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

¸ services@biopharmaspec.comò +44 (0)1534 483493

CONTACTS