Download 2. Antibody drug conjugates

the ancient pond
a frog jumps in
sound of water
—Matsuo Bashō (1686)
Bioanalysis of Antibody-Drug
Conjugates (ADCs): an overview
of current approaches and case
studies illustrating the
challenges presented by hybrid
biotherapeutic molecules
Rand Jenkins
7th JBF Symposium
Tokyo Japan, 09 March 2016
Overview
3
1
Introduction to ADCs
2
BA complexity & current approaches
3
LC-MS/MS assay options
4
Need for characterization—LC-HRAMS
5
BA practicality challenges
6
Final thoughts
Biotherapeutics innovation
peptides
mAbs
bispecifics (bsAbs, DART®s, BiTE®s)
ADCs
prodrug/carrier conjugates (albumin, PEG)
fusion proteins
messenger RNA therapeutics™
oligonucleotides (ssDNA, siRNA, mRNA)
CAR-Ts
4
ADCs
Monoclonal antibody drugs
6
Monoclonal antibody drugs in cancer therapy
●
Effective for blood cancers
−
●
Limited effectiveness for
solid tumors
−
−
7
e.g. Rituximab for B-cell
non-Hodgkin lymphoma
e.g. Herceptin for HER2+
breast cancers (20-25%)
Usually require co-therapy
with standard cytotoxic
“chemo” drugs such as
paclitaxel or doxorubicin
Creating a “magic bullet”
Small
Cytotoxic
“Drug”
8
+
Monoclonal
Antibody
(mAb)
Antibody-Drug Conjugate
9
Antibody-Drug Conjugate
Like a mAb drug
●
Seeks out & binds to
specific tumor cells
●
Long half life
ADC
ADC advantages
●
Non-toxic in circulation
●
Delivers highly toxic
payload to tumor cells
●
Toxin released inside to
“safely” kill cells
10
↖
↑
linker
drug
“payload”
drug-antibody ratio (DAR) = 6
ADC technology – how they function
11
FDA approved ADC products
NEWS RELEASE: Aug. 19, 2011 (Seattle Genetics)
FDA approves ADC to treat two types of lymphoma
“The U.S. Food and Drug Administration today approved ADCETRIS
(brentuximab vedotin) to treat Hodgkin lymphoma (HL) and a rare
lymphoma known as systemic anaplastic large cell lymphoma (ALCL)”
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncementsucm268781.htm
NEWS RELEASE: Feb. 22, 2013 (Genentech/Roche)
FDA approves new treatment for late-stage breast cancer
“The U.S. Food and Drug Administration today approved Kadcyla
(ado-trastuzumab emtansine), a new therapy for patients with
HER2-positive, late-stage (metastatic) breast cancer”
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncementsucm340704.htm
12
Kadcyla®
ADCETRIS®
S. Kaur, Land O'Lakes, ADC Bioanalysis, 07.18.13
newer
engineered
designs
ADC diversity
linkers & payloads
Examples of ADC’s and Linkers and Payloads
ADCETRIS®
Monomethyl auristatin E (MMAE)
Adcetris
Brentuximab vedotin
(SGN-35)
Seattle Genetics
Payload - Monomethyl
auristatin E (MMAE)
Cleavable payload
Kadcyla
Trastuzumab emtansine, adotrastuzumab emtansine
(T-DM1)
Genentech
Payload – Maytansinoid (DM)
Non-cleavable payload
Maytansinoid (DM)
Kadcyla®
Inotuzumab ozogamicin
(INO, CMC-544)
and Mylotarg
Pfizer
Payload - N-Ac- Calicheamicin DMH
Cleavable payload
N-Ac-
Calicheamicin DMH
2013 NBC meeting, ADC Bioanalysis Workshop, 18-May-2013 / LCMS Assays for payloads and intact ADC’s / Leo Kirkovsky (Pfizer) , [email protected]
4
Other payloads…
Pyrrolobenzodiazapine (PBD) dimers
Tubulysin analogues
16
New ADC conjugation idea: GlycoConnect™
Remon van Geel et al, Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native
mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates, Bioconjugate Chem. 2015, in press
17
New ADC conjugation idea: Fleximer polymers
18
ADC complexity
What should be measured?
S. Kaur, Land O'Lakes, ADC Bioanalysis, 07.18.13
Heterogeneity challenge for bioanalysis
Pharmacokinetic
Assay Strategy: T-DM1
® example)
(Kadcyla
T-DM1: Trastuzumab“active”
with DM1 conjugated
(1-8; DAR: 3.5)
Conjugated
ADCs
12
12
-- serum
Total Trastuzumab: unconjugated Trastuzumab plus conjugated T-DM1 -- serum
Total
mAb (with or without drugs)
DM1 with TCEP
reduction
of disulfide
– plasma
“Free”
drug
(and
otherbonds
forms)
*X
*
DM1 dimer
DM1
Other
DM1-S-S-X
*
DM1-albumin
Linker-containing catabolites: MCC-DM1, Lysine-MCC-DM1
21
*
DM1 disulfide bound
to antibody
Standard ADC bioanalytical assays
Assay
Technology
Total Antibody, DAR ≥0
Assess general mAb PK behavior—efficacy
LBA (ELISA)
Conjugated Antibody, DAR ≥1
Measure total ADC molecules—efficacy
LBA (ELISA)
Anti-Therapeutic Antibody (ATA)
Detect immune response—efficacy & safety
LBA (ELISA)
and Cell-based NAb
Unconjugated Drug
Detect “free” drug—safety
LC-MS/MS
Pharmacokinetic data
(Kadcyla® example)
Phase I Study Trastuzumab-DM1 in Patients With Breast Cancer
T-DM1
Total Trastuzumab
DM1
Mean Concentration (ng/ml)
1,000,000
100,000
10,000
1,000
100
10
1
0.1
0
100
200
300
400
500
600
Time (hours)
Fig 3. Mean levels of trastuzumab-DM1 (T-DM1), total trastuzumab, and DM1
over time are shown for patients after the first dose of T-DM1 administered at the
maximum-tolerated does of 3.6 mg/kg. Standard deviation is shown in error bars.
Adverse Events v3 (NCI CTCAE v3), except for cardiac troponin I elevation
23
and segmental
wall-motion abnormalities (not described in CTCAE v3).
in Table 1, received a cumula
doses ranging from 0.3 to 4
treated at each dose level and d
shown in Figure 2.
← Total
ThreemAb
patients were treate
0.6-, 1.2-, and 2.4-mg/kg coho
served drug-related grade ! 2 e
←enrolled
Conjugated
in the 4.8-mg/kg coh
ADCpromp
4“active”
thrombocytopenia,
dose level—3.6 mg/kg (as
schema). After six patients wer
was declared
←mg/kg
“Free”
drugthe MTD,
of 15 patients.
Study treatment was ongo
13, 2009. Fifteen patients withd
sive disease, two because of th
cause of AEs (grade 4 thrombo
grade 3 pulmonary hypertens
grade 3 muscular weakness in
ered related to study treatment;
metastatic disease in the brain]
LBA Assays for ADCs are More Complex
than LBA for mAb Therapeutics
mAb Assay
S. Kaur, AAPS, San DIego, 11.04.14
11
© 2014, Genentech
LBA Assays for ADCs are More Complex
than LBA for mAb Therapeutics
mAb Assay
Total
Antibody Assay
S. Kaur, AAPS, San DIego, 11.04.14
Conjugated
Antibody Assay
11
© 2014, Genentech
Characterize Ligand Binding Assays with Individual DARs
Identified in Plasma to Evaluate Assay performance
Anti-STEAP1 ADC Purified DAR
Assay
0
1
2
4
6
Total Antibody ELISA
(% recovery)1
88
78
86
92
92
Conjugated-Antibody ELISA
(% recovery) 1
1 Based
•
•
NA
11
102
99
64
specificity
issue
on expected nominal concentration of individual DARs spiked into serum
Used individual DAR controls from plasma to test with ELISA ligand binding reagents
No single anti-Drug mAb reagent in conjugate Ab assay appropriate for all DARs
S. Kaur, AAPS, San DIego, 11.04.14
12
© 2014, Genentech
ADC bioanalysis using LBAs
Advantages
• Traditional approach – the gold standard
• High throughput and low cost per sample
Challenges
• MD expensive and time-consuming
• Potential interference from soluble target
• Reagents may show variable response to DARs
27
ADC bioanalysis
LC-MS/MS alternatives
Alternative ADC bioanalytical assays
Assay
Technology
Total Antibody, DAR ≥0
LBA (ELISA)
*
Conjugated Antibody, DAR ≥1
Measure of conjugated antibodies only
LBA (ELISA)
*
Antibody-conjugated Drug, DAR ≥1
Measure of conjugated “active” drug load
Affinity capture LC-MS/MS
(requires cleavable linkers)
Total Antibody, DAR ≥0
Most convenient for non-clinical
Affinity capture LC-MS/MS
**
**
Develop Appropriate PK Assays:
Molar Concentration
Hybrid Binding LC-MS/MS & LBA Conjugate Assay Formats
Conjugated
Conjugated
Antibody
AntibodyAssay
Assay
Detection reagent
(Biotin antigen/SAHRP or Biotin antiCDR/SA-HRP)
ADC (carrying 1 or
more drugs)
Total-Antibody
Due to DAR 0
Conjugated-Antibody
Free Drug
Capture reagent
(anti-drug mAb)
Molar Concentration
Time
AntibodyConjugated
Drug Assay
Total-Antibody
Due to DAR Changes
Antibody-Conjugated Drug
Free Drug
LC-MS/MS detection of
released drug
Linker cleavage/
ADC digestion step
ADC and
Endogenous
IgG
Capture by
Protein A (resin)
Time
13
S. Kaur, AAPS, San DIego, 11.04.14
© 2014, Genentech
Ab-conjugated drug AC-LC-MS/MS assay
Inter-assay accuracy & precision
Total mAb universal AC-LC-MS/MS assay
(non-clinical applications)
1. Generic affinity capture
●
●
Anti-human Fc
Protein A or G
2. “On-bead” processing
●
LC-MS/MS
Typical chemistry steps:
- Denaturation
- Reduction
- Alkylation
- Trypsin digestion
Peptides
Trypsin
Anti-hFc
antibody
3. LC-MS/MS Detection
●
Human IgG peptides
32
Magnetic
bead
mAb
target
LC–MS/MS assays.
sensitivity; identify the most promising light
Unique
human
choices
The most critical
performancemAb
metric forpeptide
an IS and heavy
chain candidate peptides based upon
is how reliably the ISapplications)
tracks the analyte through- their individual performance; and combine the
(non-clinical
out bioanalytical extraction and LC–MS/MS most promising light and heavy chain candidates
Table 1. Amino acid sequences of universal peptide candidates.
Peptide
Sequence
Human heavy chain subclass
TVAAPSVFIFPPSDEQLK
SGTASVVCLLNNFYPR‡
VDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSK
NA
NA
NA
NA
TPEVTCVVVDVSHEDPEVK‡
FNWYVDGVEVHNAK
VVSVLTVLHQDWLNGK§
GFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSK
IgG1
IgG1
IgG1, IgG4
IgG1, IgG2, IgG4
IgG1
Light chain candidates†
TVAA→
SGTA→
LC-1
LC-2
LC-3
LC-4
Heavy chain candidates
HC-1
HC-2
HC-3
HC-4
HC-5
VVSV→
TTPP→
All light chain peptide candidates are in the constant region of the -class.
Peptide quantified as the S-carboxymethylcysteine derivative.
§
Peptide HC-3 was previously identified as a universal peptide [7].
†
‡
Michael T Furlong, Song Zhao, William Mylott, Rand Jenkins, Mian Gao, Vendana Hegde, James Tamura, Adrienne Tymiak &
Mohammed Jemal, Bioanalysis 2013, 5(11), 1363−1376.
future science group
33
www.future-science.com
Total mAb Method Performance Evaluation (MPE):
calibration standards and batch acceptance QCs
Run ID
1RBFE2-A
2RBFE2-A
Theoretical
Concentration
Mean
%C.V.
CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8
QC 1
(µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL)
(µg/mL)
(µg/mL) (µg/mL)
0.859
3.05
11.4
41.7
48.7
0.148
1.46
35.2
0.0509 0.105
0.203
0.835
2.98
11.4
39.0
50.7
0.144
1.39
34.6
0.0538 0.096
0.217
0.802
3.02
12.5
42.7
49.8
0.151
1.49
35.9
0.0473 0.0971 0.194
0.805
3.01
11.6
37.5
48.5
0.140
1.51
36.8
0.0500 0.100 0.200
0.0503 0.0982 0.201
0.800
0.825
3.00
3.01
12.0
11.7
40.0
40.2
50.0
49.4
0.150
0.146
1.50
1.46
37.5
35.6
3.25
3.13
0.892
0.437
4.28
-2.27
5.99
0.548
2.06
-1.15
3.33
-2.98
3.48
-2.40
2.62
-5.04
4.78
-1.82
5.87
0.545
Acceptance criteria: ±25% for the LLOQ and ±20% for all other levels
34
QC 3
0.0492 0.0946 0.191
5.51
0.579
% Bias
QC 2
references
“Dual universal peptide approach to bioanalysis of
human monoclonal antibody protein drug candidates in
animal studies”
Michael T Furlong, Song Zhao, William Mylott, Rand Jenkins, Mian Gao, Vendana
Hegde, James Tamura, Adrienne Tymiak & Mohammed Jemal, Bioanalysis 2013,
5(11), 1363−1376.
“A general LC-MS/MS method approach using a
common whole molecule SIL-IS and a common
immuno-capture for sample clean up and enrichment
that is applicable to various mAbs in different matrixes”
Li, H.; Ortiz, R; Tran, L; Hall, M; Spahr,C; Walker, K; Laudemann, J; Miller, S;
Salimi-Moosavi, H; and Lee, J.W., Anal. Chem. 2012, 84, 1267−1273.
35
ADC bioanalysis using LC-MS/MS
Advantages
• Standard approach for small molecules
• “Direct” detection of molecules or fragments
• High sensitivity and selectivity/specificity
• MD relatively rapid; universal methods possible
• Data complimentary to LBAs
Challenges
• Complex and expensive instrumentation
• Lower throughput and higher costs per sample
36
through falling snow
a spring breeze
blows
—Kobayashi Issa (1818)
ADC biotransformation
characterization needed
ADCs Can Undergo Biotransformations In Vivo:
Analytes & Assay Standard Curve May Differ
Analysis of small molecule drug transformations in vivo is well established
–
Absorption, Distribution, Metabolism and Excretion (ADME) by LCMS/MS. (E.g., cytochrome P450 induction/inhibition, metabolic profiling, plasma protein
binding and P-gp transporters)
Analysis of protein or ADC transformations in vivo is challenging
Metabolites/
catabolites
Adducts
Intact antibodies
Reference Standard
Complexes with
Antigen and other antibodies
S. Kaur, AAPS, San DIego, 11.04.14
Small catabolites/metabolites
E.g., Drug, linker, linker-drug
Metabolites/catabolites
4
© 2014, Genentech
ADC characterization needed
Because:
•
Drug-antibody (DAR) distribution in vivo is dynamic
•
Drug payload can prematurely deconjugate
•
Higher drug-loaded species undergo faster clearance
•
Standard bioanalytical assays don’t detect post-dose
changes in DAR distribution
•
PK-PD modeling may be incorrect
•
Catabolism/metabolism-related changes may impact
efficacy and/or safety
40
LC-MS ADC characterization
Assay
Technology
Drug or drug-linker metabolites
Which species exist and are important?
LC-MS/MS
Catabolites (ADC or mAb)
Which species exist and are important?
Affinity capture LC-HRAMS
DAR Distribution
Assess ADC stability (in vitro / in vivo)
Affinity capture LC-HRAMS
or HIC
Critical reagent DAR specificity
Assess LBA reagents
Affinity capture LC-HRAMS
or HIC
Major focus on DAR distribution
To:
• Evaluate ADC reference and dosing material
• Assess stability-related changes in vitro
• Assess biotransformation-related changes in vivo
• Evaluate critical reagent specificity (for LBAs)
Results are generally qualitative or semi-quantitative
42
ADC characterization
Drug-Antibody Ratio (DAR)
Affinity Capture-LC-HRAMS
workflow courtesy of
Keyang Xu, Genentech
Affinity Capture LC-HRAMS allows direct
measurement of intact ADC changes
ABIO 10293
of Pages 11, Model 5G
YABIO No.
10293
January 2011
No. of Pages 11, M
25 January 2011
Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) 4xxx–xxx
A
Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) xxx–xxx
3
A
B
TIC!
7.0e4
6.0e4
Intensity (cps)
Biotinylated target
antigen, e.g., ECD
Wash
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
Elute
Streptavidin Coated
Paramagnetic Bead
Magnet
Plasma with ADCs
Biotin-Target
Antigen
0
Incubate @ 20-25 oC
Inject onto
LC-MS
ADCs
B
Intensity (counts)
mAb
20.0
16.0
14.0
8
10
Time (min)
× ×
+
× + +
×
× + +
+
× ×
**
× + +
6.0
2000
2200
70
Magnet separation
60
+ +
2400
Deconv
MS!
×
+
+
+
×
+: DAR 0
×: DAR 1
*: DAR 2
×
×
8.0
14
×
×
10.0
12
×
×
×
C
Deglycosylation, Wash
6
12.0
2.0
C
4
Raw
MS!
18.0
4.0
D
2
×
+
* * * * * *
2600
2800
m/z (amu)
+
×
+
×
×
* * + +
* * + +×
* * *
3000
3200
148
149
×
3400
DAR 1
: streptavidin coated paramagnetic bead,
: biotinylated capture probe,
: antibody or antibody drug conjugate (ADC)
: proteins, peptides and other species in plasma
Intensity (cps)
50
40
30
DAR 0
20
DAR 2
10
143
144
145
146
147
Schematic of the affinity capture LC–MS method to analyze ADCs in plasma or serum. (A) Biotinylated target antigen (e.g., ECD) is immobilized on paramagnetic
Mass (kDa)
tavidin beads by incubating at room temperature. Excess reagents are washed away. (B) The modified beads are then incubated with plasma/serum containing ADCs at
Fig.2. Measurement
of intact
with different
temperature to enable the capture of ADCs by target antigen. The inset depicts the immunoaffinity interactions between
target antigen
and ADCs
the antibodies.
(C)drug-to-antibody ratios in plasma by affinity capture LC–MS. The results show unbiased capture of anti-M
standards
(DAR
DAR 1, and
2) spiked into
rat plasma at a fixed composition. (A) Representative TIC of the TDC mixture. A retention time window (hig
ycosylation is subsequently performed. Following extensive washing to remove unbound and nonspecifically bound proteins,
beads
are0,isolated
by DAR
magnetization.
(D)
selected to extract the mass spectrum of the TDCs. (B) Corresponding extracted mass spectrum showing characteristic charge envelopes for individual TDC
ADCs are then eluted from the beads and injected onto the capillary LC–MS system for analysis.
44
Xu K, Liu L, Saad O, et al, Anal Biochem, 412, 56–66 (2011).
Deconvoluted mass spectrum from panel B showing relative abundances of TDC species.
Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) xxx–xxx
Intensity (cps)
A
Intensity (cps)
144
145
35
30
∆ = 1203 Da
20
15
10
Intensity (cps)
44%
DAR1
*
145
146
28
24
20
16
12
∆ = 1209 Da
17%
DAR0
144
16
12
8
4
143
140
48%
DAR1
145
144
145
*
146
253
146
148
147
^
149
150
151
94%
DAR2
140
100
80
6%
DAR1
60
40
143
144
145
146
Buffer_96h
Buffer_96h
*
148
147
Mass (kDa)
149
150
151
149
150
151
Rat_96h
Rat_96h
149
150
Drug release in a plasma stability study
of anti-MUC16 TDC in vitro. An aliquot
of 100 μg/ml anti-MUC16 TDC (DAR 2)
was incubated in plasma from different
species and buffer control (PBS with
0.5% BSA) at 37° C for up to 96 h.!
151
44%
DAR2
∆ = 1206 Da
Human_96h
Human_96h
*
147
252
liquid chromatography–mass spectrometry, Anal. Biochem. (2011), doi:10.1016/j.ab.2011.01.004
^
148
∆ = 1207 Da
13%
DAR0
151
∆ = 1210 Da
147
*
251
Monkey_96h
35%
DAR2
43%
DAR1
150
Human_96h
Fig.4. Drug release in a plasma stability study of anti-MUC16 TDC in vitro. An aliquot of 100 lg/ml anti-MUC16 TDC (DAR 2) was incubated in plasma from different spec
and buffer control (PBS with 0.5% BSA) at 37 !C for up to 96 h. Samples were collected at different time points and analyzed by affinity capture LC–MS. Representati
deconvoluted mass spectra are shown here for the anti-MUC16 TDC in cynomolgus monkey plasma at 0 h (A), cynomolgus monkey plasma at 96 h (B), rat plasma at 96 h (
human plasma at 96 h (D), and buffer control at 96 h (E). Minor components due to incomplete deglycosylation were observed and labeled as ‘‘^’’. Peaks labeled as ‘‘⁄’’ are y
Da
to be identified structurally and not included in calculations. Percentage DAR values shown refer to relative ratios.
^
*
146
*
250
Monkey_96h
Please cite this article in press as: K. Xu et al., Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillar
148
147
*
28
24
20
149
40%
DAR2
*
*
8
143
Intensity (cps)
148
147
*
144
∆ = 1206 Da
249
20
∆ = 1209
16%
DAR0
4
E
*
25
143
D
146
44%
DAR2
∆ = 1207 Da
120
Monkey_ 0h
Monkey_0h
7%
DAR1
5
C
E
120
143
B
93%
DAR2
40
24
20
generate mixtures of known compositions for16
affinity capture and
13%
12
subsequent LC–MS analysis. Fig. 2A shows a representative
TIC of
DAR0
the TDC mixture from affinity capture LC–MS. 8The retention time
window (!9.0–10.3 min), representing the LC elution
of the TDCs,
4
was selected and the corresponding mass spectrum was extracted
143
144
145
160
80
43%
DAR1
28
in vitro ADC plasma stability
Intensity (cps)
he method was extensively characterized using anti-MUC
andards (DAR 0, DAR 1, and DAR 2) to mimic situations
occur in vivo.
MUC16 TDC standards were spiked into rat plasma at varyntrations (with a total TDC concentration of 30 lg/ml) to
Intensity (cps)
D
ary 2011
^
148
94%
149
150
151
Xu K et al, Anal. Biochem, 412, 56–66 (2011)
A
DAR2
100
in vivo ADC characterization
90
Intensity (cps)
80
70
day post-dose
11day
post-dose
60
50
40
∆ = 1199 Da
30
DAR1
20
10
143
B
144
145
147
148
150
149
151
DAR2
50
45
DAR1
40
Intensity (cps)
146
35
∆ = 1201 Da
30
days post-dose
7 7days
post-dose
25
20
∆ = 1200 Da
15
10
DAR0
5
143
Intensity (cps)
C
13
12
11
10
9
8
7
6
5
4
3
2
1
143
144
145
146
147
148
149
150
151
DAR1
∆ = 1213 Da
Fig.6. Drug release observed in a multipledose toxicokinetic study of anti-MUC16
TDC in vivo. Anti-MUC16 TDC (DAR 2)
was administered to cynomolgus monkeys
intravenously once every 3 weeks for a
total of four doses. Three dose groups at
6, 10, and 20 mg/kg were evaluated.
Deconvoluted mass spectra of the TDC
species of a representative animal
receiving the first dose of 6 mg/kg antiMUC16 TDC show the drug release from
DAR 2 to form DAR 1 and DAR 0 with
time: (A) 1 day postdose; (B) 7 days
postdose; (C) 21 days postdose.
∆ = 1207 Da
DAR0
DAR2
days post-dose
21 21days
post-dose
Xu K et al, Anal. Biochem, 412, 56–66 (2011)
144
145
146
148
147
Mass (kDa)
149
150
151
observed in a multiple-dose toxicokinetic study of anti-MUC16 TDC in vivo. Anti-MUC16 TDC (DAR 2) was administered to cynomolgus monkeys
every 3 weeks for a total of four doses. Three dose groups at 6, 10, and 20 mg/kg were evaluated. Deconvoluted mass spectra of the TDC species of a
al receiving the first dose of 6 mg/kg anti-MUC16 TDC show the drug release from DAR 2 to form DAR 1 and DAR 0 with time: (A) 1 day postdose; (B)
Conjugation site influences linker stability
and therapeutic activity of ADCs
Shen B-Q et al. Nature Biotechnology, 30(2), 184–191 (2012)
in vivo ADC (reduced interchain S-S) characterization
Figure 7. Summary of mass
shifts and peak asymmetry
observed in cysteine-linked
ADCs in vivo. Detection of odd
loaded species at masses higher
than expected was attributed to
modification at the deconjugated
drug-linker sites, specifically
cysteinylation, and observed
asymmetry in the deconvoluted
data over time is hypothesized
to be a consequence of
increased sample heterogeneity
due to in vivo modifications,
including hydrolysis.
Hengel SM et al. Anal. Chem. 2014, 86, 3420–3425.
49
Non-cleavable ADC with site-specific catabolism
Stability of Non-Cleavable Site-Specific ADCs
Fig 1. Stability studies of site-specific non-cleavable ADCs. a) Positions of conjugation sites on an antibody. b) Structure of the PEG6-C2-MMAD noncleavable payload conjugated to the glutamine tag on the antibody, and its cleavage product. The glutamine residue is shown in blue. c) Structure of the
PEG6-C2-Aur3377 non-cleavable payload conjugated to the glutamine tag shown in blue.
doi:10.1371/journal.pone.0132282.g001
(PEG6-C2-MMAD), a potent analogue of the antimitotic agent Dolastatin 10 [22], attached to
specific sites across the anti-M1S1 C16 antibody (Fig 1a and 1b). To evaluate linker-payload
Magdalena Dorywalska et al, Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent
stability, we performed comparative in vitro plasma stability assays by incubating conjugates in
Plasma and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015
mouse plasma, purifying them and comparing their drug-antibody ratio (DAR) to that of
untreated conjugates. Unlike the cleavable Aminocaproyl-valine-citrulline-p-aminobenzylcarbamate (C6-VC-PABC) linker [21], the non-cleavable PEG6-C2-MMAD-derived conjugates
remained stable across most of the sites tested over a 4.5 day incubation period in mouse
Non-cleavable ADC with site-specific catabolism
Stability of Non-Cleavable Site-Specific ADCs
Fig 2. Mass spectrometric analysis of non-cleavable conjugates
Fig 2. Mass spectrometric analysis of non-cleavable conjugates. The Fig labels represent experimentally observed masses for conjugates before (upper
panel) and after (lower panel) in vivo exposure. a) Intact mass deconvolution of C16 Site A-PEG6-C2-MMAD conjugate. The metabolic products of the DAR 2
species show a mass loss from either 1 x 186 Da (one payload) or 2 x 186 Da (both payloads). b) Intact mass of C16 Site I-PEG6-C2-MMAD conjugate. The
metabolic product shows a 186 Da loss from one of the conjugated payloads. c) Intact mass of C16 Site A-PEG6-C2-Aur3377 conjugate. The in vivo exposed
Magdalena
Dorywalska
et compared
al, Site-Dependent
Degradation
conjugate shows
no mass shift
to the untreated
compound. of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent
Plasma
and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015
doi:10.1371/journal.pone.0132282.g002
50
A comparative in vivo efficacy study confirmed that the position-dependent C-terminal residue clipping in certain PEG6-C2-MMAD conjugates results in a reduced potency in the
BxPC3 mouse xenograft tumor model. Following a single intravenous dose of 10 mg/kg, the
Engineered ADC (DAR4) in vitro plasma stability
Control
t=0h
DAR 4
37 C
t=72h
DAR 3
DAR 2
37 C
t=7d
51
LC-MS ADC characterization and beyond
Assay
Technology
Drug or drug-linker metabolites
Which species exist and are important?
LC-MS/MS
Catabolites (ADC or mAb)
Which species exist and are important?
Affinity capture LC-HRAMS
DAR Distribution
Assess ADC stability (in vitro / in vivo)
Affinity capture LC-HRAMS
or HIC
Critical reagent DAR specificity
Assess LBA reagents
Affinity capture LC-HRAMS
or HIC
Intact ADC quantification
Useful to measure individual DARs or
catabolites?
Affinity capture LC-HRAMS
Intact ADC quantification
PPD-ThermoFisher Scientific
collaboration
53
Raw HR mass spectrum
3066.42
100
2948.52
90
Relative Abundance
80
Engineered DAR4 ADC
(mcMMAF conjugate)
3194.13
70
2839.33
60
2737.99
50
40
30
2643.61
3332.96
2555.51
20
10
0
54
2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600
m/z
LC-HRAMS quantitation data processing
Potential options using full spectrum (FS) data acquisition
+ XICs and traditional peak integration
- select m/z charge state(s), individual or summed
- use narrow data extraction window (e.g. 5 mDa)
- integrate XIC peak area
+ Deconvoluted composite spectrum
- average/sum FS across entire chromatographic peak
- peak area = total “zero-charge” state response
+ Deconvoluted individual FS
- generate “zero-charge” state XIC
- integrate XIC peak area
55
Intact analysis
100
10.53
TIC
Relative Abundance
Relative Abundance
100
3066.42
80
60
40
9.92
20
0
6
8
10
12
40
Relative Abundance
3332.96
2500
2800 3000 3200 3400 3600
m/z
2937.40
100
SILu™Lite (IS)
80
2719.89
60
3192.73
2532.25
20
0
2643.61
20
Time (min)
40
2839.33
60
0
14
ADC
80
2500
2700
2900
3100
m/z
3300
3500
Sample: 50 µg/mL ADC standard
Method:
• 30 µL mouse EDTA plasma
• Add 2 µg/mL mAb IS
• AC with anti-hFc agarose beads
• No deglycosylation
• Elute and analyze
Summed XICs approach (with mAb IS)
Calibration Curve (XICs: m/z 2839, 2892, 2948, 3006, 3066)
Analyte/IS Area Ratio
120
100
y = 1.1969x - 0.805
r=0.997
80
60
40
20
0
0
20
40
60
ADC Conc. (ug/mL)
57
80
100
A&P - Summed XICs approach (with mAb IS)
QCs
QC 1-1
QC 1-2
QC 1-3
QC 1-4
QC 1-5
QC 2-1
QC 2-2
QC 2-3
QC 2-4
QC 2-5
QC 3-1
QC 3-2
QC 3-3
QC 3-4
QC 3-5
58
Analyte/IS
Cal. Conc.
(ug/mL)
Nominal Conc.
(ug/mL)
Accuracy
3.00
2.79
2.49
2.51
2.56
17.3
15.2
14.9
17.8
16.0
108
106
108
102
102
1.83
1.66
1.41
1.42
1.47
13.8
12.0
11.8
14.2
12.7
89.3
88.2
89.9
84.3
84.6
1.6
1.6
1.6
1.6
1.6
12.5
12.5
12.5
12.5
12.5
100
100
100
100
100
115
104
88.1
89.0
91.9
110
96.3
94.1
114
101
89.3
88.2
89.9
84.3
84.6
CV%
11.7
8.35
3.01
Deconvoluted mass spectrum
Engineered DAR4 ADC
(mcMMAF conjugate)
glycoforms
59
Deconvoluted MS approach (no IS)
Calibration curve (DAR 4 mass 153430, 2nd glycoform)
Deconvolved Signal
2.50E+08
y = 2197502x - 123876
r=0.997
2.00E+08
1.50E+08
1.00E+08
5.00E+07
0.00E+00
0
20
40
60
ADC Conc. (μg/mL)
60
80
100
A&P - Deconvoluted MS approach (no IS)
QCs
QC 1-1
QC 1-2
QC 1-3
QC 1-4
QC 1-5
QC 2-1
QC 2-2
QC 2-3
QC 2-4
QC 2-5
QC 3-1
QC 3-2
QC 3-3
QC 3-4
QC 3-5
61
Intensity
3040465
3171365
3384557
2532637
3566412
28494807
26480701
25990923
28256266
27660784
201954923
202279068
200604530
210591547
206835150
Cal. Conc.
(ug/mL)
1.44
1.50
1.60
1.21
1.68
13.0
12.1
11.9
12.9
12.6
92.0
92.1
91.3
95.9
94.2
Nominal Conc.
(ug/mL)
1.60
1.60
1.60
1.60
1.60
12.5
12.5
12.5
12.5
12.5
100
100
100
100
100
Accuracy
90.0
93.7
99.8
75.6
105.0
104.2
96.9
95.1
103.3
101.1
92.0
92.1
91.3
95.9
94.2
CV%
12.1
3.99
2.03
ADC bioanalysis
other challenges
Practicality issues for ADC bioanalysis
Resource and time intensive
• Several different assays needed for each study
• Parallel sample analyses desirable, requiring
multiple technologies, staff and instruments
Preclinical/early development
• Several species to evaluate
• Multiple ADC constructs to compare
• Restricted sample volumes
63
A work in progress...
Final thoughts
• Keeping up with biotherapeutics innovation is a
challenge for analytics (CMC) and bioanalysis
• Answering critical drug development questions
requires both LBA and LC-MS assay technologies
• Inherent ADC heterogeneity and dynamic changes
in vivo require in-depth characterization
• MS-based methods, including
AC-LC-HRAMS,
are emerging as increasingly versatile tools
• Regulators are aware and likely to increase their
expectations
65
Acknowledgements
Acknowledgements
PPD® Laboratories
• Dongliang Zhan
• Diego Cortes
• William Mylott
• Pat Bennett
•
•
•
•
•
Kwasi Antwi
Jonathan Josephs
Urban A. Kiernan
Keeley Murphy
Eric E. Niederkofler
Genentech
• Keyang Xu
• Surinder Kaur
Seattle Genetics
• Shawna Hengel
• Steve Alley
Pfizer
• Leo Kirkovsky
Also
• Jun Hosogi (JBF)
• Shinobu Kudoh (JBF)
…and many others
67
A story...
1.3 billion light years from Earth
Two black holes merging into one
Simulating eXtreme Spacetimes project
(http://www.black-holes.org)
Event GW150914
C. Henze/NASA Ames Research Center
The “sound” of two black holes merging
14 September 2015 at 09:50:45 UTC
Top panels show the measured signals in the LIGO detectors.
Bottom panels show the expected signal based on numerical simulations.
B. P. Abbott et al. (LIGO Scientific Collaboration and Virgo Collaboration), “Observation of
Gravitational Waves from a Binary Black Hole Merger,” Phys. Rev. Lett. 116, 061102 (2016)
Advanced LIGO
Hanford, Washington, USA
Laser Interferometer Gravitational-wave Observatory (LIGO)
LIGO can detect relative length variations
of order 10−21, or one ten-thousandth the
diameter of a single proton
Observatories and LIGO Scientific Collaboration (LSC)
Kamioka Gravitational Wave Detector (KAGRA), Japan
Important players in the LIGO project, from left to right: Kip Thorne of the
California Institute of Technology, France A. Córdova of the National Science
Foundation, Rainer Weiss of the Massachusetts Institute of Technology, David
Reitze of Caltech and Gabriela González of Louisiana State University.
Credit: Lexey Swall for The New York Times
Thank you