Deuterium Exchange-Mass Spectrometry to

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Application of HDX-MS to Biopharmaceutical Development Requirements: Improved sensitivity to detection of conformational changes Lea Bonnington, Ingo Lindner, Ulrich Gilles, Tobias Kailich, Dietmar Reusch, and Patrick Bulau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01670 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Analytical Chemistry

Application of HDX-MS to Biopharmaceutical Development Requirements: Improved sensitivity to detection of conformational changes Lea Bonningtona, Ingo Lindnera, Ulrich Gillesa, Tobias Kailicha, Dietmar Reuscha, and Patrick Bulaua* a

Pharma Technical Development, Roche Diagnostics GmbH, Nonnenwald 2, 82377

Penzberg, Germany

* Corresponding author: Patrick Bulau Roche Diagnostics GmbH Nonnenwald 2, Building 433, Room 215 82377 Penzberg / Germany Phone: +49 8856 60 18039 Fax: +49 8856 60 6069 mailto:[email protected]

1 ACS Paragon Plus Environment


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Abstract The usefulness of the higher-order structure information provided by hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the protein therapeutic field is undisputed, however its applicability as a method for critical quality and comparability assessment has until now not been demonstrated. Here we present results demonstrating for the first time the applicability of the HDX-MS technique to monitor structural changes due to methionine

oxidation

at

sensitivity

levels

realistic

to

the

requirements

of

biopharmaceutical research and development. For the analyzed heavy chain marker peptides deuterium uptake differences due to oxidation at the conserved methionine in position 254 were significantly verifiable at the lowest increase (1%) through spiked oxidized IgG1.

Keywords: Hydrogen/deuterium exchange; mass spectrometry; oxidation; protein conformation; higher order structure; biopharmaceutical antibody


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Oxidation of mAbs has been demonstrated to affect the in vitro interaction with Protein A, binding to the neonatal Fc and Fcγ receptors1-4 and the pharmacokinetics5,6 and as such the monitoring of oxidation levels is considered critical in the biopharmaceutical protein production process.7-13 Mass spectrometry (MS) is widely applied in the characterization of biotherapeutics and increasingly for the analysis of protein higher order structure.14-18 Many examples of hydrogen deuterium exchange-mass spectrometry (HDX-MS) applied to structural changes in biopharmaceutical proteins,19-22 including monoclonal antibodies (mAb),23-29 exposed to extreme stress conditions have been published. Differential deuterium uptake in mAbs upon extreme oxidative stress has been demonstrated with HDX-MS,23-25,27 with the peptides containing or close to the conserved HC-Met-254 (Kabat numbering position 252)30 amino acid showing significantly increased deuterium uptake as a result of oxidation. With the developed approach applied here we demonstrate for the first time the applicability of the HDX-MS technique to monitor structural changes due to chemical modifications at sensitivity levels realistic to the requirements of therapeutic protein production.



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Experimental Section Sample preparation and oxidative stress conditions The recombinant IgG1 antibody mAbX was expressed in a Chinese hamster ovary cell system. The antibody was manufactured at Roche Diagnostics, Penzberg, Germany using standard cell culture and purification technology. mAbX was formulated at a concentration of 30 mg/mL in a 20 mM His-HCl buffer system at pH 6.0. Oxidation was performed using 0.2% H2O2 (v/v), as reported previously.4,8 Quantification of chemical modification levels by liquid-chromatography massspectrometry (LC-MS) Tryptic peptide digestion, LC-MS analysis of the proteolytic peptides and data evaluation for the relative quantification of chemical modification levels was conducted as described previously.31 Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) Solutions and Buffers: Working solutions of the antibody samples were prepared by dilution to a concentration of 6.6 mg/mL with equilibration buffer ‘E’ (5 mM KH2PO4, 5 mM K2HPO4, pH7.0). The deuterium labelling was achieved by 1 in 20 dilution of the samples with the deuteriumcontaining labelling buffer ‘L’ (5 mM K2HPO4, 5 mM KH2PO4/D2O-Puffer, pD 7.0) at room temperature. Quenching of the deuterium uptake was performed by 1 in 2 dilution of the labelled sample with ice-cold quenching buffer ‘Q’ (50 mM K2HPO4, 50 mM KH2PO4, 500 mM TCEP and 4 M guanidine, pH 2.4) resulting in a final pH of 2.6 and 55 pmol mAbX on column for each sample injection.


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Preliminary HDX-MS Time Survey: A kinetic experiment comparing the two most extreme states, ‘Ref - 0% Ox. mAbX’ and ‘Expt - 100%Ox. mAbX’, was performed with seven sampling points over a 2.5 hour time course, namely: 0, 0.25, 1, 10, 30, 60 and 150 minutes. All samples were shock frozen on dry ice following quenching and stored at -80°C. The LC-MS measurement was performed on the following day. The non-deuterated (t = 0) controls were measured in triplicate, while all other time points were single-fold experiments only. Targeted HDX-MS Analysis: The 0% Ox. mAbX sample and the samples containing 1%, 5%, 10%, 20%, 40%, 60%, 80% and 100% oxidized mAbX were deuterium labelled for 1 minute, quenched and shock frozen on dry ice, in triplicate. The samples were stored at -80°C and the LC-MS measurement was performed over the two subsequent days. LC/MS Measurement: The quenched deuterated protein samples were analyzed by UHPLC-MS/MS (positive ion mode ESI) with a nanoAcquity UPLC-SynaptG2 QTOF® system (Waters Corp.) equipped with temperature controlled column chambers for online pepsin digestion and chromatographic separation. Online digestion was performed at 15°C using a Poroszyme Immobilized Pepsin Cartridge (2.1 mm x 30 mm, Applied Biosystems) eluting with 0.23% formic acid in 3% MeCN (v/v) for 3 minutes at a flowrate of 100 µL/min. The peptic peptides were trapped on a RP C18 guard column at 0°C (Waters BEH VanGuard C18, 1.7 µm, 2.1 × 5 mm, Milford, MA, USA) and desalted for 3 minutes with 0.23% formic acid in 8% MeCN, (pH 2.3, 40 µl/min). The peptides were subsequently eluted from the trap to the analytical



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column (Waters BEH C18, 1.7 µm, 1.0 mm × 100 mm) and separated with an 8–40% gradient of 0.23% formic acid in MeCN (pH 2.3) over 14 minutes (40 µl/min, 0°C). The peptic peptides were identified using collision-induced dissociation (CID) MS/MS performed with the data-independent (MSE) acquisition algorithm using 3 replicates. Mass spectra were processed using the MassLynx, PLGS and DynamX software packages© (Waters Corp.). The Student’s t-test was applied to the targeted HDX-MS data through an in-house developed excel macro. Size exclusion chromatography (SEC) SEC was carried out using a TSK-Gel® G3000SWXL column (7.8 x 300 mm, 5 µm particle size; Tosoh Bioscience, Amsterdam, Netherlands). An isocratic elution using 50 mM KH2PO4, pH 7.0 at 1.0 mL/min as solvent was used for chromatographic separation, as previously described.7 Cation exchange chromatography (CEC) CEC was performed to monitor mAbX charge variants using a ProPac™ WCX-10 analytical cation exchange column (4.0 x 250 mm; Dionex Softron GmbH, #054993). A step gradient using 25 mM MES, pH 6.1 as solvent A and 25 mM MES, 250 mM NaCl, pH 6.1 as solvent B at 0.5 mL/min was applied. Chromatographic separation was executed, as reported previously.7 Surface plasmon resonance (SPR) analysis – FcRn binding The interaction between the stressed or non-stressed mAbX and the specific target protein (FcRn) was measured by SPR using a Biacore T200™ instrument (GE Healthcare) as described previously.4


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Results and Discussion Following exposure to oxidative stress conditions the ‘100% oxidized mAbX’ sample was characterized using typical quality control assays including SEC, CEC, CD, FTIR and LC-MS peptide mapping, all of which indicated "preserved structural integrity", despite up to 99% oxidation at the most susceptible Met residue as determined by peptide mapping (Table 1). Only the SPR results showed a significant effect (loss in FcRn binding activity) from the oxidation, in agreement with previous studies on the biological relevance of Met oxidation in the constant IgG1 domains.1,4-6 Deuterium uptake differences between the 0% and 100% oxidized mAbX samples were compared over time by HDX-MS (7 time intervals: 0, 0.25, 1, 10, 30, 60 and 150 min.). The Difference Index Butterfly Plot for the peptic peptides from the conserved mAbX HC region for the non-oxidized compared with the 100% oxidized sample is shown in Figure 1 (no significant differences were observed for the peptic light chain peptides over this range; data not shown). Sequence coverage maps of the mAbX peptic peptides following data processing are shown in the supplementary Figures S1 and S2. Significant deuterium uptake differences could be identified for 4 regions of the heavy chain for the 100% oxidized mAbX sample as compared to the non-oxidized. The 100% oxidized mAbX sample showed higher deuterium uptake over the non-oxidized sample in the HC aa regions: 243-253 (largest difference at 1 minute labelling time), 309-350 and 426-448 (largest difference at 150 minute labelling time). The non-oxidized mAbX sample showed higher deuterium uptake over the oxidized in the HC aa region 150-161 (largest difference at 1 minute labelling time). The uptake plot profiles for the individual peptides are shown in Figure 2 (HC aa regions: 243/244-253) and supplementary Figures



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S3. The MS spectra showing the isotopic distribution evolution of the HC peptide aas 243-253 (LFPPKPKDTL) in the non-oxidized and 100% oxidized samples over the time course of the deuteration experiment is provided in the supplementary Figure S4. Our HDX-MS results on the structural effects of Met oxidation in the constant IgG1 domains are consistent with those reported elsewhere in the literature, varying however in degree of sensitivity.23-25,27 So far no investigation demonstrated the structural differences over the sequence range HC aas 150-161 upon oxidation, as seen here due to increased sensitivity. In previous studies uptake differences for this Fc region were also observed upon FcγRIIIa and FcRn antibody binding.24,32 We demonstrate here the increased sensitivity of our optimized workflow on the best characterized modification site (conserved heavy chain methionine in position 254) using spiking experiments. The time survey experiment enabled determination of the most sensitive deuteration time point (largest deuteration uptake difference between the two extreme states) for subsequent target analysis. Non-oxidized mAbX reference material (0%) was compared with the mAbX samples containing 1%, 5%, 10%, 20%, 40%, 60%, 80% and 100% oxidized mAbX at a labeling time of 1 minute, where the largest deuteration difference was observed for the most significant peptides (HC aas 243/4-253) as shown in Figure 2. Samples were prepared in triplicate. Changes in deuterium uptake were considered significant if they exceeded the triple standard deviation, and/or passed a two-tailed, unpaired t-test (p < 0.05 or p < 0.02).33 For the peptic HC peptides aa 243-253 and 244-253 the deuterium uptake of the oxidized dilution samples were significantly higher compared to that of the non-oxidized mAbX, even with the lowest spiked oxidized mAbX content of 1% (Figure 3, Supplementary


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Figure S5). In Table 2 the results of the Student’s t-test comparison of the deuterium uptake (average centroided) for the HC peptides aa 243-253 and 244-253 are shown. Significantly higher D uptake for the selected peptides can be clearly visualized and likewise the critical t-statistic from the Student’s t-test, which defines that the populations are statistically significantly different, is exceeded with content of spiked oxidized mAbX sample ≥ 1% (lowest tested spiking level).



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Conclusion The usefulness of the information provided by HDX-MS in the protein therapeutic field is undisputed, however its applicability to routine analysis has been until now limited due to concerns regarding sensitivity and the cumbersome and time-consuming workflow.19,34,35 We present here a targeted approach which demonstrates the ability to monitor minor changes in composition within a relatively short analysis and data processing time. We demonstrate for the first time the applicability of the HDX-MS technique to monitor structural changes due to methionine oxidation at sensitivity levels realistic to the requirements of biopharmaceutical research and development (typically between 1-5% at peptide level). For the analyzed HC marker peptides aa 243-253 and 244-253 deuterium uptake differences due to oxidation at HC-Met-254 were significantly verifiable at the lowest tested increase (1%) through spiked oxidized mAbX (3 versus 4% total). The application of specific stress conditions combined with fast and sensitive HDX-MS for direct and simultaneous monitoring of structural differences in pre-defined sequence regions (e.g. CDRs) due to chemical and post-translational modifications, represents a complementary approach for the initial CQA assessment and comparability exercises of therapeutic proteins.


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Acknowledgements We are indebted to Dr. Rune Salbo of Novo Nordisk A/S (Bagsvaerd, Denmark), Assoc. Prof. Kasper Rand and Dr. Ulrike Leurs of the Dept of Pharmacy, University of Copenhagen, (Copenhagen, Denmark), Dr. Hermann Beck, Susanne Eltner, Bernd Maier and Dr. Oxana Pester of Roche Pharmaceuticals and Dr. Aaron Wecksler of Genentech (San Fransisco, U.S.A) and all members of the laboratories at Roche Penzberg (Germany) for valuable discussions and cooperation.



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Figure Legends Fig. 1: HDX-MS Difference Index Butterfly Plot for the conserved HC region of mAbX reference and oxidized (100%) material showing the deuterium uptake differences (Da) at the six measured deuteration time points (min.): -- 0.25, -- 1.0, -- 10.0, -- 30, -- 60, -- 150, and summed as grey sticks.

Fig. 2: HDX-MS Relative deuterium uptake profile plots for the HC peptides aas 243-253 (LFPPKPKDTL) and aas 244-253 (FLFPPKPKDTL) in the non-oxidized and 100% oxidized samples over time.

Fig. 3: HDX-MS Relative deuterium uptake for the non-oxidized HC peptides aas 243253 (FLFPPKPKDTL) and aas 244-253 (LFPPKPKDTL) in mAbX samples with varying levels of spiked oxidized sample content.


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Table 1: Oxidative stress condition 0% H 2O 2

0.2% H 2O 2

0.8

1.2

3.3

98.5

1.6

79.7

% Fragment

Deuterium Exchange-Mass Spectrometry to

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