Fast, Reproducible Biopharmaceutical Peptide Mapping Profiling · Fast, Reproducible Biopharmaceutical Peptide Mapping Profiling. 2 In this presentation you will learn: 1. How to

The world leader in serving science

Global BioPharma Summit

Fast, Reproducible Biopharmaceutical Peptide Mapping Profiling

2

In this presentation you will learn:

1. How to digest biotherapeutic proteins

more efficiently and reproducibly

2. How to separate peptides reliably

with less variability

3. How a new MS platform allows you to

comprehensively map a biologic

4. How new software enables easy

correlation of both intact protein and

peptide mapping data

3

DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTN

GSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDI

ADYYCQQSHSWPFTFGSGTNLEVKGQPKANPTVTLFPPS

SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETT

KPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST

VEKTVAPTECS

Why Peptide Mapping?

DIGESTION

Regulatory

Analysis Data

4

Why Peptide Mapping?

DIGESTION

Regulatory

Analysis Data

5

Why Peptide Mapping?

DIGESTION

RegulatoryComparability Quantitation Lot Release

DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSP

RLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQ

SHSWPFTFGSGTNLEVKGQPKANPTVTLFPPSSEELQANKATLV

CLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSY

LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Identity & Purity

Analysis Data

6

Upgrade Your Maps: Our Workflow Solution

Thermo Scientific™ SMART Digest™offer extremely reproducible and rapid protein digestion

Thermo Scientific™ Vanquish™ Flex UHPLC is engineered for high resolution, reproducible peptide separations

Thermo Scientific™ Acclaim™ 120 C18 column is the perfect column choice to ensure sharp peaks during peptide mapping

Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometers are the gold standard for accurate mass measurement

Thermo Scientific™ BioPharmaFinder™ softwareis the perfect software tool for peptide identification and sequence mapping

BioPharma

Finder

7

Vanquish Flex UHPLC System

• Developed specifically for biopharma

• Fully biocompatible flow path

• Range of powerful detectors including patented

Thermo Scientific™ LightPipe™ Flow Cells

• Class leading retention time reproducibility

• Sample pre-compression for maximum peak

retention time reproducibility and column

lifetime.

• Ceramic valve technology

• New column thermostatting technology with two

modes of temperature control

• Support for latest innovations in column

technology

8

Vanquish Flex UHPLC System: Retention Time Reproducibility

2.5 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 31.3

-45.2

-37.5

-25.0

-12.5

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

112.5

125.0

137.5

150.0

162.5

175.0

187.5

201.5

mAU

min

Retention time repeatability of a peptide separation

Overlay of 13 consecutive chromatographic runs of a peptide sample separated on an analytical Acclaim™ RSLC 120

column and prepared from a mAb digested with SMART™ Digest Kit.

peak # RT (min) RSD (%)

3 3.315 0.082

9 5.231 0.065

14 6.532 0.017

15 6.937 0.023

19 10.290 0.021

23 12.013 0.012

31 14.011 0.013

39 15.177 0.012

42 15.589 0.010

51 17.511 0.007

55 17.969 0.011

61 18.546 0.010

83 20.798 0.010

85 21.095 0.012

87 22.386 0.009

96 24.774 0.012

103 26.155 0.009

106 26.155 0.009

109 27.529 0.010

Retention time

Repeatability

9

Vanquish Flex UHPLC System: Retention Time Reproducibility

2.5 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 31.3

-45.2

-37.5

-25.0

-12.5

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

112.5

125.0

137.5

150.0

162.5

175.0

187.5

201.5

mAU

min

Retention time repeatability of a peptide separation

Overlay of 13 consecutive chromatographic runs of a peptide sample separated on an analytical Acclaim™ RSLC 120

column and prepared from a mAb digested with SMART™ Digest Kit.

peak # RT (min) RSD (%)

3 3.315 0.082

9 5.231 0.065

14 6.532 0.017

15 6.937 0.023

19 10.290 0.021

23 12.013 0.012

31 14.011 0.013

39 15.177 0.012

42 15.589 0.010

51 17.511 0.007

55 17.969 0.011

61 18.546 0.010

83 20.798 0.010

85 21.095 0.012

87 22.386 0.009

96 24.774 0.012

103 26.155 0.009

106 26.155 0.009

109 27.529 0.010

Retention time

Repeatability

10

Sample Analysis By Mass Spectrometry

Q Exactive Plus

Full MS scan methodm/z 200-2000

ddTopN Method: Full MS with dd MS2 scans

Time (min)0 2 4 6 8 10 12 14 16 18

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nce

8.42419.76

10.73714.02

10.77559.94

14.51603.672.70

269.067.99

625.984.50

360.20 5.60280.66

2.22448.28

15.10899.45

4.00435.18

6.58536.28

14.02712.121.58

447.2616.41

1344.2718.72

926.51

0 2 4 6 8 10 12 14 16 18Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nce

8.42419.76

10.73714.02

10.77559.94

14.51603.67

8.41419.76

2.70269.06 11.43

593.837.99625.98 14.57

603.672.72269.06

4.50360.20

11.44593.83

5.60280.66

2.22448.28

15.12899.454.49

360.20 6.58536.28

14.02712.121.58

447.26 15.15899.45 16.49

1344.27

2.70160.04

10.75472.28

10.72472.28

10.76339.173.15

355.108.41

486.29

Black: Full MS scans

Red: MS2 scans

Base Peak Chromatograms

11

Sample Analysis By Mass Spectrometry

Q Exactive Plus

Full MS scan methodm/z 200-2000

ddTopN Method: Full MS with dd MS2 scans

Time (min)0 2 4 6 8 10 12 14 16 18

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nce

8.42419.76

10.73714.02

10.77559.94

14.51603.672.70

269.067.99

625.984.50

360.20 5.60280.66

2.22448.28

15.10899.45

4.00435.18

6.58536.28

14.02712.121.58

447.2616.41

1344.2718.72

926.51

0 2 4 6 8 10 12 14 16 18Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nce

8.42419.76

10.73714.02

10.77559.94

14.51603.67

8.41419.76

2.70269.06 11.43

593.837.99625.98 14.57

603.672.72269.06

4.50360.20

11.44593.83

5.60280.66

2.22448.28

15.12899.454.49

360.20 6.58536.28

14.02712.121.58

447.26 15.15899.45 16.49

1344.27

2.70160.04

10.75472.28

10.72472.28

10.76339.173.15

355.108.41

486.29

Black: Full MS scans

Red: MS2 scans

Base Peak Chromatograms ddTopN Method with N=5 here: MS/MS data-dependent analysis of 5 most abundant peptide ions (red) based on precursors obtained in a full scan (black)

7.985 7.990Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nce

7.99

7.98

7.98

7.99

7.997.997.99

zoom

12

Rituximab Analysis With Reducing Gradient

30 min gradient

0.4 mL min-1

600 bar

20 min gradient

0.4 mL min-1

600 bar

13 min gradient

0.6 mL min-1

850 bar

8 min gradient

1.0 mL min-1

1150 bar

5 min gradient

1.1 mL min-1

1300 bar

Standard Method

Acclaim 120 C18 column, 2.1 x 250 mm, 2 - 45%

ACN + 0.1% FA, 60 °C

5 10 15 20 25 Time [min]

14.64

20.2310.592.73

10.78

14.52

7.992.70

6.97 9.43

5.20

1.86

4.456.02

3.351.13

3.12

4.122.43

1.08

Sequence

Coverage

100%

100%

100%

100%

100%

13

BioPharm Finder: Protein Deconvolution And Peptide Mapping

• Single software package for:

• Intact and Native Mass Analysis

• Sub-Unit Mass Analysis

• Peptide Mapping

• Protein Deconvolution of isotopically resolved and unresolved spectra

• Peptide mapping of biotherapeutics & other recombinant proteins

• Automated DAR calculations

• Comparison of biosimilars

• Supports all Orbitrap & ion-trap-based instruments

14

• Lengthy multi-step protocols

• Process-induced PTMs

• Reproducibility

• Throughput/speed

• Method development ease

What’s the Problem with Protein Digestion?

15

The Fundamental Five…or is that Two?

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

16

The Fundamental Five…or is that Two?

Denaturation

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

17

The Fundamental Five…or is that Two?

Denaturation

Reduction

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

18

The Fundamental Five…or is that Two?

Denaturation

Reduction

Alkylation

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

19

The Fundamental Five…or is that Two?

Denaturation

Reduction

Alkylation

Digestion

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

20

The Fundamental Five…or is that Two?

Denaturation

Reduction

Alkylation

Digestion

Clean up

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

21

The Fundamental Five…or is that Two?

Digestion

Clean upDenaturation

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

22

The Fundamental Five…or is that Two?

Digestion

Clean upDenaturation

Primary structure

Secondary structure (H bonding)

Tertiary structure (disulphide bonding)

Alkylating agent

Enzyme

23

Thermo Scientific SMART Digest Kits

24

Thermo Scientific SMART Digest Kits

25

Thermo Scientific SMART Digest Kits

Denaturation

26

Thermo Scientific SMART Digest Kits

Denaturation

Digestion

27

Thermo Scientific SMART Digest Kits

Denaturation

Digestion

28

Thermo Scientific SMART Digest Kits

DenaturationClean up

Digestion

29

Thermo Scientific SMART Digest Kits

DenaturationClean up

Digestion

30

Thermo Scientific SMART Digest Kits

DenaturationClean up

Digestion

31

The Science

of SMART

32

Let’s Immobilize Trypsin…and Make it Heat Stable!

Simultaneously denature using

heat

Low autolysis & wrong

cleavages

Increase thermal

stability & solubility

More Enzyme

Denaturation

33

• Save time

• Reagent prep/denaturation,

reduction/alkylation

• Simplify

• Only 2 steps

• Increase robustness

• Stable, reproducible tryptic

activity

• Untrained users

• Easy method transfer

Let’s Immobilize Trypsin…and Make it Heat Stable!

Simultaneously denature using

heat

Low autolysis & wrong

cleavages

Increase thermal

stability & solubility

More Enzyme

Denaturation

34

Optimize the protein for Heat Denaturation!

Thermal denaturation of IgG

Protein

Denaturation

35

Optimize the protein for Heat Denaturation!

Thermal denaturation of IgG

Protein

Denaturation

36

Optimize the protein for Heat Denaturation!

Thermal denaturation of IgG

0 50 100

Re

sp

on

se

(P

eak

Are

a)

Digest Time (minutes)

Native IgG Digest Profile

monitoring VVSVLTVLHQDWLNGK

50°C

60°C

70°C

Protein

Denaturation

37

Optimize the protein for Heat Denaturation!

Thermal denaturation of IgG

0 50 100

Re

sp

on

se

(P

eak

Are

a)

Digest Time (minutes)

Native IgG Digest Profile

monitoring VVSVLTVLHQDWLNGK

50°C

60°C

70°C

Protein

Denaturation

38

3 Reasons to change to SMART digestion

Fast & flexible

method development

39

3 Reasons to change to SMART digestion

Fast & flexible

method development5.0 10.0 15.0 20.0 25.0 30.0

0

25

50

75

100

125

150

175

min

mAU

200

n=3

Standardized reproducibility

RSDs: RT 0.024 %; Peak Area 2.82 %

(n=5 users, based on 15 peaks)

40

3 Reasons to change to SMART digestion

Reduced complexity

Fast & flexible

method development5.0 10.0 15.0 20.0 25.0 30.0

0

25

50

75

100

125

150

175

min

mAU

200

n=3

Standardized reproducibility

RSDs: RT 0.024 %; Peak Area 2.82 %

(n=5 users, based on 15 peaks)

41

3 Reasons to change to SMART digestion

Reduced complexity

Fast & flexible

method development5.0 10.0 15.0 20.0 25.0 30.0

0

25

50

75

100

125

150

175

min

mAU

200

n=3

Standardized reproducibility

RSDs: RT 0.024 %; Peak Area 2.82 %

(n=5 users, based on 15 peaks)

Automation!

42

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

43

Trypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

44

F1

F1Trypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

45

F1 F2

F2 F1Trypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

46

F1 F2

F2 F1Trypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

47

F1 F2

F2 F1Trypsin

Chymotrypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

48

F1 F2

F3

F2 F1

F3

Trypsin

Chymotrypsin

SMART Digestion (Trypsin or Chymotrypsin) and HR-LC/MS/MS of the modified peptide

49

Reproducibility

and Precision!

You have the theory now for the real data!

50

51

52

53

Sequence Coverage Map from Infliximab using the Magnetic SMART Digest

54

Sequence Coverage Map from Infliximab using the Magnetic SMART Digest

The world leader in serving science

FPTIPLSRLF DNAMLRAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK

SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEEG IQTLMGRLED GSPRTGQIFK

QTYSKFDTNS HNDDALLKNY GLLYCFRKDM DKVETFLRIV QCRSVEGSCG F

Somatotropin Sequence

Model Therapeutic protein

56

57

58

Proteins Number of MS Peaks MS Peak Area Sequence Coverage Abundance (mol)

Somatotropin 457 90.9% 100.0% 100.00%

Unidentified 14 9.1%

How to Achieve 100% Sequence Coverage

59

Proteins Number of MS Peaks MS Peak Area Sequence Coverage Abundance (mol)

Somatotropin 205 89.6% 100.0% 100.00%

Unidentified 4 10.4%

MS Only Data Acquisition

60

Position Sequence Retention time

[Mod/Native]

Modification %

5 min

%

15 min

N149 FDTNSHNDDALLK 9.43 / 8.68 Deamidation 1.05 1.64

M170 DMDKVETFLR 9.58 / 10.31 Oxidation 0.59 0.60

M14 LFDNAMLR 10.41 / 12.28 Oxidation 1.52 1.34

M125 DLEEGIQTLMGR 12.40 / 15.48 Oxidation 0.18 0.21

D130 LEDGSPR 3.95 / 4.06 Isomerization 0.18 0.55

Chemical Modifications Present in Proteins

61

Position Sequence Retention time

[Mod/Native]

Modification %

5 min

%

15 min

N149 FDTNSHNDDALLK 9.43 / 8.68 Deamidation 1.05 1.64

M170 DMDKVETFLR 9.58 / 10.31 Oxidation 0.59 0.60

M14 LFDNAMLR 10.41 / 12.28 Oxidation 1.52 1.34

M125 DLEEGIQTLMGR 12.40 / 15.48 Oxidation 0.18 0.21

D130 LEDGSPR 3.95 / 4.06 Isomerization 0.18 0.55

Chemical Modifications Present in Proteins

62

Position Sequence Retention time

[Mod/Native]

Modification %

5 min

%

15 min

N149 FDTNSHNDDALLK 9.43 / 8.68 Deamidation 1.05 1.64

M170 DMDKVETFLR 9.58 / 10.31 Oxidation 0.59 0.60

M14 LFDNAMLR 10.41 / 12.28 Oxidation 1.52 1.34

M125 DLEEGIQTLMGR 12.40 / 15.48 Oxidation 0.18 0.21

D130 LEDGSPR 3.95 / 4.06 Isomerization 0.18 0.55

Chemical Modifications Present in Proteins

63

RT: 0.00 - 60.00

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

25.9920.752.12

23.1733.83

19.32

26.7418.64

16.5727.00

38.57

39.11

39.20

14.24

2.3731.74

13.53 36.553.79

12.774.20

8.488.40

29.92 39.638.72 51.17

48.516.25 9.22 40.72 48.03 52.50 59.3955.68

NL:2.18E8

Base Peak F: FTMS + p ESI Full ms [300.00-2000.00] MS herceptin_pep

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW

VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA

DTSKNTAYLQMNSGTQTYICNVNHKPSNTKVDKKVE

PPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

• Peptide Map• PTMs• Impurities

Sequence/PTMs unknown or needs

confirming

Peptide identification by MS and MS/MS

Fast analysis

Peptide Mapping of Therapeutic Monoclonal Antibodies

64

RT: 0.00 - 60.00

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

25.9920.752.12

23.1733.83

19.32

26.7418.64

16.5727.00

38.57

39.11

39.20

14.24

2.3731.74

13.53 36.553.79

12.774.20

8.488.40

29.92 39.638.72 51.17

48.516.25 9.22 40.72 48.03 52.50 59.3955.68

NL:2.18E8

Base Peak F: FTMS + p ESI Full ms [300.00-2000.00] MS herceptin_pep

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW

VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA

DTSKNTAYLQMNSGTQTYICNVNHKPSNTKVDKKVE

PPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

• Peptide Map• PTMs• Impurities

Sequence and PTMs known, no further information

required

Stability studies, QA/QC, batch release)

Peptide identification by unknown and reference sample chromatogram

comparison

High degree of confidence on retention

time determination is required!

Sequence/PTMs unknown or needs

confirming

Peptide identification by MS and MS/MS

Fast analysis

Method Transfer to LC-UV

Peptide Mapping of Therapeutic Monoclonal Antibodies

65

Overview of the Peptide Mapping Workflow

60 min 20 min 10 min

Combining all the components of this Peptide Mapping work flow a complete

analysis can be done in less than 90 minutes.

Can be set up and verified by MS very quickly.

A relatively inexperienced analyst can obtain reproducible results from UV with

simple sample preparation and walk up UHPLC analysis.

66

Our Workflow Solution Gives You….

Biopharma

Finder

Hugely better reproducibility and

robustness!

Massive time and labour savings!

Better sequence coverage!

More integrated software!

67

Our Workflow Solution Gives You….

Biopharma

Finder

Hugely better reproducibility and

robustness!

Massive time and labour savings!

Better sequence coverage!

More integrated software!

*Unique automated Digestion

*Class leading chromatography and

UHPLC

*Highest resolution, highest sensitivity

Orbitrap MS systems

*Integrated Industry specific Software

68

Summary

• Game changing automation of protein

digestion for ultimate precision and

reproducibility

• Robust and highest reproducibility UHPLC

system for peptide mapping

• Most sensitive and highest resolution mass

spectrometer with the Q Exactive

• Industry specific BioPharma Finder

software

• Complete workflow enables ease of use

and time/cost savings

• Easy method transfer and reduced need

for highly skilled operators