Accepted Manuscript Title: Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis Author: Marie-Claire Janin-Bussat Marina Dillenbourg Nathalie Corvaia Alain Beck Christine Klinguer-Hamour PII: DOI: Reference:
S1570-0232(14)00769-7 http://dx.doi.org/doi:10.1016/j.jchromb.2014.12.017 CHROMB 19246
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
15-9-2014 11-12-2014 17-12-2014
Please cite this article as: M.-C. Janin-Bussat, M. Dillenbourg, N. Corvaia, A. Beck, C. Klinguer-Hamour, Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.12.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis
Marie-Claire Janin-Bussat§, Marina Dillenbourg§, Nathalie Corvaia, Alain Beck*, Christine
ip t
Klinguer-Hamour*
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Centre d’Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-
These authors contributed equally to this work
an
§
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en-Genevois, France
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*Corresponding authors:
Christine Klinguer-Hamour. Centre d’Immunologie Pierre Fabre, 5 Avenue Napoleon III BP
Ac ce pt e
Tel.: +33 4 50 35 35 83
d
60497, 74164 Saint-Julien-en-Genevois, France.
e-mail:
[email protected] Alain Beck. Centre d’Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France. Tel.: +33 4 50 35 35 37
e-mail:
[email protected] Conflict of interest M.C. Janin-Bussat, N. Corvaia, A. Beck and C. Klinguer-Hamour are currently employees of the Institut de Recherche Pierre Fabre
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Abstract Antibody-drug conjugates (ADC) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the
ip t
current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been
cr
published to date. Here, we report on the improvement of an ADC peptide mapping protocol
us
to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris®), a commercial ADC with an average of 4 drugs linked to
an
interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the
M
hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-
Ac ce pt e
d
digestion and reduction of the ADC were also characterized.
Keywords
Antibody drug conjugates, brentuximab vedotin, drug-loaded peptide mapping, IdeS, liquid chromatography mass spectrometry, payload positional isomers.
Abbreviations
ADC, antibody drug conjugates; DAR, drug to antibody ratio; Fc/2, constant fragment of IgG heavy chain; Fd, variable-CH1 fragment of IgG heavy chain; HC, heavy chain; IdeS, immunoglobulin degrading enzyme of Streptococcus pyogenes; IgG, immunoglobulin G; LC, light chain; mAb, monoclonal antibody; RT, room temperature; Rt, retention time; vc-
2
Page 2 of 20
MMAE, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl-
Ac ce pt e
d
M
an
us
cr
ip t
auristatin E.
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Page 3 of 20
1. Introduction ADCs combine the potency of cytotoxic drugs with the high specificity of a monoclonal antibody (mAb) and become increasingly important as new targeted therapies in oncology. Two ADCs, brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla®)
ip t
were recently approved by the US Food and Drug Administration [1], and more than 30 are currently being investigated in clinical trials [2]. The primary sites used for protein-directed
cr
conjugation are the amino groups of lysine residues or the sulfhydryl groups of the inter-chain
us
cysteine residues, representing respectively one third and two third of ADCs in clinical trials [3]. Conjugates like brentuximab vedotin are formed through partial reduction of the antibody
an
interchain disulfide bonds, followed by alkylation with a preformed drug-linker maleimide activated species. They result in conjugates with a distribution of drug loading from 0, 2, 4, 6
M
or 8 drugs incorporated per antibody and an average drug to antibody ratio (DAR) of ~4 drugs/mAb [4]. Conjugation of drugs to mAbs increases the structural complexity of the
d
resulting molecule, which triggers the need for improved characterization methods [5] for
Ac ce pt e
analysis of DAR, drug distribution [6], size and charge variants, unconjugated drug, peptide mapping [7] and ADC biophysical properties [8]. Peptide mapping of ADC with hydrophobic drugs linked to its native cysteine residues by LC-MS analysis remains challenging due to the hydrophobicity of drug-loaded peptides [9]. This is especially true for peptides with two and more conjugated drugs.
Junutula and coll. [10] described a site-specific conjugation of maleimidocaproyl-valinecitrulline-p-aminobenzyloxycarbonyl-monomethyl-auristatin E (vc-MMAE) to an antibody through engineered cysteine substitutions at positions on light and heavy chains (ThiomAbDrug Conjugate) resulting in only tree isoforms with 0, 1 or 2 drugs and no positional isomer. Tryptic peptide mapping with LC/MS detection of these conjugates identified four drugconjugated peptides by a characteristic in-source fragmentation ion (m/z 718.5) that is
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observed in all mass spectra of molecules containing vc-MMAE. All peptides were identified as complete or partial tryptic cleavage fragments located around the engineered cysteines. Surprisingly, to our knowledge, no established protocol is currently published for endogenous mAbs cysteine residues-drug conjugates that yet represent two thirds of ADCs in clinical
cr
characterize the payloads positional isomers using LC-MS platform.
ip t
trials. Here, we report on the improvement of an ADC peptide mapping protocol to
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2. Materials and methods 2.1. Reagents and materials
an
The ADC brentuximab vedotin (Adcetris©) was produced by Millenium
Pharmaceuticals/Takeda (London, UK). Dithiothreitol (DTT), guanidine and Trizma-Base
M
were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France), trifluoroacetic acid (TFA), acetic acid and formic acid from Fluka (Saint-Quentin-Fallavier, France), acetonitrile
d
was obtained from Merck (Fontenay-sous-Bois, France), calcium chloride (CaCl2) and
Ac ce pt e
isopropanol from Prolabo (Fontenay-sous-Bois, France), and Carlo Erba Reagenti (Peypin, France), respectively. RapiGest SF was purchased from Waters (Saint-Quentin-en-Yvelines, France), and Poros R1 resin from Applied Biosystems (Cergy-Pointoise, France). All the aqueous solutions were prepared using ultra-pure water.
2.2. ADC reduction and alkylation
Brentuximab vedotin was denatured by addition of 6 M guanidine, 0.1 M Tris-Base, 2 mM EDTA, pH 8.0 buffer (v/v). Reduction was performed with 10 mM DTT for 45 min at 56°C and alkylation with 25 mM iodoacetamide 30 min at RT in the dark. Acetic acid was then added to quench the reaction.
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2.3. ADC IdeS digestion and reduction Brentuximab vedotin was digested with immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS, Fabricator®) (Genovis Lund, Sweeden) added at a ratio of 1U/µg of ADC and incubated at 37°C for 30 min. The sample was then treated as described in §2.2 without
cr
2.4. ADC IdeS fragments LC-MS analysis and isolation
ip t
alkylation.
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Reversed phase separation was performed on an ultra-high performance liquid
chromatography (UHPLC) system (Acquity UPLC, Waters, Milford, MA, USA) coupled to
an
an electrospray mass spectrometer detector (LCT Premier ESI-TOF, Waters) for LC-MS analyses and to an UV detector (TUV, Waters) for peaks isolation. The reduced samples were
M
directly injected on a PLRP-S column (2.1 x 150 mm, 8 µm, 1000Å, Agilent) heated at 80°C with a flow rate of 0.25 ml/min for analysis and a PLRP-S column (4.6 x 150 mm, 8µm, 1000
d
Å) at a flow rate of 1.2 ml/min for isolation. Elution was performed with water as eluent A
Ac ce pt e
and acetonitrile as eluent B, both containing 0.05% TFA. The following elution gradient was applied, B was raised from 5% to 30% in 8 min and then from 30 % to 50% in 40 min. The mass spectrometer was operated in positive mode and ions were scanned over a m/z range of 1000-3500. Fractions of interest were collected using UV detection at 280 nm and fragments recovered using Poros R1 resin (Applied Biosystems) following the manufacturer instructions.
2.5. Drug-loaded peptides mapping Prior to Lys-C digestion, IdeS fractions corresponding to drug-loaded fragments were evaporated to dryness, reconstituted in 30 µl of digestion buffer (50 mM Tris-HCl, 1 mM CaCl2, pH 7.1) containing 0.1% of RapiGest surfactant (Waters), heated for 15 min at 80°C
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and reduced with 20 mM DTT. Then, 10% acetonitrile was added to the sample. 1.5 µg of Lys-C was added to fractions, and digestion was performed for 3 h at 37°C. A second reduction was performed by addition of DTT to a final concentration of 35 mM followed by incubation for 45 min at 56°C. After the second reduction, isopropanol was added to the
ip t
sample to a final concentration of 40%. RapidGest was eliminated by acidification using a 10% TFA solution and incubation at 37°C for 30 min, following the manufacturer
cr
instructions. LC-MS peptide mapping analysis was performed on an UHPLC system, with
us
water as eluent A and acetonitrile as eluent B, both containing 0.1% formic acid. Digest was injected on an Acquity CSH C18 column (2.1 x 100 mm, 1.7 µm, 130Å, Waters) heated at
an
40°C. After an isocratic gradient at 0% B for 3 min, peptides were eluted from the column by increasing B to 60% in 60 min. MS analysis was performed on a LCT Premier ESI-TOF mass
M
spectrometer (Waters) in W positive mode from m/z 150 to 2500 with voltages of 2 kV, 25 V and 5V for capillary, sample cone and aperture 1, respectively. For in-source fragmentation,
Ac ce pt e
d
the aperture 1 voltage was increased to 40 V.
3. Results and discussion 3.1.
Peptide mapping improvement
First, a classical protocol of mAb peptide mapping was applied with reduction and alkylation as described in § 2.2. Exchange buffer was performed on ZebaTM Desalt Spin column (Thermo Scientific) following the manufacturer instructions. Lys-C digestion was performed as described in § 2.5 excepted that there was no addition of acetonitrile. After digestion, RapiGest was eliminated as describe in §2.5 and sample analyzed by LC-MS. The intensity of all peptide signals on the chromatogram was low due to the loss of loaded-HC and -LC. Then, the protocol was improved. The guanidine denaturation was replaced by heating without exchange of buffer before the Lys-C digestion to avoid the precipitation of loaded-LC and
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HC. To keep the loaded-peptides in solution after Lys-C digestion, 10 % acetonitrile was added to the sample before digestion and 40% isopropanol after digestion. To avoid the presence of disulfide-bound peptides, a second reduction was introduced after digestion. With
3.2. Separation and identification of ADC drug-loaded fragments
ip t
this improved protocol, all the loaded-peptides were identified on the LC-MS chromatogram.
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The LC-MS analysis of reduced and alkylated brentuximab vedotin resulted in six major
us
peaks (Figure 1A) as reported by Lyon et al. [11], but with a better resolution for peaks 4 and 5 showing satellite peaks as reported by Le et al [6]. The deconvoluted mass spectra of these
an
six peaks show masses of 24,013 Da (Rt = 13.5 min) and 25,273 Da (Rt = 18.3 min) that matched with alkylated LC (MTheo = 24,013 Da) and LC+1 payload (MTheo = 25,273 Da)
M
and masses of 50,955 Da (Rt = 19.7 min), 52,215 Da (Rt = 22.2 min), 53,475 Da (Rt = 27.7 min) and 54,734 Da (Rt = 32.1 min) that matched with alkylated HC (MTheo = 50,957 Da),
d
alkylated HC+1 payload (MTheo = 52,216 Da), alkylated HC+2 payloads (MTheo = 53,476
Ac ce pt e
Da), HC+3 payloads (MTheo = 54,735 Da). Two minor peaks were also observed and their deconvoluted mass spectra show masses of 52,216 Da and 53,475 Da that matched with alkylated HC+1 payload and alkylated HC+2 payloads, respectively. To summarize, alkylated HC+1 payload and alkylated HC+ 2 payloads fragments eluted at two different retention times (22.2 and 23.0 min) and (26.9 and 27.7 min), respectively, and could correspond to payloads positional isomers as described by Le et al. using the abundance of the different DAR species (HIC data), combined with capillary electrophoresis-based dissociation of the entire, unfractionated sample [6]. To confirm this hypothesis by LC-MS analysis, we first intended to improve the resolution of the peaks corresponding to fragments that carry the payloads. The IdeS digestion followed by reduction has already been successfully used to better characterize mAbs [12] and antibody
8
Page 8 of 20
fluorophore conjugate (AFC), which was recently proposed as ADC model [13]. The use of IdeS allows the analysis of the Fc/2 properly separated from the Fd fragment. As a result, the full glycoprofiling and demonstration of the absence of additional conjugation are easily achieved.
ip t
Here, we show for the first time that IdeS digestion of the ADC brentuximab vedotin before reduction was also possible and improved very significantly the peak separation as shown by
cr
LC-MS analysis (Figure 1B). As expected, the deconvoluted mass spectra of the peaks eluted
us
at 12.0 min, 14.7 min, 19.6 min, 22.6 min and 39.7 min matched with Fc/2 (M Theo = 25,204 Da), LC (MTheo = 23,728 Da), LC+ 1 payload (M Theo = 25,044 Da), Fd (M Theo = 25,142
an
Da) and Fd+ 3 payloads (MTheo = 29,092 Da), respectively. Confirming our hypothesis, the two peaks eluted at 26.2 min and 27.4 min having a mass of 26,458 Da (Figures 2A and 2B)
M
matched with the theoretical mass of Fd conjugated to 1 payload (Fd1) and the two peaks eluted at 32.5 min and 33.9 min having a mass of 27,775 Da (Figures 2C and 2D) matched
d
with the theoretical mass of Fd conjugated to 2 payloads (Fd2). Using these new sample
Ac ce pt e
preparation conditions, the separation of the different peaks corresponding to loaded fragments was greatly improved. Furthermore, the potential payloads positional isomers observed after reduction and alkylation of brentuximab vedotin were confirmed after IdeS digestion of the same ADC.
3.3. Identification of interchain cysteinyl-linked ADCs payload positional isomers To confirm that peaks having the same masses were payload positional isomers, peaks called Fd1a, Fd1b, Fd2a and F2b (Figure 1B) were enriched by reversed phase chromatography collection. The characterization of each fraction of interest was carried out after Lys-C digestion and LC-MS analysis. ADC peptide mapping remains a challenge due to the hydrophobicity of drug-loaded peptides [9], and no established protocol has been published
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Page 9 of 20
for interchain cysteinyl-linked ADCs. We describe here, conditions for ADC fragments Lys-C digestion, leading to hydrophobic drug-loaded peptides that were maintained in solution by adding acetonitrile and isopropanol. The drug-loaded peptides were identified by a characteristic in-source fragmentation ion (m/z 718.5) observed in all mass spectra of
drug-loaded peptides was confirmed by measuring their masses.
ip t
molecules containing vc-MMAE as previously described in [10]. The identification of these
cr
Using our sample preparation conditions, we identified the drug-loaded peptides in the Fd1
us
and Fd2 fractions after Lys-C digestion (Table 1). The peptide in Fd1a fraction (71%) presented a [M+2H] mass of 884.48 Da (Figure 3A) and was consistent with the theoretical
an
mass of the HC peptide [SCDK] conjugated to 1 payload ([M+2H] Theo = 884.48 Da) and referred to as HC L15+1 payload, its Cys220 is involved in the disulfide bond with the LC.
M
The peptide in the Fd1b fraction had a [M+3H] mass of 917.83 Da (Figure 3B) which corresponded to the HC peptide [THTCPPCPAPELLG] conjugated to 1 payload (MTheo =
d
917.82 Da) and referred to as HC L16+1 payload peptide, its Cys 226 and 229 are involved in
Ac ce pt e
the disulfide bonds between the two HC. The position of the payload in the HCL16 +1 payload peptide was not identified precisely (Cys 226 or Cys229) and could be further investigated by electron transfer dissociation (ETD). The HC L15+1 payload and HCL16 +1 payload peptides (Figures 3C and 3D) were both found in the Fd2a fraction. The peptide identified in the Fd2b fraction (83%) presented a [M+4H] mass of 1017.58 Da (Figure 3E) consistent with the L16+2 payloads peptide ([M+4H] Theo = 1017.56 Da). All the above reported data confirm that the drug was linked, as expected, to the inter-chain cysteines of the heavy and light chains. Furthermore, when characterizing the payload positional isomers by LC-MS, it was unambiguously demonstrated that the drug was linked preferentially to the HC L15 peptide on Cys220 when only one drug was bound to the HC. In
10
Page 10 of 20
contrast, when two drugs were linked to the HC, they were preferentially bound to the HC L16 peptide on Cys 226 and Cys229 (Figure 4).
4. Conclusion
ip t
In this study, we presented an improvement of ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. Brentuximab vedotin, an ADC with a
cr
heterogeneous distribution of ~4 drugs/mAb conjugated on native cysteines was submitted to
us
IdeS enzymatic digestion. The separation of the different peaks corresponding to drug-loaded fragments was greatly improved compared to usual reduction and alkylation protocol.
an
Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution by
M
addition of solvents. The drug-loaded-peptides were then characterized by LC-MS peptide mapping. This method can provide important structural information about the locations of
d
conjugation sites on cysteines residues of heterogeneous ADCs. To our knowledge, this is the
Ac ce pt e
first time that the payload positional isomers on the heavy chain of an ADC conjugated on native cysteines, were characterized by peptide mapping LC-MS analysis. Chromatographic separations of ADC peptides combined with MS analysis should also be applied to pharmacokinetic studies of ADC for characterization of their drug-loaded peptides distribution after their in vivo administration [14].
Acknowledgements
The authors thank Elsa Wagner-Rousset and Olivier Colas for advice on IdeS digestion for LC-MS analysis and Dr. Davy Guillarme, School of Pharmacy, University of Geneva for critical reading of the manuscript.
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A. Beck, J.M. Reichert, MAbs. 6 (2014) 15.
[2]
C. Klinguer-Hamour, P. Strop, D.K. Shah, L. Ducry, A. Xu, A. Beck, MAbs. 6 (2014) 18. A. Beck, J.F. Haeuw, T. Wurch, L. Goetsch, C. Bailly, N. Corvaïa, Discov.
ip t
[3]
Med. 10 (2010) 329.
J.F.Valliere-Douglass, W.A. McFee, O. Salas-Solano, Anal. Chem. 84 (2012)
cr
[4]
[5]
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2843.
F. Debaene, A. Bœuf, E. Wagner-Rousset, O. Colas, N. Corvaïa, A. Van
[6]
an
Dorsselaer, A. Beck, S. Cianférani, Anal. Chem. (in press).
L.N. Le, J.M. Moore, J. Ouyang, X. Chen, M.D. Nguyen, W.J. Galush, Anal.
[7]
M
Chem. 84 (2012) 7479.
L. Wang, G. Amphlett, W. A. Blattler, J. M. Lambert, W. Zhang, Protein
A. Beck, E. Wagner-Rousset, D. Ayoub, A. Van Dorsselaer, S. Sanglier-
Ac ce pt e
[8]
d
Science 14 (2005) 2436.
Cianférani, Anal. Chem. 85 (2013) 715.
[9]
A. Wakankar, Y. Chen, Y. Gokarn, FS. Jacobson, MAbs. 3 (2011) 161.
[10]
J.R. Junutula, H. Raab, S. Clark, S. Bhakta, D.D. Leipold, S. Weir, Y. Chen,
M. Simpson, S.P. Tsai, M.S. Dennis, Y. Lu, Y.G. Meng, C. Ng, J. Yang, C.C. Lee, E. Duenas, J. Gorrell, V. Katta, A. Kim, K. McDorman, K. Flagella, R. Venook, S. Ross, S.D. Spencer, W. Lee Wong, H.B. Lowman, R. Vandlen, M.X. Sliwkowski, R.H. Scheller, P. Polakis, W. Mallet, Nat. Biotechnol. 26 (2008) 925.
[11]
R.P. Lyon, D.L. Meyer, J.R. Setter, P.D. Senter, Methods Enzymol. 502 (2012) 123.
12
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[12]
G. Chevreux, N. Tilly, N. Bihoreau, Anal. Biochem. 415 (2011) 212.
[13]
E. Wagner-Rousset, M.C. Janin-Bussat, O. Colas, M. Excoffier, D. Ayoub, J.F. Haeuw, I. Rilatt, M. Perez, N. Corvaïa, A. Beck, MAbs. 6 (2014) 273.
[14]
S.M. Hengel, R. Sanderson, J. Valliere-Douglass, N. Nicholas, C. Leiske, S.C.
Ac ce pt e
d
M
an
us
cr
ip t
Alley, Anal. Chem. 86 (2014) 3420.
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Page 13 of 20
Figure captions
Figure captions
Figure 1: Separation of brentuximab vedotin fragments obtained after reducion and alkylation
ip t
(A) and IdeS digestion and reduction (B) using a PLRP-S column and MS detection.
Figure 2: Deconvoluted spectra of Fd conjugated to 1 payload (Fd1) at Rt of 26.2 min (A) and
us
cr
27.4 min (B) and Fd conjugated to 2 payloads (Fd2) at Rt of 32.5 min (C) and 33.9 min (D)
Figure 3: MS spectra of peptide HC L15+1 payload on Cys220 (A) peptide HC L16+1
an
payload peptide on either Cys 226 or 229 (B) isolated from fraction Fd1a and Fd1b, respectively. MS spectra of HC L15+1 payload (C), HCL16 +1 payload (D) peptides and
M
L16+2 payloads peptide (E) isolated from Fd2a and Fd2b fractions, respectively. Inserts
ed
represent the isotopic spectra of the major m/z.
fractions.
ce pt
Figure 4: Schematic representation of the positional payload isomers found in Fd1 and Fd2
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
Page 14 of 20
*Highlights
Highlights
ip t cr us an M ed ce pt
Characterization of ADC drug-loaded peptides by LC-MS analysis. Characterization of payload positional isomers of ADC conjugated on native cysteines. IdeS digestion of ADC conjugated on native cysteines
Ac
Page 15 of 20
cr
i
Figure 1
LC 13.5
A
M an
us
Figure 1
LC1 18.3
HC1 HC 19.7 22.2
100
HC2
27.7
15.00
20.00
ce pt
10.00
Fc/2 12.0
B
LC 14.7
Ac
%
100
10.00
26.9
ed
%
23.0
15.00
LC1 19.6 Fd 22.6
20.00
HC3 32.1
25.00
30.00
35.00
Fd1
Fd2
Fd1a 26.2 Fd1b 27.4
Fd2b 33.9 Fd2a 32.5
25.00
30.00
35.00
Time
40.00
Fd3 39.7
40.00
Time
Page 16 of 20
i
Figure 2
B
26458
100
Fd1b
26458
%
%
100
us
Fd1a
M an
26476 26439
26500
Fd2a
C
27775
%
ce pt
100
0 26200
mass
ed
26250
26203 26384
26514
0
Ac
27750 28000
mass
26514 26576
26400
26600
mass
Fd2b
D
27775
100
27791 27813 27671 2796728124
0
26475
%
A
cr
Figure 2
27792 27813 0 27500 27750 28000
mass
Retention Time (min)
Theoretical mass (Da)
Measured Mass (Da)
Fd1a
26.2
26,459
26,458
Fd1b
27.4
26,459
26,458
Fd2a
32.5
27,775
27,775
Fd2b
33.9
27,775
27,775
Page 17 of 20
i
Figure 3
C 100
us
cr
Figure 3
[M+2H]
884.48 884.99 885.49
[M+3H]
884.99
[M+3H]
884.48 884.98 885.48
%
884.98 885.48
ed 886.50
608.63 0
100
700
800
850
900
917.83
918.16
919.16
900
1000
1100
100
700
911.45
786.76 750
800
850
900
918.16 918.83 917.82
918.49
919.16
918.83
920.48 m/z [M+2H]
935.78
1376.71
1000.40 1169.42
1233.32
0 900
m/z
1000
1100
1200
1300
m/z
1400
m/z
935.79 937.49
0
650
D [M+3H]
918.83 919.51
Ac
918.83
710.39
919.16
918.16 918.50
[M+3H]
917.84
%
750
m/z
ce pt
B
600
885.99
590.99
650
885.98
0
m/z
590.65
600
590.99 608.30
885.99
590.32
885.50 m/z
%
100
1200
1300
[M+2H]
E
1376.74
100
m/z
1400
%
A
885.99
590.66
%
[M+2H]
M an
590.33
[M+4H] 1018.08 1018.08 1018.58 1017.83 1017.58 1019.09 1018.59
[M+3H] m/z 1357.11
1017.571018.81
1358.12 1356.41
1031.83 1032.79
0 1000
1100
1200
1300
m/z
Page 18 of 20
M an
Figure 4
us
cr
i
Figure 4
Fd1a 71%
%
LC1 19.6
Fd 22.6
15.00
20.00
Fd2
Fd1a 26.2 Fd1b 27.4
Fd2b 33.9 Fd2a 32.5
25.00
30.00
35.00
or
Fd2a 17%
or
Fd3 39.7
40.00
Time
L15 : Cys220
Cys 226 L16: Cys 229
Paylaod
Ac
10.00
ed
LC 14.7
100
Fd1
ce pt
Fc/2 12.0
Fd1b 29%
Fd2b 83%
Page 19 of 20
M an
us
cr
i
Table 1
ed
Fd1a
ce pt
Drug-loaded peptides
HC L15 + 1 payload
Fd1b
Fd2a
Fd2b
HC L16 + 1 payload
HC L15 + 1 payload and HC L16 + 1 payload
HC L16 + 2 payloads
Mass measured
1766.96 Da
2750.49 Da
1766.96 Da and 2750.46 Da
4066.32 Da
Theoretical mass
1766.95 Da
2750.44 Da
1766.95 Da and 2750.44 Da
4066.22 Da
Ac
Table 1: Summary of the drug-loaded peptides identification in the Fd1 and Fd2 fractions after Lys-C digestion and LC-MS analysis
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