Apa 2017 diego rodriguez 3

©2017 Waters Corporation 1COMPANY CONFIDENTIAL

LC-MS/MS for Bioanalytical Protein

Quantification: Life of a Biotherapeutic

Infliximab Case Study


Humira Linear

Range:

25-500 mg/mL

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

100

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

100

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

100

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

100

5132015 Blank Rat Plasma_250pt0 ugmL 151 (2.947) 1: TOF MS ES+ 2.00e52743.07842693.2227

2645.1912

2598.7388

2554.0410

2468.8767

2428.45852314.6531

2211.0151

2794.7969

2848.5042

2904.3540

2907.5510

2965.6665

3022.9172

3085.7578

3154.8474 3223.6106

3370.1011


2693.2292

2645.1558

2598.7661

2554.0063

2510.7832

2428.46562314.6995

2244.6521

2848.5764

2904.3823

2962.4216

3022.9009

3026.2302

3089.1956

3154.9912

3291.5718


2645.1282

2598.7595

2553.9998

2510.67362428.4788

2314.6135

2200.6035

2848.5479

2904.3762

2907.5068

2965.64433022.8723

3026.9216

3155.0083


2645.1138

2598.8704

2554.0063

2468.8838

2351.42552314.69952204.6226

2848.5107

2904.3823

2962.5107

3022.8560

3026.2751

3089.1726

m/z2720 2740 2760

%

0

100

m/z2720 2740 2760

%0

100

m/z2720 2740 2760

%

0

100

m/z2720 2740 2760

%

0

100

5132015 Blank Rat Plasma_250pt0 ugmL 151 (2.947)2.00e52743.0784

2739.3311

2716.3662

2746.1213

2748.4795


2738.9309

2737.7749

2746.0850

2749.1294

2750.7810


2740.7654

2737.8540

2746.0999

2748.3723

2749.6160

2751.4180


2742.1853

2716.8633

2746.1492

2748.8291

2749.8157

250 mg/mL

100 mg/mL

50 mg/mL

25 mg/mL

Generic Affinity Capture of Humira from Rat

Plasma

mass148000 148250 148500 148750

%

0

100

5132015 Humira 50 ugmL_3 147 (2.879) 1: TOF MS ES+ 5.49e5148078.0000

148221.0000

148363.0000

148510.0000

mass148000 148250 148500 148750

%

0

100

5132015 Humira 100 ugmL_4 151 (2.947) 1: TOF MS ES+ 7.53e5148078.0000

148222.0000

148370.0000

148529.0000

Deconvoluted Spectra


LC-MS/MS Protein Quantification Workflow

Protein-level clean-up (optional)

LC-MS

Results

Protein in Plasma/Serum

Peptides

Digestion

ID unique peptides & transitions

Optimize/Fine-tune MRM transitions

Peptide-level clean-up

(optional)

PurifiedPeptides

Purified Protein

DataProcessing

TargetLynx


Identification of Unique Signature Peptides

MRM Optimization of Unique Peptides

Internal Standard Options

Protein Level Sample Preparation

Digestion

Peptide Level Sample Preparation

MS Quantification

Outline


Infliximab is a chimeric human-murine anti-human tumor necrosis

factor (TNF) monoclonal antibody.

Infliximab (Remicade) Sequence

Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS

HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE

DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

http://www.drugbank.ca/drugs/DB00065

Formula: C6428H9912N1694O1987S46

Molecular Weight: ~ 149.1 kD


Infliximab LC and HC Tryptic Peptide Identification


Step 1: BLAST Search Infliximab Light Chain to

Identify Closest Related Protein in Database

Infliximab LC sequence

https://blast.ncbi.nlm.nih.gov/Blast.cgi


Closely related to LC of Human IgG

Results of BLAST search for Infliximab LC

Protein sequence differences and gaps


Step 2: Align the mAb Sequence Against Closely

Related Sequences in ExPASy SIM Alignment Tool

Paste infliximab LC sequence

Paste the identified (see step 1), closely related human IgG LC sequence

http://web.expasy.org/sim/


ExPASy SIM Alignment Results: Infliximab LC

vs. Human IgG LC

Highlights protein sequence differences while maintaining sequence alignment


Trypsin cleaves peptides on the C-terminal side of lysine

(K) and arginine (R) amino acid

– Tryptic peptides can be found using freeware, such as Skyline

and Protein Prospector , but these tools will not maintain

sequence alignment

Text editor (ex. TextPad, Notepad)

Step 3: Generate Tryptic Peptides, Maintaining

Sequence Alignment

Gaps = tryptic peptide differences, maintaining sequence alignment

“matched” non-unique peptides

“un-matched” potentially unique peptides


Infliximab, a chimeric murine mAb should have close sequence homology to mouse

Tryptic Peptide Specificity: 3 Possibilities for

Infliximab LC vs. IgG1kappa 1

too long (45 AA)

DILLTQSPAILSVSPGER: Mouse IgG peptide-Specificity in human, rat

ASQFVGSSIHWYQQR: 80% Mouse IgG peptide-Specificity in human, rat, and mouse

YASESMSGIPSR:90% Mouse IgG peptide-Specificity in human, rat, and mouse


Step 4: Assess uniqueness of the un-matched tryptic

peptides using peptide BLAST search

https://blast.ncbi.nlm.nih.gov/Blast.cgi

ASQFVGSSIHWYQQR

Long List of Possibilities


Step 4: Detailed View of Closely Related

Database Matches

Mouse

Monkey

“Unique” < 100% sequence

homology

It found infliximab (100% homology)

Unique for mouse, monkey and

human (homology of 93, 85 and

78%) , thus it could be used to

quantify in those species

ASQFVGSSIHWYQQR

Infliximab

Human


Peptide Options for Quantification:

Infliximab (Remicade)

Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota,

USA 9-13 June 2013.

Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS

HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE

DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT

KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Conserved region: blue (Human IgG1 Sequence)

Variable regions: red

CDR regions: green

Unique signature

Generic signature

http://www.drugbank.ca/drugs/DB00065

Formula: C6428H9912N1694O1987S46

Molecular Weight: ~ 149.1 kD

Final Selection of Tryptic Peptide Options

Choices for quantification are based on sensitivity

and specificity


Best Practice for Signature Peptide Choice

Once you have a selection of Unique Peptides:

Size

– Ideally 8-24 amino acids (6-7 sometimes okay)

Avoid

o Peptides with modifications (i.e. oxidation or deamidated)

o Those containing Cys, Met, N-terminus Gln, Trp, or Asn-Gly

LC/MS characteristics

– Strong MS ionization (increased sensitivity)

– Chromatographic retention, peak shape/width

– Specificity in Matrix






Digestion


MS Quantification

Outline


Developed by the MacCoss laboratory in the University of Washington

Best Practice:

Our Approach = Skyline Workflow

Start Here!


SkyLine

In silico generation of MRM transitions for peptides

Output scouting MRM method

MassLynx/Xevo TQ-XS

Acquisition of MRM traces for all proposed transitions

SkyLine

Comparison of overlaid MRM traces, to pick optimal

transitions & collision energies

Output final MRM method

MassLynx/Xevo

TQ-XS

Sample analysis

Overview of Skyline Workflow


Skyline MRM Method Creation: Infliximab LC

With Skyline, importing a

protein sequence elicits an

in silico digestion algorithm

and MRM transition

prediction


Skyline MRM Method Creation: Infliximab LC


Green light implies peak detection with great sensitivity, yellow light is peak detection with moderate sensitivity, and red light is minimal to no peak detection. HOWEVER, the lights are not always right.

First Skyline Import: Filtering Potential Infliximab

peptides


Sample Analysis with MassLynx: Skyline

imported methods Narrowed down Skyline methods

are imported into MassLynx for

LC/MS acquisition


Skyline MRM Method Optimization: CE

Optimization

The optimized method can

be exported automatically


Infliximab Skyline Optimization vs. Manual:

DILLTQSPAILSVSPGER (LC)

Infliximab Skyline

Time2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

%

0

100

29Dec2016_Infliximab_Optimized_003 14: MRM of 3 Channels ES+ 632.686 > 545.268 (Infliximab_LC DILLTQSPAILSVSPGER.3)

3.94e6Area

29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)

3.94e6Area

5.5926370

Skyline Optimization633.1>546.3

More sensitive transition!

Manual Optimization633.1>731.8

5.59140139

Alternate MRM transition






Digestion


MS Quantification

Outline



Peptide Level (added just prior to digestion)

– Labeled Peptide

– Extended Tag Labeled peptide

Protein Level (added prior to denaturation, reduction and alkylation)

– Generic Labeled protein

– Generic Unlabeled protein

– Unique Labeled protein






Digestion


MS Quantification

Outline


Sample Preparation Options

Protein Level Clean-up prior to digestion

– None

o Whole serum/plasma digestion

– Affinity-based

o Generic: Protein A/G

o Anti-human

o Specific Receptor

– MWT cut-off filters

– Depletion Plates

– Gel Filtration


100uL SPE, Protein A, 1:5 Worthington, DTT, 17hr digestion

Time7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

500ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)

6.43e4Area


6.43e4Area

8.49722


6.43e4Area

8.50345

blank_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)

6.43e4Area

8.49163

100uL SPE, Kappa

Time7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100


1.30e5Area


1.30e5Area

8.43810


1.30e5Area

8.43401


1.30e5Area

50uL SPE, whole plasma, 1:12.5 Worthington, DTT

Time7.50 8.00 8.50 9.00

%0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100

7.50 8.00 8.50 9.00

%

0

100


2.60e4Area

8.38493


2.60e4Area

8.4179


2.60e4Area


2.60e4Area

Effect of Protein Level Clean-Up: Trastuzumab

Blank PlasmaBlank Plasma Blank Plasma

50 ng/mL 50 ng/mL 50 ng/mL

100 ng/mL100 ng/mL100 ng/mL

500 ng/mL500 ng/mL

Specific:Anti-human in Rat

Generic:Protein A in Human

None:Direct Human Plasma

The level of sample prep needed will be based on the sensitivity and specificity required for the assay?

500 ng/mL8.43 3412 8.50 1707






Digestion


MS Quantification

Outline


ProteinWorks™ eXpress Digest Kits

Standardized protocols

– No Method Development

– Diverse proteins

Reliable and reproducible

High Sensitivity

Quantification Total antibody analysis for ADCs

– Detection of conjugated peptides






Digestion


MS Quantification

Outline


ProteinWorks µElution SPE Kit: Mixed-mode

Specificity

Maximizing Instrument Up-Time, Recovery & Sensitivity

Orthogonal to RP LC method!

Remove interfering buffer salts and digest reagents

Recover unique and generic signature peptides with high efficiency using a single SPE method

Minimize sample loss with µElution format

Concentrate the sample up to 15x

0

20

40

60

80

100

120

Oasis MCX

One generic protocol achieves high recovery for a diversity of tryptic peptides

Peptides

Purified Peptides

Peptide-level clean-up (optional)


Infliximab Peptide Level Clean-Up:

Mixed-Mode SPE with Oasis MCX

direct 100ug/ml

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

18Dec2014B_nospe_dir_1033 MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)

3.65e6Area

4.40;149708

18Dec2014B_spe_direct_1040 MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)

3.65e6Area

4.4173225

Direct Plasma Digestion No SPE

Direct Plasma Digestion SPE

DSTYSLSSTLTLSK Tryptic Peptide






Digestion


MS Quantification

Outline


20142014

2012

Xevo TQ-S

2010

2016

Xevo TQ Family

Data coming soon!

New data available!


High Sensitivity Infliximab Quantification: Xevo

TQ-S Triple Quadrupole

Time2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40

%

0

100

2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40

%

0

100

MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.20e4Area

4.15361

MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.20e4Area

10 ng/mL infliximab

Blank plasma

SINSATHYAESVK Unique Signature Peptide

Linear, Precise and Accurate Single digit

%CVs

LLOQ of 10ng/mL35 µL sample, Protein A, ProteinWorks digestion and MCX peptide clean-up


Segmented quadrupole

second stage –

focused ion beam –

enhanced ion

transmission to

detector

Constant RF voltage at 1st stage

across horizontal plates

-> less energetic

collisions/fragmentation – better for

labile compounds

Wider profile first stage,

more space for ion cloud –

less collision w/ plates


ionKey Microfluidics

ESI Assembly Incoming FlowEEProm Memory Electrical Connections

Analytical ColumnColumn Heater Sensor


Infliximab High Analytical Sensitivity Using

Integrated Microflow

Time5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

%

0

100

Std_21Sep_49 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK)

4.07e6


4.70e5


4.66e4


1.33e4


1.14e4

blank_21Sep_8 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK)

9.15e3

5.94

7.377.19

blank

10,000 ng/mL

1000 ng/mL

100 ng/mL

10 ng/mL

1 ng/mL

For research use only. Not for use in diagnostic procedures


m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

0

100

m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

0

100

m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

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m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

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3252015 C4 Herceptin ADC 10000X Dilution_2 156 (3.051) Cm (144:169) 1: TOF MS ES+ 6.08e32904.37552847.4702

2740.01492689.4590

2593.2161

2904.7944

2963.8701 3089.8511

3090.2375


2792.83572689.2893

2904.2434

2904.7063 3025.3850


2724.7688

2644.5042

2884.9858

2904.7285


2644.6094

2598.2388

2847.9941

2885.3372

2924.1621250 ng

25.0 ng

2.5 ng

0.25 ng

Trastuzumab ADC (De-Glycosylated) High

Sensitivity Spectra

Trastuzumab MW: 145165.1575 g/mol

Linker and Drug: 958.5322 g/mol

1 Drug + 1 Antibody: 146123.6 g/mol


Intact Mass Analysis on T-DM1

+Drug 0

+Drug 1

+Drug 2 +Drug 3

+Drug 4

+Drug 5

+Drug 6

+Drug 7+Linker

+Linker

+Linker

+Linker +Linker

Antibody with 0-7 drugs observed

Linker-only peaks observed


Conclusions

Waters offers a Total Solution

– Software tools

– Sample preparation

– LC/MS

– Service and Application support

Innovative tools for the latest performance

– Xevo TQ-XS improved Step Wave and Detector

– ionKey microfluidics

– Xevo G2-XS


Acknowledgements

Mary Lame

Nikunj Tanna

Paula Orens

Caitlin Dunning

Erin Chambers

Catalin Doneanu

Steven Calciano

Ian Edwards

Logan Umberger

Mark Wrona

Yun Alelyunas

Hua Yang

Apa 2017 diego rodriguez 3

Science

Transcript of Apa 2017 diego rodriguez 3