Professional Documents
Culture Documents
Journal of Pharmaceutical Sciences
Journal of Pharmaceutical Sciences
Pharmaceutical Biotechnology
a r t i c l e i n f o a b s t r a c t
Article history: This study investigates the effect of low levels of electrolytes on storage stability in freeze-dried sucrose-
Received 17 June 2015 based protein formulations. Both bovine serum albumin and recombinant human serum albumin were
Revised 23 September 2015 freeze dried with sucrose and alkali halides (LiCl, NaCl, KCl, RbCl, and CsCl) at selected low levels. All
Accepted 5 October 2015
formulations were stored at 50 C and 65 C up to 2 months and then assayed for protein aggregation. The
Available online 30 December 2015
data demonstrate that low levels of LiCl and NaCl enhance stability. No obvious correlations with either
protein secondary structure or global dynamics (structural relaxation time) were found. However, good
Keywords:
correlations were found between stability and both free-volume hole size via positron annihilation
protein formulation
stability
lifetime spectroscopy (PALS) and fast dynamics by neutron scattering. Volume changes on mixing and
freeze drying the partial molal volume of salt were also studied in an effort to detect decreases in free volume. These
lyophilization data did not support the hypothesis that reduction in free volume was the primary mechanism for salt-
glass dynamics induced stabilization. Finally, a positive effect of postlyophilization annealing on stability was demon-
mobility strated. In summary, we find that small amounts of LiCl and NaCl significantly stabilize these proteins,
free volume which is a result at variance with conventional formulation wisdom.
PALS © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
mean square displacement
neutron scattering
http://dx.doi.org/10.1016/j.xphs.2015.10.004
0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541 531
metal chlorides, that is, LiCl, NaCl, KCl, RbCl, and CsCl. To include proteins, a result that is at variance with conventional formulation
divalent cations and anions was judged inappropriate for the pur- wisdom.
poses of this study because those ions often have protein-specific
interactions that may significantly impact protein conformation Materials and Methods
and therefore impact stability. For example, it has been reported
that the storage stability of phosphofructokinase has been Materials
improved by adding Mg2þ,12 and coagulation factor VIII, somato-
tropin, and human serum albumin are affected by Ca2þ, Zn2þ, and Bovine serum albumin, sucrose, potassium chloride, rubidium
Cu2þ, respectively.13 Anions such as I and SCN tend to impact chloride, cesium chloride, potassium phosphate, and sodium sul-
protein structure according to the Hofmeister series, at least in fate were purchased from Sigma-Aldrich (St. Louis, MO). Lithium
aqueous solution.14,15 Hence, we used the chloride anion and chloride, sodium chloride, sodium phosphate monobasic anhy-
monovalent cations, that is, Liþ, Naþ, Kþ, Rbþ, and Csþ, for the drous, sodium phosphate dibasic heptahydrate, and potassium
purpose of studying “general effects” of ions on protein stability in phosphate monobasic were purchased from Fisher Scientific
freeze-dried solids. (Pittsburgh, PA). Recombinant HSA in sterile ultrahigh purity grade
The secondary objective was to better understand the role of was a product of Albumin Bioscience (Huntsville, AL).
electrolytes in stabilization based on two hypotheses of protein
stabilization in disaccharide matrices; one commonly discussed Preparation of Freeze-Dried Protein Formulations
mechanism is a thermodynamic stabilization mechanism, acting
by stabilizing the native conformation by having the stabilizer BSA and rHSA were dialyzed against 5-mmol/L sodium phos-
hydrogen bond to the protein as does water and is termed the phate buffer at pH 7 using a membrane with MWCO: 6-8,000
“water substitution mechanism.” Here, stabilization results from (Spectrum Laboratories, Dominguez, CA). Protein concentrations
maintaining “native-like” structure. In the present work, corre- were determined by UV-VIS spectroscopy (Cary 50 Bio, Varian) at
lations between “structure” and stability were studied using 280 nm and then filtered by 0.22 mm polyether sulfone Millex-GP
infrared spectroscopy.20,21 The other is a purely kinetic stabili- (Millipore, Billerica, MA) to prepare the protein-buffered solu-
zation mechanism which acts by suppressing molecular mobility, tions of BSA and rHSA at 50 mg/mL and 5 mg/mL, respectively.
often denoted the “glass dynamics mechanism.”3,5 Mobility was Separately, sucrose and the electrolyte, that is, LiCl, NaCl, KCl, RbCl,
studied by evaluation of both “global mobility,” as represented by or CsCl, were dissolved and also filtered before mixing with the
the structural relaxation time measured by isothermal calorim- protein solution to prepare the protein-sucrose-electrolyte solu-
etry22,23 and by evaluation of “fast dynamics” by analysis of tions at 1:1 in weight ratio for protein:sucrose with various weight
neutron scattering.16,17 A number of protein systems have been fractions of electrolyte. All percentages described in this article
studied using neutron scattering, by which one can characterize were the weight fraction or mole fraction relative to total solute
the local dynamics in the glassy solid. Generally, it has been components. After dispensed into 5-mL tubing clear glass vials
found that this local dynamics or “fast dynamics” correlates well (Schott, Lebanon, PA) and semistoppered with Daikyo Fluoroteck
with storage stability of the protein in glassy systems.16-19 In stoppers (West Pharmaceutical, Lititz, PA), lyophilization was car-
addition, recently, high-precision density measurement for glassy ried out using a laboratory freeze dryer (Dura-Stop, SP Scientific).
materials was used to characterize the system free volume, which Samples were frozen by cooling to 40 C and holding for 1 h, fol-
is potentially a predictor of molecular mobility and therefore, lowed by setting the shelf temperature to 33 C and the chamber
stability.24,25 Good correlations were found between free volume pressure to 60 mTorr (8 Pa) for primary drying. Although, given the
(i.e., density) and storage stability.3 Thus, one might expect pre- collapse temperature (Tc) is usually about 2 C higher than the glass
0
cision density measurements may be useful in understanding the transition temperature of freeze concentrate (Tg ),40 primary drying
0
role of alkali metal salts in stabilization of proteins in the glassy conditions for each formulation were designed based on each Tg in
state. To interpret variations in density in terms of free volume, an attempt to maintain the product temperature below the
we applied specific volume-based approaches; (1) volume change assumed Tc, there were some cases where the product temperature
0
on mixing and (2) partial molal volume of solute (i.e., salt) to did increase above the Tg ; yet no sign of collapse or loss of quality
evaluate free-volume changes in glassy formulations on addition was observed even in the “worst case” of 9.0% LiCl-BSA and/or
0
of electrolyte. To directly measure size of the “holes” constituting sucrose formulation with Tg of 47 C, where a maximum product
free volume, a positron annihilation lifetime spectroscopy (PALS)
temperature of 37 C was observed. Such unconventional phe-
study26-35 was carried out. In addition, we investigated the effect nomena have also been observed in recent works.41,42 That is,
0
of “post-lyophilization-annealing,” which can be executed after collapse temperature and Tg can become high, and one may
the freeze-drying process by heating dried cakes to induce significantly exceed the collapse temperature measured by
structural relaxation36,37 or densification,25 resulting in freeze-drying microscopy and still not observe collapse when
enhancement of storage stability. This thermal treatment has freeze-drying in vials. Secondary drying was conducted at 40 C for
been found to be effective not only for stabilization of small 4 h before backfilling with nitrogen and sealing.
molecules36,38 but also for stabilization of an IgG protein,39 so it All samples freeze dried without visual collapse, and the water
was deemed of interest to determine if annealing had any impact content was determined to be less than 1% by Karl Fischer titration
on stability of formulations containing low levels of added salt. (756 KF coulometer, Metrohm).
As model proteins, bovine serum albumin (BSA) was selected The glass transition temperatures (Tg) and the changes in heat
because that is a well-investigated protein, and large amounts of capacity at Tg (DCp) were measured by Modulated DSC (Q1000, TA
protein were readily available for the experiments. Additionally, as Instrument) at 1 C/min with modulating ±0.5 C every 100 s.
a highly purified protein was desirable in the protein aggregation Samples were compressed into thin disks and placed in hermeti-
stability studies, recombinant human serum albumin (rHSA) was cally sealed aluminum pans. All sample handling was carried out in
used for the purpose of “validation” of the BSA results. Monovalent a dry bag continuously purged with dry nitrogen or air (relative
electrolytes in various amounts were colyophilized with a 1:1 humidity <3%). Glass transition temperatures were taken as the
weight ratio of protein to sucrose. We found that small amounts of midpoint of the heat capacity shift. To confirm amorphous before
LiCl and NaCl, that remain amorphous, significantly stabilize these and after storage, powder X-ray diffraction (PXRD; AXS D2 Phaser,
532 H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541
Bruker) was used because cubic crystal structures, such as sodium Table 1
chloride, do not show birefringence with polarized light Density of the BSA-Sucrose (1:1) Formulation
1.3849 0.0034
Stability Studies 1.3859 0.0010
1.4051 0.0026
1.3820 0.0011 1.3915 0.0036
The BSA samples were stored at 50 C and 65 C for 2 months and
1.3980 0.0012
1 month, respectively, whereas the rHSA samples were stored at 1.3930 0.0016
50 C for 1.5 months. Samples were taken at selected times for assay.
For the annealing study, freeze-dried rHSA samples were annealed
at 75 C for 13 h before storage, and stored at 50 C for 1.5 months
ampoule, which was tightly sealed to avoid moisture exposure. The
with nonannealed samples as a reference or “control.” Storage
sample ampoule was introduced into the calorimeter controlled at
stability was evaluated by determining the increase in protein
40 C and the heat flow was measured for 72 h. The modified
aggregate as a function of the storage time. Size exclusion chro-
stretched exponential (MSE) function was fit to the data instead of
matography was performed using a TSKgel G2000SWXL column
using the Kohlrausch-Williams-Watts (KWW) equation to avoid
(7.8 mmID 30 cm, 5 mm, Tosoh Bioscience) to determine the
errors near t ¼ 0.22,23 The MSE equation can be expressed for power
percentage of aggregate at each time point. Freeze-dried samples
(heat flow), P (mW/g), with time in hours, as follows.
were reconstituted with purified water and appropriately diluted
and filtered before injection. All reconstituted samples were visu-
ally clear after reconstitution. A mobile phase of sodium phosphate Tg T $DCp b$t t b2
P ¼ 277:8 $ 1þ $ 1þ
buffer at pH 7 with NaCl was used for BSA samples, whereas po- t0 t1 t1
" b1 #
tassium phosphate buffer at pH 7 with Na2SO4 was used for rHSA t t
analysis. The flow rate of 1 mL/min and the detection system by UV exp 1þ (2)
t0 t1
absorbance at 280 nm were used. The precision for a given sample
as well as between replicate samples was ±0.1%.
1=b ðb1Þ=b
tD ¼ t0 t1 (3)
Characterization of Protein Structure in Freeze-Dried Solid (Fourier
transform infrared spectroscopy [FTIR]) We evaluated values of effective KWW-stretched relaxation
time, tbD as this parameter is more robust and not as sensitive to
Secondary structure of the protein in the solid state was inves- systematic error as the effective KWW structural relaxation time,
tigated using a Nicolet Magna-IR system 560 spectrometer. Atten- tD.22
uated total reflectance (ATR) spectra were collected using a single
bounce, diamond crystal accessory because direct observation
without sample preparation is possible, and strong peak intensity is
obtained with a relatively small amount of sample.20 Freeze-dried
samples were moderately crushed under low-humidity condition Density Measurement by Gas Pycnometer
(<3% relative humidity) and quickly placed on the stage and
scanned 128 times at a resolution of 4 cm1. As the fast analysis Densities of freeze-dried samples were measured using an
time did not allow objectionable uptake of water, scans were run in AccuPyc 1330 helium pycnometer (Micromeritics, Norcross, GA) as
ambient condition. Raw spectra were baseline corrected using the described in previous literature.45 Calibration with a standard iron
Advanced ATR Correction.43 Second derivative spectra were created sphere of known volume followed by a verification run using
using OMNIC software (Thermo Nicolet, Madison, WI); the spectra crystalline sucrose of known density was performed before sample
were baseline corrected and area normalized over the amide I re- measurement for each day. The measured value was checked for
gion (1600-1700 cm1).20 The spectrum of native structure was consistency with the density of crystalline sucrose, 1.5854 ± 0.0048
evaluated by running the protein in a buffer solution. The similarity g/cm3.25 Otherwise, the sample cup which was dried overnight at
of spectra to the native was quantitated by using the area of overlap 65 C to completely eliminate moisture was exchanged, and the
method, as described in the literature.21 The width of the alpha runs were repeated. Once the accuracy of measurement was veri-
helix peak at half height, or full width at half maximum (FWHM), as fied, the sample powder was loaded in a sample cup, the inner and
a measure of structure that was recently suggested44 was calcu- outer surfaces of which were cleanly wiped to insure freedom from
lated based on curve fitting of the peak to the Gaussian function: particulates remaining. As freeze-dried materials were mostly
bulky and fluffy, repeated compression of the powder with a metal
( ) qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðx bÞ2 rod was needed to load the desired amount of the sample. After
when; f ðxÞ ¼ a$exp 2
; FWHM ¼ 2c$ 2$lnð2Þ; confirming all surfaces of the cup being clean, the sample was
2c
placed in the chamber where helium gas of 134 kPa (19.5 psig) was
(1) filled and then equilibrated till not more than 34 Pa/min (0.005
psig/min) to give a density value by taking an average of 20 mea-
where a, b, and c represent fitting parameters. surements. The density value for amorphous sucrose was obtained
as 1.4988 ± 0.0021 g/cm3, which was consistent with the published
Investigation of Global Glass Dynamics by the Thermal Activity data.25 In addition, the density of the “base” BSA-sucrose formu-
Monitor (TAM) lation (no salt) was measured six times for different vials. As
summarized in Table 1, we obtained the average value of
Enthalpy relaxation is a measure of global dynamics and was 1.3915 g/cm3, with standard error of 0.0036, which was the same
directly measured by isothermal calorimetry (TAM model 2277, precision as found in the previous study (standard error ¼ 0.0036
Thermometric, Sweden). Working in a dry box, samples of about for amorphous sucrose),25 suggesting that the measurement pro-
200 mg were appropriately crushed and put into the stainless steel cedures are reliable for our protein-sucrose materials.
H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541 533
0
Analysis of Density Values to Characterize Free Volume 0 vFv 0 0
V2 ¼ w2 $ þ Fv ; and when Fv
vw2
When comparing densities in a series of samples with variable 0 0
chemical composition with the intent to compare trends in free ¼ intercept þ slope$lnðw2 Þ; V2 ¼ slope þ Fv (7)
volume, much if not most of the density variation can arise from
If there is a loss of free volume by adding a salt, the apparent
variation in atomic density, particularly for systems containing
specific volume should be less than the molar volume of the pure
heavy metal atoms, such as cesium. To avoid the variable atomic
salt, which we take as the salt in the crystalline form. To generate
density problem, we use density data to evaluate volume changes
partial molal volume data, Equation 7 was used. The slope of the
on mixing and apparent molal volumes and examine these data for
linear plot of apparent specific volume versus ln(w2) was com-
trends that might suggest changes in free volume, as previously
bined with the value of apparent specific volume calculated from
discussed.45 With specific volume denoted as, v0, and given by the
0 the density (Eq. 6), to generate the partial specific volume. The
reciprocal of density, d, the volume change on mixing, Dvmix , may
partial molal volume is simply the product of the partial specific
be written,
volume and the molecular weight of the solute (i.e., the salt).
Uncertainties in the calculated partial molal volumes were esti-
X
0 0
Dvmix ¼ v0 v0 ¼ ð1=dÞ wi $ 1 di;0 (4) mated from the standard error in the slope and the standard
i
error in the calculated apparent specific volume, using standard
propagation of errors methodology. As previously noted by
Chieng,45 the relation between partial molal volume and volume
0
where, v0 is the sum of the specific volumes of the pure compo-
nents i, with weight fraction of wi and its density of di,0. The vol- change on mixing may be given by,
umes of the atoms themselves, or van der Waals volumes, will then
0
cancel when forming the difference between final volume of the 0 0 0 Dvmix
fv ¼ v2* þ v2;0
F
þ (8)
mixture and the sum of the volumes of the individual components w2
in their pure states. Therefore, the change in specific volume on
mixing of two components may be related to free volume, v0 F by, That is, the qualitative trends for apparent specific volume and
volume change on mixing must be the same.
0 0 0 0
Dvmix ¼ v F w1 $v1;0
F F
w2 $v2;0 (5)
0
Positron Annihilation Lifetime Spectroscopy Measurement
where, vi;0F denotes specific free volume of component i in pure
state, and thus, the free volume in the mixture may contain changes PALS can be used as a means of direct measurement of size of
in free volume of both components when mixed. If free volume is the free-volume “holes” in the amorphous solid. Recent studies
reduced in the mixture relative to sum of the pure components, the have been expanding applications of PALS for pharmaceutical
volume change on mixing should be negative. In our study, we take materials.29,31 The measurement is based on analyzing lifetime and
component 1 as a BSA-sucrose matrix (or “solvent”) and compo- intensity of positronium, which in principle are sensitive to size and
nent 2 as a salt (or “solute”). In addition, at least as a first approx- concentration of free volume in a material.28 PALS methodology
0
imation, we can neglect the v2;0 F because the pure salt is the and procedure of measurement were briefly discussed previ-
crystalline form with (nominally) essentially zero free volume. This ously.45 Each of the collected spectra was best fitted with 3 positron
means loss of free volume in the systems corresponds to the loss of lifetimes with the third lifetime and its intensity attributed to the
free volume of the protein-disaccharide matrix, which should annihilation of positronium. The mean pore diameter was calcu-
produce a less mobile matrix and therefore, perhaps provide sta- lated based on the Tao-Eldrup model which uses the assumption of
bilization. One might argue that we need to use the specific volume spherical microvacancies.26 The measurement was repeated 3
of the pure amorphous salt instead of the crystalline salt because times per formulation.
the salt is not crystalline in the mixture. However, such data are not
readily available. Using amorphous salt as the “baseline” would of
course systematically lower volume changes on mixing.
As another method to characterize free-volume changes from Neutron-Scattering Measurements
measured densities, we may evaluate the partial molal volume of
the “solute”, or in our case, the salt dissolved in the protein:sucrose The high-flux backscattering spectrometer at NIST Center for
solvent. The partial molal volume is the volume change of the Neutron Research was used to measure <u2> as a function of
system when one mole of solute is added to an infinite amount of temperature. In the neutron-backscattering experiment, samples
solvent. A comparison of the partial molal volume to the molar were probed by incident neutrons with wavelength of 6.271 Å
volume of pure salt then allows an assessment of loss of free vol- and kinetic energy of 2.08 meV with 0.85 meV full width at half
ume in the solvent on addition of solute. We may calculate apparent maximum energy resolution. Powders of freeze-dried samples at
specific volume of component 2 (i.e., salt) in the mixture, denoted approximately 400 mg each were spread onto aluminum foil
0
Fv , with total volume of the mixture (V), volume of component 1 in and then loaded into aluminum sample cells. The sample cells
pure state (V1,0), and mass of component 2 (m2). were hermetically sealed with indium o-rings and tightened
with aluminum screws. The procedure was performed inside of
a helium glove box at room temperature, where the relative
0 V V1;0 humidity was approximately 0.1% to prevent absorption of
Fv ¼ ; or when the total mass is 1 gram;
m2 moisture by lyophilized powders. The high-flux backscattering
spectrometer was operated in the fixed-window scan mode with
0 ð1=dÞ w1 , 1 d1;0
Fv ¼ (6) the samples cooled from 324 K (51 C) to 4 K at 0.7 K per
w2
minute. The elastic scattering intensity was analyzed to extract
0
The partial specific volume of salt ðV2 Þ is calculated from the <u2>, same as previously described.45 All samples were kept
apparent specific volume data from the relationship, at 20 C before use.
534 H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541
Figure 3. Effect of electrolyte on storage stability by plotting the rate constant against the weight fraction of electrolyte; (a) BSA-sucrose-electrolyte formulations stored at 50 C for
2 months, (b) BSA-sucrose-electrolyte formulations stored at 65 C for 1 month, (c) rHSA-sucrose-electrolyte formulations stored at 50 C for one and half a month; Error bars show
standard error of the rate constant. LiCl (diamond); NaCl (square); KCl (triangle); RbCl (filled circle); CsCl (open circle).
state is given by the data in Table 2. Note that the water contents are between the protein structure and the stabilizing effect of elec-
all very low, less than 1%, so water contenteinduced variations in Tg trolyte. As summarized in Table 2, there were no large differences
between samples are not significant in the context of this discus- between the electrolytes; the range of variation in the area of
sion. Although LiCl increased the Tg, the impact of NaCl is minimal. overlap was 0.74-0.82 for BSA formulations and 0.70-0.79 for rHSA
Other electrolytes reduced Tg, the amount of decrease depending formulations, with no obvious correlation with protein stability. As
on the weight fraction. For KCl at 9.1%, the midpoint Tg was well, the width at half height of the alpha helix peak band (FWHM
decreased to 74 C. Here, the onset Tg was 66 C, which is very close in Table 2) did not indicate any systematic relationship between the
to the storage temperature of 65 C. These trends may arise from conformation and the stability; the FWHM varied “randomly” in
charge density differences between the cations and thus reflect the the range of 12.1-15.2 cm1 for the different formulations.
impact of electrostatic interactions on Tg. It seems likely that the
poor stability of the KCl systems at 9% KCl and higher is a result of
Global Mobility by TAM
the proximity to the glass transition and the enhanced mobility at
these conditions. As previously demonstrated, Tg is a significant
The effect of electrolyte on global mobility below Tg has been
factor to be considered for design and interpretation of storage
studied in the BSA formulations. Stretched relaxation times ob-
stability studies.1,8 In the following discussions, we focus on con-
tained using the MSE equation are summarized in Table 3. Before
ditions where the formulations are stored well below Tg.
data analysis, it was confirmed that no crystallization of electrolyte
occurred during the measurement (i.e., at 40 C for 72 h). The un-
Secondary Structure of Protein by FTIR certainties given are standard errors evaluated from the replicate
runs, normally duplicates. Most of the differences are within the
By comparing the similarity of the protein structure in the solid sum of standard errors and are obviously not significant. The
to the native structure, the effect of electrolyte on the protein relaxation time showed no significant trend with increasing salt
conformation in the solid was investigated. Figure 4 shows second with only two possible exceptions. For both LiCl and KCl at 9%, there
derivative spectra for the rHSA formulations. Analysis of the spec- seemed to be a small decrease in relaxation time that corresponds
troscopic data and stability data did not reveal any relation to a small systematic increase in molecular mobility. However,
536 H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541
Table 2
Effect of Electrolytes on the Glass Transition Temperatures (Tg) and the Changes of Heat Capacity at Tg (DCp) for the Protein Formulation Including Sucrose and Different
Electrolytes
Formulation Electrolyte (%w/w) Tga ( C) DCpb (J/g$K) Area of Overlap FWHM (cm1)
Formulation Electrolyte (%w/w) Tgc ( C) DCpc (J/g$K) Area of Overlap FWHM (cm1)
these changes are very minor, as well as uncorrelated with stability, Figure 5a, particularly for LiCl, NaCl, and KCl. Because all values
likely due to scatter in the data, and we conclude that the stabili- must be zero at zero molal, the shape of the curve must actually
zation effect by electrolyte cannot be attributed to changes in global be “S-shaped,” with a maximum around 0.5 molal and a distinct
dynamics. Recent studies suggest that global mobility in the glass minimum around 1.5-2.0 molal, at least for LiCl, NaCl, and KCl. Of
matrix often does not correlate well with global mobility, at least course, uncertainty in the data is significant, and had we used
0
well below the Tg.3,5 amorphous densities for the pure salts to calculate Dvmix , the
entire curve would be displaced lower (amorphous densities are
Effect of Electrolyte on Density and Its Interpretation as Free-Volume not available). However, this complex shape with both a
Change maximum and minimum would not be changed and therefore
seems to be real. Evidently, a low concentration of salt introduces
Table 4 summarizes the density data, the volume change on some free volume, but then higher levels decrease free volume
mixing the salt with the base BSA-sucrose system, true, and the until the minimum. Part of the explanation of the trend past the
calculated partial molal volumes. As expected, density increased as maximum at about 0.5 mol may involve the concept of two types
the amount of salt increased because adding salt means adding of free volume, as discussed for polymers32-35; one type of free
atoms with high atomic density. volume is multidimensionally interconnected spaces, where the
To characterize changes in free volumes, we examined volume size becomes relatively large. This type of free volume is observed
change on mixing and partial molal volume. Volume changes on in polymer crystals and termed “interstitial free volume.” The
mixing are plotted against molality in Figure 5a. Volume change other appears as many discrete and irregularly shaped holes due
on mixing should be negative if the free volume of the protein- to disordered packing in the amorphous phase. That is usually
disaccharide matrix decreases with addition of salt. Values are called “local free volume,” also denoted as “holes.” The scale of
very small (within almost ±0.010 cc/g), with an apparent slight the local free volume is subnanometer, being too small to
0
“V-shape” trend. However, given the definition of Dvmix , this contribute significantly to the transport of whole molecules, but
value must approach zero as the concentration of added solute some holes can affect the mobility of chain segments of the
(i.e., salt) approaches zero. This appears not to be the case in polymer.32,33 We note that the local free volume has been studied
H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541 537
Table 3
Effect of Electrolyte on the Structural Relaxation Time in the BSA-Sucrose-Electrolyte
Formulation at 40 C, Best Fit by MSE Equation
Table 4
Effect of Electrolyte on Density, Volume Change on Mixing, and Apparent Specific Volume of Electrolyte, in BSA:Sucrose Formulations; the Measurement Was Repeated
At Least 2 Times
0 0
Formulation; BSA:Sucrose (¼1:1) :Salt Salt (wt%) Density (g/cm3) Dvmix (cc/g) Fv (cc/g)
Density of crystalline salt: LiCl, 2.07 g/cm3; NaCl, 2.17 g/cm3; KCl, 1.988 g/cm3; RbCl, 2.8 g/cm3; CsCl, 3.988 g/cm3.46
538 H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541
Figure 5. (a) Volume change on mixing versus molality (mol/Kg-solvent); LiCl (diamond); NaCl (square); KCl (triangle); RbCl (filled circle); CsCl (open circle). (b) Partial molal
volume of salt (symbols) and each molal volume of crystal (short bars); LiCl (diamond); NaCl (square); KCl (triangle); RbCl (filled circle); CsCl (open circle). The short heavy bars
represent the molal volume of the crystalline salts, in order of LiCl, NaCl, KCl, CsCl, and RbCl (from bottom to top).
NaCl, KCl, and RbCl are slightly less than crystalline molal volume, In Figure 7, the correlation of the PALS parameters with stability
but differences are almost within standard errors. The partial molal is illustrated. There is a good correlation of mean pore diameter of
volume for CsCl is much less than molar volumes of either glass or the free volume with storage stability (R2 > 0.96), and the corre-
crystal. Rate constant for degradation increases in the order, LiCl < lation is sensible, at least in the series of the salt systems studied.
NaCl z KCl < RbCl z CsCl, the latter two salts having essentially the However, no monotonic relationship was observed between in-
same degradation rate as the system without any salt. Thus, tensity and stability. As PALS gives a direct measurement of rela-
although in several cases, addition of salt causes loss in free volume, tively small free volume (subnanometer), PALS provides a different
particularly for CsCl, this salt has no impact on stability. Thus, it is assessment of free volume than what is provided by density. That is,
clear, from analysis of both the volume changes on mixing and the PALS counts not all “free volume,” and it is the size of the “holes”
partial molal volumes that the impact of free volume as assessed by that is measured. Density (nominally) measures all free volume
density measurements is not related in any simple way to stability. accessible to helium in the gas pycnometric measurement.
Although more extensive data are needed for a definitive conclu-
sion, it does appear that PALS data may provide meaningful infor-
Free-Volume Measurements by PALS
mation on free volume that is critically linked to long-term stability
in the glassy formulations.
Physical parameters collected from PALS measurement are
summarized in Table 5. The positron lifetime as well as the mean
diameter of the free-volume pores, both of which have the same Fast Dynamics by Neutron Scattering
trend because d3 was directly derived from t3, are given. The
standard error of the triplicate data was 0.02 ns for t3. The intensity Based on previous studies,3,45 we would expect a better corre-
data are given because intensity is approximately related to the lation between stability and fast dynamics, measured by neutron
number density of “holes.” Here, we assume, as is conventional, scattering, than with global dynamics, as measured by enthalpy
that the positron lifetime is not very sensitive to chemical sur- relaxation time. This is exactly what we observe. Results from the
roundings of the vacancy and mainly reflects free-volume pore neutron scattering experiments with the rHSA:sucrose system
sizes.30,31 Although the relationship can be complex and intensity without salt and with 7% LiCl or with 7% CsCl are given in Figure 8.
may be impacted by other factors as well,30 the qualitative trend The vertical axis gives the mean square amplitude of motion on a
itself of the lifetime would not be impacted. nanosecond timescale, mostly by hydrogen-bearing groups of
atoms, and the horizontal axis is temperature. It should be noted
that the formulation without salt and the formulation with CsCl
added have essentially the same fast dynamics by this measure, but
the formulation with added LiCl shows much lower mean square
amplitude of motion, particularly in the range of room temperature
to the storage temperatures used in the stability studies conducted
in this study. The meaning is simply that the addition of LiCl
dampens the fast dynamics, or antiplasticizes mobility on the
nanosecond timescale. Note that the stability data for BSA and rHSA
systems (Figure 3) are well correlated with the fast dynamics re-
sults. Addition of LiCl stabilizes but addition of CsCl either has no
systematic effect (BSA) or destabilizes somewhat (rHSA). The PALS
data (Table 5, Figure 7) also show a trend of hole size with these
Table 5
Positron Lifetime, Intensity, and Calculated Mean Diameter of Free Volume by PALS;
the Measurement Was Repeated Three Times (±0.02 ns)
Conclusions
Figure 7. Correlation of mean diameter of the local free volume with aggregation rate
constant. Open squares, intensity (I3 ÷ 5); filled circles, diameter (d3 3). Rate con- This study has demonstrated that the addition of monovalent
stants were estimated from those for 7% salt formulations.
electrolytes (alkali chlorides) to sucrose-based protein formula-
tions can improve storage stability. The variations of aggregation
systems that is consistent with the fast dynamics results and sta- rate could not be rationalized by either protein secondary structure
bility. That is, small hole size is associated with smaller mean using FTIR or global glass dynamics detected by TAM. Changes in
square amplitude of motion and better stability. Perhaps, the cor- free volume measured by volume changes on mixing and partial
relation with fast dynamics and/or PALS hole size data arises molal volumes of salt in the protein:sucrose systems suggested
because of differing electrostatic interactions due to different ion decreases in free volume for some systems, but those changes in
size or is a reflection of “optimal volume” in modifying free volume. free volume were not well correlated with the impact of the salts on
However, the detailed mechanism is not clear. stability. In an attempt to correlate stability with an alternate
measure of free volume, PALS measurements were carried out to
examine the correlation between stability and the mean diameter
Annealing Effect on Protein Stabilization in Salt Systems of free-volume holes, as calculated from the lifetime of positro-
nium. Although the data set is sparse, hole size was well correlated
After and/or during the drying process, samples that are held at with stability data in the series: LiCl, CsCl, and no salt formulation,
a given temperature, ambient or above but below Tg, can result in suggesting that the relatively small and local free volume as
significant “annealing effects,” thereby producing a significant
decrease in global mobility as well as significant stabilization
against degradation.39 The maximum annealing effect for mobility
measured by PALS hole size may be a good predictor of storage 16. Cicerone MT, Tellington A, Trost L, Sokolov A. Substantially improved stability
of biological agents in dried form: the role of glass dynamics in preservation of
stability in glassy formulations. Fast dynamics, as characterized by
biopharmaceuticals. Bioprocess Inter. 2003;1:36-47.
the mean square amplitude of motion on a nanosecond timescale, 17. Cicerone MT, Soles CL. Fast dynamics and stabilization of proteins: binary
is consistent with the PALS data and stability; small hole size from glasses of trehalose and glycerol. Biophys J. 2004;86:3836-3845.
PALS means smaller amplitude of fast dynamics motion and better 18. Yoshioka S, Miyazaki T, Aso Y, Kawanishi T. Significance of local mobility in
aggregation of b-Galactosidase lyophilized with trehalose, sucrose or sta-
stability. Additional data are needed to support this tentative chyose. Pharm Res. 2007;24:1660-1667.
conclusion. Additionally, annealing was observed to further 19. Zhong Q. Protein Stability in Sugar-Based Glass: Using Dynamics to Sort Causality
improve stability in protein-sucrose-salt systems. The observation From Correlation. Cambridge, MA: World Lyophilization Summit; 2011.
20. Carpenter JF, Prestrelski SJ, Dong A. Application of infrared spectroscopy to
that small amounts of LiCl and NaCl can significantly stabilize these development of stable lyophilized protein formulations. Eur J Pharm Biopharm.
proteins provides new insight into the impact of sodium chloride, 1998;45:231-238.
which is commonly used in biopharmaceuticals, on product quality. 21. Kendrick BS, Dong A, Allison SD, Manning MC, Carpenter JF. Quantitation of the
area of overlap between second-derivative amide I infrared spectra to deter-
mine the structural similarity of a protein in different states. J Pharm Sci.
1996;85:155-158.
Acknowledgments 22. Liu J, Rigsbee DR, Stotz C, Pikal MJ. Dynamics of pharmaceutical amorphous
solids: the study of enthalpy relaxation by isothermal microcalorimetry.
J Pharm Sci. 2002;91:1853-1862.
The authors acknowledge partial support from NIH/NIBIB under 23. Kawakami K, Pikal MJ. Calorimetric investigation of the structural relaxation of
grant R01 EB006398-01A1. The authors are grateful to Dr. Jack amorphous materials: evaluating validity of the methodologies. J Pharm Sci.
Gromek at the Institute of Materials Science (IMS) at University of 2005;94:948-965.
24. Imamura K, Maruyama Y, Tanaka K, Yokoyama T, Imanaka H, Nakanishi K. True
Connecticut for using PXRD and also would like to express appre-
density analysis of a freeze-dried amorphous sugar matrix. J Pharm Sci.
ciation to Dr. Norman Chieng and Dr. Puneet Sharma for discus- 2008;97:2789-2797.
sions. This work used facilities supported in part by the National 25. Kikuchi T, Wang B, Pikal MJ. High-precision absolute (true) density measure-
Science Foundation under Agreement No. DMR-0944772. Certain ments on hygroscopic powders by gas pycnometry: application to determining
effects of formulation and process on free volume of lyophilized products.
trade names and company products are identified in this article to J Pharm Sci. 2011;100:2945-2951.
specify adequately the experimental procedure. Such identification 26. Itoha Y, Shimazu A, Sadzuka Y, Sonobe T, Itai S. Novel method for stratum
does not imply recommendation or endorsement by the National corneum pore size determination using positron annihilation lifetime spec-
troscopy. Int J Pharm. 2008;358:91-95.
Institute of Standards and Technology, or does it imply that the 27. Townrow S, Kilburn D, Alam A, Ubbink J. Molecular packing in amorphous
instruments, materials, or suppliers identified are necessarily the carbohydrate matrixes. J Phys Chem. 2007;111:12643-12648.
best available for the purpose. 28. Xie W, Ju H, Geise GM, et al. Effect of free volume on water and salt transport
properties in directly copolymerized disulfonated poly(arylene ether sulfone)
random copolymers. Macromolecules. 2011;44:4428-4438.
29. Dlubek G, Shaikh MQ, Ratzkeb K, Pionteckc J, Paluchd M, Faupel F. Sub-
References nanometre size free volumes in amorphous Verapamil hydrochloride: a posi-
tron lifetime and PVT study through Tg in comparison with dielectric
1. Kasper JC, Winter G, Friess W. Recent advances and further challenges in relaxation spectroscopy. Eur J Pharm Sci. 2010;41:388-398.
lyophilization. Eur J Pharm Biopharm. 2013;85:162-169. 30. Tant MR, Hill AJ. Structure and Properties of Glassy Polymers. ACS Symposium
2. Wang B, Tchessalov S, Warne NW, Pikal MJ. Impact of sucrose level on storage Series. 710. Washington DC: ACS Books; 1998.
stability of proteins in freeze-dried solids: I. Correlation of protein-sugar 31. Zelko R, Orban A, Suvegh K. Tracking of the physical ageing of amorphous
interaction with native structure preservation. J Pharm Sci. 2009;98:3131-3144. pharmaceutical polymeric excipients by positron annihilation spectroscopy.
3. Wang B, Tchessalov S, Cicerone MT, Warne NW, Pikal MJ. Impact of sucrose J Pharm Biomed Anal. 2006;40:249-254.
level on storage stability of proteins in freeze-dried solids: II. Correlation of 32. Dlubek G, Piontech J, Kilburn D. The structure of the free volume
aggregation rate with protein structure and molecular mobility. J Pharm Sci. in poly(styrene-co-acrylonitrile) from positron lifetime and pressure volume
2009;98:3145-3166. temperature (PVT) experiments. I. Free volume from the Simha-Somcynsky
4. Wang W, Singh S, Zeng DL, King K, Nema S. Antibody structure, instability, and analysis of PVT experiments. Macromol Chem Phys. 2004;205:500-511.
formulation. J Pharm Sci. 2007;96:1-26. 33. Dlubek G, Bondarenko V, Al-Qaradawi IY, Kilburn D, Krause-Rehberg R. The
5. Chang L, Shepherd D, Sun J, Tang X, Pikal MJ. Effect of sorbitol and residual structure of the free volume in poly(styrene-co-acrylonitrile) from positron
moisture on the stability of lyophilized antibodies: implications for the lifetime and pressure volume temperature (PVT) experiments. II. Local free
mechanism of protein stabilization in the solid state. J Pharm Sci. 2005;94: volume from positron annihilation lifetime spectroscopy (PALS). Macromol
1445-1455. Chem Phys. 2004;205:512-522.
6. Katayama DS, Carpenter JF, Menard KP, Manning MC, Randolph TW. Mixing 34. Dlubek G, Stejny J, Lüpke TH, et al. Free-volume variation in polyethylenes of
properties of lyophilized protein systems: a spectroscopic and calorimetric different crystallinities: positron lifetime, density, and X-ray studies. J Polym
study. J Pharm Sci. 2009;98:2954-2969. Sci. 2001;40:65-81.
7. Osterberg T, Fatouros A, Mikaelsson M. Development of a freeze-dried albu- 35. Dlubek G, Pionteck J, Ratzke K, Kruse J, Faupel F. Temperature dependence of the
min-free formulation of recombinant factor VIII SQ. Pharm Res. 1997;14:892- free volume in amorphous Teflon AF1600 and AF2400: a pressure-volume-
898. temperature and positron lifetime study. Macromolecules. 2008;41:6125-6133.
8. Yoshioka S, Aso Y. Correlations between molecular mobility and chemical 36. Abdul-Fattah AM, Dellerman KM, Bogner RH, Pikal MJ. The effect of annealing
stability during storage of amorphous pharmaceuticals. J Pharm Sci. 2007;96: on the stability of amorphous solids: chemical stability of freeze-dried mox-
960-981. alactam. J Pharm Sci. 2007;96:1237-1250.
9. Jameel F, Tchessalov S, Bjornson E, Lu X, Besman M, Pikal MJ. Development of 37. Luthra SA, Hodge IM, Pikal MJ. Effects of annealing on enthalpy relaxation in
freeze-dried biosynthetic factor VIII: I. A case study in the optimization of lyophilized disaccharide formulations: mathematical modeling of DSC curves.
formulation. Pharm Dev Technol. 2009;14:687-697. J Pharm Sci. 2008;97:3084-3099.
10. Pikal MJ, Dellerman KM, Roy ML, Riggin RM. The effects of formulation vari- 38. Wang B, Pikal MJ. The impact of thermal treatment on the stability of freeze
ables on the stability of freeze-dried human growth hormone. Pharm Res. dried amorphous pharmaceuticals: I. Dimer formation in sodium ethacrynate.
1991;8:427-436. J Pharm Sci. 2010;99:663-682.
11. Her L, Deras M, Nail SL. Electrolyte-induced changes in glass transition tem- 39. Wang B, Cicerone MT, Aso Y, Pikal MJ. The impact of thermal treatment on the
peratures of freeze-concentrated solutes. Pharm Res. 1995;12:768-772. stability of freeze-dried amorphous pharmaceuticals: II. Aggregation in an IgG1
12. Carpenter JF, Crowe LM, Crowe JH. Stabilization of phosphofructokinase with fusion protein. J Pharm Sci. 2010;99:683-700.
sugars during freeze-drying: characterization of enhanced protection in the 40. Pikal MJ, Shah S. The collapse temperature in freeze-drying: dependence on
presence of divalent cations. Biochim Biophys Acta. 1987;923:109-115. measurement methodology and rate of water removal from the glassy state. Int
13. UniProtKB, protein knowledgebase. Available at: http://www.uniprot.org/ J Pharm. 1990;62:165-186.
uniprot/. Accessed December 2, 2015. 41. Nunn S, Nishikida K. Advanced ATR Correction Algorithm. Waltham, MA: Thermo
14. Broering JM, Bommarius AS. Evaluation of Hofmeister effects on the kinetic Fisher Scientific Inc.; 2008. Application note: 50581.
stability of proteins. J Phys Chem B. 2005;109:20612-20619. 42. Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. Systematic
15. Zhang L, Tan H, Fesinmeyer R, et al. Antibody solubility behavior in monovalent investigation of the effect of lyophilizate collapse on pharmaceutically
salt solutions reveals specific anion effects at low ionic strength. J Pharm Sci. relevant proteins I: stability after freeze-drying. J Pharm Sci. 2010;99:2256-
2012;101:965-977. 2278.
H. Goshima et al. / Journal of Pharmaceutical Sciences 105 (2016) 530e541 541
43. Tchessalov S, Dixon D, Warne N. 2010. Lyophilization Above Collapse. WO 45. Chieng N, Cicerone MT, Zhong Q, Liu M, Pikal MJ. Characterization of dynamics
2010017296 A1. in complex lyophilized formulations: II. Analysis of density variations in terms
44. Xu Y, Carptenter JF, Cicerone MT, Randolph TW. Contributions of local mobility of glass dynamics and comparisons with global mobility, fast dynamics, and
and degree of retention of native secondary structure to the stability of re- PALS. Eur J Pharm Biopharm. 2013;85:197-206.
combinant human growth hormone (rhGH) in glassy lyophilized formulations. 46. National Center for Biotechnology Information. The PubChem Project. Available
Soft Matter. 2013;9:7855-7865. at: http://pubchem.ncbi.nlm.nih.gov. Accessed December 4, 2015.