescence spectra: ADC3 vs mAb3
21
Wavelength (nm)
350 400 450 500 550F
luo
resc
ence
In
ten
sity
0
200
400
600
800
1000
inotuzumab
F75626 in buffer
inotuzumab
ozogamicin
H87906 in buffer
inotuzumab
F75626 in 6M GdmCl
inotuzumab
ozogamicin
H87906 in 6M GdmCl
20 °C
Wavelength (nm)
350 400 450 500 550
Flu
ores
cen
ce I
nte
nsi
ty
0
200
400
600
800
1000
inotuzumab
F75626
inotuzumab
ozogamicin
2007B0088
inotuzumab
ozogamicin
H87906
20 °C
A mAb3
ADC3
mAb3 + payload 3
Native (formulation) ADC3 & mAb3 (peak normalized)
Denatured (6M GdmCl) ADC3 & mAb3
ADC3 vs mAb3 Native vs denatured
The fluorescence spectrum of ADC is superimposable with the peak-normalized
spectrum of the mAb, indicating the overall shape of the mAb is not altered in ADC
Using near-UV CD as a screening tool - a caveat
22
mAb1
Be cautious when interpreting the
apparent trends
-30
-25
-20
-15
-10
-5
0
0 1 2 3 4 5 6
Δe
(M
-1cm
-1) @
272 n
m
GuanidineHCl (M)
[GdmCl (M)]
PBS
mAb1 in GdmCl
@272 nm
-40
-30
-20
-10
0
10
20
260 280 300 320 340
Ele mAb 0MEle 1M mAbEle mAb 3MEle mAb 6M
A
0
272 nm
1 M
3 M
6 M
De
(M
-1cm
-1)
Wavelength (nm)
mAb1
6 M GdmCl
3 M GdmCl
FB (pH 6)
1 M GdmCl
6 M GdmCl
-40
-30
-20
-10
0
10
20
250 260 270 280 290 300 310 320
De
(M
-1cm
-1)
Wavelength (nm)
FB (pH 6)
FB + 150mM NaCl
PBS (pH 7)
Pfizer Confidential │ 23
Thermal stability: DSC profiles
0
40
80
120
160
30 40 50 60 70 80 90
Temperature (°C)
Cp
(kca
l\m
ole
\°C
)
ADC
huA1 mAb1 (IgG1)
ADC1 (Cys conj)
-5
20
45
70
95
120
145
35 45 55 65 75 85 95
Cp
(k
cal/
mo
le/
C)
Temperature ( C)
ADC1 and mAb1 exhibit different thermal
stability profiles; all Tm’s are >60ºC
Tm1 of ADC2 is lower than Tm1 of
mAb1; all Tm’s are >60ºC
mAb2 (IgG1)
ADC2 (Cys conj)
c 24
Thermal stability: DSC profiles
Temperature (°C)
30 40 50 60 70 80 90
0
50
100
inotuzumabozogamicin2007B0088
inotuzumabozogamicinH87906
inotuzumab F75626
Cp
(kcal·m
ole-
1 ·°C
-1)
ADC3 and mAb3 exhibit different thermal stability
profiles; all Tm’s are >60ºC
mAb3 (IgG4)
ADC3 (Lys conj)
Interpretations – structural perspectives
In general ─ Spectroscopic methods are non-destructive methods to assess the
HOS of proteins, reporting mainly the overall shape
─ DSC is a destructive method, applying heat to unfold proteins; it
reflects mainly the strength of side chain interactions in folded proteins
─ Proteins with similar shapes may have different side chain interaction
energies, manifest in different stabilities
25
For Pfizer ADC1, ADC2 and ADC3
─ The CD and fluorescence spectra of the ADCs are similar to that of their respective
mAbs, indicating that the protein conformations are not significantly altered by the
multi-payload conjugations
─ The DSC profiles of the ADCs are not the same as that of their respective mAbs,
indicating changes in amino acid side chain interactions due to the payload
conjugation and/or the conjugation process
─ The payload, IgG type, conjugation chemistry and conditions, payload locations,
etc., may affect the structural properties of the conjugates
Egg vs egg-
shaped rock
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Interpretations – relation to function and safety
In general ─ Cytotoxic immune responses induced by mAb require the antigen-binding and the
Fc effector functions, therefore, require intact structure of the entire mAb
─ Cytotoxicity induced by ADC requires mAb binding to the antigen, therefore,
requires intact structure of the antigen-binding region
─ Once internalized, the payloads cause the cell death; therefore, the structure of
mAb other than the antigen-binding region is not critical to the function of ADC
─ However, a mAb in ADC with significantly altered structure may pose quality and
immunogenicity (anti-drug antibodies) issues for the ADC as biotherapeutics
26
Target
cell
Cell death
mAb binding ADC internalization Cytotoxicity
Target
cell
mAb binding
Cell death
ADCC
CDC
Cytotoxicity
mAb cytotoxicity MOA ADC cytotoxicity MOA
Fc effector
functions
DNA / scaffold
destruction
For Pfizer ADC1, ADC2 and ADC3
─ The antigen-binding of each ADC is comparable to the respective mAb,
indicating the first step of the ADC function is not compromised by conjugation
─ The overall shapes (conformation) of the mAbs are not altered by the
conjugations, suggesting little or no new surface is exposed; therefore, little or
no change in the immunogenicity of the protein (anti-drug antibodies)
─ The thermal stability may be affected by the conjugation; however, all Tm’s of
these ADCs are >60ºC, suggesting the HOS is stable under the storage
conditions and at the body temperature
─ These 3 ADCs should have the expected functionalities, with low safety
concerns from the HOS perspective
27
mAb1
ADC1
Antigen-binding ELISA for the ADCs vs the mAbs
mAb3
ADC3
mAb2
ADC2
Interpretations – relation to function and safety
Wavelength (nm)
350 400 450 500 550
0
200
400
600
Flu
ores
cen
ce I
nte
nsi
ty
Wavelength (nm)
200 210 220 230 240 250
-1
0
1
2
De
(M-1
cm-1
)
Biophysical methods for product comparability
28
-100
-80
-60
-40
-20
0
20
250 260 270 280 290 300 310 320 330 340 350
De
(M-1
cm-1
)
Wavelength (nm)
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
195 205 215 225 235 245 255
De
(M-1
cm-1
)
Wavelength (nm)
30 40 50 60 70 80 90
0
20
40
60
80
100
120
140
Demo1 A
Demo 1 B
Demo 1 C
GMP0 A
GMP0 B
GMP0 C
RM ADC A
RM ADC B
RM ADC C
Cp
(kca
l/m
ole
/oC
)
Temperature (oC)
Batch 1
Batch 2
Batch 3
Batch 1
Batch 2
Batch 3
Batch 1
Batch 2
Batch 3
ADC2 ADC2 ADC2
Far-UV CD Near-UV CD DSC
Far-UV CD Fluorescence
Batch 1
Batch 2
Batch 3
ADC3
Batch 1
Batch 2
Batch 3
ADC3
Acknowledgements
Lucas Wafer
Cliff Entrican
Marek Kloczewiak
Zhaojiang Lu
Sharada Sant
Peter Richard
Jamie Lee
Tom Lerch
Jim Zobel
29
Jason Starkey
Jim Mo
Heyi Li
Scott Allen
Olga Friese
Jason Rouse
Meg Ruesch
c 30
DSC profiles for IgGs
Garber and Demarest (2007) Biohem Biophys Res Commun. 355:751-7
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