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Fast Comparative Structural Characterization of Intact Therapeutic Antibodies using Hydrogen Deuterium Exchange and Electron Transfer Dissociation Jingxi Pan, Suping Zhang, Albert Chou, Darryl Hardie, and Christoph H. Borchers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504809r • Publication Date (Web): 30 Apr 2015 Downloaded from http://pubs.acs.org on May 5, 2015
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Analytical Chemistry
Fast Comparative Structural Characterization of Intact Therapeutic Antibodies using Hydrogen Deuterium Exchange and Electron Transfer Dissociation
Jingxi Pan1, Suping Zhang1, Albert Chou1, Darryl Hardie1, and Christoph H. Borchers1,2*
1. University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada 2. Department of Biochemistry & Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada
* Corresponding author: Christoph H. Borchers, Ph.D. Department of Biochemistry & Microbiology University of Victoria-Genome British Columbia Proteomics Centre #3101-4464 Markham St., Vancouver Island Technology Park Victoria, BC V8Z 7X8, Canada Email: [email protected] Tel.: (250) 483-3221 Fax: (250) 483-3238
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Abstract Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER were determined to be located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also determined by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data analysis, make it well suited for fast comparative structural evaluation of intact antibodies.
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Analytical Chemistry
As one of the most important members of the fast-growing biopharmaceutical market, recombinant monoclonal and polyclonal antibodies have been approved for the treatment of various diseases including cancer. The proper function and therapeutic activities of antibodies are largely defined by their unique higher-order structures, thus any changes in an antibody’s higher-order structure need to be strictly monitored.1,2 This is particularly important for developing antibody biosimilars − the generic versions of originator protein drugs -- as even a small change in their higher-order structure may cause significant change in their therapeutic properties and produce unpredictable side effects in patients. Therefore, the structures of an antibody biosimilar product and the originator drug have to be closely evaluated and compared in order to get approval from the regulatory agencies.1,3 On the other hand, comparative structural characterization is also important during the manufacture of antibodies such as purification, storage, batch-to-batch quality control, and new formulation development, to ensure that the product is active and in the correct conformation.4 To fulfill these requirements, a method that can provide fast monitoring of the higher-order structural changes of antibodies is highly desirable. X-ray crystallography and NMR are two classical methods for protein higher-order structure analysis. However, the challenge in antibody crystallization and the time-consuming nature of data analysis make X-ray crystallography unsuitable for routine antibody analysis. Intact antibodies are too large and too complex for NMR. Amide hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is a powerful technique for characterizing protein higher-order structural changes.
5-7
There are currently two
workflows for experiments of this type: the traditional proteolysis-based "bottom-up" approach and the ‘top-down’ approach. The former includes quick digestion of the labeled protein under cold acidic conditions, followed by LC-MS analysis of the resulting peptides.8-10 In contrast, the top-down approach bypasses in-solution digestion and directly fragments the labeled protein using gas-phase mechanisms such as electron capture dissociation (ECD)11-15 or electron transfer dissociation (ETD)16-18. Although 3 ACS Paragon Plus Environment
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each approach has advantages, the top-down approach should be preferable in terms of biopharmaceutical characterization, as it is relatively fast, requires less manipulation of the protein sample, greatly reduces H/D back exchange, and the incorporated deuterium can often be determined at amino-acid resolution. Another unique feature of the top-down approach is that the deuterium content of the ECD/ETD fragments can be correlated directly with that of the intact protein.13,14,19 This correlation normally cannot be established with the enzymatic digestion-based approach, due to the different back exchange levels of the resulting peptides (typically 10-50%),20 and the fact that each digestion event leads to the loss of deuteration information for the two amides at the newly generated peptide's N-terminus.13 Nonetheless, probably due to the large size of antibodies, all of the HDX-MS studies on antibodies reported thus far have used the digestion-based approach,21-26 except the one from our laboratory where the light chain and heavy chain of an antibody were fragmented separately using LC-ECD.27 Here we report for the first time the application of top-down HDX-ETD to the comparative structural characterization of an intact therapeutic antibody − Herceptin (HER, 148 kDa) at the individual IgG domain level, on the LC time scale.
Scheme 1. The schematic structure of antibody HER. The light chain consists of one variable domain (VL) and one constant domain (CL), and the heavy chain consists of one variable domain (VH) and three constant domains (CH1-3). The gold-colored "sticks" represent disulfide bonds. Glycans on Asn300 are shown in dark green.
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EXPERIMENTAL SECTION
Materials and Reagents. Herceptin was expressed by Genscript USA Inc. (Piscataway, NJ). Protein L and ubiquitin were obtained from Sigma-Aldrich (St. Louis, USA), and deuterium oxide (D2O) was from Cambridge Isotope Laboratories (Andover, MA, USA). The peptide enfuvirtide was bought from Thermo Scientific (Bremen, Germany). Hydrogen/Deuterium Exchange. The complex of HER and protein-L (HER-L) was prepared by mixing 2 µL protein L (1 mM) with 20 µL of stock HER (100 µM, pH 7.4), followed by incubation for 2 h at room temperature. These concentrations should ensure close to 100% complex formation according to the very high affinity binding between protein L and various immunoglobulins (~ 1.0 × 109 M-1).28 HDX was conducted by mixing HER or HER-L (50 mM sodium phosphate buffer, pH 7.4) with D2O buffer at a ratio of 1:9 (v/v). After incubation for a specific amount of time (20 s, 5 min, 1 h, or 3 h), 15-µL aliquots were removed and quickly quenched by reducing the pH to 2.5 with 15 µL of phosphate buffer at pH 2.4. These samples were then flash frozen using liquid nitrogen and stored at -80 °C. The H/D scrambling test experiments were carried out using a two-syringe continuous-flow setup as described previously.12 Briefly, HDX of ubiquitin was initiated by mixing 20 microliter of ubiquitin (100 µM) in 10 mM ammonium acetate with 380 microliter of D2O (pD = pDread + 0.4 = 7.0). This resulted in a deuterium content of 95% in the final solution. Then the HDX sample was mixed online with 0.3% formic acid in 100% acetonitrile using two syringes, both being pushed forward by syringe pumps (Harvard Apparatus, Holliston, MA) to give a flow rate of 20 µL/min. Ubiquitin precursor ions were isolated at 750 m/z with an isolation window of 200 in the linear ion trap, which included charge states 11+ to 13+. ETD was carried out in a similar manner as was done for HER, with a reaction time of 12
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ms. ETD data was acquired for 0.1 min at HDX time of 5 min. The ETD spectrum of the unlabeled protein was obtained by using ubiquitin (5 µM) in 50% acetonitrile with 0.2% formic acid. Liquid Chromatography and Mass Spectrometry. LC-MS at -20 °C was carried out using a subzero setup described previously.27 The sample injector (Rheodyne Model 7125, sample loop volume 20 µL) was embedded in an ice bath beside the freezer. A 13-minute binary solvent gradient was used for protein elution, including a 1.5 min desalting time. Solvent A contained 35% methanol with 0.1% formic acid, while solvent B was 100% acetonitrile with 0.1% formic acid. The eluent was diverted to a waste bottle for the first 2 min to prevent salts from entering the instrument. HER was eluted using 40% solvent B. Protein L in the HER-L samples was removed by washing with 27% solvent B for 1.6 to 2.7 min. All MS experiments were conducted on an Orbitrap (Fusion) mass spectrometer equipped with ETD capability and an IonMAX ion source (Thermo Scientific, Bremen, Germany).
Basic instrumental
settings were as follows: spray voltage 3500 V (positive), transfer tube temperature 300 °C, vaporizer temperature 275 °C, sheath gas 25, auxiliary gas 10, S-lens RF level 60. The detection of intact HER ions in the LC-MS experiments was performed in the Orbitrap mass analyzer over the 600–4000 m/z mass range, and the resolving power was set at 15,000. AGC (automatic gain control) target was set at 2 ×105, and the maximum injection time was 100 ms. For the ETD MS/MS experiments, HER precursor ions were isolated at 2900 m/z with an isolation window of 300 in the linear ion trap. Fluoranthene radical anions were introduced into the ion trap over 50 ms with an ETD reagent target value of 3 ×105, and the ETD reaction time was set as 12 ms. AGC target value was 3 ×105. Fragment ions were detected in the Orbitrap over a scan range of 200−4000 m/z, with a resolution of 120,000. The Orbitrap was calibrated for the high mass range (m/z 200–4000) using the peptide drug Fuzeon (Enfuvirtide, Thermo Scientific). For the HDX experiments, frozen antibody aliquots were thawed on ice, injected instantly onto the column and analyzed by LC-MS. HPLC parameters and instrumental settings were kept the same as those used for HER without HDX. The HPLC flow rate (200 µL min−1) was decreased to 50 µL min−1
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during the HER elution to increase the time available for ETD. ETD experiments were performed from 2.9 to 6.0 min of the HPLC run for both HER and HER-L. Data Analysis. The raw MS data were processed using Xcalibur software (version 3.0.63, Thermo Scientific).
The
ETD
fragment
ions
were
identified
through
ProteinProspector
(http://prospector.ucsf.edu) using an m/z uncertainty constraint of 10 ppm, and the assignments were checked
manually.
The
sequence
of
protein
L
is
SEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG. The ETD ions used for HDX analysis of protein L were 1+: z7-z8; 2+: z21; 3+: z26, z33; 4+: z37-z40, z43-46; 5+: z49-z51, z53; 6+: z56, z58; 8+: parent ion. The centroid m/z values for ETD fragment ions were determined using an in-house written excel micro (Microsoft, Redmond, WA) spreadsheet. RESULTS AND DISCUSSION Intact Mass Measurements and Glycoform Analysis.
Herceptin (HER, also called
trastuzumab) is a humanized IgG1 antibody with high potency, used for treating breast cancer. HER consists of two light chains and two heavy chains, which are held together by 4 inter-chain disulfide bonds (Scheme 1). There is one glycosylation site on each heavy chain at residue Asn300. Shown in Figure 1 is the mass spectrum of intact HER obtained by a single 13 min LC-MS run on the Orbitrap Fusion. The charge states range from ca. 38+ to 77+, centered at ca. 51+. The clean spectrum indicates that the short LC gradient is sufficient for sample desalting and cleanup. Deconvolution of the mass spectrum gives a neutral mass of 148056.6 Da (Figure 1 inset), which is in very good agreement with the calculated molecular weight of trastuzumab, 148057.2 Da.2 The 0.4 Da difference represents the 2.7 ppm mass error in intact mass measurement for this large m/z value on our instrument. The major component corresponds to the G0 glycoform (no terminal galactose) of HER. The two minor components (148218.7 and 148380.7 Da) show a mass increase of 162 Da and 2×162 Da, compared to the major component (Figure 1 inset). They are assigned to G1 and G2 glycoforms, which have one and two extra galactose moeties, respectively, at the end of the glycan structures.2 7 ACS Paragon Plus Environment
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148056.6 Da 51+
148218.7 Da 148380.7 Da
55+ 47+
148.0 148.5 Mass (kDa)
60+ 42+
2000
2500
3000
3500
4000
m/z
Figure 1. LC-FTMS spectrum of intact HER obtained on Orbitrap-fusion. Charge states are shown as 51+, 55+ etc. The deconvoluted spectrum is shown in the inset.
The availability of the glycosylation profile of the originator HER3 gives us the opportunity to compare the glycoforms of the antibody products from different companies. Although significant batchto-batch variation in glycosylation profile was observed in the originator antibody, the relative percentage of the main glycoforms could be calculated based on their relative intensities.
A comparison of
glycoforms between the originator HER3 and Genscript HER is shown in the following table. G0
G1
G2
G3
Originator HER
25%
35%
30%
10%
Genscript HER
70%
18%
9%
< 3%
Apparently, the G0 glycoform is much more dominant in the Genscript HER sample compared to the originator drug, indicating a significantly lower extent of galactosylation. It has been reported that galactosylation can substantially enhance the binding affinity between an IgG1 antibody and its receptor FcγRIIIa,22 suggesting that a variation in glycosylation pattern may affect the activity of the antibody. 8 ACS Paragon Plus Environment
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Top-down ETD Fragmentation of HER. After obtaining good ESI-MS signals from intact HER, online ETD experiments were carried out on this antibody. Shown in Figure 2 is an ETD mass spectrum obtained in a single LC-MSMS experiment using an ETD reaction time of 12 ms. Precursor ion selection was performed in the ion trap using an isolation window of 300 m/z units centered around 2900. The spectrum is characterized by a number of distinct fragment ions with very strong intensities. Many weaker ions can also be seen. Interestingly, the most intense ions were found to be fragments of about 100 residues from the terminus of both the heavy chain and light chain (Figure 3), which were resulted from fragmentation at three hinge regions on the HER antibody. One of the hinges is located between immunoglobulin domains VL and CL (residue 89-133) on the light chain, and the other two are between VH and CH1 (residues 97-146), and CH2 and CH3 (residues 325-369) on the heavy chain (Scheme 1). The number of cleaved amides for the three hinge regions is 17, 10, and 14, respectively. The preferential ETD cleavage sites observed here are consistent with the reported fragmentation pattern of other antibodies using ETD29 or ECD30, and also support the hypothesis that the presence of disulfide bonds greatly limits protein fragmentation.31 Although more cleavage has been reported by averaging up to 40 LC runs,29 this was not attempted here because of its incompatibility with subsequent HDX experiments. The most intense fragment ions are the most useful for subsequent top-down HDX-ETD measurements, and give us the opportunity to investigate the conformational changes of intact antibodies.
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z93 5+
2098
2100
2102
c99 5+
2104
c99 7+ z86 6+ z106 8+
c99 z93 6+
z91 6+ 6+
2160 z86 5+
1400
2162
2164
2166
Light chain z106 6+
z93 5+
Heavy chain c99 5+ z86 4+
c99 8+
1200
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1600
1800
2000
2200
2400
2600
m/z
Figure 2. ETD spectrum of HER obtained in a single LC run. The ETD ions from the light chain are labeled in magenta, whereas those from the heavy chain are in blue. Two representative ETD ions are shown in the insets.
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Figure 3. ETD sites on the primary structure of HER. (Top) light chain; (Bottom) heavy chain. Only c and z ions are considered. The dotted blue lines represent intra-chain disulfide bridges. The red letters (a-e) represent the ETD fragments used to marked out the 6 domains: (a) Light chain c99(5+-7+); (b-d) heavy chain c110(7+), c111(7+) and c113(9+); (e-g) heavy chain z86(6+), z91(6+), and z93(5+), respectively.
Hydrogen/Deuterium Exchange Measurements. HDX experiments were conducted on both HER and its complex with protein L (HER-L) in order to explore the potential of top-down HDX for
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measuring structural differences between intact antibodies. The HDX samples were prepared by mixing HER or HER-L with D2O buffer at a ratio of 1:9 (v/v). The samples quenched at various time points (20 s, 5 min, 1 h, and 3 h) were first measured at the intact antibody level by LC-MS at -20 ºC. The backexchange levels of proteins at this subzero temperature were determined to be ∼ 2%.27 From the data shown in Figure 4A, it was determined that the number of exchanged amides on HER-L was a slightly lower (11±1) than that of HER-G0, indicating that protein L provided extra protection to the antibody. Considering the large molecular mass (148 kDa) and large number of amino acid residues (1326) of HER, this 11 Da difference is quite small but significant. The HDX behavior of the G1 and G2 glycoforms was also determined and was compared to that of G0 (Figure 4B). It was found that addition of one galactose (G1) or two galactose (G2) to HER’s glycan chain did not have a significant effect on the structure and dynamics of the antibody. A
B
600
Number of Deuterium
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
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400
200
0 0
10
100
1,000
10,000 0
10
100
1,000
10,000
HDX time (s)
Figure 4. Time course of deuterium uptake for intact HER as determined by LC-MS. (A) HERG0 (black circles) and HER-L (red circles). (B) HER-G0 (black circle), HER-G1 (pink triangles pointing down), HER-G2 (dark green triangles pointing up). The error bars (± 1 Da in (A) and ± 2 Da in (B)) are from data measured on three replicates. As mentioned earlier, the presence of multiple disulfide bonds in HER (16 in total, one in each IgG domain, scheme 1) limits the ETD fragmentation to the hinge regions. However, the relatively strong signal from these large fragments makes it possible to localize the structural differences down to the 12 ACS Paragon Plus Environment
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Analytical Chemistry
individual IgG domain level. Shown in Figure 5 are LC-ETD fragments from HER after HDX for 20 s. Each of the three fragments covers a single IgG domain of HER, with c99 for the VL domain (Figure 5 A and B), c111 for the VH domain (Figure 5 C and D), and z93 for the CH3 domain (Figure 5 E and F). From these results, it is apparent that protein L binding only affected the VL domain of the light chain. This result is consistent with our recent work where the protein L binding site was determined to be on the N-terminal portion of the HER light chain.27 In that work, top-down electron capture dissociation (ECD) was carried out on the light chain and heavy chain separately after disulfide reduction, so it was possible to obtain amino acid level spatial resolution even for disulfide connected regions.27 Because the current work was done on the intact HER without reduction, and because every IgG domain contained a disulfide bond, HDX information could only reach the individual domain level. 7+
c997+(VL)
z935+(CH3)
c111 (VH) A
C
E
B
D
F
HER
HER-L
1547
1549
1760
1762
2105
2108
m/z
Figure 5. Mass spectra of representative top-down ETD fragments from HER (top row) and HER-L (bottom row) after HDX for 20 s. The mass difference in fragment ion c99 from the light chain can be calculated based on the following equation32:
N =
n( m1 − m2 ) P
(1)
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where m1 and m2 are the centroid m/z values of c99 (7+) from HER and HER-L, respectively. The parameter n is the charge state of the fragment ion. Considering that the back-exchange level during the LC is 2%, the P value for the HDX experiments in 90% D2O is determined as 90% × 98% = 0.882. The N value thus calculated is found to be 5.4 Da. Because there are two identical VL domains on HER, the total difference should be 5.4 × 2 = 10.8 Da. This value is very close to that observed for intact HER, which is ∼11 Da. Similarly, the mass difference can be calculated for other fragments from the same domain, and also for fragments from the heavy chain. The results of measuring representative fragments are summarized in Figure 6. It is clear that significant differences were only observed for fragments from the light chain variable domain, but not for those from the heavy chain. Thus, the combination of intact antibody level HDX information with individual IgG domain level information suggests that the structural differences observed in the VL domain included all of the changes that occurred in the whole HER antibody. 14 c99(5+) c99(6+) c99(7+)
12 10
∆D
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8 6 4 2 c110(7+) c111(7+) 0
c113(9+)
Heavy chain VH domain
z86(6+) z91(6+) z93(5+)
Heavy chain CH3 domain
Light chain VL domain
Figure 6. HDX differences observed on representative ETD fragments from HER and HER-L. Error bars are from data measured in triplicates. The observation of HDX differences only on the VL domain, but not on other domains such as VH and CH3, indicates that H/D scrambling is insignificant under conditions of the current work.33 Nevertheless, we confirmed this conclusion by studying ubiquitin.
The N-terminal of ubiquitin is
involved in a stable β-sheet structure, whereas the C-terminal has no structure and is freely accessible to
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the solvent (Figure S1 inset).12 This characteristic makes this protein an ideal model system for testing the scrambling issue.12,18 Here, we used a simple one-step mixing setup for the ETD experiments (see Experimental Section for details). Figure S1 shows the mass spectra of the ETD ions c3 and z4 before and after HDX. Both c3 and z4 have 8 exchangeable hydrogens, but their mass shift is clearly different after 5 min of HDX. Based on equation 1 (here P=0.95), the number of deuterium atoms acquired by fragment z4 was determined as 8.0, indicating complete exchange, i.e., no protected hydrogen. In contrast, c3 acquired 6.1 deuterium atoms while 1.9 amide hydrogens were protected. On the basis of reported NMR data,12,34 the theoretical number of protected hydrogens on z4 is zero (pH 7.0, 22 °C), while on c3 it is 1.964 (residue specific values: M1 = 0, Q2 = 0, I3 = 0.984, F4 = 0.980). The excellent agreement between our ETD data and the NMR data suggests that scrambling is negligible under the conditions of our experiments. The ubiquitin experiment is very simple and easy to carry out, and thus
Amide Deuteration Status D
can be conveniently used in other laboratories for testing protein H/D scrambling on any instrument.
1.0 0.8 0.6 0.4 0.2 0.0
Protein L
1.0 0.8 0.6 0.4 0.2 0.0
L-HER
0
10
20
30
40
50
60
1.0 0.8
∆D
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
Analytical Chemistry
0.6 0.4 0.2
Difference
0.0 0
10
20
30
40
50
60
Residue Number
Figure 7. The amide deuteration status of protein L after HDX for 20 s. (Top) Protein L alone; (Middle) protein L in the presence of HER; (Bottom) the difference spectrum between Protein L and L-HER. 15 ACS Paragon Plus Environment
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After determining the effect of protein L binding on the structure of HER, we also explored the binding effect of HER on the structure of protein L. The HDX experiments were carried out on Protein L alone and also when it was part of a complex with HER (L-HER). After HDX for 20 s (the same conditions as for HER), Protein L in the complex with HER (L-HER) acquired 18 fewer deuteriums than Protein L alone, which indicated that HER binding provided extra protection for protein L. To further localize these protected residues, the deuteration status of individual amides from protein L was obtained by using online ETD fragmentation as we did for HER, using the data-analysis strategy we developed previously.27 A total of 19 ETD fragment ions were used for this 63-residue protein, representing an average spatial resolution of approximately 3 residues. As shown in Figure 7, protein L alone, in solution, had only marginal protection (amide status D < 1), e.g., residues 8-12. In contrast, formation of the complex increased the protection dramatically in several regions, such as residues 1-5, 15-23, and 2442. When these results are compared with the crystal structure data (PDB entry 1HEZ), we found that these regions strongly interact with the antibody, with residues 15-23 involved in a β-sheet and residues 24-42 involved in a long α-helix. The dramatic increase of protection on these residues suggest that complexation to HER reduced the solvent accessibility and/or the structural dynamics in these regions.
CONCLUSION In summary, this work demonstrates for the first time the application of top-down MS to the characterization of the higher-order structures of intact antibodies.
It combines HDX, ETD
fragmentation, and subzero temperature chromatography. Domain-level deuteration information was obtained for 6 out of 12 IgG domains (Figure S2), which included the important paratope regions that are crucial for antigen binding. Combining data from HDX measurements at the intact-antibody level with ETD information at individual IgG domain level makes it possible to determine the region of protein16 ACS Paragon Plus Environment
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protein interaction, as shown for HER, where the binding site of protein L was found to be located only on the VL domain of the light chain. In addition, accurate mass measurement of intact antibodies was found to be a means of determining global glycosylation profile and glycoform specific HDX properties. Although the presence of the disulfide bonds prevents the described approach from being able to obtain amino acid level structural information, the advantages such as minimal sample manipulation, fast workflow, very low back exchange, and simple data analysis make it a simple but powerful method for comparative structural evaluation of intact antibodies. This method would be particularly suitable for assessing higher-order structural comparability between antibodies, such as a biosimilar product and the originator antibody.
It may also be used as a fast screening method for monitoring antibody structural
changes, from which one can decide if further experiments are necessary to pinpoint the differences down to amino acid level.27
ASSOCIATED CONTENT Supporting Information: ETD spectrum of ubiquitin and ETD coverage mapped on the structure of HER. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS
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The authors would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support. We are also grateful to Genome Canada and Genome BC for providing Science and Technology Innovation Centre funding and support for the University of VictoriaGenome BC Proteomics Centre. We also thank Carol E. Parker for helpful discussions and careful review of this manuscript.
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Figure for TOC only:
Intact IgG
VL domain
Top-down HDX-ETD
1547
1549 m/z
References (1) Berkowitz, S. A.; Engen, J. R.; Mazzeo, J. R.; Jones, G. B. Nat. Rev. Drug Discovery 2012, 11, 527-540. (2) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Anal. Chem. 2013, 85, 715-736. (3) Beck, A.; Sanglier-Cianferani, S.; Van Dorsselaer, A. Anal. Chem. 2012, 84, 4637-4646. (4) Thompson, N. J.; Rosati, S.; Rose, R. J.; Heck, A. J. R. Chemical Communications 2013, 49, 538-548. (5) Kaltashov, I. A.; Eyles, S. J. Mass Spectrom. Rev. 2002, 21, 37-71. (6) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158-170. (7) Konermann, L.; Pan, J.; Liu, Y. Chem. Soc. Rev. 2011, 40, 1224-1234. (8) Kan, Z. Y.; Walters, B. T.; Mayne, L.; Englander, S. W. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 16438-16443. (9) Zhang, Q.; Willison, L. N.; Tripathi, P.; Sathe, S. K.; Roux, K. H.; Emmett, M. R.; Blakney, G. T.; Zhang, H. M.; Marshall, A. G. Anal. Chem. 2011, 83, 7129-7136. (10) Rand, K. D.; Zehl, M.; Jensen, O. N.; Jørgensen, T. J. D. Anal. Chem. 2009, 81, 5577-5584. (11) Rand, K. D.; Adams, C. M.; Zubarev, R. A.; Jørgensen, T. J. D. J. Am. Chem. Soc. 2008, 130, 1341-1349. (12) Pan, J.; Han, J.; Borchers, C. H.; Konermann, L. J. Am. Chem. Soc. 2008, 130, 1157411575. (13) Pan, J.; Han, J.; Borchers, C. H.; Konermann, L. J. Am. Chem. Soc. 2009, 131, 12801– 12808. 19 ACS Paragon Plus Environment
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(14) Wang, G. B.; Abzalimov, R. R.; Bobst, C. E.; Kaltashov, I. A. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 20087-20092. (15) Wang, G.; Kaltashov, I. A. Anal. Chem. 2014, 86, 7293. (16) Abzalimov, R. R.; Kaplan, D. A.; Easterling, M. L.; Kaltashov, I. A. J. Am. Soc. Mass Spectrom. 2009, 20, 1514-1517. (17) Sterling, H. J.; Williams, E. R. Anal. Chem. 2010, 82, 9050-9057. (18) Amon, S.; Trelle, M. B.; Jensen, O. N.; Jørgensen, T. J. D. Anal. Chem. 2012, 84, 44674473. (19) Pan, J.; Borchers, C. H. Proteomics 2013, 13, 974-981. (20) Kaltashov, I. A.; Bobst, C. E.; Abzalimov, R. R. Anal. Chem. 2009, 81, 7892-7899. (21) Houde, D.; Arndt, J.; Domeier, W.; Berkowitz, S.; Engen, J. R. Anal. Chem. 2009, 81, 26442651. (22) Houde, D.; Peng, Y.; Berkowitz, S. A.; Engen, J. R. Mol Cell Proteomics 2010, 9, 17161728. (23) Burkitt, W.; Domann, P.; O'Connor, G. Protein Sci. 2010, 19, 826-835. (24) Zhang, A.; Singh, S. K.; Shirts, M. R.; Kumar, S.; Fernandez, E. J. Pharm. Res. 2012, 29, 236-250. (25) Pan, L. Y.; Salas-Solano, O.; Valliere-Douglass, J. F. Anal. Chem. 2014, 86, 2657-2664. (26) Zhang, A. M.; Hu, P.; MacGregor, P.; Xue, Y.; Fan, H. H.; Suchecki, P.; Olszewski, L.; Liu, A. Anal. Chem. 2014, 86, 3468-3475. (27) Pan, J.; Zhang, S.; Parker, C. E.; Borchers, C. H. J. Am. Chem. Soc. 2014, DOI: 10.1021/ja507880w [Epub ahead of print]. (28) Nilson, B. H. K.; Solomon, A.; Bjorck, L.; Akerstrom, B. J. Biol. Chem. 1992, 267, 22342239. (29) Fornelli, L.; Damoc, E.; Thomas, P. M.; Kelleher, N. L.; Aizikov, K.; Denisov, E.; Makarov, A.; Tsybin, Y. O. Mol. Cell. Proteomics 2012, 11, 1758-1767. (30) Mao, Y.; Valeja, S. G.; Rouse, J. C.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2013, 85, 4239-4246. (31) Ganisl, B.; Breuker, K. ChemistryOPEN 2012, 1, 260-268. (32) Pan, J.; Borchers, C. H. Proteomics 2014, 14, 1249–1258. (33) Zehl, M.; Rand, K. D.; Jensen, O. N.; Jørgensen, T. J. D. J. Am. Chem. Soc. 2008, 130, 17453–17459. (34) Bougault, C.; Feng, L.; Glushka, J.; Kupce, E.; Prestegard, J. H. J. Biomol. NMR 2004, 28, 385-390.
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Article
Fast Comparative Structural Characterization of Intact Therapeutic Antibodies using Hydrogen Deuterium Exchange and Electron Transfer Dissociation Jingxi Pan, Suping Zhang, Albert Chou, Darryl Hardie, and Christoph H. Borchers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504809r • Publication Date (Web): 30 Apr 2015 Downloaded from http://pubs.acs.org on May 5, 2015
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Analytical Chemistry
Fast Comparative Structural Characterization of Intact Therapeutic Antibodies using Hydrogen Deuterium Exchange and Electron Transfer Dissociation
Jingxi Pan1, Suping Zhang1, Albert Chou1, Darryl Hardie1, and Christoph H. Borchers1,2*
1. University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada 2. Department of Biochemistry & Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada
* Corresponding author: Christoph H. Borchers, Ph.D. Department of Biochemistry & Microbiology University of Victoria-Genome British Columbia Proteomics Centre #3101-4464 Markham St., Vancouver Island Technology Park Victoria, BC V8Z 7X8, Canada Email: [email protected] Tel.: (250) 483-3221 Fax: (250) 483-3238
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Abstract Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER were determined to be located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also determined by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data analysis, make it well suited for fast comparative structural evaluation of intact antibodies.
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As one of the most important members of the fast-growing biopharmaceutical market, recombinant monoclonal and polyclonal antibodies have been approved for the treatment of various diseases including cancer. The proper function and therapeutic activities of antibodies are largely defined by their unique higher-order structures, thus any changes in an antibody’s higher-order structure need to be strictly monitored.1,2 This is particularly important for developing antibody biosimilars − the generic versions of originator protein drugs -- as even a small change in their higher-order structure may cause significant change in their therapeutic properties and produce unpredictable side effects in patients. Therefore, the structures of an antibody biosimilar product and the originator drug have to be closely evaluated and compared in order to get approval from the regulatory agencies.1,3 On the other hand, comparative structural characterization is also important during the manufacture of antibodies such as purification, storage, batch-to-batch quality control, and new formulation development, to ensure that the product is active and in the correct conformation.4 To fulfill these requirements, a method that can provide fast monitoring of the higher-order structural changes of antibodies is highly desirable. X-ray crystallography and NMR are two classical methods for protein higher-order structure analysis. However, the challenge in antibody crystallization and the time-consuming nature of data analysis make X-ray crystallography unsuitable for routine antibody analysis. Intact antibodies are too large and too complex for NMR. Amide hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is a powerful technique for characterizing protein higher-order structural changes.
5-7
There are currently two
workflows for experiments of this type: the traditional proteolysis-based "bottom-up" approach and the ‘top-down’ approach. The former includes quick digestion of the labeled protein under cold acidic conditions, followed by LC-MS analysis of the resulting peptides.8-10 In contrast, the top-down approach bypasses in-solution digestion and directly fragments the labeled protein using gas-phase mechanisms such as electron capture dissociation (ECD)11-15 or electron transfer dissociation (ETD)16-18. Although 3 ACS Paragon Plus Environment
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each approach has advantages, the top-down approach should be preferable in terms of biopharmaceutical characterization, as it is relatively fast, requires less manipulation of the protein sample, greatly reduces H/D back exchange, and the incorporated deuterium can often be determined at amino-acid resolution. Another unique feature of the top-down approach is that the deuterium content of the ECD/ETD fragments can be correlated directly with that of the intact protein.13,14,19 This correlation normally cannot be established with the enzymatic digestion-based approach, due to the different back exchange levels of the resulting peptides (typically 10-50%),20 and the fact that each digestion event leads to the loss of deuteration information for the two amides at the newly generated peptide's N-terminus.13 Nonetheless, probably due to the large size of antibodies, all of the HDX-MS studies on antibodies reported thus far have used the digestion-based approach,21-26 except the one from our laboratory where the light chain and heavy chain of an antibody were fragmented separately using LC-ECD.27 Here we report for the first time the application of top-down HDX-ETD to the comparative structural characterization of an intact therapeutic antibody − Herceptin (HER, 148 kDa) at the individual IgG domain level, on the LC time scale.
Scheme 1. The schematic structure of antibody HER. The light chain consists of one variable domain (VL) and one constant domain (CL), and the heavy chain consists of one variable domain (VH) and three constant domains (CH1-3). The gold-colored "sticks" represent disulfide bonds. Glycans on Asn300 are shown in dark green.
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EXPERIMENTAL SECTION
Materials and Reagents. Herceptin was expressed by Genscript USA Inc. (Piscataway, NJ). Protein L and ubiquitin were obtained from Sigma-Aldrich (St. Louis, USA), and deuterium oxide (D2O) was from Cambridge Isotope Laboratories (Andover, MA, USA). The peptide enfuvirtide was bought from Thermo Scientific (Bremen, Germany). Hydrogen/Deuterium Exchange. The complex of HER and protein-L (HER-L) was prepared by mixing 2 µL protein L (1 mM) with 20 µL of stock HER (100 µM, pH 7.4), followed by incubation for 2 h at room temperature. These concentrations should ensure close to 100% complex formation according to the very high affinity binding between protein L and various immunoglobulins (~ 1.0 × 109 M-1).28 HDX was conducted by mixing HER or HER-L (50 mM sodium phosphate buffer, pH 7.4) with D2O buffer at a ratio of 1:9 (v/v). After incubation for a specific amount of time (20 s, 5 min, 1 h, or 3 h), 15-µL aliquots were removed and quickly quenched by reducing the pH to 2.5 with 15 µL of phosphate buffer at pH 2.4. These samples were then flash frozen using liquid nitrogen and stored at -80 °C. The H/D scrambling test experiments were carried out using a two-syringe continuous-flow setup as described previously.12 Briefly, HDX of ubiquitin was initiated by mixing 20 microliter of ubiquitin (100 µM) in 10 mM ammonium acetate with 380 microliter of D2O (pD = pDread + 0.4 = 7.0). This resulted in a deuterium content of 95% in the final solution. Then the HDX sample was mixed online with 0.3% formic acid in 100% acetonitrile using two syringes, both being pushed forward by syringe pumps (Harvard Apparatus, Holliston, MA) to give a flow rate of 20 µL/min. Ubiquitin precursor ions were isolated at 750 m/z with an isolation window of 200 in the linear ion trap, which included charge states 11+ to 13+. ETD was carried out in a similar manner as was done for HER, with a reaction time of 12
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ms. ETD data was acquired for 0.1 min at HDX time of 5 min. The ETD spectrum of the unlabeled protein was obtained by using ubiquitin (5 µM) in 50% acetonitrile with 0.2% formic acid. Liquid Chromatography and Mass Spectrometry. LC-MS at -20 °C was carried out using a subzero setup described previously.27 The sample injector (Rheodyne Model 7125, sample loop volume 20 µL) was embedded in an ice bath beside the freezer. A 13-minute binary solvent gradient was used for protein elution, including a 1.5 min desalting time. Solvent A contained 35% methanol with 0.1% formic acid, while solvent B was 100% acetonitrile with 0.1% formic acid. The eluent was diverted to a waste bottle for the first 2 min to prevent salts from entering the instrument. HER was eluted using 40% solvent B. Protein L in the HER-L samples was removed by washing with 27% solvent B for 1.6 to 2.7 min. All MS experiments were conducted on an Orbitrap (Fusion) mass spectrometer equipped with ETD capability and an IonMAX ion source (Thermo Scientific, Bremen, Germany).
Basic instrumental
settings were as follows: spray voltage 3500 V (positive), transfer tube temperature 300 °C, vaporizer temperature 275 °C, sheath gas 25, auxiliary gas 10, S-lens RF level 60. The detection of intact HER ions in the LC-MS experiments was performed in the Orbitrap mass analyzer over the 600–4000 m/z mass range, and the resolving power was set at 15,000. AGC (automatic gain control) target was set at 2 ×105, and the maximum injection time was 100 ms. For the ETD MS/MS experiments, HER precursor ions were isolated at 2900 m/z with an isolation window of 300 in the linear ion trap. Fluoranthene radical anions were introduced into the ion trap over 50 ms with an ETD reagent target value of 3 ×105, and the ETD reaction time was set as 12 ms. AGC target value was 3 ×105. Fragment ions were detected in the Orbitrap over a scan range of 200−4000 m/z, with a resolution of 120,000. The Orbitrap was calibrated for the high mass range (m/z 200–4000) using the peptide drug Fuzeon (Enfuvirtide, Thermo Scientific). For the HDX experiments, frozen antibody aliquots were thawed on ice, injected instantly onto the column and analyzed by LC-MS. HPLC parameters and instrumental settings were kept the same as those used for HER without HDX. The HPLC flow rate (200 µL min−1) was decreased to 50 µL min−1
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during the HER elution to increase the time available for ETD. ETD experiments were performed from 2.9 to 6.0 min of the HPLC run for both HER and HER-L. Data Analysis. The raw MS data were processed using Xcalibur software (version 3.0.63, Thermo Scientific).
The
ETD
fragment
ions
were
identified
through
ProteinProspector
(http://prospector.ucsf.edu) using an m/z uncertainty constraint of 10 ppm, and the assignments were checked
manually.
The
sequence
of
protein
L
is
SEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG. The ETD ions used for HDX analysis of protein L were 1+: z7-z8; 2+: z21; 3+: z26, z33; 4+: z37-z40, z43-46; 5+: z49-z51, z53; 6+: z56, z58; 8+: parent ion. The centroid m/z values for ETD fragment ions were determined using an in-house written excel micro (Microsoft, Redmond, WA) spreadsheet. RESULTS AND DISCUSSION Intact Mass Measurements and Glycoform Analysis.
Herceptin (HER, also called
trastuzumab) is a humanized IgG1 antibody with high potency, used for treating breast cancer. HER consists of two light chains and two heavy chains, which are held together by 4 inter-chain disulfide bonds (Scheme 1). There is one glycosylation site on each heavy chain at residue Asn300. Shown in Figure 1 is the mass spectrum of intact HER obtained by a single 13 min LC-MS run on the Orbitrap Fusion. The charge states range from ca. 38+ to 77+, centered at ca. 51+. The clean spectrum indicates that the short LC gradient is sufficient for sample desalting and cleanup. Deconvolution of the mass spectrum gives a neutral mass of 148056.6 Da (Figure 1 inset), which is in very good agreement with the calculated molecular weight of trastuzumab, 148057.2 Da.2 The 0.4 Da difference represents the 2.7 ppm mass error in intact mass measurement for this large m/z value on our instrument. The major component corresponds to the G0 glycoform (no terminal galactose) of HER. The two minor components (148218.7 and 148380.7 Da) show a mass increase of 162 Da and 2×162 Da, compared to the major component (Figure 1 inset). They are assigned to G1 and G2 glycoforms, which have one and two extra galactose moeties, respectively, at the end of the glycan structures.2 7 ACS Paragon Plus Environment
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148056.6 Da 51+
148218.7 Da 148380.7 Da
55+ 47+
148.0 148.5 Mass (kDa)
60+ 42+
2000
2500
3000
3500
4000
m/z
Figure 1. LC-FTMS spectrum of intact HER obtained on Orbitrap-fusion. Charge states are shown as 51+, 55+ etc. The deconvoluted spectrum is shown in the inset.
The availability of the glycosylation profile of the originator HER3 gives us the opportunity to compare the glycoforms of the antibody products from different companies. Although significant batchto-batch variation in glycosylation profile was observed in the originator antibody, the relative percentage of the main glycoforms could be calculated based on their relative intensities.
A comparison of
glycoforms between the originator HER3 and Genscript HER is shown in the following table. G0
G1
G2
G3
Originator HER
25%
35%
30%
10%
Genscript HER
70%
18%
9%
< 3%
Apparently, the G0 glycoform is much more dominant in the Genscript HER sample compared to the originator drug, indicating a significantly lower extent of galactosylation. It has been reported that galactosylation can substantially enhance the binding affinity between an IgG1 antibody and its receptor FcγRIIIa,22 suggesting that a variation in glycosylation pattern may affect the activity of the antibody. 8 ACS Paragon Plus Environment
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Top-down ETD Fragmentation of HER. After obtaining good ESI-MS signals from intact HER, online ETD experiments were carried out on this antibody. Shown in Figure 2 is an ETD mass spectrum obtained in a single LC-MSMS experiment using an ETD reaction time of 12 ms. Precursor ion selection was performed in the ion trap using an isolation window of 300 m/z units centered around 2900. The spectrum is characterized by a number of distinct fragment ions with very strong intensities. Many weaker ions can also be seen. Interestingly, the most intense ions were found to be fragments of about 100 residues from the terminus of both the heavy chain and light chain (Figure 3), which were resulted from fragmentation at three hinge regions on the HER antibody. One of the hinges is located between immunoglobulin domains VL and CL (residue 89-133) on the light chain, and the other two are between VH and CH1 (residues 97-146), and CH2 and CH3 (residues 325-369) on the heavy chain (Scheme 1). The number of cleaved amides for the three hinge regions is 17, 10, and 14, respectively. The preferential ETD cleavage sites observed here are consistent with the reported fragmentation pattern of other antibodies using ETD29 or ECD30, and also support the hypothesis that the presence of disulfide bonds greatly limits protein fragmentation.31 Although more cleavage has been reported by averaging up to 40 LC runs,29 this was not attempted here because of its incompatibility with subsequent HDX experiments. The most intense fragment ions are the most useful for subsequent top-down HDX-ETD measurements, and give us the opportunity to investigate the conformational changes of intact antibodies.
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z93 5+
2098
2100
2102
c99 5+
2104
c99 7+ z86 6+ z106 8+
c99 z93 6+
z91 6+ 6+
2160 z86 5+
1400
2162
2164
2166
Light chain z106 6+
z93 5+
Heavy chain c99 5+ z86 4+
c99 8+
1200
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1600
1800
2000
2200
2400
2600
m/z
Figure 2. ETD spectrum of HER obtained in a single LC run. The ETD ions from the light chain are labeled in magenta, whereas those from the heavy chain are in blue. Two representative ETD ions are shown in the insets.
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Figure 3. ETD sites on the primary structure of HER. (Top) light chain; (Bottom) heavy chain. Only c and z ions are considered. The dotted blue lines represent intra-chain disulfide bridges. The red letters (a-e) represent the ETD fragments used to marked out the 6 domains: (a) Light chain c99(5+-7+); (b-d) heavy chain c110(7+), c111(7+) and c113(9+); (e-g) heavy chain z86(6+), z91(6+), and z93(5+), respectively.
Hydrogen/Deuterium Exchange Measurements. HDX experiments were conducted on both HER and its complex with protein L (HER-L) in order to explore the potential of top-down HDX for
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measuring structural differences between intact antibodies. The HDX samples were prepared by mixing HER or HER-L with D2O buffer at a ratio of 1:9 (v/v). The samples quenched at various time points (20 s, 5 min, 1 h, and 3 h) were first measured at the intact antibody level by LC-MS at -20 ºC. The backexchange levels of proteins at this subzero temperature were determined to be ∼ 2%.27 From the data shown in Figure 4A, it was determined that the number of exchanged amides on HER-L was a slightly lower (11±1) than that of HER-G0, indicating that protein L provided extra protection to the antibody. Considering the large molecular mass (148 kDa) and large number of amino acid residues (1326) of HER, this 11 Da difference is quite small but significant. The HDX behavior of the G1 and G2 glycoforms was also determined and was compared to that of G0 (Figure 4B). It was found that addition of one galactose (G1) or two galactose (G2) to HER’s glycan chain did not have a significant effect on the structure and dynamics of the antibody. A
B
600
Number of Deuterium
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
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400
200
0 0
10
100
1,000
10,000 0
10
100
1,000
10,000
HDX time (s)
Figure 4. Time course of deuterium uptake for intact HER as determined by LC-MS. (A) HERG0 (black circles) and HER-L (red circles). (B) HER-G0 (black circle), HER-G1 (pink triangles pointing down), HER-G2 (dark green triangles pointing up). The error bars (± 1 Da in (A) and ± 2 Da in (B)) are from data measured on three replicates. As mentioned earlier, the presence of multiple disulfide bonds in HER (16 in total, one in each IgG domain, scheme 1) limits the ETD fragmentation to the hinge regions. However, the relatively strong signal from these large fragments makes it possible to localize the structural differences down to the 12 ACS Paragon Plus Environment
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individual IgG domain level. Shown in Figure 5 are LC-ETD fragments from HER after HDX for 20 s. Each of the three fragments covers a single IgG domain of HER, with c99 for the VL domain (Figure 5 A and B), c111 for the VH domain (Figure 5 C and D), and z93 for the CH3 domain (Figure 5 E and F). From these results, it is apparent that protein L binding only affected the VL domain of the light chain. This result is consistent with our recent work where the protein L binding site was determined to be on the N-terminal portion of the HER light chain.27 In that work, top-down electron capture dissociation (ECD) was carried out on the light chain and heavy chain separately after disulfide reduction, so it was possible to obtain amino acid level spatial resolution even for disulfide connected regions.27 Because the current work was done on the intact HER without reduction, and because every IgG domain contained a disulfide bond, HDX information could only reach the individual domain level. 7+
c997+(VL)
z935+(CH3)
c111 (VH) A
C
E
B
D
F
HER
HER-L
1547
1549
1760
1762
2105
2108
m/z
Figure 5. Mass spectra of representative top-down ETD fragments from HER (top row) and HER-L (bottom row) after HDX for 20 s. The mass difference in fragment ion c99 from the light chain can be calculated based on the following equation32:
N =
n( m1 − m2 ) P
(1)
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where m1 and m2 are the centroid m/z values of c99 (7+) from HER and HER-L, respectively. The parameter n is the charge state of the fragment ion. Considering that the back-exchange level during the LC is 2%, the P value for the HDX experiments in 90% D2O is determined as 90% × 98% = 0.882. The N value thus calculated is found to be 5.4 Da. Because there are two identical VL domains on HER, the total difference should be 5.4 × 2 = 10.8 Da. This value is very close to that observed for intact HER, which is ∼11 Da. Similarly, the mass difference can be calculated for other fragments from the same domain, and also for fragments from the heavy chain. The results of measuring representative fragments are summarized in Figure 6. It is clear that significant differences were only observed for fragments from the light chain variable domain, but not for those from the heavy chain. Thus, the combination of intact antibody level HDX information with individual IgG domain level information suggests that the structural differences observed in the VL domain included all of the changes that occurred in the whole HER antibody. 14 c99(5+) c99(6+) c99(7+)
12 10
∆D
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8 6 4 2 c110(7+) c111(7+) 0
c113(9+)
Heavy chain VH domain
z86(6+) z91(6+) z93(5+)
Heavy chain CH3 domain
Light chain VL domain
Figure 6. HDX differences observed on representative ETD fragments from HER and HER-L. Error bars are from data measured in triplicates. The observation of HDX differences only on the VL domain, but not on other domains such as VH and CH3, indicates that H/D scrambling is insignificant under conditions of the current work.33 Nevertheless, we confirmed this conclusion by studying ubiquitin.
The N-terminal of ubiquitin is
involved in a stable β-sheet structure, whereas the C-terminal has no structure and is freely accessible to
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the solvent (Figure S1 inset).12 This characteristic makes this protein an ideal model system for testing the scrambling issue.12,18 Here, we used a simple one-step mixing setup for the ETD experiments (see Experimental Section for details). Figure S1 shows the mass spectra of the ETD ions c3 and z4 before and after HDX. Both c3 and z4 have 8 exchangeable hydrogens, but their mass shift is clearly different after 5 min of HDX. Based on equation 1 (here P=0.95), the number of deuterium atoms acquired by fragment z4 was determined as 8.0, indicating complete exchange, i.e., no protected hydrogen. In contrast, c3 acquired 6.1 deuterium atoms while 1.9 amide hydrogens were protected. On the basis of reported NMR data,12,34 the theoretical number of protected hydrogens on z4 is zero (pH 7.0, 22 °C), while on c3 it is 1.964 (residue specific values: M1 = 0, Q2 = 0, I3 = 0.984, F4 = 0.980). The excellent agreement between our ETD data and the NMR data suggests that scrambling is negligible under the conditions of our experiments. The ubiquitin experiment is very simple and easy to carry out, and thus
Amide Deuteration Status D
can be conveniently used in other laboratories for testing protein H/D scrambling on any instrument.
1.0 0.8 0.6 0.4 0.2 0.0
Protein L
1.0 0.8 0.6 0.4 0.2 0.0
L-HER
0
10
20
30
40
50
60
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∆D
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0.6 0.4 0.2
Difference
0.0 0
10
20
30
40
50
60
Residue Number
Figure 7. The amide deuteration status of protein L after HDX for 20 s. (Top) Protein L alone; (Middle) protein L in the presence of HER; (Bottom) the difference spectrum between Protein L and L-HER. 15 ACS Paragon Plus Environment
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After determining the effect of protein L binding on the structure of HER, we also explored the binding effect of HER on the structure of protein L. The HDX experiments were carried out on Protein L alone and also when it was part of a complex with HER (L-HER). After HDX for 20 s (the same conditions as for HER), Protein L in the complex with HER (L-HER) acquired 18 fewer deuteriums than Protein L alone, which indicated that HER binding provided extra protection for protein L. To further localize these protected residues, the deuteration status of individual amides from protein L was obtained by using online ETD fragmentation as we did for HER, using the data-analysis strategy we developed previously.27 A total of 19 ETD fragment ions were used for this 63-residue protein, representing an average spatial resolution of approximately 3 residues. As shown in Figure 7, protein L alone, in solution, had only marginal protection (amide status D < 1), e.g., residues 8-12. In contrast, formation of the complex increased the protection dramatically in several regions, such as residues 1-5, 15-23, and 2442. When these results are compared with the crystal structure data (PDB entry 1HEZ), we found that these regions strongly interact with the antibody, with residues 15-23 involved in a β-sheet and residues 24-42 involved in a long α-helix. The dramatic increase of protection on these residues suggest that complexation to HER reduced the solvent accessibility and/or the structural dynamics in these regions.
CONCLUSION In summary, this work demonstrates for the first time the application of top-down MS to the characterization of the higher-order structures of intact antibodies.
It combines HDX, ETD
fragmentation, and subzero temperature chromatography. Domain-level deuteration information was obtained for 6 out of 12 IgG domains (Figure S2), which included the important paratope regions that are crucial for antigen binding. Combining data from HDX measurements at the intact-antibody level with ETD information at individual IgG domain level makes it possible to determine the region of protein16 ACS Paragon Plus Environment
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protein interaction, as shown for HER, where the binding site of protein L was found to be located only on the VL domain of the light chain. In addition, accurate mass measurement of intact antibodies was found to be a means of determining global glycosylation profile and glycoform specific HDX properties. Although the presence of the disulfide bonds prevents the described approach from being able to obtain amino acid level structural information, the advantages such as minimal sample manipulation, fast workflow, very low back exchange, and simple data analysis make it a simple but powerful method for comparative structural evaluation of intact antibodies. This method would be particularly suitable for assessing higher-order structural comparability between antibodies, such as a biosimilar product and the originator antibody.
It may also be used as a fast screening method for monitoring antibody structural
changes, from which one can decide if further experiments are necessary to pinpoint the differences down to amino acid level.27
ASSOCIATED CONTENT Supporting Information: ETD spectrum of ubiquitin and ETD coverage mapped on the structure of HER. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS
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The authors would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support. We are also grateful to Genome Canada and Genome BC for providing Science and Technology Innovation Centre funding and support for the University of VictoriaGenome BC Proteomics Centre. We also thank Carol E. Parker for helpful discussions and careful review of this manuscript.
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Figure for TOC only:
Intact IgG
VL domain
Top-down HDX-ETD
1547
1549 m/z
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