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
Analytical Technologies Europe 2015
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
Analytical Technologies Europe 2015
Sanofi R&D Biotherapeutics Frankfurt: MS-Equipment
LC-ESI-QTOF (Waters Synapt G2-Si X Acquity UPLC)
MALDI TOF TOF (Bruker Ultraflextreme)
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Characterization of a monoclonal IgG1 by MS Intact and deglycosylated mAB by SEC-MS
Spectra SEC-Chromatograms
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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
Analytical Technologies Europe 2015
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
Analytical Technologies Europe 2015
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
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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
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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…)
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Characterization of a monoclonal IgG1 – peptide mapping LC-MS chromatograms after digestion with four different proteases
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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
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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
…
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*
*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
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(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
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LC-MS chromatograms of tryptic digests before and after reduction
Tryptic digest 98.6% coverage
Reduced tryptic digest 93.6% coverage
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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
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Tryptic digest
Comparison of signal intensities for disulfide bonds between RS and stressed sample
Native disulfide bonds Scrambled disulfide bonds
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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
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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
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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
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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
Analytical Technologies Europe 2015
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