Structure-Based Correlation of Light-Induced Histidine Reactivity in A

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Structure-based Correlation of Light-induced Histidine Reactivity in A Model Protein Ming Lei, Toshiro Carcelen, Benjamin Thomas Walters, Camellia Zamiri, Cynthia Quan, Yuzhe Hu, Julie Nishihara, Holly Yip, Nicholas Woon, Taylor Yonghua Zhang, Yung-Hsiang Kao, and Christian Schoneich Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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Analytical Chemistry

Structure-based Correlation of Lightinduced Histidine Reactivity in A Model Protein Ming Lei1*, Toshiro Carcelen2, Benjamin T. Walters3, Camellia Zamiri2, Cynthia Quan1, Yuzhe Hu2, Julie Nishihara1, Holly Yip1, Nicholas Woon1, Taylor Zhang1, Yung-Hsiang Kao1 and Christian Schöneich4*

1. Protein Analytical Chemistry, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080 2. Late Stage Pharmaceutical Development, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080 3. Early Stage Pharmaceutical Development, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080 4. Department of Pharmaceutical Chemistry, University of Kansas 2095 Constant Avenue, Lawrence, Kansas 66047, USA * To whom correspondence should be addressed. Email: [email protected]; [email protected]

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ABSTRACT Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in crosslink generation1 (Pattison, et al, 2012). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here we used rhDNase which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibilityrelated parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photo-reactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment. KEY WORDS RhDNase, light stress, crosslink, aggregation, hydrogen-deuterium exchange

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INTRODUCTION Light is essential for the survival and thriving of life, but at the same time, it is an energy source for many chemical reactions2-6. Proteins are often the targets of these reactions due to their abundance in biologic systems and the presence of many reactive sites. When exposed to light, proteins can undergo photo-derived transformations initiated both by direct absorption of light by several amino acids (e.g. Trp, Phe, His, Try and Cys)1,2,4 and by photo-sensitized reactions through the absorption of light by other sensitizing moieties internal (e.g. Trp) or external of the protein (e.g., vitamin B2)7-12. The photosensitized reactions involve both singlet oxygen (1O2) and radical species1,4,13. These light-induced reactions lead to many types of transformation of proteins, including oxidation7,14-16, disulfide bond reduction8,11,17, racemization18, fragmentation19 and aggregation13,15,20. Here, light-induced aggregation is particularly important for biopharmaceutical researchers as aggregates can be immunogenic21,22. Previous studies have identified various light-induced covalent crosslinks between amino acids such as Tyr-Tyr, Trp-Trp, Trp-Gly, His-His and His-Lys10,23-28. Crosslinking may occur via radical-radical reactions or the reaction of electrophilic photo-products with electrophiles. The latter appear to play a particular role for His-derived crosslinks 29,30. One reaction mechanism of light-induced His-His crosslinking was elucidated by Shen et al. using N-benzoyl-l-His23. During this reaction, light-induced 1O2 first reacts with His to form endoperoxide which subsequently leads to intermediates which are +14 Da and +32 Da higher in molecular weight. The +32 Da intermediate can undergo nucleophilic addition of a nearby His and ultimately lead to a His-His cross-link (Figure 1). This 3



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reaction is demonstrated to be pH-sensitive by a systematic study using a series of imidazole derivatives7. The photo-oxidation rate of His is significantly slower in its protonated state (pH 6). Recently, an analogous His-His crosslink was identified in a light-exposed monoclonal antibody by Zhou et al31-33, using tryptic digestion in H218O. Since the work by Zhou et al31,33 on a protein is more relevant to current study, the His-His crosslink reaction mechanism is adapted from reference-33. Despite of all the information available on His-His crosslinks, it is unclear why some His residues in proteins form cross-link after light exposure while others do not. A systematic study to elucidate the factors that influence the reactivity of His can help to identify sites that are prone to light-induced cross-linking, and ultimately predict the propensity of light-induced aggregation for a protein. Other factors, such as other lightsensitive components in the buffer and primary sequence may also affect the photoreactivity of His in protein. Besides His, there are usually many other residues (e.g. Trp, Tyr and Cys) that can cross-link upon light exposure, which poses a challenge to study the His-His crosslinking mechanism independently from other crosslinking reactions and to fully characterize the involved sites. Additionally, the solubilities of light-induced protein aggregates are often poor, which further hinders the complete characterization and understanding of the crosslinks involved. The large size of commonly studied proteins such as IgG can also pose a challenge to the analytical techniques used to elucidate light-induced reactions. Therefore, a mid-sized protein, rhDNase was chosen as a 4


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model system for His-His crosslinking studies. Clinically, rhDNase has been used to treat cystic fibrosis-related lung diseases for over two decades34. This molecule is a 260-amino acid glycoprotein that is produced by recombinant DNA technology using Chinese Hamster Ovary (CHO)-K1 cells. It contains two disulfide bonds, two fully occupied N-glycosylation sites and all residues (Trp, Try, His and Cys) that can potentially crosslink upon light treatment34,35. DNase has reported to absorb low level of UV light at wavelengths above 300 nm36, which is within the output of the light exposure condition used here. We observed that rhDNase is unusually resistant to light stress and mainly forms covalent dimers even after very intense light exposure (5 ICH (International Conference on Harmonisation) cycles, 1 ICH cycle is 1.32 million lux hours and 538 watt-hours/m2 over 24 hours at 35 ºC. This condition exceeded the dose recommended by the ICH Q1b and ICH Q5b guidelines for photostability testing37). This relative simple case allowed us to fully characterize the cross-links involved in light-induced covalent dimerization. We found that all the crosslinks are of the type His-His. We proceeded to correlate the photo--reactivity of these His residues with solvent accessible surface area (SASA), calculated from the crystal structure, and with structural flexibility measured by hydrogen-deuterium exchange (HDX) mass spectrometry (MS) experiments. Our results shed light on the significance of His solvent accessibility and protein dynamics for the formation of His-His crosslink. To our knowledge, this is the first time that such correlation has been attempted for light-exposed proteins.

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MATERIALS AND METHODS Reagents and Materials RhDNase was generated in-house at Genentech (South San Francisco, CA) formulated in 150 mM NaCl, 1 mM CaCl2 with pH at 7 with protein itself as a buffer. Information on other reagents used in this study can be found in Supporting Information. Sample Preparation for Light Exposure The light-stress experiments were carried out in an Atlas SUNTEST CPS+ light box (Atlas Material Testing Technology, Mount Prospect, Illinois), equipped with an ID65 filter, for up to 5 cycles. The Atlas SUNTEST light box has a Xenon lamp and an ID65 filter capable of producing an irradiance level of approximately 22.5 watts/m2 in the UV region between 300 nm to 400 nm and approximately 55,000 lux in the visible region between 400 nm to 800 nm6. This light condition was according to the recommendation by International conference on harmonization (ICH) guideline Q1b37. One ICH cycle is defined as cumulative exposure to 1.32 million lux hours and 538 watt-hours/m2 over 24 hours at 35 ºC37. The control samples were foil wrapped and stored side by side with the test samples in the light box throughout the 5-ICH cycles at 35 ºC. Five milliliters of 7 mg/mL rhDNase in 150 mM NaCl, 1mM CaCl2 was filled into each polyethylene terephthalate glycol-modified (PETG) containers. Half of the rhDNase samples were spiked with methionine to a final concentration of 13 mM to quench photo-generated 6


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peroxides and other reactive oxygen species such as, e.g., singlet oxygen, hydrogen peroxides, peroxyl radicals and hydroxyl radicals. The experiments used to determine the optimum amount of methionine for quenching are described in the Supporting Information.

Tryptic Digest and Peptide Mapping Samples were pretreated with EGTA to denature the protein before digestion with trypsin, and then buffer-exchanged into 100 mM Tris, pH 8.5 buffer by NAP-5 columns (GE Healthcare, Pittsburgh, PA). No reduction or further denaturation was performed. The samples were digested with trypsin for 4 hours at 30 °C. The peptides were separated on an ACQUITY UPLC BEH Shield RP18 Column, 130Å, 1.7 µm, 2.1 mm X 150 mm (Waters, Milford, MA), which was fitted to a Waters H-class UPLC system equipped with a UV detector, with the detection wavelength set at 214 nm. The mobile phase A was 0.1% TFA in water while mobile phase B was 0.08% TFA in acetonitrile. The column temperature was 45 °C, the flow rate for the separation phase was 0.35 mL/min, and the injection volume was 100 µL. The separation gradient increased mobile phase B linearly from 0 to 42% within 50 minutes.

Hydrogen-Deuterium Exchange (HDX)-MS experiments The HDX-MS experiments were performed according to a recent publication by Walters et al38. Briefly, rhDNase samples were diluted 1:15 into a solution of D2O containing 20 mM His, 50 mM NaCl, at pD 7.0, and allowed to exchange for seven time intervals from 0.5 to 1000 min. Time points taken in triplicate were randomized and collected by the 7



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LEAPv1 HX robotics platform (Leap Technologies, Carrboro, NC). Quenched samples were injected into a cold online system where they were digested at 0 °C by an immobilized pepsin column (2.1 x 30 mm, Applied Biosystems, Foster City, CA), desalted on a trap column (ACQUITY Vanguard C18), separated by reversed phase chromatography (ACQUITY UPLC BEH C18, 1.7-mm particle size, 1.0 x 50 mm), and introduced into the mass spectrometer (Thermo Orbitrap Elite). The MS measurements were performed at the resolving power of 120,000 Hz at m/z 400. Data were analyzed using the ExMS program38,39 along with custom Python scripts that combine degenerate charge states and fit uptake traces with binomials to determine the amount of incorporated deuterium and assess modality38,40.

Details for other assays for performed in this study can be found in Supporting Information.

RESULTS AND DISCUSSION Light-induced rhDNase Dimer Is Non-dissociable and Non-reducible The percentage of dimer and percentage of HMWS (high molecular weight species, larger than dimer) were determined by the Size-exclusion chromatography (SEC) assay. An overlay of SEC chromatograms of a typical light-exposed sample and a control are shown in the Supporting Information, Figure S1. The levels of dimer and HMWS were below the quantitation limit of the assay for control samples. After light treatment, increases of both the %dimer and % HMWS were observed (Table 1). The majority of 8


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the aggregates existed in covalent dimer form (3.8% for 5-ICH light cycle treatment, as defined in the Materials and Methods section) while only a small amount of HMWS (0.5% for 5-ICH light cycle treatment) were formed. The dimer and HMWS yields were lowered when methionine at a final concentration of 13 mM was present during photo-irradiation. Met is a common antioxidant excipient in biopharmaceutical products14. It quenches peroxides and other reactive oxygen species such as, e.g., singlet oxygen, hydrogen peroxides, peroxyl radicals and hydroxyl radicals. The reduction of the yields of lightinduced aggregates in the presence of Met suggested that peroxides were contributing to aggregation during light-treatment.

In order to determine the contribution of hydrophobic interactions to aggregation, we performed the SEC assay with 10% isopropyl alcohol (IPA) added into the mobile phase. The addition of IPA is used to dissociate aggregates formed by hydrophobic interaction41,42. By comparing the %dimer and %HMWS in the absence and presence of 10% IPA (Table 1), it is apparent that dissociable aggregate levels are insignificant. To further verify the nature of the light-induced aggregates and to understand if the aggregation is related to disulfide bond formation, SDS-PAGE experiments using both reduced and non-reduced conditions were performed. Under reducing conditions, the samples were incubated with dithiothreitol (DTT) to reduce the disulfide bonds before analysis. As shown in Figure 2, rhDNase monomer appears as a wide band between 25-37 kDa. The wide distribution of rhDNase monomer band is due to variations in glycosylation. A band between 50-75 kDa is only observed for light-treated sample and 9



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is consistent with the molecular weight of rhDNase dimer. No higher aggregate band was observed for control or light-treated samples. The dimer band was observed in the presence of detergent (SDS), indicating that the dimer is likely covalent in nature. No shift in band location or new band was observed (Figure 2b) when comparing the images between the reduced and non-reduced samples. The relative optical density of each band was not changed between reduced and non-reduced samples. This result indicated that the linkage between the dimers cannot be reduced by the conditions used here. The two different methods used here measures different types of aggregates. SEC is non-denaturing, hence the HMWS and dimer observed is a mixture of covalent and non-covalent species. SDS-PAGE contains denaturing agent that disrupts noncovalent (hydrophobic) interactions. Reducing SDS-PAGE results suggest that dimer formation doesn’t involve disulfide bonds. The conclusions from two orthogonal methods, SDS-PAGE and SEC, are consistent with the interpretation that the majority of light-induced rhDNase dimer is non-dissociable and not reducible.

Dimers Are Due to His-His Crosslinking When comparing the tryptic peptide maps of samples treated with 0, and 5 ICH light cycles, only two regions exhibited new peaks that increased with light treatment. These peaks were identified by mass spectrometry to be peptides containing the +14 Da and + 32 Da intermediate products of His44. Specifically, the +32 Da product peak was observed as a doublet, which is consistent with the proposed mechanism that the +32

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Da product contains two structural isomers. The tryptic peptide map chromatograms of the regions with new peaks were shown in Supporting Information Figure S2.

The relative abundance of the +32 Da product for His44 was 3.6% for the 5-ICH lighttreated sample. An error tolerant database search of LC-MS/MS data found +32 Da products on three additional peptides containing His64, His134 and His208 residues. The relative abundances of the +32 Da products for H64 and H208 were both at 0.8%, while that level was 0.1% for H134 for the 5-ICH light-treated sample .The +32 Da product was not observed for His252. The His44 residue was the most susceptible to light-induced crosslinking, as the +32 Da product of His44 was approximately 4-times more abundant than that from all other His residues.

Since rhDNase contains only five His residues that are distributed in five different tryptic peptides, it is possible to calculate the theoretical masses for all His-His crosslinked peptides. The masses of these His-His crosslinked peptides were searched manually in both the 5-ICH light cycle treated sample as well as the purified dimer fractionated by SEC. In short, three His-His crosslinked peptides T5-T5 (His44-His44), T5-T6 (His44His64) and T5-T15 (His44-His208) were observed in the light-treated samples. No other His-His crosslinked peptides were found. Other crosslinked peptides through other residues (such as Tyr-Tyr, Trp-Trp, Tyr-Trp) that can potentially be formed under light treatments were not observed by manually search their theoretical masses. The identities of these three crosslinked peptides were confirmed by MS/MS experiments (Supplemental Information Figure S2). The fragment ions corresponding to cleavages 11



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both before and after the His sites, as well as the fragment ion corresponding to the cleavage between the His-His crosslink were observed. The His-His crosslink structure (Scheme 1) was proposed by analogy to published NMR data on such His-His crosslinks23,43. The sequence and the MS information for all His containing peptides are shown in Table 2. As expected, all of these crosslinked peptides involved His44 in T5. Among them, T5-T5 is the dominant crosslinked species (>80%) while the other crosslinked peptides accounted for 40% oxidized at its most susceptible Trp residue50. This difference can be 17



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partially explained by the amount of light-induced hydrogen peroxide (H2O2) generated by rhDNase and IgG. At a similar protein concentration and ICH light condition, rhDNase after 120-hr ICH (cumulative exposure of 6.6 million lux hours and 2690 watthours/ m2) light treatment generates approximately the same amount of H2O2 as an IgG in 3 hours (cumulative exposure of 0.17 million lux hours and 67.3 watt-hours/ m2)50. However, this difference alone cannot explain the unusual light stability of rhDNase. Other factors, such as solvent accessibility of Trp residues, as well as the distance between singlet oxygen and radical generation center (e.g, Trp) to other reactive residues (e.g. His and Met) can also play a significant role. A systematic comparative study between rhDNase and other molecules can further elucidate the role of protein structure and dynamics on light-induced reactions in protein.

CONCLUSIONS As we demonstrated using rhDNase as a case study, light stress is a potential inducer for covalently crosslinked protein aggregates. Multiple crosslinked His residues were found in the light-induced rhDNase covalent dimers. The reactivity of the His residues were correlated with their solvent accessibility and structural flexibility. Solvent accessibility calculated from the crystal structure and structural flexibility measured by in-solution experiment such as HDX can help rank the reactivity of the His residues. In cases with multiple His residues of similar reactivities, complex crosslinked aggregates should be expected for light-exposed samples.

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FIGURE CAPTIONS Figure 1. Photo-induced His-His crosslink and intermediate products (adapted from ref33). The two isoforms of the + 32 Da product and the His-His crosslink product both gave rise to doublet peaks in peptide map chromatogram. Figure 2. SDS-PAGE image of non-reduced (a) and reduced (b) samples. Lane information in both figures: 1. Protein standards; 2. Sample buffer; 3. 5-ICH light cycle treated rhDNase; 4. Control. Figure 3. Three dimensional representation of the main His-His crosslinked covalent dimer species through His44-His44. The structure of the monomer is rhDNase crystal structure (PDB:4AWN). The crosslinking site His44 was labeled in red without showing the exact crosslinking chemistry. The bound metal ions were presented by orange sphere (Ca2+) and green sphere (Mg2+). The approximate location of rhDNase catalytic site and substrate binding site were also shown in this figure. Figure 4. Correlation between percent oxidation (+32 Da) at each His site and SASA based on crystal structure (a) or the natural logarithm of protection factor measured by HDX experiments (b). A linear fit and its R2 value was also shown on each figure.

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Table 1. The percentage of dimer and The percentage of HMWS measured by SEC with and without 10% isopropyl alcohol (IPA) in the mobile phase. The injection volume was 50 µL. M stands for the presence of 13 mM methionine during the light exposure.

Sample

No IPA % Dimer Control N/D* Control-M N/D 5-ICH cycles 3.8 5-ICH cycles-M 1.5

% HMWS N/D N/D 0.5 0.1

10% IPA % Dimer N/D N/D 3.2 1.2

% HMWS N/D N/D 0.4 0.1

N/D: not detected.

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Table 2. Observed mass, calculated mass, mass difference for the light-induced modifications on His-containing peptides and crosslinked peptides after 5 ICH cycles of light treatment. T15 (SH) is generated by reducing the tryptic digest by TCEP in order to reduce the size of the peptide. Crosslinked peptides are listed on the bottom of the table.

Peak name

T5 T5+14 T5+32 T6 T6+32 T13 T13+32 T15 (SH) T15 +32

His position

His44

H64 H134 H208

T17 T17+32

H252

Retention time (min)

Observed mass MH+ (Da)

Expected mass MH+(Da)

Mass difference (ppm)

12.4 14.1 11.2 25.7 27.4 45.7 47.5 34.6 35.5

927.4898 941.4734 959.4886 2629.2922 2661.2885 3371.7046 3385.6886 3194.4887 3226.4879

927.4904 941.4724 959.4878 2629.2892 2661.2911 3371.7046 3385.6999 3194.4938 3226.4833

1.1 -1.1 -0.9 1.1 1.0 0.0 3.4 1.3 -1.4

46.4

4062.0544

4062.0317

5.6

N/D

N/D

N/D

N/D

T5-T5

His44His44

15.1

1867.9550

1867.9550

0

T5-T6

His44-H64

23.2

3569.7559

3569.7549

2.8

T5-T15

His44H208

30.3

4134.9637

4134.9544

2.2

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Sequence

DSHLTAVGK DSH (+14) LTAVGK DSH (+32) LTAVGK LLDNLNQDAPDTYHYVVSEPLGR LLDNLNQDAPDTYH (+32)YVVSEPLGR EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK EFAIVPLH (+32)AAPGDAVAEIDALYDVYLDVQEK LWTSPTFQWLIPDSADTTATPTHCAYDR LWTSPTFQWLIPDSADTTATPTH(+32)CAYDR GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK GAVVPDSALPFNFQAAYGLSDQLAQAISDH (+32)YPVEVMLK DS(H+14) LTAVGK DS(H)LTAVGK DS(H+14) LTAVGK LLDNLNQDAPDTY(H)YVVSEPLGR DS(H+14) LTAVGK LWTSPTFQWLIPDSADTTATPT(H)CAYDR DS(H) LTAVGK LWTSPTFQWLIPDSADTTATPT(H+14)CAYDR


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

Figure 1

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Figure 2.

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Figure 3

(b)

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Figure 4

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Table of content graphics

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Structure-Based Correlation of Light-Induced Histidine Reactivity in A

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