protein drugs are often 
 formulated as lyophilized solids to preserve 
 their native structure and minimize the rate of degradation during 
 storage . these lyophilized powders are 
 usually amorphous rather than crystalline , a fact that has been attributed 
 to the lyophilization process and the properties of the proteins themselves . though high resolution methods such as x - ray crystallography and nmr 
 spectroscopy can be used to study protein structure in solution and 
 in crystalline solids , the methods for assessing protein structure in amorphous 
 solids are far more limited . vibrational spectroscopy methods such 
 as fourier transform infrared spectroscopy ( ftir ) and raman spectroscopy are 
 routinely used by pharmaceutical companies to probe protein secondary 
 structure in lyophilized powders , to assess the effects of formulation 
 additives (  excipients  ) , and to design formulations . such methods lack the structural resolution of x - ray crystallography 
 and nmr , however , and the data are only semiquantitative . hydrogen  deuterium 
 exchange with mass spectrometric analysis 
 ( hdx - ms ) has been widely used to study protein structure , stability , 
 and dynamics in solution . in hdx - ms , a protein 
 in aqueous solution is exposed to d2o and the rate and 
 extent of deuterium incorporation detected as a change in mass ( m / z ) . proteolytic digestion prior to ms 
 analysis allows exchange to be monitored with peptide - level resolution . to preserve the sites of labeling during analysis , the reaction 
 is usually quenched by lowering ph and temperature to inhibit the 
 reverse reaction (  back exchange  ) . together with rapid 
 sample analysis under refrigerated conditions , this typically allows 
 retention of label acquired by backbone amide groups , so that the exposure of protein sequence to d2o can be mapped . in pharmaceutical development , hdx - ms has been used 
 to map the interactions between receptors and small molecule drugs , to monitor conformational changes in protein 
 drugs mediated by post - translational modifications , and to compare batch - to - batch variation in developing biosimilars . to extend hdx - ms to lyophilized samples , deuterium exchange with mass spectrometric 
 analysis ( sshdx - ms ) to measure protein structure and excipient interactions 
 in lyophilized powders with high resolution . in sshdx - ms , lyophilized samples 
 are exposed to d2o in the vapor phase at controlled relative 
 humidity ( rh ) and temperature . samples are reconstituted under quenched 
 conditions and subjected to ms analysis with or without proteolytic 
 digestion . our previous sshdx - ms studies have shown that proteins 
 in lyophilized solids are protected from deuterium exchange in a manner 
 that depends on relative humidity , position in the protein sequence , 
 and the type and amount of excipient used . for example , when compared to hdx in solution , hdx - ms of equine 
 myoglobin ( mb ) colyophilized with sucrose showed greater protection 
 from exchange than mb colyophilized with mannitol , while samples colyophilized with guanidine hydrochloride 
 showed greater deuterium uptake than native mb in aqueous solution . when comparing different lyophilized formulations , 
 sshdx - ms often shows differences not detected by ftir . this suggests that sshdx - ms may be useful as 
 a screening tool to select formulations that best preserve protein 
 structure and so promote stability during storage . to date , however , 
 the relationship between the solid state structural information provided 
 by sshdx - ms and protein stability in lyophilized powders has not been 
 explored . in the work reported here , we used sshdx - ms and ftir 
 to monitor 
 the effects of different excipients on the conformation of mb in lyophilized 
 powders , and to relate these measures of structure to mb aggregation 
 during 1 year ( 360 days ) of storage of the lyophilized powder 
 under controlled conditions . the results demonstrate that quantitative 
 measures of deuterium exchange provided by sshdx - ms are highly correlated 
 with aggregate formation during storage and provide a better indication 
 of the propensity for aggregation than secondary structure as measured 
 by ftir . equine myoglobin ( mb ) 
 was purchased from 
 sigma - aldrich ( st . louis , mo ) . mass spectrometry grade water , acetonitrile 
 ( acn ) , and formic acid ( fa ) were 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 ) . mb was solubilized and dialyzed 
 thoroughly in 2.5 mm potassium phosphate buffer , ph 7.0 at 4 c . 
 the dialyzed mb stock solution ( 6.8 mg / ml ) the samples 
 were filtered using millex - gv 0.22 m pvdf filter ( millipore , 
 billerica , ma ) and filled as 2.2 ml into 8 ml glass vial ( nuova ompi , 
 padova , italy ) using a peristaltic pump ( flexicon pf6 , flexicon , wilmington , 
 ma ) . the vials were loaded and freeze - dried in a boc edwards lyophilizer 
 ( lyomax 0.4 ) ( boc edwards , tonawanda , ny ) . lyophilization was initiated 
 by precooling the shelves to 2 c , followed by freezing 
 at 40 c . primary drying was carried out under vacuum 
 ( 70 mtorr ) at 35 c for 75 h followed by secondary drying 
 at 25 c for 12 h and 5 c for 12 h. all vials were backfilled 
 with nitrogen prior to sealing . after lyophilization , the mb formulations 
 were analyzed for any excipient and/or lyophilization induced changes 
 using dynamic light scattering ( dls ) , size exclusion chromatography 
 ( sec ) , ftir , and sshdx - ms , as described below . the lyophilized samples 
 were then placed on stability at 25 c and 60% rh or 40 c 
 and 75% rh over a period of 1 year . samples were withdrawn at intervals 
 and analyzed for aggregate content by sec and dls as described below . to evaluate excipient 
 and/or lyophilization induced secondary structural changes in mb , 
 ftir spectra were acquired for mba , mbb , mbc , mbd , and mbe ( table 1 ) using a tensor 37 ftir spectrometer ( bruker optics , 
 billerica , ma ) . approximately 2 mg of lyophilized protein was mounted 
 onto the atr germanium crystal and the data collected with 128 scans 
 at a resolution of 4 cm . to avoid interference 
 from atmospheric water vapor , background spectra collected under similar conditions were 
 subtracted from the raw spectra acquired for each sample . data were 
 processed and analyzed using the opus software ( version 6.5 , bruker 
 optics ) . the raw spectra were first cut between 1720 and 1580 cm to analyze the amide i band . spectra were smoothed 
 using a nine - point savitsky  golay smoothing function , and baseline 
 correction was carried out using the rubberband correction method 
 with 64 baseline points . finally , spectra were centered and min / max 
 normalized before second derivatization was carried out . golay 
 smoothing function . to 
 determine the rate 
 and extent of moisture sorption by mb formulations during sshdx - ms , 
 moisture sorption was measured using a gravimetric analyzer ( q5000sa ; 
 ta instruments , new castle , de ) . approximately 34 mg of mb 
 powder was loaded onto the platinum sample pan , and the samples were 
 heated to 85 c at 0% rh for 10 min to remove any moisture present 
 before the start of the experiment . samples were then equilibrated 
 at 5 c , 0% rh and exposed to 33% rh for 24 h , with sorption 
 data collected at 4 s intervals . to determine the rate and extent 
 of deuterium incorporation for intact ( i.e. , undigested ) mb , sshdx - ms 
 was carried out at 5 c by incubating the vials in a sealed desiccator 
 containing a saturated solution of mgcl2 in d2o ( 33% rh over d2o ) . samples were removed at six different 
 time points over 240 h , and exchange was quenched by flash freezing 
 the vials in liquid nitrogen , which were then stored at 80 
 c until analysis . deuterium uptake by intact mb was measured 
 using a high performance liquid chromatography mass spectrometer ( lc / ms ) 
 ( 1200 series lc , 6520 qtof ; agilent technologies , santa clara , ca ) 
 equipped with a custom - built refrigeration unit which maintained the 
 column at 0 c . the samples were quickly reconstituted 
 in chilled 0.2% formic acid ( fa ) , and approximately 15 pmol of protein 
 was injected onto a protein microtrap ( michrom bioresources , inc . samples were desalted for 1.7 min with 15% acetonitrile , 
 85% water , and 0.1% fa and eluted in 2.3 min using a gradient to 90% 
 acetonitrile , 10% water , and 0.1% fa . mass spectra were obtained over 
 the m / z range 2003200 , and 
 the masses of both undeuterated and deuterated protein were obtained 
 by deconvoluting the spectra using masshunter workstation software 
 ( version b.03.01 , agilent technologies ) . to calculate the number of 
 deuterons incorporated in intact mb , the mass of undeuterated mb was 
 subtracted from the mass of deuterated mb at each exchange time point . using graphpad prism software version 5 ( san diego , ca ) , deuteration 
 kinetic data for intact mb in different formulations were fitted to 
 a biexponential model ( eq 1 ) which assigns deuterons 
 to  fast  and  slow  exchanging pools , 
 as described previously.1peak 
 width analysis for deuterated intact 
 mb was carried out as described previously . to determine the 
 distribution of deuterium incorporation along the mb sequence , deuterated 
 mb exchange reactions were carried out as above , the quenched 
 sample was quickly reconstituted in chilled 0.2% formic acid ( fa ) , 
 and approximately 15 pmol of protein was then injected into an immobilized 
 pepsin column . online digestion was carried out for 2 min in water 
 containing 0.1% fa at a flow rate of 0.2 ml / min . the digested sample 
 was desalted in a peptide microtrap ( michrom bioresources , inc . , auburn , 
 ca ) and eluted using a gradient of acetonitrile ( 1060% ) in 
 0.1% fa onto a reverse phase analytical column ( zorbax 300sb - c18 ; 
 agilent technologies ) at 50 l / min . the pepsin column , peptide 
 microtrap , and analytical column were located within the refrigeration 
 unit and connected through a two - position valve ( epc12cw , vici valco 
 instruments co. , inc . peptides from an undeuterated mb sample were also analyzed , and their 
 mass was measured by ms / ms analysis ( cid fragmentation ; masshunter 
 software ; agilent technologies ) . peptides indentified using undeuterated 
 mb were mapped onto subsequent deuteration experiments using prototype 
 custom software , hdexaminer ( sierra analytics , modesto , ca ) , to obtain 
 the average number of deuterons exchanged for each of the pepsin digest 
 fragments . kinetic data for deuteration of 13 nonredundant peptides 
 were fitted to a biexponential association model ( eq 1 ) which accounts for deuterons exchanging at  fast  
 and  slow  rates , as described above . data from 48 h 
 sshdx - ms samples were mapped onto the crystal structure of mb ( pdb 
 i d : 1wla ) using pymol ( pymol molecular graphics system , 
 version 1.3 , schrodinger , llc ) . peak width analysis for the peptide 
 mass envelopes was carried out using hx - express software as described 
 previously . the formation of particles 
 in the stability samples was monitored using 90 dls ( zetasizer 
 nano zs90 , malvern instruments , ltd . , westborough , ma ) . approximately 
 1 ml of the reconstituted mb sample was placed in a polystryrene cuvette 
 ( vwr ) and analyzed with a path length of 10 mm at 25 c . triplicate 
 samples for each formulation were recorded 3 times using an automatic 
 mode for the selection of best number of subruns . the z - average diameter ( nm ) was calculated from the correlation function 
 using the dispersion technology software supplied with the instrument 
 ( version 4.20 , malvern , westborough ma ) . hp - sec was used to assess the percentage 
 loss of monomeric mb during 
 the stability study . an agilent 1200 system with uv detection at 280 
 nm ( agilent technologies , palo alto , ca ) was used to monitor the loss 
 in monomeric protein . vials containing lyophilized mb for each formulation 
 were reconstituted with 2.2 ml of sterile distilled water ; samples 
 in triplicate were centrifuged ( microfuge 22r refrigerated microcentrifuge , 
 vwr ) at 14,000 rpm for 15 min to remove any insoluble protein aggregates . 25 l aliquots were then injected onto a tosoh tskgel g3000swxl 
 column ( tosoh bioscience llc , king of prussia , pa ; 3000 mm  
 7.8 mm ) and eluted with buffer ( 50 mm potassium phosphate , 100 mm 
 nacl , ph 7.5 ) at a flow rate of 0.5 ml / min . the percentage loss of 
 monomeric mb was determined using the following equation ( eq 2).2 all 
 second derivative ftir spectra showed a strong -helix band 
 at 1654 cm . a decrease in band intensity was observed for samples mbc and mbd , suggesting reduced -helix 
 content for these formulations ( figure 1 ) . overlay 
 of second derivative amide i ftir spectra of mb in formulations 
 mba , mbb , mbc , mbd , and mbe . before exchange can 
 occur in sshdx - ms , d2o must be sorbed by the solid matrix 
 from the vapor phase and diffuse into the solid . the rate of moisture 
 sorption and diffusion thus may influence the rate of exchange . the 
 effect of hydration on hdx of mb colyophilized with sucrose or mannitol 
 has been extensively characterized in our previous study . the results showed that moisture sorption is complete in a period 
 of hours while hdx continues for days , indicating that , under conditions 
 comparable to those used here , hdx in lyophilized powders is not simply 
 a measure of moisture sorption . in the 
 work reported here , the rate of moisture uptake was measured at 5 
 c , 33% rh to confirm that the rate of water vapor sorption is 
 rapid relative to the deuterium exchange rate . the rate and extent 
 of moisture absorption differed among the formulations ( figure 2 ) . the formulation with mb alone ( mbd ) reached its 
 sorption maximum absorption in < 1 h and sorbed more moisture ( 0.103 
 g water / g solid ) than the other formulations . the formulations containing 
 sucrose ( mba , mbe ) reached their sorption maxima in 20 h and 
 sorbed similar levels of moisture ( 0.085 and 0.083 g of water / g of 
 solid , respectively ) . formulations containing mannitol ( mbb ) and nacl 
 ( mbc ) sorbed the least moisture ( 0.053 and 0.064 g of water / g of solid , 
 respectively ) but reached equilibrium in 2 h. based on these 
 results , and since sshdx occurs over hundreds of hours ( see figure 3 and figure s2 in the supporting 
 information ) , it is unlikely that vapor sorption dominates 
 sshdx kinetics , except at t < 24 h for samples 
 containing sucrose ( mba , mbe ) . moisture sorption kinetics for mb formulations 
 mba , mbb , mbc , mbd , 
 and mbe . data were collected at 33% rh , 5 c for 24 h. ( a ) overlay of deconvoluted mass spectra of 
 intact mb from formulations 
 mba , mbb , mbc , mbd , and mbe following 48 h of deuterium exchange . ( b ) kinetics of 
 deuterium uptake for intact mb in formulations mba ( closed circle ) , 
 mbb ( closed triangle ) , mbc ( open circle ) , mbd ( open square ) , and mbe 
 ( closed square ) . plots of the time course of deuterium exchange were 
 fitted to an equation for two phase exponential association ( graphpad 
 prism software version 5 ( san diego , ca ) ) ( n = 3 , 
 se ) . sshdx - ms data were acquired 
 for the five formulations ( table 1 ) at six 
 different time points ( 1240 h ) . overall , mba and mbe showed less deuterium uptake than mbb , mbc , and 
 mbd ( table 2 ) . furthermore , mbe is less deuterated 
 than mba , indicating that the greater sucrose content in mbe provides 
 greater protection from exchange . formulations containing nacl ( mbc ) 
 or without excipient ( mbd ) showed the greatest deuterium uptake at 
 all time points ( figure 3 ) . the extent of deuterium 
 uptake at 48 h ( figure 3a and table 2 ) was not simply related to the extent of water 
 vapor sorption at 24 h ( figure 2 ) . for example , 
 mbc showed less moisture sorption than mbd but comparable deuterium 
 uptake . mba and mbe ( with sucrose ) showed high vapor sorption yet 
 had the greatest protection from exchange , while mbb ( with mannitol ) 
 showed the lowest vapor sorption and an intermediate extent of exchange . these results are consistent with our previous sshdx - ms study of mb 
 at 43% rh , in which mb formulations with sucrose sorbed 25% 
 more moisture and showed 20% less deuteration than those containing 
 mannitol following 48 h of hdx . values obtained from three independent 
 sshdx - ms experiments ( mean  se ) . percent deuterium uptake relative 
 to theoretical maximum by intact mb after 48 h of hdx at 5 c , 
 33% rh . nfast and nslow values were 
 determined by 
 nonlinear regression of sshdx - ms kinetic data . time course of deuterium 
 exchange for intact mb was fitted to the two phase exponential association 
 model ( eq 1 ) . peak width was measured at 20% height 
 of the deconvoluted mass spectrum of intact mb . kinetic plots of deuterium uptake 
 for intact mb were fitted to 
 a biexponential model ( figure 3b ) . on average , 
 the nfast and nslow values for mba and mbe were less than those for mbb , mbc , and mbd 
 ( table 2 ) . among the formulations , differences 
 in the nslow values were larger than differences 
 in the nfast values ( table 2 ) . this suggests that greater nslow values in mbb , mbc , and mbd may be due to the recruitment of amide 
 groups that do not exchange in mba and mbe into the slowly exchanging 
 pool . the fraction of exchanging amide groups in the rapidly exchanging 
 pool is expressed in the ratio nfast/(nfast + nslow ) ( table 2 ) , which provides another measure of the distribution 
 between the two pools . a total of 41 fragments were identified ; 
 from these , a nonredundant 
 set of 13 fragments covering 100% of the mb sequence was selected 
 for analysis ( figure s1 in the supporting information ) . the average number of deuterons exchanged for each peptide was 
 calculated , and the results from 48 h samples were mapped onto the 
 crystal structure of mb ( pdb i d : 1wla ) ( figure 4 ) . the 
 kinetics of deuterium uptake for the 13 nonredundant peptic fragments 
 showed biexponential behavior ( figure s2 in the supporting information ) , consistent with subpopulations of 
 amide groups undergoing  fast  and  slow  
 exchange . the number of amides in the  fast  and  slow  
 pools , nfast and nslow , varied with formulation and in different regions of the 
 mb molecule ( figure 5 ) . overall , the nfast and the nslow values for most of the peptides in formulations mbb , mbc , and mbd 
 were greater than those in mba and mbe , with the differences in the nslow values somewhat greater than differences 
 in the nfast values ( figure 5a , b ) . crystal structures of holo - mb ( pdb i d : 1wla ) showing the percent 
 deuterium uptake 
 for mb in formulations mba , mbb , mbc , mbd , and mbe following 48 h 
 of deuterium exchange . sshdx - ms data from 13 nonredundant pepsin digest 
 fragments were mapped onto the structure . the heme group in holo - mb 
 is shown in brown spheres ; color coding for deuteration level is as 
 per legend key indicated . comparison of nfast ( a ) , nslow ( b ) , and nfast/(nfast + nslow ) ( c ) 
 values for mb in formulations mba , mbb , mbc , mbd , and mbe . values 
 were obtained from nonlinear regression of sshdx - ms kinetic data for 
 13 nonredundant peptic peptides ( n = 3 , se ) . the sshdx - ms kinetic data also 
 differed in sucrose formulations 
 having different concentrations of excipient ( mba , mbe ) . nfast values for peptides 129 , 1429 , 2832 , 
 3355 , 70105 , 87106 , 106137 , 107123 , 
 and 138153 in formulations mba and mbe showed slight or no 
 differences , whereas nslow values were 
 generally smaller in mbe than in mba . similarly , values of the nfast/(nfast + nslow ) ratio were greater for these peptides 
 in mbe ( figure 5c ) . for regions spanning residues , 
 17 , 3040 , 5669 , and 119148 , the nfast values for mba were greater than for mbe , 
 and nfast/(nfast + nslow ) values were smaller . together , 
 the results suggest that these regions in mbe are more rigid or folded 
 than in mba . in sshdx - ms , 
 a comparison of peak widths at a similar level of deuteration is expected 
 to provide information on differences in the spatial and/or conformational 
 heterogeneity of proteins in solid samples . for intact mb , peak width steadily increased with time for formulations 
 mbb , mbc , and mbd but showed smaller increases in mba and mbe ( figure 6a ) . to establish a level of deuterium uptake for 
 comparing different formulations , peak width was plotted against percentage 
 deuterium uptake ( figure 6b ) , and 15% deuterium 
 incorporation was selected for comparison . since this value is greater 
 than the maximum deuterium uptake observed in mbe , it was omitted 
 from the comparison ( figure 6b ) . at 15% 
 deuterium uptake , mbc and mbd showed 2-fold greater peak width 
 than formulations mba and mbb , and mbb showed slightly greater peak 
 width ( 1.2 da ) than mba ( table 2 ) . thus , peak width analysis in intact mb suggests that protein in mba 
 retained a greater degree of native structure and/or was more homogeneous 
 than in mbb , mbc , or mbd . ( a ) peak width broadening for intact deuterated 
 mb as a function 
 of time . peak width at 20% height of the deconvoluted mass spectrum 
 was measured for formulations mba ( closed circle ) , mbb ( closed triangle ) , 
 mbc ( open circle ) , mbd ( open square ) , and mbe ( closed square ) . ( b ) 
 peak width broadening in formulations mba , mbb , mbc , mbd , and mbe 
 with differing levels of deuterium uptake ( n = 3 , 
 se ) . ( c ) comparison of peak width for 8 nonredundant peptic 
 peptides obtained from mb in mba , mbb , mbc , mbd , and mbe at similar 
 deuteration level . peak with at 20% peak height was plotted for residues 
 129 at 15.1% , 2832 at 6.2% , 3040 at 6.1% , 
 3355 at 17.2% , 5669 at 7.9% , 70105 at 9.3% , 
 106137 at 10.0% , and 138153 at 12.5% deuteration ( n = 3 , se ) . peak width analysis was also carried out for the peptic peptides 
 from mb using hx - express software . the eight nonredundant peptides 
 were analyzed for peak width to represent the distribution of conformations 
 of mb within the solid matrix . the extent of deuteration of the eight 
 peptides of mbe at 240 h , relative to the theoretical maximum , were 
 as follows : residues 129 , 15.1% ; residues 2832 , 6.2% ; 
 residues 3040 , 6.1% , residues 3355 , 17.2% ; residues 
 5669 , 7.9% ; residues 70105 , 9.3% ; residues 106137 , 
 10.0% ; and residues 138153 , 12.5% . peak widths for the remaining 
 formulations were compared at an extent of deuteration equal to the 
 value for that peptide in mbe at 240 h. the increase in peak width 
 and its differences among formulations are region specific ( figure 6c and figures s3 and s4 in the supporting information ) . for formulations mbc and mbd , the 
 n - terminal region ( residues 129 ) showed a greater peak width 
 ( 2.5 da ) for formulations mbc and mbd than for the remaining 
 formulations . the region spanning residues 3355 showed greater 
 peak widths ( 2.0 da ) for mbc and mbd ( 2.0 da ) and 
 for mbb ( 1.0 da ) . other regions ( i.e. , 5669 , 70105 , 
 106137 , and 138153 ) also showed greater peak widths 
 for mbc and mbd , and to a lesser extent for mbb , though the magnitude 
 of the effect is not as large . these results are consistent with a 
 greater distribution of conformations in mbb , mbc , and mbd , particularly 
 in domains 129 and 3355 . the stability of the 
 mb formulations during long - term storage was 
 characterized using dls and hp - sec , which monitor subvisible particles 
 and loss of monomeric protein , respectively . for dls analysis , the 
 lyophilized samples were reconstituted in sterile distilled water 
 and analyzed with a zetasizer nano zs90 ( malvern instruments , ltd . the reconstituted lyophilized samples showed an 
 increase in particle size relative to the prelyophilized controls , 
 with the largest particle sizes observed in mbc and mbd ( figure s5 
 in the supporting information , t = 0 ) . during storage at 25 and 40 c , particle size 
 increased with time for formulations mbc and mbd at both temperatures . mba , mbb , and mbe showed a slight increase in particle size over time 
 ( figures 7a and 7b ) . mb aggregation 
 in lyophilized formulations during stability study , 
 as monitored using dls and hp - sec . the increase in particle size for 
 formulations stored at 25 c ( a ) and 40 c ( b ) and the percentage 
 loss of monomeric mb for formulations stored at 25 c ( c ) and 
 40 c ( d ) were plotted against time ( n = 3 , 
 se ) . mba ( closed circle ) , mbb ( closed triangle ) , mbc ( open circle ) , 
 mbd ( open square ) , and mbe ( closed square ) . the effects of excipients on lyophilization - induced aggregation 
 were also characterized using hp - sec . lyophilized samples were reconstituted 
 with sterile distilled water and subjected to hp - sec , and the chromatographic 
 peak area was used to calculate the loss of monomeric mb . at t ( i.e. , immediately after lyophilization and reconstitution ) , 
 the percent loss of mb in formulations mba , mbb , mbc , mbd , and mbe 
 was 1.3  0.1 , 0.8  0.1 , 4  0.3 , 1.4  0.1 , 
 and 1.3  0.2% , respectively . to assess the effect of excipients 
 on mb stability during storage , the lyophilized mb formulations were 
 placed at 25 and 40 c for a period of 360 days and evaluated 
 at regular intervals . the rate of loss of monomeric protein was greatest 
 for mbc at 25 c and for mbb , mbc , and mbd at 40 c ( figures 7c and 7d ) . at both temperatures , the rate of aggregate formation was slowest in mba and mbe and the 
 loss of monomeric mb for these samples after 360 days was less than 
 4% . to evaluate sshdx - ms as a potential screening tool for lyophilized 
 formulations , we correlated quantitative measures of deuterium exchange 
 in freshly lyophilized formulations ( t = 0 ) to the 
 extent of aggregation during 6 months and 1 year of storage as measured 
 by hp - sec . the quantitative measures of exchange were as follows : 
 ( i ) deuterium uptake in intact mb after 48 h d2o exposure ; 
 and ( ii ) nfast/(nfast + nslow ) values determined 
 by nonlinear regression of hdx kinetic data ( eq 1 ) for intact mb . the extent of aggregation was also correlated with 
 secondary structure in freshly lyophilized samples as measured by 
 -helix band intensity and peak position in ftir ( figures 8b18b4 and figures 
 s6b1s6b4 in the supporting information ) . for both 25 and 40 c stability samples , the percent loss 
 of mb monomer after 180 and 360 days increased monotonically with 
 the percent deuterium uptake as measured by sshdx - ms at t = 0 ( figures 8a1 and 8a2 and figures s6a1 and s6a2 in the supporting 
 information ) ( table 3 and table s1 in 
 the supporting information ) , and decreased 
 monotonically with nfast/(nfast + nslow ) ( figures 8a3 and 8a4 and figures s6a3 and s6a4 in the supporting information ) ( table 3 and table s1 in the supporting information ) . in contrast , correlation of aggregation during storage with ftir 
 measures of secondary structure was poor ( figures 8b18b4 and figures s6b1s6b4 
 in the supporting information ) ( table 3 and table s1 in the supporting 
 information ) . correlation of mb aggregation during long - term storage 
 ( at t = 360 days ) with t = 0 sshdx - ms 
 ( a ) and 
 ftir ( b ) analysis . the percent loss of mb monomer after 360 days of 
 storage at 25 and 40 c as a function of percent deuterium uptake 
 ( a1 and a2 ) , nfast/(nfast + nslow ) ( a3 and a4 ) , 
 band intensity for -helix ( b1 and b2 ) , and band position for 
 -helix ( b3 and b4 ) in formulations , mba ( closed circle ) , mbb 
 ( closed triangle ) , mbc ( open circle ) , mbd ( open square ) , and mbe ( closed 
 square ) ( n = 3 , se ) . the data were subjected 
 to linear regression to obtain slope , intercept , and r values ( see table 3 ) . parameters  se determined 
 by linear fit of values obtained from ftir and hdx - ms experiments 
 at t = 0 against mb aggregation at t = 360 days . lyophilization is often used in an attempt to inhibit aggregation 
 and other types of degradation in protein drug products . though it 
 is often helpful in this regard , aggregates have nevertheless been 
 observed in lyophilized solids . in general , the extent of aggregation 
 depends on the composition of the solid and on lyophilization 
 process variables , with optimal formulation and processing conditions 
 varying from protein to protein . to develop lyophilized protein drug 
 products in a rational way , the rates of degradation should be correlated 
 to measurable properties of the formulation . identifying such properties 
 would enable a formulation to be designed to achieve 
 desired performance , rather than developed by trial - and - error in stability 
 studies requiring months or years to complete . to date , consensus 
 has not been reached on the properties that control degradation in 
 lyophilized solids , however . there have been a number of attempts 
 to correlate protein stability in the solid state with properties 
 such as glass transition temperature ( tg ) , moisture content , free volume measurement by gas pycnometry , protein secondary structure as measured by ftir , and fast local dynamics measured using neutron scattering , which have met with varying degrees of success . for example , though 
 ftir measures of secondary structure correlated well with long - term 
 storage stability in some studies , others have 
 reported a weak or unsuccessful correlation . a limitation common to these approaches is 
 that they rely on bulk properties of the matrix and/or population 
 averaged properties of protein molecule . since protein instability 
 is driven by changes occurring at the molecular or functional group 
 scale , bulk methods may lack sufficient resolution to monitor the 
 relevant molecular environment . the spatial and dynamic heterogeneities 
 of amorphous solids further complicate the picture . to address 
 the need for molecular - scale characterization of proteins 
 in lyophilized solids , our group has developed sshdx - ms to assess 
 protein conformation and excipient interactions in the solid state 
 with peptide - level resolution . in the current study , we used sshdx - ms to correlate protein conformation 
 in solid samples with the extent of aggregation during long - term storage . five different mb formulations were produced and characterized for 
 process- or formulation - induced structural changes prior to evaluating 
 long - term storage stability . the studies tested the hypothesis that 
 quantitative measures of protein conformation provided by sshdx - ms 
 are correlated with the extent of mb aggregation during storage , and 
 that the correlation is superior to those based on ftir peak intensity 
 or position . initial characterization of solid samples using 
 ftir showed retention 
 of -helix band intensity in formulations mba , mbb , and mbe , 
 reflecting a high degree of native structure , while decreased band 
 intensity in mbc and mbd indicated structural perturbation . these 
 results suggest that formulations with sucrose ( mba and mbe ) or mannitol 
 ( mbb ) are likely to be more stable . ftir spectra did not reveal differences 
 among formulations mba , mbb , and mbe at t = 0 , however , 
 though the three formulations showed different levels of aggregation 
 on stability . in addition , the extent of aggregation was poorly correlated 
 to quantitative measures of secondary structure ( i.e. , peak intensity , 
 peak position ) obtained from ftir spectra ( figures 8b18b4 ) . in contrast , 
 sshdx - ms provided quantitative measures of protein 
 conformation that were highly correlated with the extent of aggregation 
 during storage . in sshdx - ms analysis , mb in formulations mbb , mbc , 
 and mbd showed greater deuterium uptake throughout the molecule than 
 formulations containing sucrose ( mba , mbe ) ( figures 35 ) . formulations that were 
 indistinguishable by ftir ( mba , mbb , mbe ; figure 1 ) showed measurable differences in the extent of sshdx in 
 intact mb after 48 h of d2o exposure ( figures 3a and 4 ) , which were highly 
 correlated with aggregation during storage ( figures 8a1 and 8a2 ) . studies of sshdx kinetics 
 allowed the number of amide groups in rapidly and slowly exchanging 
 pools to be quantified ( nfast , nslow ; table 2 ) . while 
 a definitive structural assignment of the nfast and nslow pools can not be made , it is 
 reasonable to expect that the nslow amide 
 groups correspond to regions that are protected from exchange , either 
 through folding of mb or through interactions with the solid matrix . in contrast , the nfast amides are expected 
 to be present on the surface of protein molecule or to have limited 
 interaction with the matrix . the fraction in the rapidly exchanging 
 pool ( nfast/(nfast + nslow ) ) was also highly correlated 
 with the extent of aggregation on storage ( figures 8a3 and 8a4 ) . thus , the results of these 
 studies support the hypothesis that sshdx - ms results are correlated 
 with the extent of mb aggregation during storage , and that the correlation 
 is superior to those based on ftir peak intensity or position . previous 
 studies in solution have shown that cold - unfolded mb retains a significant 
 amount of secondary structure and easily aggregates at moderate temperatures 
 through partially unfolded forms . if 
 partial unfolding is also involved in mb aggregation in the lyophilized 
 samples studied here , sshdx - ms may be superior to ftir as a measure 
 of aggregation potential because it can detect partial unfolding when 
 secondary structure is largely retained . however , it should be noted 
 that the results presented here can not justify the poor correlation 
 observed in earlier ftir studies , since protein formulations and experimental conditions 
 used in those studies differ . with pepsin digestion , sshdx - ms 
 provided additional , higher resolution 
 information on the effects of formulation on mb conformation and stability . data from the digests show that portions of the mb b - c helices ( fragments 
 2832 , 3040 ) and g helix ( fragment 107123 ) 
 are relatively unaffected by solid composition and show low deuterium 
 uptake in all five formulations ( figures 4 , 5 ) . in solution at low ph and temperature , mb forms 
 an equilibrium molten globule involving helices a , g , and h , together 
 with part of the b helix ; the molten globule is thought to be an intermediate 
 on the mb folding pathway . that the regions 
 most protected from exchange in lyophilized samples correspond to 
 the equilibrium molten globule suggests that this domain is folded 
 in all five lyophilized formulations , and that formulation primarily 
 affects folding in other mb domains . interestingly , residues 17 
 from the n - terminus ( a helix ) and residues 138153 from the 
 c - terminus ( h helix ) are less protected from exchange in solid samples 
 than other helices in the equilibrium molten globule ( figures 4 and 5 ) , though the reasons 
 for this are not clear . whether the heme group is intact in the lyophilized 
 samples is also unclear , since it is not retained during lc / ms analysis 
 and patterns of exchange near the heme pocket are not conclusive ( figure 4 ) . nevertheless , the results demonstrate that high 
 resolution structural information can be obtained for proteins in 
 lyophilized solids using sshdx - ms . together , the results support 
 the use of sshdx - ms as a screening 
 tool in developing stable lyophilized formulations of protein drugs . the sshdx - ms studies reported here were completed in 48 h ; with sample 
 preparation and data analysis , the process required 710 days 
 in total . in room temperature ( 25 c ) stability studies , differences 
 among the formulations became clear only after 180 days ( 6 months ) 
 of storage ( figure 7c , d ) . accelerated ( 40 c ) 
 stability data at earlier time points were not indicative of the ultimate 
 rank - ordered performance of the formulations at room temperature ( figure 7c , d ) . though mb is a well - characterized protein , 
 sshdx - ms can also be applied to new and incompletely characterized 
 proteins if the amino acid sequence is known . while we have used the 
 known x - ray crystal structure of mb to map the sites of exchange in 
 the solid state , sshdx - ms can be applied as a stand - alone method in 
 the absence of detailed structural information . in fact , the results 
 presented here demonstrate that storage stability is highly correlated 
 with the extent of hdx in the intact protein , data apart from other 
 structural characterization . the strong correlation of solid stability 
 with sshdx - ms results reported here supports its wider adoption . however , 
 establishing the utility of sshdx - ms as a formulation tool will ultimately 
 require that it be applied to many proteins and formulations sshdx - ms provided quantitative measures of the conformation of 
 mb in lyophilized solids which were highly correlated to aggregation 
 during 6 months and 1 year of storage in the solid state . in contrast , 
 spectral band intensity and position as measured by ftir in lyophilized 
 solids was poorly correlated with aggregation during storage . the 
 results support the use of sshdx - ms as a rapid screening tool to predict 
 the propensity for aggregation during storage and to discriminate 
 among candidate formulations .
solid 
 state amide hydrogen / deuterium exchange with mass spectrometric 
 analysis ( sshdx - ms ) was used to assess the conformation of myoglobin 
 ( mb ) in lyophilized formulations , and the results correlated with 
 the extent of aggregation during storage . 
 mb was colyophilized with 
 sucrose ( 1:1 or 1:8 w / w ) , mannitol ( 1:1 w / w ) , or nacl ( 1:1 w / w ) or 
 in the absence of excipients . 
 immediately after lyophilization , samples 
 of each formulation were analyzed by sshdx - ms and fourier transform 
 infrared spectroscopy ( ftir ) to assess mb conformation , and by dynamic 
 light scattering ( dls ) and size exclusion chromatography ( sec ) to 
 determine the extent of aggregation . 
 the remaining samples were then 
 placed on stability at 25 c and 60% rh or 40 c and 75% 
 rh for up to 1 year , withdrawn at intervals , and analyzed for aggregate 
 content by sec and dls . in sshdx - ms of samples immediately after lyophilization 
 ( t = 0 ) , mb was less deuterated in solids containing 
 sucrose ( 1:1 and 1:8 w / w ) than in those containing mannitol ( 1:1 w / w ) , 
 nacl ( 1:1 w / w ) , or mb alone . 
 deuterium uptake kinetics and peptide 
 mass envelopes also indicated greater mb structural perturbation in 
 mannitol , nacl , or mb - alone samples at t = 0 . 
 the 
 extent of deuterium incorporation and kinetic parameters related to 
 rapidly and slowly exchanging amide pools ( nfast , nslow ) , measured at t = 0 , were highly correlated with the extent of aggregation 
 on storage as measured by sec . 
 in contrast , the extent of aggregation 
 was weakly correlated with ftir band intensity and peak position measured 
 at t = 0 . 
 the results support the use of sshdx - ms 
 as a formulation screening tool in developing lyophilized protein 
 drug products .