Infliximab life of a biotherapeutic comments v5

©2017 Waters Corporation 1COMPANY CONFIDENTIAL

LC-MS/MS for Bioanalytical Protein Quantification: Life of a Biotherapeutic

Infliximab Case Study


Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification

Outline

Kelly Doering

I think a slide for "Why do we use the surrpgate peptide approach" and a couple of references showing its adoption would be welcome and would help set the stage. Maybe after your case study goal slide?

Kelly Doering

column selection?SW? Maybe that could be a slide at the end that summarizes all SW used. A thought.


Case Study Goal

How do we get there?

Let’s take a look at the overall LC/MS workflow for protein quantification via the surrogate peptide approach…

The goal of this study is to develop a discovery bioanalytical method for the quantification of infliximab in rat and mouse plasma, with the intent to extend to human at a later date


Why the Surrogate Peptide Approach?


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



Outline

Kelly Doering

do generic peptides need to be addressed?


Find similar sequences of light and heavy chains (LC and HC)– Protein BLAST against the NCBI database

Align the mAb sequence against closely related LC/HC sequences– SIM alignment tool (ExPASy Proteomics Server)

Generate proteotypic peptides in-silico while maintaining sequence alignment– Trypsin used in this study, cleaves at lysine and arginine– Text editor (ex. TextPad, Notepad)

Assess uniqueness of the un-matched tryptic peptides from therapeutic mAb– Peptide BLAST against the NCBI database

Identification of Unique Proteotypic Peptides

Kelly Doering

for consistency, label these as Steps 1, 2, etc (per Slide 8, etcOR, if you wanted to label the steps in your outline as Steps 1, 2 etc, the steps for ID of unique sig peptides could be labeled as Step 1A, Step 1B, etc....This could be depicted as a flow diagramWhat about spealling out the "Why?" at each step?


Infliximab is a chimeric human-murine anti-human tumor necrosis factor (TNF) monoclonal antibody.

Infliximab (Remicade) Sequence

Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

Formula: C6428H9912N1694O1987S46Molecular Weight: ~ 149.1 kD

Light Chain

Heavy

Cha

in


Step 1: BLAST Search Infliximab Light Chain to Identify Closest Related Protein in Database

Protein BLAST search screen

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

BLAST Result for LC

Protein sequence differences and gaps


Step 2: Align the mAb Sequence Against Closely Related Sequences in ExPASy SIM Alignment Tool

SIM Protein Alignment for Infliximab LC

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

ExPASy SIM Protein Alignment for 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

Infliximab LC: Tryptic Peptides

Gaps = tryptic peptide differences, maintaining sequence alignment

“matched” non-unique peptides

“un-matched” potentially unique peptides


Step 4: Assess uniqueness of the un-matched tryptic peptides using peptide BLAST search


ASQFVGSSIHWYQQR

Peptide Level BLAST

Infliximab LC

Long List of Possibilities


Step 4: Detailed View of Closely Related Database Matches

Mouse

Monkey

Goal: BA method for rat and mouse with extension to human= ID peptides unique in rat, mouse, human

“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 speciesThe alignment and gaps show the differences

ASQFVGSSIHWYQQR

Infliximab

Human

Kelly Doering

bullet these points?


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, ratASQFVGSSIHWYQQR: 80% Mouse IgG peptide-Specificity in human, rat, and mouseYASESMSGIPSR:90% Mouse IgG peptide-Specificity in human, rat, and mouse

Infliximab LC Tryptic Peptides

Erin Chambers

what is this slide trying to show?

Steven Calciano

What I am taking away from this slide is that we have three possibile tryptic peptides, all with varying degrees of specificity depending on the matrix.

Kelly Doering

including a statement for the takeaway would be helpful


Follow same process as LC:

Infliximab HC Tryptic Peptide Identification

BLAST search full chain

SIM Alignment of Infliximab HC

BLAST Search Peptides from Infliximab HC

Generate Tryptic Peptides In-silico

Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique Infliximab HC Tryptic Peptides


List of Unique Infliximab Tryptic Peptides

Light Chain– DILLTQSPAILSVSPGER (human & rat)– ASQFVGSSIHWYQQR (human, rat &

mouse)– YASESMSGIPSR (human, rat & mouse)– LC conserved region human IgG1

peptides (no specificity in human plasma)o DSTYSLSSTLTLSK

Heavy Chain– LEESGGGLVQPGGSMK (rat & human)– GLEWVAEIR (rat & human)– SINSATHYAESVK (mouse, rat &

human)– HC conserved region human IgG1

peptides (no specificity in human plasma)o GPSVFPLAPSSKo STSGGTAALGCLVKo DTLMISRo VVSVLTVLHQDWLNGK

Erin Chambers

are the species in parentheses the ones where the peptide is unique?

Steven Calciano

Which peptides did we choose to move forward with? Or will that depend on the model we are working in?


Best Practice for Signature Peptide Choice

Once you have a selection of Unique Peptides:Size

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

– Ideally rule out: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

Kelly Doering

again, should we address generic peptides?



Outline


Skyline Software: Download to Acquisition PC

https://skyline.ms/project/home/software/Skyline/begin.view


Developed by the MacCoss laboratory in the University of Washington

Best Practice:Our Approach = Skyline Workflow

Start Here!


SkyLine

In silco 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

Kelly Doering

silico

Kelly Doering

I like this slide bc it is more visual than a list


Skyline MRM Method Creation: Infliximab LC

With Skyline, importing a protein sequence elicits an in silico digestion algorithm and MRM transition prediction

Skyline Protein Import


Skyline MRM Method Creation: Infliximab LC

Skyline> MassLynx Method Export


Method Conditions

LC System: ACQUITY UPLCMS System: Waters Xevo TQ-S, ESI+

Column: ACQUITY UPLC Peptide BEH C18, 300Å, 1.7 µM, 2.1 mm x 150 mm

Column Temperature: 55 °CSample Temperature: 10 °CMobile Phase A: 0.1 % Formic Acid in waterMobile Phase B: 0.1 % Formic Acid in waterGradient: 1 min hold at 10% B,

then 10-55% B in 6 minutes

Skyline LC/MS Conditions

MS ConditionsCapillary (KV): 3.00Cone (V): 30Source Temperature (°C): 150Desolvation Temperature (°C): 1000Cone Gas Flow (L/Hr): 150Desolvation Gas Flow (L/Hr): 1000Collision Gas Flow (mL/min): 0.15Nebuliser Gas Flow (Bar): 7.00

Kelly Doering

could there be a brief slide that describes column selection?

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiarY6Ju-DRAhWFVyYKHfQhBZkQjRwIBw&url=http://www.waters.com/waters/en_US/Original-UPLC-UHPLC-system-with-sub-2-micron-particle-technology-for-separations/nav.htm?cid=514207&psig=AFQjCNECjtxTz8KBkOsqVoV0b08n1D0XYg&ust=1485542429028585


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

Kelly Doering

it took me a second to see that the stoplight assignment referred to the left hand side. Can that be enlarged and/or the arrows made more pronounced? My eyes were drawn to the peaks on the right.


Skyline MRM Method Creation: Narrowing Down MRMs

Skyline Protein Import


Sample Analysis with MassLynx: Skyline imported methods

Narrowed down Skyline methods are imported into MassLynx for LC/MS acquisition

MassLynx imported Skyline Methods


Skyline MRM Method Optimization: CE Optimization

Transitions are optimized by collision energy and the results are visualized

The optimized method can be exported automatically

Skyline MRM CE Optimization


DILLASQ

DSTY

YASE

Final Optimized Skyline Method for Instrument Import: Peptides, Precursors, and Fragments


Exporting a Final MRM Method to MassLynx

When you’ve narrowed down your best optimized transitions, it is important to check back in the transitions settings to make sure the “Use optimization values when present” is checked. This allows Skyline to choose the best CE per transition to export into your method instead of the original predicted CE.

Options include transition or precursor.


Optimized Skyline MassLynx Method: Generic and signature peptides

Optimized MRM method for HC and LC Infliximab tryptic peptides

Individual MRMs for DILL peptide

Kelly Doering

Generic - I think this is the 1st time it is mentioned


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>546.3More sensitive transition!

Manual Optimization633.1>731.8

5.59140139

Alternate MRM transition


Infiximab Unique Signature PeptidesInfliximab 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

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

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

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

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


6.51e5Area

29Dec2016_Infliximab_Optimized_003 11: MRM of 3 Channels ES+ 598.629 > 631.307 (Infliximab_LC ASQFVGSSIHWYQQR.3)

3.49e6Area

4.42;114896

29Dec2016_Infliximab_Optimized_003 15: MRM of 3 Channels ES+ 642.798 > 359.204 (Infliximab_LC YASESMSGIPSR.2)

6.19e6Area

3.64;246053

29Dec2016_Infliximab_Optimized_003 7: MRM of 2 Channels ES+ 515.924 > 576.281 (Infliximab_HC LEESGGGLVQPGGSMK.3)

4.93e6Area

3.88;170856

29Dec2016_Infliximab_Optimized_003 8: MRM of 2 Channels ES+ 536.793 > 171.113 (Infliximab_HC GLEWVAEIR.2)

9.39e6Area

5.21;329917

29Dec2016_Infliximab_Optimized_003 5: MRM of 3 Channels ES+ 469.569 > 603.791 (Infliximab_HC SINSATHYAESVK.3)

5.03e7Area

3.38;1735473

5.58;22653

DILLTQSPAILSVSPGER-LC

ASQFVGSSIHWYQQR-LC

YASESMSGIPSR-LC

LEESGGGLVQPGGSMK-HC

GLEWVAEIR-HC

SINSATYAESVK-HC

633.1>731.8

598.6>631.3

642.8>359.2

515.9>576.3

536.8>171.7

469.6>603.8


Infliximab Generic, Human IgG1, Signature Peptides

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

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

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

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

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

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

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 2: MRM of 4 Channels ES+ 418.221 > 506.276 (Infliximab_HC DTLMISR.2)

1.73e7Area

3.90;549369

29Dec2016_Infliximab_Optimized_003 9: MRM of 4 Channels ES+ 559.939 > 708.849 (Infliximab_HC FNWYVDGVEVHNAK.3)

2.02e7Area

4.67;650255

29Dec2016_Infliximab_Optimized_003 12: MRM of 3 Channels ES+ 603.34 > 805.439 (Infliximab_HC VVSVLTVLHQDWLNGK.3)

5.12e6Area

29Dec2016_Infliximab_Optimized_003 3: MRM of 2 Channels ES+ 419.755 > 654.382 (Infliximab_HC ALPAPIEK.2)

2.99e7Area

3.84;1004329

29Dec2016_Infliximab_Optimized_003 16: MRM of 3 Channels ES+ 643.841 > 359.204 (Infliximab_HC EPQVYTLPPSR.2)

9.18e5Area

3.64;31544

29Dec2016_Infliximab_Optimized_003 19: MRM of 2 Channels ES+ 848.715 > 764.357 (Infliximab_HC GFYPSDIAVEWESNGQPENNYK.3)

4.71e6Area

29Dec2016_Infliximab_Optimized_003 13: MRM of 2 Channels ES+ 625.312 > 234.145 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)

1.99e6Area

29Dec2016_Infliximab_Optimized_003 1: MRM of 3 Channels ES+ 394.729 > 588.335 (Infliximab_HC SLSLSPGK.2)

6.02e6Area

3.63;215260

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

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

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

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

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

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

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


5.12e6Area

29Dec2016_Infliximab_Optimized_003 3: MRM of 2 Channels ES+ 419.755 > 654.382 (Infliximab_HC ALPAPIEK.2)

2.99e7Area

3.84;1004329

29Dec2016_Infliximab_Optimized_003 16: MRM of 3 Channels ES+ 643.841 > 359.204 (Infliximab_HC EPQVYTLPPSR.2)

9.18e5Area

3.64;31544

29Dec2016_Infliximab_Optimized_003 19: MRM of 2 Channels ES+ 848.715 > 764.357 (Infliximab_HC GFYPSDIAVEWESNGQPENNYK.3)

4.71e6Area

29Dec2016_Infliximab_Optimized_003 13: MRM of 2 Channels ES+ 625.312 > 234.145 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)

1.99e6Area

29Dec2016_Infliximab_Optimized_003 1: MRM of 3 Channels ES+ 394.729 > 588.335 (Infliximab_HC SLSLSPGK.2)

6.02e6Area

3.63;215260

29Dec2016_Infliximab_Optimized_003 17: MRM of 4 Channels ES+ 751.883 > 234.092 (Infliximab_LC DSTYSLSSTLTLSK.2)

1.32e6Area

4.81;50235

29Dec2016_Infliximab_Optimized_003 10: MRM of 3 Channels ES+ 593.827 > 418.23 (Infliximab_HC GPSVFPLAPSSK.2)

1.96e7Area

4.76;737428

5.30;162081

5.55;64156

5.86;166012

DSTYSLSSTLTLSK

GPSVFPLAPSSK

DTLMISR

FNWYVDGVEVHNAK

VVSVLTVLHQDWLNGK

ALPAPIEK

EPQVYTLPPSR

GFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSK

SLSLPGK

751.9>234.1

593.8>418.2

418.2>506.3

559.9>708.8

603.3>805.4

409.7>654.4

643.8>359.2

848.7>764.3

625.3>234.1

394.7>588.31


Infliximab Specificity in Matrix: Generic Signature VVSVLTVLHQDWLNGK (HC)

remicade buffer 50

Time4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20

%

0

100

4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20

%

0

100

4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20

%

0

100

11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)

4.84e5Area

5.54;16050

03May2016_Infliximab_AutomationReagentStability_016 MRM of 10 Channels ES+ 603.3 > 1110.6 (Generic VVSVLTVLHQDWLNGK)

4.31e3Area


2.34e5Area

5.53;8028

remicade buffer 50

Time4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20

%

0

4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20%

0

4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20

%

0

100


4.84e5Area

5.54;16050


1.14e7Area

5.53;391409


1.26e7Area

5.52;430796

Infliximab 50 µg/mL Buffer Digest

Blank Rat Plasma

50 µg/mL Rat Plasma

Blank Human Plasma

50 µg/mL Human Plasma

Generic Human IgG PeptideVVSVLTVLHQDWLNGK

Steven Calciano

include pop-up explaining that the VVSV peptide will be incorporated into the endogenous VVSV present in human plasma?Just to make it dummy proof.


Infliximab Specificity in Matrix: Unique Signature DILLTQSPAILSVSPGER(LC)

remicade buffer 50

Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%

0

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%

0

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%

0

100

11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)

2.67e6Area

5.32;71906

03May2016_Infliximab_AutomationReagentStability_016 MRM of 10 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)

1.53e6Area


6.75e5Area

5.32;19165

remicade buffer 50

Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%

0

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%0

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

%

0

100


2.67e6Area

5.32;72436


4.30e5Area


8.76e6Area

5.32;264330

Infliximab 50 µg/mL Buffer Digest

Blank Rat Plasma

50 µg/mL Rat Plasma

Blank Human Plasma

50 µg/mL Human Plasma

Unique Signature PeptideDILLTQSPAILSVSPGER



Outline


Internal Standard Options

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


Peptide Level IS: TrastuzumabC13N15 Labeled Trastuzumab (FTISADTSK) peptide with terminal

overhang– Added just prior to digestion

Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

%

0

100

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

%

0

100

7pt5ugmL_2hr_bicarb_070214_002 MRM of 8 Channels ES+ 489.2 > 729.4 (FTISADTSK(13C15N) HC)

4.33e57.41

7pt5ugmL_2hr_bicarb_070214_002 MRM of 8 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)

4.33e5

7.41

8.65

C13N15 FTISADTSK Trastuzumab PeptideInternal Standard

FTISADTSK Trastuzumab Peptide

Kelly Doering

subscript


Labeled Antibody Internal Standard: SILu™Mab

SILuMab Heavy ChainEVQLVESGGGLVQPGGSLRLSCVAS GFTLNNYDMHWVRQGIGKGLEWVSK IGTAGDRYYAGSVKGRFTISRENAKD SLYLQMNSLRVGDAAVYYCARGAGR WAPLGAFDIWGQGTMVTVSS|ASTKG PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

SILuMab Light ChainQSALTQPRSVSGSPGQSVTISCTGT SSDIGGYNFVSWYQQHPGKAPKLMI YDATKRPSGVPDRFSGSKSGNTASLT ISGLQAEDEADYYCCSYAGDYTPGV VFGGGTKLTVL|GQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Labeled Peptides:DTLMISR Heavy Chain (IgG1, IgG2, IgG3, IgG4)FNWYVDGVEVHNAK Heavy Chain (IgG1)VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4)NQVSLTCLVK Heavy Chain (IgG1, IgG2, IgG3, IgG4)GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4)AGVETTTPSK Light Chain (lambda)YAASSYLSLTPEQWK Light Chain (lambda)


direct 50ug/ml SPE*

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

10018Dec2014B_spe_direct_1037 MRM of 10 Channels ES+

603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)3.07e5

prot a spe blk plasma silumab

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

10015Jan2015_HerceptinProtA_spe_1009 MRM of 10 Channels ES+

423.225 > 516.284 (SILUMAB IS-DTLMIS[R].HEAVY-3)8.58e5

Chromatography: SILuMab IS Labeled Antibody Drug Signature Peptides

1 2 3

mAb PeptideRetention Time

(min)4 Unique Herceptin FTISADTSK HC 3.315 Generic STSGGTAALGC[+57.0]LVK HC 4.056 Generic DSTYSLSSTLTLSK LC 4.397 Generic GPSVFPLAPSSK HC 4.508 Unique Remicade DILLTQSPAILSVSPGER LC 5.179 Generic VVSVLTVLHQDWLNGK HC 5.41

4 5 6 7 8 9

SILUMAB IS PeptideRetention Time

(min)1 DTLMIS[R] HC 3.562 YAASSYLSLTPEQW[K] HC 4.773 VVSVLTVLHQDWLNG[K] HC 5.41

SILuMabInternal Standard

Antibody Drug Unique and Generic Signature Peptides


Intact Murine mAb Mass Check Standard (Waters)

Murine mAb Light chain:DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Murine mAb Heavy chain:QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQPPGQGLEWLMGIWGDGSTDYNSALKSRISITKDNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQYFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTDSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG

Conserved region: blue Variable regions: red Signature peptides: BOXED

Unique SignatureGeneric Signature


Generic Internal StandardWorkflow check Standard

prot A spe blk ISTD murine mab

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

10014Aug2015_RemicadeSPE_ProteinA_01004 MRM of 11 Channels ES+

983.95 > 397.21 (Murine 4)4.91e6

prot a spe 50 ug/ml

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

10014Aug2015_RemicadeSPE_ProteinA_01038 MRM of 11 Channels ES+

633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)8.35e6

1

2

3

4

5

6

7

8

9

Murine mAbInternal Standard

Infliximab Unique and Generic Signature Peptides

Retention Time(min)

1 MNSLQTDDTAK 4.062 VNSAAFPAPIEK 5.073 NTQPIMDTDGSYFVYSK 5.374 SVSELPIMHQDWLNGK 5.66

Murine mAb IS Peptides

Retention Time(min)

5 SINSATHYAESVK* 4.226 DSTYSLSSTLTLSK 5.437 GPSVFPLAPSSK 5.488 DILLTQSPAILSVSPGER* 6.029 VVSVLTVLHQDWLNGK 6.16

mAb Peptides

Intact Murine mAb Mass Check Standard (Waters)



Outline


Sample Preparation Options

Protein Level Clean-up prior to digeston (Optional)– None

o Whole serum/plasma digestion– Affinity-based

o Generic: Protein A/G with binding affinity to various immunoglobulins in various species

o Anti-human: kappa with binding affinity to light chains on all human immunoglobulins

o Specific Receptor: anti-idiotypic binding– MWT cut-off filters– Depletion Plates– Gel Filtration

Kelly Doering

typoDOn;t think you need (Optional) bc you are listing the options, and one is "None"

Kelly Doering

should there be any guidance as to options for solid supports for ab conj? Beads, etc, Kingfisher, manual, etc.


spe 10 ug/ml

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

16Oct2015_4mAb_HumanPL_3St_ProtA_1038 MRM of 15 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

1.28e7Area

4.12;486885

16Oct2015_4mAb_HumanPL_3St_direct_1035 MRM of 15 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

1.28e7Area

4.1195194

GE Multi-Trap Protein A Clean-Up of Infliximab from Plasma

Direct Digestion of Plasma

Protein A Clean-up Prior to Digestion

Infliximab SINSATHYAESVK Tryptic Peptide


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 Plasma Blank Plasma Blank Plasma

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

100 ng/mL100 ng/mL100 ng/mL

500 ng/mL 500 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

Kelly Doering

I asusme we don't have equivalent data for infliximab?



Outline


ProteinWorks™ eXpress Digest Kits

Key AttributesStandardized protocols

– Eliminates Method Development for Discovery Studies– Digestion and quantification of a diversity of proteins– 3 and 5-Step Digestion to accommodate variety of proteins and signature

peptide typesScalable, flexible formatReliable and reproducibleHigh SensitivityQuantification Total antibody analysis for ADCs

– Detection of conjugated peptidesNo capital investment required

Kelly Doering

"minimizes" or "significantly reduces"

Kelly Doering

maybe a bullet could be about applications and include - diversity of proteins- high sensitivity- total mAb


Peptide

Std. Curve Range (mg/mL) Weighting

Linear fit (r2)

Mean % Accuracy

of all points

DILLTQSPAILSVSPGER 0.25-500 1/x 0.998 100.01

QC Conc (ug/mL)Mean Cal. Conc

(ug/mL) Std. Dev. %CV Mean AccuracyDILLTQSPAILSVSPGER* 0.35 0.33 0.02 5.80 93.2SILUMAB-DTL(IS) 3.50 3.79 0.02 0.49 108.2

35.00 39.58 0.17 0.44 113.1350.00 350.02 3.09 0.88 100.0

QC Conc (ug/mL)DILLTQSPAILSVSPGER* 0.35 0.34 0.00 0.89 96.5SILUMAB-VVSV (IS) 3.50 3.65 0.05 1.47 104.2

35.00 36.51 0.73 2.01 104.3350.00 350.80 3.90 1.11 100.2

* Signature

Accurate and Precise Quantification using ProteinWorksInfliximab Direct Plasma Digestion

Linear, Precise and

Accurate

Single digit %CVs



Outline


Peptide Level Clean Up:

Peptides

Purified Peptides

Target AnalyteSensitivity

Interfering Digest Reagents, Buffer and Matrix Salts,

Phospholipids

Achieving Recovery of Target Peptides

High Levels of Unwanted

Endogenous Peptides

Should I clean up my

peptides?

Peptide-level clean-up (optional)

System Robustness

Kelly Doering

I like the inclusion of this piece of the overall flow. I think this tactic could be used more throughout the deck; keeps the viewer on top of where they are in the workflow.


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

020406080

100120

Oasis MCX

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


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

Kelly Doering

these two slides are good. Can you include a statement that calls out the impact, i.e. less background or signal improvement x-fold?



Outline


Place Holder for MS Introduction

Options for TQ or Accurate MassPros and ConsData for TQ, G2-XS


High Sensitivity Infliximab Quantification:Xevo TQ-S Triple QuadrupoleLinear, Precise and Accurate

Single digit %CVs

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

Kelly Doering

I thought this was repeated on the TQ-XSCould there be a workflow intro - there was affinity, correct?sample type, PW, etc


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


2.20e4Area

10 ng/mL infliximab

Blank plasma

SINSATHYAESVK Unique Signature Peptide


Coming soon

G2-XS Data


Conclusions

Erin Chambers

Need to change these to match seminar, infliximab example specifically


Acknowledgements

Mary LameNikunj TannaPaula OrensCaitlin DunningErin ChambersCatalin DoneanuSteven Calciano Ian EdwardsLogan UmbergerMark WronaYun AlelyunasHua Yang


Supplemental Information


Skyline Optimization vs. Manual Optimization - DILL

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


3.94e6Area

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

3.94e6Area

5.5926370

Skyline Optimization

Manual Optimization

5.59140139


High Sensitivity Antibody Quantification: Remicade (infliximab)

Linear, Precise and Accurate

Single digit %CVs


High Sensitivity Antibody Quantification: Remicade (infliximab)

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


2.20e4Area

4.15361


2.20e4Area

LLOQ of 10ng/mL35 µL sample, Protein A affinity sample clean-up and

digestion

10 ng/mL infliximab

Blank plasma

SINSATHYAESVK Unique Signature Peptide


Protein BLAST: Infliximab HC

Protein BLAST search screen

Infliximab HC sequence


Protein BLAST: Infliximab HCBLAST

Result for HCProtein sequence differences and gaps


Align the mAb sequence against closely related sequences: ExPASy SIM Alignment

SIM Protein Alignment for Infliximab HC


ExPASy SIM Alignment Results: Infliximab HC

ExPASy SIM Protein Alignment for

Infliximab HC vs. Human IgG HC

Protein sequence differences maintaining sequence alignment


Generate Tryptic Peptides Maintaining Sequence Alignment: Infliximab HC

Infliximab HC: Tryptic Peptides

Trypsin cleaves peptides on the C-terminal side of lysine (K) and arginine (R) amino acid


Assess uniqueness of the un-matched tryptic peptides from therapeutic mAb: Infliximab HC

GLEWVAEIR: Variable and Complimentary Determining Region (CDR)

LEESGGGLVQPGGSMK: Variable Region

SINSATHYAESVK:Variable and Complimentary Determining Region (CDR)

This a chimeric murine mAb and variable region should have very close sequence homology to mouse

Peptide-Level BLAST

Infliximab HC100% sequence homology mouse

~85% sequence homology mouse & human

100% sequence homology to mouse


Tryptic Peptide Specificity: Trastuzumab HC vs. IgG1kappa 1

Heavy Chain

too long (25 AA)

LEESGGGLVQPGGSMK and GLEWVAEIR : Mouse IgG peptides-Specificity in humanSINSATHYAESVK:~85% Mouse IgG peptide-Specificity in mouse, rat and humna

This a chimeric murine mAb and variable region should have very close sequence homology to mouse


Skyline Optimization vs. Manual Optimization - GLEW

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 8: MRM of 2 Channels ES+ 536.793 > 488.283 (Infliximab_HC GLEWVAEIR.2)

8.47e6Area

5.21;292371

29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 536.793 > 773.43 (GLEWVAEIR)

8.47e6Area5.21

250364


Manual Optimization


Skyline Optimization vs. Manual Optimization - GPSV

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 10: MRM of 3 Channels ES+ 593.827 > 418.23 (Infliximab_HC GPSVFPLAPSSK.2)

1.96e7Area

4.76;737428

29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 593.83 > 699.4 (Generic GPSVFPLAPSSK)

1.96e7Area4.77

606398


Manual Optimization


Infliximab Mary

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


5.12e6Area

29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 603.3 > 1110.6 (Generic VVSVLTVLHQDWLNGK)

5.12e6Area

Skyline Optimization vs. Manual Optimization - VVSV


Manual Optimization

5.86175812

5.866451


Internal Standard Comparison

Best curve fit from generic mab IS using peptide with RT closest to analyte– Curve fit from extended tag peptide IS very close

Average accuracy of standard curve points comparable between extended peptide IS (9%) and mab IS (12%)

Worst fit and accuracy from mab IS using peptide with very different RT

Curve FitAvg Acc of Std Curve Points RSD of IS

Extended Tag Pep IS 0.988 9% 17%

SILuMab IS, close RT 0.998 12% *8%

SILuMab IS, diff RT 0.979 33% 19%

*mab IS 8% RSD through entire workflow, includes protein A and digestion



LC-MS/MS Protein Quantification Challenges Identification and LC/MS optimization of unique surrogate peptides is complex

and laborious, – Workflow can be challenging, particularly for small molecule bioanalytical scientists

Currently no end-to-end informatics platform exists to helps a scientist navigate and method develop for the surrogate peptide approach

Simplification and LC/MS platforms with improved informatics platforms are need– Current approaches require multiple informatics free-ware

o BLAST protein search (NCBI database)o ExPASY SIM Alignmento Protein Prospectoro Skylineo MassLynx BioLynx Application/UNIFI…

Many possible sample preparation workflow options– Lengthy method development times– Many steps to optimize– Lack of standardization– Sensitivity and reproducability issues


Manual Optimization Process


Protein Prospector (UCSF)– In silico tryptic digestion DILLTQSPAILSVSPGER (LC)– Prediction of precursors and MRM fragments

Possible Approach: Manual Optimization

Possible Precursors Potential fragment ions

http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct

DILLTQSPAILSVSPGER (LC)


Confirmation of Precursor Presence and Retention Time

DILL 2nd Charge State

Time3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00

%

1

3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00

%

1

26Jan2017_DILLMRM_005 1: MRM of 1 Channel ES+ 632.69 > 632.69 (DILL 3rd Charge State)

1.81e7Area

5.92;710710

26Jan2017_DILLMRM_006 1: MRM of 1 Channel ES+ TIC (DILL 3rd Charge State)

1.81e7Area

5.92;710710

Infliximab digested in buffer

Blank buffer digest

DILL Peptide 632.73+


MassLynx Manual Optimization: Infliximab DILLTQSPAILSVSPGER (LC)

MS/MS Spectra of MH+3Precursor

50 µg/mL Infliximab DILL

m/z200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975 1000 1025 1050 1075

%

0

100

20Jan2017_Infliximab_ManualOpScans_013 237 (5.937) Cm (237) Daughters of 633ES+ 9.28e5610.8

575.8

474.6230.5

215.2

458.3

342.5

308.9

261.5

388.9

423.1

526.4

509.2

495.5

541.3

563.0

732.0

645.3

658.8

706.9676.2

844.2

798.7

783.2752.9

1029.7

925.9

907.5

889.1

959.0

939.7 1007.0

b2 +1H

b3 +1H MH+3

y7 +1H

633.5

b17 +3H

y8 +1H

b11 +3Hy9 +1H

y4 +1H

y6 +1H

y10 +1H545.3y5 +1H

DILL 2nd Charge State

Time3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00

%

1

3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00

%

1

26Jan2017_DILLMRM_005 1: MRM of 1 Channel ES+ 632.69 > 632.69 (DILL 3rd Charge State)

1.81e7Area

5.92;710710

26Jan2017_DILLMRM_006 1: MRM of 1 Channel ES+ TIC (DILL 3rd Charge State)

1.81e7Area

5.920


MassLynx Manual Optimization: Infliximab DILLTQSPAILSVSPGER (LC)

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

5 10 15 20 25 30 35

Peak

Are

a

CE Ramp of DILL

632.69>458.24 Area

632.69>545.27 Area

632.69>731.37 Area

MRM CE Optimization of MH+3Precursor


Optional ExPasy Peptide Level BLAST searching


This can be done in NCBI data base too. See notes section

Infliximab Peptide level BLAST:ExPASY example ASQFVGSSIHWYQQR

Mouse

Rat

Monkey

Human

http://web.expasy.org/blast/


Optional Visualization


Confirming Species Specificity Multiple Sequence Alignment Tool: Example below is mAb, mouse, human and monkey

Infliximab Peptide level BLAST:NCBI example ASQFVGSSIHWYQQR

Mouse

Rat

Monkey

Human

http://web.expasy.org/blast/

InfliximabMouseHumanMonkey



Additional Scrren Shot Detail from Skyline


Reasons why you want BLAST searching and filtering to narrow down peptide options

Text in box is why you import multiple methods– Each method is one peptide, one precursor, with its predicted fragments

Skyline Exporting MRM Method: Fragment Determination

Too many transitions for 11 minute method. Doesn’t allow enough points (≈1.0) across the peak for useful data. So we must narrow it down even further.


Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

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%

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%

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%

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0

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%

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%

0

100

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%

0

100

December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 402.718 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)

1.54e55.19


2.65e55.18


4.31e35.203.64

3.56 3.895.15

5.31 6.18 6.39


3.14e55.18


2.79e45.19


2.38e45.18

3.55 5.21


5.92e35.18

5.23


1.65e35.18


3.76e45.193.91

3.56 4.02 5.05Time

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 147.113 (Infliximab_HC VVSVLTVLHQDWLNGK.3)

4.93e43.13 5.44

3.77 4.66


5.75e36.073.783.71

2.715.534.22

4.66 5.31 6.46 6.636.95


2.00e55.44

5.47


1.53e45.42

5.48


5.89e36.50

5.443.88 6.04 6.55


1.83e55.43


1.19e55.44

5.49


4.37e35.43

5.46


3.40e33.140.04 4.774.644.273.92 5.455.76

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

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%

0

100

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%

0

100

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%

0

100

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%

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%

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%

0

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%

0

100

December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 246.181 (Infliximab_HC SINSATHYAESVK.3)

2.25e53.13


1.47e63.13


2.32e43.12

6.695.695.535.21 6.566.16


2.32e43.12

6.695.695.535.21 6.566.16


7.64e53.13


1.33e43.13

3.17


226.88


1.61e43.13

2.58 6.794.113.773.22 5.664.71 5.57 6.695.75 7.01


8.73e43.13

If not for Skyline…

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

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%

0

100

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%

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%

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%

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%

0

100

December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 1441.765 (Infliximab_LC DILLTQSPAILSVSPGER.2)

3.17e45.25


9.31e35.26

5.30 6.326.08 6.95


9.86e35.24

5.223.74 3.89 6.375.89


9.31e35.26

5.30 6.326.08 6.95


2.96e36.535.275.114.84 6.035.83 6.84


1.87e45.26

5.28


1.32e55.25


5.28e35.265.10

5.30

December2016_InfliximabMD_001 MRM of 32 Channels ES+ TIC (Infliximab_LC DILLTQSPAILSVSPGER.2)

8.47e55.25

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

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%

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%

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%

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%

0

100

December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 472.288 (Infliximab_LC YASESMSGIPSR.2)

6.79e43.40


4.20e43.41

3.44


4.06e36.274.25

3.39 4.68 4.955.17 5.59

6.70


1.11e43.42

3.92 4.854.25 6.02


1.66e43.40


2.02e53.40


2.96e53.40


3.78e53.41


3.70e53.41

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

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%

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%

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%

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%

0

100

December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 418.74 (Infliximab_LC DSTYSLSSTLTLSK.2)

2.43e44.49

4.54 5.24


1.05e44.49

4.56 6.37


1.74e54.49


3.91e44.49

4.47 4.52


2.24e54.49


1.05e44.49

4.56 6.37


3.09e34.51

4.39 5.224.71 6.77


8.49e34.50

4.53


6.16e54.49

You’d have to individually pick through 2000+ chromatograms (only 4 peptides) for the most sensitive fragments.


These transitions will be optimized further for collision energy. Repeat for each peptide and each charge state.

2nd Charge State: from 19 > 6 transitions

Importing Data: Filtering data/Exclusion

Science

Infliximab life of a biotherapeutic comments v5