ARTICLES

Freezing-Induced Phase Separation and Spatial Microheterogeneity in Protein Solutions

Jinping Dong,†,§ Allison Hubel,‡ John C. Bischof,‡ and Alptekin Aksan*,‡

Characterization Facility, Institute of Technology, UniVersity of Minnesota, Minneapolis, Minnesota 55455, andMechanical Engineering Department, 111 Church Street Southeast, UniVersity of Minnesota, Minneapolis,Minnesota 55455

ReceiVed: NoVember 3, 2008; ReVised Manuscript ReceiVed: May 11, 2009

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complexorganisms have not yet been fully established. One major bottleneck is the inability to probe into and controlthe molecular level interactions. The molecular interactions are responsible for the significant differences inthe outcome of the preservation processes.1 In this communication, we have utilized confocal Ramanmicrospectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid)in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), whichexperienced different degrees of supercooling. Detailed quantitative spectral analysis was performed acrossthe ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was establishedthat the characteristics of the microstructures observed after freezing depended not only on the concentrationof trehalose in the solution but also on the degree of supercooling. It was shown that, when samples werefrozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase.Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase.Also, it was observed that induction of freezing after a high degree of supercooling of high trehaloseconcentrations resulted in aggregation of the sugar and the protein.

Introduction

The worldwide market for the 75 FDA approved therapeuticproteins is expected to reach $70 billion by the end of 2008.Currently, approximately 500 therapeutic proteins are in thedevelopment or approval stages to be used in the treatment ofvarious conditions ranging from cancer (e.g., monoclonalantibodies and interferons),2 heart attack and stroke (e.g., growthfactors and antiplatelet factors),3 anemia (erythropoietin toimprove red blood cell production),4 and hemophilia (bloodclotting factor VII).5 Proteins also find widespread use asbiocatalyzers, and in bioreactors and biosensors.6-9

The very high costs associated with the production, isolation,and purification of the therapeutic proteins necessitate that theproteins should be successfully stabilized at high concentrations,and stored with minimum loss of activity.10-12 The majority ofthe therapeutic proteins are stabilized and preserved by drying,freezing, or freeze-drying.13-17 Stabilization of a protein byfreezing and freeze-drying involves addition of buffering andstabilization/preservation agents into the solution,16,18,19 andremoval/separation of the liquid water from the solution in theform of ice. The presence of chemical agents in the solutionand the freezing/freeze-drying processes change the chemicalpotential of water, which directly alters protein structure.20 Thestructure of the protein is directly related to its activity after

reconstitution. Aggregation, gelation, and cold-denaturation areexamples of processing and storage-induced physical changesthat result in the loss of protein stability, and therefore loss ofits activity.11,21

Freezing of water induces solute rejection, creating regionsof high solute concentration. Freezing-induced partitioning ofthe solution into different thermodynamic phases (an ice phaseand a freeze-concentrated liquid (FCL) phase) induces segrega-tion of the protein, exposing it to different microenvironmentalconditions within the same medium. The local microenviron-ment that the protein is exposed to continues to evolve as themedium is cooled further and eutectic or crystalline phases formor the FCL region vitrifies. A similar microsegregation phe-nomenon is also observed during room-temperature desiccationof protein solutions, even though the physical mechanisms aredifferent.22

Phase separation and microsegregation in protein solutionsduring desiccation, freezing,23 and lyophilization24 has beenpreviously studied in bulk solutions, mainly by thermal, kinetic,and calorimetric analyses,12,25-30 X-ray diffractometry,31 andinfrared (IR) spectroscopy30,32,33 of the bulk product. Theultrastructure of the processed products has been visualized usingscanning electron microscopy (SEM).34,35 However, it is yet notknown how the protein is distributed among the different regionsin the frozen medium, and how its structure is affected by thefreezing-induced partitioning and temperature dependent evolu-tion of these different regions.12,13

Simply, the question we set to answer is: “In a frozenmedium, is the protein even “seeing” the stabilization agent tobenefit from its protection?” Previous studies used SEM or

* Correspondingauthor.E-mail:aaksan@me.umn.edu.Phone:612.626.6618.Fax: 612.624.5230.

† Characterization Facility.‡ Mechanical Engineering Department.§ Present address: Characterization Facility, Institute of Technology, 12

Shepherd Laboratory, 100 Union St. S.E., Minneapolis, MN 55455.

J. Phys. Chem. B 2009, 113, 10081–10087 10081

10.1021/jp809710d CCC: $40.75 2009 American Chemical SocietyPublished on Web 07/02/2009

electron spectroscopy for chemical analysis (ESCA) to answerthis question in dried and freeze-dried protein formulations anddemonstrated that protein and the stabilizing agents frequentlydo not colocalize.17,22 In this study, we employed confocalRaman microscopy (CRM) with frozen protein formulations toobtain 3-D high spatial resolution (∼200-300 nm) chemicalmaps of the system to quantify the extent of microsegregationof proteins into the coexistent thermodynamic phases formedduring freezing. We measured the spatial distribution of theexcipients in the frozen medium, identified the conditions thatyield to aggregation, and determined the changes in thesecondary structure of the model protein as a function of thefreezing protocol applied and the composition of the medium.

Results and Discussion

Isotonic Saline Solution. Buffers such as phosphate bufferedsaline solution (PBS), divalent cations (Zn2+), as well as basicamino and dicarboxylic acids have commonly been used in thecryo-/lyopreservation formulations developed for proteins, vac-cines, as well as micro-organisms and mammalian cells.16,19,36,37

However, it is also known that sodium and potassium-basedbuffers, upon freezing, may destabilize proteins38,39 due tocrystallization of the buffer and the significant change of pH.Therefore, one should be extremely cautious when using a bufferwith a high concentration of sodium-based salts, although, inpractice, PBS is used very commonly in lyophilized biophar-maceutical products.16

Solidification patterns of the PBS solution are complex(Figure 1) and continue to evolve with time as ice crystalscoalesce and grow (white arrows in Figure 1). Two characteristicpeaks located in the ν-OH band (2600-3600 cm-1) of theRaman spectra were used to identify the thermodynamic phaseof the water (solid vs liquid) within the frozen sample at veryhigh spatial resolution (lateral plane, ∼200-300 nm; verticalplane, ∼500-600 nm). The peak characteristic of liquid waterwas centered at 3430 cm-1, and the sharp peak located at 3145cm-1 corresponded to ice.40 The spatial change in the ratio ofthe integrated areas under these two peaks was used to generatethe 2-D CRM images that were used in the analysis. Note thatthe laser that was used in Raman spectroscopy is of low power(∼2mW) and the integration time (laser exposure at each pixelscanned) was less than 0.1 s for image acquisition and less than1 s for spectral analysis. Therefore, heating of the sample bythe laser was not expected. However, we also conductedexperiments where a single spot inside the ice domain of thesample was exposed to prolonged irradiation (>10 min). Nospectral changes indicative of temperature or phase changeeffects were observed. Note that the spectral characteristics ofthe Raman peaks of water change with temperature and also

with phase (ice spectrum is different than that of liquid waterat the same temperature).41 However, we did not observe anychange during prolonged exposure studies.

In the spontaneously frozen 1×PBS solution (i.e., highsupercooling (HS), with Tfreezing ∼ -20 °C), large ice crystals(daverage ∼ 10-20 µm) with a relatively uniform size distributionwere observed (Figure 1). The smooth, round boundaries of theice crystals indicated spherulitic morphology characteristic ofthe hexagonal ice phase formed under equiaxed freezingconditions.42 The crystals were surrounded by narrow channelsand lacunae that were devoid of ice (i.e., the FCL regions). FCLregions were formed as a result of the depression of the freezingtemperature of the solution, which now had a higher solutecontent than the original solution due to the presence of thesolutes rejected by the nucleating and growing ice phase. Whenthe frozen 1×PBS solution was held at a constant temperature(T ) -26 °C), the ice and the FCL regions rearranged slowlywith time: ice crystals coalesced (white arrows in Figure 1),while the distinct FCL regions decreased in number butincreased in size. The recrystallization process continued forapproximately 60 min at T ) -26 °C.

The 1×PBS solutions manually nucleated at T ) -3 °C(freezing with low supercooling (LS)) and cooled down to T )-26 °C (dT/dt ) 10 °C/min) showed phase separation similarto what was observed in the HS samples. However, the icecrystal size distribution was much broader (daverage ∼ 2-25 µm).Luyet and Rapatz42 report similar observations that in frozenaqueous solutions the resulting crystalline microstructures areinfluenced by the composition of the solution, thickness of thesolution layer, and the freezing temperature.

Low Trehalose Concentration Lysozyme Solutions (100mg/mL TRE:20 mg/mL LYS). For the majority of proteins,successful stabilization (by freezing, freeze-drying, vitrification,or desiccation) requires the use of specific stabilizing agents.10-12

These agents preserve the protein structure, eliminate/minimizeaggregation, and ensure protein activity after reconstitution.13-15,43

Sugars are commonly used to improve protein stability,44,45 andthere has been considerable interest specifically in the nonre-ducing disaccharide trehalose (TRE) as a stabilizing agent.46-48

The thermodynamic mechanism of stabilization offered bysugars such as TRE has been established.49 Recent studies havedemonstrated that the addition of TRE to a conventionalcryopreservation solution can change the interfacial free energyof the ice/cell/liquid system and influence partitioning of cellswith respect to the solid phase.50

In order to quantify the effect of TRE on the solutionfreezing behavior and phase separation, 1×PBS solutionscontaining TRE and a model protein, lysozyme (LYS), wereeither allowed to spontaneously freeze during cooling (HS)

Figure 1. CRM images of frozen 1×PBS solution (HS: high degree of supercooling). Scale bar ) 5 µm.

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down to T ) -26 °C (dT/dt ) 10 °C/min) or initially seededwith ice at T ) -3 °C (LS) and then cooled down to T )-26 °C (dT/dt ) 10 °C/min). Spontaneously frozen (HS)TRE-LYS solutions formed irregular ice crystals with abroad size distribution (daverage ∼ 2-20 µm; Figure 2, A1).The total concentration of the organic matter (TRE + LYS)in different regions within the frozen samples was determinedusing the integrated intensities of a group of peaks locatedat 2900-3000 cm-1 (Figure 3), which correspond to the ν-CHbands originating both from the protein and the sugar.51 Inthe long tortuous channels separating the ice domains (FCLregion), the liquid water peak (3430 cm-1) showed very lowcontrast relative to the ν-CH peak (Figure 2, A1), indicatingthe presence of a very low concentration of liquid water inthis phase as compared to the unfrozen solution. Note that,if we assume that the phase diagram of 8% w/w NaCl solutionis similar to that of 1×PBS, in the presence of >100 mg/mLTRE at T ) -26 °C, under equilibrium conditions, therewould only be ice and FCL phases present in the system,which would be devoid of any crystallized salts.

In addition to the tortuous channels separating the icedomains, there were round lacunae located within the icedomains where high concentrations of TRE and LYS ac-cumulated (white arrows in Figure 2, A1). It was determinedby scanning several layers in the vertical direction that the roundinclusions were very small (as compared to the tortuouschannels) with a vertical dimension of 1-2 µm (which was

similar to their lateral dimensions), suggesting that they hadspherical geometry. In the HS TRE-LYS samples, timedependent coalescence of ice crystals, which was observed inthe frozen 1×PBS solution, was absent and no significant changewas observed in the microstructure over a 60 min period oftime. However, in some prolonged experiments (>4 h), it wasfound that the tortuous channels slowly broke into smallersegments and eventually transitioned to small lacunae.

A high resolution Raman scan across the tortuous channelbetween the two ice regions (along the white line shown in

Figure 2. CRM images and line scan analyses of frozen TRE + LYS in 1×PBS solution. Column A, 20 mg/mL LYS + 100 mg/mL TRE solution(HS); column B, 20 mg/mL LYS + 100 mg/mL TRE solution (LS); column C, 20 mg/mL LYS + 300 mg/mL TRE solution (HS); column D, 20mg/mL LYS + 300 mg/mL TRE solution (LS). Row 1: CRM images of the samples. Row 2: Spatial variation of ice concentration (dashed line),TRE hydration (solid blue line), and LYS/TRE concentration ratio (solid red line). Row 3: Spatial variation of LYS R-helix (solid red line) and�-sheet (solid blue line) content, and LYS hydration (solid green line). Note: The white line in the first image shows the location of the line scanin the first sample (HS, high degree of supercooling; LS, low degree of supercooling).

Figure 3. Typical Raman spectra collected in the ice, FCL, and ice/FCL interface regions in a frozen 20 mg/mL LYS + 100 mg/mL TREsolution (HS).

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Figure 2, A1) was performed in the HS samples to analyze thecompositions of the ice and the FCL regions as well as theinterface between them (Figure 3). The spatial distribution ofLYS with respect to TRE in different regions was assessed bymeasuring the ratio of the integrated area of the tryptophan peaklocated at 1552 cm-1 to that of the C-C-C ring deformationpeak of TRE located at 536 cm-1.52 It was determined that theice regions (even outside the lacunae) were not composed ofpure water but contained low concentrations of LYS and TRE.This was evident from the characteristic peaks of LYS and TREobserved in the spectra obtained in the ice region (Figure 3).Additionally, it was observed that the LYS/TRE ratio was 2-4times higher in the ice phase (Figure 2, A2), which indicatedthat, during ice nucleation and growth, the ice phase preferen-tially rejected more TRE than LYS. Entrapment of LYS andTRE in the ice phase is very unusual. Under equilibriumconditions, the partition coefficient (defined as the concentrationof a given solute in the solid phase divided by the concentrationof a given solute in the liquid phase) of most solutes in the icephase is extremely small (∼10-6, see Korber et al.53 for areview). On the other hand, the presence of detectable levels ofLYS and TRE in ice is consistent with rapid ice interfacepropagation, resulting from the high supercooling.54 Rapidpropagation of the solid/liquid interface has been associated withan increase in the partition coefficient and, correspondingly, anincrease in the concentration of solutes (in this case, LYS andTRE) in the solid phase.55 A previous study also showed thatthe inclusion of solutes in the ice was dependent on the icegrowth rate and the direction of the thermal gradient. Eventhough it was not possible by CRM analysis to preciselydetermine how TRE and LYS molecules were distributed inthe ice phase, it is highly likely that both solutes were occludedin the ice phase in very small lacunae. Generally AgCl or similarmolecules are capable of being incorporated into the ice lattice,56

and the complex structures and large sizes of LYS and TREmake integration into the ice lattice unlikely.

To quantify the absolute concentrations of TRE and LYS ineach region (FCL and ice phases), the changes in the peakintensities at 1552 cm-1 (for LYS) and 536 cm-1 (for TRE)with concentration were calibrated using spectra obtained frombinary and ternary solutions containing 20-400 mg/mL TREand/or 20-100 mg/mL LYS. The LYS/TRE ratio in each phasewas calculated using area-averaged spectra collected from theFCL and ice regions. The volume of each phase was estimatedfrom the areas in the 30 × 30 µm2 CRM images (first row inFigure 2). Table 1 lists the average concentrations of TRE andLYS in the ice and FCL regions for all conditions tested. In the100 mg/mL TRE HS samples, the concentration of TRE in the

FCL region was 705.6 mg/mL (7 times its concentration inthe solution), while LYS was 133.1 mg/mL (approximately 6.7times more concentrated in FCL). The measured concentrationof TRE in FCL was in the lower end of the range (705-833mg/mL) reported in the literature for frozen TRE solutions.57

This was attributed to the presence of LYS in our experimentalsystem. In the ice phase, the TRE concentration was about 1/10

of its concentration in the solution, while LYS was about 1/7

less concentrated (Table 1).Depending on their immediate microenvironment (i.e., en-

trapped in the ice region or in the FCL region), TRE and LYSpresented significantly different spectral features than those insolution. The major differences were observed at the two doubletpeaks located at 1440-1460 and 1060-1080 cm-1, whichcorresponded to the δ-CH2 and the combination ν-C-O, ν-C-C,and δ-COH vibrations, respectively.58 The doublet at 1450 cm-1

has contributions from both TRE and LYS; therefore, the relativeintensities of the Gaussian peaks in the doublet located at 1070cm-1 were used to quantify the hydration level of TRE. Thereare different definitions of the “hydration level” used in theliterature. The definition we adopted is the one offered byKacurakova and Mathlouthi,58 where the hydration level isevaluated using the ratio of the 1080 cm-1 peak (ν-C-C andδ-COH) to the 1060 cm-1 peak (ν-C-O). In a separate study,we have also confirmed the hydration sensitivity of thesedoublets using FTIR spectroscopic analysis of concentratedsugar solutions at low temperatures.59

Figure 2, A2 (blue line, solid circles), shows the variation inTRE hydration (degree of hydrogen bonding) across the ice andFCL phases (ice concentration is shown as the dashed line).Overall, TRE was approximately 3 times more hydrated in theFCL region than in the ice phase. The high hydration level ofTRE in the FCL region combined with the observation of alower LYS/TRE ratio in the same region point to three distinctpossibilities: (1) preferential binding of TRE to the remainingunfrozen water molecules (which is in accord with the prefer-ential exclusion hypothesis,60,61 (2) increase in the TRE-TREintermolecular interactions in the FCL region, and/or (3)preferential binding between the TRE and LYS (which is inaccord with the water replacement hypothesis62).

To determine the responsible phenomena, we quantifiedthe changes in the secondary structure of LYS within differentregions. The peak at ca. 1260 cm-1 in the amide III regionoriginates from R-helical structures, while the peak at ca.1238 cm-1 originates from �-sheet structures.52,63 Figure 2,A3, shows the variation in the R-helix (the red line, the opensquares) and the �-sheet (the blue line, the full triangles)content of LYS across the different regions (note that theice concentration profile is the same as that shown by thedotted line in Figure 2, A2).

With reduction in the hydration level of LYS (as seen in theice region), the R-helix content decreases, while the �-sheet andthe random coil contents increase.63,64 The R-helix and �-sheetcontents of the LYS changed considerably between differentregions, with the R-helix content reaching its maximum (show-ing native-like secondary structure) in the FCL region. Thecorresponding drop in the �-sheet content in the FCL regionsupported this observation. Increase in the �-sheet content ofLYS during freezing and desiccation was confirmed in parallelexperiments performed with FTIR spectroscopy (data notshown) and therefore was attributed to the loss of native-likestructure. The native-like structure of LYS in the FCL regioncould be attributed to the absence of ice, and a lower LYS/TRE ratio, and possibly to a specific interaction of TRE with

TABLE 1: Average Concentrations of TRE and LYS inDifferent Phases

concentration (mg/mL)

solutionfreezing

condition soluteFCL

regionice

region Ka

100 mg/mL TRE + HS LYS 133.1 3.1 0.02320 mg/mL LYS TRE 705.6 9.5 0.0135

LS LYS 143.7 4.7 0.033TRE 778.6 16.1 0.021

300 mg/mL TRE + HS LYS 40.1 9.2 0.2320 mg/mL LYS TRE 729.3 68.9 0.09

LS LYS 51.3 3.9 0.078TRE 790.6 47.3 0.06

a The partition coefficient; K ) concentration in ice/concentrationin FCL.

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LYS (by either exclusion or binding), preserving its structure.Previous reports in the literature show that LYS secondarystructure is relatively stable during temperature change but issensitive to the change in the hydration state of its sidechains.65,66 To further quantify the difference in the hydrationlevels of LYS in different regions, hydration of LYS wasanalyzed by plotting the intensity ratio of the peaks at 1032cm-1 (the phenyl ring of the phenylalanyl residue) and 1013cm-1 (the tryptophanyl residue).52 The higher the intensity ratioof the peak located at 1032 cm-1 to that at 1013 cm-1, the higherthe hydration level of LYS.67 The analysis revealed that LYSwas 2-3 times more hydrated in the FCL phase than in the icephase (green line in Figure 2, A3, full circles).

In the LS samples cooled down to T ) -26 °C after manualice seeding at -3 °C (Figure 2, B1), the ice crystals were veryuniform, and exhibited a plate-like geometry with a high degreeof anisotropy (with dimensions of 5-10 µm by 10-20 µm inthe x-y plane and much smaller dimensions in the verticalplane). These structures were stable and did not exhibitsignificant coarsening/coalescence over a 60 min period whenthe samples were kept at T ) -26 °C. As expected, TRE andLYS were concentrated in the FCL phase between the icecrystals. In the ice phase, the absolute concentrations of bothLYS and TRE were about 1.5 times higher than those measuredin the HS samples (Table 1). These results showed that moreLYS and TRE were entrapped in the ice phase in LS samplesthan HS. This is counterintuitive, since increasing the temper-ature at which the solution nucleates reduces the interfacevelocity of the ice crystal during the initial nucleation/crystalgrowth phase, reducing the effective partition coefficient of thesolutes. However, in this case, the partition coefficient increasedwith lower supercooling. In the LS samples, LYS structuralvariation across different regions (red and blue curves in Figure2, B3) followed a similar trend as the HS samples with a native-like structure in the FCL region. The only significant differencebetween the two freezing protocols was that the overall hydrationlevel of LYS was lower in the LS samples (green curve in Figure2, B3).

High Trehalose Concentration Lysozyme Solutions (300mg/mL TRE:20 mg/mL LYS). In order to quantify the effectsof increasing the sugar concentration in the solution, freezingexperiments were carried out with sugar/protein solutions thatcontained a higher concentration of TRE (300 mg/mL). In theHS samples, relatively uniform and plate-like ice crystals wereobserved. However, these crystals were much smaller in size(∼2 × 5 µm, Figure 2, C1) than those obtained in the low TREconcentration solutions (∼5 × 15 µm, Figure 2, B1). Contraryto the relatively homogeneous distribution of TRE and LYS inthe FCL region that was observed in the low TRE solutions, at

high TRE concentrations, small aggregates of pure organicmatter were observed in the FCL region (white arrows in Figure4B). Analysis at a higher spatial resolution (Figure 4C) showedthat these aggregates containing TRE and LYS preferentiallyconcentrated near the ice/FCL interface (white arrows in Figure4C). Note that Figure 4D shows the total solute (TRE + LYS)concentration profile along the line shown in Figure 4C.Aggregation of LYS indicates a higher degree of denaturation,which is often irreversible due to the presence of high levels ofnon-native, intermolecular �-sheet structures.68 Accumulationof solutes at the ice/FCL interface has previously been observedby transmission electron microscopy of bacteria in a freeze-dried substrate.69

Similar to those observed in the low TRE solutions, the LYS/TRE ratio (red curve in Figure 2, C2) was higher in the icephase (Table 1). The hydration levels of TRE in both the iceand FCL phases were lower than those in the low TREconcentration cases (blue curve in Figure 2, C2). This findingimplied that TRE hydration depended mainly on its specificinteractions with LYS. The protein structure in the ice phasehad significantly higher �-sheet content (blue curve in Figure2, C3) even though the LYS/TRE ratio was lower (more TREmolecules should be available per LYS molecule). Similarly,the hydration level of LYS was lower in the ice and higher inFCL regions (green curve in Figure 2, C3).

As compared to the high TRE HS samples, in the high TRELS samples, larger (4 × 15 µm) plate-like ice crystals with amore uniform distribution were observed (Figure 2, D1).However, the distribution of TRE and LYS in the FCL regionwas more uniform without any significant aggregation at theice/FCL interface (red curve in Figure 3, D2). The LYS/TREdistribution was significantly different with a higher concentra-tion found in the center of the ice phase, which graduallydecreased toward the interface. The more gradual change in theLYS/TRE concentration across all regions measured in the LSsamples could be attributed to the functional difference in thetemperature dependence of the partition coefficients of the twosolutes.

Conclusion

Solidification Microstructures. The solidification micro-structures observed in the presence of TRE and LYS exhibiteda high degree of anisotropy in contrast to the spheruliticmorphology observed in the frozen 1×PBS solutions. Low TREconcentration solutions that were allowed to freeze spontane-ously (HS) had the most irregular ice shapes (non-plate-likestructures) and a wide size distribution. As the TRE concentra-tion in the solution increased, the ice crystal size became smaller;the ice crystals became plate-like with a more uniform shape

Figure 4. (A, B) CRM images of frozen 20 mg/mL LYS + 300 mg/mL TRE solution (HS). (C) High resolution CRM image (the white squarein image B). (D) Line scan (across the black line in image C) of the same solution showing aggregation of TRE and LYS in the FCL region.

Freezing-Induced Phase Separation J. Phys. Chem. B, Vol. 113, No. 30, 2009 10085

and size distribution. Previous studies have shown similarphenomena of lyophilization-induced phase separation usingSEM.35

Purity and Homogeneity of the Phases. The ice phase wasnot pure, but low levels of TRE and LYS were present in theice crystals either in the form of large lacunae or smallerinclusion bodies. The LYS/TRE distribution was relativelyuniform in the FCL regions (except in the 300 mg/mL TREHS solution where organic aggregates formed). However, thedistribution of TRE and LYS hydration was not uniform,reaching their highest levels at the center of the tortuous FCLchannels. This might be attributed to the relatively heterogeneousdistribution of water even in the FCL region (see the ice profilesin Figure 2, A2-D2). Note also that the LYS/TRE distributionwas significantly lower in the high TRE samples across allregions.

With freezing, the ice phase preferentially rejected solutes;however, the concentration of TRE entrapped in the ice phasewas higher in all of the solutions tested. Since the size of TREis much smaller than LYS, this could only be explained bypreferential ice-LYS interactions. It is known that LYS has avery high surface affinity toward air/liquid interfaces.70 Itssurface adsorption kinetics are also affected by the specific sugartype and its concentration in the solution.71,72 A similar affinityfor the ice interface might have caused the increased LYSconcentration within the ice region.

Hydration of Molecules and Structure. The hydration levelof TRE in the FCL phase decreased with increasing TREconcentration in the solution (Figure 2, A2-D2). Presumably,at high concentrations, TRE preferentially interacted with LYS(which was obvious in the spontaneously frozen samplesforming the aggregates) to satisfy hydrogen bonding require-ments. The higher native-like structural content of LYS in theFCL region also shows that TRE interacts more with LYS,preserving its structure. A direct correlation among TRE andLYS hydration levels and the R-helix content of LYS strengthensthis conclusion.

In this research, we have utilized CRM with frozen solutionsto determine the effects of freezing kinetics on the segregationbehavior of proteins among different coexisting thermodynamicphases. Enhancing our understanding of phase separation andthe effects of the interactions between the protein and the iceinterface on protein structure and aggregation will permit us toimprove the stabilization of proteins. The findings presented inthis research open new avenues for exploration not only tounderstand the mechanisms of biostabilization but also forprotein isolation and purification processes and developmentof highly concentrated, stable therapeutic protein suspensions(which currently presents a road block for the medical advancesbased on protein therapies73).

Materials and Methods

Hen egg white lysozyme was purchased from Sigma (Sigma-Aldrich Corp., St. Louis, MO). High purity trehalose dihydratewas purchased from Pfanstiehl (Ferro Pfanstiehl LaboratoriesInc., Waukegan, IL). Other chemicals were purchased fromSigma and used as received.

Confocal Raman microspectroscopy (CRM) was conductedwith a WITec Alpha 300R confocal Raman microscope (WITecInstruments Corp., Germany). The Raman microscope wasequipped with a UHTS200 spectrometer and a DV401 CCDdetector. A 100× Nikon air objective (NA ) 0.90) was usedfor all measurements. A 514.5 nm AR-ion laser at a maximumpower of 50 mW (operated at 2 mW) was used for excitation.

High throughput optics of the microscope enabled 3-D confocalchemical mapping at a spatial resolution of 150-300 nmlaterally and 500 nm vertically.

A cold water cooled Peltier stage (Agilent Technologies Inc.,Tempe, AZ) was used for controlled rate cooling of the samples.A 1 µL portion of the experimental solution was placed on analuminum surface, and a small piece of thin polystyrene (PS)film (∼2 µm) was placed on top of the liquid droplet to preventevaporation or sublimation during freezing and data acquisition.The sample and the Peltier stage were mounted on a piezoelec-tric scanning table placed under the Raman microscope. Theexperimental solutions were either cooled down to T ) -26°C at a rate of dT/dt ) 10 °C/min with manual ice seeding atT ) -3 °C (low supercooling, LS) by touching the sample witha liquid N2 chilled needle or allowed to spontaneously nucleatein the range -20 °C < T < -26 °C (high supercooling, HS).3-D CRM images were collected at T ) -26 °C at differentdepths (∼2-6 µm) below the PS film by raster scanning. Anarray of spectra (e.g., 80 × 80 corresponding to a sample areaof 30 × 30 µm2) was collected using identical integration timesat each pixel. CRM images were generated by integrating oneor more of the characteristic spectral peaks.

Calibration experiments were conducted with solutions ofvarying TRE and LYS content to validate that the tryptophanand the glycosidic link band peaks used in the analysis are notdependent on the level of hydration but concentration. We havealso carefully calibrated our measurements and assessed the errorin our concentration measurements as follows: Raman chemicalmaps were collected in 25 µm × 25 µm areas (80 pixels by 80pixels) in pure TRE or LYS solutions of different concentration.The concentration of the solute (TRE or LYS) was calculatedat each pixel using the characteristic Raman peak. The standarddeviation of the measurement was calculated on the basis ofthe values obtained in each pixel in the homogeneous solution.It was found that the measurement error (i.e., % standarddeviation from the average) was inversely correlated to con-centration. This was expected, since at lower concentrations theRaman peak size was smaller. For TRE solutions, the error wasbelow 1% when the TRE concentration was higher than 700mg/mL, and was about 15% when the concentration was below10 mg/mL.

Acknowledgment. This research was supported by a grantfrom the Institute for Engineering in Medicine at the Universityof Minnesota.

Supporting Information Available: Raman spectra obtainedfrom pure solutions (100 mg/mL TRE in 1×PBS and 20 mg/mL LYS in 1×PBS) at 20 and -26 °C. This material is availablefree of charge via the Internet at http://pubs.acs.org.

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