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The world leader in serving science
Jon Bardsley, Thermo Fisher Scientific, Chromatography Consumables
Simple solution for complex analysis; an assessment of Heat-stable trypsin for surrogate peptide quantitation
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Reproducibility summarySummary
• Overview of workflows
• Overview of SMART digest
• Data examples
• Quantitative assay – a generic approach
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Snap-shot of the Workflow
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• Lengthy multi-step protocols
• Reproducibility issues
• Low throughput/speed
• Method development ease and transfer
Getting the Digest Right Each Time
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Reproducibility summaryQuantitative Analysis Workflow Solution
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Immobilized Heat Stable Enzyme: Reproducible Protein Digestion Within Minutes
• Scientific™SMART Digest™ kits is a heat-stable immobilized enzyme
digestion kit;
• Proteins are heat-denatured for digestion,
• Additional denaturing agents or reduction and alkylation is not required
• High-throughput and automation-compatible formats
• 96 x PCR tubes pre-packed with resin
• Bulk resin format
• Magnetic bulk resin format
• Additional post digestion clean up options:
• 96 well filter plate
• Thermo Scientific™ SOLAµ™ SPE plate
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Easy to Use Sample Preparation
Thermo Scientific™
SMART Digest™Thermo Scientific™
Vanquish™ Flex UHPLC
Thermo Scientific™
Acclaim™ 120 C18 column
Thermo Scientific™
Q Exactive™ PlusHRAM-MS
Thermo Scientific™
BioPharma Finder™ informatics platform
Sample preparation: Easy to use
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Thermal denaturation of IgG
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50000
100000
150000
200000
250000
0 20 40 60 80 100R
esp
on
se (
Peak A
rea)
Digest Time (Minutes)
Native IgG digest profile
monitoring VVSVLTVLHQDWLNGK
50°C
60°C
70°C
Accelerated Protein Digestion
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Simplifying the Sample Preparation
Denaturation
Reduction
Alkylation
Digestion
Clean up
Primary structure
Secondary structure (H bonding)
Tertiary structure (disulphide bonding)
Alkylating agent
Enzyme
Clean up
Digestion
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Reproducibility summaryReplicate Digests for Consistency
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50
100
10 20 30 40 50 60
RT (min)
10 12 14 16 18 20 22 24 26 28 30 32
RT (min)
10 replicate SMART digest preps of
Cetuximab followed by LC-MS analysis on
Vanquish Flex Binary and Q-Exactive Plus on
2.1 × 250 mm Acclaim Vanquish C18
Excellent retention time and peak area
reproducibility between samples, 100%
sequence coverage when searched in BF3.0
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Reproducibility summaryReplicate Digests for Consistency
0
50
100
10 20 30 40 50 60
RT (min)
10 12 14 16 18 20 22 24 26 28 30 32
RT (min)
Whatever of end goal is -
reproducibility is key
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Reproducibility summaryQuantitative Workflows
• Surrogate peptide analysis
• Pick 1 or 2 suitable peptides to
focus on
• Same principle applies –
reproducibility is key
• Post-digest activities are
simplified
0
50
100
10 20 30 40 50 60
RT (min)
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Reproducibility summaryQuantitative Workflows
Straight to digest
Or
IA capture then digest?
SMART digest ImmunoAffinity
Immobilised enzyme
PLUS
Capture moiety
• Streptavidin, ProA or ProG
The world leader in serving science
Development of Generic Methodology for Humanized mAb
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Reproducibility summaryGeneric Assay for Quantitation of Humanised mAbs in non-Human matrix
• Analytical method for the determination of humanised mAb from rat
plasma
• Suitable extraction method that could work as a start point for multiple
assays of this nature
• Inclusive of sample preparation and LC-MS/MS endpoint
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Reproducibility summaryPeptide Selection
• Typically from the CDR (complimentary-determining region) to be
specific to our target
However
• A lot of therapeutic Mabs have similar Fab and Fc regions to human
IgG in order to decrease immune response
• To reduce method development time we can use this, provided we are
in a matrix that does not interfere with specificity
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Reproducibility summaryPeptide Selection
>RM 8671 NIST mAb heavy chain
QVTLRESGPALVKPTQTLTLTCFSFSLSTAGMSVGWIRQ
PPGKALEWLADWWDDKKHYNPSLKDRLTISKDTSKNQ
VVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVT
VSSASTKGPSVFPLAPSSKSTGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
>Rituximab heavy chain
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWV
KQTPGGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSS
STAYMQLSSLSEDSAVYYCARSTYYGGDWYFNVWGAG
TTVTVSAASTKGPSVPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVKKAEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQSLSLSPGK
• Overlap of Tryptic peptides in the Heavy chain region of two examples
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Reproducibility summaryLiquid Chromatography Conditions following Tryptic Digestion
System: Vanquish Horizon UHPLC
Column: Acclaim 120 C18
Col. Temp: 50 ºC
Col. Mode: still air
Flow : 0.5 mL/min
Gradient:10 – 45 % B over 3 minutes
MPA: 0.1% formic acid in water
MPB: 0.1% formic acid in acetonitrile
Detection: MS/MS
Injection: 20 µL
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Reproducibility summaryInternal Standard Purity
13C6, 15N4-labeled arginine and 13C6, 15N2-labeled lysine
• Heavy labelled IgG
used as an internal
standard
• R and K labelled so
there should always
be a heavy tryptic
peptide
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Reproducibility summarySample Preparation
Capture Wash Digest
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Reproducibility summarySample Preparation
Capture Wash Digest
Antibody selection
Bulk prep of beads
Capture time optimisation
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Reproducibility summarySample Preparation
Capture Wash Digest
Antibody selection
Bulk prep of beads
Capture time optimisation
We used anti human IgG
(Fc) for a generic
approach
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Reproducibility summaryIA Capture Step – Time Course Experiment
• Optimised capture
times by monitoring
chosen peptides to
completion
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Reproducibility summaryRecovery Levels from IA capture Step
0
20
40
60
80
100
120
Digested Std IA capture from Water IA capture from Plasma
• Recovery levels
tested from plasma
and water compared
to a digested standard
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Reproducibility summarySample Preparation
Capture Wash Digest
Wash with
supplied buffer
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Reproducibility summarySample Preparation
Capture Wash Digest
Optimise digestion time
Use pre-supplied buffer
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Reproducibility summaryTryptic Digestion – Time Course Experiment
0
50000
100000
150000
200000
250000
300000
0 10 20 30 40 50 60 70 80 90 100
Digestion Time (mins)
• Optimised digest
times by monitoring
chosen peptides to
completion
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Reproducibility summaryRecovery Comparisons
• Assay was tested on
Rituximab, NIST mAb and
on human IgG from water
and rat plasma
• Similar performance across
the test cases
• Good assay start point
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Reproducibility summaryQuantitative data
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Reproducibility summaryQuantitative data
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Reproducibility summaryQuantitative data
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Reproducibility summaryWhat if I am in Human Matrix?
• Monitor signature peptides from CDR (complimentary-determining
region)
• Specific anti-body for IA capture
• Physically, the process is the same!
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Thermo Scientific™ KingFisher™ Duo
Heating plate
Plate HolderLocation PinElution Block
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Summary
SMART Digest™ Kits are easy to use,
reproducible
ImmunoAffinity versions available for capture
and digestion on the same bead
Ability to perform generic assays, or start points
for more selective assays
Simple step-by-step method development and
option to automate