in-depth characterization of biotherapeutics by mass ... · in depth analysis of quality attributes...

In-depth characterization of biotherapeutics by mass spectrometry - a case study

Analytical Technologies Europe 2015 Dr. Udo Roth. Sanofi FF SCP Biologics

Agenda

• MS Laboratory

• Case study - Characterization of a therapeutic monoclonal IgG1 by MS:

o Mass determination of intact and deglycosylated mAB by SEC-MS

o Analysis of reduced antibodies by SEC-MS

o Peptide Mapping and detection of chemical or postranslational modifications

o ETD for differentiation of iso-Asp and Asp after asparagin deamidation

o Structure elucidation of native and scrambled disulfide bonds by LC-MS

o Determination of antibody termini by MALDI ISD

o Profiling of the glycan structures by MALDI TOF MS/MS

• Summary

Analytical Technologies Europe 2015

Goal of BioR: ● Development of therapeutic monoclonal antibodies

In depth analysis of quality attributes required

High-resolution mass spectrometry as ideal tool for studying

antibodies on the molecular level

Presented here is a case study of the MS-based characterization of a specific monoclonal IgG1antibody


Molecular antibody features and quality attributes

G

G

D O

O D

D

pyro-E

K

G

G

D O

O D

D

pyro-E

K

pyro-E D

O

G

K

Pyro-Glu Deamidation Methionine oxidation Glycation

High mannose. G0. G1. G1. G2 Sialylation

C-term Lys

Adapted from S. Kozlowski. P. Swann (2006). Advanced Drug Delivery Reviews. Vol. 58. 707


Sanofi R&D Biotherapeutics Frankfurt: MS-Equipment

LC-ESI-QTOF (Waters Synapt G2-Si X Acquity UPLC)

MALDI TOF TOF (Bruker Ultraflextreme)


Characterization of a monoclonal IgG1 by MS Intact and deglycosylated mAB by SEC-MS

Spectra SEC-Chromatograms


Analytical conditions: Deglycosylation overnight with PNGase F; SEC column (4.6 mm x 300 mm 200Å. 1.7 µm) at 30°C and isocratic elution with 0.1% TFA / 0.1 % FA / 30 % ACN

Intact

Deglycosylated

2xG0F

(G0F+G1F)

2xG0F

deglyco

G0F+G1F

SEC-MS of the same IgG1 mAb stored for six months at 25 °C to study stability characteristics


Spectra SEC-Chromatograms

stressed sample

reference STD

oligomer(s) Fab fragments

LC

Characterization of a monoclonal IgG1 by MS Analysis of reduced mAB by SEC-MS


SEC-Chromatogram

heavy chain light chain

Analytical conditions: Reduction with DTT (10 mM, 10 min@70°C). SEC column (4.6 mm x 300 mm. 200Å. 1.7 µm) at 30°C and isocratic elution with 0.1% TFA / 0.1 % FA / 30 % ACN

HC HC

G0F

G1F Man5 G0F-GlcNAc

Spectra

LC LC

Characterization of a monoclonal IgG1 by MS Analysis of reduced / deglycosylated mAB by SEC-MS

SEC-Chromatogram

heavy chain light chain

Analytical conditions: Deglycosylation overnight with PNGase F; then reduction with DTT (10 mM, 10 min@70°C). SEC the same as for „reduced antibody“)

HC LC

Spectra


Characterization of a monoclonal IgG1 by MS Summary „Mass of intact and reduced antibody“

Calculated mass Exp. determined mass

Glycosylated intact mAb

2G0F 147373 147378

G0F+G1F 147535 147540

Deglycosylated mAb 144486 144491

Calculated

mass

Exp. determined

mass

Reduced HC

G0F 50219.1 50219

G1F 50382.1 50381

G0F-GlcNAc 50016.0 50015

Man5 49991.0 49992 Deglycosylated reduced HC

48775.1 48776

possibly glycated HC 48937.1 48938

Heavy chain

Calculated mass Exp. determined

mass

Reduced LC 23484.1 23484

Possibly glycated LC 23647.1 23646

Light chain

Deglycosylated reduced LC

23484.1 23484

possibly glycated LC 23647.1 23646

Whole mAb


Characterization of a monoclonal IgG1 by MS Peptide mapping by LC-ESI-MSE

LC-MS chromatogram of a tryptic mAb digest Example spectra of selected LC peak

• mAbs are digested with different proteases (e.g. trypsin)

• Peptides are separated by C18 RP chromatography using TFA (!) as modifier

• Analysis of LC peaks by UV and MSE

• Bioinformatic assignment of peptide and fragment ions (BiopharmaLynx SW)

Creation of sequence coverage maps

Information on possible peptide modifications (oxidation, deamidation…)


Characterization of a monoclonal IgG1 – peptide mapping LC-MS chromatograms after digestion with four different proteases


Trypsin

Asp-N

Glu-C

Chymotrypsin

Peptide mapping summary

Protease Sequence coverage

Trypsin 96.5 %

Asp-N 98.9 %

Glu-C 77.0 %

Chymotrypsin 93.1 %

Combined 100.0%

Q296/(N298) EEQYNSTYR (Glycosylation G0F) 2:T023 none 98.8 1x deamidation 1.2

Q312/N316 VVSVLTVLHQDWLNGK 2:T024 none 81.4 1x deamidation 18.6 2x deamidation 0

N362/Q363 NQVSLTCLVK 2:T034

none 98 1x deamidation 1.9 2x deamidation 0.1

N385/Q387 /N390/N391 GFYPSDIAVEWESNGQPENNYK 2:T035

none 75.3 1x deamidation 24.6 2x deamidation n.d.

Q419/Q420 /N422/N435/ Q439

WQQGNVFSCSVMHEALHNHYTQK 2:T039 none 96.3 1x deamidation

3.7

Sequence coverage over all four proteases

Report on peptide modifications, e.g. deamidation on different HC sites

Asn/Gln position Peptide Label Modification % intensity


A deeper look into the PENNYK peptide: ETD of peptides for detection of iso-aspartate The „PENNYK“ peptide covers a well known Fc hot spot for protein deamidation of the heavy chain

The precise deamidation position can be determined and relative ratios between the deamidated and

native peptide can be estimated

CID cannot differentiate between Asp and isp-Asp formation (peaks 1 and 3), ETD however provides

reporter ions for differentiation

…


*

*Ni W. Dai S. Karger BL. Zhou ZS. Anal Chem. (2010) 82:7485

1 2

3 Asn

Asp / Iso-Asp?

ETD sequencing of the PENNYK peptide after direct infusion of standards


(1)

(2)

(3)

Determination of disulfide linkages in mABs Sample preparation protocol

Alkylation of possibly free Cys residues with iodoacetamide in denaturing conditions

Buffer exchange to 0.1 M Tris-HCl (pH 7.5)

Addition of RapiGest™ SF

Digestion with trypsin or LysC

IAA


LC-MS chromatograms of tryptic digests before and after reduction

Tryptic digest 98.6% coverage

Reduced tryptic digest 93.6% coverage


2:T12-2:T13

2:T12 2:T13

Comparison of signal intensities for disulfide bonds between RS and stressed sample

Native disulfide bonds Scrambled disulfide bonds


Tryptic digest

Comparison of signal intensities for disulfide bonds between RS and stressed sample

Native disulfide bonds Scrambled disulfide bonds


LysC digest

Determination of the N- and C-termini by MALDI-TOF ISD MS

• mAB cleavage by TCEP and desalting by ultrafiltration • Preparation with matrix (SDHB or DAN) on MALDI target • Analysis with MALDI MS by in-source-decay fragmentation (ISD) • Data processing using Biotools SW Confirmation of the heavy and light chain termini

Terminal sequences assigned to HC with pyro-N terminus sequence without pyro-N terminus sequence


Characterization of a monoclonal IgG1 by MS Identification of N-glycans by MALDI TOF MS

• N-glycan release by PNGase F and purification using graphitized carbon SPE • Preparation with SDHB matrix on a ground steel MALDI target • Analysis by MALDI MS(/MS) and Protein Scape SW (Bruker)

Identification of neutral N-glycan structures, no sialylated species detected Semi-quantitative profiling of N-glycans


Glycan m/z exp. m/z calc. Area Man5+Na 1.257.46 1.257.4 312 G0F-GlcNAc+Na 1.282.49 1.282.5 353 G0+Na 1.339.51 1.339.5 160 G0+K 1.355.67 1.355.6 85 G0F+Na 1.485.58 1.485.5 11803 G0F+K 1.501.56 1.501.6 1342 G1F+Na 1.647.64 1.647.6 664

Identified N-Glycans

Characterization of a monoclonal IgG1 by MS Identification of the 1282 m/z peak as G0F-GlcNAc

Fragment m/z meas. Mr calc. Δ MH+ [Da] 1B 168,0538 167,0082 0,0383 1B 185,0118 184,0347 -0,0302 1B 208,0053 207,0508 -0,0528

1^{0,4}A_{Man} 210,0242 209,0188 -0,0018 2Y 226,045 225,0613 -0,0236 2Y 244,0666 243,0719 -0,0126

1Y1^{1,5}X_{Fuc} 272,0434 271,0668 -0,0307 1C1Y 347,053 346,0875 -0,0419 1Z 372,1249 371,1192 -0,0016 1B 388,1243 387,1141 0,0029

1C1^{3,5}X_{Man} 418,0981 417,077 0,0138 2Y 447,1481 446,1513 -0,0105

1Y1^{1,5}X_{Man} 475,1645 474,1462 0,011 1C1Z 492,1341 491,1138 0,0131 1C1Y 509,1747 508,1403 0,0271

1Y1^{3,5}A_{GlcNAc} 547,5065 547,14 -0,6408 1C1Z 550,1698 549,1669 -0,0044

1B1^{1,5}X_{Man} 578,0327 577,1619 -0,1365 1Y 593,2102 592,2092 -0,0063

1^{1,5}X_{Man} 621,1539 620,2041 -0,0575 1B 712,2899 711,2197 0,0628 1C 730,3129 729,2303 0,0753

1B1^{1,5}X_{GlcNAc} 740,2517 739,2147 0,0297 2Y 753,304 752,2463 0,0503 2Y 771,2861 770,2569 0,0219

1^{3,5}A_{GlcNAc} 786,2976 785,2565 0,0338

1^{0,2}A_{GlcNAc} 832,3605 831,262 0,0912 1C 915,4677 914,2991 0,1612 2Y 933,4966 932,3097 0,1797 2Y 939,9085 938,2992 0,602

1^{1,5}X_{Man} 945,3243 944,3097 0,0073

1Z1^{2,5}X_{Fuc} 955,4029 954,2941 0,1015 1Y 1079,677 1078,368 0,3024 1Y 1120,83 1119,394 0,4286 1Y 1136,774 1135,389 0,3774

1^{1,3}X_{GlcNAc} 1181,818 1180,399 0,411

1^{1,3}X_{Fuc} 1222,853 1221,426 0,4198 1264,844 1263,436 0,4005


Annotated LIFT-CID spectrum

Fragment ion table

Summary • A biotherapeutic IgG1 antibody was characterized by different high resolution mass spectrometry

methods

• Identity was confirmed by determination of the intact and deglycoslyated molecular masses

• A middle-down approach (cleavage of heavy and light chain):

o confirmed the correct molecular masses of the the HC and LC

o facilitated the identification of different glycoforms

o revealed possible glycation sites (HC and LC)

• Peptide mapping experiments allowed:

o Confirmation of the correct amino acid sequence and discovery of a prominent intrinsic

deamidation site

o ETD fragmentation helped to differentiate Asp and iso-Asp deamidation products of this site

o Determination of native and scrambled disulfide bridges

• MALDI-ISD facilitated the confirmation of the pyro-form at the N-terminus of the heavy chain

• G0F was found as predominant structure after analysing the N-glycan profile with MALDI TOF MS


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