Critical Quality Attribute Assessment by Peptide Mapping ... · Critical Quality Attribute...

Critical Quality Attribute Assessment by Peptide Mapping Using LC/MS with Superficially Porous Columns

1

For Research Use Only. Not for use in diagnostic procedures.

Analytical groups are tasked with method development, sample analysis, and method transfer

Centrifuge

Biomarker ID & validation

DNA Vector

HT Clone Screening &

Selection

mAb Generation Platform e.g. transgenic mice

Cell Culture Scale-up & Production

Human gene

Establishment of genetically engineered cells (e.g. CHO) that

produce desired product

Analytical GroupsDevelop analytical methods; analyze samples from other functions, methods transfer

QA/QC

SamplesSamples Samples Samples

SamplesSamples

Methods

Market Manufacturing Pre-clinicalClinical ChromatographyColumn #1

ChromatographyColumn #2

ChromatographyColumn #3

2


Pyro-Glutamate

Deamidation/Oxidation

Fragmentation(Hinge)

Glycosylation(G0, G1, G2)

Truncation(Lys 0, 1, 2)

DisulfideShuffling

Aggregation

HILIC

IEC

IEC

IEC

RP

SEC

RP

LC/MSIntact massFragment massIdentificationPTM ID & locationsGlycan ID

Critical Quality Attributes & Testing Methods

Not just used for monoclonal antibodies – same techniques are used for any potentially therapeutic protein

3


Peptide Mapping Workflow Solutions to Accelerate Biomolecule Characterization

4

�Delivery of Results

Reporting

Sequence MatchingDetermine Post Translational

Modifications

Data Acquisition

Sample Preparation

Chromatographic Separation

Complete Agilent Workflow Solution

B(6

7-7

3)

B ( 6 0 - 6 6 )

A(1

03

-10

6)

B(3

30

-33

7)

B(2

78

-29

1)

Peptide mapping chromatogram Peptide ID by MS/MS


Sample Preparation

Denaturation, Reduction, &

Alkylation

Digestion

Enrichment & Cleanup

Sample Preparation for Peptide MappingDepletion of matrix proteins, enrichment of target protein,

dialysis or desalting

Reduce disulfide bonds, and block to prevent re-formation

Trypsin is the most common digestion enzyme, but others may

be called for depending on the protein and experiment.

Sample concentration, enrichment for specific PTMs, or desalting

A time consuming, labor-intensive process to do manually, with multiple steps that can

be optimized.

5


AssayMAP Bravo – Sample Prep Automation

6

Peptide mapping

Detect


AssayMAP Sample Prep Portfolio

FractionationAntibody

Purification

Immunoaffinity

Purification

Phosphopeptide

EnrichmentN-glycan

Sample Prep

Protein

Digestion

Peptide Clean

Up

Protein Clean

Up

7


General Considerations for Peptide Mapping Column Selection

Instrument capabilities and requirements- UV vs MS detection or both- Pressure capabilities

Mobile phase requirements- High pH required for sample- TFA vs Formic acid

Sample- Hydrophilic vs hydrophobic peptides- Larger polypeptides present

Column dimensions- Generally prefer longer columns, especially for more complicated maps

- 50, 150, and 250 mm lengths available

- Use 2.1 mm i.d. for MS-sensitivity- 3.0 and 4.6 mm i.d. also available

- Smaller pore sizes ideal, usually 100-150 Å

8


Detectors for Peptide Mapping

UV

• Often used in routine process measurements, QA/QC labs

• No peptide mass or structural information; relies on reproducibility of RT, peak area, and peak area ratios

• Traditionally uses TFA modifier

MS

• Often used in discovery/early development stage, R&D labs

• MW and structural information (MS/MS) to assist with co-elution challenges, verify sequence coverage, and to ID unknown peaks when they appear

• Generally prefer formic acid modifier to avoid ion suppression from TFA

9


Mobile Phases for Peptide Mapping

Ion pairing agents used for RP LC

• Increase retention of small, poorly retained peptides• Block exposed silanol sites on silica-based columns• Acid helps solubilize and ionize peptide and protein samples

TFA works well on “traditional” silica-

based columns, but formic acid does not

• Formic acid produces broad, asymmetric peaks• Poor resolution and poor peak capacity

Need column solution for formic acid

modifier

• AdvanceBio Peptide Plus• Charged surface chemistry solves the

problems associated with formic acid

10


Agilent Bio-LC Column PortfolioAgilent Bio-LC Columns

Affinity

Bio-Monolith Protein A

Bio-Monolith Protein G

Multiple Affinity Removal System

Reversed Phase

AdvanceBio Peptide Plus

AdvanceBio Peptide Mapping

AdvanceBio RP mAb

AdvanceBio

Oligonucleotides

AdvanceBio Desalting-RP

PLRP-S

ZORBAX RRHD 300Å, 1.8 μm

AdvanceBio Amino Acid Analysis

ZORBAX Amino Acid Analysis

ZORBAX 300SB

Poroshell 300

HILIC

AdvanceBio Glycan Mapping

ZORBAX RRHD 300-HILIC

Size Exclusion

AdvanceBio SEC

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF-250

ZORBAX GF-450

Ion Exchange

Bio mAb

Bio IEX (SAX, SCX, WAX, WCX)

PL SAX

PL SCX

Bio-Monolith (QA, DEAE, SO3)

11


Agilent Bio-LC Column Portfolio

Agilent Bio-LC Reverse Phase Columns for Protein Analysis

Intact Proteins (& Fragments)

PLRP-S, 5 µm, 1000 Å

AdvanceBio RP mAb

ZORBAX RRHD 1.8 µm, 300 Å

ZORBAX 300SB

Poroshell 300

AdvanceBio Desalting-RP

Peptides


AdvanceBio Peptide Mapping

ZORBAX Eclipse Plus C18

ZORBAX 300SB*

Amino Acids

AdvanceBio Amino Acid Analysis

ZORBAX Amino Acid Analysis

12

*unusually large pore size for peptide mapping, but some people really like it nonetheless



Poroshell particleUHPLC performance at lower pressure

Unique charge hybrid/C18 bonded phase Improved peak shape and peak capacity for peptide

analysis with formic acid and MS detection

To provide AdvanceBio Peptide Plus that gives benefits in accuracy and reproducibility of results

for characterization of mAb identity, PTMs

13


Columns Individually Tested for Efficiency

14

AdvanceBio Peptide Plus Column


Column Lots QA Tested Specifically for Peptide Separations

15

Peptides Standard

(p/n G2455-85001) AdvanceBio Peptide Plus Column For Research Use Only. Not for use in diagnostic procedures.

LC/MS Instrumentation

16

• 1290 Infinity II LC system

• 6545XT AdvanceBio LC/Q-TOF

• BioConfirm B.08 for intact protein

and peptide mapping data analysis


6545XT Features for Peptide Mapping

• Sensitive peptide detection featuring Agilent Jet Stream

• Quick-start peptide mapping method

• Access low intensity peptides/PTMs with the new IterativeMS/MS mode

• Sub-ppm mass accuracy with 50k resolution from improved beam optics

• Peptide fragmentation performance verification at install

• Ease of maintenance with vent-free capillary removal



Method Conditions

18

Parameter 1290 Infinity II LC

Column AdvanceBio Peptide Plus 2.1 × 150mm, 2.7µm, 120Å

Mobile phase A: 0.1%FAB: 0.1% FA in ACN

Gradient

Time (min) %B0 31 331 4033 9534 9534.1 3

Post time 10 minInj vol 1 µLSample thermostat 5 °CPost time 5 minColumn temp 55 °CFlow rate 0.5 ml/minSample trastuzumab digest (1ug/ul)

Parameter 6545XT AdvanceBio LC/Q-TOF

Ion mode Positive ion mode, dual AJS ESI (profile)

Drying gas temp 325 °C

Drying gas flow 13 L/min

Sheath gas temp 275 °C

Sheath gas flow 12 L/min

Nebulizer 35 psi

Capillary voltage 4000 V

Fragmentor voltage 175 V

Skimmer voltage 65 V

Oct RF Vpp 750 V

Acquisition parameters Data were acquired at 2 GHz Extended Dynamic

Range

MS mass range 100–1700 m/z

MS/MS mass range 50 – 1700

MS scan rate (spectra/second): 8

MS/MS scan rate (spectra/second): 3

Ramped collision energy

Charge state Slope Offset

2 3.1 1

3 and > 3 3.6 –4.8

Data analysis BioConfirm software B.08.08

All results shown are with formic acid mobile phase


Effect of Flow Rate on Peak Capacity


19

TIC

• Increasing the flow rate from 0.1 mL/min to0.5 mL/min leads to an increase of twice thepeak capacity

8x10

0

0.5

1

1.5

8x10

0

0.5

1

8x10

0

0.5

1

8x10

0

0.5

1

8x10

0

0.5

1

1.5

8x10

0

0.5

1

1.5

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

0.1 ml/min

0.2 ml/min

0.3 ml/min

0.4 ml/min

0.5 ml/min

0.6 ml/min


1ug trastuzumab digest

Effect of Gradient Length on Peak Capacity


20

• Increasing the gradient time from 0 min to 60 minleads to an increase 2.5 times peak capacity,

• After 30min, no significant increase in peak capacity

TIC

8x10

0

0.5

1

1.5

8x10

0

0.5

1

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

8x10

0

0.4

0.8

1.2

Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

10 min

20 min

30 min

40 min

50 min

60 min



Effect of Gradient Slope on Peak Capacity


21

8888x10x10x10x10

0.50.50.50.5

1111

1111 2222 3333 4444 5555 6666 7777 8888 9999 10101010 11111111 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 20202020 21212121 22222222 23232323 24242424 25252525 26262626 27272727 28282828 29292929 30303030

8888x10x10x10x10

1111

Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)

2222 4444 6666 8888 10101010 12121212 14141414 16161616 18181818 20202020 22222222 24242424 26262626 28282828 30303030 32323232 34343434 36363636 38383838 40404040 42424242 44444444 46464646 48484848 50505050 52525252 54545454 56565656 58585858 60606060

0.50.50.50.5

0-40% in 30min

Slope 1.3%/min

0-40% in 60min

Slope 0.6%/min

• Higher efficiency → sharper peaks• Increased sensitivity• Peak capacity = 402• Sequence coverage = 96.69%

• Improved resolution• Peak capacity = 590• Sequence coverage = 94.59%

TIC



Effect of Temperature on Peak Capacity


22

LC/MS Chromatogram

• Higher temperature produces narrower peaks, improving resolution• Change in temperature changes selectivity • ~20% more peaks resolved by increasing the column temperature to 550C

6666x10x10x10x10

0000

1111

2222

6666x10x10x10x10

0000

1111

2222

3333

Counts vs. Acquisition Time (min)Counts vs. Acquisition Time (min)Counts vs. Acquisition Time (min)Counts vs. Acquisition Time (min)

1111 2222 3333 4444 5555 6666 7777 8888 9999 10101010 11111111 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 20202020 21212121 22222222 23232323 24242424 25252525 26262626 27272727 28282828 29292929 30303030 31313131 32323232 33333333 34343434 35353535 36363636 37373737 38383838 39393939 40404040

45oC

55oC

Pc = 504

Pc = 600



Back Pressure Characterization


23

Elevated temp produces narrower peak bands and lower pressure

Temperature

*pressure reading observed (max)


2.1 x 150 mm

600 bar pressure maximum

Good Peak Shape & Retention Time Stability with High Mass Load

24

Sample: 3-peptide mixture in 0.1%FA

• Bradykinin

• Angiotensin II

• Venin substrate

All 2.1 x 50 mm columns

Key for transferring methods between LC/MS and LC/UV


AdvanceBio Peptide Plus Competitor 1

Competitor 2 Competitor 3

Highly Reproducible Separations

25

n = 10 %RSD

Peak no. RT FWHM Area Height

1 0.20 1.13 0.31 1.36

2 0.06 1.57 5.76 2.54

3 0.06 1.55 4.27 1.39

4 0.06 1.02 4.60 1.23

5 0.07 0.91 3.41 1.53

6 0.05 0.86 2.69 0.74

7 0.06 3.22 5.79 3.33

8 0.04 0.99 3.81 1.12

9 0.02 0.92 2.66 1.31

10 0.02 0.95 4.22 1.80

8888x10x10x10x10

0000

1111

2222

3333

4444

5555


1111 2222 3333 4444 5555 6666 7777 8888 9999 10101010 11111111 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 20202020 21212121 22222222 23232323 24242424 25252525

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Hydrophilic Hydrophobic

• Consistent chromatography

• RSD values demonstrate the robustness of the methodand precision of the LC system

• Especially crucial for LC/UV peptide mapping

1

2

3

45

6

7

8

9

10



Lifetime Testing

26

Plates dropped < 10%

Rt dropped < 10%

Retention time %

Plates count %

• The column has subjected to 1000injections and lost no more than 10%of the initial plate and retention valueat the end of the test

• Good chemical stability under formicacid conditions

Column: AdvanceBio Peptide PlusSample: 10 peptide standard (5190-0583)Injection: 3 µLFlow rate: 0.5 mL/minMobile phase: A – 0.1%FA

B – 0.1% FA in ACN

% P

late

co

un

t


8888x 10x 10x 10x 10

0000

0 . 50 . 50 . 50 . 5

1111

1 . 51 . 51 . 51 . 5

2222

2 . 52 . 52 . 52 . 5

3333

3 . 53 . 53 . 53 . 5

4444

4 . 54 . 54 . 54 . 5

5555

5 . 55 . 55 . 55 . 5

6666

8888x 10x 10x 10x 10

0000

0 . 50 . 50 . 50 . 5

1111

1 . 51 . 51 . 51 . 5

2222

2 . 52 . 52 . 52 . 5

3333

3 . 53 . 53 . 53 . 5

4444

4 . 54 . 54 . 54 . 5

5555

5 . 55 . 55 . 55 . 5

6666

C ounts v s. Acquisition Time (min)C ounts v s. Acquisition Time (min)C ounts v s. Acquisition Time (min)C ounts v s. Acquisition Time (min)

1111 2222 3333 4444 5555 6666 7777 8888 9999 10101010 11111111 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 20202020 21212121 22222222 23232323 24242424 25252525 26262626 27272727 28282828 29292929 30303030 31313131 32323232

12

35

4

67

8

9 10AdvanceBio Peptide Mapping


1

2

3 5

4

67 8

9

10

Column Options for Alternate Selectivity

27

10 Peptide Standard (p/n 5190-0583)

TIC

6. Renin substrate porcine 7. [Ace-F-3,-2 H-1] Angiotensinogen (1-14)8. Ser/Thr Protein Phosphatase (15- 31) 9. [F14] Ser/Thr Protein Phosphatase (15-31) 10. Melittin (honey bee venom)

1. Bradykinin frag 1-7 2. Bradykinin 3. Angiotensin II (human) 4. Neurotensin5. Angiotensin I (human)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

FW

HM

Peptide peak

AdvBio Peptide Mapping

AdvBio Peptide Plus

Be

tte

r


mAb Peptide Mapping

28

LC/MS of mAb tryptic digest on 2.1 x 150mm AdvanceBio Peptide Plus Column

Hydrophilic peptide retention

Narrow Peaks with baseline resolution

Hydrophobic peptide elution

Reduced and fast analysis time

Critical and desired peptide mapping

components to achieve fast, selective,

and highly efficient peptide separations

across a wide dynamic range.

8888x10x10x10x10

0000

1111

2222

3333

4444

5555


1111 2222 3333 4444 5555 6666 7777 8888 9999 10101010 11111111 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 20202020 21212121 22222222 23232323 24242424 25252525

Co

un

ts

Co

un

ts

Co

un

ts

Co

un

ts

Hydrophilic Hydrophobic


Tryptic Peptides of Trastuzumab


29

LC/MS Peptide Map

30

sequence coverage 99.25%mass accuracy of 5 ppm



Sample Prep Separation Detection Data Processing & Report

MassHunter BioConfirm for Peptide Mapping


Separation of Oxidized Peptides

32

5555x10x10x10x10

0000

1111

2222

3333

4444

5555

6666

8888


4444 5555 6666 7777 8888

7777

Co

un

ts

Co

un

ts

Co

un

ts

Co

un

ts

Oxidized peptide

DLTMISR peptide

Relative % of Oxidation = 1.69%EIC

2x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

5.5

5.75

6

6.25

6.5

6.75

7

7.25

7.5

7.75

8

TTTT74.060474.060474.060474.0604

L,IL,IL,IL,I86.096586.096586.096586.0965

100.8496100.8496100.8496100.8496126.0552126.0552126.0552126.0552

144.0666144.0666144.0666144.0666

171.0775171.0775171.0775171.0775

175.1200175.1200175.1200175.1200

189.0863189.0863189.0863189.0863199.0725199.0725199.0725199.0725

bbbb2222217.0827217.0827217.0827217.0827

229.0918229.0918229.0918229.0918

245.1374245.1374245.1374245.1374

yyyy2222262.1507262.1507262.1507262.1507

bbbb3333----H2OH2OH2OH2O

312.1557312.1557312.1557312.1557

bbbb3333330.1666330.1666330.1666330.1666

yyyy3333375.2340375.2340375.2340375.2340

409.7209409.7209409.7209409.7209

443.1929443.1929443.1929443.1929

yyyy4444506.2769506.2769506.2769506.2769

575.3696575.3696575.3696575.3696602.3394602.3394602.3394602.3394

yyyy5555619.3600619.3600619.3600619.3600

619.6263619.6263619.6263619.6263

Counts v s. M ass-to-Charge (m/z)

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

bbbb 4444----H2OH2OH2OH2Oyyyy 2222 ----NH3NH3NH3NH3

yyyy1111

RRRR

3x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

L,IL,IL,IL,I

86.096086.096086.096086.0960

88.037788.037788.037788.0377

111.0555111.0555111.0555111.0555

144.0642144.0642144.0642144.0642

yyyy1111----NH3NH3NH3NH3

158.0920158.0920158.0920158.0920

175.1176175.1176175.1176175.1176

189.0875189.0875189.0875189.0875


199.0721199.0721199.0721199.0721

bbbb2222

217.0824217.0824217.0824217.0824

229.6381229.6381229.6381229.6381

yyyy2222262.1503262.1503262.1503262.1503

286.1798286.1798286.1798286.1798


312.1563312.1563312.1563312.1563

330.1614330.1614330.1614330.1614

351.6818351.6818351.6818351.6818

yyyy3333375.2351375.2351375.2351375.2351

394.2188394.2188394.2188394.2188

417.2133417.2133417.2133417.2133

426.2170426.2170426.2170426.2170

458.2711458.2711458.2711458.2711

478.2066478.2066478.2066478.2066502.2897502.2897502.2897502.2897

yyyy4444

522.2694522.2694522.2694522.2694

571.3618571.3618571.3618571.3618

yyyy5555635.3547635.3547635.3547635.3547

Counts vs. Mass-to-Charge (m/ z)

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

DLTMISR

MS/MS spectra

Increase in mass (16Da)

Unmodified peptide

Unmodified peptide

Oxidized peptide

DLTMISR



Separation of Deamidated Peptides

33

ASQDVNTAVAWYQQKPGK peptideNTAYLQMNSLR peptide

Relative % of Deamidation = 0.21% – 15.9%Relative % of Deamidation = 9.1%

EIC

NTAYLQMNSLR 15.9%

NTAYLQMNSLR 5.5%

NTAYLQMNSLR 0.23%

NTAYLQMNSLR 0.97%

NTAYLQMNSLR 2.5%

NTAYLQMNSLR 0.24%

NTAYLQMNSLR 0.21%

NTAYLQMNSLR

Deamidated peptides

non-deamidated

peptide

NTAYLQMNSLR

Deamidated peptide

Non-deamidated peptide

Relative % deamidation = 9.1%

ASQDVNTAVAWYQQKPGK



Pyro-Glutamate

Deamidation/Oxidation

Fragmentation(Hinge)

Glycosylation(G0, G1, G2)

Truncation(Lys 0, 1, 2)

DisulfideShuffling

Aggregation

HILIC

IEC

IEC

IEC

RP

SEC

RP

LC/MSIntact massFragment massIdentificationPTM ID & locationsGlycan ID

Critical Quality Attributes & Testing Methods

Not just used for monoclonal antibodies – same techniques are used for any potentially therapeutic protein

With a Multi-Attribute Method, peptide mapping can monitor many of these CQAs simultaneously.

34


Multi-Attribute Method (MAM)

35


Traditional Silica Column AdvanceBio Peptide Plus

• Determination of deamidated peptides is an important goal of peptide mapping. Chromatographic resolution is important. MS/MS confirms which peak corresponds to which deamidation site.

• AdvanceBio Peptide Plus enhances analysis because it has higher selectivity for deamidated forms of peptide over the normal form.

Separation of Deamidated Peptides

VVSVLTVLHQDWLnGK or VVSVLTVLHqDWLNGKm/z 904.9984, doubly charged peptide

Traditional silica columns don’t resolve deamidationas well as charged surface columns.


Choosing a Column Chemistry

For LC/MS work, AdvanceBio Peptide Plus with formic acid is the first choice.

Best peak shape with formic acid, high peak capacity, high mass load tolerance, excellent PTM resolution (especially deamidation), peak shape and recovery of hydrophobic peptides, unique selectivity

Charged surface chemistry


“Regular” silica chemistry

AdvanceBio Peptide MappingEclipse Plus RRHDZORBAX 300

vs

37


Exception 1: Sample is especially rich in small, hydrophilic peptides.

Exception 2: Different selectivity is needed to separate a key pair.

Poor retention of small, hydrophilic peptides is a feature common to charged surface columns. In this case, try AdvanceBio Peptide Mapping or Eclipse Plus C18 first.

Choosing a Column Chemistry


38

Sample Detection First Choice of column Alternative column

Peptides

LC/MS (FA) AdvanceBio Peptide Plus AdvanceBio Peptide Mapping

LC/UV AdvanceBio Peptide Mapping AdvanceBio Peptide Plus

ZORABX RRHD 300SB-C18 or Diphenyl

For maximum flexibility to transfer methods across instrument platforms, use AdvanceBio Peptide Plus with formic acid.

Method Development Tips & Reminders

39

• Bonded Phase: C18 is routinely used. Try AdvanceBio Peptide Plus first; for alternate selectivity or highly hydrophilic samples try AdvanceBio Peptide Mapping or a ZORBAX column.

• Column dimension: Good start with 2.1mm × 150mm, for higher resolution use longer column

• Gradient: Acetonitrile : Water with 0.1% formic acid, other organic solvent substitutions can be used for different selectivity

Starting gradient time: 0-40% in 30min (1.3%/min)

• Temperature: Higher column temperature can dramatically improve both resolution and recovery (60 °C max)

• Desalt peptide mixture prior to injection

• Sample handling may cause artifacts (peptide modifications)


Troubleshooting – Reasons for < 100% Sequence Coverage

Question to ask: Looking at the protein sequence, what’s missing?

• Very small and/or very hydrophilic peptides?• If using AdvanceBio Peptide Plus, try AdvanceBio Peptide Mapping for better retention and/or

resolution.

• Very large and/or very hydrophobic peptides?• Can you go to a higher percent organic mobile phase?

• Are peptides sticking to sample tubes or otherwise lost during sample preparation? Or on the LC?

• If using AdvanceBio Peptide Mapping, try AdvanceBio Peptide Plus

- May even need to consider a C8 column rather than C18

• Very large, or very small peptides of any hydrophobicity?• Consider a different digestion enzyme


40

More Information

Application Notes:

LC/MS Analysis of Peptide Mapping with Formic Acid Ion-Pairing Agent (5991-7574EN)

Enhancing the Quality of Peptide Mapping Separation for the Analysis of Post-Translational Modifications (5991-7875EN)

How-to Guide

41


Standards & Reagents

42

10 Peptide Standard (p/n 5190-0583)HSA peptides Standard

(p/n G2455-85001)

1. Bradykinin frag 1-7 2. Bradykinin 3. Angiotensin II (human) 4. Neurotensin5. Angiotensin I (human) 6. Renin substrate porcine 7. [Ace-F-3,-2 H-1] Angiotensinogen (1-14)8. Ser/Thr Protein Phosphatase (15- 31) 9. [F14] Ser/Thr Protein Phosphatase (15-31) 10. Melittin (honey bee venom)

7777x 1 0x 1 0x 1 0x 1 0

0000

0 . 20 . 20 . 20 . 2

0 . 40 . 40 . 40 . 4

0 . 60 . 60 . 60 . 6

0 . 80 . 80 . 80 . 8

1111

1 . 21 . 21 . 21 . 2

1111 2222 3333 4444 5555 6666 7777 8888 9999 1 01 01 01 0 1 11 11 11 1 1 21 21 21 2 1 31 31 31 3 1 41 41 41 4 1 51 51 51 5 1 61 61 61 6 1 71 71 71 7 1 81 81 81 8 1 91 91 91 9 2 02 02 02 0 2 12 12 12 1 2 22 22 22 2 2 32 32 32 3 2 42 42 42 4 2 52 52 52 5

Co

un

ts

Co

un

ts

Co

un

ts

Co

un

ts

A cquisition Time (min)A cquisition Time (min)A cquisition Time (min)A cquisition Time (min)

1

23

5 67

10

9

8

4

Blank

1. AAFTE[CAM][CAM]QAADK 2. YLYEIAR 3. LVNEVTEFAK 4. KVPQVSTPTLVEVSR 5. RP[CAM]FSALEVDETYVPK 6. AVMDDFAAFVEK 7. HPYFYAPELLFFAK

Trypsin Digest Methylated BSA Standard

(p/n G1990-85000)Trypsin


Summary

43

• High efficiency, high resolution separation of peptides using AdvanceBio Peptide Plus column

• MS-compatible AdvanceBio Peptide Plus column delivers high peak capacity with sharp and narrow

peaks using MS-friendly formic acid modifier mobile phase conditions.

• AdvanceBio Peptide Plus and AdvanceBio Peptide Mapping are superficially porous columns that yield

low back pressures & STM-like performance, allowing peptide mapping separations to be run on both

HPLC and UHPLC systems

• Well-resolved PTM modified peptide peaks enabled reliable mAb peptide maps and development of

Multi-Attribute Methods

• High quality separations in ~30 minutes with AdvanceBio Peptide Plus make peptide mapping more

time-effective

• Multiple column options for every customer’s unique needsFor Research Use Only. Not for use in diagnostic procedures.

Critical Quality Attribute Assessment by Peptide Mapping ... · Critical Quality Attribute...

Documents

Transcript of Critical Quality Attribute Assessment by Peptide Mapping ... · Critical Quality Attribute...