Download Poster Multi-level MS charact of ADCs in a regulated

Multi-level mass spectrometric characterisation of
Antibody-Drug Conjugates in a regulated environment
Eric Largy; Anicet Catrain; Arnaud Delobel
Quality Assistance sa - Technoparc de Thudinie 2 - 6536 DONSTIENNES (Belgium)
[email protected]
Antibody-Drug Conjugates (ADCs) are antibodies engineered to deliver a cytotoxic agent specifically to tumor cells expressing a defined antigen. Their physico-chemical characterisation
requires a large number of assays aimed at verifying their sequence identity, post-translational modifications, and range/average number of conjugated drugs (Drug-to-Antibody Ratio: DAR).
Cysteine and lysine-linked ADCs are two classes of ADCs that differ by the nature of the amino-acids conjugated, and hence call for well-adapted assays.
Using state-of-the-art technologies (Waters Xevo G2-XS QTOF with 1D and 2D-UPLC) and software (UNIFI 1.8), we developed fast and efficient workflows that offer a comprehensive,
unambiguous physico-chemical characterisation of ADCs. The use of various orthogonal methods ensures the reliability of the results, and provides a larger range of information (average
DAR, distribution of drugs, positional isomers, etc.).
Characterisation of Antibody-Drug Conjugates: an analytical challenge
Analysis of ADCs subunits
• Antibody-Drug Conjugates are drug-biologic hybrids,
consisting in a cytotoxic drug coupled to a monoclonal Drug to antibody ratio distribution depending on
the coupling technology
antibody.
• This analysis can be performed
on both Lys- and Cys-conjugated
ADCs (due to heterogeneity, LC
separation on Lys-ADCs is not
optimal).
• The most common coupling chemistries are lysine and
cysteine conjugation.
• All the fragments obtained
have a MW ~ 25 kDa, which
facilitates MS analysis.
• Two ADCs are marketed: Kadcyla® (trastuzumab emtansine,
lysine-conjugated) and Adcetris® (brentuximab vedotin,
cysteine conjugated).
• With a 45’ one-pot sample
preparation without further
purification,
valuable
information on drug load and
localisation of conjugation sites
(LC, Fc/2, Fd) can be obtained.
• Conjugation increases the heterogeneity of the product due
to:
– Variability of drug-to-antibody ratios (DAR)
– Heterogeneity of conjugation sites (especially for lysine
conjugates)
• Site-specific conjugation technologies are under development
to circumvent these issues and avoid too high payloads.
• State-of-the-art analytical methodologies have to be
implemented to deal with the high complexity of ADCs.
• This methodology is a good
alternative to intact mass MS and
is much easier to implement (no
need to maintain the integrity
of non-covalent complexes
during chromatography and
MS detection)
From Lin et al. Pharmaceutical Research 06/2012; 29(9):2354-66.
Determination of DAR at the intact ADC level
• Mass spectrum of the intact ADC allows the determination of the drug-to-antibody ratio (DAR).
• Deglycosylation is optional, but it can facilitate data processing and DAR calculation.
• Drug load distribution can not
be determined.
• Cysteine-linked ADCs have to be analysed under non-denaturing conditions (aka. native MS) in order to avoid
disruption of non-covalent interactions. This requires fine tuning of analytical conditions.
• Data processing and DAR calculations are automated within UNIFI.
Mass spectrum (left) and deconvoluted spectrum (right) of
The DAR values obtained are in good
deglycosylated Kadcyla®
DAR 4
agreement with what can be obtained
DAR 3
DAR 5
DAR 2
using other analytical techniques (UV
DAR 6
spectroscopy for Kadcyla®, HIC/UV for
DAR 1
DAR 7
Adcetris®).
957 Da
DAR 8
DAR 0
Mass spectrum (left) and deconvoluted spectrum (right) of
deglycosylated Adcetris®
DAR 4
DAR 2
DAR 6
DAR 0
DAR 8
When applied to lysine-conjugated ADCs,
this method has the advantage to give
information on both the mean value and
distribution of DAR.
Principle of IdeS digestion performed in a reducing buffer
IdeS
2 x LC
2x Fc/2
2 x Fc/2
UV chromatogram and corresponding mass spectra obtained for Adcetris®
after IdeS-digestion (reducing conditions) & deglycosylation
Fc/2
Fd + 3 drugs
LC
LC +1 drug
Fd
Fd + 1 drug
Fd + 2 drugs
ADC characterisation by peptide mapping
• As for other therapeutic proteins, peptide mapping is a method of choice for: (1) Confirmation of primary sequence
; (2) Characterisation of PTMs, including glycosylation ; (3)Targeted purity profiling / Monitoring of degradation
(oxidation, deamidation …)
• For ADCs, peptide maps can provide additional information such as the precise localisation of conjugation sites
and the determination of site occupancy (based on MS data)
• The « classical » method uses a double digestion Trypsin + Glu-C (overnight), but peptide mapping of Lys-linked
ADCs requires specific conditions to determine conjugation sites occupancy, as trypsin cannot cleave after
conjugated lysine residues
• 2 digestions are required to get both good sequence coverage and site occupancy information : Trypsin + Glu-C
and Asp-N + Glu-C
The methods presented here are quick
and easy to implement, and can be used in
a regulated environment for batch testing
and stability studies.
• Optimisation of the method was necessary as regards the choice of the enzyme, the mobile phase, the gradient
and the MS conditions, in order to be able to detect all peptides, even the most hydrophobic ones.
• UPLC combined with high resolution MS and dedicated software such as UNIFI allow the use of this methodology
in routine work.
UV chromatograms and corresponding sequence coverage obtained for peptide mapping of Kadcyla®
Trypsin + Glu-C
Coverage: 99 %
• Cysteine-conjugated ADCs are present as a mixture The HIC method that can be used in routine (batch
of different DAR, and different positional isomers for testing, stability studies, batch characterisation) can be
each DAR.
hyphenated to mass spectrometry for peak identification
• DAR values of Cys-conjugated ADCs are commonly with a 2D-LC configuration.
Unambiguous peak identification was done for all peaks
detected on the UV chromatogram.
• Mobile phase in HIC is not compatible with MS
Reduction
Analysis and reporting can be fully
automated within UNIFI software.
Determination of DAR and positional isomers by 2D HIC - RP with UV and MS detections
calculated by HIC/UV
2 x Fd
F(ab’)2
• A HIC/UV method was developed for Adcetris®, and Minor amounts of DAR 1 and DAR 3 species were
2D-HPLC/MS was used for identification of the peaks detected and successfully identified.
(reversed-phase as second dimension for desalting This method can be applied to stressed samples for
and separation of sub-units).
degradation products identification.
HIC/UV chromatogram obtained for Adcetris (A) with 2D-HPLC/MS data
corresponding to DAR 4 / isomer a (B), DAR 4 / isomer b (C) and DAR 8 (D)
A
C
B
D
Asp-N + Glu-C
Coverage: 95 %
Trypsin + Glu-C: excellent sequence coverage
Asp-N + Glu-C: possibility to determine site occupancy
• A fingerprint of conjugated peptides can be obtained
by extraction of signature ions corresponding to
specific fragments of the drug. This can be used for the Specific detection of conjugated peptides of Kadcyla®
based on the detection of signature ions in the high
assessment of batch-to-batch consistency.
energy MSE trace
• ETD fragmentation (Electron Transfer Dissociation) can
be used to localise the site of conjugation on peptides
containing several lysine residues. This cannot be done
Loss of 1 drug
No sequence info
by regular MS/MS (CID, Collision Induced Dissociation)
Fragmentation of a conjugated peptide of Kadcyla® by
ETD to localise the conjugation site
Peptide KHKVYACE
OH
O
O
HN
O
HN
O
O
H OH
OH
O
Cl
O
H
O O
N
O
N
N
O
NH
O
Cl
S
O
O
O
O
N
N
OH
HCl
N
O
O
+
Chemical Formula: C27H34ClN2O4
Exact Mass: 485,2202
O
O
+
Chemical Formula: C28H36ClN2O7
Exact Mass: 547,2206
Release date: 20170227