Towards the Standardization of High Performance Protein ...€¦ · ©2015 Waters Corporation 12 Blood Oncology Endocrine Anti-infective CNS Rheumatology Modified slide from McKinsey

©2015 Waters Corporation 1

Towards the Standardization of

High Performance Protein Quantification

Workflows

Paula Orens

Applications Scientist

October 2016


PEPTIDES AND PROTEINS ARE NOT SMALL

MOLECULES

Analyzing large molecules is one of the

greatest challenges that a bioanalytical

scientist faces!


Topics

Challenges in Workflow Options and Typical Steps

– Protein Digestion Optimization Complexity

Antibodies and Choice of Representative Tryptic Peptide

– Impact of 3 vs. 5-Step Digestion Protocol

Effect of Protein and Peptide Level Clean-Up

Addressing the Challenges

– Kit-based approach

Examples of Quantification

– Small-mid size proteins

– mAbs

– ADCs


Complexity of Protein Bioanalysis Workflows: Surrogate Peptide Approach


Method Development Challenges

Protein Digestion


Protein Digestion: Method Development Challenges

Optimization of Protein

Denaturation

Testing Digestion

Reproducibility

Up to 5 STEPS •Denaturation •Reduction •Alkylation •Enzymatic Digestion •Quench

Optimization of Reduction/Alkylation Agent Concentrations, Time, Temp, Reagent;

Or..Omit?

Choosing the Enzyme, Vendor, Protein:Enzyme

Ratio, Temp

Peptides Digestion

Protein

Optimization of Digestion Time


There is a sweet spot for reducing agent concentration

Optimization Complexity: Reducing Agent Concentration

%Area

Decreasing Concentration

4 variables to optimize! One must carefully choose the specific reducing

agent, the concentration of each reagent, and the time and temperature


There is a sweet spot for reducing agent concentration

Optimization Complexity: Reducing Agent Concentration

%Area

Decreasing Concentration

%Area Normalized to 80C

Reducing Agent 1 60°C Reducing Agent 2 60°C

4 variables to optimize! One must carefully choose the specific reducing

agent, the concentration of each reagent, and the time and temperature


Optimization Complexity: Enzyme

native peptide

13C15N peptide

Increasing Digestion Time

20

30

40

50

60

70

80

90

100

10 20 30 50 100

Protein to trypsin ratio

Digestion Efficiency

native peptide

13C15N peptide

Increasing Protein:Trypsin Ratio

Time Ratio


Optimization Complexity: Enzyme

native peptide

13C15N peptide

Increasing Digestion Time

20

30

40

50

60

70

80

90

100

10 20 30 50 100

Protein to trypsin ratio

Digestion Efficiency

native peptide

13C15N peptide

Increasing Protein:Trypsin Ratio

Type A

($150/mg)

Type A

($0.3/mg)

Type B

($9.2/mg)

TypeA

($473/mg)

Type C

($0.5/mg)

Type B

($0.2/mg)

Type C

($0.7/mg)

Type B

($820/mg)

Vendor #1 Vendor #2 Vendor #3 Vendor #4

Type A

($1030/mg)

Time Ratio

Not all enzymes are created equal


Topics










– mAbs

– ADCs


Blood

Oncology

Endocrine

Anti-infective

CNS

Rheumatology

Modified slide from McKinsey and Company Data Source: Evaluate Pharma

2015 2014 2013 2012 2011 2010

PEGIntron

Erbitux

NovoRapid/NovoLog

4.3 Lantus Neulasta

Avonex Rebif

NovoSeven Kogenate Procrit/Eprex

Tysabri

Pegasys

5.7

Avastin Rituxan Herceptin Remicade

6.1

2020 2019 2018 2017 2016

Enbrel

Humira

Levemir

Norditropin SimpleXx

NovoMix

Humalog

Monoclonal Ab Other protein

US Patent Expiration Date

Large Molecule Drugs

2021

2009 sales ($Billion)

1.1 0.8


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 variable regions: red CDR regions: green

Unique signature

Generic signature

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

Formula: C6428H9912N1694O1987S46

Molecular Weight: ~ 149.1 kD

Multitude of Tryptic Peptide Options

Choices for quantification are based on sensitivity

and specificity


Optimization Complexity: Peptide Considerations for Quantification Trastuzumab (Herceptin)

Conserved region Surrogate Peptides

Anti-HER2 Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-HER2 Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Formula: C6470H10012N1726O2013S42

Molecular Weight: ~ 145.5 kDa (claims are 148 package insert)

http://www.drugbank.ca/drugs/DB00072

Unique Signature

Generic Signature

Multitude of Tryptic Peptide Options

Choices for quantification are based on sensitivity

and specificity


Eliminate reduction/alkylation (3 Step)?

Peptide Choice: Impact on Protocol for Unique Variable Region Peptides

%Area

For unique variable region signature peptides from 4 monoclonal antibody drugs, 3 step protocol works well

For variable region peptides, one may not

need to reduce and alkylate

*Normalized to 5 step method

3 step*


Peptide Choice: Impact on Protocol for Generic Conserved Region Peptides

%Area

3 step, normalized to 5 step protocol

Eliminate reduction/alkylation (3 Step)?

For generic conserved region signature peptides from 4 monoclonal antibody drugs, 5 step protocol is more efficient, more often than not

For conserved region peptides, reduce and alkylatation increases

performance


Topics










– mAbs

– ADCs


Protein Level Sample Clean-Up: Options

Protein Clean-Up Techniques

General Proteins

Monoclonal Antibodies Discovery Development Sensitivity

None x x x low

MW cutoff filters x x x x low

Depletion Plates x x x x low

PPT (Pellet Digestion) x x x x medium

Protein A/G x x x medium

Kappa (anti-human) x x high

Specific Capture Reagent x x x high

An Ideal Digestion Approach Adapts to Any One of These Clean-up Techniques

Protein in Plasma/Serum

Purified Protein


Clean up at the Protein Level: Effects of Matrix Interferences

MEOH 1:1

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

20.000

40.000

60.000

80.000

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

25Jan2015_Herceptin_dilution_MEOH_1002 MRM of 24 Channels ES+ 485.2 > 608.3 (Herceptin-FTISADTSK HC)

7.03e5

5.18

4.59 5.90

25Jan2015_Herceptin_dilution_ACN_1014 MRM of 24 Channels ES+ TIC

2.22e8

8.696.07

5.56

4.48

4.244.94

6.33

7.14

6.65

7.84

8.12

25Jan2015_Herceptin_dilution_MEOH_1002 % ARange: 900.02

Trastuzumab (100ug/mL)

Albumin Peptides

% Aqueous

Human Serum Albumin makes up the largest protein fraction in plasma

(~55%)

Removing or minimizing matrix interferences through

sample clean-up would be advantageous


Simple, Cost-Effective Method: Protein Precipitation (PPT) Albumin Depletion and Herceptin Recovery

PPT with Solvent 4 helps remove half of the HSA peptide, increasing

sensitivity and specificity


Sensitivity and Sample Prep: What is Enough and How do I Get There?

Blank Plasma Blank Plasma

Blank Plasma

50 ng/mL

50 ng/mL 50 ng/mL

100 ng/mL 100 ng/mL

100 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


Complexity of Protein Bioanalysis Workflows: Surrogate Peptide Approach

A kit-based approach could simplify method development and create some level of standardization.


Topics










– mAbs

– ADCs


Development of Kit-Based Approach: What is Needed From a Standardized Method?

Accommodates all types of protein-level pre-treatment:

– PPT, generic and specific affinity, etc.

Flexibility to include or omit reduction/alkylation steps

Range of protein types (small, large, ADC’s)

Range of starting sample volumes

Sensitivity

Accuracy

Reproducibility

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjMj9mYiOHKAhXFVz4KHXs9BykQjRwIBw&url=http://processma.com/resource/msa.php&psig=AFQjCNEY5xnd98qfQ6ahFP7KH-Ovt2fl3A&ust=1454776805980361


Solution to the Complexity: ProteinWorks™ eXpress Digest Kits

Key Attributes

Standardized protocols

– Eliminates Method Development for Discovery Studies

– Digestion and quantification of a diversity of proteins

– 3 and 5-Step Digestion

Scalable, flexible format

Reliable and reproducible

High Sensitivity

Quantification Total antibody analysis for ADCs

– Detection of conjugated peptides

No capital investment required


Kit Performance: Intra and Inter-Kit

5 lots Trypsin, 2 prep days, 2 analysts

Kit-to-Kit eXpress Direct Digest kit,

avg 7.7% CV

Raw Area Counts, No Internal Standard

Within kit eXpress Direct Digest kit,

avg 1.9% CV


Standardization, while ensuring performance and

reproducibility

Protein Quantification Digestion Kit


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

DILLTQSPAILSVSPGER* 0.25-500 1/x 0.995 101.26

* Quantified using SILUMAB-VVSV (IS)

QC Conc (ug/mL)

Mean Cal. Conc

(ug/mL) Std. Dev. %CV Mean Accuracy

DILLTQSPAILSVSPGER* 0.35 0.33 0.02 5.80 93.2

SILUMAB-DTL(IS) 3.50 3.79 0.02 0.49 108.2

35.00 39.58 0.17 0.44 113.1

350.00 350.02 3.09 0.88 100.0

QC Conc (ug/mL)

Mean Cal. Conc


DILLTQSPAILSVSPGER* 0.35 0.34 0.00 0.89 96.5

SILUMAB-VVSV (IS) 3.50 3.65 0.05 1.47 104.2

35.00 36.51 0.73 2.01 104.3

350.00 350.80 3.90 1.11 100.2

* Signature

Accurate and Precise Quantification with a Standardized Protocol: Remicade (infliximab)

Linear, Precise and Accurate

Single digit %CVs


Achieving High Sensitivity with a Standardized Protocol: Remicade (infliximab)

Time 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

2.20 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 )

2.20e4 Area

4.15 361

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

2.20e4 Area

10 ng/mL infliximab

Blank plasma

SINSATHYAESVK Unique Signature Peptide

LLOQ of 10ng/mL 35 µL sample, Protein A affinity sample clean-up, digestion with

ProteinWorks eXpress Digest Kit, and MCX SPE peptide level clean-up.


Accurate and Precise Quantification with a Standardized Protocol: Avastin (bevacizumab), QC Statistics

Peptide QC Conc (ug/mL)

Mean Cal. Conc


GPSVFPLAPSSK 0.35 0.34 0.03 9.88 96.4

SILUMAB-DTL(IS) 3.50 3.84 0.06 1.67 109.8

35.00 37.88 1.49 3.94 108.2

*350.00 368.26 1.29 0.35 105.2

QC Conc (ug/mL)

Mean Cal. Conc


STSGGTAALGC[+57]LVK 0.35 0.35 0.02 6.80 100.6

SILUMAB-DTL(IS) 3.50 3.62 0.10 2.73 103.4

35.00 38.20 1.52 3.99 109.1

350.00 341.18 17.14 5.02 97.5

QC Conc (ug/mL)

Mean Cal. Conc


DSTYSLSSTLTLSK 0.35 * * * *

SILUMAB-DTL(IS) 3.50 3.36 0.17 5.06 96.0

35.00 37.60 1.50 3.99 107.4

350.00 381.10 8.99 2.36 108.9

* Outside of curve dynamic range

Excellent Accuracy and Precision

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiu1qOOouHKAhWIbj4KHQycBCkQjRwIBw&url=http://www.nature.com/nm/journal/v16/n10/fig_tab/nm1010-1107_F1.html&bvm=bv.113370389,d.cWw&psig=AFQjCNGbORP8nHqt2P6kmhxHvA3uUw9tQw&ust=1454783793311183


Flexibility : Across different mAbs & multiple plasma volumes

Protein Peptide Linear fit (r2) with 1/x

weighting Mean % Accuracy

Std. Curve Range Standard Curve Range 5.0-50.0 (µg/mL)

15 µL plasma

35 µL plasma

70 µL plasma

15 µL plasma

35 µL plasma

70 µL plasma

Infliximab SINSATHYAESVK 0.999 0.999 0.997 100.00 99.99 99.99

DILLTQSPAILSVSPGER 0.999 0.998 0.994 99.99 100.00 100.01

Trastuzumab

FTISADTSK 0.997 0.993 0.998 100.00 100.01 99.99

DTYIHWVR 0.995 0.995 0.996 100.00 100.02 100.02

IYPTNGYTR 0.998 0.996 0.991 99.98 99.98 98.79

Bevacizumab STAYLQMNSLR 0.999 0.998 0.995 100.00 100.01 99.99

FTFSLDTSK 0.999 0.999 0.993 100.02 100.00 100.00

Adalimumab APYTFGQGTK 0.994 0.997 0.995 99.99 99.99 99.99

NYLAWYQQKPGK 0.997 0.998 0.999 99.99 100.02 100.01

Linear, precise and accurate with multiple plasma volumes for multiple mAbs


Compound name: MIFAGIK

Correlation coefficient: r = 0.998906, r̂ 2 = 0.997814

Calibration curve: 7119.39 * x + -13715.7

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x̂2, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Res

pons

e

-0

200000

400000

600000

800000

1000000

1200000

1400000

Quantification of Small Proteins: Cytochrome C

MW 12,327

35 µL sample

10 minute digestion

MGDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFSYTDANKNK

GITWGEETLMEYLENPKKYIPGTKMIFAGIKKKGEREDLIAYLKKATNE

Peptide Std. Curve

Range (µg/mL)

Limit of Detection (µg/mL)

Weighting Linear fit

(r2)

Mean % Accuracy of all points

TGPNLHGLFGR 2.0-250 2.0 1/x2 0.996 100.01

MIFAGIK 2.0-250 0.5 1/x2 0.998 100.86

EDLIAYLK 2.0-250 0.5 1/x2 0.999 99.99

GITWGEETLMEYLENPKK 2.0-250 0.5 1/x2 0.999 100.00

QC mean accuracies between 90-108% with single digit % CV’s


Compound name: MIFAGIK

Correlation coefficient: r = 0.998906, r̂ 2 = 0.997814

Calibration curve: 7119.39 * x + -13715.7

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x̂2, Axis trans: None

Conc-0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Res

pons

e

-0

200000

400000

600000

800000

1000000

1200000

1400000

Quantification of Small Proteins: Cytochrome C

MW 12,327

35 µL sample

10 minute digestion

MGDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFSYTDANKNK

GITWGEETLMEYLENPKKYIPGTKMIFAGIKKKGEREDLIAYLKKATNE

Peptide Std. Curve

Range (µg/mL)

Limit of Detection (µg/mL)

Weighting Linear fit

(r2)

Mean % Accuracy of all points

TGPNLHGLFGR 2.0-250 2.0 1/x2 0.996 100.01

MIFAGIK 2.0-250 0.5 1/x2 0.998 100.86

EDLIAYLK 2.0-250 0.5 1/x2 0.999 99.99

GITWGEETLMEYLENPKK 2.0-250 0.5 1/x2 0.999 100.00

QC mean accuracies between 90-108% with single digit % CV’s


0%

20%

40%

60%

80%

100%

120%

140%

0 10 20 30 40 50 60

Pe

rce

nta

ge c

om

par

ed

to

15

min

Time (min)

Time course of Apo a1 peptides (1 mg/ml) in rat plasma (3-Direct kit)

AKPA 2

ATEH 3

DLAT 3

VQPY 4

LLDN 3

Quantification of Mid-Size Proteins: Apolipoprotein a1 MW 28,300

15 µL sample

Less than 30 minute digestion


Apo a1 peptide: Standard Curve and QC Statistics

Peptide Std. curve range

(mg/mL)

Weighting Linear fit (r2)

Mean % Accuracy of all

points

LLDNWDSVTSTFSK 10 - 1500 1/x2 0.988 99.99

Peptide Apo A1 QC conc.

(mg/mL) Mean Cal. Conc.

(mg/mL) Std. Dev. %CV

Mean %Accuracy

# of QCs passed

18 16.81 1.33 7.91% 93.40% 3 out of 3

LLDNWDSVTSTFSK 180 190.74 7.33 3.84% 105.97% 3 out of 3

1200 1322.72 64.64 4.89% 110.23% 3 out of 3

Excellent Accuracy and Precision


Waters Performance vs. Competitor


Higher Area Counts for Better Sensitivity: Unique Signature Peptides

ProteinWorks Outperforms the Competition Higher area counts across unique signature peptides of 4 different monoclonal antibodies


Higher Area Counts for Better Sensitivity: Generic Signature Peptides

0

25

50

75

100

125

Generic VVSVLTVLHQDWLNGK Generic GPSVFPLAPSSK Generic DSTYSLSSTLTLSK

Area

WatersWaters 3 and 5-Step vs. Competitor *

% Area Generic Signature Peptides

Waters 3-Step

Waters 5-Step

Competitor

* Normalized to Waters 3-Step 35 uL Plasma

ProteinWorks Outperforms the Competition Higher area counts across generic signature peptides of 4 different monoclonal antibodies


Improving Sensitivity and Specificity

Peptide Level Clean-Up


Matrix Effects at the Signature Peptide Level

1 nM trastuzumab in Solvent A

human serum digest

1 nM trastuzumab in serum digest

~1500 area counts

~500-700 area counts = 2-3X lower!

Addressing the problem with sample prep………


ProteinWorks µElution SPE Kit

Maximizing Instrument Up-Time, Recovery & Sensitivity

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


Peptide Level Clean-UP: Mixed-Mode SPE Protein A & Oasis MCX Incremental Improvement in Signal

Time 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

1: MRM of 2 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)

6.23e5 Area

7.09 3185


6.23e5 Area

7.10 14439


6.23e5 Area

7.09 27490

Direct plasma digestion: no clean-up

Protein A plasma clean-up

Protein A plasma clean-up, Oasis MCX digest clean-up


ProteinWorks Digest Kits

ADC Quantification


Trastuzumab Emtansine (T-DM1)

Bioconjugate Chemistry 2014, 25, 1223-1232

DM1 + MCC (Drug + Linker) 956.3644 Da

MCC (Linker) 219.0895 Da

Drug Maytansinoid

Trastuzumab based

90 lysines

SMCC linker

Two-step conjugation

Hydrophobic

Data courtesy of Liuxi Chen


Trypsin Asp N

Unconjugated peptide

Conjugated peptide

Quantitation Relative abundance of conjugated peptides

Relative site occupancy ratio

Lysine Conjugated ADC: Choice of digestion and tryptic peptides are critical

K

K

“miscleavage”

Slide courtesy of Liuxi Chen

If lysine is occupied by a small molecule drug (payload), there is potential for miscleavage during digestion if trypsin is used.


Peptide Std. curve range

(µg/mL)

Weighting Linear fit (r2)

Mean % Accuracy

of all points,

duplicate curves

IYPTNGYTR (T-DM1/trastuzumab Signature peptide)

0.25 - 500 1/x 0.998 100.00

GPSVFPLAPSSK (Generic mAb peptide)

0.1 - 500 1/x 0.998 100.00

FTISADTSK (Typical tratuzumab

Signature peptide, T-DM1 with no drug)

0.25 - 500 1/x 0.999 100.00

T-DM1 Summary Standard Curve Statistics: Direct Plasma Digest, 35 µL

All simple fit with excellent linearity (r2 0.99) and great accuracy (100%)


T-DM1 and Trastuzumab Quantification Using Trastuzumab standard curve: direct digest, 35 µL plasma

Peptide Trastuzumab

QCs conc. (mg/mL)

Mean Cal. Conc.


Mean %Accuracy

# of QCs passed

0.65 0.64 0.03 4.58% 99.77% 3 out of 3 3.5 3.25 0.19 5.96% 92.90% 3 out of 3

IYPTNGYTR 6.5 6.83 0.16 2.29% 105.13% 3 out of 3 35 36.41 0.42 1.16% 104.03% 3 out of 3

65 63.31 2.18 3.44% 97.40% 3 out of 3

350 345.64 18.66 5.40% 98.73% 3 out of 3

Peptide T-DM1

QCs conc. (mg/mL)

Mean Cal. Conc.


Mean %Accuracy

# of QCs passed

0.65 0.65 0.05 6.94% 100.50% 3 out of 3 3.5 3.36 0.24 7.10% 95.87% 3 out of 3

6.5 7.10 0.05 0.66% 109.20% 2 out of 3 IYPTNGYTR 35 34.51 1.09 3.17% 98.57% 3 out of 3

65 59.74 3.72 6.22% 91.90% 3 out of 3

350 324.72 17.06 5.25% 92.80% 3 out of 3

MRM transition: 542.77 > 808.40


Blank rat plasma digest, w/ IS, Direct 5 step, no SPE, 1

Time12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

0

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.21

14.60

12.21

13.2729006

13.3923826

Blank rat plasma digest, w/ IS, Direct 5 step, no SPE, 1

Time12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

3

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

3

12.87

12.09

12.88

12.10

14.082899

13.942489

ProteinWorks Performance: Detection and Confirmation of Conjugated T-DM1 peptides

Blank Rat Plasma

TDM-1 350.0 µg/mL

Trastuzumab 350.0 µg/mL

GPSVFPLAPSSKSTSGGTAALGCLVK Miscleavage peptide of T-DM1

MRM: 1149.22 > 547.20

FTISADTSKNTAYLQMNSLR Miscleavage peptide of T-DM1

MRM: 1073.17 > 547.20


ADC, QC, 350 ug/ml, Direct 5 step, no SPE, 2

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50

%

0

11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50

%

1

110415_WAA678_CD_070b Sm (SG, 2x3) MRM of 17 Channels ES+ 1073.167 > 485.22 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 2)

1.93e5Area

13.38;7282

110415_WAA678_CD_070b Sm (SG, 2x3) MRM of 17 Channels ES+ 1073.167 > 547.2 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 1)

5.82e5Area13.39;24966

13.27;9539

ProteinWorks Performance: Monitoring miscleaved peptide FTISADTSKNTAYLQMNSLR with the small molecule drug attached

Two MRM transitions confirm that the correct peptide is being monitored.

1073.167 > 485.22

1073.167 > 547.20

13.27;29901


For all of the peptides evaluated, ProteinWorks Direct Digest Kit, using a

3 and/or 5-Step protocol performance (using peak area) was better than

competitor

Waters vs. Competitor Kit ADC Performance: Signal Comparison Summary

ProteinWorks Outperforms the Competition Higher area counts across multiple herceptin tryptic signature peptides.


Blank rat plasma digest, Direct 5 step, no SPE, 2

Time12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

1

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

1

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

3

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

2

120815_WAA678_CD_009 Sm (SG, 2x3) MRM of 17 Channels ES+ 1073.167 > 547.2 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 1)

7.59e4

12.87

13.9113.7113.3213.07

13.57


1.56e5Area

13.406327


2.56e5Area

13.37;11950


6.49e5Area

13.35;30915

Blank rat plasma digest, Perfinity, no SPE, 2

Time12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

17

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

39

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

36

12.80 13.00 13.20 13.40 13.60 13.80 14.00

%

28


2.24e4Area


2.63e4Area


2.48e4Area


4.77e4Area

13.25442

Rat plasma blank

50 µg/mL

100 µg/mL

250 µg/mL

Waters vs. Competitor Kit Performance: Conjugated peptide FTISADTSKNTAYLQMNSLR with the small molecule drug attached

Competitor

ProteinWorks Direct Digest 5-Step

The signal is concentration dependent. Detection of conjugated peptides is questionable even at higher

concentrations with competitor kit.


Conclusions

Protein BA workflows are complex and laborious…but they don’t

have to be!

Waters ProteinWorks eXpress Digest kits provide standardized,

reproducible, and fully flexible solutions for accurate and precise

quantification of a diversity of proteins.

– One optimized protocol

– Accommodating different types of sample pre-treatment

– Range of plasma volumes

– Small and large proteins

o 10 min to 2 hr digestion times

– ADC (T-DM1) Quantification

o Detection of multiple conjugated peptides


Acknowledgements

Erin Chambers

Mary Lame

Hua Yang

Liuxi Chen

Jay Johnson

Henry Shion

Gregory Roman

James Murphy

Weibin Chen

John Gebler

Steven Calciano

Sherri Naughton

Catalin Doneanu

Documents

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