Pharmaceutical Research, Vol. 14, No. 8, 1997
`
`Review Article
`
`Rational Design of Stable Lyophilized Protein Formulations:
`Some Practical Advice
`
`John F. Carpenter,1•2•6 Michael J. Pikal,3 Byeong S. Chang,4 and Theodore W. Randolph1•5
`
`Received March 7, 1997; accepted May 15, 1997
`
`KEY WORDS: protein drugs; design of fonnulations; lyophilization; stabilization of proteins.
`
`WHY USE LYOPHILIZATION TO PREPARE
`STABLE PROTEIN DRUG PRODUCTS?
`
`Early in the development of a protein therapeutic 1t 1s
`essential to design a formulation that is stable during shipping
`and long-term storage. Obviously, an aqueous liquid formula(cid:173)
`tion is the easiest and most economical to handle during manu(cid:173)
`facturing, and is the most convenient for the end user. However,
`many proteins are susceptible to chemical (e.g., deamidation
`or oxidation) and/or physical degradation (e.g., aggregation and
`precipitation) in liquid formulations ( 1,2). It may be possible
`to design an aqueous formulation to slow protein degradation
`adequately, under controlled storage conditions (i.e., constant
`temperature and minimal agitation). However, during shipping,
`when precise control of conditions is not always feasible, prod(cid:173)
`ucts can be subjected to numerous stresses that denature pro(cid:173)
`teins. These include agitation, high and low temperatures, and
`freezing (2). Furthermore, although a formulation and shipping
`system might be designed to circumvent damage from these
`stresses, it still may not be possible to inhibit damage suffi(cid:173)
`ciently during long-term storage. For example, there are cases
`where conditions that minimize chemical degradation foster
`physical damage and vice versa (1,2). Then, conditions that
`provide a compromise affording the requisite long-term stability
`cannot be found.
`All of these difficulties theoretically can be avoided with
`a properly prepared lyophilized formulation. In the dried solid,
`degradative reactions can be avoided or slowed sufficiently,
`such that the protein product remains stable for months or
`years at ambient temperatures (3-6). Furthermore, short-term
`
`1 University of Colorado Center for Phannaceutical Biotechnology,
`School of Phannacy, University of Colorado Health Sciences Center,
`Denver, Colorado 80262.
`2 Department of Pharmaceutical Sciences, School of Phannacy, Univer(cid:173)
`sity of Colorado Health Sciences Center, Denver, Colorado 80262.
`3 School of Phannacy, University of Connecticut, Storrs, Connecti(cid:173)
`cut 06269.
`4 Amgen, Inc., Thousand Oaks, California.
`5 Department of Chemical Engineering, University of Colorado, Boul(cid:173)
`der, Colorado.
`6 To whom correspondence should be addressed. (e-mail: John.
`Carpenter@UCHSC.edu)
`
`excursions in temperature control during shipping are usually
`not damaging to a lyophilized protein (6). Even in cases where
`two, or more, degradative pathways require different conditions
`for maximum thermodynamic stabilization, the reduced reaction
`rates in a dried product can allow for long-term stability. Thus,
`in general, whenever preformulation studies indicate that suffi(cid:173)
`cient protein stability cannot be achieved in aqueous liquid
`formulations,
`lyophilization provides
`the most attractive
`alternative.
`However, lyophilization requires sophisticated processing
`and is time consuming and expensive, relative to simply filling
`vials with a liquid formulation (3-10). Also---0f greatest con(cid:173)
`cern for the current review-without appropriate stabilizing
`excipient(s) most protein preparations are at least partially dena(cid:173)
`tured by the freezing and dehydration stresses encountered dur(cid:173)
`ing lyophilization (2,3-6, 11-16). The result is often irreversible
`aggregation of a fraction of the protein population, either imme(cid:173)
`diately after processing or after storage (e.g., 15,16). Because
`most protein drugs are delivered parenterally, only a few percent
`of aggregated protein will be unacceptable. Finally, simply
`designing a formulation that allows the protein to survive the
`lyophilization process does not assure stability during long(cid:173)
`term storage in the dried solid (6,13-16). A poorly formulated
`lyophilized product, in which the protein is sufficiently reactive
`to require storage at subzero temperature, should not be consid(cid:173)
`ered a success.
`The purpose of this mini-review is to provide some practi(cid:173)
`cal guidelines for designing formulations that protect proteins
`during freezing and drying, and that are stable during shipping
`and long-term storage at ambient temperatures. Also, as will
`be discussed briefly, formulations must be designed with consid(cid:173)
`eration of the physical constraints on processing conditions
`needed to obtain a proper final cake with a low residual mois(cid:173)
`ture. All of these issues have been reviewed previously in detail
`(3-10). Furthermore, relevant new reviews by us and others
`will appear this year in books edited by Vincent Lee and Louis
`Rey. We will not discuss the design and optimization of lyophili(cid:173)
`zation cycles. Nor will we digress from practical advice about
`excipient choices to address the debates about the mechanisms
`by which these compounds stabilize proteins (see 2,5,6, 11-16).
`The pharmaceutical scientist who has had extensive experience
`bringing lyophilized protein products to market may not benefit
`
`969
`
`0724-8741/97/0800-0969$12.50/0 © 1997 Plenum Publishing Corporation
`
`AMNEAL EX. 1011
`
`

`

`970
`
`Carpenter, Pikal, Chang, and Randolph
`
`greatly from this review. Rather, our goal is to provide a starting
`point for the researcher for whom design of stable lyophilized
`protein formulations is still a new and major challenge.
`
`WHAT CONSTRAINTS GOVERN THE DESIGN OF
`THE FORMULATION
`
`There are so many factors to consider when designing a
`proper lyophilized formulation, that the task when viewed as
`a whole can appear overwhelming. This need not be the case,
`if the major constraints governing success are well understood.
`
`Protein Stability
`
`First, it must be remembered that the whole reason for
`lyophilizing the product is because the protein of choice is
`unstable. The most sensitive element in the formulation is the
`protein, and the primary concern in formulation design must
`be the choice of excipients that provide optimal stability. This
`is the issue on which we will focus in detail below.
`
`Final Product Configuration
`
`Secondly, the final product configuration must be clearly
`defined prior to starting formulation efforts. Issues to be consid(cid:173)
`ered include route of administration, which is often parenteral,
`other agents to be co-administered to the patient, product vol(cid:173)
`ume, protein concentration, and whether the product can be
`lyophilized in vials or whether alternative systems such as
`syringes must be employed. Also, if the final product is intended
`for multi-use, it will necessary to include a preservative in the
`formulation, which may reduced protein stability.
`
`Formulation Tonicity
`
`In choosing excipients, designing an isotonic solution
`might be a concern. Mannitol or glycine are usually good
`choices as tonicity modifiers. As explained below, these excipi(cid:173)
`ents are often preferable to NaCl, which due to its relatively
`low eutectic melting and glass transition temperatures, can make
`a formulation more difficult to lyophilize properly (3-10, 17-20).
`Also, if the product has a relatively low mass of protein per
`vial, often it will necessary to have a bulking agent in the
`formulation to prevent the protein from being lost from the vial
`during drying (e.g., 4-6). Mannitol or glycine can also serve
`this role because they usually crystallize to a substantial degree
`during lyophilization and form a mechanically strong cake
`(4-6). However, it must be realized that crystalline excipients
`when used alone will usually not provide adequate stability to
`most proteins during processing or storage in the dried solid
`(12-14).
`
`Cake Structure
`
`Finally, the dried product must have an elegant cake struc(cid:173)
`ture, which is mechanically strong and has not undergone any
`collapse and/or eutectic melting and in which the residual mois(cid:173)
`ture is relatively low (ca. 1 g H2O/100 g dried solid). If the
`product collapses, it will not only be aesthetically unacceptable,
`but also it could have excessively high residual moisture, and
`reconstitution time will be prolonged (3-6,17,18).
`
`Product Glass Transition Temperature
`
`Also, to assure long-term stability of the protein in the
`dried solid, the glass transition temperature (Tg) of the amor(cid:173)
`phous phase in the product, which contains the protein, must
`exceed the planned storage temperature ( 4-6, 14-16). Since
`water is a plasticizer of the amorphous phase (3-6,9, 17, 18),
`low residual moisture is needed to insure that Tg is greater
`than the highest temperature encountered during shipping and
`storage (usually greater than 40°C).
`
`Product Collapse Temperature
`
`In general, achieving these goals requires maintaining the
`product temperature below its glass transition temperature dur(cid:173)
`ing the lyophilization cycle (3-10, 17, 18). During primary dry(cid:173)
`ing, when ice is sublimed, the product must be maintained
`below the collapse temperature, which usually coincides with
`the thermotropic transition that has been referred to the glass
`transition temperature of the maximally freeze-concentrated
`amorphous phase of the sample (Tg') or as the softening temper(cid:173)
`ature of the amorphous phase (Ts) (3-10,17,18). Also, it is
`necessary to keep the product temperature below the eutectic
`melting temperature of any crystalline component. In practice,
`these temperatures can be determined using either differential
`scanning calorimetry (DSC) or freeze-drying microscopy. The
`ability to determine collapse temperature is essential to formula(cid:173)
`tion development (19 ,20).
`Drying a product below the collapse temperature carries
`a price (3-10). The lower the sample temperature, the slower
`and more expensive the drying cycle becomes. In general,
`freeze-drying below -40°C is not practical (3-10). Also, there
`are physical limits in the temperatures to which samples can
`be reduced, which are dependent on the lyophilizer and sample
`configuration (3-10). As the formulation is being developed, the
`pharmaceutical scientist should work closely with the process
`engineers, who will be designing the lyophilization cycles. It
`is especially important to know how the large-scale lyophilizers,
`which will be used for commercial production, compare to the
`research-scale unit that is used during formulation development.
`Often, the large units do not have the same level of control of
`process parameters as do the small research units, and in part
`due to the large size of a production unit, intervial variation in
`product temperature during the process may be greater. Finally,
`input from a researcher knowledgeable in the physics of freeze(cid:173)
`drying will help prevent the formulation scientist from arbi(cid:173)
`trarily rejecting useful formulations. There are ways (see
`reviews 3-10) in which the process parameters can be manipu(cid:173)
`lated such that relatively rapid and controlled drying can be
`achieved with products that have relatively low collapse
`temperatures.
`It is clear that one goal of formulation design is to provide
`the highest collapse temperature that is practical, within the
`constraints of maintaining protein stability. The collapse tem(cid:173)
`perature (i.e., the Tg') of the product will be dictated primarily
`by the formulation composition. If the protein is present at a
`level exceeding about 20% (wt/wt) of all solute it can have a
`relatively large effect on Tg'. Although it is often difficult to
`measure the Tg' of pure protein solutions with DSC, it has
`been found that adding increasing amounts of protein to most
`formulations leads to a higher Tg' (S.D. Allison, B.S. Chang,
`T.W. Randolph, M.J. Pikal and J.F. Carpenter, unpublished
`
`AMNEAL EX. 1011
`
`

`

`Design of Stable Lyophilized Protein Formulations
`
`971
`
`observations). By extrapolation it appears that pure protein
`solutions have a Tg' of about - l0°C, which is much higher
`than that of most pure excipient solutions (e.g., Tg' of sucrose
`is about - 32°C). Thus, from a process economy viewpoint,
`one desires a high ratio of protein to stabilizer in the formulation
`(cf. 6). However, stability normally increases as the weight
`ratio of stabilizer to protein increases, so typically a compromise
`must be made between providing a high collapse temperature
`and adequate protein stabilization ( e.g., 2,4-6, 11, 16). Also, as
`will be discussed below, protein resistance to freezing damage
`often improves as the protein concentration increases (2,6,22).
`Thus, in general stability is best at both high protein concentra(cid:173)
`tion and high weight ratio of stabilizer to protein. Therefore,
`in tum, stability optimization may lead to very high total solids
`content, which creates processing difficulties. Formulations
`with total solids in excess of 10% (w/w) may be difficult to
`process (3,7-10).
`Also, the manner in which the formulation is treated prior
`to applying a vacuum can alter the Tg'. Usually such treatment
`involves an annealing step, which results in removing some
`fraction of a given component from the amorphous phase (6).
`For example, if glycine is used as a crystalline bulking agent,
`depending on the freezing protocol, a significant fraction of
`the glycine molecules may remain in the amorphous phase of
`the sample (6). Glycine has a relatively low Tg' (e.g., ca.
`-42°C; 6,20). Thus, it is important to crystallize as much as
`possible, which in turn should increase the Tg' of the amorphous
`phase and make drying more rapid and economical. To design
`the optimum protocol for excipient crystallization, DSC can be
`used to simulate the processing conditions used during freezing
`and annealing. This approach is described in Carpenter and
`Chang (6).
`
`AT WHAT STEPS IS STABILIZATION OF THE
`PROTEIN REQUIRED?
`
`Essentially every step from vial filling to final reconstitu(cid:173)
`tion of the dried product can damage the protein and require
`formulation components to inhibit degradation ( 1,2,6, 11,21,22).
`During the rapid steps (e.g., filling, freezing, drying and rehy(cid:173)
`dration) the major problem is usually physical damage, which
`is typically manifested as formation of oligomeric and/or precip(cid:173)
`itated protein molecules (1,2,6,15,21,22). Normally the transi(cid:173)
`tion from solution to solid slows the rate of physical changes
`more than it slows chemical changes, so chemical degradation
`in the dried solid is often the more serious storage stability
`problem (e.g., 6,15,16). However, protein aggregates can form
`during storage/reconstitution (e.g., 6, 13-16). These degradative
`processes can be minimized if protein unfolding (here, meaning
`even a small fraction of the total molecular population) is
`inhibited during the most damaging stresses of freezing and
`drying (6,15,16). Thus, a primary focus of formulation design
`should be protecting the protein during these steps, so that the
`dried formulation immobilizes the native protein in a chemically
`inert solid matrix having both high Tg and low residual mois(cid:173)
`ture (5,6,14,16).
`
`Stabilization During Freezing
`
`stabilizers (see below) in the formulation. In general, the three
`most important parameters to consider are protein concentra(cid:173)
`tion, buffer choice, and freezing protocol (2,6,21-24).
`Increasing protein concentration leads to increased resis(cid:173)
`tance to denaturation during freezing (2,6,22,24). This phenom(cid:173)
`enon can be demonstrated by simply determining the percentage
`protein aggregated after freeze-thawing, which varies inversely
`with protein concentration (e.g., 22). Normally, it would be
`expected that increasing protein concentration would increase
`aggregation, and this would be the case if the fraction of protein
`molecules unfolded during freezing were independent of con(cid:173)
`centration. However, it is now thought that increasing protein
`concentration directly
`reduces
`freezing-induced protein
`unfolding. It has been speculated that damage during freezing
`involves protein denaturation during formation of the ice-water
`interface (21,22). Assuming that only a finite number of protein
`molecules can be denatured at this interface, then increasing
`the initial protein concentration will lead to a smaller percentage
`of damaged molecules. For practical purposes, it is sufficient
`simply to consider protein concentration as an important vari(cid:173)
`able to examine, and to include the highest possible concentra(cid:173)
`tion in testing during formulation development.
`Buffer choice can also be critical. The main culprits here
`are sodium phosphate and potassium phosphate, which can
`undergo drastic changes in pH during freezing and annealing
`(6,23,24). With sodium phosphate, the dibasic form will readily
`crystallize, resulting in a frozen sample in which the pH in the
`remaining amorphous phase (containing the protein) can be
`reduced to 4 or lower (23,24). With potassium phosphate, the
`dihydrogen salt crystallizes, giving a final pH near 9 (23,24).
`The risk of alteration in pH and its damage to proteins can be
`minimized by increasing the initial cooling rate, limiting the
`duration of annealing steps and minimizing the buffer concen(cid:173)
`tration, all of which reduce opportunity for salt crystallization
`(6,24). Rapid freezing, without annealing also limits the length
`of exposure of protein to denaturing conditions in the frozen
`state 6,24). Although other excipients can aid in inhibiting the
`pH change (24), the best approach is to avoid using sodium
`phosphate or potassium phosphate buffers. Buffers that have
`minimal pH change upon freezing include citrate, histidine and
`Tris (22,24; T.J. Anchordoquy and J.F. Carpenter, unpub(cid:173)
`lished observations).
`In studies in which complications due to buffer pH changes
`have been avoided, it has been found that the degree of protein
`damage during freezing correlates directly with cooling rate,
`with more damage found at higher cooling rates where surface
`area of ice is larger (21,22). It has been speculated that this is
`due to protein denaturation during the formation of the ice(cid:173)
`water interface (21,22). More rapid cooling leads to smaller
`ice crystals, which have a greater surface area to volume ratio
`than larger crystals. Since cooling rates will usually be dictated
`by the physical constraints of the Iyophilizer, excessively rapid
`cooling probably will not be a problem (3-10). However, some
`proteins are so sensitive to freezing, that even with slow, con(cid:173)
`trolled cooling they will be denatured (e.g., 21,24).
`
`Stabilization During Drying and Storage in the Dried
`Solid
`
`Whether a given protein is susceptible to freezing damage
`depends of many factors, beyond the inclusion of the appropriate
`
`Even if the entire population of protein molecules survives
`the freezing step, there will be denaturation during subsequent
`
`AMNEAL EX. 1011
`
`

`

`972
`
`Carpenter, Pikal, Chang, and Randolph
`
`dehydration, unless the appropriate stabilizers are added
`(15, 16,25-28). Simply stated, removal of the protein molecule's
`hydration shell, which occurs during lyophilization, destabilizes
`the native conformation (15, 16,25-28). To date, infrared spec(cid:173)
`troscopic studies with dozens of proteins have shown that, in
`the absence of the appropriate stabilizer(s) (e.g., sucrose) pro(cid:173)
`teins will be unfolded in the dried solid (15,16,25-28). If sam(cid:173)
`ples are rehydrated immediately, the degree of damage (e.g.,
`percent of aggregation) correlates directly with how "non(cid:173)
`native" the infrared spectrum of the dried protein appeared
`(15,16,25-28). Thus, reducing post-rehydration damage is
`dependent on minimizing the unfolding during freezing and
`drying. Moreover, even if 100% native molecules are recovered
`in samples rehydrated immediately, there can be a substantial
`fraction ofunfolded molecules in the dried solid (15,16,25-28).
`Intramolecular refolding during rehydration can dominate the
`intermolecular interactions leading to aggregation, thereby giv(cid:173)
`ing 100% native protein on reconstitution.
`Fortunately, appropriate excipients can prevent or at least
`minimize unfolding, and the success of the formulation can
`be judged immediately by examining the protein secondary
`structure
`in
`the dried solid with
`infrared spectroscopy
`(15,16,25-28). More importantly, in the few studies published
`to date, it has been shown that stability during long-term storage
`in the dried solid is dependent of retention of native protein
`during freeze-drying (15, 16). Even for samples stored at temper(cid:173)
`atures well below the formulation Tg, damage arose rapidly
`(e.g., within weeks) if the protein was unfolded in the dried
`solid. Therefore, infrared spectroscopy, which can be used
`immediately after lyophilization
`to determine
`if protein
`unfolded has arisen, should be considered another essential tool
`for the protein formulation scientist.
`
`WHICH EXCIPIENTS ARE THE BEST FIRST
`CHOICES?
`
`After this review of all of the dangers of lyophilization
`and all the factors to be considered it might seem that rapid
`development of a stable lyophilized formulation would be an
`impossible task. Fortunately with a rational approach to formu(cid:173)
`lation design, most formulation problems are quickly resolved.
`Here we will provide the rationale for the initial choices of
`formulation components. In some cases, the "initial formula(cid:173)
`tion" may be all that is needed for the final marketed product.
`The composition to be given, with various minor modifications,
`has already been used with success with protein drugs (e.g.,
`16). We wish to stress that for any lyophilized formulation, the
`minimum number of components necessary for protein stability
`and cake structure should be used. No excipient should be
`added unless there are data to document that it has a beneficial
`role in the formulation.
`
`Specific Conditions for Stability of a Given Protein
`
`Before choosing the appropriate "general" stabilizers,
`which are effective at protecting most proteins, it is absolutely
`essential that the formulation be optimized for the specific
`factors that increase the physical and chemical stability of a
`given protein. For example, simply avoiding extremes in pH
`can drastically reduce the rate of deamidation (1). Moreover,
`it has been found that the resistance of a protein to unfolding
`
`during freeze-drying can be dramatically increased by optimiz(cid:173)
`ing the pH of solution (e.g., 15). Also, other specific ligands
`that increase protein stability (e.g., by increasing the free energy
`of unfolding) should be investigated. The stabilizing effects of
`heparin and other polyanions on growth factors (e.g., 29) pro(cid:173)
`vide a good example. Another important factor to be considered
`is the effect of ionic strength on protein unfolding and aggrega(cid:173)
`tion. It must be recognized that during freezing, the ionic
`strength may increase SO-fold as ice formation concentrates
`all solutes (5,6,9,19). The persons responsible for bulk drug
`purification and pharmaceutical preformulation often already
`have insight into these issues. Thus, it is imperative that the
`these people, prior to
`formulation scientist confer with
`embarking of design of a lyophilized formulation.
`Even with specific solution conditions optimized for pro(cid:173)
`tein stability, it probably will be necessary to add other protec(cid:173)
`tive excipients, if the protein is to survive lyophilization and
`long-term storage in the dried solid. First, let us consider some
`compounds that have been used for lyophilized protein formula(cid:173)
`tion but which do not provide stability and may actually foster
`damage during storage. We will then provide an outline of a
`simple, but effective formulation, and the rationale for the
`choice of the components will be discussed.
`
`Excipients that Can Fail to Stabilize Proteins
`
`With the goal of obtaining a strong cake structure during
`a rapid lyophilization cycle, polymers such as dextran and
`hydroxyethyl starch, which have relatively high collapse tem(cid:173)
`peratures, are attractive excipients. Also, the Tg of the final
`dried product will be high (e.g., >90°C) with these polymers
`( 15). Unfortunately, these polymers do not inhibit protein
`unfolding during lyophilization and they typically fail to provide
`stability during subsequent storage (15,30). The failure to inhibit
`lyophilization-induced denaturation is presumably because the
`polymers are too bulky to hydrogen bond to the protein in the
`place of the water that is lost during dehydration and/or because
`the polymers form a separate amorphous phase from the protein
`(5,6). Although when used alone such polymers are not good
`choices as stabilizers, as described below, they could be prove
`useful
`in combination with certain disaccharide protein
`stabilizers.
`Among the numerous compounds tested it appears that
`the most effective stabilizers during the lyophilization cycle
`are disaccharides (2,5,6,11,15,16,25-28). However, one group
`of compounds that should be avoided are the reducing sugars.
`These compounds may effectively inhibit protein unfolding
`during the lyophilization cycle, but during storage in the dried
`solid they have the propensity to degrade proteins via the Mail(cid:173)
`lard reaction between carbonyls of the sugar and free amino
`groups on the protein (31). The result can be a brown syrup
`containing degraded protein instead of a white cake containing
`active protein drug. Usually, the only way to slow this process
`significantly is to store the product at subzero temperatures,
`which defeats the purpose of a lyophilized product. Compounds
`in this undesirable category include glucose, lactose, maltose
`and maltodextrins.
`As noted earlier, crystalline bulking agents such as manni(cid:173)
`tol and glycine do not provide protection during lyophilization
`(6, 11, 12,25). However, some effective lyophilized formulations
`employing mixtures of these two agents have been developed
`
`AMNEAL EX. 1011
`
`

`

`Design of Stable Lyophilized Protein Formulations
`
`973
`
`and marketed. In these cases the appropriate ratio of manni(cid:173)
`tol:glycine led to a significant fraction of the compounds
`remaining amorphous (e.g., 30). Presumably this amorphous
`fraction was sufficient to inhibit protein unfolding during lyoph(cid:173)
`ilization and to provide stability during long-term storage. How(cid:173)
`ever, we caution against such an approach because achieving
`just the right processing conditions, in combination with the
`appropriate excipient ratio, can be time consuming and tricky.
`
`Rational Choice of Stabilizing Excipients
`
`So what are appropriate, rational choices for excipients?
`To provide a concrete example, let's make the following
`assumptions about a fictitious case. 1) The protein drug will
`be formulated at 2 mg/ml. 2) The major routes of degradation are
`aggregation immediately after lyophilization/rehydration and
`deamidation during storage in the dried solid. 3) Optimizing
`specific conditions (e.g., using a citrate buffer at pH 6.0) only
`reduces aggregation upon freeze-drying and reconstitution to
`about 10% and deamidation still proceeds at an unacceptably
`rapid rate during storage, even when the product is stored 20°C
`below its Tg. 4) A crystalline bulking agent (e.g., mannitol) is
`desired to form a mechanically strong and elegant cake.
`At this point, the major component missing is a nonreduc(cid:173)
`ing disaccharide, which forms an amorphous phase with the
`protein in the dried solid and serves as the primary stabilizer.
`The main choices are sucrose or trehalose (5,6). These com(cid:173)
`pounds are relatively effective at protecting proteins during
`freezing and usually excellent at inhibiting unfolding during
`dehydration (5,6,15,16,25-28). Freezing protection depends on
`the initial bulk concentration of the sugar, and sometimes con(cid:173)
`centrations exceeding 5% (wt/vol) are needed to maximize
`stabilization (2,6). In contrast, protection during drying depends
`on the final mass ratio between the sugar and the protein (5,6).
`Generally, a weight ratio of sugar to protein of at least I: 1 is
`required for good stability, with optimal stability being reached
`at around 5: 1. In practice, it is not necessary to determine the
`most appropriate concentration for each type of protection.
`Rather, with the protein concentration held constant, a range
`of sugar concentrations can be tested during formulation screen(cid:173)
`ing to discern the optimal concentration for retention of native
`protein in the dried solid and the resultant reduction in aggrega(cid:173)
`tion upon rehydration.
`In general, the optimal sugar concentration for stabilizing
`the protein during lyophilization will also provide storage stabil(cid:173)
`ity, if the final dried powder has a Tg well above the storage
`temperature (5,6, 15, 16). For example, assuming that a high
`room temperature of about 30°C is the maximum intended
`storage temperature, then a product containing a native protein
`and with a Tg of > 50°C should be stable. Since residual
`moisture lowers Tg, the condition Tg > 50°C must apply to
`the maximum water content allowed by the product specifica(cid:173)
`tions. It is imperative that DSC be used to measure the Tg of
`each product to be certain that this goal is achieved.
`Both sucrose and trehalose have advantages and disadvan(cid:173)
`tages. 1) Trehalose has a higher Tg at any moisture content
`and, thus, is more easily lyophilized (32). In addition, the condi(cid:173)
`tion Tg > 50°C will hold at higher residual water contents for
`trehalose. However, a skilled process engineer should be able
`to design economical, effective cycles for either sugar. Also,
`in products with a relatively high protein concentration, the
`
`protein could contribute to an increased Tg, which serves to
`minimize the advantages of trehalose. 2) Trehalose is also more
`resistant than sucrose to acid hydrolysis. Hydrolysis of these
`disaccharides produces reducing sugars, which must be avoided.
`Usually this is not a problem, unless very low pH's of around
`4 or lower are employed. 3) Sucrose appears to be more effective
`at inhibiting unfolding during lyophilization (unpublished
`observations). This difference has been most obvious when
`there is a relatively high protein concentration and a need to
`employ a relatively high initial concentration of sugar. This
`need can develop when the protein is very unstable during
`freezing and/or there is a relatively high protein content in
`the dried solid. Evidence to date indicates that less effective
`stabilization by trehalose is due to the greater propensity of
`this sugar to phase separate from polymers (e.g., the protein in
`a formulation) during freezing and drying (S.D. Allison, T.W.
`Randolph, B.S. Chang and J.F. Carpenter, unpublished observa(cid:173)
`tion). Whether this is a problem with a given formulation cannot
`be predicted. Hence, the capacity to protect a protein must be
`examined for each formulation. 4) Sucrose is commonly used
`in parenteral products that are approved by the Food and Drug
`Administration (33). In contrast, trehalose has not yet been
`used in an approved product, probably because to date there
`has not been sufficient practical bendit to justify using this
`sugar in place of sucrose. Safety of t:rehalose will most likely
`not be a concern. Thus, if there is a clear advantage of trehalose
`over sucrose in a given product, use of trehalose should not
`hinder regulatory approval.
`At this point, our example formulation might be complete,
`as is the case for many proteins. However, let's assume that
`even with sucrose completely inhibiting detectable protein
`unfolding, as assessed with structural analysis of the dried solid
`with infrared spectroscopy, after rehydration there is still about
`1 % aggregated protein. Since there are no aggregates in the
`starting material, it is assumed that at some point during freezing
`and drying a very small fraction of the protein population was
`unfolded. During rehydration some of these molecules refolded,
`but others formed aggregates. This actually appears to be a
`common problem, as is the format