Solid-State HydrogenâDeuterium Exchange Mass Spectrometry

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

Solid-State Hydrogen−Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations Balakrishnan S. Moorthy,† Isidro E. Zarraga,‡ Lokesh Kumar,§ Benjamin T. Walters,∥ Pierre Goldbach,⊥ Elizabeth M. Topp,† and Andrea Allmendinger*,⊥ †

Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States Late State Pharmaceutical Development, Genentech (A Member of the Roche Group), South San Francisco, California 94080, United States § Pharmaceutical Processing and Technology Development, Genentech (A Member of the Roche Group), South San Francisco, California 94080, United States ∥ Early State Pharmaceutical Development, Genentech (A Member of the Roche Group), South San Francisco, California 94080, United States ⊥ Late-stage Pharmaceutical and Processing Development, Pharmaceutical Development and Supplies, Biologics, Europe, Hoffmann-La Roche, CH-4070 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: Solid state hydrogen−deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D2O(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen−deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection. KEYWORDS: mass spectrometry, monoclonal antibody formulations, lyophilization, hydrogen−deuterium exchange, stability

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INTRODUCTION

The growth of monoclonal antibody (mAb) therapeutics has been substantial in the past decade, representing about half of the worldwide biopharmaceuticals market in 2013.1,2 In 2014, there were 47 mAbs on the market for different therapeutic indications, and with over 300 mAbs in development, worldwide sales are projected to reach $125 billion by 2020.1 © XXXX American Chemical Society

Received: June 16, 2017 Revised: August 23, 2017 Accepted: October 20, 2017

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DOI: 10.1021/acs.molpharmaceut.7b00504 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Composition and Physical Properties of the Lyophilized mAb1 Formulations F1−F4 composition basic composition F1 F2 F3 F4

50 mg/mL mAb1; 20 mM L-His/HisHCl pH 6.0; 0.02% (w/v); polysorbate 20

physical properties mannitol (mg/mL)

sucrose (mg/mL)

Tg (°C)

residual moisture (%)

reconstitution time at t = 0 (s)

32 53

40 80 8 27

84 72 69 80

0.4 0.7 1.1 0.8

155 139 60 73

ssHDX-MS could accurately screen lyophilized formulations of a full length mAb for their solid-state stability behavior, including aggregation propensity and chemical degradation (>2.5 years). In the studies reported here, the group used ssHDX-MS to compare and correlate the stability of the formulations, which contained different concentrations of sucrose and mannitol. Investigators performing ssHDX-MS in the Topp lab initially were blinded to formulation composition and relative stability. Deuterium incorporation measured by ssHDX-MS was correlated with formulation stability obtained at various conditions for more than 2.5 years. This allowed the formulations to be rank-ordered on the basis of ssHDX-MS studies conducted over several weeks, rather than in storage stability studies conducted over several years.

Today, mAb products are approved for a wide variety of diseases, although a substantial and growing portion of this drug class is focused on oncology and autoimmune diseases. The complex structure of proteins, especially high molecular weight proteins such as mAbs, together with their susceptibility to chemical and physical degradation, makes it challenging to produce drug products that are stable at 2−8 °C for a typical shelf life of 2−3 years.3 Generally, a liquid protein formulation is preferred, as it is easier to process during manufacturing and is convenient for the final user. However, owing to the susceptibility of a number of mAb formulations to chemical (e.g., isomerization, deamidation) and/or physical degradation (e.g., aggregation) in the solution state, alternative formulation strategies must be used to limit their degradation during shelf storage. Lyophilization is a common alternative approach.4 In lyophilized formulations, degradation can be slowed significantly due to the significantly lower molecular mobility and lower moisture content in the lyophilized solid than in solution. This allows the formulation to be stable for the intended shelf life of the product.4,5 To design an effective lyophilized mAb formulation, careful selection of excipients is required. Stabilizing agents such as low molecular weight disaccharides, e.g., sucrose and trehalose, are typically used to protect against mAb degradation during freezing and/or drying.6 To determine the optimal proportion of excipients to produce a stable lyophilized product, a design of experiments (DoE) approach is usually used and candidate formulations are assessed for stability at the desired storage temperature (e.g., 2−8 °C to room temperature) and/or at an elevated temperature (e.g., 40 or 50 °C; generally below the glass transition temperature Tg of the lyophilized product). However, in both the real-time and the elevated temperature stability studies, a significant period of time must elapse (e.g., 3−6 months) before a clear read-out and differentiation among the candidate lyophilized formulations can be obtained. As a result, there has been increased interest in recent years in predictingat least qualitatively, if not quantitativelythe rank order of long-term stability of lyophilized formulations. One such technique developed by Topp et al. at Purdue University is solid state hydrogen−deuterium exchange coupled with mass spectrometry, or ssHDX-MS.3,7 Using a carefully selected list of different lyophilized formulations for myoglobin (Mb; average molecular weight 16.7 kDa), the ssHDX-MS method was observed to correlate with Mb aggregation during one year of storage at 25 and 40 °C.7 While promising, the results of this study were limited in the relatively short duration of storage (especially with regard to desired shelf life), in that aggregation was the only form of instability considered, and in the use of a model protein that is not representative of most protein drug products, particularly mAbs. To extend the work on myoglobin to proteins of greater commercial and therapeutic interest, scientists from RocheGenentech collaborated with the Topp lab to evaluate whether

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EXPERIMENTAL SECTION Materials. Deuterium oxide (D2O) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Mass spectrometry grade water, acetonitrile (ACN), and formic acid (FA) were purchased from ThermoFisher Scientific (Waltham, MA). Pepsin from Sigma-Aldrich was immobilized on Poros AL resin (Applied Biosystems, Foster City, CA) and packed into a high performance liquid chromatography (HPLC) column (50 × 2.1 mm, Grace Davison Discovery Sciences, Deerfield, IL). LHistidine/L-histidine-hydrochloride was obtained from Ajinomoto Inc. (Louvain-la-Neuve, Belgium), polysorbate 20 was from Croda (Edison, NJ), and sucrose and mannitol were purchased from Ferro Pfanstiehl (Waukegan, IL) and Roquette Freres (Lestrem, France), respectively. All other chemicals were at least reagent grade and used as received. Lyophilization of mAb1 Formulations. Purified monoclonal antibody mAb1 (IgG1, pI ∼ 8.4, 149 kDa) was provided by Hoffman-La Roche Ltd. (Basel, Switzerland). 10 mL of four different mAb1 formulations (Table 1) were lyophilized in 20 cm3 clear Fiolax glass vials (SCHOTT, Müllheim, DE) on a LyoStar laboratory scale freeze-dryer (SP Scientific, Stone Ridge, NY) using 20 mm Lyo-stoppers D777-1 (DAIKYO Seiko Ltd., Tokyo, JPN) and put on stability. Two different lyophilization cycles were used in this study (Table 2). Formulations F3 and F4, having mannitol, were subjected to an additional annealing step for efficient crystallization prior to drying. All the vials were backfilled with nitrogen prior to sealing. The lyophilized samples were placed on stability at 5, 25, 40, and 50 °C over a period of 960 days. Samples were withdrawn at regular intervals and analyzed for both physical and chemical degradation by size exclusion chromatography (SEC), ion exchange chromatography (IEC), and liquid chromatography mass spectrometry (LC−MS) as described below. For ssHDX-MS and ssFTIR studies, 200 μL of the formulations were lyophilized in 2 cm3 glass vials using a VirTis Advantage Plus freeze-dryer (SP Scientific, Gardiner, NY). Physical Properties of Lyophilized mAb Formulations. Residual moisture in the lyophilized formulations was B


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Molecular Pharmaceutics

Global Solid-State Hydrogen/Deuterium Exchange Mass Spectrometry (ssHDX-MS) of mAb Formulations. ssHDX-MS at the intact protein (global) level was carried out by incubating the unstoppered vials under 11% RH at 22 °C in a sealed glass desiccator. Conditions were selected at sufficiently low relative humidity in order to maintain cake structure as confirmed by SEM data and sufficiently low temperature to maintain stability during sample preparation. Triplicates of vials were withdrawn at regular intervals between 2 h and 15 days, and the reactions were quenched by immersing the vials in liquid nitrogen. Deuterated samples were analyzed using an LC−MS instrument (1200 series LC, 6520 qTOF; Agilent Technologies, Santa Clara, CA) with the column maintained at ∼0 °C inside a custom-built refrigeration unit. Samples were quickly reconstituted in ice cold buffer (buffer A) containing 0.2% formic acid (FA) and 5% methanol. Disulfide reduction was carried out by diluting the solution to 10× in buffer (buffer B) containing 0.2 M glycine, 0.5 M TCEP, and 6 M guanidine hydrochloride. The mixture was incubated on ice with 30 s vortexing at every 30 s for 3 min. Samples were further diluted to 2× in buffer B. The reconstituted solution was injected onto a protein microtrap (Michrom Bioresources, Inc., Auburn, CA). Desalting was carried out for 1.7 min with 10% acetonitrile, 90% water, and 0.1% FA followed by elution in 8 min using a gradient to 80% acetonitrile, 20% water, and 0.1% FA. Data were acquired by setting the m/z range to 200− 3200. The masses of light chain (LC) and heavy chain (HC) were obtained by deconvoluting the spectra using the MassHunter Workstation Software (Version B.03.01, Agilent Technologies). The masses of undeuterated LC and HC were subtracted from the masses of deuterated LC and HC, respectively, to obtain the number of deuterons incorporated. The percentage deuterium uptake by mAb at each exchange time point was calculated using eq 1. As all the deuterated samples took a similar amount of time for reconstitution and MS analysis, the values obtained were not corrected for back exchange.

Table 2. Freeze-Drying Cycle for the Four Different mAb1 Formulations process step/formulations

F1 and F2

F3 and F4

equilibration (temp/hold time) ramp to freezing freezing (temp/hold time) ramp to annealing annealing (temp/hold time) ramp to freezing freezing (temp/hold time) vacuum ramp to primary drying primary drying (temp/hold time) ramp to secondary drying secondary drying (temp/hold time)

+5 °C/1 h 1 °C/min −40 °C/3 h na na na na 75 mTorr 0.5 °C/min +5 °C/51 h 0.2 °C/min +25 °C/6 h

+5 °C/1 h 1 °C/min −40 °C/2 h 1 °C/min −15 °C/6 h 1 °C/min −40 °C/3 h 75 mTorr 0.5 °C/min +5 °C/46 h 0.2 °C/min +25 °C/6 h

determined using coulometric oven-based Karl Fischer titration, glass transition temperature (Tg) was measured by modulated differential scanning calorimetry (DSC), crystallinity of the formulations (F3, F4) was determined by X-ray powder diffraction (XRPD), and the reconstitution times of the lyophilized formulations were measured according to previously published methods.8,9 Secondary Structure Measurement. To compare the secondary structure content of mAb in lyophilized formulations F1, F2, F3, and F4, measurements were carried out using a Tensor 37 FTIR spectrometer (Bruker Optics, Billerica, MA). Approximately 1−2 mg of the lyophilized formulations was loaded separately onto the ATR germanium crystal and the data collected at a resolution of 4 cm−1 from 128 scans. The instrument was constantly purged with nitrogen to avoid interference from atmospheric moisture. Raw spectra acquired for each formulation were subtracted with background spectra collected under similar conditions. The spectra were processed and analyzed using the OPUS software (version 6.5, Bruker Optics) as described previously.7 Dynamic Vapor Sorption (DVS). Moisture sorption kinetics were determined using a gravimetric analyzer (Q5000SA; TA Instruments, New Castle, DE) to determine the extent of D2O sorption of the mAb formulations during ssHDX. Approximately 1−2 mg of the lyophilized formulations was separately loaded onto the platinum sample pan and equilibrated at 40 °C, 0% RH for 60 min to remove any loosely bound moisture. Samples were then equilibrated at 22 °C, 0% RH for 60 min, and moisture sorption was carried out at 22 °C, 11% RH, under conditions similar to those used for ssHDX. Data were collected at 4 s intervals until the signal reached a plateau within the experimental time frame. Scanning Electron Microscopy (SEM). To evaluate any moisture sorption induced changes in cake morphology, SEM analysis was carried out for lyophilized formulations before and after exposure to D2O at 11% RH, 22 °C for 5 days. Lyophilized cakes were sputtered with gold using a Cressington 108auto sputter coater to increase the conductivity of the sample. Parameters were set to 120 s of sputtering with a current intensity of 30 mA under an argon flow at 0.1 bar. SEM images were acquired on a Sigma VP system (Zeiss, Oberkochen, DE). The secondary electron detector under high vacuum was used for the image acquisition with an acceleration tension of 3 kV. A line averaging of 17 scans per frame was applied to reduce the noise in the images, resulting in a full acquisition time of 44.6 s per image.

deuterium uptake (%) =

deuterium uptake (LC + HC) × 100 exchangeable amides (LC + HC)

(1)

The deuteration kinetics for mAb in different formulations was fitted to a monoexponential model (eq 2) using Graph Pad Prism software version 5 (San Diego, CA). deuterium uptake = Dmax (1 − e−kt )

(2)

where Dmax is the maximum deuteration at long time, k is the rate constant, and t is the deuteration time (h). Local ssHDX-MS of mAb Formulations. To identify sites of deuterium incorporation and to compare local differences among the mAb1 formulations, ssHDX was coupled with online pepsin digestion before MS analysis to allow peptic fragments to be analyzed. Exchange reactions and quenching were carried out as described above under global ssHDX-MS. The quenched samples were reconstituted in ice cold buffer A, followed by disulfide reduction and dilution, as above. Approximately 170 pmol of mAb was injected onto an immobilized pepsin column housed inside an oven at 23 °C, and digestion was carried out in water containing 0.1% FA at a flow rate of 0.2 mL/min. The peptides were then desalted in a peptide microtrap (Michrom Bioresources, Inc., Auburn, CA) for 4.3 min, and a gradient elution of 10−60% acetonitrile C


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hydrochloride, pH 6.0. Samples were then incubated with 100 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma-Aldrich, Steinheim, DE) at 25 °C for 20 min to reduce the disulfides. Subsequent cysteine modification was carried out by alkylation of the free thiol groups by incubation with 1 M Nethylmaleimide (NEM, Sigma-Aldrich, Steinheim, DE) for 20 min at 25 °C. Approximately 240 μg of protein was loaded onto a PhyTips desalting column (PhyNexus Inc., San Jose, CA) and eluted with buffer 20 mM L-histidine/L-histidine hydrochloride, pH 6.0. Trypsin digestion was carried out by incubating the reduced and alkylated mAb1 with 5 μg of trypsin at 37 °C overnight. The digested mAbs were separated by reversedphase high-performance liquid chromatography (RP-HPLC) using a Waters ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 μm particle size) on a Waters ACQUITY UPLC system. The ACQUITY UPLC was directly coupled to a Waters LCT Premier XE mass spectrometer equipped with an electrospray ionization source. The semiquantitative evaluation of the oxidative modifications of the samples was performed by integrating the specific ion chromatograms of the respective peptides. The data are presented as the sum of percentage Met oxidation of the solvent exposed Met258 and Met434 in the HC.

containing 0.1% FA was carried out in a C18 analytical column (Zorbax 300SB-C18; Agilent Technologies). To minimize back exchange, the peptide microtrap and analytical column were housed within a refrigeration unit and activated through a twoposition valve (EPC12CW, VICI Valco Instruments Co., Inc., Houston, TX). Data were acquired by setting the m/z range to 100−1700. Peptides from an undeuterated mAb were analyzed in triplicate, and their mass was identified using the MassHunter Software (Agilent Technologies). Identifications were considered only if the fragments were present in all three replicate injections. Peptides with identical masses were confirmed by MS/MS analysis (CID fragmentation; MassHunter Software; Agilent Technologies). Peptides identified from the undeuterated mAb were mapped onto subsequent deuteration experiments using the HDExaminer software (Sierra Analytics, Modesto, CA) to obtain the deuteration level for each peptic fragments. Deuterium uptake values for the mAb formulations following 120 h of ssHDX were mapped onto the mAb1 structure using PyMOL (PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC). Stability of mAb Formulations. Size Exclusion High Performance Liquid Chromatography (SE-HPLC). To assess monomer loss and aggregation propensity during storage, SEHPLC was performed on an Alliance 2695 HPLC instrument (Waters Corporation, Baden-Daettwil, Switzerland) equipped with a UV detector (Waters Corporation, Baden-Daettwil, Switzerland). Prior to analysis, lyophilized samples were reconstituted and diluted to 0.5 mg/mL with mobile phase and stored at 30 °C for at least 24 h in the auto sampler. 100 μL of diluted samples were injected into a TSK G3000 SWXL, 7.8 × 300 mm column (Tosoh Bioscience, Stuttgart, DE) maintained at 25 °C and eluted with buffer containing 200 mM K2HPO4/KH2PO4 and 250 mM KCl (pH 6.2) at 0.5 mL/ min. Data were collected at a wavelength of 280 nm. The percent peak area of high molecular weight species (HMWs) relative to the total peak area was obtained using the Empower 3 Chromatography Data System software (Waters Corporation, Baden-Daettwil, Switzerland). Ion Exchange Chromatography (IEC). Charge heterogeneity was characterized by IEC using a Dionex ProPac WCX10L, 4.0 × 250 mm column (Thermo Scientific) on an Alliance 2695 HPLC instrument (Waters Corporation, Baden-Daettwil, Switzerland) equipped with a UV detector (Waters Corporation, Baden-Daettwil, Switzerland). The column was preequilibrated with 70% solvent A (20 mM ACES, pH 6.5) and 30% solvent B (200 mM NaCl in solvent A) at 40 °C. Prior to analysis, samples were diluted to 1 mg/mL with solvent A and incubated with 1% w/w of a carboxypeptidase B solution (1 mg/mL in solvent A) for 20 min at 37 °C. The samples were then stored at 5 °C in the auto sampler during analysis. 50 μL of sample was injected into the column, and a step gradient elution of 30−43% of solvent B for 25 min, followed by 43− 100% of solvent B for 28 min, was carried out at 0.5 mL/min. Data were collected at a wavelength of 280 nm. The percentage peak area of main peak and acidic and basic species relative to the total peak area was reported using the Empower 3 Chromatography Data System software (Waters Corporation, Baden-Daettwil, Switzerland). Liquid Chromatography−Mass Spectroscopy (LC−MS). Oxidation of solvent exposed amino acids (HC Met258 + HC Met434) was analyzed by LC−MS. The lyophilized mAbs were reconstituted and denatured with buffer containing 200 mM L-histidine/L-histidine hydrochloride, 8 M guanidine

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RESULTS Physical Properties of mAb1 Formulations. Lyophilized mAb1 formulations with different sucrose/mannitol concentrations (Table 1) were characterized for glass transition temperature (Tg), moisture content, reconstitution time, and crystallinity. Formulations F1 and F3 showed the lowest (0.4%) and highest (1.1%) residual moisture content, respectively. The Tg values of the formulations were between 69 °C (F3) and 84 °C (F1). In general, Tg is dependent on the residual moisture, with lower Tg values typically observed for formulations with higher residual moisture. Tg values often also depend on the relative sucrose concentration, with higher Tg values observed at lower sucrose concentrations.10−12 Reconstitution time was dependent on the sugar concentration, with faster reconstitution observed for the mannitol containing formulations than for those lyophilized only with sucrose. XRPD measurements showed that formulations F3 and F4 have crystalline character, presumably due to mannitol (Figure S1), whereas F1 and F2 are completely amorphous (data not shown). Secondary Structure of mAb1. The second-derivative FTIR spectra of the mAb1 formulations showed major peaks at ∼1617 cm−1, ∼1637 cm−1, and ∼1690 cm−1 for β-sheet and ∼1670 cm−1 for β-turns (Figure 1). Formulations F1 and F2 showed similar band intensity at ∼1637 cm−1, whereas for formulations F4 and F3, the intensity decreased, in keeping with the decrease in sucrose and increase in mannitol concentrations. This suggests reduced β-sheet content for these formulations. The formulation with the least mannitol (F3) showed the lowest signal for β-sheet. Moisture Sorption Kinetics and Cake Morphology. To study the effect of moisture content on deuterium incorporation, moisture sorption was carried out for the mAb1 formulations under ssHDX conditions (Figure 2). Formulations F2 and F3 adsorbed similar levels of moisture (18 mg water/g solid), whereas formulations F1 and F4 adsorbed 20 and 14 mg water/g solid, respectively. Formulations F1, F3, and F4 reached a plateau in less than 2 h, whereas F2 with the highest sucrose concentration reached a plateau only after 8 h. D


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Figure 1. Secondary structure measurements using ssFTIR. Overlaid second-derivative amide I infrared spectra of lyophilized mAb1 formulations.

Figure 3. Global ssHDX kinetics for mAb1 formulations. (A) Percent deuteration is plotted as a function of time and fitted to a one-phase exponential model using GraphPad Prism software version 6 (San Diego, CA) (n = 3, ±SE). (B) Regression parameters for global ssHDX kinetics of lyophilized mAb1 formulations. aData from 6 h to 10 days were fitted to monoexponential model (eq 2).

(charge variants most likely via the deamidation/succinimide formation pathway), and the level of Met oxidation upon longterm storage of the lyophilized formulations was related to various deuterium incorporation and to various physicochemical properties of the solids. Data were correlated to FTIR band intensity and to Tg, residual moisture, and sucrose concentration, as well as to global deuterium uptake obtained by ssHDX-MS. At regular intervals, the lyophilized mAb1 formulations were analyzed for protein integrity by SE-HPLC (HMW) and IEC (charge variants differentiated in acidic regions, main peak, and basic regions) over a period of up to ∼2.5 years (960 days) for storage at 5, 25, and 40 °C and up to 6 months at 50 °C (180 days), respectively. All formulations showed an initial level of ∼3% HMWs. HMWs increased over storage time with increasing storage temperature (Figure S3). After 180 days of storage at 50 °C, formulations F1, F3, and F4 showed 8.6%, 24.6%, and 12.4% HMWs, respectively. Similarly, after 960 days of storage at 40 °C, F1, F3, and F4 were 9.0%, 26.2%, and 12.3% aggregated, respectively. Similar trends were observed for formulations stored at 5 and 25 °C. In contrast, formulation F2 did show almost no change in the level of aggregation under all temperature conditions after 960 days (∼3.8%). IEC was used to measure the relative level of charge variants in mAb1 formulations. An increase in both acidic and basic regions was observed for formulations with increase in storage temperature over storage time (Figure S4). As for HMW formation, the extent of acidic and basic regions increased in the order F2 < F1 < F4 < F3 under all temperature conditions. ESI LC−MS was used to characterize methionine and tryptophan oxidation for formulations stored at 5, 25, and 40 °C for 960 days. Tryptic fragments containing residues Met4 in light chain and Met34, Trp47/50, Met83, Met258, Trp283, Trp319, Met364, Trp387, and Met434 in heavy chain were analyzed to measure the relative amount of oxidation (Tables S2 and S3). Met258 and Met434 in the heavy chain showed

Figure 2. Moisture sorption kinetics for mAb1 formulations at 11% RH and 22 °C. Data were fitted to a one-phase exponential model using GraphPad Prism software version 6 (San Diego, CA). Inset shows raw data during the early stage (0−1.5 h) of moisture sorption.

To identify any moisture-induced cake collapse during ssHDX, SEM analysis was carried out for formulations before and after exposure to D2O vapor at 11% RH, 22 °C for 5 days. No significant difference in cake morphology was observed between formulations with and without D2O exposure (Figure S2). ssHDX-MS of mAb1 Formulations at the Global Level. ssHDX Kinetics. The kinetics of deuteration for all the formulations were best fitted to a one-phase association model (Figure 3A,B; R2 of 0.98−0.99). Formulations F1 and F2, containing only sucrose, showed lower deuterium uptake than formulations with both sucrose and mannitol (F3 and F4), and deuterium incorporation was generally lower for formulations with higher sucrose concentration. The rate (k) and extent (Dmax ) of deuterium uptake for the four formulations increased in the order F2 < F1 < F4 < F3. Differences in deuterium uptake among the formulations were observed during both the initial and plateau phases (Dmax). Correlation of mAb1 Stability with Structural Measurements (FTIR and Deuterium Uptake) and Physical Properties. The extent of deuterium incorporation in ssHDX-MS has been recently reported as superior to FTIR in estimating the aggregation behavior of myoglobin upon long-term storage.7 In order to further investigate whether the deuterium uptake of the different mAb formulations is correlated to their stability behavior, protein aggregation (HMWs), chemical degradation E


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Figure 4. Correlation of mAb1 aggregation stored at 40 °C for 960 days with band intensity for β-sheet (1637.5 cm−1) obtained using ssFTIR (A), amount of sucrose in formulations (B), percent initial residual moisture in formulations (C), and the glass transition temperature (Tg) (D).

Figure 5. Correlation of deuterium uptake (% Dmax) with HMWs of mAb1 formulations stored at 5 °C for 960 days (A), 25 °C for 960 days (B), 40 °C for 960 days (C), and 50 °C for 180 days (D). The data were fitted to a linear model to obtain the slope, intercept, and R2 values.

increases in oxidation for formulations stored at 40 °C (Figure 5C and Table S2) presumably due to their solvent exposure, and were thus used for correlation with deuterium incorporation by ssHDX-MS. However, overall oxidation was found to be low, and the extent of oxidation (sum of Met258 and Met434) for the mAb1 formulations increased in the order F2 < F1 < F4 < F3. The extent of mAb1 HMW formation was correlated with the band intensity for β-sheet at wavenumber 1637.5 cm−1 obtained by FTIR (Figure 4A). FTIR measures the band intensity associated with secondary structure, which correlated partially with the extent of aggregation during storage (R2 = 0.87). In contrast, the FTIR band intensity from formulations

F3 and F4 lyophilized with both sucrose and mannitol correlated well with the formation of HMWs, and formulations F1 and F2 with varying sucrose concentrations showed similar band intensity for β-sheet, but different HMW formation (Figures 4A and S5A/BI). Though the amount of sucrose used in the formulations did not correlate linearly with HMW formation, a decrease in mAb1 HMW formation was observed with an increase in sucrose concentration (Figures 4B and S5A/ BII) in line with a decrease in specific surface area measured by BET (data not shown). The percent residual moisture and the glass transition temperature (Tg) in freshly lyophilized samples correlated poorly with mAb1 aggregation, with R2 = 0.61 and 0.21, respectively (Figures 4C/D and S5A/BIII/IV). The F


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Dmax by ssHDX-MS was also correlated with the level of chemical degradation (charge variants and methionine oxidation) upon storage >960 days. Figures 6 A−6C show the correlation of acidic regions of the different formulations upon storage with Dmax, with correlation coefficients of R2 = 0.88, R2 = 0.99, and R2 = 0.90 for 5, 25, and 40 °C storage, respectively. Similar to acidic regions, the level of basic regions and main peak show good correlation with Dmax (Figure S7). Figure 6 D shows the correlation of Dmax with methionine oxidation with R2 = 0.77, indicating the correct rank order of the formulations but only partial correlation. Overall oxidation was low with 4−7% (Met258) and 1−2% (Met434) after 40 °C storage >2.5 years, and the extent to which Dmax is correlated to oxidation requires additional evaluation in a separate study with higher oxidation levels. ssHDX-MS of mAb1 Formulations at Peptide Level. Deuterium uptake was measured for 110 overlapping peptides from light and heavy chains with sequence coverage of ∼90% (Table S1, Figure 7). The differences in the level of deuterium uptake and the rank order of the formulations are consistent with the intact level analysis. Overall, the deuterium uptake for almost all the peptides from mAb1 is greatest for F3, while F2 shows protection from deuterium uptake throughout the molecule. The average deuterium uptake values for all peptic fragments show that formulation F2 is ∼10% more protected than F3 after 10 days of ssHDX. Compared to F1, formulation F2 showed ∼4% more protection from deuteration, presumably due to the 2× fold higher sucrose concentration. Comparison of deuterium incorporation in F1 and F4 showed that, with the exception of residues 116−123, most of the regions are ∼1% more deuterated in F4. Following 10 days of ssHDX, overlapping fragments 116−122, 116−123, and 117−123 in the CL domain are ∼4% less deuterated in F4 than in F1. The maximum deuterium uptake values for light chain and heavy chain are consistent over the deuteration time course for the different formulations (Figure S8). Overall, the heavy chain showed slightly greater deuterium uptake (∼3%) than the light chain for all formulations. The average deuterium uptake following 5 days of ssHDX was calculated and mapped onto the modeled mAb1 structure (Figure 8). Peptides 144−148 and 153−157 in the CL domain and 104−114 and 106−115 in the loop region connecting VL to CL showed the greatest deuterium uptake in all formulations. These regions showed ∼33%, ∼22%, ∼40%, and ∼34% deuterium incorporation following 10 days of ssHDX for formulations F1, F2, F3, and F4 respectively. Also, peptic fragments spanning residues 247−258 in the CH2 domain and 340−354 in the CH3 domain showed higher deuterium uptake than other regions. Fragments spanning residues 167−176 in the CL, 11−18, 72−92, and 84−110 in the VH, and 307−312 in the CH2 domain showed less than 10% deuterium uptake consistently in all formulations.

increase in residual moisture was also measured at the end of stability testing and determined negligible with roughly 0.1% for all formulations taking into account the variability of the shelf position as well as of the method for moisture determination. The correlation of stability data with the protein melting temperature of mAbs is controversially discussed in the literature.13,14 For our formulations, the protein melting temperature as determined by microdifferential scanning calorimetry15 did not reveal the same rank order as suggested by long-term stability data (data not shown). Table 3 summarizes the correlation parameters obtained for Figures 4, 5, and 6 defining in particular poor, partial, or good correlation depending on R2. Table 3. Parameters Obtained from Correlation of Either FTIR Band Intensity, Sucrose Content, Residual Moisture, Tg, or Percent Dmax (t = 0) with Level of mAb1 Aggregation and Chemical Degradation during Long-Term Storage parameters

R2 b

figure

correlation param

slope

intercept

A B C D

FTIR band sucrose level residual moisture Tg

20877.00 −0.28 25.78 −0.63

177.68 23.65 −6.52 60.51

0.86 0.79 0.61 0.21

A B C D

Dmax Dmax Dmax Dmax

0.21 0.71 3.18 2.95

−0.09 −5.91 −35.71 −32.67

0.99 0.99 0.99 0.99

A B C D

Dmax Dmax Dmax Dmax

0.36 1.46 3.20 0.59

21.44 10.74 −1.54 −2.33

0.87 0.99 0.90 0.77

4a

5a

6a

a

Refer to Figures 4,5, and 6 for storage conditions and variables used. Figures 4 and 5 present the correlation with aggregation (HMWs %) and Figure 6 with chemical degradation (acidic species) and oxidation. b 2 R values ≥0.90, 0.71−0.89, and ≤0.70 represent good, partial, and poor correlation, respectively.

ssFTIR measurements were also correlated with the level of acidic regions (Figures S6I-III A−C) and methionine oxidation during storage (t = 960 days, Figure S6I-III D). Secondary structure measures from FTIR showed partial correlation with acidic regions and Met oxidation (Figure S6I) with R2 = 0.86− 0.88 and 0.86, respectively. The levels of charge variants and Met oxidation were indirectly proportional to the amount of sucrose used for the formulations (Figure S6II). The residual moisture and Tg showed poor correlation with mAb1 acidic regions and Met oxidation under all storage conditions (Figure S6III/IV). In the same way, the deuterium incorporation by the antibody as determined by ssHDX-MS measurements performed at the initial time point were correlated with the physical stability of mAb1 formulations upon long-term storage (Figure 5). The maximum deuterium uptake (Dmax) obtained from the intact level analysis for formulations stored at 25, 40, and 50 °C correlated very well with HMW formation, with correlation coefficients of R2 = 0.99, and in particular at the intended storage temperature of 5 °C with R2 = 0.998.

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DISCUSSION Lyophilizing therapeutic protein formulations is widely used in the industry in an attempt to ensure an acceptable shelf life, especially when significant stability risks are encountered in solution formulations. While lyophilization significantly reduces protein molecular mobility and can improve stability, proteins nevertheless can degrade even in the solid state.16−18 The stability of proteins in lyophilized powders often depends on the retention of protein native structure during lyophilization and subsequent storage. Measures of protein structural G


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Figure 6. Correlation of deuterium uptake (% Dmax) with percent acidic regions in mAb1 formulations stored at 5 °C (A), 25 °C (B), and 40 °C (C) for 960 days. Deuterium uptake (% Dmax) correlated with percent Met oxidation (HC Met258 + HC Met434) for formulations stored at 40 °C (D) for 960 days.

Figure 7. Deuterium uptake plots for the peptic fragments from mAb1 in formulations F1 (A), F2 (B), F3 (C), and F4 (D). The peptide number in x-axis represents the 110 overlapping peptides obtained from pepsin digestion of light (LC) and heavy chain (HC). Domain locations and ssHDX time points are denoted at the top of the figure. Amino acid sequence and the corresponding peptide numbers are provide in Table S1. Values were obtained from average of three independent experiments.

mobility, as Tg is a parameter associated with the transition to viscous flow. In a few instances, Tg has been observed to have good correlation with protein stability, but in other cases poor

integrity, as well as mobility in the solid state, can therefore be useful in predicting long-term stability. Historically, Tg values measured calorimetrically have been used as a measure of global H


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neither the intensity nor the position of the α helix band, as measured by solid state Fourier transform infrared spectroscopy (FTIR), correlated with the loss of monomer. In the studies reported here, we applied ssHDX-MS to lyophilized formulations of a monoclonal antibody mAb1 (∼150 kDa), which currently represent the largest segment of recently approved parenteral drugs as well as of biologics in development.2,26 Long-term stability data (>2.5 year) were obtained for these formulations at intended storage as well as accelerated storage conditions. As with the myoglobin formulations, the FTIR band intensity and position of the mAb1 formulations did not correlate well with the increase in HMWs during storage, but the extent of deuterium incorporation (% D) did (Figures 4, 5). Other parameters not previously evaluated for the Mb formulations such as sucrose level, Tg, and residual moisture (both determined at initial time point and at end of stability testing) were also evaluated for mAb1 and showed partial or poor correlation with HMW increase compared to % D, with Tg having the weakest correlation. This suggests that deuterium exchange captures in a relatively short time a physicochemical property of the lyophilized protein that is related to protein aggregation propensity in the long term. If ssHDX-MS had been used initially as a tool in formulation selection, the four mAb1 formulations studied here could have been correctly rankordered for their aggregation propensity in 2−4 weeks, rather than the >2.5 year storage stability study that was actually used in formulation development. The attentive reader might argue that, for this set of formulations, the rank order was already established after 3 month storage time at elevated temperature, such as 50 °C. However, even if for this set of formulations this was successful, degradation mechanisms at significantly higher temperatures close to the glass transition temperature pose the risk that degradation pathways might change, not being representative for the intended storage temperature. In fact, the rank order of glass transition temperatures of the formulations Tg is different in this study compared to the rank order of their aggregation propensity as shown in Figure 4D. Moreover, ssHDX is performed on purpose at conditions selected with the goal to not impair protein structure and, thus, offers the advantage to reflect the degradation mechanism at intended storage temperature. Interestingly, % D values measured by ssHDX-MS were also well correlated with the percent of acidic and basic species in lyophilized mAb1 formulations (Figures 6 and S6/7). The relationship between ssHDX-MS results and chemical instability in lyophilized solids has not been examined previously. The observed correlation suggests that some chemical and physical degradation mechanisms, perhaps related to protein conformation and/or the local accessibility of the protein to residual moisture, are captured adequately by exposure to D2O vapor in the ssHDX-MS technique. Recently, solid state mAb formulations were also shown to exhibit covalent modifications of the mAb with buffer and excipient molecules on heat-stress stability.27 Evidently, further investigation of different buffer and excipient species, as well as testing over a broad pH range, are needed to determine their impact on the correlation between D2O uptake and the corresponding increase in charged variants on stability (and chemical degradation in general), and is the subject of ongoing research. The extent of Met oxidation was less strongly correlated to % D than the acidic and basic variants (Figure 6), perhaps because oxidative degradation reactions are less dependent on these

Figure 8. Percent deuterium uptake mapped onto the model structure of mAb1 for formulations F1 (A), F2 (B), F3 (C), and F4 (D) following 240 h of ssHDX. The structure of mAb1 was generated by homology modeling using 1IGY as a template.

correlation has been observed.19,20 Other techniques to study protein structure in solid state typically used for proteins are NMR or X-ray crystallography.21 These are high resolution techniques, but require samples in the crystalline state and thus are not applicable to most protein formulations, which contain a variety of excipients and form amorphous cake structures when lyophilized. FTIR,22 Raman spectroscopy, and NIR23 are analytical techniques that can study secondary structure of proteins even in the amorphous state. However, these techniques have low resolution and analyze global changes only. In particular for FTIR, it was shown that the band intensity for β-sheet resulted only in poor correlation with longterm stability data in some cases.7,11,24 In this light, solid state hydrogen−deuterium exchange coupled with mass spectrometry (ssHDX-MS) was developed by Topp and co-workers as a high resolution method to study protein structure in amorphous solid samples.7 HDX-MS is a known technique and conventionally applied for analysis in solution to study protein structure as well as protein dynamics.25 In regions where hydrogen bonding is weak, hydrogen atoms of the amide backbone exchange rapidly with deuterium when exposed to D2O. In contrast, protected regions show slower exchange that can depend on the dynamics of the protein in solution. The ssHDX-MS method presented in this manuscript applies this technique to the solid state. In contrast to spectroscopic techniques, ssHDX-MS offers the potential to elucidate the conformation and interactions of a lyophilized protein with a sugar matrix: (i) quantitatively and (ii) at peptide-level resolution. In addition, a kinetic study using ssHDX-MS may allow rank ordering of storage stability of lyophilized formulation. In a previous study, the relatively small protein myoglobin (Mb, ∼17 kDa) was studied in formulations containing different excipients.7 Results of ssHDX-MS experiments performed over the course of 10 days were shown to predict protein aggregation during long-term storage (up to 1 year). Both deuterium uptake and the fraction of amide groups undergoing fast exchange correlated well with monomer loss as measured by size exclusion chromatography. In contrast, I



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properties. Since solvent accessibility is clearly different in solution and solid states, it may be useful in future studies to examine whether specific chemical degradation reactions (e.g., deamidation) correlate with local deuterium exchange as measured by peptide level analysis. While the mechanisms responsible for relationship between % D in ssDHX-MS and stability are not known, it is highly likely that the hydrogen bonds of the backbone amide (peptide) bonds of the protein are involved, since this functional group is known to be the subject of solution state HDX measurements.11,28−30 We propose that ssHDX-MS interrogates the participation of these peptide bonds in the broader network of hydrogen bonds in the lyophilized solid, which includes both the intramolecular hydrogen bonds that contribute to higher order structure of the protein (e.g., α-helix, β-sheet) and the intermolecular hydrogen bonds between the protein and the surrounding matrix. In an ssHDX-MS experiment, deuterium incorporation then is likely to occur most readily for the peptide bonds that do not participate in this hydrogen bond network, e.g., because they are on the protein surface and interact with residual moisture rather than matrix excipients. Deuterium incorporation may also occur, though less readily, for peptide bonds involved in relatively weak intra- or intermolecular hydrogen bonds, with which sorbed D2O can compete effectively to insert deuterium. Greater protection from exchange in ssHDX-MS thus is consistent with a greater number and/or greater strength of inter- and intramolecular hydrogen bonds. It is reasonable to expect that these interactions would contribute to a reduced propensity for aggregation on storage, reflecting a protein that is more fully folded and/or with greater interactions with the surrounding matrix. That chemical degradation as measured by the production of acidic and basic variants is also correlated with deuterium incorporation suggests that rates of reactions that depend on proton transfer, such as deamidation and other hydrolytic reactions, may also be related to the “exchangeability” of protons as measured by ssHDX-MS. Conversely, that oxidative degradation is less correlated with % D than the production of acidic and basic species (Figure 6) may suggest that oxidation is less dependent on the hydrogen-bond network, and that the electron transfer processes involved in oxidation can occur through other means.

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00504. Additional figures and tables discussed in the text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Hoffmann-La Roche, Grenzacherstr. 124, CH-4070 Basel, Switzerland. E-mail: [email protected]. ORCID

Benjamin T. Walters: 0000-0001-5400-0696 Andrea Allmendinger: 0000-0002-8250-0978 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank F. Hoffmann-La Roche for providing the mAb1 formulations. The authors also acknowledge Pascal Chalus (F. Hoffmann-La Roche, Basel) for supporting the SEM analysis, Anja Bathke and Patrick Heim (F. Hoffmann-La Roche, Basel) for the analysis of global mAb oxidation using LC−MS, Vanessa Haefliger, Jasmin John, Severine Ughetti, and Christina Häu ser (F. Hoffmann-La Roche, Basel) for supporting the SEC, IEC, μDSC, and BET analysis, and Peter Stärtzel (Vetter, Ravensburg) for supporting the preparation of the freeze-dried formulations as well as for the physical characterization of the lyophilized samples. Financial support for the project and for ssHDX-MS studies performed at Purdue University was provided by Roche-Genentech.

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REFERENCES

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CONCLUSIONS

Solid-state hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) was applied to lyophilized monoclonal antibody (mAb1) protein formulations. The extent of deuterium incorporation in the solid state was linearly correlated with the aggregation propensity and chemical degradation rates during storage (>2.5 years) for the different formulations, and was superior to other biophysical measures (ssFTIR, Tg, sucrose content) in correlating with protein stability. It is the subject of ongoing research to confirm the applicability of ssHDX-MS for mAb formulations over a broader pH range, dependent on different buffer species, and different lyo-and kryoprotectants used in formulations of biologics. We conclude that ssHDX-MS can be used in the development of lyophilized mAb products to allow an early read-out of expected differences in long-term stability among candidate formulations. Applying this method may enable the best candidate formulations to be identified earlier in development, reducing workload and cost during long-term stability studies. J


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