Characterization and Detection of Erythropoietin Fc Fusion Proteins

Oct 13, 2017 - Doping Center, Elizavetinskiy per., 10/1, Moscow, 105005, Russian .... After that, 200 μl of mouse anti-EPO monoclonal antibodies (clo...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: J. Proteome Res. 2018, 17, 689−697

pubs.acs.org/jpr

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry Natalia V. Mesonzhnik,† Pavel V. Postnikov,*,‡ Svetlana A. Appolonova,† and Grigory I. Krotov§ †

Institute of Pharmacy and Translational Medicine, Sechenov First Moscow State Medical University, 2-4 Bolshaya Pirogovskaya Street, 119991 Moscow, Russia ‡ Anti-Doping Center, Elizavetinskiy per., 10/1, 105005 Moscow, Russian Federation § NRC Institute of Immunology FMBA of Russia, 24 Kashirskoye Highway, Moscow 115478, Russia S Supporting Information *

ABSTRACT: Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performanceenhancing 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−tandem MS methods. KEYWORDS: bottom-up, middle-up, intact mass spectrometry analysis, IdeS, fusion protein identification, signature peptide, EPO-Fc

■

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 erythropoietin (EPO) through the genetic and 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 composed of monomeric or dimeric recombinant 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 with native or rhEPO. The Fc-fused EPOs can potentially be used in clinical practice for anemia 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 © 2017 American Chemical Society

complete amino acid sequences of the available EPO-Fc 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 physicochemical 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. Liquid chromatography−mass spectrometry (LC−MS) represents an attractive alternative to the traditional techniques for protein drug analysis, such as 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 for the analysis of 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 Received: October 17, 2017 Published: December 2, 2017 689

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research

Intact and Middle-Up and 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 EPOFc 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 analyzed 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 Cross-Linked with Anti-EPO 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, 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. A total of 240 μL of each magnetic protein A beads cross-linked with mouse IgG2A antiEPO 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. A total of 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). A total of 5.5 μL of 500 mM IAA was used as an alkylation agent (30 min,

In 2012, the possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (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 the determination of EPOFc using electrophoretic techniques were developed.18 The goal of this study is to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides). The 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). 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). 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 of sequencing grade, endoproteinase Lys-C of sequencing grade, sequencing-grade modified trypsin, Magne Protein G beads (20% slurry), and Magne Protein A beads (20% slurry) were obtained from Promega Corporation (Madison, WI).The phosphate-buffered saline (PBS) tablets were purchased from Amresco (Solon, OH). A steriflip microfiltration device was purchased from Millipore (Billerica, MA). Mouse IgG2A anti-EPO antibodies (clone AE7A5) were obtained from R&D Systems (Minneapolis, MN). The recombinant human EPO-Fc chimeric protein was received from Cell Sciences Technology (Canton, MA). 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 at pH 8.5 (for Lys-C digestion) containing 5 μg of EPO-Fc and 15 μL of 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. Next, 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 analyzed by LC−MS/MS. 690

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research Table 1. IgG2-Characteristic Tryptic Peptides Found in EPO-Fc Samples score

−10 LgP

hinge 100.0

113.5

99.9

99.7

99.9

69.2

99.9

73.4

CH2 CH3 CH3*

mass

m/z

ppm

RT

protein

k.C(+57.02)C(+57.02)VEC(+57.02)PPC(+57.02)PAPPVAGPSVFLFPPKPK.d 2907.3943 −0.6 727.8554 34.0 sp|P01859|IGHG2_HUMAN r.TPEVTC(+57.02)VVVDVSHEDPEVQFNWYVDGVEVHNAK.t 3796.7678 2.3 1266.5995 35.4 sp|P01859|IGHG2_HUMAN k.TTPPMLDSDGSFFLYSK.l 1904.8866 1.0 635.9701 34.4 sp|P01859|IGHG2_HUMAN k.GFYPSDIAVEWESNGQPENNYK.t 2543.1240 −2.1 1272.5667 33.2 sp|P01857|IGHG1_HUMAN

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), and 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 of 250 °C, heat block temperature of 400 °C, interface temperature of 300 °C, nebulizer gas flow rate of 3 L/ min, heating gas flow rate of 10 L/min, and drying gas flow rate of 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 of carbamidomethylation (C) and variable modifications of deamidation (N or Q), oxidation (H, W or M), and carbamylation. In all cases, the fragment ion assignments of newly reported peptides were manually inspected.

room temperature in the 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 of 1:40 to each sample. After overnight incubation (18−20 h, 37 °C, 450 rpm, Thermomixer Comfort), samples were analyzed 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 EPO-Fc 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/H2O (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 three 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 IdeStreated 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. 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

■

RESULTS AND DISCUSSION

Bottom-Up

The commercially available recombinant human EPO-Fc (Cell Science Technology (Canton, MA) 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 analyzed using nanoscale liquid chromatography coupled with electrospray ionization highresolution 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 data set 691

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research obtained from HCD-based tandem high-resolution mass spectrometry 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 high-resolution tandem mass spectrometry results. It was found 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. 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 and lysine) to the EPO and 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-to-sequence 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 observed as double and triple ion pairs at m/z 554.74372+ and 370.16493+ (Table 2 and 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 fourth and sixth amino acids can be explained according to the exact mass as a

Table 2. Peptides Related to Fusion Area Identified after Glu-C and Lys-C Proteolysis in EPO-Fc Samples m/z (z) RT, min 13.5

14.5

elemental composition

observed

theoretical

e.ACRTGDRGSE.r, Glu-C bicarbonate C40H69N17O18S 554.7437 (2+) 554.7436 370.1649 (3+) 370.1648 k.LYTGEACRTGDRGSERK.c, Lys-C C78H130N28O29S 978.4720 (2+) 978.4712 652.6497 (3+) 652.6499 489.7401 (4+) 489.7393 392.9934 (5+) 392.9929

error, ppm

(2+) (3+)

0.2 0.3

(2+) (3+) (4+) (5+)

0.8 −0.3 1.6 1.3

combination of aspartic acid and glycine (−2.3 ppm). Thus, the derived sequence covers the C-terminal region of EPO (P01588) starting from position Ala18 and ending at the Nterminal glutamic acid of IgG2 (P01859) hinge region at position 99. These are connected to each other by a 2-amino acid linker, [GS]. It is hypothesized that the cleavage of the leader sequence and the last amino acid Arg by post-translational modification yields the mature EPO.24 According to our examination EPOFc 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 hEPO25 and EPO biosimilar products.26 The findings were further confirmed using Lys-C endoproteinase. After the 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 multiply charged 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 and 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 yions and a small number of less intensive complementary bions. 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 Scientific, purity of >98%, Figures S4 and S5). Summarizing the data from bottom-up experiments, we have suggested that the protein of interest is a chimera of EPO Ctermini joined to the hinge N-terminus of IgG2 through the mono dipeptide glycyl-seryl linker.27,28 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 analyze the whole Fc-fusion EPO molecule with 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 692

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research

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 fragment assignment of peptide related to fusion area found in EPO-Fc Glu-C-digest.

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, β-elimination, and 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 four cysteines (two disulfide bonds), and IgG2 Fc contains eight cysteines (four 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. 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 nonreducing conditions, IdeS digestion of EPO-Fc should generate three polypeptide chains: two of reduced Fc fragments (Fc/2)

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, O-glycosidase, neuraminidase, β1−4 galactosidase, and β-N-acetylglucosaminidase).29 The raw deglycosylated EPO-Fc that was analyzed 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 and 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. Because 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 α O-glycosidic bond of Nacetylglucosamine (GlcNAc) and N-acetylgalactosaminoglycans (GalNAc) can be reached by affecting of several exoglycosidases. This is in contrast to the deglycosylation of N-linked 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 and Thr of the EPO-Fc molecule can block the action of O-glycosidases31,32 due to 693

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research

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

matched to the 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.

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 (Figures 4 and 5). The first eluting peak at 20.75 min (23 789.7 Da) corresponds to the Fc/2 fragment of IgG. The utilized highresolution mass-spectrometry allowed us to obtain the monoisotopic mass of the analyzed fragment. As shown in Figure 4b (insert), the measured monoisotopic mass of the Fc/ 2 was determined to be 23 774.8262 Da. The measured MWs are consistent with the lysine clipping and S → A substitution with sub-parts per million MW errors. 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 IdeS-reduced EPO-Fc fragment and

Determination of EPO-Fc in Human Serum Samples

The 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 behavior 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 nonredundant and SwissProt/UniProt databases. The peptide shares 70% identical amino acids with EPO human (P01588). A 70 to 88% query 694

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research

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

Figure 4. (a) Full MS spectrum and (b) deconvolution results 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+ to 14+ charge states) SIM mode, was deconvoluted using the Xtract algorithm, obtaining an accurate monoisotopic mass (insert).

Figure 5. (a) Full MS spectrum and (b) deconvolution results of the deglycosylated IdeS-proteolytic fragment, EPO-hinge.

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 antiEPO antibodies. Next, eluates were digested using Lys-C protease and assayed by the 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. 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 695

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research

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 (gray). MRM TIC is the sum of the signal for 489.7 → 560.6 (y153+) and 489.7 → 614.9 (y163+).

EPO-Fc analysis in biological samples. However, further improvements in sensitivity are required.

ORCID

CONCLUSIONS 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. 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.

Notes

Pavel V. Postnikov: 0000-0003-3424-0582

■

■

The authors declare no competing financial interest.

■

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

■

(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. U. S. A. 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. (5) Gillies, S.; Way, J.; Lo, K.-M. Fc-erythropoietin fusion protein with improved pharmacokinetics. US Patent 20050202538, A1, October 11, 2017. (6) Wang, H.; Yong, D.; Zhang, R.; Xu, J.; Liu, L. Recombinant human EPO-FC fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo. US Patent 8431132, B2, October 13, 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.; Yang, S. I.; Chung, Y. K. A long-acting erythropoietin fused with noncytolytic human Fc for the treatment of anemia. Arch. Pharmacal Res. 2012, 35 (5), 757−759.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.7b00739. A table showing human IgGs alignment. Figures showing C-terminal lysine clipping in EPO-Fc, high-resolution full scan spectrum of the Glu-C “spacer” peptide and the Lys-C “spacer” peptide, and comparisons of HCD spectra. (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +7-4992677320. 696

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697

Article

Journal of Proteome Research (9) Recombinant Human EPO-alpha Fc Chimera. Cell Sciences Datasheet. http://www.cellsciences.com/PDF/CSI20107.pdf (accessed October 8, 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: Boca Raton, FL, 2009, 47, pp 1−30. (12) Gillette, M. A.; Carr, S. A. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 2012, 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 Spectrom. Rev. 2017, 36, 734. (14) van den Broek, I.; Niessen, W. M.; van Dongen, W. D. Bioanalytical LC−MS/MS of protein-based biopharmaceuticals. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 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 immuno-affinity 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, 7, 999−1008. (19) Ellison, J.; Hood, L. Linkage and sequence homology of two human immunoglobulin gamma heavy chain constant region genes. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 1984−1988. (20) Vidarsson, G.; Dekkers, G.; Rispens, T. IgG subclasses and allotypes: from structure to effector function. Front. Immunol. 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.; Patel, R.; Cheng, G.; Kita, A. Z. 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. (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) Han, K.-Y.; Kim, Y.-J.; Lee, J.-I.; Lee, J.-G. Polypeptide linker and method of analyzing target material using the same. US Patent 8911954B2, October 13, 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. https:// worldwide.promega.com/-/media/files/resources/protocols/productinformation-sheets/n/protein-deglycosylation-mix-protocol.pdf (accessed September 26, 2017). (30) Sergeevna, S. M.; Aleksandrovna, L. O.; Nikolaevna, K. T.; Vladimirovna, B. A.; Valer’evna, Z. M.; Aleksandrovna, L. A.; Konstantinovna, K. J.; Mikhajlovna, E. E.; Semenovna, M. E.; Valer’evna, S. A., et al. Method of detecting O-glycosylated proteins in cell homogenates prepared for proteomic and phosphoproteomic analysis. Patent RU 2 509 807 C1, October 13, 2017. (31) Prozyme Inc. Enzymatic Deglycosylation Kit for N-Linked & Simple O-Linked Glycans. https://prozyme.com/products/gk80110 (accessed October 2, 2017). (32) Brockhausen, I.; Schachter, H.; Stanley, P. 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: Cold Spring Harbor, NY, 2009; pp 115−127. (33) Vincents, B.; von Pawel-Rammingen, U.; Bjö r ck, 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.

697

DOI: 10.1021/acs.jproteome.7b00739 J. Proteome Res. 2018, 17, 689−697