Probing the Conformation of an IgG1 Monoclonal Antibody in

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen−Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS) Ehab M. Moussa,† Satish K. Singh,‡,§ Michael Kimmel,‡ Sandeep Nema,‡ and Elizabeth M. Topp*,† †

Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States ‡ BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, Missouri 63017, United States S Supporting Information *

ABSTRACT: Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen−deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient. KEYWORDS: aggregation, conformation, interactions, lyophilization, monoclonal antibody, solid-state

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INTRODUCTION Monoclonal antibodies (mAbs) constitute one the fastest growing classes of therapeutic proteins.1 In an effort to improve long-term storage stability of these large and complex macromolecules, mAbs are often formulated as lyophilized solids.2 The freezing and drying processes, however, impose a number of stresses on protein molecules including cold denaturation, pH changes, formation of an ice−water interface, freeze-concentration of solutes, and dehydration.3 Accordingly, stabilizing additives are used to preserve protein structure during and after lyophilization.4 Chief among these are disaccharides, which can act as both cryo and lyoprotectants. Stabilization by amorphous excipients, especially disaccharides, has been attributed to their ability to form hydrogen bonds with proteins in the amorphous solid-state5 to replace those lost upon removal of water, and/or to the immobilization of proteins in a glassy amorphous matrix6 in which the rates of degradation reactions are slowed by the reduced molecular mobility. To guide the development of stable lyophilized protein formulations, longterm stability in different lyophilized formulations has been related to the retention of protein higher order structure and/or to the molecular mobility in the matrix, with varying degrees of success.7 © XXXX American Chemical Society

In the solid-state, structure and dynamics can be probed using various analytical methods including vibrational, fluorescence, dielectric relaxation, and nuclear magnetic resonance spectroscopies.8 Correlations between aggregation and secondary structure retention of mAbs as measured by solid-state Fouriertransform infrared (ssFTIR) spectroscopy have been thoroughly investigated in a wide range of formulation compositions.7,9,10 Similarly, the secondary structure of an IgG mAb as measured by solid-state Raman spectroscopy has been correlated with aggregation in lyophilized and spray dried formulations.11 Aggregation of a model mAb in different lyophilized formulations has also been correlated with tertiary structure changes measured using solid-state fluorescence spectroscopy.10 Moreover, the effects of mAb dynamics on aggregation have been probed by measuring structural relaxation using dielectric relaxation6 and NMR10 spectroscopies. One limitation of these techniques is that they provide only global information regarding the structure and dynamics of Received: August 12, 2017 Revised: December 21, 2017 Accepted: January 9, 2018

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DOI: 10.1021/acs.molpharmaceut.7b00696 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics proteins. In solution, hydrogen−deuterium exchange with mass spectrometric analysis (HDX-MS) provides high-resolution analysis of protein structure and dynamics12 by measuring the kinetics of deuterium uptake in both the intact protein and its peptic fragments, and is hence well suited to analyze many different proteins with few limitations in size.13 Recently, our group has adapted HDX-MS to probe the conformation of proteins in the amorphous solid-state. The solid-state HDX-MS (ssHDX-MS) method involves exposing a solid powder containing the protein to D2O vapor at controlled vapor pressure and temperature, and monitoring the extent of deuterium incorporation by the protein and its peptic fragments as a function of time using mass spectrometry.8,14 Using this method, site-specific effects of calcium15,16 as well as several carbohydrate excipients17 on the conformation of lyophilized calmodulin were elucidated. Additionally, ssHDXMS has been used to study the effect of carbohydrate excipients and linear polymers on different proteins.18 More recently, we have used ssHDX-MS to map and quantify the kinetics of local hydration in lyophilized myoglobin formulations,19,20 and have shown that deuterium incorporation is highly correlated with long-term storage stability for two different proteins.21,22 In the present study, the effects of various excipients on the conformation and matrix interactions of a lyophilized IgG1 mAb were compared using ssHDX-MS and were related to the aggregation rate. The results demonstrate a correlation between the extent of ssHDX and aggregation rate on storage at 40 °C in formulations of different excipients. Next, we investigated the effect of histidine as a single amorphous excipient or in combination with sucrose on deuterium incorporation in ssHDX as well as aggregation. Interestingly, adding histidine to a base sucrose formulation decreased aggregation of the mAb at different pH values. However, the protective effects of both excipients were not correlated with the extent of deuterium incorporation in ssHDX, suggesting that histidine stabilizes the mAb via a mechanism not detected by this method. Finally, ssHDX-MS analysis of peptic digests showed that in the low molecular weight sugar formulations (i.e., sucrose, trehalose or mannitol), observed differences in deuterium incorporation are global rather than local, whereas site-specific protection from ssHDX is evident in a histidine matrix.

Table 1. Composition and Physical Properties of the Single Excipient Formulations at pH 6.8 formulation

a

H2 H3 M1 M3 NE S1 S2.7 S3 S6 T1 T3

excipient

excipient/ mAb ratio (w/w)

moisture contentb

moisture sorbed at 11% RHc

Tgd

histidine histidine mannitol mannitol none sucrose sucrose sucrose sucrose trehalose trehalose

2:1 3:1 1:1 3:1 NA 1:1 2.7:1 3:1 6:1 1:1 3:1

0.99 ± 0.34 1.10 ± 0.08 0.83 ± 0.21 0.76 ± 0.05 0.56 ± 0.06 1.10 ± 0.03 1.37 ± 0.32 1.34 ± 0.07 1.40 ± 1.40 1.00 ± 0.21 0.99 ± 0.12

NM 1.02 NM 1.20 NM NM NM 1.88 1.74 NM 2.43

105 100 ND ND ND ND NM 70 63 103 98

a

All formulations were prepared in 2.5 mM potassium phosphate buffer at pH 6.8. bResidual moisture content was measured by Karl Fischer titration. cThe w/w ratio of water to dry solid measured at the plateau of thermogravimetric analysis of water sorption by the dry cakes at 11% RH. NM, not measured. dTg, the glass transition temperature of the dried solid. ND, no transition could be detected by modulated differential scanning calorimetry (MDSC). Values are reported to the nearest unit.

Table 2. Composition and Physical Properties of the Sucrose/ Histidine Formulations at Different pH Conditions formulation 5H2 5S1 5S1H0.5 5S1H1 6H2 6S1 6S1H0.5 6S1H1 H2 S1 S1H0.5 S1H1

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a

b

pH

5 5 5 5 6 6 6 6 6.8 6.8 6.8 6.8

sucrose/mAb ratio (w/w)

histidine/ mAb ratio (w/w) 2:1

1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1

0.5:1 1:1 2:1 0.5:1 1:1 2:1 0.5:1 1:1

moisture contentc

Tgd

2.82 ± 1.31 2.72 ± 0.24 1.67 ± 0.77 2.57 ± 0.01 2.79 ± 0.67 2.01 ± 0.22 1.60 ± 0.39 1.95 ± 0.38 0.99 ± 0.34 1.10 ± 0.03 2.55 ± 0.13 2.12 ± 0.76

94 ND 90 90 106 93 95 101 105 ND 94 98

a In these abbreviations, the first number corresponds to the formulation pH, the first letter refers to the first excipient, the second number refers to the weight ratio of the first excipient to other excipients, the second letter refers to the second excipient, the third number is the weight ratio of the second excipient to other excipients. H, histidine. S, sucrose. bAll formulations were prepared in 2.5 mM potassium phosphate buffer. pH values were adjusted with phosphoric acid. cResidual moisture content was measured by Karl Fischer titration. dTg, the glass transition temperature of the dried solid. ND, no transition could be detected by modulated differential scanning calorimetry (MDSC). Values are reported to the nearest unit.

EXPERIMENTAL SECTION Formulation. An IgG1 mAb was provided by Pfizer Biotherapeutics (St. Louis, MO) as a 19.3 mg/mL solution. The protein solution was dialyzed extensively at 4 °C into different formulation buffers (Tables 1 and 2) using Slide-ALyzer dialysis cassettes 30K MWCO (Thermo Scientific, Rockford, IL). The pH was adjusted to 6.8 when necessary using phosphoric acid. Dialyzed mAb solutions were diluted in the respective formulation buffers to a final mAb concentration of 10 mg/mL (Tables 1 and 2). Diluted samples were then filtered using Millex-GV 0.1 μm PVDF filters (Millipore, Billerica, MA), and 200 μL were dispensed into 2 mL borosilicate Type 1 glass vials (Wheaton Inc., Millville, NJ). Hereinafter, the formulations are referred to using the abbreviations listed in the left-most column of Tables 1 and 2, in which the letter refers to the excipient and the number that follows refers to the excipient/ mAb weight ratio. The formulations with no leading number were lyophilized from protein solutions at pH 6.8. For the formulations lyophilized from protein solutions at pH 5 and 6, the leading number refers to the pH value.

Lyophilization. Lyophilization of all formulations (except S1, T1, and M1) was carried out in a benchtop lyophilizer (VirTis Advantage, SP Scientific, Gardiner, NY). The shelf temperature was ramped from room temperature to −2 °C and held isothermally for 15 min. The shelf-temperature was then ramped to −40 °C and held isothermally for 60 min. Lyophilization of the S1, T1, and M1 formulations was carried out in a laboratory-scale lyophilizer (Revo, MillRock Technology, Kingston, NY). The shelf temperature was ramped from room temperature to 5 °C, held isothermally for 15 min, ramped to −5 °C, held isothermally B


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Molecular Pharmaceutics for 15 min, and then ramped to −40 °C and held isothermally for 60 min. For all formulations, primary drying was carried out at a shelf temperature of −35 °C and a chamber pressure of 70 mTorr for 24 h, followed by secondary drying at a shelf temperature of 25 °C and a chamber pressure of 70 mTorr for 12 h. Different lyophilization batches were used for structural and conformational analyses and for the stability studies. Karl Fischer Titration. The moisture content in the lyophilized samples was determined by the Karl Fischer titration method using a 831 KF Coulometer (Metrohm, Riverview, FL). The samples were reconstituted with 1 mL of hydranal-methanol dry or 1 mL of a 1:1 mixture of hydranal-methanol dry and hydranal-formamide dry (Fluka, St. Louis, MO), and the suspension was titrated with Riedel-de Haën Hydranal Coulomat reagent (Hoechst Celanese Corp., Germany) until the end point was reached. Two vials were analyzed for each formulation. Modulated Differential Scanning Calorimetry. To measure the glass transition temperature of the dry solid (Tg), 1−3 mg was loaded into a Tzero aluminum pan (TA Instruments, New Castle, DE). The pans were hermetically sealed under nitrogen in a glovebox maintained below 5% RH. The samples were equilibrated at −5 °C for 1 min followed by a temperature ramp to 140 °C at 5 °C/min using a Q2000 calorimeter (TA Instruments). Superimposed temperature modulation was carried out at ±1 °C every 60 s. The Tg value was calculated as the midpoint temperature of the transition in the reversing heat flow signal. The DSC instrument was calibrated at the specified heating rate using indium. Solid-State Fourier Transform Infrared Spectroscopy. The secondary structure of the mAb in the different lyophilized matrixes was analyzed using ssFTIR spectroscopy in attenuated total reflectance (ATR) mode. Spectra were collected using a Nicolet Nexus spectrometer (Thermo Scientific, Waltham, MA) equipped with a Smart iTR accessory. The solid powder was loaded on the diamond cell and pressed against the diamond using a metal anvil. Spectra were recorded over a range of 800− 4000 cm−1 with 120 scans and 4 cm−1 resolution. All spectra were corrected for moisture content and CO2. Spectra were then processed using OPUS software (Bruker, Billerica, MA), baseline corrected, smoothed, and normalized before calculating the second derivative. Measurements were conducted for samples from three different vials. For histidine formulations, spectra of lyophilized excipients were subtracted manually. Solid-State Fluorescence Spectroscopy. The tertiary structure of the mAb in the different lyophilized samples was analyzed using a Cary-Eclipse spectrofluorometer (Agilent Technologies, Santa Clara, CA). Lyophilized cakes were pulverized and loaded into the solid-state sample holder as described previously.23 Measurements were carried out at an incident angle of 25°, a photomultiplier tube (PMT) voltage of 600, and a slit width of 5 nm. The samples were excited at 280 nm, and emission spectra were monitored from 280 to 450 nm. To facilitate visualizing peak shifts, the spectra were intensitynormalized as described previously.24 In this analysis, it is assumed that the peak intensity in solid-state protein samples is not a function of concentration.24 Solid-State Hydrogen−Deuterium Exchange with Mass Spectrometric Analysis: Intact Protein. Vials containing lyophilized cakes were incubated at 22 °C in a sealed desiccator containing a saturated solution of LiCl in D2O to achieve the equivalent of 11% RH in D2O. Vials were removed at different time points over 240 h, and HDX was quenched by placing the vials on dry ice and storing them at −80 °C.

Deuterium uptake by the intact mAb was measured using high performance liquid chromatography coupled to a mass spectrometer (LC/MS) (1200 series LC, 6520 qTOF; Agilent Technologies) and equipped with a custom-built refrigeration unit kept at ∼0 °C to minimize back exchange. Hereinafter, “intact mAb” refers to ssHDX-MS samples analyzed without pepsin digestion to provide the overall number of deuterons incorporated into the mAb structure, in contrast to “peptide level” or “digest level” analysis. At the time of analysis, the vials stored at −80 °C were reconstituted in ice-cold quench buffer (0.1% formic acid (FA), pH ≈ 2.5) and then injected onto a protein microtrap (Michrom Bioresources, Inc., Auburn, CA). The samples were desalted for 1.7 min with 10% acetonitrile, 90% water, and 0.1% FA and eluted in 7 min using a gradient of 90% acetonitrile, 10% water, and 0.1% FA. Mass spectra were acquired over the m/z range 200−20,000, and the masses of both undeuterated and deuterated mAb were obtained by deconvoluting the acquired spectra using the maximum entropy function in MassHunter Workstation Software Version B.04 (Agilent Technologies). Deuterium uptake values as a function of time were fitted to a biexponential association model as described previously.15 D(t ) = Nfast(1 − e−k fastt ) + Nslow(1 − e−kslowt )

(1)

where D(t) is the total number of deuterons incorporated into the mAb at time t, Nfast and Nslow are the numbers of amide hydrogens in the rapidly and slowly exchanging pools, respectively, and kfast and kslow are the respective exchange rate constants. Solid-State Hydrogen−Deuterium Exchange with Mass Spectrometric Analysis: Peptide Level. Deuterated lyophilized mAb samples were reconstituted as described above and injected onto an immobilized pepsin column kept at 25 °C in a column oven within a custom-built LC column refrigeration unit. Injected samples were digested for 2 min in 0.1% FA at an isocratic flow rate of 0.2 mL/min. The protein digest was then desalted in a peptide microtrap (Michrom Bioresources) and eluted using a gradient of 10−60% acetonitrile in 0.1% FA onto a reverse phase analytical column (Zorbax 300SB-C18; Agilent Technologies) at flow rate of 50 μL/min. The pepsin column, peptide microtrap, and analytical column were all placed inside the refrigeration unit and were connected through a two-position valve (EPC12CW, VICI Valco Instruments Co., Inc., Houston, TX). The total time between reconstitution and MS analysis was approximately 12 min. Mass spectra were acquired over the m/z range 100−1700. Peptic fragments were identified by accurate MS using time-of-flight (TOF) mass spectrometry (Agilent Technologies) and/or collision-induced dissociation (CID) tandem mass spectrometry using a hybrid linear ion trap Orbitrap mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher). HDExaminer software version 2.2 (Sierra Analytics, Modesto, CA) was used to analyze the raw data and obtain the average number of deuterons exchanged for each peptic fragment. For each fragment, one charge state was consistently compared across all samples. Size Exclusion Chromatography. Size exclusion chromatography (SEC) was used to determine the monomer and aggregate content in formulations after reconstitution at time zero and after 45 and 90 days (formulations in Table 1) or following 60 days (formulations in Table 2) of storage at 40 °C and 22% RH. Samples were centrifuged at 10,000g for 10 min to remove large aggregates, and supernatants containing soluble aggregates were then analyzed using a G3000 SWXL column (7.8 C


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Figure 1. Second-derivative ssFTIR spectra of the mAb lyophilized in formulations containing excipients at different excipient/mAb weight ratios: M3, mannitol 3:1. M1, mannitol 1:1. S6, sucrose 6:1. S3, sucrose 3:1. S2.7, sucrose 2.7:1. S1, sucrose 1:1. T3, trehalose 3:1. T1, trehalose 1:1. H3, histidine 3:1. H2, histidine 2:1. NE, excipient-free.

mm, 30 cm, 5 μm) fitted with a security guard column (Tosoh Bioscience, King of Prussia, PA) placed in an Agilent 1200 high performance liquid chromatography (HPLC) system (Agilent Technologies). One hundred microliters of each sample was analyzed using 0.1 M sodium phosphate buffer containing 0.25 M NaCl and 0.05% NaN3 (pH 6.8) as a mobile phase at a flow rate of 1 mL/min for 15 min. The aggregate content of each formulation was calculated by measuring the percentage decrease in the area of the monomer peak, with the assumption that all loss is attributable to aggregation. For the stability study comparing histidine to sucrose (formulations in Table 2), relative stability (Rs) was calculated using eq 2 as described previously.25 Rs =

ΔA° − ΔAH ΔA°

Effect of Different Excipients on Secondary Structure at Prelyophilization pH 6.8. ssFTIR spectra of the buffered mAb solution were in approximate agreement with the typical spectra of IgG,26 with bands at ∼1615 cm−1 (β-sheet or side chain), ∼1637 cm−1 (β-sheet), ∼1661 and ∼1677 cm−1 (turns), and ∼1690 cm−1 (β-sheet). The major β-sheet band is at ∼1635 cm−1 in the buffered solution and ranged from ∼1635 to ∼1640 cm−1 in the different lyophilized formulations (Figure 1). In general, peak intensities in IR spectra of proteins are used as a measure of the fraction of the various secondary structural components, whereas peak position may indicate the strength of hydrogen bonding interactions.27 Among the different formulations, the spectra of the H2 and H3 formulations appear to be the most perturbed, with decreases in the intensity of the major peak and increases in the intensity of the peak at ∼1615 cm−1 relative to the solution control. Effect of Different Excipients on Tertiary Structure at Prelyophilization pH 6.8. Tertiary structure of the mAb in the solid-state was characterized using front surface fluorescence spectroscopy (Figure 2) to identify whether the observed differences in ssHDX are the result of different interactions with excipients or differences in protein structure. A red shift in the peak maximum indicates an increase in the hydrophilicity of the environment of tryptophan residues and is consistent with partial unfolding. The peak maximum of tryptophan in a completely unfolded state in the solid-state has been reported to be 334 nm.23 In all sucrose, trehalose, and mannitol formulations, and in H3, the peak maximum was red-shifted by ∼2 nm compared to S3 (S6 formulation was not tested), indicating a more folded structure in S3. In H2, the peak was red-shifted by ∼4 nm, whereas in the excipient-free formulation the peak was redshifted by ∼6 nm, indicating more pronounced unfolding. Effect of Different Excipients on Deuterium Incorporation at Prelyophilization pH 6.8. ssHDX kinetics in formulations of different excipient/mAb weight ratios were compared. Increasing the sucrose/mAb ratio from 1:1 to 6:1 led to a decrease in deuterium incorporation (Figure 3) and slowed the rate of deuterium incorporation in the fast exchanging pool,

(2)

where ΔA° and ΔAH represent the increase in the percent of soluble aggregates in the base sucrose formulation (5S1, 6S1, or S1) and the corresponding histidine (H) containing formulation, respectively.

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RESULTS Physical Characterization of the Lyophilized Formulations. Lyophilized formulations were first characterized for physical appearance, crystallinity, and glass transition. No cake collapse, shrinkage, or visual defects were observed in the cakes for any of the formulations. The formulations were completely amorphous, except for the mannitol containing formulation (M3, Table 1), in which delta polymorph crystals of mannitol were detected (Figure S1); a glass transition was not observed for this formulation. Higher Tg values were observed for the trehalose and histidine formulations than for the sucrose-rich formulations (S3 and S6) (Table 1). For the NE, 5S1, S1, and M1 formulations, no glass transition was observed due to a lack of sufficient sensitivity. For each formulation containing histidine, the Tg of the powder was greatest at pH 6 and least at pH 5 (Table 2). At pH 6, adding histidine to sucrose at 0.5:1 and 1:1 weight ratios increased Tg by 2 and 8 °C, respectively (Table 2). D


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Table 3. Nonlinear Regression Parameters of Deuterium Uptake Kinetics (11% RH, RT) in the Intact mAb in Different Formulations at pH 6.8a formulation

Nfast

kfast (h−1)

Nslow

kslow (h−1)

H2 H3 M1 M3 NE S1 S2.7 S3 S6 T1 T3

56(11) 120(28) 180(11) 200(30) 200(26) 88(8) 67(13) 62(24) NF 119(8) 81(19)

0.75(0.72) 0.05(0.01) 0.18(0.02) 0.16(0.04) 0.18(0.04) 0.21(0.04) 0.18(0.06) 0.06(0.02) NF 0.19(0.03) 0.09(0.03)

170(10) 130(150) 175(9) 186(26) 163(22) 134(7) 135(12) 118(14) NF 180(28) 129(13)

0.036(0.004) 0.003(0.007) 0.009(0.002) 0.016(0.005) 0.016(0.005) 0.012(0.002) 0.011(0.004) 0.008(0.003) NF 0.006(0.002) 0.009(0.004)

a See eq 1 and associated text for regression equation and parameter definitions. Values in parentheses represent standard errors of the regression parameters. NF indicates that an acceptable fit could not be obtained. Values of Nfast and Nslow are rounded.

Figure 2. Intrinsic fluorescence spectra of the mAb lyophilized in formulations containing excipients at different excipient/mAb weight ratios: M3, mannitol 3:1. M1, mannitol 1:1. S3, sucrose 3:1. S1, sucrose 1:1. T3, trehalose 3:1. T1, trehalose 1:1. H3, histidine 3:1. H2, histidine 2:1. NE, excipient-free.

consistent with limited protection from exchange provided by mAb−crystalline−mannitol interactions (M3), by mAb−mAb interactions (NE), and/or the loss of intramolecular hydrogen bonds in the partially unfolded mAb (NE, Figure 2). S2.7, T3, and H3 formulations had similar overall deuteration kinetics (Figure 3) indicating similar extent of matrix interactions and/or folded structure, the latter also indicated by similar fluorescence spectra (Figure 2). Correlation of ssFTIR Peak Intensity and the Extent of ssHDX with Aggregation at Prelyophilization pH 6.8. Aggregation of the mAb in the different formulations (see Table 1) stored for 90 days at 40 °C was monitored using SEC. NE samples had the greatest amount of aggregates, followed in order

as indicated by decreases in kfast values (Table 3). The decrease in deuterium uptake, however, seems to plateau on increasing the ratio from 3:1 to 6:1 presumably due to saturation of mAb− sucrose interactions. Increased protection was also observed upon increasing the trehalose/mAb ratio from 1:1 to 3:1 or histidine/mAb ratio from 2:1 to 3:1. In contrast, increasing the mannitol/mAb ratio from 1:1 to 3:1 led to an increase in deuterium uptake. On comparing formulations containing different excipients, the rate and extent of ssHDX were greater in M3 and NE formulations than in the rest of the formulations (Figure 3, Table 3). The similar deuteration kinetics in these two formulations is

Figure 3. Time course of deuterium incorporation by the mAb lyophilized in different formulations following exposure to D2O vapor at 11% RH and 22 °C. M3, mannitol 3:1. M1, mannitol 1:1. S6, sucrose 6:1. S3, sucrose 3:1. S2.7, sucrose 2.7:1. S1, sucrose 1:1. T3, trehalose 3:1. T1, trehalose 1:1. H3, histidine 3:1. H2, histidine 2:1. NE, excipient-free. Ratios represent excipient/mAb weight ratio. Lines represent the best fit obtained using eq 1. E


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formulations refer to the S1 and H2 formulations, respectively, lyophilized from solutions at different pH values (Table 2). At all pH values, ssFTIR spectra of the base sucrose formulations had an intense β-sheet peak at ∼1635 cm−1 (Figure 5A). In the base histidine formulations, the intensity of the major peak was slightly less than in the base sucrose formulations (Figure 5). At all pH values, the intensity of the ∼1615 cm−1 band was greater in the base histidine than in the base sucrose formulation. At pH 5, adding histidine to the base sucrose formulation resulted in a significant increase in intensity of the peaks at ∼1662 and ∼1677 cm−1 (Figure 5C,D), and increasing the histidine/sucrose ratio led to a decrease in the intensity of the major peak. At pH 6, adding histidine to the base sucrose formulation led to a slight decrease in the intensity of the major peak and a minimal increase in that of the peak at ∼1615 cm−1. At pH 6.8, no changes were observed in the ssFTIR spectra on adding histidine to sucrose. Changes in secondary structure indicated by ssFTIR were not associated with changes in tertiary structure as measured by solid-state intrinsic fluorescence, however (Figure S4). Only at pH 6.8, the peak maximum in the base histidine formulation appears to be red-shifted by 2 nm relative to the rest of the formulations, consistent with partial unfolding. Effect of Histidine on Deuterium Incorporation at Different Prelyophilization pH Values. The extent of ssHDX in the base histidine formulation was significantly greater than in all sucrose-containing formulations regardless of the pH value (Figure 6). At pH 5, adding histidine to the base sucrose formulation resulted in only a slight decrease in the extent of ssHDX (Figure 6A), whereas at pH 6 and 6.8 no effect on the extent of ssHDX is evident (Figure 6B,C). In general, the rate and extent of ssHDX increased with increasing pH (Figure S5). This is consistent with the effect of pH on the intrinsic rate of HDX in solution. While such dependence is linear in solution, the nature of this effect remains unknown in the solid-state. Therefore, it was not possible to deconvolute the effect of conformation on the rate of exchange across pH values. On adding histidine to the base sucrose formulation, the extent of uptake at pH 6 approached that at pH 6.8 (Figure S5B). This effect became more pronounced as the histidine/sucrose weight ratio increased (Figure S5C). Interestingly, in the base histidine formulations, more deuterons were incorporated at pH 6 than that at pH 6.8 after 10 days of D2O exposure (Figure S5D). Adding Histidine to Sucrose Decreases Aggregation of the mAb at Different Prelyophilization pH Values. The aggregate content after storage for 60 days at 40 °C was used as a measure of the physical stability of the mAb (formulations in Table 2). For each formulation composition, relative stability was greatest at pH 6 and was least in the base histidine formulations regardless of the pH value. Adding histidine to the base sucrose formulation at a histidine/sucrose weight ratio of 0.5:1 increased relative stability at all pH conditions. Increasing the histidine/ sucrose weight ratio from 0.5:1 to 1:1 increased relative stability only at pH 5, however. Although trends were observed, statistical analysis was not feasible because only two vials were tested per formulation. Regardless of the pH value, the greater rate and extent of ssHDX (Figure 6) as well as the significant decrease in the intensity of the major peak in the ssFTIR spectra in the base histidine formulations compared to the sucrose-containing formulations (Figure 5) were associated with negative relative stability (Figures 6D). However, there is no correlation between

by M3 and M1. In these formulations, a significant increase in dimer and oligomer content was observed (Figure S3). In the rest of the formulations, dimer content increased but oligomers were not observed. The aggregation rate was poorly correlated with the decrease in the intensity of the major peak in the second derivative of the ssFTIR spectra relative to that in buffered solution (Figure 4A). Interestingly, the aggregation rate in the different formulations was linearly correlated with the extent of ssHDX at 10 days (Figure 4B) when NE is excluded.

Figure 4. Relationship between the apparent aggregation rate constant of the mAb in lyophilized formulations during storage at 40 °C for 90 days, and the decrease in ssFTIR peak intensity relative to buffered solution (A) and the extent of ssHDX after 10 days (B). M3 mannitol 3:1 (black triangle). M1, mannitol 1:1 (black circle). S3, sucrose 3:1 (red triangle). S2.7, sucrose 2.7:1 (red inverted triangle). S1, sucrose 1:1 (red circle). T3, trehalose 3:1 (blue triangle). T1, trehalose 1:1 (blue circle). H3, histidine 3:1 (green triangle). H2, histidine 2:1 (green hexagon). NE, excipient-free (magenta square). Ratios represent excipient/mAb weight ratio. The solid black line represents the best fit line of linear regression when NE is excluded (B).

Effect of Histidine on the Secondary and Tertiary Structure at Different Prelyophilization pH Values. Histidine possesses unique molecular properties compared to disaccharides including small size, ability to form electrostatic interactions, and the different ionization states of the imidazole ring at different pH conditions. Accordingly, the effects of histidine as a single amorphous excipient or in combination with sucrose on the structure and conformation of the mAb were investigated further. In this study, the base sucrose and histidine F


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Figure 5. Secondary structural changes in sucrose and histidine formulations at different pH conditions. Values represent the second derivative of the ssFTIR spectra of the mAb at 3 pH values: (A) 1:1 sucrose/mAb. (B) 2:1 histidine/mAb. (C) 1:0.5:1 sucrose/histidine/mAb. (D) 1:1:1 sucrose/ histidine/mAb; ratios are w/w(/w). Numbers on the graph represent peak positions.

Figure 6. Deuterium uptake profiles and relative stability of the mAb in different histidine-containing formulations. Deuterium uptake profile at pH 5 (A), pH 6 (B), and pH 6.8 (C). In parts (A−C), values represent the increase in the mass of the mAb in each formulation as a function of incubation time with D2O vapor, error bars represent one standard deviation of the mean (n = 2−8), and lines represent best-fit lines obtained by fitting the data using eq 1. In part D, values represents the relative stability of the mAb after storage for 60 days at 40 °C and 22% RH (see eq 2) in the histidine-containing formulations compared to the base sucrose formulation at each pH value, and error bars represent SEM (n = 2). S1, 1:1 sucrose/mAb. S1H0.5, 1:0.5:1 sucrose/histidine/mAb. S1H1, 1:1:1 sucrose/histidine/mAb. H2, 2:1 histidine/mAb. Ratios are weight per weight.

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Figure 7. Percentage of deuterium uptake after 5 days at 11% RH and 22 °C by individual peptic fragments in sugar formulations of the mAb. Error bars represent one standard deviation of the mean. M3, 3:1 mannitol. S3, 3:1 sucrose. T3, 3:1 trehalose. Ratios represent excipient/mAb weight ratio. VH, CH1, CH2, and CH3 represent the variable, constant 1, constant 2, and constant 3 domains of the heavy chain, respectively. VL and CL represent the variable and constant domains of the light chain, respectively.

The local effects of the four excipients on deuterium exchange were mapped using 45 reporter peptides that cover approximately 83% of the mAb primary sequence. Sucrose provided greater protection from exchange than did trehalose, whereas mannitol provided less protection, presumably due to crystallization. These differences appear across the different peptides with little evidence of site-specificity (Figure 7), indicating that the differences in the extent of interaction with these excipients are experienced similarly across the mAb structure. On comparing H3 and T3, however, differences in deuterium uptake across the mAb structure were bidirectional (Figure 8). That is, some peptides were more protected from ssHDX in the trehalose matrix, whereas others were more protected in the histidine matrix, suggesting more site-specific interactions with the mAb. Figure 9 provides a color-coded representation of the average percentage of deuterium uptake in each formulation after 5 days of exposure to D2O vapor, mapped onto a homology model of the mAb structure.

relative stability and these parameters on comparing the sucrosecontaining formulations at each pH value, suggesting that the stabilization observed on adding histidine to sucrose is not due to preservation of the mAb structure nor due to a greater extent of hydrogen bonding interactions with the matrix. Effect of Different Excipients on Deuterium Incorporation by Individual Peptides at Prelyophiization pH 6.8. The mass increase in the peptic fragments was measured to map local deuterium incorporation across the mAb structure and to investigate potential site-specific protective effects of the different excipients used in this study. For this experiment, four formulations were compared to determine whether the observed differences in ssHDX are global or local. S3 has lower Tg than T3 (Table 1), more folded structure as indicated by fluorescence spectra (Figure 2), and lower extent of deuterium incorporation (Figure 3). H3 has similar Tg (Table 1), similar folded structure (Figure 2), and similar extent of deuterium incorporation (Figure 3) to T3. Histidine, however, has small molecular size and fewer hydrogen bonding groups than sucrose and trehalose but is capable of forming electrostatic interactions at the formulation pH 6.8. M3 shows crystallinity and exhibits rapid global ssHDX kinetics similar to that of the NE formulation (Figure 3) but retains similar tertiary structure to T3 and H3 as indicated by solid-state fluorescence (Figure 2).

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DISCUSSION Drying removes much of the hydration shell that surrounds proteins in solution, which may disrupt protein structure and facilitate aggregation.3 In this vein, the stabilizing effects of excipients in amorphous solids have been attributed to the H


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Figure 8. Difference plot of the percent deuterium uptake at different time points at 11% RH and 22 °C by individual peptic fragments in mAb formulations containing 3:1 trehalose or 3:1 histidine to mAb weight ratio. Positive values represent greater deuterium uptake in the histidine formulation; negative values represent greater deuterium uptake in the trehalose formulation. VH, CH1, CH2, and CH3 represent the variable, constant 1, constant 2, and constant 3 domains of the heavy chain, respectively. VL and CL represent the variable and constant domains of the light chain, respectively.

Figure 9. Homology model of the mAb structure showing the percentage deuterium uptake by the peptic fragments in the sucrose (A), trehalose (B), mannitol (C), and histidine (D) matrixes at 5 days. All formulations are 3:1 excipient/mAb weight ratio. Gray represents areas where there is no HDX information (∼17% of the mAb sequence).

formation of a glassy matrix that spatially separates protein molecules and limits their molecular motions.6 An alternative hypothesis suggests that amorphous excipients, especially polyols, form hydrogen bonds with the protein molecule to

replace those lost upon the removal of water, hence preserving the protein structure.5 Numerous studies have addressed these hypotheses to understand the determinants of long-term stability of therapeutic I


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after lyophilization, and the maximum amount of moisture sorbed at the 11% RH conditions used for D2O exposure did not exceed 3%. As a result, no shrinkage or collapse was observed in the cakes after exposure to D2O for 10 days. In addition, moisture content and moisture sorption were not correlated with the percentage of deuterium incorporation observed. A contribution of moisture sorption kinetics to deuteration kinetics is also unlikely because the half-life of moisture sorption is approximately one-order of magnitude faster than that of deuterium uptake, in keeping with earlier results with myoglobin by Sophocleous et al.20 In our study, the rate of aggregation is linearly correlated with the extent of ssHDX when the excipient-free formulation is excluded (Figure 4B). Although fluorescence measurements suggest that the mAb is more unfolded in NE than in M3 (Figure 2), the extent of ssHDX is greater in M3. This may be because mAb−mAb interactions protect the mAb from exchange in NE, which may also explain the greater aggregation rate in NE compared to M3. Mannitol, even in its crystalline form, can spatially separate the mAb molecules and prevent interactions that may lead to aggregation, whereas there are few barriers to mAb−mAb interactions in the excipient-free formulation. Such correlation of the extent of ssHDX with aggregation is in agreement with a previous study from our group21 in which the storage stability of lyophilized myoglobin correlated with deuterium uptake. As in that work,21 ssFTIR peak intensity was poorly correlated with aggregation; this is especially true of the histidine formulations in the present study (Figure 4A). Weak correlations of aggregation with mAb secondary structure, as measured by ssFTIR in lyophilized, spray dried and vacuumdried solids, have also been reported previously.9 Taken together, these results suggest that greater interaction with excipients stabilizes against aggregation by limiting mobility and/or blocking protein−protein interactions, rather than by preserving secondary structure. As such, the results presented here support the use of ssHDX-MS to predict aggregation of lyophilized mAbs on storage and suggest that additional studies are warranted in this area. A robust correlation with long-term chemical and physical stability (i.e., 1−2 years) was beyond the scope of this study and has been addressed in recent work from our laboratory.22 The H3 formulation had many interesting properties: relatively high Tg, similar extent of ssHDX to T3, and an aggregation rate comparable to T3 and S3. These properties motivated further investigation of the effects of histidine on conformation and stability of the lyophilized mAb. In the pH range 5−7, the carboxylic acid and primary amine groups of histidine are charged.37 The imidazole ring is doubly protonated and positively charged below pH 6 and is a neutral tautomer near pH 7.37 Accordingly, histidine can form hydrogen bonds as well as electrostatic interactions with proteins to varying extents at different pH values and hence has been investigated as stabilizer for different lyophilized proteins. In one study, Osterberg et al. developed a formulation containing histidine and sucrose to stabilize factor VIII during lyophilization and storage.38 More recently, Izutsu et al. reported that lyophilizing lactate dehydrogenase (LDH) with a high concentration of histidine resulted in the retention of most of the enzyme’s activity.39 Similarly, histidine was found to stabilize lyophilized LDH by acting as a buffer as well as an amorphous excipient.40 Moreover, Sane et al. reported that increasing the histidine/mAb ratio resulted in greater protection of secondary structure and improved stability in lyophilized formulations.11 To investigate

proteins in lyophilized formulations. In one study, Cleland et al. showed that a specific sugar-to-protein molar ratio provided long-term stability for an IgG mAb by preventing unfolding during lyophilization.28 In keeping with these results, Pikal and co-workers investigated the mechanism of stabilization of a model IgG1 mAb by sugars and showed that preservation of protein structure, rather than molecular mobility in the glassy matrix as measured by alpha-relaxations, correlated with both physical and chemical stability.7 In a follow-up study, they showed that this observation depends on formulation composition: in protein-rich formulations, stability was correlated with secondary structure retention, whereas in sugar-rich formulations, stability was correlated with differences in molecular mobility associated with fast dynamics.9 Along similar lines, Park et al. showed that the stability of an IgG1 mAb in sucrose/mannitol formulations was equally dependent on the preservation of secondary and tertiary structure, and on glassy state mobility and dynamics as measured by NMR relaxation times.10 More recently, Stärtzel et al. found that structural relaxation times, measured using MDSC, in different formulations of an IgG1 mAb lyophilized with different sucrose/arginine ratios did not correlate with long-term physical stability.29 In light of these studies, the importance of characterizing mAb structure in lyophilized solids cannot be overstated.30 Accordingly, predicting the stability of lyophilized mAbs using FTIR7,28,31−33 and Raman11 spectroscopies has been thoroughly investigated. One limitation of both techniques is that the intensity of the spectra reaches a maximum at a certain mAb-tosugar ratio11,31,33 suggesting that mAb stabilization by sugars may not be completely explained by vibrational spectroscopy across the full range of relevant compositions. Another limitation is that the aggregation of proteins may not necessarily be mediated by secondary structure changes in all cases. To address these limitations and to provide higher resolution structural characterization for a mAb in lyophilized solids, we used ssHDX-MS to probe the conformation and/or matrix interactions of a lyophilized IgG1 mAb and investigated the relationship between ssHDX-MS parameters and aggregation upon storage for 90 days. In solution, HDX-MS has been successfully used to study the conformational stability of mAbs under different conditions.34 In principle, ssHDX is similar to solution HDX in that it measures protein conformation, albeit with certain provisos.19 In particular, while HDX in solution reflects the conformational dynamics of the protein, ssHDX can be influenced by interactions between the protein and the surrounding environment as well as by protein conformation. In ssHDX-MS, the lyophilized cake is exposed to D2O at controlled relative humidity and temperature,8,14 deuteration incorporation kinetics are monitored with mass spectrometry, and the resulting time course is fitted to a biexponential association model that assumes the presence of rapidly and slowly exchanging pools of amide hydrogen atoms.15,20 Typically, mass transport and moisture sorption are complete before the first deuteration time point.20 Here, we carried out the experiments at low relative humidity to avoid provoking structural changes in the cake. An increase in moisture content of lyophilized IgG1 mAbs has been associated with a decrease in Tg,35 an observation consistent with the plasticizing effects of water. At moisture levels less than 5%, lyophilized samples containing an IgG1 mAb and sucrose have been shown to maintain a Tg significantly greater than the room temperature conditions35 used in our ssHDX-MS experiments. Also, in our study, the moisture content in all formulations was less than 3% J


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the zwitterionic histidine underlie this pattern. It is also possible that the different size and flexibility of both molecules underlie this observation. However, mechanistic and structural reasons for these differences remain unclear and require further investigation.

this stabilizing mechanism, Tian et al. demonstrated using isoperibol calorimetry where histidine interacts directly with the mAb in lyophilized solids.36 In a follow-up study, the authors demonstrated that the native structure of the mAb in solution was partially retained when lyophilized with a high concentration of histidine.41 In the present study, adding histidine to sucrose increased the Tg of the formulation at pH 6, although it decreased the weight fraction of the protein. Increasing the histidine/sucrose ratio also increased Tg at pH 6 and 6.8 but had no effect at pH 5. The different behavior observed at pH 5 may be due to different hydrogen bonding interactions of the ionized imidazole with the phosphoric acid in the formulation. A similar pH-dependent increase of the Tg of a histidine/citric acid formulation system of LDH has been reported previously.39 Here, it is possible that neutral zwitterionic histidine forms an interaction network with phosphoric acid in a manner similar to arginine.42 Adding histidine to a sucrose formulation perturbed the secondary structure of the mAb at pH 5 and to a lesser extent at pH 6, but had no effect at pH 6.8 (Figure 5). Since no perturbation was observed in the base sucrose formulation at pH 5, the observed perturbation can be attributed to histidine, probably to the positively charged imidazole ring, rather than the effect of pH per se. Similar perturbation of the secondary structure of LDH by positively charged histidine has been reported previously.43 Accordingly, the observed stabilization of the mAb by adding histidine to sucrose is not mediated by preserving the secondary structure. ssHDX-MS data presented here (Figure 3) as well as ssHDX data reported by others using ssFTIR44 show that increasing the excipient/mAb ratio generally results in greater protection from deuterium incorporation. However, adding histidine to sucrose resulted in little or no protection from deuteration compared to the base sucrose formulation, suggesting that in a matrix containing two excipients that interact differently with the protein molecule, the protective effect of one excipient from ssHDX may dominate. It is, therefore, reasonable that aggregation did not correlate with the extent of ssHDX in these formulations, indicating that other mechanisms may be contributing to stabilization of the mAb by adding histidine to sucrose. Recently, Chieng et al. reported a correlation between protein stability and the available free volume in the amorphous matrix.45 In this vein, Forney-Stevens et al. postulated that in amino acid/ sucrose formulations of two model proteins a possible stabilizing mechanism is that the amino acid fits into the free volume of the protein−sucrose matrix and suppresses fast dynamics.25 It is possible that the improved stability of the mAb in the sucrose/ histidine formulation follows, at least in part, this mechanism. At the peptide-level, the differences in deuterium uptake in the sugar-based formulations (Figure 7) appear to be global (i.e., distributed across the protein sequence) rather than local (i.e., occurring preferentially at one or more specific sites). In the solid-state, global effects are reasonable given that amorphous sugars such as trehalose and sucrose likely form hydrogen bonds with the protein in a similar manner,46 while more limited interactions occur with crystalline excipients such as mannitol.47,48 In contrast, ssHDX-MS data at the peptide level show that histidine provides more protection from ssHDX in some regions of the mAb structure, while trehalose is more protective in others (Figure 8), suggesting that there are local differences in the extent of the interaction of the two excipients with the mAb in the solid-state. It is possible that electrostatic interactions with

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CONCLUSIONS In the present study, we report conformational analysis of a model IgG1 mAb in lyophilized solids using ssHDX-MS. The extent of ssHDX is correlated with aggregation of the mAb in different lyophilized formulations, suggesting that ssHDX-MS can be useful in predicting the stability of lyophilized formulations of mAbs. Moreover, adding histidine to base sucrose formulations at different pH conditions decreased aggregation, but perturbed the secondary structure and did not result in greater protection from ssHDX, suggesting that the added stability might be due to ionic interactions or suppressed dynamics rather than hydrogen bonding. Additionally, we report peptide-level HDX-MS analysis of the structure of an IgG1 mAb in the solid-state and show that protection from exchange by sugars occurs globally rather than locally. In contrast, on comparing histidine to trehalose, we observe site-specific protection from deuterium uptake in certain peptic fragments, consistent with local differences in excipient interactions with the mAb.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00696. Supplementary methods, results, and figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Industrial and Physical Pharmacy College of Pharmacy, Purdue University 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States. E-mail: topp@ purdue.edu. Phone: 765-496-7760. Fax: 765-494-6545. ORCID

Elizabeth M. Topp: 0000-0003-1734-0223 Present Address §

Lonza AG, Basel CH-4002, Switzerland.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support and the IgG1 mAb used in this study were provided by Pfizer, Inc. (Chesterfield, MO). This work was also supported in part by a Purdue Research foundation grant and a McKeehan Graduate Fellowship awarded by Purdue University to E.M.

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REFERENCES

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Probing the Conformation of an IgG1 Monoclonal Antibody in

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