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Physical Characterization of Pharmaceutical Formulations in Frozen andFreeze-Dried Solid States: Techniques and Applications in Freeze-DryingDevelopmentJinsong Liu a

a DSM Pharmaceuticals Inc., 5900 NW Greenville Boulevard, Greenville, NC

Online Publication Date: 01 February 2006

To cite this Article Liu, Jinsong(2006)'Physical Characterization of Pharmaceutical Formulations in Frozen and Freeze-Dried SolidStates: Techniques and Applications in Freeze-Drying Development',Pharmaceutical Development and Technology,11:1,3 — 28

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Physical Characterization of Pharmaceutical Formulations in Frozen and Freeze-Dried Solid States: Techniques and Applications in Freeze-Drying Development

Techniques and Applications in Freeze-DryingJinsong LiuDSM Pharmaceuticals Inc., 5900 NW Greenville Boulevard, Greenville, NC 27834

Physical characterization of formulations in frozen and freeze-dried solid states provides indispensable information for rationaldevelopment of freeze-dried pharmaceutical products. This articleprovides an overview of the physical characteristics of formulationsin frozen and freeze-dried solid states, which are essential to both for-mulation and process development. Along with a brief description oftechniques often used in physical characterization for freeze-dryingdevelopment, applications of and recent improvements to these tech-niques are discussed. While most of these techniques are used con-ventionally in physical characterization of pharmaceuticals, sometechniques were designed or modified specifically for studies infreeze-drying. These include freeze-drying microscopy, freeze-drying X-ray powder diffractometry and cryoenvironmental scanningmicroscopy, which can be used to characterize the physical propertiesof the formulation under conditions similar to the real vial lyophiliza-tion process. Novel applications of some conventional techniques,such as microcalorimetry and near infrared (NIR) spectroscopy,which facilitated freeze-drying development, receive special atten-tion. Research and developmental needs in the area of physical char-acterization for freeze-drying are also addressed, particularly the needfor a better understanding of the quantitative correlation between themolecular mobility and the storage stability (shelf life).

Keywords freeze-drying, lyophilization, physical characterization,formulation, amorphous solids

INTRODUCTION

Freeze-drying or lyophilization is a drying technologywidely utilized in the pharmaceutical industry. It provides

products with improved stability and/or desired physico-chemical properties, such as enhanced dissolution ratesand bioavailability. In recent years, with the advent ofbiotechnology, development and manufacture of biophar-maceutical products such as proteins and peptides isincreasing. However most proteins and peptides sufferfrom marginal stability and have to be freeze-dried toobtain the desired stability for long-term storage (shelflife). Essentially, a freeze-drying process comprises threestages: freezing (solidification), primary drying (ice subli-mation), and secondary drying (moisture desorption). Thisis known as the freeze-drying cycle. The success and theefficiency of each stage in a freeze-drying cycle dependupon process parameters such as pressure, temperature,and duration, and should be designed according to thephysical properties of the formulation. The physical prop-erties of formulations in frozen and freeze-dried solidstates should be characterized using advanced techniques.

A development loop for a freeze-dried product isillustrated in Fig. 1. Characterization of the physical prop-erties of a formulation in the frozen state (1) provides criti-cal information for formulation development (A) as wellas for freeze-drying cycle design and optimization (B). Forexample, a pharmaceutical formulation having a glasstransition temperature (Tg′) for the freeze-concentratedsolution lower than −40°C is not practically suitable forfreeze-drying, even though it may have good stability. Inthis case, modification of the formulation is necessary inorder to improve the Tg′. The Tg′ can be modified by low-ering the buffer concentration if possible, and/or adding acollapse modifier to the formulation.[1] Here, a collapsemodifier refers to a high molecular weight excipient thatremains in the amorphous phase and causes the glass tran-sition temperature of the amorphous mixture to increase.Even if a given formulation has the potential to become astable product in the dried solid state, only an appropri-ate freeze-drying process may realize this potential. An

Received 1 April 2005, Accepted 1 October 2005.Address correspondence to Jinsong Liu, Product Development,

American Pharmaceutical Partners, Inc., 2045 N. Cornell Avenue,Melrose Park, IL 60160; E-mail: [email protected]

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4 J. Liu

optimized cycle should not only lead to an efficient andyet robust freeze-drying manufacturing process, but alsoensure, in conjunction with an optimized formulation, afinal product with desired quality attributes, such asacceptable long-term stability (shelf life) and aestheticappearance. For a formulation to be practically suitable forfreeze-drying, results from physical characterization in thefrozen state are crucial to rational freeze-drying cycledesign, which is based upon mass and heat transfer princi-ples. [2–4] Historically, in the pharmaceutical industry, thedevelopment of freeze-drying cycles was, and in manycases still is, an empirical practice.[5]

A formulation may be freeze-dried with minimaldeterioration and acceptable appearance, but it is not nec-essarily an acceptable product. A freeze-dried formulationhas to be evaluated (2) by characterizing its various physi-cal attributes (for instance, cake appearance, moisturecontent, crystallinity, glass transition temperature, pH,reconstitution time and so on), as well as chemical proper-ties, such as product stability. While chemical assays forlong-term stability are time-consuming and labor inten-sive, physical characterization of a formulation in thefreeze-dried solid state may generate stability-indicatingdata in a quick manner, which provides a guideline for theformulation and process development scientists to furtherimprove the freeze-drying cycle (C) and/or to further opti-mize the formulation (D). Therefore, characterization offormulations in the frozen and the freeze-dried solid statesis critical for both formulation and process development.

In recent years, a number of excellent books,[6–9] bookchapters, [10–13] and journal review articles[4,14,15] on pharma-ceutical lyophilization/freeze-drying have been published.These books and articles generally present fundamental prin-ciples and practices of freeze-drying, applied aspects offormulation, process development, and scale-up in pharma-ceutical and biological industries. However, it appears thatphysical characteristics of the formulations for freeze-dryingand/or the related characterization techniques have not beencomprehensively discussed in the literature. This articleattempts provide a general overview of the physical charac-teristics of formulations in frozen and freeze-dried solid

states and their relevant characterization techniques. Empha-sis is placed on thermal and microstructural properties and thechanges during and after freeze-drying. With extensive andup-to-date references, progress in understanding the physicalproperties of formulations, applications, and recent improve-ment of characterization techniques are discussed. Thisreview should prove useful to readers, particularly the begin-ners in the field of freeze-drying formulation and processdevelopment. Most of the content in this article appliesequally to small molecules and proteins, while more discus-sion is committed to protein formulations. However, biophys-ical and biochemical properties of proteins in formulationsand their characterization are not included, though they arecertainly of paramount significance in the development offreeze-dried biopharmaceuticals. Extensive review articles onthose aspects can be found in the literature.

ESSENTIAL PHYSICAL CHARACTERISTICS OF FORMULATIONS IN THE FROZEN STATE

Super-Cooling, Ice Nucleation, and Ice Crystal Growth

A freezing process is generally the first stage in afreeze-drying cycle. In this step, most of the water sepa-rates into ice crystals throughout a matrix of glassy and/orcrystalline solutes. Therefore, most of the desiccation of aformulation solution occurs at the freezing stage. Whilethis stage is generally controlled by the shelf-cooling rate,holding temperatures, and holding times in a freeze-dryer,the freezing rate of the formulation solution is not neces-sarily related to the shelf-cooling rate.

The freezing of a formulation solution starts with icenucleation followed by ice crystal growth. The tempera-ture at which the solution starts forming ice crystals isknown as the ice nucleation temperature. The ice nucle-ation temperature is stochastic and dependent upon a num-ber of process and formulation variables as well as surfaceproperties of the container.

The microstructures of both the ice crystals and thesolute formed during freezing determine the characteristicsof the subsequent primary drying and secondary dryingstages in a freeze-drying cycle, and consequently, thequality of the final product. Therefore, freezing is a criticalstage in a freeze-drying process. While many studies onfreezing and its impact on the biophysical properties havebeen published in cryobiology and biophysics, fewer char-acterization studies[16–18] on this stage have been reportedas compared to the number of publications on the primarydrying stage in pharmaceutical lyophilization.

Since pharmaceutical injectable solutions are filteredand essentially free of suspended impurities, supercoolingusually occurs during freezing and is often in the range of

Figure 1. Physical characterization for freeze-drying:integrating formulation and process development.

Freeze-drying

processFreeze-dried

product

Characterization in

freeze-dried solid state

Formulation development /optimization

1

A

B

D

Cycle

design/optimization

Characterization in

frozen state

Formulation in

solution

2

C


Techniques and Applications in Freeze-Drying 5

10 to 15°C or more below the equilibrium freezingpoint.[19,20] Also, ice nucleation temperatures and icegrowth rates in a solution filled in vials for freeze-dryingare largely heterogeneous, since the ice nucleation may beinitiated by nucleation sites either in the solution or on thesurface of the container. This heterogeneity of ice-forma-tion in a batch causes large heterogeneity in the primarydrying rate and also affects the secondary drying rate to alesser extent. A lower ice nucleation temperature leads tosmaller sized of ice crystals, which means smaller poresizes and thus a longer primary drying time and a shortersecondary drying time.[21] Recent work by Searles et al.[22]

showed that a decrease of 1°C in ice nucleation tempera-ture caused about a 3% increase in the primary drying (icesublimation) time. Therefore, in the development stage,and particularly in the scale-up stage of a pharmaceuticalfreeze-drying process, the formulation solution prepara-tion and freeze-drying development batch should be car-ried out in an environment as clean as possible to mimicthe clean environment at the sterile production operationsite. This is to minimize the potential for contamination ofparticulates to affect the ice nucleation. On the other hand,some approaches, such as ice annealing[23] and “ice-fog”nucleation[24] can be used in the design of the freezing pro-cess to reduce the heterogeneity of ice nucleation.

There are very few suitable techniques to directlycharacterize ice nucleation and ice crystal growth underreal conditions of vial freeze-drying. Visual observation isabout the only direct technique currently available. Mostcharacterization work was conducted by studying the“porous cake” left behind by ice crystals after drying. Thesizes and the shapes of ice crystals formed during freezingessentially determine the sizes and morphology of theresulting pores. Scanning electronic microscopy (SEM) isgenerally a useful technique to examine cake morphology.[20]

Searles et al.[22] found, based upon SEM observation thatthe degree of supercooling and the composition of thesolution determined the ice crystal morphology. Therecent work by Rambhatla et al.[21,24] demonstrated thatBET specific surface measurement is a useful method for aquantitative measurement of surface area, which charac-terizes the efficiency of a freezing step. However, thismethod should be used with caution when cakes haveexperienced micro- or partial collapse, since the measuredsurface areas in these cakes do not precisely reflect thequantity of ice crystal occupation during the freezing.

Glass Transition, Eutectic Melting, and Structure Collapse

In the freezing stage, solutes may crystallize. In prac-tice, the solute usually nucleates and crystallizes only after

supercooling at about 10–15°C below the equilibriumfreezing point. Those solutes that do not crystallize areconverted to amorphous solids when the temperaturedrops below the glass transition temperature (Tg′) of themaximally concentrated solute (or freeze concentrate).Therefore, this process is also called solidification. In thefreeze-drying of protein formulations, proteins, in general,do not crystallize during freezing, but are transformed intorigid amorphous (glassy) solids at Tg′, which is oftenknown as glassification or vitrification.

In freeze-drying, the notation Tg′ is specifically usedfor the glass transition temperature of the “maximally con-centrated solutes” or “freeze concentrates” formed duringfreezing. In general, the glass transition temperature forthe amorphous solid is denoted as Tg, which depends uponthe moisture content in the system. Fig. 2 illustrates asupplemented phase diagram of a typical solute (such assugar)/water system for freeze-drying.

If the product temperature during primary dryingexceeds the eutectic melting temperature (Te) or the glasstransition temperature (Tg′), the eutectic crystalline solids

Figure 2. Supplemented phase diagram for a binaryformulation system where the solute (S), such as sugar, does notcrystallize during freeze-drying. Arrows indicate the freeze-drying process. A represents formulation aqueous solution in thefill for freeze-drying; Tm(W) and Tm(S) denote meltingtemperatures of water and solute, respectively; Tg(W) and Tg(S)denote glass transition temperatures of water and solute,respectively; Tg′ and Tg(P) denote glass transition temperaturesof freeze concentrate of the formulation and the freeze-driedproduct, respectively; Te denotes the eutectic temperature. Asthe temperature is lowered, the solute S does not crystallize atTe, due to high viscosity from concentration of solute and lowtemperature, so that freeze-concentration proceeds beyond Teand goes through a viscous liquid/glass state transition at Tg′.

Equilibriumfreezing curve

Equilibriu

m

solubility curve

Tg’

GlassGlass transition curveIce and solution

TeRubber

Tg (S)

Tm(S)

Unsaturated solution

Super-saturated solutio

n

Weight Fraction of Solute

erutarepmeT

Tm (W)

A

Primarydrying

Secondary

dryingSuper-cooling

rF eezing

Tg (W)

Tg (P)

(Ice crystallization & growth)(Nucleation)

0 1


6 J. Liu

melt down or the viscosity of the amorphous solute phasedecreases. Both phase changes will cause sufficient flowin the formulation system to result in the loss of porestructure in the dried matrix. Visually, the lose of structurecauses “cake collapse” in a freeze-dried product, regard-less of crystalline melting or amorphous collapse. How-ever, historically, collapse temperature (Tc) is onlyapplied to amorphous systems, analogous to the eutecticmelting temperature in crystalline systems. Collapsetemperature for an amorphous system refers to the temper-ature, above which the dried region adjacent to ice loses itsstructure.[25]

Primary drying is frequently the most time-consumingstage in a freeze-drying process. An increase in producttemperature during primary drying would greatly reducethe duration of primary drying, provided that the chamberpressure is fixed. Therefore, an optimized process mustoperate near the maximum allowable product temperature,i.e. Tc for an amorphous system or Te for a crystalline sys-tem, and yet, the product is left in the form of a porouscake that has good dehydration properties and acceptableappearance. Collapse of cake leads to high residual mois-ture in a product with resultant low stability, and/or pro-longed reconstitution time.[26,27] Sometimes, high in-process product degradation was observed in the collapsedsamples, due to high molecular moiety in the system.[28]

However, Wang et al.[29] recently reported exceptionalresults in which collapsed samples of recombinant factorVIII and α-amylase showed no significant differences oreven improved stability than did the noncollapsed samplesat different temperatures, which led to an argument thatcollapse is not necessarily detrimental to long-term stabil-ity of freeze-dried proteins.

However, in this author’s view, Wang’s results do notnecessarily contradict the common concept that cake col-lapse in general compromises storage stability. In wang’swork, the secondary drying times were very long(20 hours), resulting in very low (less than 2%) moisturecontent in both collapsed and noncollapsed samples,though the former was slightly higher than the latter. Par-ticularly, after 2-month storage, the moisture content inboth types of samples reached essentially the same level,due to the moisture pick up from the stoppers. The samelevel of moisture content in both collapsed and noncol-lapsed samples led to the insignificant difference in theobserved storage stability in this case. Nevertheless, col-lapse of cake normally results in the rejection of productvials simply due to lack of elegance or prolonged reconsti-tution times.

Characterization of Te, Tg′, and/or Tc is crucial forthe design of freezing and primary drying conditions.A number of techniques, such as differential scanning cal-orimetry (DSC), differential thermal analysis (DTA),

dielectric analysis (DEA), and freeze-drying microscopy(FDM), have been used. These techniques and their appli-cations will be discussed later in detail. Tg′ (Te) and Tc ofselected model proteins and some common excipients forproteins in finished dosage form are summarized in Table 1.More data can be found in the literature.[10,14,30,41]

Annealing

Annealing, or thermal treatment, in frozen solution isa process to facilitate crystallization, in general, of a bulk-ing agent such as glycine or mannitol. Conditions forannealing (temperature and duration) should be deter-mined by the crystallization kinetics of bulking agents.Optimal conditions should ensure the completion of crys-tallization of the bulking agent during the annealing stagebefore entering primary drying. Otherwise, one may losequantitative control over the physical state of the bulkingagent during primary drying, secondary drying, and/orstorage. That is, the crystallinity of the bulking agent mayvary. Consequently, the reproducibility of the quality, andin particular, the stability of the finished product can becompromised. A typical phenomenon reported in case ofuncompleted crystallization of mannitol was “vial break-age” due to the volume expansion resulting from contin-ued crystallization of mannitol during the dryingstages.[43–45]

Crystalline solids of a bulking agent generated byannealing not only provide elegant cake appearance, butalso may accelerate the freeze-drying process. For such apartially crystalline formulation system, the eutectic tem-perature of the crystalline solid (Te) is in general muchhigher than the glass transition temperature of the freezeconcentrate (Tg′) in the amorphous phase of the formula-tion; therefore, primary drying can be carried out at aproduct temperature between Te and Tg′. In this case, amicroscopic form of collapse is developed in the driedmaterial while the main large pores formed during freez-ing remain unchanged and the crystalline matrix retains agood cake structure. This phenomenon is termed micro-collapse. The drying time can also be substantiallyreduced due to the higher product temperature and the lowresistance to vapor flow resulting from micro-collapse.[32]

However, the molecular mobility at a temperaturehigher than Tg′ is greatly accelerated, which in principleleads to higher degradation rates of active molecules.Therefore, in case such in-process degradation becomessignificant at a temperature between Te and Tg′, the producttemperature in the primary drying stage should be kept yetbelow Tg′.[46] Sometimes, the presence of the crystallineform of the bulking agent leads to a more unfavorable situ-ation for the protein stability in the formulation. This is



probably due to the interface of protein with the bulk agentcrystals that causes more protein aggregation duringfreeze-drying and storage. In such a case, the use of a crys-talline bulking agent can be questioned.[27]

One special application of annealing is annealing ice,which has been increasingly applied in pharmaceuticaldevelopment. That is, after freezing to a very low tempera-ture for solidification, the frozen formulation is broughtback to a higher temperature between the Tg′ and the icemelting point, and held for a certain period of time beforerefreezing below the Tg′. At such a high temperature, theunfrozen water will diffuse through the frozen matrix and

the ice crystals will grow in size due to a “ripening effect.”This may also create channels due to the ice crystal inter-connections. Consequently, the primary drying time may beshortened.[47,48] This effect has been demonstrated experi-mentally in some published work.[18,23,49] However, the det-rimental effects of annealing cannot be ignored. First, theannealing process does take time and the optimal conditions(temperature and length) still need to be determined empiri-cally. Secondly, high molecular mobility during the anneal-ing may cause more significant problems of instability, suchas crystallization of components, pH shifts, phase separa-tion, and destabilization of proteins.[17,21]

Table 1 Glass Transition and Collapse Temperatures of Selected Proteins and Common Excipients for Proteins in Finished Dosage Form

Component Tg′/°C Tc/°C Tg/°C

ProteinsCatalose −28 [28] −29 [28]

Galactosidase −29 [28] −15 [28]

Bovine serum albumin (BSA) −11 [30]

Ovalbumin −11 [30] −10 [31]

Lactate dehydrogenase −9 [30]

rhuMAB HER −20 [32]

SugarsSucrose −32 [30] −31 [26] 75 [33]

Trehalose −29 [34] −28.5 [26] 118 [33]

Lactose −28 [30] −30.5 [26] 114 [33]

Maltose −30 [35] 100 [33]

Amino acidsGlycine −62 (−3.5)* [36,37] 30 [27]

β-Analine −65 [30]

Arginine 42 [38]

Histidine −33 [30] 37 [38]

PolyolsGlycerol −65 [35] −93 [35]

Sorbitol −46 [34] −54 [26] −1.6 [39]

Mannitol −35 (−1.0)* [30,37] −1.4 [26] 13 [39]

PolymersPolyethlene glycol (6KD) −13 [31]

Dextran (70K) −11 [26]

Dextran (40K) 101 [40]

Polyvinylpyrrolidone (40K) −20.5 [41] −24 [26] 180 [42]

Buffer components and saltsSodium acetate −64 [30]

Sodium citrate −41 [30]

KH2PO4 −55 [30]

K2HPO4 −65 [30]

Tris · base −51 (−4)* [30]

Tris · HCl −65 (−13)* [30]

NaCl · 2H2O −60 (−23)* [30]

CaCl2 −95 (−52)* [30]

ZnCl2 −88 [30]

*Eutectic temperature (Te).


8 J. Liu

The DSC experiments of annealing a formulation pro-vide data for rational design of an annealing step in freeze-drying cycle development. For example, ice nucleation,glass transition of the freeze concentrate, glycine crystalli-zation, and ice melting in a formulation mixture of 60%(w/w) sucrose + glycine (2:5, by weight) can be detectedby DSC as shown in Fig. 3A. The Tg′ of the amorphousphase comprising both sucrose and glycine was −51°C.After annealing at −25°C for 30 minutes, which is about10°C higher than the onset temperature of glycine crystalli-zation, the rerun DSC thermogram showed that the Tg′ was−33°C (Fig. 3B), approximately equal to the Tg′ of sucrosealone, confirming that glycine crystallized out from theamorphous phase completely after the annealing step.

Freeze-drying X-ray powder diffractometry is a valuedtechnique to quantitatively characterize the polymorphs of abulking agent in the formulation formed by annealing.[50]

SEM is often the method of choice to illustrate the dif-ference in microstructure of the product resulting fromannealing.[23] These techniques will be discussed later inthis article.

Phase Separation

In the development of freeze-dried parenteral prod-ucts, it is always desirable to make a formulation as simpleas possible. However, many drug formulations, particu-larly for proteins, contain excipients such as stabilizer,bulking agent, salt, surfactant, and so on. In these multi-component formulation systems, phase separation canoccur during a freeze-drying process. Three types of phaseseparation during the freeze-drying process have beenreported: crystallization of amorphous solids, separationinto different amorphous phases, and amorphization fromcrystalline solids.

Crystallization of amorphous solutes can occur tobuffer salts, stabilizers, and bulking agents. Buffer saltcrystallization during freezing is a common phenomenon,which often weakens the buffering capacity and causes pHshift.[51] However, buffer salt crystallization is difficult topredict since other excipients, particularly the proteinitself, in the formulation may inhibit the salt crystalliza-tion. Freezing may also induce crystallization of some sta-bilizers, such as polymers. Izutsu et al.[52] reported thatpolyethylene glycol (PEG) crystallized in frozen solutionsand lost its stabilization effect. While crystallization ofbuffer salts and stabilizers is hostile to the protein stabilityand thus should be prevented, the crystallization of bulkingagents, such as mannitol or glycine, is a phase separationthat should be promoted, since they should exist in thecrystalline form to achieve the desired functions.[53] How-ever, the crystallization features are determined by bothformulation and process conditions, such as freezing tem-perature and freezing rate,[54–56] which was discussed ear-lier (Annealing). The crystallization of bulking agentsduring the freezing stage and/or primary and/or secondarydrying may also lead to different polymorphs in the finalproduct,[57–59] and the uncompleted crystallization of bulk-ing agents may even continue during the storage of thefinal product.

During freezing, the most common phase separationphenomenon occurring to stabilizers is the separation ofactive drug molecules (e.g., proteins) from excipients(e.g., stabilizers) into different amorphous phases.[60,61]

This type of freeze-induced phase separation can directlyor indirectly affect the drug’s stability by changing thecomponent interaction. Understanding and manipulatingthe component’s miscibility and its effects are of signifi-cant importance in formulation design.[60] Interestingly,

Figure 3. DSC characterization of sucrose + glycine mixture(60% w/w, 2:5 by weight) for designing an annealing step in afreeze-drying cycle (author’s unpublished data). A) Icenucleation, glass transition, crystallization of glycine, and ice-melting during a cooling/heating process without annealing;B) Glass transition temperature after annealing at −25°C for30 minutes.

-2

0

2

4

-60 -50 -40 -30 -20 -10 0

Temperature (oC)

Hea

t fl

ow

(w

/g)

Glycine crystallization

Ice nucleation

Ice melting

-0.06

-0.04

-0.02

0

-57 -47 -37Temperature (

oC)

Hea

t fl

ow

(w

/g)

Tg': -51 oC

A Exothermic

-0.05

-0.04

-0.03

-50 -40 -30 -20

Temperature (oC)

Heat

Flo

w (

W/g

)

Tg': -33 oC

B

Exothermic



phase separation into multiple amorphous phases as aresult of freeze-concentration does not appear to be limitedto polymers or other high molecular weight compounds.For example, DSC thermograms for frozen solutions ofglycine and NaCl showed multiple glass transitions, indi-cating multi-amorphous phases formed.[62]

Amorphization from crystalline solids (buffer compo-nents or stabilizers) could also occur during a freeze-dryingprocess. This type of phase transition was revealed bySuryanarayanan and his coworkers, using a freeze-dryingX-ray device.[63,64] For example, disodium hydrogen phos-phate crystallized as the dodeca-hydrate by freezing,which was completely transformed into the amorphousanhydrate form during the subsequent primary dryingstage. Similar phenomena were also observed in thefreeze-drying of a raffinose-protein (lactate dehydrogenase,LDH) formulation solution. During an annealing at −10°C,raffinose pentahydrate crystallized from the protein-raffi-nose amorphous phase, which then dehydrated back toamorphous solids during the subsequent primary dryingstage. It was found that the phase separation of the protein(LDH) from the amorphous raffinose in the freeze-dryingprocess resulted in a significant reduction in the recoveryof LDH activity, even though the finished product wascompletely amorphous.

Phase separation in the frozen solutions and freeze-dried solids is a critical issue to the product quality, partic-ularly, the stability during processing and storage.[60,65,66]

Characterization of phase separation in formulations forfreeze-drying provides crucial information for formulationdesign and process development. Differential scanningcalorimetry is a common technique to study the crystalli-zation of excipients during the freezing stage.[55] However,it is much more difficult for DSC to detect the separationof amorphous phases than crystallization events becauseof the small thermal signals in the former.[62] An SEManalysis demonstrated a useful technique to indicate phaseseparation as a result of freeze-induced concentration.[66]

Fourier-transform infrared spectrometry (FTIR) was alsoused to detect protein structure differences betweenphases.[67] The recently developed freeze-drying X-raypowder diffractometry is credited as a unique technique toobtain more insight by detecting the in-process crystalliza-tion or amorphization during freeze-drying.[63,68]

Non-Aqueous Solvents in Formulations for Freeze-Drying

Many formulations contain small percentages ofsolvents as carryovers from previous processing steps.While nonaqueous solvents have also been used toincrease stability of bulk solutions and the dried products

of small molecules, they generally destabilize proteins insolution. However, at low concentrations certain nonaqueoussolvents may have a stabilization effect.[69] In fact, polyhy-dric alcohols, such as glycerol, are among the commonlyused and effective cryoprotectants. Nonaqueous solventsare also increasingly used, particularly in developmentwork, to modify the physical properties of formulations, andconsequently, to increase the wettability and/or the solubil-ity of the molecule of interest, or to facilitate the process byincreasing the sublimation rate in freeze-drying.[70,71]

Nonaqueous solvents present in the formulation mayfreeze or remain as unfrozen liquid residues distributedthroughout the ice matrix. This results in substantially dif-ferent thermal and structural properties of formulations inthe frozen state, and thus affects the rate of drying andinfluences the choice of plant design, the productioncapacity, and the product quality.[72,73] In a recent review,Teagarden and Baker[74] discussed extensively the practi-cal aspects of using nonaqueous solvents in formulationsfor freeze-drying. Characterization of thermal and struc-tural properties is of vital importance in developing formu-lations containing nonaqueous solvents for freeze-drying.Among others, thermal analysis [DSC/thermogravimetricanalysis (TGA)], freeze-drying microscopy, scanningelectronic microscopy, and surface area measurement arethe most common techniques used for these studies.[75-78]

ESSENTIAL PHYSICAL CHARACTERISTICS OF FORMULATIONS IN THE FREEZE-DRIED SOLID STATE

Desorption Isotherm and Residual Moisture

After primary drying, the entire formulation matrix ina vial passes the frozen state and enters a dried state, andcontains a fairly high amount of “unfrozen water” (Fig. 2).The unfrozen water may be absorbed on the surface ofcrystalline solids or in the solute phase, either as hydratewater in a crystalline hydrate or dissolved in amorphoussolids to form a solid solution. The latter is the mostcommon case in freeze-dried biopharmaceuticals. Theunfrozen water is greatly reduced in the secondary dryingstage (Fig. 2).

Because the glass transition temperature (Tg) of afreeze-dried formulation is a function of the moisture con-tent, the Tg changes sharply with the decrease of moistureduring the ramp from primary drying to secondary drying,and during secondary drying. To avoid structural shrink-age or deformation of the cake, the product temperatureshould be lower than the Tg during the ramp. Therefore,knowledge of the Tg as a function of the moisture content,moisture desorption isotherm, and desorption kinetics is


10 J. Liu

required for secondary drying optimization. Someadvanced techniques, such as dynamic vapor sorption(DVS), allow one to characterize the moisture sorptionproperties accurately and efficiently.

The optimal moisture content for a given freeze-driedformulation has yet to be established by empirical stability/potency studies. From the point of view of glassy dynamics,a freeze-dried product should be the dryer, the better. Thishas been found true for formulations of small molecules,but not always for proteins or other biologics, most likelydue to the high structural complexity of the latter.[79,80]

The role of moisture in protein stability was addressed insome excellent reviews.[81–83]

Most decomposition reactions are minimal at or belowthe (Brunauer, Emmett, and Teller (BET) monolayerlevel of hydration, generally 5%–9% water content,[81,84]

due to the low availability of water and limited dynamicactivity of the protein. However, the oxidation of proteinscan be an exception. It has been found that many oxidationreactions actually showed increased rates at or below themonolayer.[81] In these systems, water acts as an antioxi-dant by diluting trace catalysts and facilitating the recombi-nation of radicals. In consideration of the water substitutionmechanism, the concept of monolayer water coverage isuseful in the development of freeze-dried protein formula-tions. The water sorption monolayer of pharmaceuticalproteins can be determined by various methods.[84]

However, if stabilizers are properly used, proteins inthe freeze-dried amorphous formulation solids show betterstability at a moisture level (<1%) much lower than themonolayer level.[85,86] In these dried formulations, the sta-bilizer molecules are able to retain the protein conforma-tion via substituting the monolayer water molecules, whilethey also lower the mobility of the protein molecules byforming a glassy matrix. Both effects result in improvedprotein stability in the amorphous solid state.[87]

Since the water content plays a key role in the molec-ular mobility in the dried solid matrix, as well as in reten-tion of the conformational and/or chemical states ofproteins, an accurate and precise method for moisturedetermination is critical in the development of freeze-driedprotein formulations. A wide variety of techniques havebeen utilized, including “loss on drying,” thermogravimet-ric analysis (TGA), gas chromatography (GC), and mostcommonly, Karl Fischer titration. These methods havebeen extensively discussed in great detail in the litera-ture.[88,89] Among others, one of the common problemsencountered with those methods is reabsorbing moisturefrom the environment. Even for the Karl-Fischer titrationusing dry solvent extraction in an unopened sample vial,the solvent, methanol for instance, can pick up water fromits surroundings, and it is difficult to accurately compen-sate for this moisture contamination in the blank.[89] An

emerging nondestructive technique, near-infrared reflec-tance (NIR) spectroscopy, may be the solution to thisproblem. The use of NIR to measure the moisture contentof freeze-dried products will be discussed later.

Molecular Mobility: Glass Transition and Structural Relaxation

At the end of a freeze-drying process, the freeze-driedformulation exits as an amorphous (glassy) or partiallyamorphous system. The glass transition temperature of afreeze-dried formulation is determined by components ofthe formulation. Glass transition temperatures of somecommon excipients for proteins in the finished dosage formare included in Table 1. Residual water is always a compo-nent of the freeze-dried formulation, and it is regarded as aplasticizer in glassy formulation solids. The plasticizationof water in freeze-dried formulation solids can be predictedby, for example, the Gordon-Taylor equation.[90]

At a temperature under the glass transition temperature(Tg), the mobility of drug molecules, such as proteins,which are dispersed in an inert glassy matrix, is greatlyreduced.[91] However, the molecular mobility is not deter-mined by the glass transition temperature alone. Thoughthe molecular mobility slows greatly under the glass transi-tion temperature, Tg, it is not zero. To achieve zero mobility,the freeze-dried product should be stored at a temperature(T0), which is in general much lower than the Tg,[92,93] asshown in Fig. 4. As a rule of thumb, it has been suggestedthat the freeze-dried amorphous product should be kept at50°C below its Tg during the shelf life. While this rulemight be approximately right for many pharmaceutical

Figure 4. Molecular mobility in a freeze-dried amorphoussystem: structural (enthalpy) relaxation from amorphous state Ato B during storage at TS. T0 denotes the zero-mobilitytemperature; Tg and Tm denote glass transition temperature andmelting temperature, respectively.

Liquid

(Melt)

Liquid

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ooled)

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mgla

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lpy

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B

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systems with high fragility, the difference between Tg andT0 is a function of the fragility of the system and thus doesdepend upon the formulation system.[94]

Characterization of the mobility of freeze-dried for-mulation systems in terms of relaxation time has gainedincreasing interest. The formulation system in the amor-phous state, which is a metastable physical state, has thetendency to move towards a stable (equilibrium) stateduring storage at a temperature Ts, under the glass tran-sition temperature Tg.[90] As an amorphous systemmoves towards the equilibrium state, the structural orderin the system becomes greater, i.e., the entropydecreases. This process is called structural relaxation.The structural relaxation time is the time for amorphoussolids relaxing from the nonequilibrium state toward theequilibrium supercooled liquid state. As the initial stateis of higher enthalpy than the equilibrium state, heat isgiven off during the structural relaxation process, and thisprocess is also known as enthalpy relaxation (Fig. 4). Liuet al.[95] studied the enthalpy relaxation of variousfreeze-dried pharmaceutical systems, revealing that thestructural relaxation time depends upon a number of for-mulation and freeze-drying process variables as well asstorage conditions, such as nature of material, tempera-ture, moisture content, thermal history (processing his-tory), and so on.

Physical stability is clearly related to viscosity, andtherefore, to structural relaxation.[96] This correlation hasbeen experimentally demonstrated by studies of variousactive pharmaceutical ingredients and formulations usingdifferent techniques, such as NMR,[97,98] DSC,[99] andmicrocalorimetry.[100]

The relationship between relaxation (mobility) andchemical stability as yet has not been very clearly eluci-dated. Unlike physical stability, chemical stability may besubjected to additional factors. However, the mobilityassociated with relaxation provides the potential for reac-tions, thus leading to the instability of drugs in the glassysolids. The coupling between the chemical stability, mea-sured as degradation or aggregation, and the enthalpyrelaxation or NMR-derived structural relaxation below Tgwas found in several freeze-dried protein or peptides for-mulations.[94,101–103] While more experimental work isneeded to further explore such correlations, mobility char-acterization of freeze-dried formulations would provideguidance for rational design of formulations and freeze-drying processes as well as a basis for predicting the sta-bility trend.

The most conventional technique for characterizationof glass transition temperatures, enthalpy recovery, andrelaxation is differential scanning calorimetry (DSC), inparticular, the modulated temperature DSC.[94,99,104,105]

Other thermal methods, such as microcalorimetry,[95]

nuclear magnetic resonance (NMR),[97,98] and dielectricanalysis (DEA)[106] also have been used for characterizingstructure relaxation. However, relaxation times fromthermal methods are not directly related to the onesfrom NMR or DEA. More work is needed in order to gaininsight to their interrelation,[106] and in particular, theextent of coupling between these relaxation times andstability of drugs (such as proteins) in freeze-driedformulations.

Recrystallization of Freeze-Dried Products during Storage

The storage stability of a freeze-dried product is agreat challenge. Phase transitions can occur in a freeze-dried product during storage, affecting its physical andchemical stability.

The phase transitions during storage include thetransformation of crystal forms of a bulking agent andthe recrystallization of amorphous formulation compo-nent(s). While the former might result in changes ofphysicochemical properties, such as solubility, the latteris the major concern. In general, the crystallization ofamorphous drug molecules might increase the storagestability, but physical properties such as solubility willbe compromised. While freeze-dried proteins areunlikely to crystallize during storage, the crystallizationof stabilizers during storage is not uncommon, evenunder Tg,[107,108] which has significant impact on stabil-ity of the freeze-dried protein products. While crystalliz-ing to an anhydrate, the stabilizer is partially orcompletely removed from the drug (amorphous) phase,losing its ability to function as a stabilizer. Furthermore,the moisture associated with the amorphous solids of thestabilizer will be taken up by the remaining amorphousphase. That can, thereby, increase the moisture contentof the drug-containing amorphous phase, and conse-quently, decrease the stability.[109] However, if an excip-ient in the amorphous phase crystallizes as a hydratebelow the glass transition temperature during storage,the recrystallization will remove a substantial amount ofplasticising water and thus increase the Tg of theremaining amorphous phase. This recrystallization is, tosome extent, favorable to the drug stability during stor-age. Based upon this type of potential recrystallization, aconcept of a self-stabilizing formulation was recentlyproposed.[110,111] The recrystallization of stabilizers dur-ing storage can be inhibited by other excipients[112,113] orthe protein itself,[114] and therefore, can be too compli-cated to be predicted and has to be characterized bytechniques, such as DSC, X-ray powder diffractometry,and microcalorimetry.


12 J. Liu

TECHNIQUES FOR PHYSICAL CHARACTERIZATION OF FORMULATIONS IN THE FROZEN AND THE FREEZE-DRIED SOLID STATES

A great number of techniques for physical character-ization have been explored and applied in the developmentof freeze-drying, some of which are quite conventional insolid-state characterization while some are innovative andemerging. In this section, techniques used for characteriza-tion of thermophysical properties, microstructures, mor-phology, and crystallinity of the formulations during andafter the freeze-drying process, as well as techniques forwater desorption and moisture content are brieflydescribed. Recent developments and applications of thesetechniques in freeze-drying are discussed. The emphasis isplaced on the techniques and instruments designed ormodified specifically for freeze-drying development, andon the novel applications of some conventional techniquesin freeze-drying development.

Techniques for Characterization of Thermophysical Properties

Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC)

Differential Thermal Analysis (DTA) and DifferentialScanning Calorimetry (DSC) are two classical thermalanalysis techniques, which have often been used in freeze-drying development. Differential thermal analysis (DTA)measures the temperature difference between a sample anda thermally inert reference material as a function of time orsample temperature while DSC measures the energychange (heat flow) instead of temperature. For both tech-niques, temperature conditions of the sample container(for instance, the DSC pan) can be controlled similar to afreeze-drying process. In some cases, DTA can detect thephase changes under conditions (such as the sample sizeand containers) that mimic the actual freeze-dryingprocess more closely which has been regarded as anadvantage.[115] While DTA is continuously used in somelaboratories,[116] DSC is a much more popular instrumentthan DTA in the pharmaceutical industry.

In recent years, for rational development or optimiza-tion of freeze-drying processes, DSC has been exten-sively explored to characterize thermal properties offormulations in the frozen state.[30,117] These propertiesinclude degree of supercooling, glass transition tempera-ture of freeze concentrate (Tg′), or eutectic temperature(Te), crystallization temperature, degree of crystalliza-tion, unfreezable water content, melting point, and so on.

Among others, the Tg′ is the most critical physicalproperty that is related to product cake collapse. How-ever, in some cases, the Tg′ of protein formulations can-not be detected easily by DSC, because of the relativelylow sensitivity of DSC. In many cases, this difficulty canbe resolved by using fast heating rates or concentratedsolutes, since the Tg′ is independent of the formulationconcentration.[34]

If the difficulty in detection of Tg′s is due to the over-lapping of thermal events, operation of the DSC under themodulated temperature mode (i.e., MDSC) can be a solu-tion, since the MDSC enables one to separate the heatflows resulting from reversible events (such as glass tran-sition) and nonreversible events, for example, crystalliza-tion and enthalpy recovery.[118] Note that the Tg′ valueevaluated from reversible heat flow is generally 2 to 5°Chigher than the one evaluated from the total heat flow.[119]

In the characterization of formulations in the frozenstate, two glass transition events were commonly observedfrom the DSC curves, particularly for the solutions con-taining a high concentration of sugars. Different mecha-nistic interpretations of these events are suggested in theliterature. While controversial opinions remain,[120,121] it isgenerally agreed that the high-temperature events corre-spond to collapse phenomenon in freeze-drying.[122–124]

Since characterization of a thermal event as reversibleor irreversible provides more insight into its nature, theMDSC technique has demonstrated unique advantages inthe solid-state characterization of the freeze-dried prod-ucts, such as determination of glass transition temperatureand enthalpy recovery,[104,105,125] recrystallization temper-ature and kinetics.[126] Fig. 5 exemplifies the separationadvantage of MDSC in characterization of amorphoussolids.[118]

Figure 5. MDSC thermoanalytical curve of polyethyleneterephthalate (PET) showing the total, reversing, andnonreversing heat flow signals. Reprinted from Ref. 118, withpermission from Elsevier.



Themoelectric Analysis

Thermoelectric analysis (TEA) or electrokinetic anal-ysis is a technique used to measure electric resistance as afunction of temperature. The resistance to electric currentin the frozen state is high because mobility of charge-car-rying species is limited. As the sample is warmed to thaw,the electrical resistance drops significantly while ionicmobility increases. Therefore electric resistance measure-ments provide a means for characterization of the thermalproperties of a formulation in the frozen state. Though theinstrument for thermoelectric analysis is not as popular asDSC, some freeze-dryers are featured with in-situ electricresistance (or eutectic) measurement capabilities.

Back in the 1960s, the TEA technique was used tocharacterize some formulations for freeze-drying by somepioneering scientists, such as Rey[127] and Deluca.[128]

However, this technique has not been systematically eval-uated as a tool for formulation and process development.Her’s work[129] has shown that TEA is a sensitive methodfor measuring the onset of eutectic melting for single-com-ponent solutions of some inorganic salts and mannitol, butTEA thermograms of frozen solutions containing amor-phous solute alone only show a gradual change in slopeover the temperature range of interest, with no inflectionpoint that corresponds to Tg’. However, they further dem-onstrate that addition of low level (ca. 0.1%) of electrolyteto the amorphous solute could make the glass transitionregion detectable (Fig. 6). In a recent work, Ma et al.[116]

used this technique to characterize a monoclonal antibodyformulation. Interestingly, they were able to detect theglass transition temperature of freeze concentrate, theeutectic crystallization temperature, and the ice melting

temperature with agreement to the values determined withDTA. While more work is needed to explore the applica-tion of TEA in the characterization of thermal propertiesof amorphous formulations, the application of TEA is lim-ited by their nonstandard nature. In particular, the responseof TEA is very dependent on frequency, but there is noconsistency between different instruments with respect totheir frequency of use.

Dielectric Analysis (DEA)

Dielectric properties are related to the ability of amaterial to polarize when placed in an electromagneticfield. The polarization is a dynamic process that dependsupon the structure and molecular properties of the mate-rial. Therefore dielectric analysis provides the possibilityfor characterization of these properties. The principle andapplications of dielectric analysis in pharmaceuticals werepresented in some recent reviews.[130,131]

Among others, the nonisothermal method is one of theexperimental approaches widely used to determine thedielectric properties in pharmaceuticals. Dielectric thermalanalysis is the simplest nonisothermal technique, in whicha single frequency is used for dielectric analysis and thecomplex permittivity is measured as a function of temper-ature. A relaxation is manifested as an increase in real per-mittivity with increasing temperature and an associatedloss peak. This technique has been used to determinethe collapse temperature of formulations for freeze-drying.[132,133] Fig. 7 showed the low temperature transi-tions in sucrose solution evaluated by DEA.[134] Theauthors[133,134] developed a take-off frequency (TOF)

Figure 6. TEA curves of 10% A) sorbitol, B) sucrose, C)trehalose, D) ficoll, and E) dextran doped with 0.1% ammoniumnitrate. Reprinted from Ref. 129 with kind permission fromSpringer Science and Business Media.

Figure 7. Low temperature transitions of sucrose evaluated byDEA. Reprinted from Ref. 134 with permission from TAInstruments, Inc.


14 J. Liu

method to determine the collapse temperature which washighly consistent with the literature value. This techniquecan also be used to discriminate between frequency-independent first-order transitions and frequency-depen-dent higher-order transitions occurring in the frozenstate.[135] Therefore, DEA is one of the favorable tech-niques to measure macroscopic thermal behavior to facili-tate the development of freeze-drying cycles.

In the characterization of molecular mobility of amor-phous formulation solids, the use of DEA is reported as apromising technique.[136] For different lyophilized formu-lation mixtures of sugar and drug, significant differencesin dielectric relaxation kinetics, and activation energywere observed by El Moznine et al.,[137] and these relax-ation kinetics data were found to correlate with the amountof product degradation. In the future, applications of DEAneed to be further explored in the development of freeze-dried formulations with high storage stability.

Thermally Stimulated Current (TSC) Spectrometry

Thermally stimulated current spectrometry includesboth thermally stimulated depolarization current (TSDC)and thermally stimulated polarized current (TSPC) methods.This is a technique related to dielectric thermal analysis,which exploits the relationship between molecular mobilityand temperature, and thus, provides a temperature/timeprofile comprising well-resolved relaxation times and acti-vation energies.

Since the sensitivity of TSC is proportional to thestimulus, it was proven a sensitive tool to measure glasstransition temperature and to observe relaxation in theglass state,[138] and has been used to investigate the molec-ular dynamics of heterogeneous and complex polymermaterials. Though TSC is not a very familiar technique inthe community of pharmaceutical scientists, in recentyears, it has found increasing applications in characteriz-ing freeze-dried/amorphous formulations. With a highersensitivity than DSC, TSC is a very convenient techniquenot only to study molecular mobility and to determine thefragility index in glass-forming systems, but also to detectthe secondary relaxation, which was not detectable byother techniques, such as DSC.[139,140] In studies of somefreeze-dried solid systems, Collins et al.[141] found notonly a good agreement between TSC and DSC results oncrystallization and melting temperatures, but also TSC’scapability of providing information supplementary to DSCon molecular mobility in protein systems.

While TSC found valuable applications in the charac-terization of dried solid systems in the development of sta-ble formulations, very little work has been reported on theuse of TSC to characterize formulations in the frozen state.In this aspect, more studies need to be performed in order

to establish relationships between phase behavior of thefrozen solutions and the TSC measurement results.[141]

Thermal Mechanical Analysis (TMA) and Dynamic Mechanical Thermal Analysis (DMTA)

In thermal mechanical or thermomechanical analysis(TMA), the deformation resulting from a constant stress(load) is measured as a function of temperature. It hasbeen used as a tool for food scientists to detect the glasstransitions of complex food products in both frozen anddried amorphous states. These transitions were expected tooccur over a broad temperature range and could not berevealed by conventional DSC,[142] In an effort to optimizeprotein lyophilization, Chang and Randall[30] found thatTMA is a very useful technique to complement DSC in thestudy of physical changes of formulations under subambi-ent conditions. A most interesting and unique applicationof TMA in freeze-drying is to study dimensional changes infrozen solutions as a function of temperature, particularlythose changes that might cause vial breakage. Williamset al.[45] used TMA to detect the expansion occurring in afrozen mannitol solution (Fig. 8), which was found relatedto the observed vial breakage. This work and the mostrecent work by Hirakura et al.[143] suggested that TMA canbe used as a tool for screening formulations that mightcause vial breakage during freeze-drying or for developingan appropriate process to prevent the vial breakage fromoccurring.

Dynamic mechanical thermal analysis (DMTA)[144]

measures the viscoelastic properties (mechanical stiffnessand damping) of materials as they are deformed underperiodic stress at variable frequency. This technique

Figure 8. TMA warming thermogram of 5% w/v mannitol.The sample size had a significant effect on the amplification ofthe dimensional changes. Reprinted from Ref. 45 withpermission from PDA, Inc.



permits the study of the changes in mechanical propertiesinduced by the state transitions, and thus, enables one toobtain a better understanding of the physical evolution inthis temperature range. Though underused, DMTA hasbeen proven useful at detecting glass transitions of materi-als that were difficult to measure using DSC, such asproteins.[145] For measurements of sucrose[145,146] the dif-ference between DSC-derived and DMTA-derived Tg′values was about 3 to 5°C. However, it has been arguedthat the DMTA loss peaks do not correspond with the trueTg, but to the softening temperature Ts.[147]

Isothermal Microcalorimetry

Isothermal microcalorimetry (IMC) is a technique tomeasure heat evolution rates and extents of chemical/phys-ical/biological processes under isothermal conditions. Thistechnique has been proven a useful tool to monitor thedecomposition of pharmaceuticals, and thus, to assess thestability of drugs in the solid state.[148,149] Advantages ofisothermal microcalorimetry include high sensitivity andcontrollability of experimental conditions, i.e., temperatureand humidity can be controlled like product storage condi-tions. Therefore, the IMC should be a useful tool to screenfreeze-dried formulations in the development stage, thoughthis application has not been widely explored.[150,151]

A novel application of IMC is to characterize theglassy dynamics of freeze-dried formulations. In an effortto explore the correlation between the physical/chemicalstability and molecular mobility, the IMC was mostrecently used to directly monitor the rate of enthalpy relax-ation of amorphous solid samples during aging experi-ments.[95] Taking advantage of high sensitivity, one canobtain the relaxation time for a given sample in a muchshorter time and much less labor than using DSC. More-over, materials that relax very slowly (such as trehalose)and/or release little heat can be studied by this techniquewhile it is practically impossible to study them by usingDSC. An example of microcalorimetric measurement ofenthalpy relaxation of freeze-dried trehalose is given inFig. 9. This methodology was also extended by Kawakamiand Ida[152] to study enthalpy relaxation and recovery pro-cesses of freeze-dried maltose-based formulations. Thecalorimetry-derived relaxation times can also be used topredict the physical stability of freeze-dried products, asshown by Rambhatla et al.[100] From another perspective,Lechuga-Ballesteros et al.[153] demonstrated that microcal-orimetric measurements of the moisture-induced thermalactivity traces, resulting from water vapor interactionswith amorphous solids, could also provide a meaningfulindicator of long-term physical stability of amorphous for-mulation solids.

By measuring the recrystallization heat flow underdifferent relative humidity conditions, isothermal micro-calorimetry was also a sensitive technique to detect differentsolid-state structures (amorphous states) formed in differ-ent processes, which could not be distinguished by the X-ray power diffraction (XRPD) method (154). Therefore, thistechnique can be an alternative method for detectingbatch-to-batch variations caused by up-scaling the pro-cess, changing the formulation, or changing the processparameters for lyophilized products.

Thus far, isothermal microcalorimetry has not beenexplored to study phase transitions of formulations in thefrozen state. Given the fact that some microcalorimetersystems can be operated at subambient temperatures andeven are featured with a temperature-scanning mode, itshould be possible to study phase transitions, such as crys-tallization in the annealing stage of formulations in thefrozen state.[155] The advantage is that the formulation canpossibly be contained in the same vial as in a freeze-dryer,and the sensitivity is higher than that of DSC. Therefore,quantitative results can be obtained under conditions simi-lar to the conditions in a real freeze-drying process.

Techniques for Characterization of Microstructure, Morphology, and Crystallinity

Polarized Light Microscopy and Freeze-Drying Microscopy

Polarized light microscopy (PLM) is simple, quick,and sensitive technique to detect the crystallinity inpharmaceutical solids, including freeze-dried products,through the presence of birefringence in the sample. This

Figure 9. Relaxation of freeze-dried trehalose at 50°C asmonitored by an isothermal microcalorimeter (TAM). Reprintedfrom Ref. 95 with permission from Wiley-Liss, Inc., a subsidiaryof John Wiley & Sons, Inc.

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16 J. Liu

technique is widely used in the pharmaceutical industry.When viewed through crossed polarizing materials, mostcrystalline materials appear bright or colored while amor-phous materials lack birefringence.[156]

By mounting a cryo-stage, in connection with a con-trollable cooling system and a vacuum system, a micro-scope can be used to directly observe the microstructuresof the formulation solution sample under conditions offreezing and freeze-drying, and thus, this technique istermed freeze-drying microscopy (FDM).

In the early days, freeze-drying microscopes werehomemade by some pioneering scientists.[157–159] Thedesign of freeze-drying stage, which is the most criticalpart of the system, varied widely. While in most of thefreeze-drying stages the temperature is nearly constantacross the sample, some of them were specially designedsuch that a temperature gradient existed across the sample;the aim was to mimic the situation encountered in a real

freeze-drying process.[160,161] While some new develop-ment work on freeze-drying microscopy has been reportedby Nail and his co-workers,[162,163] freeze-drying micro-scopic systems are commercially available and increas-ingly used in pharmaceutical development laboratories.

In general, freeze-drying microscopy (FDM) isregarded as the best technique for the determination of col-lapse temperature of the product, since the loss of struc-ture, i.e., cake collapse, can be visually observed underexperimental conditions that are controlled to simulate thereal freeze-drying process (Fig. 10). However, one shouldbear in mind that the collapse temperature is not a uniqueproperty of the solute material, rather it depends on themeasurement methodology and the rate of water removalfrom the glassy state.[25] To minimize the variationbetween laboratory measurement of collapse temperatureand production behavior, the collapse temperature measure-ments should be conducted using solute concentrations

Figure 10. Observation of structure changes in a protein formulation matrix during sublimation under a freeze-drying microscope(Linkam). A. −31°C: structure retained; B. −30°C: collapse developed; C. −28°C: structure collapsed. Author’s unpublished data.



comparable to the concentrations of ultimate interest inproduction. Differences in resolution and time of observa-tion may cause small differences between collapse temper-ature measured by the microscopic method and collapseobserved in a product freeze-dried in a vial. In general, thelatter is slightly higher than the former by 1 to 3°C.[25]

Collapse temperatures of selected proteins and excipientsare included in Table 1, along with Tgs. Because ofdynamic effects, collapse temperature (Tc) and glass tran-sition temperature (Tg’) as measured by the thermal analy-sis are not identical. In most cases, Tc > Tg’ (see Table 1),provided the experimental temperature ramp rates arecomparable for both Tc and Tg’ measurements, althoughthe difference may be negligible. The FDM technique,when used along with thermal methods, provides valuableinformation about the relevance of each of the transitionsto the optimal freeze-drying process and the quality offreeze-dried products.[164,165]

Another important application of freeze-dryingmicroscopy is for examining the influence of formulationcomposition and freezing history on the morphology ofice,[166] which is associated with ice sublimation rate dur-ing primary drying and the water desorption rate during thesecondary drying stage as well. However, as discussed ear-lier, freezing is a very complicated process, therefore, thefreezing behavior and the morphology of the formed iceobserved by microscopy, in some cases, can be quite differ-ent from the ones in a vial under freeze-drying conditions.

According to a recent report by Zhai et al.,[167] theFDM procedure may even be a rapid and visual methodfor determination of the diffusion coefficient for watervapor through the dried cake formed by sublimation.Though they found the coefficient obtained by this tech-nique was valid for the studied case, the general feasibilityof the approach is under question, because the frozenmatrix in the microscopic scale can be very different fromthe one formed inside a vial for freeze-drying.

Scanning Electronic Microscopy and Cryoenvironmental Scanning Electronic Microscopy

Scanning electronic microscopy (SEM) uses focusedelectron beams instead of light; therefore, it is more pow-erful than optical microscopy, and capable of high magni-fication by which the structure of 10–20 nm can beobserved. This technique is now widely used in pharma-ceutical development to address questions about particle-to-particle interaction, surface characteristics, and otherdetails encompassed by the suboptical to macromolecularsize range.

Since the early 1960s,[168] SEM has been extensivelyused to examine the morphology (microstructures) offreeze-dried samples in relation to formulation compositions

and freeze-drying process parameters and to determine theproduct quality. Essentially, the microstructure of the driedcake observed by the SEM was determined by the size of icecrystals and their distribution in the solid solute matrixformed during freezing. Therefore, characterization withSEM can evaluate the impact of formulation compositionsand the freezing processes. For example, Kasraian andDeluca[76] observed with SEM that the cake of lyophilizedTBA-containing formulation consisted of long straightchannels, which were occupied by needle-like ice crystalsduring freezing and were responsible for the facilitated icesublimation rate during primary drying. Heller et al.[67] usedSEM to illustrate the phase separation induced by freezing.Searles et al.[23] found that the freezing and annealing pro-cesses affected the morphology of the cakes, as observed bySEM. However, the microstructure formed in the freezingcould also further change in the drying stages due to micro-collapse, for instance. The micro-collapse was observed asthe development of small holes in the main porous structureof the cake (Fig. 11), which led to a decreased resistance tovapor flow during primary drying.[32,169]

Since in the preparation of SEM samples, the lyo-philized cake is removed from the vial and cut to revealthe desired cross section, moisture absorption to the cakecan occur, which can affect the morphology of the sample.In order to avoid misleading morphology observations it isadvised that the sample should be prepared as much aspossible in a dry nitrogen or argon environment.[170] Alter-natively, some researchers[171–173] modified the traditionalsample preparation methods in order to minimize the pos-sible alteration of microstructure during the preparation.

Figure 11. Microstructure of lyophilized trehalose formulationobserved under SEM: Small holes (diameter 2–8 μm) in theplates were formed by micro-collapse during the primary dryingprocess, while the large pores (diameter ∼100 μm) were leftbehind by the ice crystals formed during the freezing process.Reprinted from Ref. 32 with permission from Wiley-Liss, Inc., asubsidiary of John Wiley & Sons, Inc.


18 J. Liu

Extensive discussion of the use of SEM for lyophilized pro-teins can be found in an excellent review by Overcashier.[174]

Scanning electron microscopy can also be used in anenvironmental mode which does not need sample coating,to observe the structure of the sample surface. This type ofSEM was recently adapted by Meredith et al.[175] to studyfreeze-drying processes in a manner similar to FDM. Thecryoenvironmental scanning electronic microscopy(CESEM) that they developed involved the installation of apeltier stage through which very cold and dry nitrogen gascan be passed. The environmental conditions and coolingrates, which can be applied in-situ, were comparable tothose attainable in commercial freeze-drying equipment.The spatial resolution of this microscope is theoretically thesame as that of conventional SEM. It was shown that thetechnique was capable of identifying temperatures at whichmicrostructural changes occur that are detrimental to thefinal product. Fig. 12 shows an example of CESEM obser-vations of microstructure changes of a poly-D, L-lactide-co-glycolide/acid sample during the primary drying process.

Since SEM is fundamentally a technique for observa-tion of the sample surface, a CESEM study of freeze-dryingis limited to observations of the development of the samplesurface texture, and thus, must be used together with othercharacterization techniques.

X-ray Powder Diffractometry and Freeze-Drying X-ray Powder Diffractometry

X-ray powder diffractometry (XRPD) is perhaps thegold standard for qualitative determination of crystallinity.This technique can not only confirm the presence of a crystal-line phase, but also identify polymorphs of crystals. Unam-biguous experimental results can be obtained by XRPD in aquick manner. Comprehensive treatment of the theory ofXRPD and its applications in physical characterization ofpharmaceutical solids can be found in the literature.[176]

X-ray powder diffractometry has been a method ofchoice for characterization of the physical state of freeze-dried solids. Documenting the physical state of a productboth immediately after freeze-drying and periodically dur-ing stability studies is often overlooked, but it can providevaluable insight into possible physical or chemical stabil-ity problems. The advanced feature of controlling the tem-perature and humidity in the X-ray chamber enables one tostudy readily the physical stability of freeze-dried productsin relation to the storage conditions.

However, using X-ray diffractometry to investigatethe phase transitions during a freeze-drying process wasnot a practice until a recent work by Suryanarayana andhis co-workers.[50] By attaching a vacuum pump to thecryo-stage of an X-ray diffractometer, an entire freeze-drying process can be carried out in the sample chamber of

the XRPD. The phase changes that occur during the pro-cess can be monitored in-situ. It was termed freeze-dryingX-ray powder diffractometry (FDXRPD). Since the physi-cal state of a solute is influenced in part by the processconditions, knowledge of the crystalline-related phasetransitions of the solute during the freeze-drying cycle canbe obtained.[56,63,68] This enables the identification of thecritical process variables and leads to a consistent productthat meets regulatory requirements. In the example shownin Fig. 13, FDXRPD provided in-situ direct evidence ofcrystallization of sodium nafcillin during an annealing stepin the freeze-drying process.[50] The FDXRPD has alsorevealed some phase transitions, which have not beenobserved by other techniques, such as amorphization in

Figure 12. Cryoenvironmental scanning electron microscopy(CESEM) micrograph of a poly-D,L-lactide-co-glycolide/aceticacid sample during the primary drying process at A) −40°C.Small amounts of environmental ice can still be seen, but theunderlying microstructure does not appear to contain any solidsolvent; B) All surface ice was removed and the remainingmaterial was organized into sheets of thin layers containingsmall voids. Reprinted from Ref. 175 with permission fromWiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.



the drying stage from the crystalline solids that formed inthe freezing stage.[63,64] These observations provide moreinsight about the fundamentals of freeze-drying. However,the FDXRPD lacks the sensitivity of DSC and is unsuit-able for the characterization of transitions in the amor-phous phase, such as glass transitions. Using a combinedsystem of XRD-DSC under freeze-drying conditions is,therefore, a very useful method for the optimization offreeze-dried pharmaceuticals.[177]

Nuclear Magnetic Resonance

The application of solid-state nuclear magnetic reso-nance (NMR) in pharmaceuticals has been amply demon-strated.[178] However, NMR has not been widely used inpharmaceutical freeze-drying development. In most studiesrelated to freeze-drying, low-field pulsed NMR, or relax-ametry, has been used. Here the NMR does not providechemical shift information, but measures molecular mobilityfor frozen or freeze-dried solid systems through the spin-lattice (T1) and spin-spin (T2) relaxation times. In contrastto DSC or dielectric relaxation spectrometry, NMR allowsidentification of the origin of molecular motion, and thus,determination of molecular mobility of the drug and excip-ients in a freeze-dried formulation.

Thus far, very few reports have been found in the lit-erature on using NMR to characterize pharmaceutical for-mulations in the frozen state. Izutsu et al.[113] used NMR tomeasure mobility of water and solutes in frozen solutions,

and to investigate their relevance to glass transition of sol-utes. Monteiro-Marques et al.[179] used a low-resolutionpulse NMR to monitor freeze-drying processes. Theydemonstrated that estimations of relaxation times T1 andT2 using rapid methods can be useful for detection of theend point of ice sublimation.

In recent years, it has been demonstrated that solid-state NMR is a promising tool to characterize molecularmobility in freeze-dried formulation solids, and thus, toelucidate the possible correlation between stability andmobility of the proteins. In studies of molecular mobilityof proteins in freeze-dried systems, using solid state NMR,Separovic and co-workers[180,181] found that the relaxationtimes (T1) were correlated with changes in aggregationand activity of the protein, and that stabilizers decreasedthe relaxation rates in the protein-sugar systems, whilehydration increased the rates. Extensive research in thisarea was carried out by Yoshioka and co-workers. Theyfound that aggregation rates of protein in freeze-dried for-mulations could be correlated to relaxation times,T2[101,182] or the critical mobility temperatures (Tmc),which were derived from NMR relaxation measure-ments.[183,184] By using high-resolution 13C solid-stateNMR,[185] they also demonstrated that molecular mobilityof protein molecules in freeze-dried formulations did linkto the molecular mobility of excipients, supporting thecurrent protein stabilization theory. In a recent review,[186]

Yoshioka summarized the NMR methods that they usedfor detecting the molecular mobility in freeze-dried formu-lations and also discussed the effects of molecular mobil-ity on the storage stability of freeze-dried formulations.

Techniques for Characterization of Water Desorption and Moisture Content

As previously discussed, both thermal properties andmicrostructure of freeze-dried solids are affected by theresidual moisture in the product. Residual moisture isdetermined by the water desorption process duringsecondary drying. Here, selected techniques for character-ization of moisture desorption/sorption behavior and mois-ture content are discussed.

Dynamic Vapor Sorption System and Symmetrical Gravimetric Analyzer

Traditionally, the moisture sorption isotherm is mea-sured simply using desiccators containing P2O5 powder orvarious saturated salt solutions for controlled humid-ity.[116] However, this method requires a large number ofsamples for one isotherm, and very long equilibrationtimes. Therefore, this method is very labor- and space-

Figure 13. XRD patterns of a frozen aqueous solution ofsodium nafcillin (22% w/w) at different temperatures asmeasured by freeze-drying X-ray powder diffractometry. Theintense peaks observed at 22.5, 24.0, 25.6 and 33.5°2θ areattributed to hexagonal ice. While heating to −4°C did not resultin crystallization of sodium nafcillin, annealing for 30 minutescaused its crystallization (characteristic peaks marks with anasterisk). Reprinted from Ref. 50 with kind permission fromSpringer Science and Business Media.


20 J. Liu

intensive. Moreover, sorption/desorption kinetics cannotbe measured.

Pikal et al.[187] determined sorption isotherms and sorp-tion kinetics gravimetrically using a vacuum microbalanceprocedure in a homemade device. Similar devices also havebeen setup and used by other scientists. In the mid-1990s aninstrument for such measurements, dynamic vapor sorption(DVS), was introduced to the market by Surface Measure-ment Systems (London, UK). This device utilizes verysmall sample sizes combined with a dynamic flow ofhumidified gas, by which the moisture sorption isothermcan be measured in a matter of hours or days at a tempera-ture range from 5°C to 80°C. Another similar characteriza-tion instrument is the symmetrical gravimetric analyzer(SGA) marketed by VTI Corporation (Florida, USA). TheSGA is a continuous vapor flow sorption instrument forobtaining water and organic vapor isotherms at temperaturefrom 0°C to 80°C at ambient pressure or subambient pres-sure down to very high vacuum.

Costantino et al.[188] used SGA to study water sorptionbehavior of freeze-dried protein-sugar systems, and thewater monolayer was calculated. The resultant data sup-ported the view that amorphous sugars interact with phar-maceutical proteins in the solid state. In an effort to developa mathematical model for the water distribution in freeze-dried solids, Chan et al.[189] utilized an SGA instrument tostudy the sorption isotherms of freeze-dried protein formu-lation solids at ambient temperature under partial vacuum.

Since water plays a very critical role in the stability offreeze-dried product, characterization of water sorption/desorption is not only important for developing an effi-cient drying process, but is also significant in elucidatingthe stabilization mechanism of a drug in the freeze-driedformulation matrix, which has yet to be fully understood.

Near-Infrared Reflectance Spectroscopy

The use of near-infrared reflectance (NIR) spectros-copy to determine residual moisture content in freeze-driedsucrose powder through intact glass vials was first reportedby Kamat et al.[190] This method offers rapid, noninvasiveand nondestructive measurements, and thus may avoid thereabsorbing of moisture from the environment, which wasencountered in other moisture detection techniques, includ-ing Karl Fischer titration. In recent years, it has beenexplored in the development of freeze-dried products, inparticular, for screening purposes.[191,192]

Because of its nondestructive nature, the very samesamples used for NIR moisture determination may also beused for other tests, such as the stability assay. This isattractive in the development of freeze-dried formulations.For example, in searching for a suitable specification forthe residual moisture in a freeze-dried product, using the

NIR technique, Derksen et al.[193] only used one-fifth ofthe number of samples that were needed for determinationin the traditional way. In the traditional way, a large numberof samples are generally required for generating the profileof stability vs. residual moisture, because of the high intra-batch variability of the residual moisture.

For predication of the moisture content of freeze-dried formulations, the NIR data must be correlated withan accepted residual moisture technique, in general, theKarl Fischer method, to generate a standard curve for theanalysis. In some cases, such predication needs precau-tions. A calibration for residual moisture analysis is spe-cific for a fixed formulation and product configuration.According to Lin and Hsu’s work,[194] small physical vari-ations, such as porosity, or small chemical variations, suchas surfactant and buffer concentrations, can be accommo-dated by a standard NIR calibration. However, significantformulation changes, such as the concentration of stabi-lizer, may cause spectral feature alteration, and conse-quently impair the measurement accuracy.[194]

Measurements of moisture contents by this techniquemay also be very useful in improving efficiency in freeze-drying manufacturing. Suzuki et al.[195] reported that ahigh quality IR inspection system was used for sorting outdefective product and for mapping moisture distribution ofproduct in a freeze-dryer. Recently, in a new adventure,Brulles[196] demonstrated that the NIR technique can beused to in-situ monitor the freeze-drying process bothqualitatively and quantitatively. This monitoring tech-nique can provide new information about the process, suchas the end point of ice-sublimation as well as the rate ofthe desorption process that was not possible to detect withconventional process monitoring.

RESEARCH AND DEVELOPMENT NEEDS IN PHYSICAL CHARACTERIZATION FOR FREEZE-DRYING

Physical characterization of formulations in both fro-zen and freeze-dried solid states is indispensable for therational development of both the formulation and the pro-cess to produce a successful freeze-dried product.

Techniques, such as DSC, which have already beenwidely used in pharmaceutical development, have beenapplied to characterize either formulations in the frozenstate or final products in the freeze-dried solid state. Mean-while, some conventional techniques, including microcalo-rimetry and NIR, have found some novel applications inthe freeze-drying development. Furthermore, special modi-fications or improvements made in some techniques, suchas microscopy, SEM, and XPD, enable one to character-ize the physical properties and their changes during a



freeze-drying process in-situ, which facilitates the rationaldevelopment of formulation and freeze-drying process.However, the properties that can be characterized in-situare mainly limited to microstructure and crystallinity.Additional in-situ physical characterization techniques willbe greatly appreciated. Table 2 summarizes the techniquesfor physical characterization of formulations during each

stage in a freeze-drying process and of final products rightafter processing and during storage.

Thus far, the applications of physical characterizationdata to freeze-drying process development have essentiallyfocused on the optimization of primary drying, i.e., shorteningthe primary drying time without deteriorating the productquality. Comparably, much less work has been done on the

Table 2 Physical Characterizations for Freeze-drying

Freeze-drying stages (States of the formulation)Physical properties/in-process

transitionsCharacterization

techniques Key references

Freezing Super-cooling, ice crystallization DSC 30,117(Frozen state) DTA 115,116

FDM 166Annealing/crystallization of

bulking agentDSC 32,164

FD-XRD 50,64Annealing of ice crystals DSC 23,49Morphology of ice crystals FDM 166

CE-SEM 175Volume expansion due to

crystallizationTMA 45,143

Primary Drying (Partially frozen and partially dried state)

Glass transition temperature (Tg′)/Eutectic temperature

DSC/MDSC 117,119

TEA 116,129TMA 30,142DMTA 145,146

Collapse temperature (Tc) FDM 159,162DEA 132,133

Mobility in frozen state NMR 113,179In-process morphology change CESEM 175In-process crystallinity change FD-XRD 50,63Ice sublimation NIR (in-situ) 196

Secondary Drying (Dried state)

Water desorption DVS/SGA 188,189

Desorption rate NIR (in-situ) 196Freeze-dried product Glass transition temperature DSC/MDSC 118,119(Freeze-dried solid state) Residual moisture NIR (non-

destructive)190,194

Crystallinity XPD 176PLM 156

Morphology SEM 174Recrystallization during storage IMC 154

DSC/MDSC 126Mobility DSC 104,105

DEA 136,137TSC 139,140IMC 95,152NMR 180,186

Phase separation (Formed during freeze-drying)

SEM 65,66

DSC 55,62


22 J. Liu

physical characterization related to both the freezing and thesecondary drying processes, particularly in product vials.

Continued efforts in both theoretical and experimentalwork on the physical characterization of products in thefreeze-dried solid state are needed. Currently, the routinephysical characterization of the freeze-dried formulationsolids, in most cases includes only moisture content andglass transition temperature. The determination of optimalmoisture content for a given formulation in the freeze-driedsolid state is still an empirical practice based upon stabilitydata. Further advances in the theoretical understanding ofthe role of water in the formulation solids, with the aid ofadvanced characterization techniques, would help to deter-mine the optimal moisture content with reduced lab work.Long-term stability (shelf life) has been always a greatchallenge in freeze-dried product development.[197] It isnow well accepted that storage of a product below the glasstransition temperature does not ensure an adequate shelflife. Unfortunately, the shelf life has to be determined byreal-time assay. This stability challenge can be partiallyaddressed by understanding of the coupling betweenmolecular mobility and stability of the freeze-dried formu-lation solids. While some stimulating work on exploringthis correlation has been reported, showing the promise ofpredicting the long-term stability based upon the structuralrelaxation,[198–201] more extensive work on the physicalcharacterization of glassy dynamics of formulations in thefreeze-dried solid state and its correlation to the stability, inparticular the chemical stability, is needed in the future.

ACKNOWLEDGMENTS

The author would like to thank Professor MichaelJ. Pikal (University of Connecticut) for his encouragementand valuable discussion in the preparation of this manu-script, and colleagues at DSM Pharmaceuticals Inc.,Dr. Paresh Dalal (currently with Pfizer, Inc.), Dr. ElaineMorefield (currently with FDA) and Ms. Lisa Stevens, fortheir help in reading the manuscript and their comments.

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