CYS National Annex to CYS EN 1998-1:2004 Eurocode 8: Design of ...
FINAL mAb poster - BioPharmaSpecIdeS Digest Analysis MS Analysis: Reduced Cys-linked ADC ...
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IdeS Digest Analysis MS Analysis: Reduced Cys-linked ADC m/z % 0 100 718.52 977.75 977.70 971.58 935.63 935.57 902.22 719.52 871.22 869.23 842.21 977.92 1010.40 1010.48 1016.88 1059.17 1098.17 1098.23 1148.05 2.36e5 400 600 800 1200 1400 1600 1800 2000 1000 2200 2400 Immunoglobulin-degrading enzyme from S. pyrogenes (IdeS), is a cysteine protease which cleaves at the hinge region of IgG subclass antibodies. is enzyme is being increasingly used for characterisation of domain -specific protein modifications, e.g drug conjugation. Deconvoluted MS Analysis: IdeS of Cys-linked ADC m/z % 0 100 1.79e5 718.51 1262.62 1202.52 1147.90 1147.81 1098.03 1097.96 719.51 1052.33 1010.32 720.52 784.48 1262.64 1270.74 1328.97 1337.52 1337.55 1337.62 1402.75 1402.81 1412.45 1485.23 1485.29 400 600 800 1200 1400 1600 1800 2000 1000 200 2200 2400 LC-MS is used to identify the F(ab’) 2 and Fc digest fragments. Deconvoluted ese fragments can be further separated by reduction into their LC, Fd and Fc/2 regions for MS analysis. Using this information the Drug : Ab ratio (DAR) can be calculated to give:- Total DAR of 2.4. LC Fd LC + drug Fd + drug Fd + 2 drug mass % 0 100 6 e 6 4 . 3 25235 23443 24759.5 23465.5 23997 24781 25397 26700 25420 26720.5 28016.5 28038 22500 23000 23500 24000 24500 25000 25500 26000 26500 27000 27500 28000 28500 29000 25383 mass % 100 8.06e6 25396 25234 23993 22828 23969 24756 25086 25557 26695 22500 23000 23500 24500 25000 25500 26000 26500 24000 22000 0 Fc/2 + G0F Fc/2 + G2F Fc/2 + G1F % 50793 50468 50058 51112 51413 52006 51566 51616 53217 52859 52120 52815 53461 53634 54917 54606 54170 54061 54766 Fab´ 100 53391 51452 6.20e5 0 50500 51000 51500 52500 53000 53500 54000 54500 52000 50000 mass ADC Peptide Mapping Proteolytic digestion of protein and glycoprotein biopharmaceuticals followed by RP-HPLC and tandem ES-MS/MS provides confirmation of the predicted primary amino acid sequence. Mapping is also the principal method for characterisation of protein modifications such as drug conjugation. Given the inherent heterogeneity of monoclonal antibodies arising from production and storage processes and variable region differences, structural elucidation including protein modifications is essential both for the efficacy and safety of the ADC product. % 0 100 4 e 4 9 . 4 559.94 541.33 453.76 447.76 420.18 454.25 529.29 484.75 485.25 560.27 560.60 668.35 563.30 577.81 615.81 616.32 654.39 668.85 839.41 708.85 669.34 670.35 709.35 709.85 765.88 816.43 840.40 904.48 841.39 m/z 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 3+ 2+ y˝ 12 2+ 2+ y˝ 11 y˝ 13 2+ MS Analysis: HC digest of ADC 121 ASTKG PSVFP LAPSS KSTSG GTAAL GCLVK DYFPE PVTVS WNSGA LTSGV HTFPA VLQSS 181 GLYSL SSVVT VPSSS LGTQT YICNV NHKPS NTKVD KKVEP KSCDK THTCP PCPAP ELLGG 241 PSVFL FPPKP KDTLM ISRTP EVTCV VVDVS HEDPE VKFNW YVDGV EVHNA KTKPR EEQYN 301 STYRV VSVLT VLHQD WLNGK EYKCK VSNKA LPAPI EKTIS KAKGQ PREPQ VYTLP PSREE 361 MTKNQ VSLTC LVKGF YPSDI AVEWE SNGQP ENNYK TTPPV LDSDG SFFLY SKLTV DKSRW Amino acid Sequence of Herceptin Heavy Chain Peptide mapping of domain-specific antibody drug conjugates (ADCs) provides conjugation site specific-characterisation. Protein digest fragments are identified from MS or MS/MS data, by cross-correlating the peptide charge state, associated b/y˝ ions, and any potential modifications with the predicted amino acid sequence. FNWYVDGVEVHNAK 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 9.46 9.12 9.04 9.20 8.94 Absorbance pI pI marker icIEF data of Antibody-Drug Conjugate 8.94 9 .04 9 .12 9 .20 9.46 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 pI 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Absorbance pI marker icIEF data of Herceptin reference material e UV response is used to estimate relative amounts of isoforms present for comparative batch to batch and reference material to biosimilars or ADCs. e data show that each of the mAbs have a number of isoforms. For a Cys linked ADC the data as expected show no apparent change on drug conjugation. Dedicated systems such as the iCE3 analyser are used for accurate and reproducible separation and semi-quantitation of protein and glycoprotein pI isoforms. Imaged Capillary IsoElectric Focusing (icIEF) Identification of charge state differences relating to PTMs such as: - deamidation - phosphorylation - sialylation Or resulting from linker/drug conjugation to a Lysine residue. INTRODUCTION Monoclonal antibodies are glycoproteins, and for regulatory purposes the carbohydrate portion of the molecule needs to be characterised to the extent possible. Confirmation of the glycosylation site and structure of the carbohydrate chain and oligosaccharide profile is performed using LC- and/or GC-MS methods. Oligosaccharide profiling for example as illustrated above involves: - Protease digestion - Glycan release using PNGaseF - Sep-Pak purification - Application of 2-AB label - LC/MS analysis + fluorescence detection 2- AB labelled N-linked oligosaccharides G0 G1F Man-5 G2F G0F Time EU x 10e4 0.000 50000.004 100000.008 150000.016 200000.016 250000.016 300000.031 350000.031 400000.031 450000.031 500000.031 550000.063 m n 0 2 4 m E , 0 3 3 x E A h C R L F Y T I U Q C A ) 1 ( 4 4 4 8 7 5 : e g n a R 2.78 2.35 1.90 3.73 3.18 5.10 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 e power of Q-TOF Mass Spectrometric technology has been amply demonstrated over the past 20 years since its inception 1 , both for accurate intact mass analysis of biopolymers up to 200kD and for the essential task of peptide and protein sequencing 2 . Combined with the earlier development of peptide mapping strategies utilising specific proteolytic digestion protocols 3 , mass spectrometry is now an essential tool in biopharmaceutical characterisation for manufacturing and regulatory purposes. Of particular importance is the ease of assignment of co- or post-translational modifications of proteins (PTMs) using this methodology, including Glycosylation 4 , Acylation 5 and Disulphide Bridge configuration 6 . e conjugation of cytotoxic drugs to Antibodies whether directly or through various linkers to produce ADCs is conceptually simply another example of post translational modification of the carrier protein and the MS strategies developed for native PTM identification are equally applicable to this exciting field of medical discovery. For ADC development, this analysis is performed to show that the chemistry used to attach the drug is not having an impact on glycosylation. ss ss ss ss Reduction of intermolecular disulphide bridges mAb prior to linker and cytotoxic drug attachment ADC Conjugation of drug to Cysteine residues ADC ANALYSIS Carbohydrate Characterisation Structural and Physico-Chemical Characterisation of Antibody-Drug Conjugates (ADCs) Christina Morris, Sam Williams, Georgina Sawyer, Alex Leach, Daniel Binet, M.J. O’Connor BioPharmaSpec Ltd, Suite 3.1, Lido Medical Centre, St. Saviour, Jersey, JE2 7LA, UK REFERENCES 1. Morris, H.R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R.S.; Hoyes, J. & Bateman, R.H. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Communications in Mass Spectrometry 10, 889-896 (1996). 2. van der Wel, H.; Morris, H.R.; Panico, M.; Paxton, T.; North, S.J.; Dell, A.; omson, J.M. & West, C.M. A non-golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium. Journal of Biological Chemistry 276, 33952-33963 (2001). 3. Morris, H.R.; Panico, M. & Taylor, G.W. FAB Mapping of Recombinant-DNA Protein Products. Biochemical and Biophysical Research Communications 117, 299-305(1983). 4. Tissot, B., North, S.J., Ceroni, A., Pang, P.C., Panico, M., Rosati, F., Capone, A., Haslam, S.M., Dell, A. and Morris, H.R. (2009) Glycoproteomics: Past, Present and Future. FEBS Lett 583, 1728-1735 5. Stein, T.; Vater, J.; Kruſt, V.; Wittmann-Liebold, B.; Franke, P.; Panico, M.; McDowell, R.M. & Morris, H.R. Detection of 4-phosphopantetheine at the thioester binding site for L-Valine of Gramicidin Synthetase-2. FEBS Lett 340, 39-44 (1994). 6. Morris, H.R. & Pucci, P. A new method for rapid assignment of S-S bridges in proteins. Biochemical and Biophysical Research Communications 126, 1122-1128 (1985). ACKNOWLEDGEMENTS We would like to thank ADC BIO Ltd. for supplying the ADC material for these analyses. https://www.biopharmaspec.com ¸ [email protected] ò +44 (0)1534 483493 CONTACTS
Transcript of FINAL mAb poster - BioPharmaSpecIdeS Digest Analysis MS Analysis: Reduced Cys-linked ADC ...
IdeS Digest Analysis
MS Analysis: Reduced Cys-linked ADC
m/z
%
0
100 718.52
977.75
977.70
971.58
935.63
935.57902.22
719.52
871.22
869.23
842.21
977.92
1010.40
1010.481016.88
1059.17
1098.17
1098.23
1148.05
2.36e5
400 600 800 1200 1400 1600 1800 20001000 2200 2400
Immunoglobulin-degrading enzyme from S. pyrogenes (IdeS), is a cysteine protease which cleaves at the hinge region of IgG subclass antibodies. This enzyme is being increasingly used for characterisation of domain -specific protein modifications, e.g drug conjugation.
Deconvoluted
MS Analysis: IdeS of Cys-linked ADC
m/z
%
0
100 1.79e5718.51
1262.621202.52
1147.90
1147.81
1098.03
1097.96719.51
1052.33
1010.32720.52
784.48
1262.64
1270.74
1328.971337.521337.551337.62
1402.751402.81
1412.45
1485.231485.29
400 600 800 1200 1400 1600 1800 20001000200 2200 2400
LC-MS is used to identify the F(ab’)2 and Fc digest fragments.
Deconvoluted
These fragments can be further separated by reduction into their LC, Fd and Fc/2 regions for MS analysis.
Using this information the Drug : Ab ratio (DAR) can be calculated to give:-Total DAR of 2.4.
LC
Fd
LC + drug
Fd + drug
Fd + 2 drug
mass
%
0
100
6e64.3 25235
23443
24759.5
23465.5 23997 24781
25397
26700
25420 26720.528016.5
28038
22500 23000 23500 24000 24500 25000 25500 26000 26500 27000 27500 28000 28500 29000
25383
mass
%
1008.06e6
25396
25234
23993
22828 23969
24756 2508625557
26695
22500 23000 23500 24500 25000 25500 26000 2650024000220000
Fc/2 + G0F
Fc/2 + G2F
Fc/2 + G1F
%
50793
50468
5005851112
51413
52006
5156651616
53217
528595212052815
53461
53634 54917546065417054061
54766
Fab´
100
53391
51452
6.20e5
050500 51000 51500 52500 53000 53500 54000 545005200050000
mass
ADC Peptide Mapping
Proteolytic digestion of protein and glycoprotein biopharmaceuticals followed by RP-HPLC and tandem ES-MS/MS provides confirmation of the predicted primary amino acid sequence. Mapping is also the principal method for characterisation of protein modifications such as drug conjugation.
Given the inherent heterogeneity of monoclonal antibodies arising from production and storage processes and variable region differences, structural elucidation including protein modifications is essential both for the efficacy and safety of the ADC product.
%
0
1004e49.4 559.94
541.33453.76
447.76420.18
454.25529.29
484.75485.25
560.27
560.60
668.35
563.30
577.81615.81
616.32654.39
668.85
839.41708.85669.34
670.35
709.35
709.85765.88 816.43
840.40
904.48841.39
m/z350 400 450 500 550 600 650 700 750 800 850 900 950 1000
3+
2+y 1̋2
2+ 2+y1̋1
y 1̋32+
MS Analysis: HC digest of ADC
121 ASTKG PSVFP LAPSS KSTSG GTAAL GCLVK DYFPE PVTVS WNSGA LTSGV HTFPA VLQSS 181 GLYSL SSVVT VPSSS LGTQT YICNV NHKPS NTKVD KKVEP KSCDK THTCP PCPAP ELLGG 241 PSVFL FPPKP KDTLM ISRTP EVTCV VVDVS HEDPE VKFNW YVDGV EVHNA KTKPR EEQYN 301 STYRV VSVLT VLHQD WLNGK EYKCK VSNKA LPAPI EKTIS KAKGQ PREPQ VYTLP PSREE 361 MTKNQ VSLTC LVKGF YPSDI AVEWE SNGQP ENNYK TTPPV LDSDG SFFLY SKLTV DKSRW
Amino acid Sequence of Herceptin Heavy Chain
Peptide mapping of domain-specific antibody drug conjugates (ADCs) provides conjugation site specific-characterisation.
Protein digest fragments are identified from MS or MS/MS data, by cross-correlating the peptide charge state, associated b/y˝ ions, and any potential modifications with the predicted amino acid sequence.
FNWYVDGVEVHNAK
8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8
0.00
0.05
0.10
0.15
0.20
0.25
0.30
9.46
9.12
9.04
9.20
8.94
Absorbance
pI
pI marker
icIEF data of Antibody-Drug Conjugate
8.94
9 .04
9.12
9.20
9.46
8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8pI
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Absorbance
pI marker
icIEF data of Herceptin reference material
The UV response is used to estimate relative amounts of isoforms present for comparative batch to batch and reference material to biosimilars or ADCs.
The data show that each of the mAbs have a number of isoforms. For a Cys linked ADC the data as expected show no apparent change on drug conjugation.
Dedicated systems such as the iCE3 analyser are used for accurate and reproducible separation and semi-quantitation of protein and glycoprotein pI isoforms.
Imaged Capillary IsoElectric Focusing (icIEF)
Identification of charge state differences relating to PTMs such as: - deamidation - phosphorylation - sialylation Or resulting from linker/drug conjugation to a Lysine residue.
INTRODUCTION
Monoclonal antibodies are glycoproteins, and for regulatory purposes the carbohydrate portion of the molecule needs to be characterised to the extent possible. Confirmation of the glycosylation site and structure of the carbohydrate chain and oligosaccharide profile is performed using LC- and/or GC-MS methods.
Oligosaccharide profiling for example as illustrated above involves: - Protease digestion - Glycan release using PNGaseF- Sep-Pak purification- Application of 2-AB label- LC/MS analysis + fluorescence detection
2- AB labelled N-linked oligosaccharides
G0
G1F
Man-5G2F
G0F
Time
EU
x 1
0e4
0.000
50000.004
100000.008
150000.016
200000.016
250000.016
300000.031
350000.031
400000.031
450000.031
500000.031
550000.063
mn 024mE,033xE AhC RLF YTIUQCA )1( 444875 :egnaR 2.78
2.35
1.90
3.73
3.185.10
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
The power of Q-TOF Mass Spectrometric technology has been amply demonstrated over the past 20 years since its inception1, both for accurate intact mass analysis of biopolymers up to 200kD and for the essential task of peptide and protein sequencing2. Combined with the earlier development of peptide mapping strategies utilising specific proteolytic digestion protocols3, mass spectrometry is now an essential tool in biopharmaceutical characterisation for manufacturing and regulatory purposes.
Of particular importance is the ease of assignment of co- or post-translational modifications of proteins (PTMs) using this methodology, including Glycosylation4, Acylation5 and Disulphide Bridge configuration6. The conjugation of cytotoxic drugs to Antibodies whether directly or through various linkers to produce ADCs is conceptually simply another example of post translational modification of the carrier protein and the MS strategies developed for native PTM identification are equally applicable to this exciting field of medical discovery.
For ADC development, this analysis is performed to show that the chemistry used to attach the drug is not having an impact on glycosylation.
s s
s ss s
s s
Reduction of intermoleculardisulphide bridges
mAb prior to linker and cytotoxic drug attachment
ADCConjugation of drug to Cysteine residues
ADC ANALYSIS
Carbohydrate Characterisation
Structural and Physico-Chemical Characterisation of Antibody-Drug Conjugates (ADCs)Christina Morris, Sam Williams, Georgina Sawyer, Alex Leach, Daniel Binet, M.J. O’Connor
BioPharmaSpec Ltd, Suite 3.1, Lido Medical Centre, St. Saviour, Jersey, JE2 7LA, UK
REFERENCES1. Morris, H.R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R.S.; Hoyes, J. & Bateman, R.H. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel
quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Communications in Mass Spectrometry 10, 889-896 (1996).2. van der Wel, H.; Morris, H.R.; Panico, M.; Paxton, T.; North, S.J.; Dell, A.; Thomson, J.M. & West, C.M. A non-golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.
Journal of Biological Chemistry 276, 33952-33963 (2001).3. Morris, H.R.; Panico, M. & Taylor, G.W. FAB Mapping of Recombinant-DNA Protein Products. Biochemical and Biophysical Research Communications 117, 299-305(1983).4. Tissot, B., North, S.J., Ceroni, A., Pang, P.C., Panico, M., Rosati, F., Capone, A., Haslam, S.M., Dell, A. and Morris, H.R. (2009) Glycoproteomics: Past, Present and Future. FEBS Lett 583, 1728-17355. Stein, T.; Vater, J.; Kruft, V.; Wittmann-Liebold, B.; Franke, P.; Panico, M.; McDowell, R.M. & Morris, H.R. Detection of 4-phosphopantetheine at the thioester binding site for L-Valine of Gramicidin
Synthetase-2. FEBS Lett 340, 39-44 (1994).6. Morris, H.R. & Pucci, P. A new method for rapid assignment of S-S bridges in proteins. Biochemical and Biophysical Research Communications 126, 1122-1128 (1985).
ACKNOWLEDGEMENTSWe would like to thank ADC BIO Ltd. for supplying the ADC material for these analyses. � https://www.biopharmaspec.com
¸ [email protected]ò +44 (0)1534 483493
CONTACTS
