Stablizing Theory ALL

Advanced Drug Delivery Reviews 173 (2021) 1–19
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/adr
Stabilizers and their interaction with formulation components in frozen

and freeze-dried protein formulations q
Seema Thakral a,b, Jayesh Sonje a, Bhushan Munjal a, Raj Suryanarayanan a,⇑
a
Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States
b
Characterization Facility, University of Minnesota, Minneapolis, MN 55455, United States
a r t i c l e i n f o a b s t r a c t
Article history: This review aims to provide an overview of the current knowledge on protein stabilization during freez-
Received 8 December 2020 ing and freeze-drying in relation to stress conditions commonly encountered during these processes. The
Revised 6 February 2021 traditional as well as refined mechanisms by which excipients may stabilize proteins are presented.
Accepted 3 March 2021
These stabilizers encompass a wide variety of compounds including sugars, sugar alcohols, amino acids,
Available online 17 March 2021
surfactants, buffers and polymers. The rational selection of excipients for use in frozen and freeze-dried
protein formulations is presented. Lyophilized protein formulations are generally multicomponent sys-
Keywords:
tems, providing numerous possibilities of excipient-excipient and protein-excipient interactions. The
Protein formulation
Aggregation
interplay of different formulation components on the protein stability and excipient functionality in
Frozen storage the frozen and freeze-dried systems are reviewed, with discussion of representative examples of such
Freeze-drying interactions.
Stabilizers Ó 2021 Elsevier B.V. All rights reserved.
Cryoprotectants
Lyoprotecants
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Processing-induced stresses and stabilization mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Freezing and frozen storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Drying and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Classes of stabilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1. Sugars and sugar alcohols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.3. Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.4. Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.5. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.6. Additional excipients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4. Interaction of formulation components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1. Excipient- excipient interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1.1. Sugars with other excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1.2. Bulking agents with other excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.2. Protein-excipient interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Abbreviations: AA, amino acids; BSA, bovine serum albumin; CD, cyclodextrin; CMC, critical micelle concentration; DP, drug product; DS, drug substance; HES,
hydroxyethyl starch; HPbCD, hydroxypropyl b cyclodextrin; HSA, human serum albumin; KGF-2, keratinocyte growth factor-2; LDH, lactate dehydrogenase; mAb,
monoclonal antibody; MHH, mannitol hemihydrate; PEG, polyethylene glycol; PVP, polyvinylpyrrolidone; rhGH, recombinant human growth hormone.
q
This review is part of the Advanced Drug Delivery Reviews theme issue on ‘‘Biological Solids”.
⇑ Corresponding author.
E-mail address: surya001@umn.edu (R. Suryanarayanan).
https://doi.org/10.1016/j.addr.2021.03.003
0169-409X/Ó 2021 Elsevier B.V. All rights reserved.
S. Thakral, J. Sonje, B. Munjal et al. Advanced Drug Delivery Reviews 173 (2021) 1–19
4.2.1. Effect of protein on excipient phase behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4.2.2. Effect of excipient phase behavior on protein stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1. Introduction 2. Processing-induced stresses and stabilization mechanisms
Protein-based therapeutics are becoming increasingly impor- The stresses to which a protein is exposed during the freezing
tant in the treatment of human diseases. The primary reason for and drying steps of freeze-drying are generally considered
their popularity is the high target specificity and broad applicabil- together. Both processes involve removal of water, such as by ice
ity [1]. The active pharmaceutical ingredient in these products may crystallization during freezing and by sublimation/desorption dur-
include a monoclonal antibody (mAb), antibody-drug conjugate, ing drying. However, there is a subtle difference with respect to the
Fc-fusion protein, enzyme, hormone, interferon or interleukin [2]. unfrozen water associated with the solutes during each step. While
The production of therapeutic proteins and their formulation the unfrozen water is retained in the freeze-concentrate during
into drug products can be complex and expensive. In order to freezing, the process of drying leads to its removal. As a result,
optimize capacity use, bulk protein solutions are often produced the stresses experienced by a protein during these steps are funda-
in manufacturing campaigns. This protein drug substance (DS) mentally different [10]. Hence in this review, the stresses and
may be then stored for long time periods before being formulated mechanisms of stabilization by an excipient during the freezing
into a drug product (DP). The bulk DS are often stored in the fro- step and frozen storage are grouped together while those during
zen state with the assumption that the protein stability will be drying and storage are discussed separately.
preserved at the low temperature of storage and will increase
the shelf-life. In such situations, the DS, usually frozen in the
2.1. Freezing and frozen storage
presence of suitable stabilizers, is thawed before it is formulated
into the DP. The frozen state offers additional advantages, such as
The native (folded) structure of the protein is responsible for its
the reduced possibility of microbial growth and alleviation of
therapeutic activity and is thermodynamically the most stable
foaming or shaking issues during transport [3]. Frozen storage
form with a lower free energy when compared to the unfolded
has also been used for commercial protein products such as Jet-
state. The energy barrier for a native protein to unfold can be char-
reaÒ, cell and gene therapy products including GlyberaÒ, IMLY-
acterized by its free energy of unfolding (DGunf). Under physiologic
GICÒ, and LuxturnaÒ [4].
conditions, the free energy difference between the native (biologi-
A large fraction of the marketed protein formulations are
cally active) and folded (inactive) protein conformation can be very
administered parenterally. From both economic and ease-of-use
low (~5 to 20 kcal/mol). Hence a minor change in the external con-
considerations, a ready-to-use liquid formulation is desired. How-
ditions such as temperature, pH, ionic strength among others, can
ever, many proteins are susceptible to chemical (e.g. deamidation
result in unfolding of the protein [5].
or oxidation) and physical degradation (e.g. aggregation and pre-
The freezing stage conceptually involves two mutually non-
cipitation) in a liquid formulation [5]. If a solution dosage form
exclusive physical events, namely supercooling and ice crystalliza-
with the desired shelf-life cannot be developed, freeze-drying pro-
tion resulting in the formation of maximally freeze-concentrated
vides an avenue to prepare stable formulations. In the freeze-
solution (Fig. 1). Accordingly, the stresses experienced by protein
drying process, a solution containing the DS and the excipients is
and the possible cryoprotection mechanisms by which the excipi-
subjected to three sequential steps - freezing, primary drying pre-
ents stabilize the native protein state during each event can be
dominantly to remove ice by sublimation, and secondary drying to
distinguished.
remove the sorbed water (more details in Fig. 1). Right before use,
Supercooling. When a solution containing the DS and excipients
the lyophilized powder is reconstituted into a solution for admin-
is cooled, spontaneous ice crystallization may not be observed and
istration. Interestingly, ~30% of currently marketed biologics are
the system tends to supercool. This situation generally persists up
available as lyophilized products [6].
to 10 to 12 °C in the case of uncontrolled freezing [12].
While frozen storage and lyophilization are intended to pre-
The factors determining the degree of supercooling include cooling
serve and stabilize a biologic formulation, a protein may be sub-
rate and the type and concentration of solutes. Cold denaturation is
jected to several stresses both during freezing and drying. These
the process by which protein DS can denature due to unfolding at
stresses can lead to reversible or irreversible changes and may also
low temperatures prior to ice crystallization. However, cold denat-
result in a loss in its biological activity. In order to ‘protect’ the pro-
uration is not a major concern during freeze-drying. Protein
tein from these stresses, it is a common practice to add stabilizing
unfolding is relatively slow when compared to the timescales of
excipients. These encompass a wide variety of compounds includ-
freeze-drying [13,14]. Interestingly, the presence of additives (such
ing sugars, polyols, polymers, surfactants, and amino acids [7]. An
as sucrose and trehalose) and high protein (b-lactoglobulin) con-
understanding of these stresses and functional mechanisms of sta-
centration, lower the cold denaturation temperature [4].
bilizers is critical to the design of stable biologic formulations.
Ice crystallization and freeze-concentration. Further lowering of
The present review focusses on the rational selection of excipi-
temperature eventually induces the formation of ice nuclei fol-
ents for use in frozen and freeze-dried formulations. The various
lowed by crystal growth. Ice crystallization creates ice-water and
types of stabilizers and the stabilization mechanism will be dis-
ice-air interfaces which can induce stress leading to protein desta-
cussed. We have attempted to summarize the important research
bilization [15,16]. Water removal (as ice) concentrates the solution
findings of the past decade. We will refrain from discussion of sev-
(containing the solutes dissolved in the unfrozen water) in the
eral related and important topics, such as lyophilization cycle opti-
interstitial region, and may alter its ionic strength, viscosity and
mization and packaging design. Some of these topics have been
pH. Additionally, in multicomponent systems, this process may
covered in excellent recent publications [7–9].
lead to separation of phases differing in their solute composition.
2
Fig. 1. Typical steps involved in lyophilization of biologics. The y-axis represents temperature (color-coded). The temperature range, during different stages of freeze-drying,
broadly follows the color scheme. Rectangles with sharp corners represent intermediate stages while rectangles with rounded edges represent the final product. The
alphabets adjacent to the arrows represent the possible mechanisms of protein stabilization during each stage (any of the possible mechanisms may be operative; more
details in the text). All the solutes are assumed to remain amorphous, both in the frozen DS/DP and lyophilized DP. A typical freeze-drying process consists of three stages;
freezing, primary drying, and secondary drying. Freezing is an efficient desiccation step where most of the solvent is separated from the solutes to form ice. As freezing
progresses, the solute phase becomes highly concentrated and is termed the ‘freeze concentrate’. During primary drying, ice is transferred from the product to the condenser
by sublimation. The primary drying stage is generally the longest and its optimization has a large impact on process economics. During secondary drying, water is desorbed
from the freeze concentrate, usually at elevated temperatures and low pressures. Secondary drying normally takes only a few hours. Typically, drying is conducted below Tg’
(glass transition temperature of the freeze-concentrate) during primary drying and below Tg (glass transition temperature of the lyophile) during secondary drying [11].
The attendant changes have the potential to destabilize the pro- restricted mobility in the glassy state makes the protein relatively
teins [13,14]. stable in the timescales of practical interest in freeze-drying
Frozen storage. As mentioned above, protein DS are often stored [14,17]. However, the long-term storage can show some mobility
frozen. The product is often stored in large containers and the cool- based on the storage temperature and consequent excipient crys-
ing rate can be uncontrolled. The optimal storage temperature can tallization leading to protein instability (Section 4.2.2).
be determined by characterizing the protein before and after stor- Water replacement (substitute) hypothesis (or preferential solute
age at different temperatures [3]. In the context of frozen storage, interaction): As a result of freeze-concentration, sufficient water
the stresses due to: (i) freezing and thawing, and (ii) extended stor- molecules may not be available to adequately hydrogen bond with
age in the frozen state mandate careful consideration. The long- the polar protein surfaces. This allows selective interaction of
term storage can induce crystallization of some excipients leading solutes with the protein, suggesting another plausible mechanism
to protein instability (Section 4.2.2). known as the water replacement (substitute) hypothesis or ‘preferen-
A wide variety of chemically diverse compounds, known as cry- tial solute interaction’ [18]. An investigation on cryopreservation of
oprotectants, can protect proteins from freezing-induced stresses. cells using in situ Raman suggested that the cryoprotective action
Multiple mechanisms have been proposed to explain their stabiliz- of sucrose could be attributed to its direct interaction with the cell
ing effects in frozen systems. membrane [19]. It encompasses the concept that the hydroxyl
Preferential exclusion (also known as solute exclusion or prefer- groups (of the sugar) hydrogen bonds with the protein, thereby
ential hydration): In aqueous solutions, the water molecules inter- replacing hydrogen bonds between water and the protein. This
act with the polar groups on the protein surface making the protein replacement of hydrogen bonds enables the protein’s native con-
preferentially hydrated. In the initial stages of the freezing process, formation to be maintained.
the cryoprotectant molecules are selectively excluded from the The protein stabilization during the freezing process may occur
immediate vicinity of the protein surface. This exclusion of solute through any of these mechanisms (Fig. 1). In particular, preferen-
molecules increases the free energy of unfolding (DGunf), thus tial exclusion is expected to be predominant in the initial stages
favoring and stabilizing the native state [10]. of the freezing process, while vitrification and water replacement
Vitrification hypothesis (or glassy matrix theory): Ice crystalliza- hypotheses may take over as the solution freeze-concentrates
tion and freeze-concentration eventually drives the system to [15]. For the stabilizer to be effective, it must be amorphous and
approach a glassy state having restricted mobility. The presence a part of the freeze-concentrate.
of cryoprotectants such as sugars and polymers result in an
increase in viscosity of the freeze-concentrate, which reduces 2.2. Drying and storage
molecular mobility, thereby slowing down all dynamic processes.
The protein becomes virtually immobilized. Since mobility is a During the drying stage of lyophilization, ice is removed by sub-
pre-requisite for denaturation and degradation reactions, the limation (primary drying) and unfrozen ‘bound’ water by desorp-
3
tion (secondary drying). Since hydrogen bonding between water speed up a-relaxation in the glass while slowing down b-
and protein is critical to the thermodynamic stability of protein, relaxation (local mobility). Adding small amounts of antiplasticizer
removal of water constitutes the major stress during drying and (sorbitol or water) to sucrose-antibody formulations resulted in a
can cause irreversible loss of biological activity for some labile pro- monotonic decrease in Tg and global mobility (a-relaxation), but
teins [20]. Many effective cryoprotectants fail to retain their stabi- optimal stabilization was obtained at an intermediate levels of
lizing effect during drying. Stabilizers that can protect the protein added sorbitol or water [26]. Interestingly, local mobility (b-
during freeze-drying as well as storage are often referred as lyopro- relaxation) changed with antiplasticizer concentration in the same
tectants [21]. Furthermore, the physical state of the protein during monotonic way as did stability, suggesting coupling of the faster
both drying and storage is similar, with the only difference being molecular dynamics to protein degradation rates. The aggregation
the water content of the amorphous phase in these conditions. or chemical degradation rate constant for >100 proteins freeze-
From this perspective, it is postulated that the fundamental mech- dried in sucrose or trehalose glasses, was inversely proportional
anisms governing stability during drying also govern stability dur- to b-relaxation time [25,27,28]. In Fig. 2A, the glass transition tem-
ing storage of the dried product [21]. The additional stress factors perature of two protein formulations [recombinant human growth
during storage include unintended temperature excursions and hormone (rhGH) or keratinocyte growth factor-2 (KGF-2)] in sugar
excipient phase separation during the product shelf-life. In addi- matrices is presented. The right y-axis is the change upon
tion, the protein degradation via chemical processes such as lyophilization of the IR a-helical peak width at half height,
deamidation and oxidation are observed during storage [22]. Dw1/2. As the sucrose (or trehalose) mass fraction increased, the
The mechanisms of excipient-induced protein stabilization dur- formulations showed more structural similarity to native protein
ing drying and storage have some parallel with that of the frozen (lower Dw1/2). This was accompanied by a decrease in glass transi-
storage. The two widely-accepted mechanisms of stabilization dur- tion temperature (Tg) of the formulation. On the other hand, the
ing drying and storage are ‘vitrification’ and ‘water replacement’ aggregation constant for lyophilized rhGH correlated with the
which result in the preservation of the protein structure, by reduc- mean square displacement (<u2>) of hydrogen atoms, a measure
ing molecular mobility and preventing changes in protein structure of local ‘‘b-fast” dynamics (as determined by neutron backscatter-
respectively. Although the underlying mechanisms differ, both ing). This was evident following storage both at 40 and 50 °C. In
hypotheses require the protein and the stabilizer to be in the same Fig. 2B, reciprocal of mean square displacement, <u2>1, is plotted.
amorphous phase [23,24]. Smaller the <u2>1, higher the local mobility. [29–31]. While most
As mentioned above, the vitrification theory is based on the of the work on b-relaxation measurements was performed with
concept of immobilizing the protein in a rigid, amorphous glassy neutron backscattering, this technique cannot be used for routine
sugar matrix. The restriction of translational and relaxation pro- characterization [28].
cesses is thought to inhibit protein unfolding [20]. The dynamics Anchorage hypothesis. The dynamics of the protein are coupled
of amorphous sugars are usually characterized by global mobility, to dynamics of the sugar matrix via a water bridge that is hydrogen
measured as a-relaxation. This is the slowest dynamic process and bonded to both [32,33]. A number of simulation and experimental
exhibits a strong temperature dependence. Near and above the studies suggest that interfacial water can play an important role in
glass transition temperature (Tg), where there is a rapid change coupling the dynamics of the glassy host (sugar) with the protein.
in a-relaxation dynamics with temperature, the kinetic immobi- While the water hydrogen bonds to both protein and sugar, inter-
lization and the associated stabilizing power of the sugar are lar- facial water anchors more effectively to trehalose glasses than to
gely lost [21]. On the other hand, the ‘water replacement’ theory sucrose glasses. This may be attributed to the higher propensity
hypothesizes that good stabilizers interact with the protein as does of trehalose to form intermolecular rather than intramolecular
water, promote the native conformation, and therefore stabilize hydrogen bonds.
the protein during drying by replacing the water that is removed. Packing density. As a further refinement of water replacement
FTIR (Fourier-transform infrared spectroscopy) measurements of theory, smaller and molecularly more flexible saccharides are bet-
lyophilized protein–sugar matrix established that sugars prevent ter able to stabilize the protein during storage after lyophilization,
dehydration-induced unfolding by hydrogen bonding to the dried than larger and molecularly more rigid counterparts. The smaller
protein in place of the lost water. Theoretically, a sugar monolayer sugar molecules are less sterically hindered in interacting with
around the protein molecule should be adequate to retain com- the protein enabling the formation of hydrogen bonds. The stron-
plete protein activity by replacing water at all hydrogen bonding ger interactions and a tighter packing leads to an increased density
sites [21,22]. and thus a decreased free volume of these formulations [27,34].
However, this has not been realized in practice. It has been well In addition, it is generally accepted that for effective stabiliza-
documented that formulation Tg often fails to predict protein sta- tion, the protein and sugar must be in the same phase. The sugar
bility. The basic assumption that all the motion relevant to stability can phase separate either by forming a distinct amorphous region
is strongly coupled with the Tg, is not universally valid. Though or by crystallization. This process causes a loss of necessary inter-
there is much evidence for a correlation between near-native con- actions, coupled with induction of shear stresses on the protein
formation in the glass and stability against degradation, this rela- [35].
tion has not been quantifiable, and does not extend over the full These mechanisms are not mutually exclusive and it is often
range of protein–sugar compositions [21,25]. The past decade has difficult to attribute stabilization to one specific mechanism. How-
witnessed a significant improvement in our understanding of ever, an understanding of these mechanisms enables the formula-
solid-state protein stability specifically in amorphous sugar tor to try different approaches for designing stable lyophilized
matrices. formulations.
Relevance of local molecular mobility: Recent studies showed
that stability of proteins sequestered in sugar glasses is directly
correlated with the relatively high frequency b-relaxation pro- 3. Classes of stabilizers
cesses or the local mobility, rather than global mobility (a-
relaxation) of the sugar matrix. This is presumed to be derived In light of the various stresses experienced by protein during
from coupling of b-relaxations to local protein motions and to dif- freeze-drying process and storage, suitable excipients need to be
fusion of small molecule reactive species in the glass. The hypoth- added to the formulation to prevent protein aggregation and
esis was further confirmed using anti-plasticizers, additives which degradation. A protein formulation is a multicomponent system
4
Fig. 2. (A) Influence of sugar content on the glass transition temperature (vertical bars; left y-axis) and native secondary structure retention (change of IR a-helical peak
width at half height (Dw1/2, cm1) as lines; right y-axis) in the lyophilized protein formulations. Protein was either recombinant human growth hormone (rhGH) (solid
lines/filled markers) or keratinocyte growth factor-2 (KGF-2) (dashed lines/hollow markers). The smaller the change in the width of IR peak, the higher the retention of native
protein secondary structure after freeze-drying. (B) Influence of sugar content on local mobility (vertical bars; left y-axis) and the aggregation rate constant (lines; right y-
axis) for rhGH formulations stored either at 40 °C (solid bars/solid lines/filled marker) or at 50 °C (dashed bars/dashed line/hollow markers). The <u2> is a measure of the
mean square displacement of hydrogen atoms. Smaller the <u2>1, higher the local mobility. Each formulation contained 1 mg/mL protein and 5% w/v excipients, which was
hydroxyethyl starch with either trehalose (blue bars and lines) or sucrose (red bars and lines). Data adapted from Xu et al [29], Devineni et al [30] and Chieng et al [31]. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
usually consisting of a cryo- or lyo-protectant, buffer, bulking either sucrose or trehalose (compare red and blue lines respec-
agent and surfactant. A combination of these excipients in certain tively) [29]. In some cases however, sucrose might provide better
protein: solute ratios has been shown to be a useful strategy to sta- protection than trehalose as observed in lyophilized lysozyme
bilize the protein. [38]. The difference in the protective action between the two sug-
ars was attributed to the greater tendency of trehalose to phase
3.1. Sugars and sugar alcohols separate when compared to sucrose [39,40]. In contrast, molecular
simulation studies suggest that trehalose should be more efficient
Many sugars or polyols are used as protein stabilizers in solu- than sucrose in preventing hGH unfolding in the solid state. Tre-
tion, in the frozen state, during freeze-thawing, freeze-drying or halose molecules protect proteins by forming a dense, compact
product storage. The level of stabilization afforded depends on hydrogen bonding network between themselves, as well as with
their concentration. Generally a weight ratio of sugar to protein the protein [41,42]. In fact, the enzymatic activity of ß-
of at least 1:1 is required for good stability, with optimal stability galactosidase decreased more slowly in lyophile containing tre-
around 3 to 5:1 [20]. Further increase in sugar concentration may halose (2:1 excipient/protein weight ratio) when compared to
reach the limit of stabilization or even destabilize a protein during those containing sucrose [43]. To summarize, the ability of each
freeze-drying and storage. The minimal effective concentration of of these sugars to provide lyophilization and storage-induced sta-
sugar is employed, because of the effect of protein: sugar ratio bilization for a given protein should be determined on a case-by-
on the glass transition temperatures [17,27]. case basis [20].
Sucrose and trehalose are the most popular stabilizers. Their In general, disaccharides (such as sucrose, trehalose, lactose,
physico-chemical properties, likely of relevance in their stabilizing maltose; ~Tg 60, 110, 114, 100 °C respectively) are reported to be
activity, are presented in Table 1. more effective lyoprotectants than monosaccharides (glucose,
As is evident from the comparison in Table 1, sucrose and tre- galactose, fructose, mannose; ~ Tg 32, 38, 13, 33 °C respectively)
halose are characterized by several unique physical and chemical [44]. This may be due to their higher Tg, though the increased steric
properties, conferring each with distinct advantages as stabilizers hindrance may interfere with the intimate hydrogen bonding with
(Table 2). Likewise, there can also be serious limitations with the a dried protein. Therefore, the excipient selection needs balancing
use of these sugars. Of particular interest for biopharmaceutical the formation of a high Tg glass with the strength of hydrogen
applications is the propensity of trehalose to crystallize during bonding [44]. Similarly, raffinose, a tri-saccharide, was a less effec-
freezing. Upon annealing frozen solutions, trehalose can crystallize tive enzyme stabilizer than sucrose during storage, in spite of its
as a dihydrate. The aggregation in frozen protein solutions, follow- higher Tg (109 °C) [45]. Upon annealing the frozen solution at
ing long term storage at ~20 °C, has been attributed to trehalose 10 °C, raffinose can crystallize as pentahydrate, which dehydrates
crystallization [37]. While trehalose crystallizes as a dihydrate in during drying to yield an amorphous lyophile. Raffinose crystal-
the frozen state, upon drying, the dihydrate converts into the lization during freeze-drying is accompanied by a significant loss
amorphous anhydrate. Importantly, the dihydrate crystallization, of protein activity [46].
dehydration and amorphization can occur in the course of the Sugar alcohols such as sorbitol, xylitol and lactitol also protect
freeze-drying process and thus may not be obvious when charac- proteins from heat-induced denaturation in aqueous solutions
terizing the final product. through preferred exclusion. In the frozen state, sorbitol is tradi-
It is worthwhile to compare the effectiveness of sucrose and tre- tionally known to be an amorphous, non-crystallizing solute, mak-
halose in a therapeutic protein formulation. Fig. 2B (Section 2.2) ing it amenable to lyophilization. However, its low Tg’ (~44 °C)
shows that the aggregation rate constant of KGF-2 upon storage and consequently low collapse temperature, make it a poor choice
at 40 and 50 °C was similar for the protein co-lyophilized with for efficient freeze-drying process [47]. Sorbitol (or glycerol) along
5
Table 1
Comparison of physico-chemical properties of sucrose and trehalose. The properties of relevance as a stabilizer for lyophilized formulations are included in the table. Compiled
from the discussion in Ref. [36]. Properties conferring relative advantage are in bold while the properties which may limit the use (i.e. potentially disadvantageous) are italicized.
S. Property of interest Implications Sucrose Trehalose

No.
A. Molecular structure
1 Composition Glucose + fructose Glucose + glucose
(monosaccharides)
Conformational flexibility Less flexible or more rigid More flexible or less rigid
2 Type of glycosidic linkage b-link between C1 on the glucosyl subunit and a-link between C1s on both glucosyl
C2 on the fructosyl unit subunits
(i) Chemical stability (i) Prone to hydrolysis ? pH 5 (i) Resistant to hydrolysis, even at
Yields reducing sugars ? glycation of proteins high temperatures and under
through Maillard reaction acidic conditions
(ii) Rotation around the (ii) less flexibility (more rigid) ii) more flexibility
bond ? molecular confirmation
flexibility
3 Number of intramolecular 2 1
hydrogen bonds
Capacity to hydrogen bond with water Comparatively lower
4 Rigidity Rigidity ? packing efficiency in solid More rigid ? less efficient packing ? higher Less ? efficient packing ? lower free
state ? free volume ? glass transition free volume ? lower Tg volume ? higher Tg
temperature
B. Behavior an aqueous solution

5 Hydration number Lower Higher
6.3 8
6 Extent of intermolecular Viscosity of aqueous solution Exist as small clusters ? lower viscosity at RT Exist as interconnected continuous
association in solution aggregates ? higher viscosity
7 Dependence of viscosity on Influence of freeze concentration Pronounced increase in viscosity at low Pronounced increase in viscosity at
solute concentration and temperature ? glass formation low temperature ? glass formation
temperature Processing of solution Lower viscosity facilitates High viscosity can pose a challenge to
Processing mixing/filtration and other processes.
8 Aqueous solubility Concentration used in Higher (in comparison to trehalose) Lower
prelyophilization solution as
protectant
9 Change in solubility with Influence of freeze concentration Almost independent of temperature Pronounced dependence ? decreases
temperature on reducing temperature
Propensity to crystallize on freezing Low Higher ? can crystallize as dihydrate
on annealing frozen solution
C. Solid state properties

10 Crystalline forms One Three anhydrous (a-, b- and d-) and
one hydrate (dihydrate)
Possibility of unintended phase No Yes, Complex phase transformations

transition during processing
11 Glass transition 65–75 °C 110–120 °C

temperature of the
amorphous form (dry Tg) Recommended storage temperature Lower Higher
12 Moisture sorption behavior Propensity to sorb water (on exposure High propensity to crystallize following water Can act as a water ‘‘sink” and
to high water vapor pressure) and sorption ? crystallization accompanied by exhibit resistance to
crystallize release of sorbed water crystallization.
with sucrose demonstrated synergistic effect on protein stabiliza- requirement of a more aggressive drying for its removal, and (iii)
tion [26,48]. if retained in the final lyophile, water released due to dehydration
Mannitol, another sugar alcohol, is one of the most popular during storage, can have a significant impact on the physical and
excipients in lyophilized formulations. When retained amorphous, chemical stability of the product [53–56].
mannitol can serve as a cryoprotectant [49,50]. Mannitol is
traditionally used as a bulking agent due to its high propensity to 3.2. Amino acids
crystallize in frozen solutions. Mannitol generally crystallizes as
the anhydrous a-, b- or d- form, though the physical form in the Amino acids (AAs) have a long history of stabilizing native pro-
final lyophile is influenced by the other formulation components tein structure in liquid formulations. [57]. However, only a few
[51,52]. Depending upon the formulation and processing parame- studies have investigated the stabilizing role of AAs in freeze-
ters, mannitol can also crystallize as the metastable mannitol dried protein formulations [58]. Some representative examples
hemihydrate (MHH). The formation of MHH during freezing and are compiled in Table 3.
its retention in the final lyophile warrants careful consideration AAs are typically used with sucrose or trehalose and have been
due to (i) its usually inconsistent formation during freezing, (ii) rarely used alone in lyophilized formulations. This is possibly
6
Table 2
Selection criteria of use of sucrose or trehalose as stabilizer in the frozen and lyophilized protein formulations.
Process Sucrose Trehalose

Frozen storage Effective cryoprotectant Effective cryoprotectant
High solubility and low viscosity of solution facilitates solution Lower solubility and high viscosity complicates solution processing.
processing
Undergoes acid hydrolysis in solution with pH < 5. To be avoided if No risk of acid hydrolysis
there is risk of freezing-induced pH shift.
Remains amorphous in frozen solution. Propensity to crystallize as trehalose dihydrate when frozen solutions
annealed for long duration at 20 °C (above Tg’ 29 °C) leading to loss
of cryoprotectant activity.
Freeze-drying Remains amorphous and imparts lyoprotection. Minimum ratio of Remains amorphous and imparts lyoprotection. Minimum ratio of
protein: sucrose required. protein: trehalose required.
To be avoided for prelyophilization solution with pH < 5. Sucrose No risk of acid hydrolysis.
hydrolysis observed in lyophilized solids.
Considerations for using with bulking agent mannitol: Considerations for using with bulking agent mannitol:
Concentration dependent inhibitory effect on crystallization of bulking Upon annealing the frozen solution, behavior of both mannitol and
agents. For mannitol-sucrose system with sucrose: mannitol trehalose highly dependent on trehalose to mannitol ratio R.
ratio > 1:1, mannitol was retained amorphous. R = 3:1: inhibition of mannitol crystallization
R = 1:1, mannitol facilitated the crystallization of trehalose as trehalose
dihydrate.
R = 1:3, mannitol crystallized, trehalose was retained amorphous.
Tg of lyophilized formulation lower than one containing trehalose. Tg of lyophilized formulation higher than one containing sucrose.
Advantage: Even with a high residual water content, the formulation
Tg is likely to be higher than the product storage temperature.
Table 3
Amino acids used as stabilizers in lyophilized protein formulations.
Amino acid as stabilizer Protein Comments Reference

Arginine mAbs, BSA, Better stabilizing effect of arginine/sucrose than sucrose alone. [59–63]
Humanized anti-IL8
IgG1
15 AAs rHSA Protein :sucrose: AA (1:1:0.3 w/w) [57]

a-Chymotrypsin Improved the long-term storage stability of freeze-dried disaccharide-based protein
formulations
Serine, phenylalanine and glycine IgG Stabilization of IgG [64]

Trehalose more potent than the AA.
Effectiveness of AA, serine > phenylalanine and glycine
Histidine LDH Histidine showed a pH and concentration dependent improvement in the stability of LDH. [65]
Maximum stabilization was achieved at pH 6 (which retained histidine amorphous) and at
histidine concentrations 10 mg/ml.
Arginine, glutamic acid and Recombinant factor Mixture of three AAs stabilized the lyophilized protein [66]
isoleucine mixture VIII, GreenGene F
Histidine with aspartic acid or mAbs Aspartic acid and glutamic acid as a counterion improved the stabilization property of [67]
glutamic acid, and/or arginine histidine and arginine
Arginine, glycine, lysine, proline, Human IgG1, AAs with sugars improved the stability and reconstitution properties of the high [68]
alanine mAb concentration protein formulations
Arginine IgG1 mAb Arginine formulations with high residual moisture levels (2.5% w/w) showed better protein [69]
stabilization than sucrose alone formulations
Arginine with phenylalanine IgG1 mAb The combination yielded elegant cake and superior protein stability in comparison to [70]
arginine-mannitol, arginine-sucrose and sucrose based formulations.
Note: BSA (bovine serum albumin), rHSA (recombinant human serum albumin), LDH (lactate dehydrogenase)
because the stabilizing effect of AAs are lower than that of the con- [72]. Several strategies have been attempted to resolve this issue.
ventional sugar cryoprotectants [64]. However, in almost all these i) Use of alternative counterions such as citric, phosphoric, succinic
studies, the addition of AAs enhanced the stabilizing effect of sug- and lactobionic acids, increased the Tg’ but were found inferior to
ars, thus making them a suitable secondary stabilizer in the the HCl salt in terms of protein stabilization [59,60,69,71,73]. ii)
formulation. High protein concentration prevented cake collapse by increasing
Arginine (generally used as the hydrochloride salt) has been the the Tg’ [59,73]. iii) The addition of sucrose led to improved cake
most studied AA in lyophilized formulations [71,72]. Its positive appearance but failed to prevent collapse [59,73]. iv) Mannitol or
effect on the solubility of proteins, and tendency to lower the vis- phenylalanine was used as a crystallizing bulking agent to yield
cosity of the bulk and reconstituted solutions are additional advan- an elegant cake. From the perspective of protein stability, pheny-
tages. However, the addition of arginine lowers the collapse lalanine was superior to sucrose or mannitol [70]. In a more com-
temperature, thereby necessitating conservative drying cycles plex matrix (trehalose + arginine HCl + mannitol), arginine lowered
7
the degree of protein aggregation by 2–2.5 times (than trehalose factant interacts with protein molecules to form a surfactant–pro-
alone or trehalose + mannitol) [63]. tein complex preventing the protein from further interactions [76].
In addition, histidine [65,67], alanine, lysine, proline [68], serine The use of non-ionic surfactants has been a successful strategy to
[64] and phenylalanine [64] have also been reported to enhance prevent or inhibit surface-induced aggregation in both liquid and
protein stability in lyophilized products. In an interesting study, freeze-dried formulations. Polysorbates (20 and 80) and poloxamer
the use of acidic AAs as counterions for arginine and histidine were P188 are surfactants widely used in protein formulations [23,24]. In
found to enhance their stabilizing effect [67]. While histidine has spite of the advantages and wide use of polysorbates in protein for-
already gained popularity as a buffer, its utility as a stabilizer mulations, they are known to undergo auto-oxidation [77]. The per-
may warrant more systematic investigations. oxides formed as a result have been shown to cause oxidation of
The mechanisms by which AAs stabilize proteins in the solid- proteins [78]. In addition, based on solution environment, polysor-
state are not well understood. These may include, but are not lim- bates are also known to undergo hydrolysis of the long chain fatty
ited to, preferential interaction with protein molecules [72,74], acids. Use of refined polysorbates and storing solutions at low tem-
alteration in free volume [57] and molecular mobility [59,75]. Nev- peratures in a nitrogen atmosphere and protected from light, can at
ertheless, the utility of AAs as stabilizers of proteins in lyophilized least partially prevent degradation [77].
formulations can expand the excipient options. The critical micelle concentration (CMC) of polysorbate 20 and
80 in water (25 C ± 0.5 °C) is ~0.007% and ~0.0017% w/v respec-
3.3. Surfactants tively [79]. When a surfactant solution is cooled, the ice crystalliza-
tion will cause pronounced concentration and result in micelle
Proteins are usually surface active and vulnerable to aggrega- formation in the freeze concentrate. The increase in CMC as a func-
tion due to surface-induced denaturation [76]. Adsorption or bind- tion of freeze-concentration has not been thoroughly investigated.
ing can occur at various interfaces, for example, at the air–liquid In this context, the surfactant–protein interaction based on the ini-
interface due to mixing of liquid formulation components and at tial surfactant concentration in protein formulation can be a good
ice-liquid or ice-air interfaces during freezing and thawing or dry- reference (Fig. 3). Generally, polysorbates are used at concentra-
ing. In this context, addition of surfactants to a protein formulation tions <0.1% (w/v). On freeze-concentration, the surfactant concen-
may stabilize proteins via two possible mechanisms: (1) Preferen- tration increases exponentially. Thus, regions 4 and 5 in Fig. 3
tial binding of surfactant molecules at interfaces. Thus, smaller sur- would possibly reflect the mechanism by which surfactants pre-
factant molecules outcompete larger protein molecules and bind vent protein denaturation. In order to maximize stability, a good
to hydrophobic surfaces. The adsorbed surfactant molecules form idea is to start with minimum polysorbate concentrations. Other
a coating at the interface and prevent protein adsorption. (2) Sur- surfactants such as Triton-XTM and BrijÒ (polyoxyethylene lauryl
Fig. 3. Graphical representation of increasing surfactant concentration and prevention of protein denaturation. Reproduced from Lee et al [76] with permission of Elsevier.
8
ether 35 and 78) have also been explored as alternative non-ionic type and concentration of buffer, the initial solution pH, and the
surfactants [80]. concentration of other formulation components (Fig. 4). For exam-
ple, citrate buffer works well for solutions with initial pH of 5.0 or
3.4. Buffers 6.0 but exhibits a pH shift of ~+2 units when the initial pH is 4.0.
Similarly, histidine buffer shows a pH shift of +2 units when used
Solution pH can be one of the determinants of protein stability. at pH 5.5, but not at pH 6.0 and 7.0. Succinate buffer is an example,
As a result, prelyophilization protein formulation compositions where the direction of pH shift is affected by the initial pH (~+1 at
invariably contain a buffer, to control the pH of (i) the pre-lyo solu- pH 4.0 and 5.0 vs ~1.0 at pH 6.0). At pH 6.0, the basic buffer com-
tion, (ii) the maximally freeze-concentrated system, and (iii) the ponent (monosodium succinate) crystallizes, while the acidic com-
reconstituted solution. Similarly, protein solutions which are ponent (b-succinic acid) crystallizes at lower pH values [87].
stored in the frozen state for prolonged time periods are often buf- The buffer concentration can have a pronounced influence on
fered, both for maintaining pH in the ‘‘frozen state” and in the sub- both the magnitude and the nature of pH shift. For example, the
sequently thawed solution. The most commonly used buffer magnitude of pH shift for sodium phosphate buffer pH 7.4, was
systems in protein formulations include - histidine, sodium phos- reduced from 4.1 (at 100 mM) to 2.8 (at 10 mM). Succinate buffer
phate, potassium phosphate, citrate, tris and succinate buffers [6]. displayed a more complex behavior with higher concentration
The selection of buffer for a pharmaceutical protein formulation (200 mM) showing a ‘pH swing’ wherein the pH first increased
is usually based upon the desired pH range, buffering capacity and from 4.0 to 8.0 and then decreased to 2.2 attributed to sequential
the possibility of buffer-specific catalysis [81]. Ideally, the use of a crystallization of buffer components. At lower concentrations (50
buffer is desired at pH values ± 1 unit of the acid dissocaition con- and 100 mM), only unidirectional shifts of a smaller magnitude
stant (pKa), with the highest buffering capacity at pH = pKa. The (~1 pH unit) were observed [87]. It is therefore judicious to use
knowledge of pKa aids in buffer selection. For example, when the the buffers at their lowest effective concentration. Another suc-
desired pH is ~7.0, phosphate buffer (pKa ~ 7.2) is widely employed, cessful approach involves inhibition of buffer crystallization by for-
while histidine (pKa ~ 6.1) is the buffer of choice at pH ~ 6.0 [6]. The mulation components, thereby attenuating any pH shift. Non-
effective range is broadened for buffers with multiple pKas, such as crystallizing solutes such as sucrose are known to inhibit buffer
sodium phosphate buffer (pKas ~ 2.1, 7.2 and 12.3) [81]. Addition- crystallization [93]. Sometimes, the protein itself may inhibit buf-
ally, for lyophilized and frozen formulations, the crystallization fer crystallization, thereby preventing pH shift [92]. This has been
propensity of buffer components during freeze-concentration, discussed in detail in Section 4.
becomes an important consideration [82]. The selective crystalliza-
tion of a buffer component during freezing may lead to pH shifts, 3.5. Polymers
thereby affecting protein stability. Sodium phosphate buffer is a
widely-studied example wherein crystallization of disodium phos- Polymers have been used to stabilize proteins in solution, and
phate dodecahydrate (Na2HPO412H2O; the basic component of also both during freezing (or thawing) and freeze-drying. Polyethy-
phosphate buffer) during freezing lowers the pH of the freeze con- lene glycol (PEG), dextran and polyvinylpyrrolidone (PVP) are com-
centrate and may alter the stability of proteins [83–85]. Hence, it is monly used to cryopreserve proteins [10]. PEG, a crystalline
desirable that the buffer components are retained amorphous dur- polymer, however provides no protection to protein during drying.
ing freezing. However, as exemplified by citrate buffer, significant Dextran and PVP, amorphous polymers, can stabilize proteins in
changes in ionization and apparent pH may be observed even the dried state, by increasing the formulation Tg [24]. However,
when buffer components remains amorphous [86]. the polymers are usually unable to interact with the surface polar
The crystallization behavior of buffers and the associated pH groups of protein. In fact, the inability of dextran to protect enzyme
shifts during freezing depends upon several factors including the catalase during dehydration, in spite of its high Tg, established that
Fig. 4. pH shift shown by different buffer solutions upon freezing, plotted in increasing order (from left to right) of the initial pH. Left panel: buffers showing shifts >1 pH unit
and right panel: buffers showing shifts 1 unit. Filled circles denote the initial pH value of the solution at ~25 °C (also mentioned in the adjacent box) for each buffer solution.
Arrows point the direction of pH shift during freezing and the tip of the arrow marks the final pH after freezing the solution to ~25 °C. Succinate buffer 200 mM displayed a
pH swing and only the first pH shift has been shown in this figure. Data adapted from Bhatnagar et al [88], Kolhe et al [89], Sundaramurthi and Suryanarayanan [90,91] and
Thorat et al [92].
9
the formation of amorphous glassy state, though a necessary con- ride to sucrose-based BSA or rHSA formulations improved their
dition, is not sufficient for protein stabilization [10,21]. storage stability. These results are in contradiction with conven-
There is advantage in using the high Tg polymers, for example tional formulation wisdom [101].
dextran, in combination with sugars. The high Tg of the final lyo-
phile may permit long-term room temperature storage of the for-
4. Interaction of formulation components
mulation. Dextran (40 and 70 kDa) is approved for parenteral use
as a plasma expander. For stabilizing an mAb formulation, a mix-
As is evident from the discussion above, lyophilized protein for-
ture of dextran 40 with sucrose (weight ratio 1:1) provided the
mulations are typically multi-component systems, providing
best formulation, both in terms of efficient freeze-drying cycle
numerous possibilities of excipient-excipient and protein-
and product stability after storage at 40 °C (14 days). Use of high
excipient interactions. Such interactions can influence the physical
molecular weight dextrans (150 and 500 kDa) resulted in unac-
form of some excipients during the different stages of the freeze-
ceptably long reconstitution times, while low molecular weight
drying process as well as in the final drug product. The physical
dextran (1 kDa) led to formation of acidic species in the lyophile
form can influence the excipient functionality and thereby the sta-
[94]. When dextran is used alone, the terminal glucose moiety
bility of the active. The active ingredient being a protein, is likely to
can induce antibody glycation at elevated temperatures [94]. In
be retained amorphous. In this section, we highlight representative
addition, the polymer flexibility will also influence its ability to sta-
examples of excipient-excipient and protein-excipient
bilize proteins as demonstrated by better stabilization with the
interactions.
molecularly-flexible inulin as compared to rigid dextran [34].
3.6. Additional excipients 4.1. Excipient- excipient interactions
Functionalized cyclodextrin (CD) such as hydroxypropyl b cy- 4.1.1. Sugars with other excipients
clodextrin (HPbCD) and sulfobutylether bCD are approved for par- Sugars being the most extensively used excipients in protein
enteral use and their potential use in freeze-dried protein formulations have shown potential influence on the phase behav-
formulations has been investigated. When used at a concentration ior of cosolutes. Both sucrose and trehalose are widely used stabi-
of ~0.1% w/v, HPbCD provided stabilization against interfacial lizers in freeze-dried formulations and their effect on other
stress during freezing-thawing and reconstitution, in a manner excipients is often expected to be qualitatively similar. However
similar to non-ionic surfactants [95]. At higher concentrations, as mentioned in Section 3.1, when frozen solutions are annealed
HPbCD stabilizes protein possibly by water replacement and vitri- often for longer durations, trehalose can crystallize as trehalose
fication. Evidence for water-replacement was provided by compar- dihydrate [37,102,103]. As a result there may be remarkable differ-
ison of lyoprotectant activity between parent and functionalized ences in the way these two sugars affect the cosolutes. Trehalose
CDs. Parent CDs form intramolecular hydrogen bonds which hinder crystallization has the potential to compromise its stabilizing func-
them to hydrogen bond with other molecules. These are likely to tionality. Some examples of the interaction of sugars with other
exhibit little degree of water replacement. Interestingly, the rela- cosolutes, in frozen solutions and during lyophilization, have been
tive activity of LDH lyophilized with hydroxypropylated CDs tabulated (Table 4). A few case studies are discussed in detail
(HPaCD, HPbCD or HPcCD) was higher than that of LDH lyophilized below.
with parent CDs (aCD, bCD or cCD). The increased activity Sugar(s) with bulking agent(s). A sugar-bulking agent combina-
observed for functionalized CDs was attributed to their additional tion offers an avenue to obtain robust lyophilized products with
hydroxyl moieties, possibly contributing to hydrogen-bonding short cycle times [11,53,111]. The sugar remains amorphous and
[96]. CDs usually yield amorphous glasses; for example, freeze- serves as a stabilizer. The bulking agent, on the other hand, crystal-
dried bCD with interleukin 2 had a Tg ~ 108 °C (residual moisture lizes and gives a rigid structure to the cake, thus enabling rapid
2–3% w/w), which might enhance protein stability during storage lyophilization (short cycle time) and often improving the reconsti-
[95]. When used alone, the amount of trehalose or HPbCD, required tution properties [112]. However, the physical state of the two
for the stabilization of immunoglobulin during freeze-drying and excipients and hence their functionality in frozen solution has been
subsequent storage at 40 °C for 3 months, was ~4:1 (stabilizer: pro- found to depend upon the ratio of sugar to bulking agent (R, w/w).
tein) [97]. On the other hand, when used in combination, in an Sugars have been shown to exhibit a concentration dependent
optimized weight ratio of 3.3:1 (trehalose: CD), with stabilizer: inhibitory effect on the crystallization of bulking agents like man-
protein ratio of ~1:1, no protein aggregation was observed in the nitol and glycine [104–107]. An inappropriate sugar to bulking
lyophile even after storage for 2 months at 45 °C. Thus, there agent ratio poses the risk of retaining the bulking agent amorphous
was evidence of synergistic effect between trehalose and HPbCD or facilitating sugar crystallization, thereby resulting in loss in their
[98]. desired functionalities. This is evident from the following
Sodium chloride (NaCl), commonly used to adjust the osmotic examples.
pressure of parenteral solutions, is not desired in lyophilized for- A high sucrose to glycine ratio (R > 4:1), inhibited glycine crys-
mulations, due to the low eutectic temperature (21 °C). In frozen tallization, retaining it predominantly in the amorphous state
solutions, NaCl crystallization is suppressed by amorphous solutes (Fig. 5) [106,113]. Annealing (at 15 °C for 90 min) also failed to
such as sucrose [99] and by crystallizing solutes, such as mannitol crystallize glycine under these conditions. On the other hand, at
and glycine [100]. This inability of NaCl to crystallize completely low sucrose to glycine ratios (R < 2:3), crystallization of glycine
lowers the collapse temperature of the system. In addition, NaCl was complete even without annealing. Intermediate compositions
also inhibits crystallization of mannitol in frozen solutions [100]. retained glycine in a partially crystalline state in the final lyophile
Metal ions, specifically divalent cations like zinc, copper, or cal- [106]. Such an interaction became even more complex, when tre-
cium, can bind to specific protein binding sites and may stabilize halose was used with mannitol [108]. A high trehalose to mannitol
(but in some cases also destabilize) the native protein conforma- ratio (R = 3:1) completely inhibited mannitol crystallization in the
tion [24]. As an example, calcium chloride is commonly used as a frozen state, retaining it amorphous. At R = 1:1, mannitol facili-
complexing agent in lyophilized products containing coagulation tated the crystallization of trehalose as trehalose dihydrate. How-
factors, whose activity and stability is promoted in presence of cal- ever, at a much lower ratio, R = 1:3, mannitol crystallized, but
cium ions [6]. In an interesting study, the addition of sodium chlo- trehalose did not [108]. An ideal scenario would be where the bulk-
10
Table 4
Examples of interaction of sugars with bulking agents (R = ratio of sugar to bulking agent, w/w) and buffers.
Effect Sugar Excipient Comment Reference

A. Sugar with bulking agent
Inhibition of crystallization of Sucrose Mannitol R > 1:1 Inhibition of mannitol crystallization in frozen solutions. Amorphous lyophile without [104]
the bulking agent annealing. With annealing, presence of crystalline mannitol in lyophile.
Sucrose inhibited mannitol crystallization in a concentration dependent manner. Mannitol [105]
crystallized only at R < 1:2.
Sucrose Glycine Sucrose inhibited glycine crystallization in a concentration dependent manner. Glycine [106]
remained substantially crystalline at R < 2:3, amorphous at R > 4:1, and partially crystalline at
intermediate compositions.
Trehalose Mannitol The addition of trehalose inhibited mannitol crystallization (upon sub Tg’ annealing) in frozen [107]
solutions when R 7:3. The inhibitory effect was lost during primary drying.
Trehalose inhibited mannitol crystallization in frozen solution at R = 3:1, while mannitol [108]
induced trehalose crystallization at R = 1:1. At R = 1:3, trehalose was retained amorphous,
while mannitol crystallized.
Alteration in the solid form of Sucrose Mannitol R = 1:4, lyophilized product (without annealing) contained crystalline mannitol with MHH [104]
the bulking agent being the major crystallizing form
Induction of sugar Sucrose Mannitol The water released by dehydration of MHH in the lyophile (R = 1:4) promoted sucrose [55]
crystallization crystallization.
Trehalose Mannitol R = 1:1, mannitol induced the crystallization of trehalose dihydrate in frozen solutions. [108]
Trehalose, Mannitol Mannitol facilitated the crystallization of trehalose dihydrate when solution containing [109]
Sucrose trehalose and mannitol (R = 2:1), were annealed at 18 °C. A fraction of the dihydrate was
retained in the final dried solid as well. Sucrose (R = 2:1) inhibited the crystallization of
trehalose dihydrate.
B. Sugar with buffers
Inhibition of buffer Sucrose, Succinate At a 50 mM buffer concentration, sugars (2% w/v) inhibited buffer crystallization and [93]
crystallization and Trehalose buffer associated pH shift, irrespective of pH. However, at higher buffer concentration (200 mM),
subsequent pH shift sugars inhibited buffer crystallization when the initial pH was 6.0 but failed to do so at pH 4.0.
Sucrose or trehalose (5.5% w/v) inhibited phosphate buffer (160 mM) crystallization and [110]
decreased the magnitude of pH shift.
Change in ionization constant Trehalose Citrate The extent of protonation of the indicator dye (bromophenol blue) was lower in trehalose– [86]
upon dehydration buffer citrate lyophiles than in citrate samples
Another interesting implication of the sugar-bulking agent

interaction is the emergence of the cryoprotective ability of the
bulking agent. Glycine when retained amorphous, in presence of
sucrose, exerted additional stabilizing effect on the protein during
freezing. While sucrose remained the primary stabilizing agent for
the protein in the solid state, inclusion of some amorphous glycine
caused a significant increase in stability [114]. This could be a
potentially useful approach if the bulking agent crystallizes com-
pletely during the subsequent drying steps. Otherwise, from the
perspective of physical stability, retention of the bulking agent in
the amorphous state in the lyophile is not desirable. The amor-
phous bulking agent may crystallize, during storage or transport,
thereby releasing the associated sorbed water. This released water
may interact with the other formulation components [108].
To summarize, the formulator must judiciously select the
weight ratio of sugar to bulking agent and optimize the processing
conditions so as to achieve a stable freeze-dried product with the
Fig. 5. The effect of sucrose concentration and annealing on the crystallinity of desired attributes.
freeze-dried glycine. Reproduced from Bai et al [106] with permission from Elsevier. Sugars with buffers. Sugars can inhibit buffer salt crystallization
in frozen solutions, thereby attenuating any pH shift brought about
ing agent crystallizes, but the stabilizer is retained amorphous, i.e. by selective crystallization of buffer components. The magnitude of
R < 2:3 and 1:3, for sucrose-glycine and trehalose-mannitol com- this effect depends upon the individual concentrations of sugar and
binations respectively. buffer, and the initial formulation pH. Trehalose and sucrose, in a
Similarly, a sucrose to mannitol ratio of 1:4, has been used to concentration dependent manner, inhibited the pH shift associated
successfully stabilize numerous proteins in lyophilized formula- with crystallization of phosphate buffer (160 mM, initial pH 7.2)
tions [53,55]. However, sucrose can affect the physical form of during freezing [110]. The pH shift was completely inhibited when
the crystallizing mannitol. At least a fraction of mannitol crystal- the molar concentration of sugar (5.5% w/v; ~160 mM) and buffer
lizes as the metastable MHH. As mentioned above, MHH is not were identical. In another case study, sucrose and trehalose effec-
desired in the freeze-dried cake, since its dehydration can lead to tively inhibited succinate buffer component crystallization irre-
water release. The released water may interact with the other for- spective of pH, when the molar concentrations of the buffer and
mulation components. For example, the water released by the sugar were about the same (50 mM buffer with 2% w/v sugar;
dehydration of MHH in the lyophile (prepared from solution con- ~58.5 mM). However, at a higher buffer concentration (200 mM
taining 4% w/w of mannitol and 1% w/w of sucrose) during storage buffer with 2% w/v sugar), the outcome was pH dependent. Both
induced sucrose crystallization [55]. Hence, it is desirable to cause trehalose and sucrose inhibited buffer crystallization when the
complete MHH dehydration during secondary drying. initial pH was 6.0 but failed to do so at pH 4.0. At the lower pH,
11
crystallization of succinate buffer component induced trehalose of d-mannitol, at higher concentrations, b-mannitol was obtained
crystallization. Sucrose, on the other hand, degraded, yielding crys- [120].
talline decomposition products thus failing to prevent pH shift With sodium chloride. In the presence of sodium chloride, man-
[93]. Such interplay warrants caution while selecting the buffer nitol may be retained amorphous during freezing. The effect was
and sugar concentration. Specifically, at low buffer concentrations evident even when the NaCl content in the prelyophilization solu-
(50 mM), the inhibitory effect of sugars is expected to assist in tions were as low as 0.5% w/v. However, annealing near Tg’ led to
retaining the buffer amorphous in frozen solutions, hence prevent- crystallization of both the components. Increasing the NaCl con-
ing loss of its functionality. Additionally, sugars can reduce the centration to 1% w/v made mannitol crystallization significantly
freeze-drying induced changes in ionization and apparent pH more difficult. Inhibition of NaCl crystallization with non-
when the buffer remains amorphous [86]. crystallizing sugars such as sucrose is well documented. Interest-
ingly, mannitol which is a crystallizing solute, also inhibited NaCl
4.1.2. Bulking agents with other excipients crystallization. These interactions can have significant implications
Bulking agents are generally used for low dose protein formula- on Tg’ and hence the primary drying temperature [100]. NaCl also
tions that per se do not have the necessary bulk to support their inhibited the crystallization of mannitol during freezing in formu-
own structure. Though mannitol and glycine are the most common lations containing mannitol and sucrose, though both mannitol
bulking agents, glucose, sucrose, lactose, dextran and amino acids and NaCl crystallized during lyophilization [104]. Neutral glycine
are other examples. In the final lyophile, a bulking agent may be solutions containing sodium chloride also showed no glycine crys-
crystalline or amorphous [115]. For crystallizing agents, such as tallization [117].
mannitol and glycine, their crystallization propensity as well as
the polymorphic form is influenced by other formulation 4.2. Protein-excipient interaction
components.
With buffers: As already mentioned, selective crystallization of a While rational design of formulations with respect to excipients
buffer component in frozen solution can result in a pronounced pH is a central theme, it is equally important to be aware of the effect
shift. Importantly, bulking agent and buffers may influence the of protein on excipient phase behavior (Table 5). Representative
crystalline propensity of each other. It is important to understand examples which highlight the interplay of proteins and excipients
the influence of cosolutes on buffer crystallization, since this will can be classified into the following categories.
influence the freeze-concentrate pH and hence the protein
stability. 4.2.1. Effect of protein on excipient phase behavior
When solutions containing sodium phosphate buffer (10 or Inhibition of sugar alcohol crystallization. In multicomponent sys-
100 mM) and glycine (0.8% w/v) were frozen, crystallization of tems, non-crystallizing solutes (lyoprotectant and protein) can
both the components was facilitated. Though this did not affect influence the crystallization behavior of bulking agents or other
the functionality of glycine as a bulking agent, the pronounced crystallizable solutes. Mannitol crystallization was inhibited by a
crystallization of the buffer salt led to pH shifts upon freezing mAb, the effect being concentration dependent and pronounced
[116]. Interestingly, the presence of glycine increased the pH shifts at mAb concentrations >20 mg/mL [105]. A similar effect was
upon freezing, for both 10 and 100 mM sodium phosphate buffers. observed when a Fc-fusion protein at concentrations 80 mg/mL
However, at low concentrations (0.4% w/v), glycine prevented resulted in inhibition of sorbitol crystallization [121]. The potential
any pH shifts in phosphate buffers upon freezing. However, at such implications of such crystallization inhibition are discussed in
a low concentration, glycine might not provide the bulk and cake Table 5.
structure. Thus, judicious selection of both the formulation compo- Inhibition of buffer crystallization. As discussed above, sodium
nents and their composition is needed to ensure the functionality phosphate buffers are known to undergo pH shift as a consequence
of both the components and obtain a stable and elegant formula- of selective buffer component crystallization. At low sodium phos-
tion. Solutions containing glycine (2% w/v) and 50 mM sodium phate buffer concentration (10 mM), BSA and b-galactosidase
phosphate buffer were annealed at 20 °C. This annealing step (10 mg/mL) inhibited buffer salt crystallization and attenuated
led to the desired glycine crystallization along with minimal buffer the pH shift. When the buffer concentration was increased to
salt crystallization [117]. The study demonstrated a successful 100 mM, the protein did not significantly inhibit buffer crystalliza-
approach to retain functionality of both the components at phar- tion. The consequent pH shift resulted in protein aggregation [92].
maceutically relevant concentrations, i.e. crystallization of bulking Modification of the physical form of excipients. Bulking agents,
agent, but not of the buffer component. such as mannitol and glycine, are expected to crystallize during
Similarly, mannitol crystallization was also inhibited by phos- lyophilization. While at higher protein: excipient ratios, proteins
phate buffer (concentration 100 mM) [118]. When mannitol can inhibit the crystallization of bulking agent, at lower ratios they
and glycine were used in combination, the phosphate buffer salts appear to influence the polymorphic form of bulking agent. For
significantly inhibited the crystallization of both of these solutes example, in freeze-dried mannitol-lysozyme formulations, with
in frozen solutions. The resultant lyoprotectant action of the increasing protein concentration, the prevalence of mannitol poly-
mannitol-glycine combination was a function of the fraction of morphic form shifted from b- to d-mannitol [122]. Importantly, the
the solutes that were retained amorphous in the lyophile. An polymorphic form of anhydrous mannitol does not seem to have
increase in buffer concentration decreased the crystallinity of man- any measurable impact on the quality attributes of the drug pro-
nitol and glycine and this, in turn, translated to a higher retention duct, while formation of the metastable MHH can have significant
of enzyme activity [119]. In general, only an amorphous cosolute implications. It is not possible to generalize the manner in which
inhibited buffer crystallization. However, when the co-solute crys- MHH formation is influenced by proteins. With an Fc-fusion pro-
tallized, buffer crystallization was also observed. tein, the maximum MHH content was observed at an intermediate
With Surfactant. When mannitol solutions, with progressively protein concentration, with higher protein content inhibiting man-
increasing concentrations of polysorbate 80 were lyophilized, as nitol crystallization. Addition of sucrose to the formulation also
expected, there was a progressive increase in specific surface area facilitated MHH formation [123]. Liao et al have shown that
of the lyophile. The surfactant concentration also influenced the MHH formation was inhibited in the presence of an albumin
physical form of mannitol in the final lyophile. While a low fusion protein [124], while Cao et al reported that a Fc-fusion
polysorbate concentration led to the preferential crystallization protein facilitated MHH formation in a concentration dependent
12
Table 5
Effect of excipient crystallization on protein stability. The experimental conditions of crystallization and corresponding excipient crystallizing are in bold font.
Protein (Concentration) Sugar/ Excipients Process Comments Reference

Polyol conditions
A. Frozen system
Fc-fusion protein Sorbitol Sodium acetate buffer (10 mM) Stored at 20, At 30 °C, sorbitol crystallized and protein [127]
(2 mg/mL) (300 mM) pH 4 and 5 30 or 70 °C aggregated
for 1 year
IgG1 and IgG2 Sorbitol Sodium acetate buffer (10 mM) Stored at 20, At 30 °C, Sorbitol crystallized and IgG [121]
(0.1–120 mg/mL) (~300 mM) pH 4 and 5 30 or 70 °C aggregated. At IgG concentration 40 mg/mL,
inhibition of sorbitol crystallization; decreased
protein aggregation.
IgG2 Trehalose Histidine HCl buffer (20 mM), Stored at 10, At 20 °C, IgG aggregation, hypothesized due to [128]
(20 mg/mL) dihydrate polysorbate 80 (0.2 mg/mL), 20 or 40 °C trehalose crystallization
(84 mg/ disodium edetate dihydrate
mL) (0.1 mg/mL) pH 5.5
mAb (25 mg/mL) Mannitol, NaP buffer(51 mM), polysorbate Stored at 20° mAb aggregation attributed to mannitol or [103]
trehalose 20 (0.04% w/v) C for 28 days trehalose crystallization
or sucrose Sucrose retained amorphous and no mAb
(6% w/w) aggregation.
mAb (25 and 100 mg/mL) Trehalose NaP buffer (51 mM), polysorbate Stored at mAb 25 mg/mL and trehalose
(0–34.2% 20 0.04% (w/v) 20 °C for concentration 1.7% w/w.: mAb aggregated
w/v) 1 year and trehalose crystallized
mAb 100 mg/mL and trehalose concentration
3.4% w/w.: Trehalose partially crystallized
and no mAb aggregation.
Stored at
40 °C for Storage below Tg’ prevented trehalose
1 year crystallization and protein aggregation
B. Freeze thaw
monomeric b - galactosidase – NaP or KP buffer Combination of NaP: Aggregation and pronounced pH shift [116]
(2 mg/mL) (10 or 100 mM) slow and fast due to disodium salt crystallization*
cooling and KP: No aggregation. The pH shift much less with
thawing monopotassium salt crystallization
tetrameric b - galactosidase – NaP or KP buffer NaP: Aggregation and pronounced pH shift
(25 mg/mL) (10 or 100 mM) due to disodium salt crystallization*
No aggregation. The pH shift much less with
monopotassium salt crystallization.
BSA (10 mg/mL) or b - galactosidase – NaP buffer (10 or 100 mM) or KP Uncontrolled NaP 10 mM: No aggregation, crystallization of [92]
(10 mg/mL) buffer (100 mM) ramp rate disodium salt inhibited in the presence of
protein
NaP 100 mM: Aggregation and disodium salt
crystallization
KP: Monopotassium crystallization did result in
protein aggregation
5% NaP buffer 100 mM Cellobiose inhibited disodium salt
Cellobiose crystallization and in turn prevented protein
aggregation
C. Freeze-dried (unannealed)
monomeric b - galactosidase (2, 25, None NaP buffer (10 or 100 mM) or KP Protein concentration did not have any effect on [136]
100 and 1000 mg/mL) or buffer (10 or 100 mM) buffer salt crystallization.
tetrameric b - galactosidase (2, Activity recovery lower in NaP (100 mM) buffer
25, 100 and 1000 mg/mL) solution due to disodium salt crystallization*
Monopotassium crystallization did result in
protein aggregation. Activity recovery lower
after freeze-drying due to other stresses
monomeric and tetrameric b - Sucrose NaP buffer (100 mM) Optimum activity recovery when sucrose was
galactosidase (100 or used as a lyoprotectant
(1 mg/mL) 500 mM)
Sucrose NaP buffer (10 mM), PEG 8000
(100 mM) (1%)
Sucrose NaP buffer (10 mM), Tween 80
(100 mM) (0.1%)
Note: NaP: sodium phosphate; KP potassium phosphate; *The disodium salt crystallization as disodium phosphate dodecahydrate (Na2HPO412H2O).
manner (upon annealing at 20 °C) [125]. In a mannitol-sucrose 4.2.2. Effect of excipient phase behavior on protein stability
(1:1) formulation, increasing lysozyme or BSA content pro- Unintended crystallization. Freezing is a common unit operation
moted MHH formation. On the other hand, with mannitol to in freeze-drying and frozen storage in the manufacturing of bio-
sucrose ratio 4:1, increasing the protein content reduced MHH logic formulations. The protein DS in presence of stabilizers is often
formation [126]. stored frozen for months prior to its formulation into a drug
13
product. From an economical and practical standpoint, 20 °C halose (Fig. 7a). The lower solubility of mannitol and trehalose
(±5°C) is a convenient storage temperature [4]. However, most of translated to their much higher crystallization propensity as well
the protein formulations are not completely frozen at this temper- as mAb aggregation. These formulations were stored at 20 °C
ature and can exist as a mixture of ice and a freeze-concentrate. for 28 days and seeded with respective excipients to accelerate
The storage temperature is critical in determining the long-term crystallization. No aggregation was detected in sucrose formula-
stability of frozen systems. tions, while aggregates increased by 1 and 3% in trehalose and
An Fc-fusion protein (2 mg/mL) containing sorbitol (5% w/v) mannitol formulations respectively (Fig. 7) [103]. The relationship
was stored frozen at 20, 30 and 70 °C for >1 year. Storage at between mannitol crystallization and protein aggregation is well-
the intermediate temperature of 30 °C resulted in least stability known. Mannitol has a high propensity to crystallize during freez-
[127]. At 30 °C, which is 15 °C higher than the Tg’ (-45 °C) of ing, on annealing and on long-term storage which can induce pro-
the matrix, unintended sorbitol crystallization resulted in loss of tein aggregation [49,118,129,130].
its cryoprotectant activity. In a follow-up study, two mAbs (IgG1 In addition, the effect of varying trehalose and mAb concentra-
and IgG2) were kept frozen in solutions containing 274 mM sor- tions were investigated to determine the optimum trehalose to
bitol and 10 mM sodium acetate buffer at concentrations ranging mAb ratio that can result in a stable formulation with minimum
from 0.1 to 120 mg/mL. When IgG2 was stored at 30 °C, a protein aggregation on 12-month storage at 20 °C. Trehalose crystalliza-
concentration dependent inhibition of sorbitol crystallization was tion was observed at all concentrations of mAb from 0 to 100 mg/
observed. Sorbitol crystallization and hence protein aggregation mL. However, the fraction of trehalose that crystallized reduced
was observed at concentrations <60 mg/mL. However, at higher from 53% at 25 mg/mL mAb concentration to 9% at 100 mg/mL
concentrations 80 to 120 mg/mL, sorbitol crystallization was inhib- mAb concentration. It was concluded that trehalose to mAb ratio
ited preventing protein aggregation (Fig. 6). in the range of 0.2:1 to 2.4:1 is required for optimum stability. At
An IgG2 mAb (20 mg/mL) solution containing trehalose (8.4% w/ ratios <0.2:1, trehalose concentration was not sufficient to act as
v) and other formulation components was stored for 12 months at a cryoprotectant and at ratios >2.4:1, the high trehalose concentra-
10, 20 and 40 °C. Aggregation of mAb was highest at 20 °C. It tion resulted in its crystallization [103].
was hypothesized that, trehalose crystallization at a temperature Crystallization of other formulation excipients such as buffers
above the Tg’ (29 °C) of the matrix resulted in loss in the cryopro- can result in pH shift during freezing and consequent protein
tectant activity at 20 °C. Although trehalose crystallized at aggregation, especially if the protein stability is pH dependent.
10 °C, the mobility in the system was high leading to refolding When cooled from RT to 25 °C, crystallization of disodium phos-
of the protein resulting in lesser aggregation. At 40 °C which is phate in 10 mM and 100 mM sodium phosphate buffers (inital pH
well below the Tg’ of the system, trehalose was retained amor- 7.0) was shown to cause pH shift of 1.5 and 3.2 units respectively.
phous and no aggregation was observed. Notably, crystallization Monomeric and tetrameric b- galactosidase (2 mg/mL to 1000 mg/
of trehalose at 10 °C did not result in loss in cryoprotection. This mL) did not have an inhibitory effect on disodium phosphate crys-
study highlighted that crystallization of cryoprotectant alone is not tallization. The recovered protein activity was lower in solutions
responsible for protein aggregation. Factors such as storage tem- with higher buffer concentration. On the other hand, in spite of
perature, ice-interface denaturation, mobility in the frozen system crystallization of monopotassium phosphate in 10 and 100 mM
along with cold denaturation effects should be thoroughly investi- potassium phosphate buffer, the pH shift in the frozen state was
gated [128]. only 0.1 and 0.3 units respectively. Aggregation of monomeric
The effect of temperature on the aqueous solubility of mannitol, and tetrameric b- galactosidase in this case was lower when com-
trehalose and sucrose were compared and irrespective of temper- pared to sodium phosphate buffer. The lower aggregation however
ature, sucrose had a much higher solubility than mannitol and tre- was attributed to factors other than buffer crystallization such as
ice-interface induced denaturation or cold denaturation [131,132].
Intended crystallization. In high concentration protein formula-
tions (>50 mg/mL), a bulking agent is not often required. However,
the ability of mannitol to crystallize on annealing in formulations
with 100 mg/mL recombinant protein, resulted in a dramatic
reduction in reconstitution times. Although increasing the protein
concentration inhibited mannitol crystallization, even the crystal-
lization of a small fraction of mannitol facilitated rapid reconstitu-
tion [112]. When a mAb solution (100 mg/mL) containing arginine
and glycine was freeze-dried, the cake was partially crystalline. It
showed shorter constitution time when compared to cakes that
were amorphous [133].
In protein formulations containing mannitol and sucrose, man-
nitol crystallization is desirable for it to act as a bulking agent.
However, annealing induced crystallization of mannitol led to
decrease in LDH activity recovery and sucrose did not act as a
lyoprotectant. LDH activity recovery increased on addition of
Tween 80, suggesting instability due to interfacial phenomenon
which can be due to ice or mannitol crystallization [134]. In
trehalose-mannitol formulations, at lower BSA concentrations
Fig. 6. IgG2 (40 to 120 mg/mL) aqueous solutions with 274 mM sorbitol in 10 mM
sodium acetate buffer and 0.004% w/v polysorbate 20 were stored at 30 °C for (4% w/w), lyophiles contained crystalline mannitol as both hemi-
24 months. Aggregate formation (%) at respective time points as a function of IgG2 hydrate and anhydrous forms whereas trehalose crystallized as
concentration are shown. The filled symbols denote crystallized sorbitol whereas the dihydrate form. At a higher BSA concentration (10% w/w), the
empty symbols denote amorphous sorbitol. At concentrations 60 mg/mL, sorbitol lyophiles contained only the d-mannitol and trehalose was
crystallized resulting in higher % aggregates whereas, at concentrations 80 mg/mL
sorbitol crystallization was inhibited. Slightly higher % aggregates observed at
retained amorphous [135]. In this case, at higher BSA concentra-
higher IgG2 concentration maybe due to other factors including protein–protein tions, trehalose will act as an effective stabilizer while mannitol
interactions. Reproduced from Piedmonte et al [121] with permission of Elsevier. will be an effective bulking agent.
14
Fig. 7. a) Solubility of sucrose (blue), trehalose (red) and mannitol (green) as a function of temperature. b) Bar plot of percentage high molecular weight aggregates (HMWS)
of mAb2 in formulation containing 6% w/v sucrose, trehalose or mannitol before freezing (red) and after 28 days of storage at 20 °C (blue). Reproduced from Connolly et al
[103] with permission from Elsevier. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
From a formulation point of view, the above studies show that The effect of storage temperatures and time, freezing rates,
crystallization of a stabilizer, such as trehalose or sorbitol, can sig- excipient and protein concentrations needs further investigation
nificantly destabilize protein formulations. for optimizing DS stability. Frozen DS, stored at relatively higher
storage temperatures (20 °C), possess adequate molecular mobil-
5. Future directions ity to induce changes in excipient phase behavior, eventually com-
promising stability. Stability testing for frozen DS should be carried
With an improved understanding of the stability behavior of out in real time at temperatures above and below Tg’. This will help
proteins in a multi-component DP, protein formulation develop- establish excipient effect on protein stability.
ment has evolved, from a predominantly trial-and-error process, Prediction of solid-state stability of lyophilized protein formula-
to a more systematic science-driven process. Nonetheless, a num- tion relies upon identifying key properties influencing protein sta-
ber of challenges still exist in the formulation development pro- bility. Novel analytical techniques, such as micro-Raman
cess. As is evident from the discussion above, the stresses spectroscopy [42], small-angle scattering [138], broadband coher-
experienced by proteins during freezing/freeze-drying and mecha- ent anti-Stokes Raman scattering [139], solid-state hydrogen/deu-
nisms of excipient-induced protein stabilization are not com- terium exchange mass spectrometry [140] and modelling tools
pletely understood. Multiple mechanisms have been proposed, such as energy landscape model [141], molecular dynamic simula-
but their validity seems to depend upon the type of biomolecule tions [142] can help in further establishing the connection. An
(s) and the process conditions. A broad generalization may not improved understanding of stabilization mechanisms will assist
be appropriate. However, recent efforts in the field of molecular in effective protein stabilization and reduce developmental
dynamics and simulations look promising in identifying excipient timelines.
ratios as well as determining effect of stresses during freezing on With the increasing popularity of high-concentration protein
protein stability. formulations, the identification of excipients with multifunctional
There is a growing interest in studying previously unidentified properties will enable the formulator to decrease the number
stresses experienced by protein during freezing, such as air bub- and concentration of excipients. On the other hand, identifying
bles formed on the ice crystallization front, local pressure and novel stabilizers might be a potential avenue, though the approach
mechanical stresses due to volume expansion during ice formation would be associated with the hurdles of toxicity and regulatory
[4]. It is hypothesized that the quasi-liquid layer, i.e., a thin film of approval process [143].
liquid water formed on ice crystals, plays an important role in pro-
tein stability. The physical microenvironment of a protein confined 6. Summary
within this layer may be substantially different than in the bulk.
The freeze-induced destabilization might be attributed to the pre- The selection of the appropriate excipients provides the avenue
ferred partitioning of proteins into the layer and hence inability of to stabilize proteins sensitive to freezing and/or drying stresses.
stabilizer to provide cryoprotection [137]. More research in this While the stabilization mechanism is incompletely understood, it
area can provide mechanistic insights into properties and func- is evident that the stabilizers encompass a wide variety of com-
tional relevance of this layer and to help identify mitigation strate- pounds, including sugars/sugar alcohols, amino acids and poly-
gies to overcome the freezing-associated stresses. mers. A sugar to protein weight ratio 1 is generally
An understanding of surfactant-induced cryoprotection war- recommended. The final formulation may also require incorpora-
rants further investigations, especially with respect to the potential tion of buffers, surfactants and bulking agents. Since the protein
for freeze-concentration to increase the surfactant concentration can influence the physical properties of the excipients and vice
above CMC. The strategies to circumvent degradation and toxicity versa, the formulation composition should be based on sound
issues associated with polysorbates are of interest. experimentation and appropriate stability studies.
15
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Advanced Drug Delivery Reviews 173 (2021) 1–19

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Stabilizers and their interaction with formulation components in frozen

4.2.1. Effect of protein on excipient phase behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction 2. Processing-induced stresses and stabilization mechanisms

S. Property of interest Implications Sucrose Trehalose

B. Behavior an aqueous solution

C. Solid state properties

Possibility of unintended phase No Yes, Complex phase transformations

11 Glass transition 65–75 °C 110–120 °C

Process Sucrose Trehalose

Amino acid as stabilizer Protein Comments Reference

15 AAs rHSA Protein :sucrose: AA (1:1:0.3 w/w) [57]

Serine, phenylalanine and glycine IgG Stabilization of IgG [64]

3.6. Additional excipients 4.1. Excipient- excipient interactions

Effect Sugar Excipient Comment Reference

Another interesting implication of the sugar-bulking agent

Protein (Concentration) Sugar/ Excipients Process Comments Reference

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