Charakterisierung der Primärstruktur - Agilent

Charakterisierung der Primärstruktur

rekombinanter Proteine mittels „Peptide

Mapping“ und „Intact Protein Analysis“

Moritz Wagner

Waldbronn

mAb Structure

Light Chain

Fc

Fab

Antigen

binding

Hinge

Glycosylation

siteTruncation

(lysine)

Disulfide

shuffling

Pyroglutamate

Deamidation/oxidation

Heavy Chain

Fucose – 146 Da

Mannose – 162 Da

N-Acetylglucosamine – 203 Da

Galactose – 162 Da

1299.3G0

1445.4G0F

1607.5G1F

1769.6G2F

Average massStructureGlycan

The Agilent Toolbox for Biologic Characterization

Intact Antibody characterization and sizing (accurate mass)

• Instrumentation: Q-TOF, TOF, CE, CE-MS

• HPLC Columns: Poroshell 300, Zorbax SB 300

Peptide mapping (resolution, resolution per time, Bioconfirm SW)

• Q-TOF- 1290 Infinity, Q-TOF-1260 RRLC, RRLC-UV

• HPLC Columns: Eclipse plus or Poroshell 120

Charge variants (resolution of charge variants)

• Bio-inert HPLC-UV, CE

• Bio HPLC Columns: Bio Mab WCX, Bio IEX

Aggregate analysis (resolution of monomers and multimers)

• Bio-inert HPLC UV, CE

• HPLC Columns: Bio SEC

Glycan analysis (confirmation of glycan pattern, detection and separation of variant)

• CE,CE-MS, mAB-Glyco-Chip, LC-MS, LC

Agilent’s Biologic Characterization and QA/QC Solutions

Glycan Analysis

mAb-Glyco-Chip + Chip LC/MS

Capillary ElectrophoresisHPLC/FLDMALDI MS

Oligonucleotides

Reversed Phase or Ion Exchange columns + LC/UV

or LC/MS

Amino Acids

Reversed Phase columns , derivitization , eluents and

standards + LC/UV or LC/MS

Peptide Characterization/

Mapping

LC/MS RP Columns + LC/UV

Oxidation

LC/MS or CE/MS

HIC and Reversed Phase Columns + LC/UV or LC/MS

Charge Variants

Ion Exchange Columns + LC/UV

Agilent CE + CE Kits

Agilent CE/MS + CE Kits

Biologic Molecular Weight Determination

Reversed Phase Columns + LC/UV or LC/MS

CE/MS

Aggregation

Size Exclusion Columns + HPLC/UV

Agilent’s LC/MS systems for biopharma applications

•Intact protein analysis

•Peptide mapping

•PTM analysis

•Intact protein analysis

•Peptide mapping

•PTM analysis

•Glycan analysis

Intact Protein Analysis

Accurate Mass TOF, QTOF

1290 Infinity HPLC, nano LC with HPLC-chip

Measure intact mAb molecular weight using Q-TOF

Accurate Mass Measurement on Agilent TOF/Q-TOF

Mcalc Mexp Error (ppm)

Intact

major glycoform148,811.95 148,812.81 5.8

deglycosylated 145,923.24 145,924.41 8

Light chain 23,746.63 23,746.50 5.5

Heavy chain

major glycoform50,675.47 50,675.58 2.2

Fc

major glycoform52,755.62 52,755.64 0.4

FAB 48,046.18 48,046.09 1.8

Delta mass ruler label the distance between peaks as mass, amino

acid or modifications. It helps the user to quickly decipher the

relationship of the two peaks.

Delta mass ruler / amino acid ruler / modification ruler

Peptide Mapping and PTM

Characterization Using Agilent Q-TOF

and BioConfirm Software

Accurate Mass TOF, QTOF

1290 Infinity HPLC, nano LC with HPLC-chip

MassHunter Acq for TOF/Q-TOF B.04.00

Improved Decision Engine for Data-Dependent MS/MS

• The scan speed (no. of transients/spectrum) is varied based on the MS1

precursor ion abundance

• “Target counts/spectrum” refers to the MS2 TIC. A higher value will

increase the accumulation time



• If “Use MS/MS accumulation time limit” is checked, the accumulation of

transients will stop, even if the selected “Target counts / MSMS spectrum”

requires longer accumulation

• If “Reject precursors that cannot reach target TIC within time limit” is

checked, MS1 precursor ion abundances that cannot create the selected

“Target counts / MSMS spectrum” within the time limit, will be ignored and

instead the next precursor chosen by the decision engine will be fragmented.



• The new “Purity” parameter determines the intensity of a precursor ion

candidate relative to the total ion intensity in the selected isolation

window (1.3, 4.0, 9.0 amu).

4 amu



• There is now more flexibility in setting collision energies for both data-

dependent and targeted MS/MS experiments.

• With the “Use Formula” feature, any function for collision energy over

mass can be defined, based on different charge states.

• The picture below shows an example for data dependent MS/MS

acquisition with 5 different collision energies for +2 charged ions,

depending on their m/z value.



• The “Use Fixed Collision Energies” or the “Use Formula” option can

be used to set up automatic acquisition at 3 collision energies for

automated PCDL search for data dependent, targeted and the new

directed MS/MS acquisition *)

• “Directed MS/MS” is a special mode of data-dependent MS/MS, where

the decision engine will ONLY do MS/MS experiments on precursor

ions in the preferred list. RT and delta RT setting will be honored ! *)

100% sequence coverage of heavy and light chain

Peptide MappingPeptide Map of mAb tryptic digest using Poroshell 120 column

Thin layer of

porous silica on a

solid core enables

peptides and

proteins to elute as

narrow bands at

high flow rates

Peptide Mass Accuracy

light chain A(112-130) 2101.1245 2101.1208 1.76

light chain A(113-130) 1945.0199 1945.0197 0.09

light chain A(131-146) 1796.8922 1796.888 2.38

light chain A(150-173) 2676.2669 2676.2627 1.55

light chain A(150-187) 4160.01 4160.0033 1.61

light chain A(154-173) 2134.9657 2134.9615 1.99

light chain A(193-211) 2140.0778 2140.0735 1.99

light chain A(195-211) 1874.9254 1874.9197 3.06

Sequence

NameLabel Mass

Target

Sequence

Mass

Match

Difference

(ppm)

light chain A(1-19) 1996.088 1996.0841 1.98

light chain A(26-47) 2438.1986 2438.1979 0.31

light chain A(48-56) 992.5656 992.5655 0.15

light chain A(64-93) 3388.5238 3388.5194 1.31

light chain A(94-107) 1538.7191 1538.7154 2.43

Sequence Name Label MassTarget Sequence

Mass

Match Difference

(ppm)

heavy chain A(1-19) 1880.0511 1880.048 1.65

heavy chain A(20-38) 2126.9587 2126.9554 1.56

heavy chain A(39-67) 2927.4492 2927.4414 2.68

heavy chain A(39-65) 2714.3216 2714.3188 1.05

heavy chain A(44-65) 2233.0584 2233.0539 2

heavy chain A(44-67) 2446.1802 2446.1765 1.51

heavy chain A(66-72) 835.4663 835.4664 -0.11

heavy chain A(68-72) 622.3431 622.3439 -1.28

heavy chain A(73-87) 1768.8812 1768.8778 1.93

heavy chain A(77-87) 1324.6816 1324.6809 0.51

heavy chain A(88-98) 1317.5937 1317.5911 2

heavy chain A(99-105) 714.4018 714.4024 -0.94

heavy chain A(106-129) 2531.2534 2531.2479 2.2

heavy chain A(107-129) 2375.1509 2375.1468 1.74

heavy chain A(130-141) 1185.6426 1185.6394 2.72

heavy chain A(142-155) 1320.6705 1320.6708 -0.21

heavy chain A(156-218) 6712.309 6712.3072 0.27heavy chain A(156-221) 7054.4995 7054.4975 0.28

heavy chain A(156-222) 7182.5962 7182.5925 0.52

heavy chain A(227-256) 3333.643 3333.6349 2.45

heavy chain A(231-256) 2843.4544 2843.4503 1.45

heavy chain A(257-263) 834.4274 834.4269 0.55

heavy chain A(264-282) 2138.0249 2138.0202 2.22

heavy chain A(283-296) 1676.7985 1676.7947 2.25

heavy chain A(297-309) 3115.3418 3115.3315 2.13

heavy chain A(310-325) 1807.0038 1806.9992 2.51

heavy chain A(310-328) 2227.2034 2227.2001 1.5

heavy chain A(335-342) 837.496 837.496 0

heavy chain A(347-368) 2509.3347 2509.3289 2.32

heavy chain A(349-368) 2310.1993 2310.1968 1.08

heavy chain A(353-363) 1285.6677 1285.6667 0.79

heavy chain A(353-368) 1871.9648 1871.9629 1.03

heavy chain A(369-378) 1160.6228 1160.6223 0.4

heavy chain A(379-400) 2543.1289 2543.1241 1.9

heavy chain A(401-417) 1872.9184 1872.9146 2.06

heavy chain A(423-447) 3043.3964 3043.393 1.12

heavy chain A(425-447) 2800.2679 2800.2598 2.89

heavy chain A(448-454) 659.3488 659.349 -0.35

Average Error = 1.3ppm

Extract MS/MS spectraUnder find by MFE, results tab, a

new section of extracting MS/MS

spectrum:

MS score

contribution

MS/MS score

contribution

Contribution

to the

MS/MS

score

Under match sequences, a new

tab „scoring‟ is added

MassHunter BioConfirm Software

•Allows multiple enzyme digest

•Missed cleavage

•More than 80 predefined

modifications in the chemical data

dictionary

•Allows user defined modifications

•Allows C13, N15, O18, D labeled

amino acid

MassHunter Bioconfirm Software

Matching rules allow incomplete

digest, predicted modifications

and DNA point mutation

Table view and spectrum view

Product ion assignment

Product ion label

MassHunter BioConfirm B.04.00 More confidence with MS/MS support

Digest sequence: TSPYVLPVPFLNVLNGGSHAGGALALQEFMIAPTGAK, match compound at 3752 Da

with 1* Oxidation predicted modification, with MSMS support, we can identify the location of predicted

modification, find out the Oxidation is on M at A171, not on H at A160 because of MSMSScore(70 vs. 49)

Unique ions

confirm Oxidation

at Met at pos. 171

Identify the predicted

modification location

Delta mass ruler label the distance between peaks as mass,

amino acid or modifications. It helps the user to quickly

decipher the relationship of the two peaks.

Delta mass ruler / amino acid ruler / modification ruler

MassHunter BioConfirm B.04.00 Compare two samples to find and visualize the difference

Coloring whether

found in Sample,

Reference or both

Mirror plot of TIC,

ECC, BPC, TCC

Compare MS and

MS/MS spectra

underneath each

other

Visualize sequence

coverage in both

samples

Forced Oxidation Study

Using Agilent Q-TOF

Mass Profiler Pro and BioConfirm Software

Comparison at Peptide Digest Level

Using Mass Profiler Pro to Analyze Large Batch of Samples

PCA plot of different oxidation conditions

•Principal component analysis (PCA) is a clustering tool often applied to reduce the dimensionality of complex data sets.

•The result for the oxidation samples demonstrates correct grouping of the technical replicates and clear separation between different concentrations of t-BHP treated mAbs.

•It is clearly observed that control (cyan) and 0.1% (red) are well separated as compared with other oxidized samples.

Using BioConfirm Qualitative Comparison to

Compare Sample One to One

Common

featuresReference Sample

R

S

R

S

SR

mirror

Table

MS spectra

MS/MS spectraChromatograms

Unmodified peptide

R

S

R

S

SR

Single oxidation

R

S

R

S

SR

Double oxidation

R

S

R

S

SR

MassHunter BioConfirm B04

Improve result quality with confidence

•Combining MS mass accuracy and MS/MS product ion assignment to reduce FP

•Improve MaxEnt deconvolution for intact protein analysis

Help user to predict the unexpected modifications

•Protein truncation

•Modification and amino acid caliper

•Mass/subsequence search

•Mix/match predicated modifications

•Non-specific enzyme digestion

Comparative analysis

•Allow easy comparison between reference control and multiple samples

•Peptide centric table

•Mirror plot for entire sample as well as individual peptide

This information is subject to

change without notice

© Agilent Technologies, Inc. 2011

Published in Germany, July 2011

Documents

Charakterisierung der Primärstruktur - Agilent