Bioanalysis of therapeutic proteins and biomarkers by LC

Department of Pharmacy
Analytical Biochemistry
Bioanalysis of therapeutic proteins and
biomarkers by LC-MS/MS – beyond ligand
binding assays
Rainer Bischoff
www.biomac.nl
DKML-Mannheim, September 29, 2016
Overview
• Protein bioanalysis: LBAs and LC-MS/MS
• In vivo biotransformation of Trastuzumab
• Determination of anti-drug antibodies against alpha-Glucosidase
1. Bults, P.; Bischoff, R.; Bakker, H.; Gietema, J. A.; van de Merbel, N. C. LC-MS/MS-based monitoring of in vivo protein biotransformation: quantitative
determination of trastuzumab and its deamidation products in human plasma. Anal Chem 2016, 88, 1871-1877.
2. Bronsema, K. J.; Bischoff, R.; Pijnappel, W. W. M. P.; van der Ploeg, A. T.; van de Merbel, N. C. Absolute Quantification of the Total and Antidrug AntibodyBound Concentrations of Recombinant Human α-Glucosidase in Human Plasma Using Protein G Extraction and LC-MS/MS. Anal Chem 2015, 87, 43944401.
3. Bults, P.; Van De Merbel, N. C.; Bischoff, R. Quantification of biopharmaceuticals and biomarkers in complex biological matrices: A comparison of liquid
chromatography coupled to tandem mass spectrometry and ligand binding assays. Expert Review of Proteomics 2015, 12, 355-374.
Protein Bioanalysis: LBAs and LC-MS/MS
• Ligand Binding Assays (LBAs) have been the only option to measure
proteins in complex, protein-rich biological matrices until recently.
• Accuracy and Precision of LBAs depend on the specificity of the
antibodies (or other kinds of affinity binders), the level of non-specific
binding and the level of interference from other binders.
• The read-out from LBAs is indirect (e.g. color, fluorescence,
chemoluminescence), so the assay provides no direct chemical
information about the analyte that led to the observed signal.
• Recent developments in LC-MS/MS provide an alternative to LBAs.
3
Developments in LC-MS/MS
• Introduction of high-speed, high-resolution and high-sensitivity mass
spectrometers
• Introduction of high-speed, high-resolution LC columns and
instruments with the option for miniaturization
• Introduction of LC-MS/MS assays (SRM and PRM) in proteomics
• Introduction of LC-MS/MS in clinical biomarker analysis
• Introduction of LC-MS/MS in regulated bioanalysis of therapeutic
proteins
4
LBAs and LC-MS/MS
• There are only a few comparisons between LBAs and LC-MS/MS
assays for protein bioanalysis in the literature.
• Quite often the results from LBAs and LC-MS/MS differ.
5
Proteins are no Single Species
Modifications:
CH3
S
CH3
S
C=O
OH
C=O
NH2
native
CH3
S
C=O
NH2
deamidation
CH3
S=O
proteolysis
CH3
S
C=O
NH2
oxidation
C=O
NH2
3D change
6
Protein Binding may Interfere with LBAs
Binding:
CH3
S
CH3
S
C=O
NH2
C=O
NH2
unbound
CH3
S
target-bound
CH3
S
CH3
S
C=O
NH2
aggregated
C=O
NH2
C=O
NH2
ADA-bound
7
Measurement Principles Differ
LBA
LC-MS/MS
8
LBA versus LBA
Influence of capturing antibody: monoclonal vs polyclonal
Example: ocrelizumab
Concentrations in patient plasma ± 2–fold lower with mAB than with pAB
pAB
mAB
Fischer et al, mAbs, 4 (2012) 623-631 Reproduced with permission, copyright Taylor & Francis, 2012
9
LBA versus LBA
Pharmacological target CD20 and monoclonal capturing antibody bind to
the same epitope of ocrelizumab
• no (or reduced) analyte recognition by ELISA when target-bound
Polyclonal capturing antibody binds to multiple epitopes
• also (partial) analyte recognition by ELISA when target-bound
10
In vivo Biotransformation of Trastuzumab
Asn55
11
LC-MS/MS Assay to follow Biotransformations
Influence of signature peptide: stable vs unstable
Asn55
Trastuzumab heavy chain
1
61
121
181
241
301
361
421
EVQLVESGGG
ADSVKGR FTI
ASTKGPSVFP
GLYSLSSVVT
GPSVFLFPPK
NSTYRVVSVL
ELTKNQVSLT
WQQGNVFSCS
LVQPGGSLRL
SADTSK NTAY
LAPSSKSTSG
VPSSSLGTQT
PKDTLMISRT
TVLHQDWLNG
CLVKGFYPSD
VMHEALHNHY
PGKGLEWVAR
GDGFYAMDYW
WNSGALTSGV
PKSCDKTHTC
WYVDGVEVHN
SKAKGQPREP
VLDSDGSFFL
IYPT NGYTR Y
GQGTLVTVSS
HTFPAVLQSS
PPCPAPELLG
AKTKPREEQY
QVYTLPPSRD
YSKLTVDKSR
SCAASGFNIK
LQMNSLRAED
GTAALGCLVK
YICNVNHKPS
PEVTCVVVDV
KEYKCKVSNK
IAVEWESNGQ
TQKSLSLSPG
DTYIHWVRQA
TAVYYCSRWG
DYFPEPVTVS
NTKVDKKVEP
SHEDPEVKFN
ALPAPIEKTI
PENNYKTTPP
K
ITCRASQDVN
EDFATYYCQQ
PREAKVQWKV
LSSPVTKSFN
TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS
HYTT PPTFGQ GTKVEIKRTV AAPSVFIFPP
DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
RGEC
Trastuzumab light chain
1
61
121
181
DIQMTQSPSS
RFSGSRSGTD
SDEQLKSGTA
LSKADYEKHK
LSASVGDRVT
FTLTISSLQP
SVVCLLNNFY
VYACEVTHQG
12
Deamidation Mechanism
L Asn peptide
O
NH2
O
H
N
- a - r- roV l Ty P
Ala
O
NH3
O
O
O
-
O
O
O
H
N
O
L Asp peptide
N
H
+H2O
+H2O
N
O
L Imide peptide
-
O
O
O
L isoAsp peptide
13
Bischoff & Kolbe, J. Chrom. B, 662, 261-278 (1994)
Probing Deamidation at Asn55
FTISADTSK
IYPTNGYTR
IYPTNGYTR
IYPTsuccGYTR
IYPTisoDGYTR
IYPTDGYTR
14
Stability Study in Plasma
15
In vivo Biotransformation
160
Concentration (µg/mL)
140
120
100
FTISATDSK
IYPTNGYTR
80
ELISA
60
IYPTisoDGYTR
IYPTDGYTR
40
20
0
0
200
400
600
Time after first dose (days)
800
16
Determination of Antibodies against
Alpha-Glucosidase (ADAs)
• Virtually all biopharmaceutical proteins illicit an immune response
• Anti-drug antibodies (ADAs) may
• Interfere with activity
• Affect in vivo half-life
• Reduce therapeutic efficacy
17
Enrichment of ADA-bound
Alpha-Glucosidase
Protein-G binds IgGs along with the
bound antigens
ADA-bound glucosidase is recovered in
an elution step at low pH and quantified
by SRM
18
Control of the Enrichment Procedure
A signature peptide of IgG-1 was monitored
• IgG-1 capture is always above 99% (specific binding)
A signature peptide of human serum albumin (HAS) was
monitored
• HAS capture is always below 5% (non-specific binding)
19
Effect of ADA Levels
LOW ADA level
HIGH ADA level
total rhGAA
ADA-bound rhGAA
20
Application to Patient Samples
Patient with reduced
response to therapy
High
Patient with normal
response to therapy
Low
Closed symbols: total concentration rhGAA
Open symbols: ADA-bound concentration rhGAA
21
Conclusions
• Systematic differences of results obtained by LBAs and LC-MS/MS are
often due to interference of anti-drug antibodies or other ligands
that compete for binding with the ELISA antibodies
• Biotransformation of biopharmaceuticals may affect the response in
LBAs and/or LC-MS/MS differently
• The level of anti-drug antibodies correlates with therapeutic efficacy
of alpha-glucosidase during long-term replacement therapy
• LC-MS/MS without the use of antibodies can rival ELISA in terms of
sensitivity
22
Acknowledgements
University of Groningen
Trastuzumab
Peter Bults
Nico C. van de Merbel
Hilde Bakker
Jourik A. Gietema
Alpha-Glucosidase
Kees Bronsema
Nico C. van de Merbel
Pim Pijnappel
Ans T. van der Ploeg
Grant T3041
Date 1/28/2014 | 24
Thank you for your attention