Characterization and detection of EPO-Fc fusion proteins using LC-MS

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Characterization and detection of EPO-Fc fusion proteins using LC-MS Natalia V. Mesonzhnik, Pavel V. V. Postnikov, Svetlana A. Appolonova, and Grigory I. Krotov J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00739 • Publication Date (Web): 02 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Journal of Proteome Research

Characterization and detection of EPO-Fc fusion proteins using LC-MS Natalia V. Mesonzhnik1, Pavel V. Postnikov2*, Svetlana A. Appolonova1, Grigory I. Krotov3 1

Institute of Pharmacy and Translational Medicine, Sechenov First Moscow State Medical University,

2-4 Bolshaya Pirogovskaya St., 119991 Moscow, Russia 2 Anti-Doping 3

Center, Elizavetinskiy per., 10/1, Moscow, 105005, Russian Federation.

NRC Institute of Immunology FMBA of Russia, 24 Kashirskoye highway, 115478, Moscow, Russia

*

Corresponding author: Dr. Pavel V. Postnikov, Departament of peptide doping and blood analysis, Anti-

Doping Center, Elizavetinskiy per., 10/1, Moscow, 105005, Russian Federation. Phone: +7 499 2677320. E-mail: [email protected]. ORCID: 0000-0003-3424-0582

KEYWORDS “Bottom-up”, “middle-up”, intact mass spectrometry analysis, IdeS, fusion protein identification, signature peptide, EPO-Fc. ABSTRACT EPO-Fc fusion proteins are potential drug candidates that have been designed for the treatment of anaemia in humans by stimulating erythrocyte production. Such compounds can be considered performance enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid-chromatography coupled with mass-spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that ‘spacer peptides’ can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-MS/MS methods. INTRODUCTION Numerous forms of recombinant erythropoietin have quickly become misused as doping agents by athletes in endurance sports to increase blood oxygen capacity. Some strategies have been offered to improve the pharmacological properties of EPO through the genetic and/or chemical modification of the native EPO protein. These proposed strategies include conjugation with polyethylene glycol (PEG), formation of a homodimer of two EPO molecules using peptide or chemical cross-linkers, and linking the EPO molecule to a carrier protein (e.g., human albumin) or immunoglobulins (IgGs) [1–4]. The EPO-Fcs are fusion proteins comprised of monomeric or dimeric recombinant erythropoietin (EPO) and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. The two hinge regions of IgG are covalently linked by disulfide bonds. The EPO molecules and the Fc portion of IgG can be attached through a linker, which may consist of between 2 to 16 amino acid residues [5–7]. It has been shown that recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared to native

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or rhEPO. The Fc-fused EPOs can potentially be used in clinical practice for anaemia treatment. For example, a phase I clinical trial with EPO-hyFc is currently ongoing in Korea [8]. Despite the lack of information about successful clinical trials with drugs based on EPO-Fc, they are offered by a number of biotechnological companies for research purposes. The complete amino acid sequences of the available EPO-Fс proteins are usually unknown and proprietary. Some general information can be found in patents and other official documents [5, 6, 9]; these mostly contain the complementary determining regions, the variable domain sequences or linker descriptions. In fact, marketed versions of EPO-Fc can vary significantly depending on the manufacturer’s bioproduction. These could affect the final physico-chemical characteristics and function of the produced product. To the best of our knowledge, there is no reference EPO-Fc approved in the EU. There is an urgent need to obtain primary structure information and to develop a reliable analytical method for the determination of EPO-Fc abuse in sport. LC-MS represents an attractive alternative to the traditional techniques for protein drug analysis like ligand-binding assays. It is widely used in pharmacokinetics and toxicokinetic studies of protein therapeutics because of its high selectivity, sensitivity and specificity, as well as its rapid and relatively cost-effective method development. Additionally, LC-MS offers a great potential to analyse proteins that are both intact and proteolyzed. The latest approach represents the clear majority of LC/MS applications that are commonly performed for pharmacokinetic and toxicokinetic studies [10-16]. In 2012 the possibility of EPO-Fc detection using nano-HPLC-MS/MS was already demonstrated [17]. However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In addition to this approach some new approaches for determination of EPO-Fc using electrophoretic techniques were developed [18]. The goal of this study is to identify peptides covering unknown fusion breakpoints (below, ‘spacer’ peptides). Identification of ‘spacer’ peptides will allow us to unequivocally confirm the presence of exogenous EPO-Fc in human biological fluids. Together with the data obtained at the protein level, these findings provide the first insights into the primary structure of EPO-Fc fusion proteins. EXPERIMENTAL PROCEDURES Chemicals All solutions and buffers were prepared with Milli-Q water (Millipore). The formic acid, trifluoroacetic acid (ACS reagent grade), D, L-dithiotreitol (DTT), ammonium bicarbonate, iodoacetamide (IAA), dimethyl pimelimidate dihydrochloride (DMP), ethylenediaminetetreacetic acid (EDTA, free acid), sodium azide and human AB serum were purchased from Sigma-Aldrich (St Louis, MO, USA). Glacial acetic acid, hydrochloric acid (36%), methanol and acetonitrile were obtained from Merck (Darmstadt, Germany). Borate buffer (pH 8.5) was received from Pierce/Thermo Scientific (Rockford, IL, USA). Protein LoBind sample tubes (0.5 and 1.5 ml), Thermomixer Comfort were from Eppendorf (Hamburg, Germany). Urea and tris(hydroxymethyl)aminomethane (PlusOne) were purchased from GE Healthcare (Uppsala, Sweden). The IdeS protease, protein deglycosylation mix, Glu-C protease sequencing grade, endoproteinase Lys-C sequencing grade, sequencing grade modified trypsin, Magne™ Protein G Beads (20 % slurry) and Magne™ Protein A Beads (20 % slurry) were obtained from Promega Corporation


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(Madison, WI, USA).The phosphate-buffered saline (PBS) tablets were purchased from Amresco (Solon, Ohio, USA). Steriflip microfiltration device was purchased from Millipore (Billerica, MA). Mouse IgG2A anti-EPO antibodies (clone AE7A5) was obtained from R&D Systems (Minneapolis, MN, USA). The recombinant human EPO-Fc chimeric protein was received from Cell Sciences Technology (Canton, MA, USA). Sample processing Bottom-up To reduce the disulfide bonds, 2.5 μL of 200 mM DTT (in water) was added to 30 μL of 100 mM ammonium bicarbonate buffer (for tryptic and Glu-C digestion) or 100 mM Tris-HCl pH 8.5 (for Lys-C digestion) containing 5 μg of EPO-Fc and 15 μL 8 M urea. The reaction mixtures were incubated for 60 min at 37 °C and spun at 450 rpm. Then, 4 μL of 500 mM iodoacetamide was added and incubated for 30 min in the dark while spinning at 450 rpm. To stop the alkylation reaction, an additional 1 μL of 500 mM DTT was added. For proteolytic digestion, the solution was diluted 3-fold with the appropriate buffer and a solution of protease was added at a 1:40 ratio. Then, the mixtures were incubated overnight at 37 °C and while spinning at 450 rpm. The samples were diluted with mobile phase solution at a ratio of 1:4 (v:v) and immediately analysed by LC-MS/MS. Intact and middle-up / deglycosylation Purification of the Fc portion from IdeS-treated EPO-Fc was done with Magne™ Protein G beads (Promega). First, 3 μL of protein deglycosylation mix (Promega) was added to 5 μg of an EPO-Fc in PBS buffer (45 μL, pH 7.4). This was incubated overnight at 37 °C while spinning at 450 rpm. Then, 2.5 μL of IdeS protease stock solution (12.5 U) was mixed with deglycosylated EPO-Fc samples. After the reaction mixture was incubated for 1 h at 37 °C, the reaction volume was adjusted to 500 μL with PBS (pH 7.4) in a 1.5 mL Eppendorf LoBind tubes. Magnetic Protein G beads (15 μL; Promega) were washed 3 times with 200 μL of PBS using a magnetic tripod and added to the reaction volume. The tubes containing the reaction mixture were incubated at RT for 2 h to bind the Fc portion of EPO-Fc. Then, the samples were washed 4 times with PBS using a magnetic rack, and the Fc portion was eluted by adding 100 μL of 2 % CH3COOH in water. Finally, the samples were analysed by LC-MS. The same protocol was used for the MW measurement of whole deglycosylated fusion protein without the Ide-S digestion step. Purification of the EPO-Fc from human serum with Protein A magnetic beads crosslinked with antiEPO antibodies First, 500 μl of Magne™ Protein A beads 20% slurry (Promega) was washed 5 times with 1 mL of PBS using a magnetic tripod to remove storage solvent. Then, it was resuspended in 300 μl of PBS (1.5 mL Eppendorf LoBind tubes). After that, 200 μl of mouse anti-EPO monoclonal antibodies (clone AE7A5, R&D Systems) were added and the tubes were rotated for 3 h at 37 °C (Stuart rotator; 40 rpm). The supernatant was discarded and the beads were washed 3 times with 500 μl of PBS and twice with 1 mL of 50 mM borate buffer (pH 8.5). Magnetic protein A beads were resuspended in 1 mL of freshly prepared 25 mM DMP in 50 mM borate buffer (pH 8.5), incubated for 45 min at room temperature (Stuart rotator; 40 rpm), washed twice with 50 mM Tris-HCl buffer (pH 7.5) and rotated at room temperature for 30 min. Finally,



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the supernatant was discarded and the beads were washed 3 times with 1 mL of PBS while stirring on a vortex for 1 min before being resuspended in 500 μl of storage buffer (PBS with 0.05% NaN3, 2-8 °C) before the experiment. For the experiment, 500 ng of EPO-Fc was spiked with 150 μl of microfiltered (Steriflip) human serum (Sigma-Aldrich) and 400 μl of PBS (in 1.5 mL Eppendorf LoBind tubes). Samples were taken in duplicate. 240 μl of each magnetic protein A beads cross-linked with mouse IgG2A anti-EPO antibodies was washed 3 times with 500 μl of PBS using a magnetic tripod. The final bead volume was adjusted with PBS to the starting volume of the slurry. 60 μl of each kind of magnetic beads was added to each of the four samples. Model samples were shaken well and rotated for 3 h at 37 °C (Stuart rotator; 40 rpm). Next, beads were separated from the serum using a magnetic tripod and washed 5 times with 500 μl of PBS. To elute bound EPO-Fc, the beads were treated with 50 μl of 2% acetic acid (in water) while rotating on a vortex for 3 min. Eluates were collected in 0.5 mL Eppendorf protein LoBond tubes and neutralized with 15 μl of 2 M Tris-HCl buffer (pH 8.5). 45 μl of 100 mM Tris-HCl buffer (pH 8.5) was added to each sample. For denaturing and reducing disulfide bonds, 3.5 μl of 500 mM DTT was added into each sample, and the samples were incubated for 30 min at 60 °C (Thermomixer Comfort, Eppendorf, 450 rpm). 5.5 μl of 500 mM IAA was used as an alkylation agent (30 min, RT in dark). The total volume of the samples was adjusted to 150 μl with 100 mM Tris-HCl buffer (pH 8.5). Next, the total amount of protein in the samples was measured using a NanoDrop-2000 spectrophotometer (Thermo Scientific), and endoproteinase Lys-C was added at a ratio 1:40 to each sample. After overnight incubation (18-20 h, 37 °C, 450 rpm, Thermomixer Comfort), samples were analysed by LC/MS-MS. Liquid chromatography – mass spectrometry The Ultimate 3000 nano-LC system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for intact and fragmented MS analysis. LC separation of the analytes was performed using an Acclaim PepMap 100A, C-18 (75 μm × 2 cm, 3 μm) trap column and an Agilent Zorbax 300SB-C18 (75 μm × 15 cm, 3.5 μm) analytical column. The deglycosylated or deglycosylated/IdeS digested EPO-Fc samples were eluted for 20 min, and the proteolytic-digested EPOFc samples were eluted for 40 min using a linear gradient (5 % B–95 % B) at a flow rate of 0.350 μL/min. Solvent A was HPLC-grade water with 0.1% FA, and solvent B was 0.1% FA in ACN/H 2O (80/20 v/v). The ion source parameters were as follows: capillary temperature of 275 °C, spray voltage of 2.1 kV, and the S-lens level of 50 (peptides) or 80 (proteins). Injection volume was 1 μL. To determine the proteolytic peptides, the Q Exactive was operated in top 3 data-dependent acquisition mode: the full MS resolution was 35 000 (at m/z 200), and the scan range was 300 – 1500 m/z. Each targeted precursor was isolated using a 3 m/z unit window and fragmented by HCD (NCE of 30%). The MS/MS scans were acquired at a resolution of 17 500 (at m/z 200), with a starting mass of m/z 100. The full MS data from 1000 to 3000 m/z were collected for the MW determination of deglycosylated EPO-Fc or IdeS-treated EPO-Fc the lowest resolution settings at 17 500 (at m/z 200). The instrument was operated with an AGC target setting of 3e6 and a maximum IT setting of 150 ms for 10 microscans. The monoisotopic mass of the Fc/2 fragment was obtained using multiplexed (5-plex) target-SIM experiment on five selected charge states (10+ - 14+) at a resolution of 140 000 (at m/z 200). The AGC target was 5E5, maximum ion injection time was 500 ms, and the isolation width for SIM scan was 4 amu.


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The qualitative analysis of EPO-Fc in serum samples was performed using Nexera X3 LC (Shimadzu Corporation, Japan) coupled with LCMS-8050 triple quadrupole (Shimadzu Corporation, Japan). Nexera X3 LC system consisted of two LC-30AD pumps, GRU-20A5R degasser, SIL-30AC autosampler, CTO-20AC column oven and CBM-20A control module. The separation was performed using a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm; 1.7 μm) and a gradient elution with mobile phase A (0.1 % formic acid aqueous solution) and mobile phase B (acetonitrile with 0.1% formic acid aqueous solution). The gradient was as follows: 0 min (4 % B), 5 min (10 % B), 20 min (50 % B), 24 min (90 % B), 29 min (90 % B), 30 min (4 % B), 40 min (90 % B). Total run time for each sample analysis was 40 min with a constant flow rate of 0.2 mL/min. The injection volume of the sample was 3.0 μL. Mass spectra were obtained using positive electrospray ionization and the multiple reaction-monitoring (MRM) mode. Nitrogen was used as the collision gas. MS operating parameters were optimized as follows: desolvation temperature: 250 °C; heat block temperature: 400 °C; interface temperature: 300 °C; nebulizer gas flow rate: 3 L/min; heating gas flow rate: 10 L/min and drying gas flow rate: 10 L/min. Data analysis Data were acquired using latest version of Thermo Scientific™ Xcalibur™ software. ProMass Deconvolution software (Thermo Scientific) was used to deconvolute deglycosylated EPO-Fc and IdeS proteolytic fragments spectra that contained unresolved isotopic clusters in production of an average zero charge masses of the proteins. The deconvolution of the msxSIM spectrum of Fc/2 was performed using the Xtract algorithm (Xcalibur, Thermo Scientific) operated in monoisotopic mass mode by averaging 20 scans across the most abundant portion of the LC peak of the protein fragment. Database search was performed with PEAKS Studio software (version 7.0, Bioinformatics Solutions, Waterloo, Canada). The MS/MS data were used to search the UniProtKB database release 17.0. Search constraints were as follows: trypsin, Lys-C cleavage or Glu-C protease (bicarbonate); three missed cleavage allowed; tolerances (10 ppm for precursor ions, and 0.05 Da for MS/MS fragments ions); FDR PSM, 0.1%, FDR PS, 1.0%; fixed modification: carbamidomethylation (C) and variable modifications: deamidation (N or Q), oxidation (H, W or M), carbamylation. In all cases, the fragment ion assignments of newly reported peptides were manually inspected. RESULTS AND DISCUSSION Bottom-up The commercially available recombinant human EPO-Fc (Cell Science Technology (Canton, MA, USA) protein was purchased from the vendor’s web site and stored according to the supplier’s recommendations. The EPO-Fc was subjected to digestion using multiple proteases, and the resulting surrogate peptides were individually analysed using nanoscale liquid chromatography coupled with electrospray ionization high resolution tandem mass spectrometry. The fusion EPO-Fc protein was originally characterized using tryptic hydrolysis. Several obtained peptide spectra were used to examine the structure of the EPO-Fc molecule. The dataset obtained from HCD-based tandem HRMS acquired in data-dependent scan mode was processed using PEAKS7 software searching against the UniProt human database in parallel with a de novo sequencing algorithm. Obtained specific sequences were verified by manually inspecting the corresponding HRMS/MS spectra. It was found



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that the tryptic digest derived from the EPO-Fc sample includes characteristic peptides relevant to human EPO (P01588) and human IgG2 (P01859). Sequence coverage with recognized peptides was equal to 53% for EPO (62% without Met1-Gly27 signal peptide) and 67% for IgG2 (96% without Ala1-Val98 CH1 region). The characteristic tryptic peptides representing the hinge region, CH2 and CH3 domains of IgG2 are shown in Table 1. Table 1. IgG2-characteristic tryptic peptides founded in EPO-Fc sample Score Hinge

-10 LgP

Mass

ppm

m/z

RT

Protein

k.C(+57.02)C(+57.02)VEC(+57.02)PPC(+57.02)PAPPVAGPSVFLFPPKPK.d 100.0

CH2

113.5

2907.3943

-0.6

727.8554

34.0

sp|P01859|IGHG2_HUMAN

r.TPEVTC(+57.02)VVVDVSHEDPEVQFNWYVDGVEVHNAK.t 99.9

99.7

CH3

3796.7678

2.3

1266.5995

35.4


k.TTPPMLDSDGSFFLYSK.l 99.9

69.2

CH3*

1904.8866

1.0

635.9701

34.4


k.GFYPSDIAVEWESNGQPENNYK.t 99.9

73.4

2543.1240

-2.1

1272.5667

33.2


The exception was the peptide k.GFYPSDIAVEWESNGQPENNYK.t that refers to CH3 domain of human IgG1 (254-275 position of UniProt entry P01857). Both subclasses share more than 95% homology in the amino acid sequences of the Fc regions, but they also show major differences in the amino acid composition of the hinge region (Table S1). The amino acid sequence of the mentioned peptide IgG1 differs from that of IgG2 in one position: 261(A→S). At the same time, the presence of an alanine to serine substitution is described as a sequence conflict in position 257 of IgG2 sequence matched to three of the four known isoallotypic variant of IgG2 at that position (UniProt entry P01859) [19,20]. Therefore, this tryptic peptide cannot discriminate two IgG subclasses. The tryptic digest of EPO-Fc reveals C-terminal lysine clipping by the presence of a C-terminal glycine residue (Figure S1). A single Lys-containing peptide was also detected. It has been shown that the lysine residue is processed in vivo by an endogenous carboxypeptidase B. Recombinant IgG molecules that are produced in mammalian cell cultures can contain from zero to two C-terminal Lys residues. The roles of these truncated proteins remain to be addressed [21-23]. Unique peptides from the linkage area of fusion proteins were not observed. This is presumably due to the close location of trypsin cleavage sites (arginine, lysine) to the EPO/IgG2 docking region. As mentioned above, neither of the tryptic peptides derived from EPO and IgGs could be used as specific markers of EPO-Fc due to their endogenous nature. The proteases Glu-C and Lys-C were used to perform hydrolysis in the other manner, which probably can release spacer peptides from fusion region of EPO-Fc. In the case of Glu-C, protein hydrolysis was carried out with a bicarbonate buffer solution to digest preferentially at the C-terminus of glutamic acid residues located close to the C- and N-termini of EPO and IgG2 hinge region. Following spectrum-tosequence assignment, the number of peptide sequences was determined for both EPO and IgG2. Evaluation of the mass spectrometry data revealed the presence of the expected Glu-C ‘spacer peptide’ of the fused area of EPO-Fc. The described peptide was eluted at 13.5 min within the nano-LC profile and


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observed as doubly/triply ion pair at m/z 554.74372+/370.16493+ (Table 2, Figure S2). Analysis of the product-ion spectrum generated from the doubly protonated peptide allowed the identification of a nearly complete b and y ion series consistent with the sequence ‘e.ACRTGDRGSE.r’, as highlighted in Figure 1. The intensive formation of [bn−1+H2O] at m/z 490.2230 (1.43 ppm) was observed due to loss of C-terminal glutamate residue. The gap between the 4th and 6th amino acids can be explained according to the exact mass as a combination of aspartic acid and glycine (-2.3 ppm). Thus, the derived sequence covers the Cterminal region of EPO (P01588) starting from position Ala18 and ending at the N-terminal glutamic acid of IgG2 (P01859) hinge region at position 99. These are connected to each other by a 2-amino acid linker [GS].

Figure 1. HCD spectra (NCE of 30%) obtained from doubly charged precursor presented at full mass range (top panel) and split mass ranges of (middle and bottom panels) and b/y fragments assignment of peptide related to fusion area found in EPO-Fc Glu-C-digest It is hypothesized that the cleavage of the leader sequence and the last amino acid Arg by posttranslational modification yields the mature EPO [24]. According to our examination EPO-Fc still contains the C-terminal arginine that was observed in the ‘spacer’ Glu-C peptide. Moreover, the existence of Arg 166 was reported both for urinary hEPO [25] and EPO biosimilar products [26].



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Table 2. Peptides related to fusion area identified after Glu-C and Lys-C proteolysis in EPO-Fc sample RT, min

Elemental composition

m/z (z) Observed

Theoretical

Error, ppm

e.ACRTGDRGSE.r, Glu-C bicarbonate 13.5

C40H69N17O18S

554.7437 (2+)

554.7436 (2+)

0.2

370.1649 (3+)

370.1648 (3+)

0.3

k.LYTGEACRTGDRGSERK.c, Lys-C

14.5

C78H130N28O29S

978.4720 (2+)

978.4712 (2+)

0.8

652.6497 (3+)

652.6499 (3+)

-0.3

489.7401 (4+)

489.7393 (4+)

1.6

392.9934 (5+)

392.9929 (5+)

1.3

The findings were further confirmed using Lys-C endoproteinase. After hydrolysis of EPO-Fc by the enzyme, the fragments containing the C-terminal lysine were cleaved off, and we found 17 amino acid ‘spacer’ peptide catching the core hinge portion of IgG2 and portion from the C-end of EPO. The multiplycharged ion spectrum of the Lys-C peptide at RT 14.5 min gave a series of multicharged ions at m/z 978.47202+, 652.64973+, 489.74014+, and 391.99345+ (Table 2, Figure S3). Based on multiplexed HCD spectra, the peptide was assigned as k.LYTGEACRTGDRGSERK.c. The fragmentation pattern presented at Figure 2 provides an intense series of y-ions and a small number of less intensive complimentary b-ions.


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Figure 2. Multiplexed HCD spectra (NCE of 30%) obtained from 2+, 3+, 4+ charged precursors presented at split mass ranges and b/y fragments assignment of peptide related to fusion area found in EPO-Fc LysC-digest The peptide derived from Lys-C contains four basic amino acid residues. Basic side chains might lead to rearrangement, and cyclization reactions result in a larger number of fragment ion types and additional ion series that complicate data interpretation. Confirmation of both the ‘spacer’ peptide sequences was achieved using synthetic standards (Thermo Fisher Sci, purity >98%, Figures S4, S5). Summarizing the data from bottom-up experiments, we have suggested that the protein of interest is a chimera of EPO C-termini joined to the hinge N-terminus of IgG2 through the mono dipeptide glycylseryl linker [27, 28].



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Intact and middle-up LC-MS of deglycosylated EPO-Fc The intact and IdeS middle-up LC-MS analysis of deglycosylated EPO-Fc was employed to corroborate the postulated structure. An initial attempt to analyse the whole Fc-fusion EPO molecule on LC-MS was unsuccessful. This was due to high heterogeneity of the molecule glycosylation that resulted in the inability to acquire any resolvable spectrum. Commercially available EPO preparation is usually expressed in mammalian cell lines like Chinese hamster ovary (CHO) cells. EPO expressed in CHO cell lines can contain different patterns and compositions of glycan structures (e.g., number of glycan residuals or amount of sialic acid in the glycan tree) compared to human serum EPO. In addition, the EPO-Fc molecule contains the glycosylation site in the CH2 domain of IgG [6]. To simplify the interpretation of the intact MW measurements, the heterogeneity was reduced using a protein deglycosylation mix from Promega (a mixture of PNGase F, Oglycosidase, neuraminidase, β1-4 galactosidase, β-N-acetylglucosaminidase) [29]. The raw deglycosylated EPO-Fc that was analysed by LC-MS showed the presence of main peak at RT=24.2. ESI conditions yielded multiply charged ions with m/z ratios between 1500-3000. The mass spectrum is presented in Figure 3a. The results from mass spectra deconvolution (Figure 3b) revealed two mass shifts with ~ 657 Da each. Since glycosylated species were detected, it is assumed that the deglycosylation process was incomplete. The absence of side nonspecific reactions and much milder conditions are the advantages of the enzymatic methods of removing glycans. Complete cleavage of the alpha O-glycosidic bond of N-acetylglucosamine (GlcNAc) and N-acetylgalactosaminoglycans (GalNAc) can be reached by affecting of several exoglycosidases. This is in contrast to the deglycosylation of Nlinked glycans, which can be carried out with a certain N-glycanase. In this case, the reaction yield is close to 100% [30]. GlcNAc and GalNAc are linked through the OH-group with hydroxyamino acids serine or threonine of polypeptide chain of mammalian glycoproteins, as is the case of EPO-Fc isolated from Chinese hamster ovary (CHO) cells. However, the presence of bulky GalNAc and sialic acids (Neu5Ac) in the structure of glycan Neu5Ac-Gal-GalNAc(Neu5Ac)-O-Ser/Thr of the EPO-Fc molecule, can block the action of O-glycosidases [31, 32] due to steric and conformational obstacles, and the yield of the desired reaction products is very low [30]. Thus, it may be necessary to conduct more severe chemical deglycosylation, like periodate oxidation, beta-elimination, the use of anhydrous hydrazine and trifluoromethanesulfonic acid (TFMS) to remove O-glycan more completely. The observed mass of 88,175.2 Da that was obtained after spectral deconvolution was assigned as EPO-Fc aglycone. It correlated well with the theoretically calculated mass of 88,175.6 Da after considering previously discovered modifications in the amino acid sequence of EPO-Fc and preprocessing changes. The presence of the [GS] linker increased the mass a total of 126 Da to the summarized masses of EPO and IgG2 Fc chains. EPO contains 4 cysteines (2 disulfide bonds) and IgG2 Fc contains 8 cysteines (4 disulfide bonds). Thus, a total of 12 Da per chain should be subtracted during MW calculation. The clipping of the C-terminal lysine results in a mass decrease of 128 Da per chain, and the amino acid substitution with Ala at position Ser 257 of IgG2 gives a 16 Da theoretical difference per chain. The three asparagine residues in the EPO molecule and the single site in the Fc undergo deamidation to yield aspartic acid during the deglycosylation process.


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a

b

Figure 3. Full MS spectrum (a) and deconvolution results (b) of the deglycosylated EPO-Fc The use of immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) is a straightforward approach to fast mass-spectrometry characterization of antibodies and antibody drug conjugates. IdeS specifically cleaves the human IgGs Fc-hinge domain between Gly-236 and Gly-237 [33, 34]. Under non-reducing conditions, IdeS digestion of EPO-Fc should generate three polypeptide chains: two of reduced Fc-fragments (Fc/2) and one of disulfide-linked EPO, containing both hinge residual and glycyl-serine linker (EPO-hinge). The resulting fragments were well resolved by chromatography and identified according to their MS profile (Figure 4, 5). The first eluting peak at 20.75 min (23,789.7 Da) corresponds to the Fc/2 fragment of IgG. The utilized high-resolution mass-spectrometry allowed us to obtain the monoisotopic mass of the analysed fragment. As shown in Figure 4b (insert), the measured monoisotopic mass of the Fc/2 was determined to be 23774.8262 Da. The measured MWs are consistent with the lysine clipping and S→A substitution with sub ppm MW errors.



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a

b

Figure 4. Full MS spectrum (a) and deconvolution results (b) of deglycosylated IdeS-proteolytic fragment, Fc/2. The Fc/2 fragment, detected at 17,500@200 resolution, was deconvoluted with Promass providing average masses. The Fc/2, acquired at a resolution setting of 140,000@200 in multiplexed (5-plex of 10+ - 14+ charge states) SIM mode, was deconvoluted using the Xtract algorithm, obtaining an accurate monoisotopic mass (insert). The deconvoluted mass spectrum (Figure 5b) of the second chromatographic peak at 25.2 min displays the same profile as observed for intact EPO-Fc. The mass of 40,631.9 Da corresponds to the IdeSreduced EPO-Fc fragment and matched to calculated mass with 0.7 Da error. These findings suggested that the glycan modification can be located on the EPO-hinge region of EPO-Fc. a

b

Figure 5. Full MS spectrum (a) and deconvolution results (b) of deglycosylated IdeS-proteolytic fragment, EPO-hinge. Determination of EPO-Fc in human serum samples Specificity of Glu-C protease depends strongly on the experimental conditions. The exchange of buffer solution or a change in pH can change the endoproteinase’s behaviour and result in undesirable cleavages of Asp in position P6. It could lead to significant challenges in the development of a method suitable for routine analysis. Lys-C protease has high activity and specificity for lysine residues, and it does not exhibit the mentioned disadvantages. The sequence LYTGEACRTGDRGSERK was submitted for a BLAST search against the NCBI non-redundant and SwissProt/UniProt databases. The peptide shares 70% identical amino acids with EPO


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human (P01588). 70 to 88% query similarity with ‘synthetic constructs’ was found. These findings indicated that this ‘spacer’ peptide is unique for EPO-Fc. The analytical interference of selected EPO-Fc peptide in serum was examined by the “spike-in” experiments. EPO-Fc were captured from blank and spiked serum samples using Protein A magnetic beads cross-linked to mouse IgG2A anti-EPO antibodies. Next, eluates were digested using Lys-C protease and assayed by LC-MS/MS method. To achieve the highest selectivity, all characteristic ions had m/z with value greater than the respective parent. The final MRM pairs of spacer peptides were two y-ions (y153+, y163+) with better MS response than b-ions due to the presence of basic amino acids. Representative MRM chromatograms of the EPO-Fc ‘spacer’ peptides are shown in Figure 6.

Figure 6. LC-MS/MS representative MRM TIC chromatograms of unique “spacer” peptide derived from EPO-Fc spiked human serum sample (black), and from blank human serum (grey). MRM TIC is the sum of the signal for 489.7→560.6 (y153+) and 489.7→614.9 (y163+)

No analyte ions were found in the blank serum samples. This indicated that serum matrices do not interfere with the determination of the EPO-Fc fusion protein. This application of LC-MS/MS demonstrates the utility of this technology for EPO-Fc analysis in biological samples. On the other hand, further improvements in sensitivity are required. CONCLUSION In conclusion, we demonstrated an approach to characterize EPO fusion proteins with unknown structures. The strategy included comparative digestion with different prototypic proteases and MW measurements at the protein level. We elucidated the primary structure of EPO-Fc, which is characterized as a chimaera with two EPOs linked to the Fc portion of IgG2 through the mono dipeptide glycyl-seryl linker. Peptides covering unknown fusion breakpoints were obtained and confirmed using synthetic standards. In addition, intact MW measurements of deglycosylated EPO-Fc and its reduced fragments contributed to the full sequence assignments. High resolution mass spectrometry helped to accurately measure Fc/2 fragment proved its nature and modification occurred in the sequence.



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The spike-in experiments revealed the possibility of using ‘spacer’ peptides for the determination of EPO-Fc fusion proteins in human matrices. The analysis of individual fragments of EPO-Fc by LC-MS/MS can be an alternative approach to the traditional ligand-binding assays with advantages to evaluate the specific type of EPO-Fc fragment.

ACKNOWLEDGEMENTS The authors thank Dr Natalia Moskaleva from Institute of Pharmacy and Translational Medicine for her technical support.

SUPPORTING MATERIALS The following files are available free of charge at ACS website http://pubs.acs.org. Table S1. Human IgGs alignment. Figure S1. C-terminal lysine clipping in EPO-Fc. The EICs (5 ppm mass tolerance window ) and HCD spectra of SLSLSPGK ([M+2H]2+) (bottom) and its truncated form SLSLSPG ([M+H]+) (top). Figure S2. High resolution full scan spectrum of the Glu-C ’spacer’ peptide related to EPO-Fc fusion area (RT 13.5). The EICs of [M+2H]2+ and [M+3H]3+ species in 5 ppm mass tolerance window. Figure S3. High resolution full scan spectrum of the Lys-C ‘spacer’ peptide related to EPO-Fc fusion area (RT 14.5). The EICs of peptide multiply ion species in 5 ppm mass tolerance window. Figure S4. Comparison of HCD spectra (NCE of 30%) obtained from doubly-charged precursors of peptide ACRTGDRGSE related to fusion area found in EPO-Fc Glu-C-digest (top panel) and synthetic standard (bottom panel) after IAA alkylation. Figure S5. Comparison of HCD spectra (NCE of 27%) obtained from four fold charged precursors of peptide LYTGEACRTGDRCSERK related to fusion area found in EPO-Fc Lys-C-digest (top panel) and synthetic standard (bottom panel) after IAA alkylation.

CONFLICT OF INTEREST The authors have no conflict of interest to declare.


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REFERENCES 1. Kochendoerfer, G. G.; Chen, S. Y.; Mao, F.; Cressman, S.; Traviglia, S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.; Carnevali, M.; Gueriguian, V.; Keogh, P. J.; Porter, H.; Stratton, S. M.; Wiedeke, M. C.; Wilken, J.; Tang, J.; Levy, J. J.; Miranda, L. P.; Crnogorac, M. M.; Kalbag, S.; Botti, P.; Schindler-Horvat, J.; Savatski, L.; Adamson, J. W.; Kung, A.; Kent, S. B.; Bradburne, J. A. Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein. Science 2003, 299, 884–887. 2. Sytkowski, A. J.; Lunn, E. D.; Davis, K. L.; Feldman, L.; Siekman, S. Human erythropoietin dimmers with markedly enhanced in vivo activity. Proc. Natl. Acad. Sci. USA 1998, 95, 1184–1188. 3. Sytkowski, A. J.; Lunn, E. D.; Risinger, M. A.; Davis, K. L. An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties. J. Biol. Chem. 1999, 274, 24773– 24778. 4. Shi, X.; Yang, J.; Zhu, H.; Ye, L.; Feng, M.; Li, J.; Huang, H.; Tao, Q.; Ye, D.; Sun, L. H.; Sun, B. N.; Sun, C. R.; Han, G.; Liu, Y.; Yao, M.; Zhou, P.; Ju, D. Pharmacokinetics and pharmacodynamics of recombinant human EPO-Fc fusion protein in vivo. PloS One 2013, 8(8), e72673. DOI: 10.1371/journal.pone.0072673. 5. US Patent 20050202538, A1. Fc-erythropoietin fusion protein with improved pharmacokinetics. Available at: http://www.google.com/patents/US20050202538 [11 October 2017]. 6. US Patent 8431132, B2. Recombinant human EPO-FC fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo. Available at: http://www.google.com/patents/US8431132 [13 October 2017]. 7. Dalle, B.; Henri, A.; Rouyer-Fessard, P.; Bettan, M.; Scherman, D.; Beuzard, Y.; Payen, E. Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo. Blood 2001, 97, 3776– 3782. 8. Yang, S. H.; Sang, I. Y.; Chung, Y. K.. A long-acting erythropoietin fused with noncytolytic human Fc for the treatment of anemia. Arch. Pharm. Res. 2012, 35(5), 757–759. 9.

Recombinant

Human

EPO-alpha

Fc

Chimera.

Cell

Sciences

Datasheet.

Available

at:

http://www.cellsciences.com/PDF/CSI20107.pdf [08 October 2017]. 10. Chen, G.; Liu, Y.-H.; Pramanik, B. N. LC/MS analysis of proteins and peptides in drug discovery. In HPLC for Pharmaceutical Scientists; Kazakevich, Y., LoBrutto, R. Eds.; Wiley: New York, 2007; pp. 837– 899. 11. Chen, G.; Mirza, U. A.; Pramanik, B. N. Macromolecules in drug discovery: mass spectrometry of recombinant proteins and proteomics. In Advances in Chromatography; Grushka, E., Grinberg, N. Eds.; CRC Press, 2009, 47, pp. 1–30.



Page 16 of 24

12. Gillette, M. A.; Steven, A. C. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nature methods 2013, 10(1), 28–34. 13. Qu, M.; An, B.; Shen, S.; Zhang, M.; Shen, X.; Duan, X.; Balthasar, J. P.; Qu, J. Qualitative and quantitative

characterization

of

protein

biotherapeutics

with

liquid

chromatography

mass

spectrometry. Mass spectrometry reviews 2016, DOI: 10.1002/mas.21500. 14. van den Broek, I.; Niessen, W. M.; van Dongen, W. D. Bioanalytical LC–MS/MS of protein-based biopharmaceuticals. J. Chromatogr. B. 2013, 929, 161–179. 15. van den Broek, I.; van Dongen, W. D. LC–MS-based quantification of intact proteins: perspective for clinical and bioanalytical applications. Bioanalysis 2015, 7(15), 1943–1958. 16. Kang, L.; Camacho, R. C.; Li, W.; D`Aquino, K.; You, S.; Chuo, V.; Weng, N.; Jian, W. Simultaneous catabolite identification and quantitation of large molecular therapeutic protein at intact level by immunoaffinity capture liquid chromatography-high resolution mass spectrometry (LC-HRMS). Anal. Chem. 2017, 89(11), 6065–6075. 17. Reichel, C.; Thevis, M. Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing. Drug Test. Anal. 2012, 4, 818–829. 18. Postnikov, P.; Krotov, G.; Mesonzhnik, N.; Efimova, Yu.; Rodchenkov, G. Fc-fragment removal allows the EPO-Fc fusion protein to be detected in blood samples by IEF-PAGE. Drug Test. Anal. 2015, 11–12, 999–1008. 19. Elison, J. W. Linkage and sequence homology of two human immunoglobulin gamma heavy chain constant region genes. PNAS 1982, 79, 1984–1988. 20. Vidarsson, G.; Dekkers, G.; Rispens, T. IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology 2014, 5, 520. 21. Tang, L.; Sundaram, S.; Zhang, J.; Carlson, P.; Matathia, A.; Parekh, B.; Zhou. Q.; Hsieh, M. C. Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry. mAbs 2013, 5(1), 114–125. 22. Harris, R. J. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J. Chromatogr. A 1995, 705, 129–134. 23. Liu, H.; Ponniah, G.; Zhang, H. M.; Nowak, C.; Neill, A.; Gonzalez-Lopez, N.; Andrien, B. In vitro and in vivo modifications of recombinant and human IgG antibodies. mAbs 2014, 6(5), 1145–1154. 24. Recny, M. A.; Scoble, H. A.; Kim, Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin. J. Biol. Chem. 1987, 262(35), 17156–17163.


Page 17 of 24 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


25. Lai, P. H.; Everett, R.; Wang, F. F.; Arakawa, T.; Goldwasser, E. Structural characterization of human erythropoietin. J. Biol. Chem. 1986, 261(7), 3116–3121. 26. Okano, M.; Sato, M.; Kageyama, S. Mass spectrometric characterisation of darbepoetin alfa biosimilars with C‐terminal arginine residues. Drug Test. Anal. 2016, 8(11–12), 1138–1146. 27. US Patent 8911954 B2. Polypeptide linker and method of analyzing target material using the same. Available at: http://www.google.sr/patents/US8911954 [13 October 2017]. 28. Grünberg, R.; Ferrar, T. S.; van der Sloot, A. M.; Constante, M.; Serrano, L. Building blocks for protein interaction devices. Nucleic acids Res. 2010, 38(8), 2645–2662. 29. Promega Corporation. Protein Deglycosylation Mix. Available at: https://worldwide.promega.com//media/files/resources/protocols/product-information-sheets/n/protein-deglycosylation-mix-protocol.pdf [26 September 2017]. 30. Patent RU 2 509 807 C1. Method of detecting O-glycosylated proteins in cell homogenates prepared for proteomic and phosphoproteomic analysis. Available at: http://www.freepatent.ru/images/patents/508/2509807/patent-2509807.pdf [13 October 2017] 31. Prozyme Inc. Enzymatic Deglycosylation Kit for N-Linked & Simple O-Linked Glycans. Available at: https://prozyme.com/products/gk80110 [02 October 2017]. 32. I. Brockhausen; H. Schachter; P. Stanley. O-GalNAc Glycans, Chapter 9. In Essentials of Glycobiology. 2nd Ed.; Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Eds.; Cold Spring Harbor: NY, 2009, pp. 115–127. PMID: 20301232. 33. Vincents, B.; von Pawel-Rammingen, U.; Björck, L.; Abrahamson, M. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry 2004, 43, 15540–15549. 34. von Pawel-Rammingen, U.; Johansson, B. P.; Björck, L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J. 2002, 21, 1607–1615.



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FOR TOC ONLY

The structure of the monomeric EPO-Fc chain and proteotypic peptides which are formed by the bonding region.


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Figure 1. HCD spectra (NCE of 30%) obtained from doubly-charged precursor presented at full mass range (top panel) and split mass ranges of (middle and bottom panels) and b/y fragments assignment of peptide related to fusion area found in EPO-Fc Glu-C-digest

ACS Paragon Plus Environment


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Figure 2. HCD spectra (NCE of 30%) obtained from 2+, 3+, 4+ charged precursors presented at split mass ranges and b/y fragments assignment of peptide related to fusion area found in EPO-Fc Lys-C-digest


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a

b

Figure 3. FullMS spectrum (a) and deconvolution results (b) of deglycosylated EPO-Fc



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b a

Figure 4. FullMS spectrum (a) and deconvolution results (b) of deglycosylated IdeSproteolytic fragment, Fc/2 The Fc/2 fragment, detected at 17,500@200 resolution, was deconvoluted with Promass providing average masses (b).The Fc/2, acquired at a resolution setting of 140,000@200 in multiplexed (5-plex of 10+ - 14+ charge states) SIM mode, was deconvoluted using the Xtract algorithm, obtaining an accurate monoisotopic mass (insert).


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a

b

Figure 5. FullMS spectrum (a) and deconvolution results (b) of deglycosylated IdeSproteolytic fragment, EPO-hinge



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Figure 6. LC-MS/MS representative chromatograms of unique “spacer” peptide derived from EPO-Fc spiked human serum sample at a concentration of 500 ng/ml, and from blank human serum


Characterization and detection of EPO-Fc fusion proteins using LC-MS

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