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  1. 1. International Journal of Pharmaceutics 203 (2000) 1 – 60 www.elsevier.com/locate/ijpharm Review Lyophilization and development of solid protein pharmaceuticals Wei Wang* Biotechnology, Bayer Corporation, 800 Dwight Way, Berkeley, CA 94701, USA Received 9 September 1999; received in revised form 21 December 1999; accepted 4 April 2000Abstract Developing recombinant protein pharmaceuticals has proved to be very challenging because of both the complexityof protein production and purification, and the limited physical and chemical stability of proteins. To overcome theinstability barrier, proteins often have to be made into solid forms to achieve an acceptable shelf life as pharmaceu-tical products. The most commonly used method for preparing solid protein pharmaceuticals is lyophilization(freeze-drying). Unfortunately, the lyophilization process generates both freezing and drying stresses, which candenature proteins to various degrees. Even after successful lyophilization with a protein stabilizer(s), proteins in solidstate may still have limited long-term storage stability. In the past two decades, numerous studies have beenconducted in the area of protein lyophilization technology, and instability/stabilization during lyophilization andlong-term storage. Many critical issues have been identified. To have an up-to-date perspective of the lyophilizationprocess and more importantly, its application in formulating solid protein pharmaceuticals, this article reviews therecent investigations and achievements in these exciting areas, especially in the past 10 years. Four interrelated topicsare discussed: lyophilization and its denaturation stresses, cryo- and lyo-protection of proteins by excipients, designof a robust lyophilization cycle, and with emphasis, instability, stabilization, and formulation of solid proteinpharmaceuticals. © 2000 Elsevier Science B.V. All rights reserved.Keywords: Aggregation; Cryoprotection; Denaturation; Excipient; Formulation; Freeze-drying; Glass transition; Stability; Lyopro-tection; Residual moisture1. Introduction both the complexity of protein production and purification, and the limited physical and chemi- Developing recombinant protein pharmaceuti- cal stability of proteins. In fact, protein instabilitycals has proved to be very challenging because of is one of the two major reasons why protein pharmaceuticals are administered traditionally * Tel.: +1-510-7054755; fax: + 1-510-7055629. through injection rather than taken orally like E-mail address: wei.wang.b@bayer.com (W. Wang). most small chemical drugs (Wang, 1996). To over-0378-5173/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 5 1 7 3 ( 0 0 ) 0 0 4 2 3 - 3
  2. 2. 2 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60come the instability barrier, proteins often have to cal and chemical instabilities and stabilization ofbe made into solid forms to achieve an acceptable proteins in aqueous and solid states (Manning etshelf life. al., 1989; Cleland et al., 1993); chemical instability The most commonly used method for preparing mechanisms of proteins in solid state (Lai andsolid protein pharmaceuticals is lyophilization Topp, 1999); various factors affecting protein sta-(freeze-drying). However, this process generates a bility during freeze-thawing, freeze-drying, andvariety of freezing and drying stresses, such as storage of solid protein pharmaceuticalssolute concentration, formation of ice crystals, pH (Arakawa et al., 1993); and application ofchanges, etc. All of these stresses can denature lyophilization in protein drug development (Pikal,proteins to various degrees. Thus, stabilizers are 1990a,b; Skrabanja et al., 1994; Carpenter et al.,often required in a protein formulation to protect 1997; Jennings, 1999). Nevertheless, it appearsprotein stability both during freezing and drying that several critical issues in the development ofprocesses. solid protein pharmaceuticals have not been fully Even after successful lyophilization, the long- examined, including various instability factors,term storage stability of proteins may still be very stabilization, and formulation of solid proteinlimited, especially at high storage temperatures. In pharmaceuticals.several cases, protein stability in solid state has To have an up-to-date perspective of thebeen shown to be equal to, or even worse than, lyophilization process and more importantly, itsthat in liquid state, depending on the storage application in formulating solid protein pharma-temperature and formulation composition. For ceuticals, this article reviews the recent investiga-example, a major degradation pathway of human tions and achievements in these exciting areas,insulin-like growth factor I (hIGF-I) is oxidation especially in the past 10 years. Four interrelatedof Met59 and the oxidation rate in a freeze-dried topics are discussed sequentially, lyophilizationformulation in air-filled vials is roughly the same and its denaturation stresses; cryo- and lyo-pro-as that in a solution at either 25 or 30°C tection of proteins by excipients; design of a ro-(Fransson et al., 1996). Similarly, the oxidation bust lyophilization cycle; and with emphasis,rate of lyophilized interleukin 2 (IL-2) is the same instability, stabilization, and formulation of solidas that in a liquid formulation containing 1 mg protein pharmaceuticals.ml − 1 IL-2, 0.5% hydroxypropyl-b-cyclodextrin(HP-b-CD), and 2% sucrose during storage at 4°C(Hora et al., 1992b). At a high water content 2. Lyophilization and its denaturation stresses( 50%), the degradation rate of insulin is higherin a lyophilized formulation than in a solution 2.1. Lyophilization processwith similar pH-rate profiles in both states(Strickley and Anderson, 1996). The glucose-in- Lyophilization (freeze-drying) is the most com-duced formation of des-Ser relaxin in a mon process for making solid protein pharmaceu-lyophilized formulation is faster than in a solution ticals (Cleland et al., 1993; Fox, 1995). Thisduring storage at 40°C (Li et al., 1996). These process consists of two major steps: freezing of aexamples indicate that stabilizers are still required protein solution, and drying of the frozen solidin lyophilized formulations to increase long-term under vacuum. The drying step is further dividedstorage stability. into two phases: primary and secondary drying. In the past two decades, numerous studies have The primary drying removes the frozen water andbeen conducted in the areas of protein freezing the secondary drying removes the non-frozenand drying, and instability and stabilization of ‘bound’ water (Arakawa et al., 1993). The amountproteins during lyophilization and long-term stor- of non-frozen water for globular proteins is aboutage. Many critical issues have been identified in 0.3–0.35 g g − 1 protein, slightly less than thethis period. These studies and achievements have proteins’ hydration shell (Rupley and Careri,been reviewed elsewhere with emphasis on physi- 1991; Kuhlman et al., 1997). More detailed analy-
  3. 3. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 3sis of each lyophilization step is provided in Sec- cluding formation of dendritic ice crystals, in-tion 4. creased ionic strength, changed pH, and phase Lyophilization generates a variety of stresses, separation; and (3) drying stress (removing of thewhich tend to destabilize or unfold/denature an protein hydration shell).unprotected protein. Different proteins toleratefreezing and/or drying stresses to various degrees. 2.2.1. Low temperature stressFreeze-thawing of ovalbumin at neutral pH did The first quantitative study on low-temperaturenot cause denaturation (Koseki et al., 1990). Re- denaturation of a model protein was conductedpeated (three times) freeze-thawing of tissue-type presumably by Shikama and Yamazaki (1961).plasminogen activator (tPA) did not cause any They demonstrated a specific temperature rangedecrease in protein activity (Hsu et al., 1995). in which ox liver catalase was denatured duringSome proteins can keep their activity both during freeze-thawing. Cold denaturation of catalase atfreezing and drying processes, such as a1-an- 8.4 mg ml − 1 in 10 mM phosphate buffer (pH 7.0)titrypsin in phosphate – citrate buffer (Vemuri et started at − 6°C. Loss of catalase activity reachedal., 1994), porcine pancreatic elastase without ex- 20% at − 12°C, remained at this level betweencipients (Chang et al., 1993), and bovine pancre- − 12°C and near − 75°C, then decreased gradu-atic ribonuclease A (RNase A, 13.7 kD) in the ally from − 75 to − 120°C. There was almost nopresence or absence of phosphate (Townsend and activity loss between − 129 and − 192°C. SimilarDeLuca, 1990). results were also obtained for ovalbumin by However, many proteins cannot stand freezing Koseki et al. (1990). Incubation of frozen ovalbu-and/or drying stresses. Freeze-thawing caused loss min solution caused structural change of ovalbu-of activity of lactate dehydrogenase (LDH) min, as monitored by UV difference spectra,(Nema and Avis, 1992; Izutsu et al., 1994b; An- which increased with decreasing temperature be-dersson and Hatti-Kaul, 1999), 60% loss of L-as- tween −10 and − 40°C. Further decrease inparaginase (10 mg ml − 1) activity in 50 mM incubation temperature to − 80°C caused lesssodium phosphate buffer (pH 7.4) (Izutsu et al., structural change, and no change at − 192°C.1994a), and aggregation of recombinant Perlman and Nguyen (1992) reported that inter-hemoglobin (Kerwin et al., 1998). Freeze-drying feron-g(IFN-g) aggregation in a liquid mannitolcaused 10% loss of the antigen-binding capacity formulation was more severe at −20°C than atof a mouse monoclonal antibody (MN12) (Ress- − 70, 5 and 15°C during storage. To preventing et al., 1992), more than 40% loss of bilirubin freezing-induced complication in studying coldoxidase (BO) activity in the presence of dextran or protein denaturation, cold and heat denaturationpolyvinylalcohol (PVA) (Nakai et al., 1998), loss of RNase A has been conducted under high pres-of most b-galactosidase activity at 2 or 20 mg sure (3 kbar). Under this condition, RNase Aml − 1 (Izutsu et al., 1993, 1994a), complete loss of denatured below −22°C and above 40°C (Zhangphosphofructokinase (PFK) and LDH activity in et al., 1995). All these examples are clear indica-the absence of stabilizers (Carpenter et al., 1986, tion of low temperature denaturation rather than1990; Prestrelski et al., 1993a; Anchordoquy and a freezing or thawing effect.Carpenter, 1996), and dissociation of Erwinia L- The nature of cold denaturation has not beenasparaginase tetramer (135 kD) into four inactive satisfactorily delineated. Since solubility of non-subunits (34 kD each) in the absence of any polar groups in water increases with decreasingprotectants (Adams and Ramsay, 1996). temperature due to increased hydration of the non-polar groups, solvophobic interaction in2.2. Denaturation stresses during lyophilization proteins weakens with decreasing temperature (Dill et al., 1989; Graziano et al., 1997). The The lyophilization process generates a variety decreasing solvophobic interaction in proteins canof stresses to denature proteins. These include (1) reach a point where protein stability reaches zero,low temperature stress; (2) freezing stresses, in- causing cold denaturation (Jaenicke, 1990). While
  4. 4. 4 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60normal or thermal denaturation is entropy-driven, centration upon freezing may drastically acceler-cold denaturation is enthalpy-driven (Dill et al., ate protease-catalyzed protein degradation.1989; Shortle, 1996). Oligomeric proteins typicallyshow cold denaturation, i.e. dissociation of sub- 2.2.3. Formation of ice-water interfaceunit oligomers, since association is considered to Freezing a protein solution generates an ice-wa-be a consequence of hydrophobic interaction ter interface. Proteins can be adsorbed to the(Jaenicke, 1990; Wisniewski, 1998). Theoretically, interface, loosening the native fold of proteins andthe calculated free energy of unfolding (DGunf) for resulting in surface-induced denaturationproteins has a parabolic relationship with temper- (Strambini and Gabellieri, 1996). Rapid (quench)ature. This means that a temperature of maximum cooling generates a large ice-water interface whilestability exists, and both high and low tempera- a smaller interface is induced by slow coolingture can destabilize a protein (Jaenicke, 1990; (also see Section 4.2). Chang et al. (1996b)Kristjansson and Kinsella, 1991). ´ demonstrated that a single freeze–thaw cycle with quench cooling denatured six model proteins, in-2.2.2. Concentration effect cluding ciliary neurotrophic factor (CNTF), gluta- Freezing a protein solution rapidly increases the mate dehydrogenase (GDH), interleukin-1concentration of all solutes due to ice formation. receptor antagonist (IL-1ra), LDH, PFK, andFor example, freezing a 0.9% NaCl solution to its tumor necrosis factor binding protein (TNFbp).eutectic temperature of −21°C can cause a 24- The denaturation effect of quench cooling wasfold increase in its concentration (Franks, 1990). greater or equivalent to that after 11 cycles ofThe calculated concentration of small carbohy- slow cooling, suggesting surface-induced denatu-drates in the maximally freeze-concentrated ma- ration. This denaturation mechanism was sup-trices (MFCS) is as high as 80% (Roos, 1993). ported by a good correlation (r= 0.99) foundThus, all physical properties related to concentra- between the degree of freeze-induced denaturationtion may change, such as ionic strength and rela- and that of artificially surface-induced denatura-tive composition of solutes due to selective tion. The surface was introduced by shaking thecrystallization. These changes may potentially protein solution containing hydrophobic Teflondestabilize a protein. beads. In a similar study, a correlation coefficient Generally, lowering the temperature reduces the of 0.93 was found between the tendency of freezerate of chemical reactions. However, chemical denaturation and surface-induced denaturationreactions may actually accelerate in a partially for eight model proteins, including aldolase, basicfrozen aqueous solution due to increased solute fibroblast growth factor (bFGF), GDH, IL-1ra,concentration (Pikal, 1999). Due to solute concen- LDH, maleate dehydrogenase (MDH), PFK, andtration, the rate of oligomerization of b-glutamic TNFbp (Kendrick et al., 1995b). However, thereacid at −20°C was much faster than at 0 or 25°C was no significant correlation (r=0.78) betweenin the presence of a water-soluble carbodiimide, freeze denaturation and thermal denaturation1-ethyl-3-(3-dimethylaminopropyl) carbodiimide temperature (Chang et al., 1996b).(EDAC) (Liu and Orgel, 1997). The increase in the rate of a chemical reaction 2.2.4. pH changes during freezingin a partially frozen state could reach several Many proteins are stable only in a narrow pHorders of magnitude relative to that in solution range, such as low molecular weight urokinase(Franks, 1990, 1994). (LMW-UK) at pH 6–7 (Vrkljan et al., 1994). At The reported oxygen concentration in a par- extreme pHs, increased electrostatic repulsion be-tially frozen solution at −3°C is as high as 1150 tween like charges in proteins tends to causetimes that in solution at 0°C (Wisniewski, 1998). protein unfolding or denaturation (Goto andThe increased oxygen concentration can readily Fink, 1989; Volkin and Klibanov, 1989; Dill,oxidize sulphydryl groups in proteins. If a protein 1990). Thus, the rate of protein aggregation issolution contains any contaminant proteases, con- strongly affected by pH, such as aggregation of
  5. 5. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 5interleukin 1b (IL-1b) (Gu et al., 1991), human separation due to polymers’ altered solubilities atrelaxin (Li et al., 1995a), and bovine pancreatic low temperatures. Freezing-induced phase separa-RNase A (Townsend and DeLuca, 1990; Tsai et tion can easily occur in a solution containing twoal., 1998). Moreover, the solution pH can signifi- incompatible polymers such as dextran and Ficollcantly affect the rate of many chemical degrada- (Izutsu et al., 1996). During freezing of recombi-tions in proteins (Wang, 1999). nant hemoglobin in a phosphate buffer containing Freezing a buffered protein solution may selec- 4% (w/w) PEG 3350, 4% (w/w) dextran T500, andtively crystallize one buffering species, causing pH 150 mM NaCl, liquid–liquid phase separationchanges. Na2HPO4 crystallizes more readily than occurred and created a large excess of interface,NaH2PO4 because the solubility of the disodium denaturing the protein (Heller et al., 1997). Addi-form is considerably lower than that of the tion of 5% sucrose or trehalose could not reversemonosodium form. Because of this, a sodium the denaturation effect in the system (Heller et al.,phosphate buffer at pH 7 has a molar [NaH2PO4]/ 1999a).[Na2HPO4] ratio of 0.72, but this ratio increases Several strategies have been proposed to miti-to 57 at the ternary eutectic temperature during gate or prevent phase separation-induced proteinfreezing (Franks, 1990, 1993). This can lead to a denaturation during freezing. These include use ofsignificant pH drop during freezing, which then alternative salts (Heller et al., 1999a), adjustmentdenatures pH-sensitive proteins. For example, of the relative composition of polymers to avoidfreezing of a LDH solution caused protein denat- or to rapidly pass over a temperature regionuration due to a pH drop from 7.5 to 4.5 upon where the system may result in liquid–liquidselective crystallization of Na2HPO4 (Anchordo- phase separation (Heller et al., 1999c), and chemi-quy and Carpenter, 1996). LDH is a pH-sensitive cal modification of the protein such as pegylationprotein and a small drop in pH during freezing (Heller et al., 1999b).can partially denature the protein even in thepresence of stabilizers such as sucrose and tre- 2.2.6. Dehydration stresseshalose (Nema and Avis, 1992). The pH drop Proteins in an aqueous solution are fully hy-during freezing may also explain why freezing drated. A fully hydrated protein has a monolayerbovine and human IgG species in a sodium phos- of water covering the protein surface, which isphate buffer caused formation of more aggregates termed the hydration shell (Rupley and Careri,than in potassium phosphate buffer, because 1991). The amount of water in full hydration ispotassium phosphate buffer does not show signifi- 0.3–0.35 g g − 1 protein (Rupley and Careri, 1991;cant pH changes during freezing (Sarciaux et al., Kuhlman et al., 1997). Generally, the water con-1998). tent of a lyophilized protein product is less than The pH drop during freezing can potentially 10%. Therefore, lyophilization removes part ofaffect storage stability of lyophilized proteins. the hydration shell. Removal of the hydrationLyophilized IL-1ra in a formulation containing shell may disrupt the native state of a protein andphosphate buffer at pH 6.5 aggregated more cause denaturation. A hydrated protein, whenrapidly than that containing citrate buffer at the exposed to a water-poor environment during de-same pH during storage at 8, 30 and 50°C (Chang hydration, tends to transfer protons to ionizedet al., 1996c). Similarly, the pH drop of a succi- carboxyl groups and thus abolishes as manynate-containing formulation from 5 to 3 –4 during charges as possible in the protein (Rupley andfreezing appeared to be the cause of less storage Careri, 1991). The decreased charge density maystability for lyophilized IFN-g than that contain- facilitate protein–protein hydrophobic interac-ing glycocholate buffer at the same pH (Lam et tion, causing protein aggregation.al., 1996). Water molecules can also be an integral part of an active site(s) in proteins. Removal of these2.2.5. Phase separation during freezing functional water molecules during dehydration Freezing polymer solutions may cause phase easily inactivates proteins. For example, dehydra-
  6. 6. 6 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60tion of lysozyme caused loss of activity apparently Lyophilization may induce several potentialdue to removal of those water molecules residing changes in the IR spectra of proteins. Disruptionfunctionally in the active site (Nagendra et al., of hydrogen bonds in proteins during lyophiliza-1998). tion generally leads to an increase in frequency Lastly, dehydration during lyophilization may and a decrease in intensity of hydroxyl stretchingcause significant difference in moisture distribu- bands (Carpenter and Crowe, 1989). Unfolding oftion in different locations of a product cake. The proteins during lyophilization broadens and shiftsuneven moisture distribution may lead to possible (to higher wave numbers) amide I componentlocalized overdrying, which may exacerbate dehy- peaks (Prestrelski et al., 1993b; Allison et al.,dration-induced protein denaturation (Pikal and 1996). Lyophilization often leads to an increase inShah, 1997). b-sheet content with a concomitant decrease in a-helix content. Conversion of a-helix to b-sheet2.3. Monitoring protein denaturation upon during lyophilization has been observed in manylyophilization proteins such as tetanus toxoid (TT) in 10 mM The most common method for monitoring sodium phosphate buffer (pH 7.3) (Costantino etprotein denaturation upon lyophilization appears al., 1996), recombinant human albumin (rHA) into be infrared (IR) spectroscopy, although other different buffer solutions at different pHsmethods have been used such as mass spec- (Costantino et al., 1995a), hGH at pH 7.8, andtroscopy (Bunk, 1997), and Raman spectroscopy seven model proteins in water, including bovine(Belton and Gil, 1994). In the following section, pancreatic trypsin inhibitor (BPTI), chymotrypsi-IR methodology is discussed in monitoring nogen, horse myoglobin (Mb), horse heart cy-protein denaturation upon lyophilization followed tochrome c (Cyt c), rHA, porcine insulin, andby a discussion on reversibility of protein RNase A (Griebenow and Klibanov, 1995).denaturation. An increase in b-sheet content during lyophilization is often an indication of protein2.3.1. Infrared (IR) spectroscopy aggregation and/or increased intermolecular inter- IR (or FTIR) is probably the most extensively action (Yeo et al., 1994; Griebenow andused technique today for studying structural Klibanov, 1995; Overcashier et al., 1997).changes in proteins upon lyophilization (Susi and Lyophilization-induced increase in b-sheet contentByler, 1986; Dong et al., 1995; Carpenter et al., seems to be a rather general phenomenon as1998, 1999). The lyophilization-induced structural lyophilization or air-drying of unordered poly-L-changes can be monitored conveniently in the lysine induced structural transition to a highlyamide I, II, or III region. For lyophilized protein ordered b-sheet (Prestrelski et al., 1993b; Wolkerssamples, residual water up to 10% (w/w) does not et al., 1998b). Such transition has also been ob-interfere significantly in the amide I region, a served in proteins during lyophilization such asfrequently used sensitive region for determination human insulin in water (pH 7.1) (Pikal and Rigs-of secondary structures (Dong et al., 1995). How- bee, 1997). The b-sheet structure after lyophiliza-ever, IR studies on proteins in an aqueous solu- tion shows a higher degree of intermoleculartion need either subtraction of water absorptionor solvent replacement with D2O (Goormaghtigh hydrogen bonding because polar groups must sat-et al., 1994). To make reliable subtraction, high isfy their H-bonding requirement by intra- orprotein concentrations ( 10 mg ml − 1) are rec- intermolecular interaction upon removal of water.ommended to increase protein absorption signal, The intermolecular b-sheet is characterized by twoand a CaF2 (or BaF2) cell with a path length of 10 major IR bands at about 1617 and 1697 cm − 1 inmm or less should be used to control the total solid state, which can be used to monitor proteinsample absorbance within 1 (Cooper and Knut- denaturation (Allison et al., 1996). Similarly, theson, 1995). relative intensity of a-helix band also can be used
  7. 7. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 7in this regard (Yang et al., 1999; Heller et al., upon lyophilization (Carpenter et al., 1993), while1999b). loss of BO activity in a PVA-containing formula- The extent of changes in overall IR spectrum of tion was at least partially reversible (Nakai et al.,a protein upon lyophilization reflects the degree of 1998). Using IR spectroscopy, Prestrelski et al.protein denaturation. The changes relative to a (1993b) demonstrated that lyophilization-inducedreference spectrum can be measured using a corre- structural changes were irreversible for bFGF,lation coefficient (r) as defined by Prestrelski et al. IFN-g, and bovine a-casein, but essentially re-(1993a), or the extent of spectral area overlap versible for G-CSF and bovine a-lactalbumin.(Heimburg and Marsh, 1993; Allison et al., 1996; The extensive aggregation and precipitation ofKendrick et al., 1996). Using the correlation co- IFN-g and casein upon rehydration confirmed theefficient, Prestrelski et al. (1993b) were able to irreversibility in structural changes. Therefore,measure the relative freeze-drying stability of sev- lyophilization of proteins may lead to three typeseral model proteins, including bFGF, bovine a- of behavior, (1) no change in protein conforma-lactalbumin, bovine a-casein, IFN-g, and tion; (2) reversible denaturation; or (3) irreversiblerecombinant granulocyte colony-stimulating fac- denaturation.tor (rG-CSF) in the presence of different sugars. In many cases, IR-monitored structural changesNevertheless, Griebenow and Klibanov (1995), during lyophilization seem to be reversible.after analyzing secondary structures of seven Griebenow and Klibanov (1995) demonstratedmodel proteins upon lyophilization, concluded that lyophilization (dehydration) caused signifi-that the correlation coefficient was not highly cant changes in the secondary structures of sevensensitive to structural alterations in proteins. In- model proteins in the amide III region (1220–stead, comparison of overlapping area-normalized 1330 cm − 1), including BPTI, chymotrypsinogen,second-derivative or deconvoluted spectra seemed Mb, Cyt c, rHA, insulin, and RNase A. Themore reliable and objective. structure of almost all proteins became more or- Recently, IR has been used in real-time moni- dered upon lyophilization with a decrease in thetoring of freezing and dehydration stresses on unordered structures. Nevertheless, all these struc-proteins during lyophilization. By this method, tural changes were reversible upon reconstitution.glucose at 10% was shown to protect lysozyme Other examples of reversible changes in the sec-both during the freezing and drying processes ondary structures of proteins upon lyophilization(Remmele et al., 1997). include rHA (Costantino et al., 1995a), Humicola lanuginosa lipase (Kreilgaard et al., 1999), IL-22.3.2. Re6ersibility of freezing- or (Prestrelski et al., 1995), and lysozyme (Allison etlyophilization-induced protein denaturation al., 1999). Many proteins denature to various extentsupon freezing, especially at low concentrations( B 0.1 mg ml − 1). Freezing-induced denaturation 3. Cryo- and lyo-protection of proteins bymay or may not be reversible. Freezing lysozyme stabilizersor IL-1ra caused reversible denaturation(Kendrick et al., 1995a). In contrast, recombinant As discussed before, both freezing and dehydra-factor XIII (rFXIII, 166 kD) was irreversibly tion can induce protein denaturation. To protect adenatured upon freezing, and loss of native protein from freezing (cryoprotection) and/or de-rFXIII at 1 mg ml − 1 increased linearly with the hydration (lyoprotection) denaturation, a proteinnumber of freeze –thaw cycles (Kreilgaard et al., stabilizer(s) may be used. These stabilizers are1998b). also referenced as chemical additives (Li et al., Similarly, lyophilization-induced denaturation 1995b), co-solutes (Arakawa et al. 1993), co-sol-can be either reversible or irreversible. In the vents (Timasheff, 1993, 1998), or excipientsabsence of stabilizers, PFK at 25 mg ml − 1 at pH (Wong and Parascrampuria, 1997; Wang, 1999).7.5 and 8.0 was fully and irreversibly inactivated In the following section, a variety of protein
  8. 8. 8 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60stabilizers are presented for cryo- and lyo-protec- For example, freezing rabbit muscle LDH in wa-tion, followed by discussions of their possible ter caused 64% loss of protein activity, and in thestabilization mechanisms. presence of 5, 10 or 34.2% sucrose, the respective losses were 27, 12, and 0% (Nema and Avis,3.1. Stabilizers for cryo- and lyo-protection 1992). Other sugars or polyols that can protect LDH during freeze-thawing to different degrees Nature protects life from freezing or osmotic include lactose, glycerol, xylitol, sorbitol, andshock by accumulating selected compounds to mannitol, at 0.5–1 M (Carpenter et al., 1990).high concentrations ( 1 M) within organisms. Increasing trehalose concentration gradually in-These accumulated compounds are known as cry- creased the recovery of PFK activity duringoprotectants and osmolytes, which are preferen- freeze-thawing and the recovery reached a maxi-tially excluded from surfaces of proteins and act mum of 90% at about 300 mg ml − 1 (Carpenter etas structure stabilizers (Timasheff, 1993). How- al., 1990). A similar stabilizing trend was alsoever, since the dehydration stress is different from observed for sucrose, maltose, glucose, or inositolthose of freezing, many effective cryoprotectants (Carpenter et al., 1986).or protein stabilizers in solution do not stabilize Since freezing is part of the freeze-drying pro-proteins during dehydration (drying). Some even cess, high concentrations of sugars or polyols aredestabilize proteins during lyophilization. For ex- often necessary for lyoprotection. These examplesample, CaCl2 stabilized elastase (20 mg ml − 1) in include the protection of chymotrypsinogen in the10 mM sodium acetate (pH 5.0), but caused the presence of 300 mM sucrose (Allison et al., 1996),lyophilized protein cake to collapse and lose activ- complete inhibition of acidic fibroblast growthity (Chang et al., 1993). factor (aFGF) aggregation by 2% sucrose (Volkin Similarly, effective lyophilization stabilizers (ly- and Middaugh, 1996), increase in glucose-6-phos-oprotectants) may or may not stabilize proteins phate dehydrogenase (G6PDH) activity from 40effectively during freezing. Therefore, in cases to about 90% by 5.5% sugar mixture (glu-when a single stabilizer does not serve as both a cose:sucrose=1:10, w/w) (Sun et al., 1998), com-cryoprotectant and a lyoprotectant, two (or more) plete recovery of LDH by either 7% sucrose or 7%stabilizers may have to be used to protect proteins raffinose, a trisaccharide (Moreira et al., 1998),from denaturation during lyophilization. significant improvement of PFK recovery by 400 mM trehalose (Carpenter et al., 1993), and com-3.1.1. Sugars/polyols plete protection of four restriction enzymes by Many sugars or polyols are frequently used 15% trehalose (Colaco et al., 1992). More exam-nonspecific protein stabilizers in solution and dur- ples can be found in Table 2.ing freeze-thawing and freeze-drying. They have Lower concentrations of sugars or polyols maybeen used both as effective cryoprotectants and or may not have any significant effect. At 5 to 100remarkable lyoprotectants. In fact, their function mM, neither trehalose nor glucose could protectas lyoprotectants for proteins has long been LDH or PFK to a significant level duringadopted by nature. Anhydrobiotic organisms (wa- lyophilization (Carpenter et al., 1993). To deter-ter content B1%) commonly contain high con- mine the minimum sugar concentration that offerscentrations (up to 50%) of disaccharides, the maximum stabilization effect, Tanaka et al.particularly sucrose or trehalose, to protect them- (1991) studied the lyoprotective effect of saccha-selves (Crowe et al., 1992, 1998). rides on the denaturation of catalase during The level of stabilization afforded by sugars or lyophilization. They demonstrated that saccha-polyols generally depends on their concentrations. rides protected the protein by direct interactionA concentration of 0.3 M has been suggested to with the protein and a concentration of saccha-be the minimum to achieve significant stabiliza- rides sufficient to form a monomolecular layer ontion (Arakawa et al., 1993). This has been found the protein surface was the minimum to achieveto be true in many cases during freeze-thawing. the maximum stabilization. Therefore, the stabi-
  9. 9. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 9lization of catalase was found to depend not on buffer (pH 7.4) (Table 2), mannitol for LDH in 50the bulk concentration of maltose but on the mM sodium phosphate buffer (pH 7.4) (Izutsu etweight ratio of maltose to catalase (Tanaka et al., al., 1994b), and myo-inositol for PFK during1991). Maximum stabilization of catalase was at a freeze-thawing and freeze-drying (Table 2). Again,ratio of about 0.4. A recent study showed that the decreased protein recovery is probably due tomaximum protection (about 75% recovery) of L- crystallization of these excipients at highasparaginase at 1.45 mg ml − 1 during lyophiliza- concentrations.tion was reached at a saccharide concentration of The level of protein protection afforded byabout 0.5 mg ml − 1, which was about the calcu- different sugars or polyols can be either similar orlated monosaccharide concentration required to significantly different, depending on the formula-interact with all exposed highly polar residues of tion composition, concentration and physicalthe protein (Ward et al., 1999). At this concentra- properties of the stabilizer, and its compatibilitytion, the weight ratio of saccharide to L-asparagi- with the protein. Ward et al. (1999) found thatnase is 0.34, which is coincidentally very close to several saccharides, including trehalose, lactose,that of maltose to catalase. maltose, sucrose, glucose, and mannitol, displayed On the other hand, increasing sugar/polyol con- similar level of protection towards tetrameric L-centration to a certain level may eventually reach asparaginase (1.45 mg ml − 1) during lyophiliza-a limit of stabilization or even destabilize a tion at saccharide concentrations up to 0.1%. Atprotein during freeze-drying. For example, actin 2%, glucose or lactose protected L-asparaginasewas maximally stabilized during lyophilization in from dissociation during freeze-drying, but man-the presence of 5% (w/v) sucrose and a further nitol did not, possibly due to its crystallizationincrease in sucrose concentration to 10% did not and loss of intimate interaction with the proteinimprove the protein stability significantly, which (Adams and Ramsay, 1996). Probably for thewas apparently attributable to sticky, pliable, and same reason, mannitol at 88 mM inhibited thecollapsed formulation structure (Allison et al., formation of insoluble hGH aggregates in phos-1998). Increasing trehalose concentration to 150 phate buffer (pH 7.4) at a freezing rate of 50°Cmg ml − 1 in a PFK formulation (at 50 mg ml − 1, min − 1, but accelerated hGH aggregation at lowerpH 8.0) increased the freeze-drying recovery of freezing rates of 0.5 and 5°C min − 1 (Eckhardt etPFK activity to about 65%, but further increases al., 1991). In a different study, however, Tanakain trehalose concentration caused a gradual de- et al. (1991) demonstrated that both mannitol andcrease in recovery of the protein activity (Carpen- sorbitol could increase the recovery of catalaseter and Crowe, 1989). At a trehalose activity during lyophilization to a similar level asconcentration of 400 mg ml − 1, basically no PFK that offorded by maltose. They also showed thatactivity was left after freeze-drying. Since tre- different sugars (maltose, glucose, and mal-halose at 400 mg ml − 1 protected about 90% of totriose) at 1 mg ml − 1 could increase the recoverythe protein activity after freeze-thawing, the desta- of catalase activity to the same level (from 35 tobilization of PFK at high concentrations of tre- 90%), but maltopentaose, maltohexaose, and mal-halose occurred in the dehydration step, possibly toheptaose were not as effective (Tanaka et al.,due to crystallization of trehalose, preventing req- 1991). The ineffectiveness of larger saccharidesuisite hydrogen bonding to the dried protein (see suggests that protein stabilization by sugars maySection 3.2) (Carpenter and Crowe, 1989). A sim- depend on their glucoside chain lengths, and ailar trend was observed in the stabilization of long chain length may interfere with intermolecu-several other proteins during lyophilization in the lar hydrogen-bonding between stabilizing sugarspresence of increasing concentrations of excipi- and proteins.ents, including mannitol for L-asparaginase (10 mg In many cases, disaccharides appear to be theml − 1) in 50 mM sodium phosphate buffer (pH most effective and universal stabilizers among7.4) (Izutsu et al., 1994b), mannitol for b-galac- sugars and polyols (Arakawa et al., 1993; Carpen-tosidase (2 mg ml − 1) in 10 mM sodium phosphate ter et al., 1997). For example, the disaccharides
  10. 10. 10 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60trehalose, sucrose, maltose, and lactose, were all 1998b), while sucrose was a better stabilizer thanessentially equivalent to or more effective than trehalose during freeze-drying of Humicola lanugi-monosaccharides such as glucose in stabilizing nosa lipase, a hydrophobic protein (Kreilgaard etPFK during lyophilization (Crowe et al., 1993b). al., 1999). Trehalose at 20 mg mg − 1 protein wasTrehalose at 400 mM increased the recovery of more effective than sucrose in stabilizing H+-AT-PFK activity to greater than 60% during Pase during lyophilization, but at 5 10 mg mg − 1lyophilization whereas glucose at the same con- protein, sucrose was more effective (Sampedro etcentration only recovered less than 5% of the al., 1998). In stabilizing G6PDH duringprotein activity (Carpenter et al., 1993). Similarly, lyophilization, both the glucose/trehalose (1:10,the activity of H+-ATPase upon lyophilization w/w) and glucose/sucrose (1:10, w/w) systemswas increased from 4 to 100, 91, and 84% in the were shown to be equally effective (Sun andpresence of disaccharides trehalose, maltose, and Davidson, 1998).sucrose, respectively, but only 72 and 37% in the Not all proteins can be stabilized by sugars/presence of monosaccharides glucose and galac- polyols. This is still an unsolved puzzle (Carpentertose at 20 mg sugar per mg protein (Sampedro et et al., 1999). For example, sucrose at concentra-al., 1998). tions from 0.1 to 0.5 M showed little effect on the Among disaccharides, sucrose and trehalose ap- aggregation of recombinant hemoglobin in PBSpear to be the most commonly used. In compari- during freeze–thaw cycles (Kerwin et al., 1998).son to sucrose, trehalose seems to be a preferable Addition of 5% sucrose in MN12 formulation didlyoprotectant for biomolecules, because it has a not show significant stabilizing effect duringhigher glass transition temperature (Crowe et al., lyophilization (Ressing et al., 1992). Trehalose at1992, 1996). The higher glass transition tempera- 5% actually increased the loss of LDH activity inture of trehalose arises at least partly from the water from 64 to 74% during freezing (Nema andformation of trehalose – protein – water microcrys- Avis, 1992). Although the pH of the trehalosetals, preventing water plasticizing the amorphous solution decreased during freezing, the pH changephase (Librizzi et al., 1999). Other properties of during freezing could not explain the destabiliza-trehalose are also considered to be advantageous, tion of LDH by trehalose because sucrose, whichwhich include (1) less hygroscopicity, (2) an ab- stabilized LDH, also caused the same pH change.sence of internal hydrogen bonds, which allows Therefore, the type of sugar and its subunit orien-more flexible formation of hydrogen bonds with tation might have caused the difference in stabiliz-proteins, and (3) very low chemical reactivity ing LDH (Carpenter et al., 1986).(Roser, 1991). To support these arguments, Roser In a few cases, sugars have to be used with(1991) demonstrated that 35 air-dried restriction another excipient(s) to achieve satisfactory proteinand DNA-modifying enzymes are maximally sta- stabilization. Carpenter et al. (1986) demonstratedbilized by 0.3 M trehalose in comparison to other that freezing rabbit skeletal muscle PFK in liquidnon-reducing sugars, including sucrose, sorbitol, nitrogen for 30 s completely inactivated themannitol, galactitol, etc. as well as reducing sug- protein. Inclusion of 1 mM ZnSO4 or 50 mMars, including glucose, mannose, galactose, mal- sugars (trehalose, sucrose, or maltose) helped totose, lactose, etc. These advantages of using retain less than 13 or 10% of the initial proteintrehalose were later challenged by Levine and activity after freeze-thawing, while a combinationSlade (1992), who contended that sucrose could of 1 mM ZnSO4 and 50 mM trehalose (sucrose orbe equally effective in protecting biomolecules. In maltose) resulted in retention of more than 80%reality, the relative stabilization effect of these two protein activity. More than 85% of protein activ-sugars seems to be depend on both the protein ity was recovered when ZnSO4 was used withand sugar concentration. For example, trehalose glucose or inositol. Thus, sugars and metal ionsat 30 mg ml − 1 was more effective in inhibiting had a synergistic effect in stabilizing PFK duringIL-6 aggregation during lyophilization than su- freezing. Similarly, neither 10 mM sugar (tre-crose at the same concentration (Lueckel et al., halose, lactose or mannitol) nor 1% PEG could
  11. 11. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 11improve the lyophilization recovery (46%) of In addition to albumin, other polymers alsoLDH at 2 mg ml − 1 in phosphate buffer (pH 7.5). have been used. The level of protein stabilizationHowever, combined use of 1% PEG and 10 mM afforded by these polymers depends on structurelactose completely protected the protein from in- and concentration of both the polymer and theactivation (Prestrelski et al., 1993a). Also, com- protein. For example, dextran (5%), PVA (2.5%),bined use of 1% PEG and sugar ( 25 mM hydroxypropyl methylcellulose (HPMC) (1%), ortrehalose or glucose) almost completely protected gelatin (0.5%) reduced the loss of rabbit musclethe activity of PFK during lyophilization (Car- LDH activity in water during freezing from 64 topenter et al., 1993). PEG in these formulations 24%, 24, 18, and 9%, respectively (Nema andserved as a lyoprotectant, while the sugars were Avis, 1992). LDH activity was also protectedused against dehydration denaturation. during lyophilization in the presence of different concentrations of polyethyleneimine (Andersson and Hatti-Kaul, 1999). While ovalbumin at 0.01%3.1.2. Polymers had little effect on the stability of catalase (8.4 mg Polymers have been used to stabilize proteins in ml − 1 in 10 mM phosphate buffer, pH 7.0) duringsolution and during freeze-thawing and freeze- freezing, gelatin at the same concentration com-drying (Arakawa et al., 1993). One of the favor- pletely protected the protein activity (Shikamaable polymers used in the history of protein drug and Yamazaki, 1961). Polyvinylpyrrolidone (PVP)development was serum albumin. It has been used (40 kD) increased both the freeze-thawing andboth as a cryoprotectant and lyoprotectant. For freeze-drying recovery of LDH in a concentra-example, bovine serum albumin (BSA) at 1% tion-dependent manner (Anchordoquy and Car-completely protected the activity of rabbit muscle penter, 1996). Addition of 2% dextran (192 kD)LDH in water during freezing (Nema and Avis, into a sucrose-containing actin formulation sig-1992). At much lower concentrations between nificantly increased the protein stability during0.05 and 0.1% (w/v), human serum albumin lyophilization (Allison et al., 1998). Hydroxyethyl(HSA), due to its effective inhibition of protein cellulose (HEC) at 1% completely inhibitedsurface adsorption and general stabilization of lyophilization-induced aggregation of aFGF atproteins during lyophilization, was used in formu- 100 mg ml − 1 in PBS containing 33 mg ml − 1lating freeze-dried hydrophobic cytokines, such as heparin, although reconstitution time was in-interleukin-1a (IL-1a), IL-1b, IL-3, and creased significantly (Volkin and Middaugh,macrophage colony stimulating factor (MCSF) 1996).(Dawson, 1992). Increasing BSA concentrations Stabilization of proteins by polymers can gener-to 0.05% gradually increased the activity recovery ally be attributed to one or more of these polymerof LDH at 25 mg ml − 1 from about 30 to 100% properties: preferential exclusion, surface activity,during freeze-thawing and to about 80% during steric hindrance of protein–protein interactions,freeze-drying (Anchordoquy and Carpenter, and/or increased solution viscosity limiting1996). Many protein products on the market, protein structural movement. In recent years, ad-such as Betaseron®, Epogen®, Kogenate®, and ditional properties of polymers have been impli-Recombinate™ contain albumin (Physicians’ cated in stabilizing proteins during freeze-thawingDesk Reference, 1999). However, the ever-increas- and freeze-drying. Polymers such as dextran haveing concern about the potential contamination of been reported to stabilize proteins by raising theserum albumin with blood-borne pathogens limits glass transition temperature of a protein formula-its future application in protein products. There- tion significantly and by inhibiting crystallizationfore, rHA has been recommended recently to of small stabilizing excipients such as sucrosereplace serum albumin as a protein stabilizer (Skrabanja et al., 1994). PEG 3350 or dextran(Tarelli et al., 1998). Nevertheless, the ultimate T500 at 4% (w/w) has been found to inhibit a pHsolution is to develop albumin-free formulations drop during freezing of a phosphate-buffered so-for protein pharmaceuticals. lution by inhibiting crystallization of disodium
  12. 12. 12 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60phosphate (Heller et al., 1997). Probably by the 1996). For example, increasing initial concentra-same mechanism, BSA or PVP (40 kD) at 10% tion of rhFXIII from 1 to 10 mg ml − 1 increaseddramatically inhibited the pH drop during freez- the recovery of native rhFXIII during repeateding of a buffered LDH solution (Anchordoquy freeze-thawing (Kreilgaard et al., 1998b). The re-and Carpenter, 1996). At least partly due to this covery of LDH activity gradually increased frominhibition effect, both BSA and PVP increased the 6% at a protein concentration of 10 mg ml − 1 tofreeze –thaw recovery of the protein in a concen- about 65% at concentrations above 175 mg ml − 1tration-dependent manner. The inhibition of crys- after freeze-thawing (Carpenter et al., 1990). Uptallization of small molecules is apparently due to to about 90% LDH activity was recovered whenpolymer-induced viscosity increase (Slade et al., the concentration was increased to 500 mg ml − 11989). (Anchordoquy and Carpenter, 1996). Koseki et al. On the other hand, polymers may cause phase (1990) demonstrated that increasing the ovalbu-separation during freezing, adversely affecting min concentration in the range 0.5–2.5 mg ml − 1protein stability (see Section 2.2). Certain poly- at pH 1.9 decreased the structural changes of themers may destabilize proteins during lyophiliza- freeze-treated (−40°C) protein, as measured bytion due to steric hindrance, preventing efficient UV.hydrogen bonding with proteins. Dextran (40 kD) Similarly, the lyophilization recovery of PFKat concentrations of up to 100 mg ml − 1 failed to activity at 25 and 40 mg ml − 1 was 34 and 64%,inhibit dehydration-induced unfolding of respectively, in the presence of 200 mM trehaloselysozyme because of its inability to form adequate (Carpenter et al., 1987). Increasing the concentra-hydrogen bonding with the protein (Allison et al., tion of rabbit muscle LDH from 10 to 500 mg1999). Similarly, this compound could hardly pre- ml − 1 gradually increased the activity recoveryvent formation of b-sheets in poly-L-lysine during from less than 20% to about 60% duringdehydration (Wolkers et al., 1998b). In fact, Dex- lyophilization (Anchordoquy and Carpenter,tran (162 kD) at 5% (w/v) was shown to destabi- 1996). Increasing the concentration of bovine andlize Humicola lanuginosa lipase during human IgG species markedly decreasedlyophilization, as determined by IR (Kreilgaard et lyophilization-induced protein aggregation (Sar-al., 1999). ciaux et al., 1998). Certain proteins, however, do not show this concentration-dependent protec-3.1.3. Protein itself tion. The percentage of lyophilization-induced de- Protein aggregation in solution is generally con- naturation of catalase in the absence of acentration-dependent. It has been suggested that stabilizer was determined to be about 65%, inde-increasing protein concentration to higher than pendent of the protein concentration in the range0.02 mg ml − 1 may facilitate potential protein 1–5000 mg ml − 1 (Tanaka et al., 1991).aggregation (Ruddon and Bedows, 1997). Increas- The mechanisms of proteins’ self-stabilizationing protein concentration increases aggregation of during freezing and/or lyophilization have notmany proteins in solution, such as LMW-UK in been clearly delineated. Proteins are polymers,the range 0.2–0.9 mg ml − 1 (Vrkljan et al., 1994), and therefore, at least some of the above-dis-IL-1b in the range 100 – 500 mg ml − 1 (Gu et al., cussed stabilization mechanisms for polymers may1991), apomyoglobin in the range 4 – 12 mg ml − 1 be applicable to proteins’ self-stabilization. Re-in the presence of 2.4 M urea (De Young et al., cently, two hypotheses have been reiterated to1993), and insulin (Brange et al., 1992a). explain the concentration-dependent protein sta- In contrast, proteins at higher concentrations bilization upon freezing (Allison et al., 1996).are often more resistant against both freezing- First, unfolding of proteins at high concentrationsand lyophilization-induced protein denaturation/ during freezing may be temporarily inhibited byaggregation. The activity recovery of many labile steric repulsion of neighboring protein molecules.proteins after freeze-thawing correlates directly Second, the surface area of ice-water interfacewith initial protein concentration (Allison et al., formed upon freezing is finite, which limits the
  13. 13. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 13amount of protein to be accumulated and dena- the driving force of protein adsorption and/ortured at the interface. In addition, favorable aggregation at these interfaces. Low concentra-protein–protein interactions (possible formation tions of nonionic surfactants are often sufficientof dimers or multimers) may contribute to the to serve this purpose due to their relatively lowincreased protein stability at high concentrations, critical micelle concentrations (CMC) (Bam et al.,as observed for thermophilic proteins (Mozhaev 1995). Other stabilization mechanisms were alsoand Martinek, 1984). proposed, such as assistance in protein refolding during thawing and protein binding, which may3.1.4. Non-aqueous sol6ents inhibit protein–protein interactions (Carpenter et Non-aqueous solvents generally destabilize al., 1999).proteins in solution. At low concentrations certain Tween 80 is one of the commonly used surfac-non-aqueous solvents may have a stabilizing ef- tants for protein stabilization during freezing.fect. These stabilizing non-aqueous solvents in- Tween 80 at concentrations of ] 0.01% protectedclude polyhydric alcohols such as PEGs, ethylene both LDH and GDH from denaturation duringglycol, and glycerol and some polar and aprotic quench freezing and thawing (Chang et al.,solvents such as dimethylsulphoxide (DMSO) and 1996b). The freeze–thaw recovery of LDH activ-dimethylformamide (DMF) (Volkin and ity was increased from 36 to 57 and 65% in theKlibanov, 1989; Carpenter et al., 1991). presence of 0.002 and 0.005% Tween 80, respec- In fact, polyhydric alcohols are among the com- tively (Nema and Avis, 1992). Maximum freeze–monly used and effective cryoprotectants. For thaw recovery (about 80%) of LDH activity wasexample, in the presence of 0.2 M PEG 400, the reached in the presence of 0.05% Tween 80.loss of rabbit muscle LDH activity upon freezing Tween 80 at concentrations from 0.005 to 0.01%was reduced from 64 to 15% (Nema and Avis, also protected several other proteins from freezing1992). LDH can also be protected from freeze- denaturation, including TNFbp, IL-1ra, bFGF,thawing denaturation to different degrees by eth- MDH, aldolase, and PFK (Kendrick et al.,ylene glycol or 2-methyl-2,4-pentanediol 1995b).(Carpenter et al., 1990). PEG at 1 – 10% (w/v) Other nonionic and ionic surfactants have alsocompletely protected both LDH and PFK at 25 been reported in cryoprotection of proteins. Themg ml − 1 (at pH 7.5 and 8.0, respectively) during following surfactants protected LDH from freez-freeze-thawing, although they were not effective ing denaturation to various degrees, Brij 35; Brijstabilizers during freeze-drying (Carpenter et al., 30 (polyoxyethylene lauryl ether); Lubrol-px; Tri-1993). Glycerol at 0.3% (v/v) prevented freezing ton X-10; Pluronic F127 (polyoxyethylene-poly-denaturation of ovalbumin (0.1%) (Koseki et al., oxypropylene copolymer); and SDS (Nema and1990) and at 1 M, increased the recovery of Avis, 1992; Chang et al., 1996b). SDS at 0.5 mMcatalase activity upon freezing from 80 to 95% increased the activity recovery of catalase (8.4 mg(Shikama and Yamazaki, 1961). ml − 1 in 10 mM phosphate buffer, pH 7.0) from Cryoprotection of proteins by these non- 80 to 90% upon freezing (Shikama and Yamazaki,aqueous solvents may be pH-dependent. Ethylene 1961).glycol stabilized RNase A at pH 2.3 but destabi-lized it at pH 5.5 (Arakawa et al., 1991). This is 3.1.6. Amino acidspartly because proteins may tolerate freezing de- Certain amino acids can be used as cryoprotec-naturation to different degrees at different pHs. tants and/or lyoprotectants. For example, freezing rabbit skeletal muscle PFK in liquid nitrogen for3.1.5. Surfactants 30 s inactivated the protein completely, and sev- The formation of ice-water interfaces during eral amino acids, including glycine, proline, or 4freezing may cause surface denaturation of hydroxyproline, significantly increased the recov-proteins (see Section 2.2). Surfactants may drop ery of the protein activity (Carpenter et al., 1986).surface tension of protein solutions and reduce Glycine at low concentrations has been shown to
  14. 14. 14 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60suppress the pH change in 10 or 100 mM sodium Recently, Ramos et al. (1997) demonstratedphosphate buffer during freezing (Pikal and Car- that 2-O-b-mannosylglycerate at 500 mM in-penter, 1998). Therefore, amino acids may protect creased the freeze-drying recovery of LDH activ-proteins from freezing denaturation at least partly ity (at 50 ug ml − 1) from 12 to 85% whileby reducing the rate and extent of buffer salt trehalose only increased the recovery to 54%.crystallization. As lyoprotectants, several amino acids in- 3.2. Mechanisms of protein stabilization duringcreased the lyophilization recovery of LDH from lyophilization22 to about 39–100%, including proline, L-serine,sodium glutamate, alanine, glycine, lysine hydro- Since freezing and drying stresses imposed onchloride, sarcosine, g-aminobutyric acid (Carpen- proteins during lyophilization are different, mech-ter et al., 1990). Glycine alone or in combination anisms of protein stabilization by excipients arewith mannitol inhibited aggregation of an anti- not the same in the two stages of lyophilization.body-vinca conjugate during lyophilization (Royet al., 1992). LDH activity was increased by 20% 3.2.1. Mechanisms of cryoprotectionduring vacuum-drying in the presence of pheny- One of the most widely accepted protein stabi-lalanine:arginine:H3PO4 (1:1:0.5 molar ratio) lization mechanisms in liquid state is preferential(Mattern et al., 1999). interaction. Preferential interaction means that a protein prefers to interact with either water or an3.1.7. Miscellaneous excipients excipient(s) in an aqueous solution. In the pres- Salts and amines have been used as cryoprotec- ence of a stabilizing excipient, the protein preferstants. LDH activity can be protected to various to interact with water (preferential hydration) anddegrees upon freezing in the presence of potas- the excipient is preferentially excluded from thesium phosphate, sodium acetate, ammonium sul- domain of the protein (preferential exclusion). Infate, magnesium sulfate, sodium sulfate, this case, proportionally more water moleculestrimethylamine N-oxide, or betaine (Carpenter et and fewer excipient molecules are found at theal., 1990). Increasing the potassium phosphate surface of the protein than in the bulk. Therefore,concentration from 10 mM to 1 M increased the preferential exclusion of an excipient is usuallyrecovery of LDH upon freezing from less than associated with an increase in the surface tension20% to more than 80% (Arakawa et al., 1993). of water. Detailed discussion of this stabilization Metal ions can protect certain proteins during mechanism can be found elsewhere (Arakawa etlyophilization. In the presence of 100 mM sugars al., 1991, 1993; Timasheff, 1993; Lin andsuch as trehalose, maltose, sucrose, glucose or Timasheff, 1996; Timasheff, 1998).galactose, some divalent metal ions improved the The preferential interaction mechanism appliesrecovery of PFK activity (at 40 mg ml − 1 in 1 mM equally well during freeze–thaw processes (Car-sodium borate, pH 7.8) during lyophilization in a penter et al., 1991; Arakawa et al., 1993; Crowe etconcentration-dependent manner. The relative ef- al., 1993b). Protein stabilizers, which are excludedfectiveness of these metal ions was apparently in from protein surface in solution, can also stabilizethe following order: Zn2 + Cu2 + Ca2 + , proteins during freezing. Nema and Avis (1992)Mn2 + Mg2 + (Carpenter et al., 1987). examined the stabilizing effect of 13 cryoprotec- The activity of LDH can be protected to differ- tants on the recovery of rabbit muscle LDH activ-ent degrees during lyophilization in the presence ity, including trehalose (5%), mannitol (5%),of some amphiphilic excipients, including HP-b- sucrose (5, 10, 34.2%), Brij 30 (polyoxyethyleneCD, 3-[(3-cholamidepropyl)-dimethylammonio]-1- lauryl ether, 0.05%), Tween 80 (0.002– 1%),propanesulfate (CHAPS), sodium cholate, sucrose Pluronic F127 (1%), HPMC (1%), PVP (2.5%),monolaurate (Izutsu et al., 1995). Combinations PEG 400 (0.2 M), gelatin (0.5%), BSA (1%),of sucrose and these amphiphilic excipients in- b-cyclodextrin (0.9%) and dextran (5%). Theycreased the protein stability synergistically. found that the cryoptotectants that increased the
  15. 15. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 15stability of LDH in solution at room temperature activity at all during lyophilization (Ramos et al.,also improved the recovery of protein activity 1997).after freeze– thaw. However, no apparent correla- One major mechanism of protein stabilizationtion was found between the increase in surface by lyoprotectants is the formation of an amor-tension induced by the cryoprotectants and their phous glass during lyophilization (Roser, 1991;protective effect on protein recovery during Franks, 1994; Fox, 1995). Formation of a glassfreeze –thaw cycles. Based on this study, several increases the viscosity to 1012 Pa s (1013 P)other stabilization mechanisms were postulated, (Angell, 1995). It is the extreme viscosity at theincluding modification of the size of ice crystals, glassy state, that increases protein stability byreduction (instead of elevation) of surface tension, slowing down interconversion of conformationaland restriction of diffusion of reacting molecules. substates and conformational relaxation of aSupposedly, reduction of surface tension is how protein (Hagen et al., 1995, 1996). This stabiliza-most surfactants stabilize proteins during freezing. tion mechanism explains the retention of G6PDH Besides polymers, many cryoprotectants can in- activity during freeze-drying (Sun et al., 1998).crease the viscosity of a solution, restricting diffu- Amorphous materials are structurally more simi-sion of reacting molecules. In fact, the difference lar to a liquid than crystalline materials (Taylorin solution viscosity has explained why trehalose and Zografi, 1998b). Freeze-dried amorphous in-is apparently more effective than sucrose, maltose, sulin is far more stable than crystalline insulinglucose, or fructose in stabilizing liquid pyrophos- against deamidation and dimer formation at dif-phatase and G6DPH (Sola-Penna and Meyer-Fer- ferent water contents up to 15% (Pikal and Rigs-nandes, 1998). On top of this, concentration of all bee, 1997). Izutsu et al. (1995) studied the effect ofsolutes during freezing increases the solution vis- amphiphilic excipients on freeze-drying of LDHcosity rapidly. Therefore, the rate of a chemical and found that only those that remain amorphousreaction may increase initially due to concentra- in the solid state protected the enzyme duringtion of all solutes but then drops gradually as the freeze-drying. These excipients, including HP-b-viscosity increases (Pikal, 1999). The rate of a CD, CHAPS, sodium cholate, sucrose monolau-chemical reaction is minimized at the glassy state rate, showed a concentration-dependentwhen the viscosity is increased to 1012 Pa·s (An- stabilization effect during freeze-drying.gell, 1995). In addition to viscosity increase, some A glass can be roughly divided into two types:of these cryoprotectants stabilize proteins by sup- fragile and strong. The viscosity of a fragile glasspressing pH changes during freezing (Anchordo- increases more deeply than a stronger glass for aquy and Carpenter, 1996). given temperature drop below the glass transition The preferential interaction mechanism does temperature (Angell, 1995). Therefore, excipientsnot fully explain protein cryoprotection by poly- forming fragile glasses are better stabilizing agentsmers or by proteins themselves at high concentra- (Hatley, 1997). Both sucrose and trehalose cantions. These different mechanisms have been form a fragile glass (Hatley, 1997; Duddu et al.,addressed in Section 3.1. 1997). Another interrelated stabilization mechanism is3.2.2. Mechanisms of lyoprotection the water replacement hypothesis (Crowe et al., During lyophilization, the preferential interac- 1993a; Allison et al., 1996, 1998). This mechanismtion mechanism is no longer applicable because involves the formation of hydrogen bonds be-the hydration shell of proteins is removed (Car- tween a protein and an excipient(s) at the end ofpenter et al., 1993; Crowe et al., 1993b; Allison et the drying process to satisfy the hydrogen bond-al., 1996). Thus, many excipients that stabilize ing requirement of polar groups on the proteinproteins in solution do not offer the same effect surface (Carpenter and Crowe, 1989; Carpenter etduring lyophilization. For example, KCl at 500 al., 1990). These excipients preserve the nativemM effectively protected LDH from thermal in- structures of proteins by serving as water substi-activation at 50°C, but did not protect the protein tutes (Carpenter et al., 1990; Arakawa et al., 1991;
  16. 16. 16 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60Carpenter et al., 1993; Prestrelski et al., 1995). In because of their structural differences. Sucrose hasthis way, intra- or interprotein hydrogen bonding been found to form hydrogen bonds with lysozymemay be prevented during dehydration (Leslie et al., to a greater extent than trehalose (Allison et al.,1995; Cardona et al., 1997). Therefore, stabilization 1999) and with PVP than both trehalose andof proteins requires hydrogen bonding with an raffinose (Taylor and Zografi, 1998b). The differ-excipient(s) during freeze-drying or dehydration ence among sugars in stabilization of proteins may(Carpenter and Crowe, 1989; Arakawa et al., 1991; be partially due to the difference in the extent andCarpenter et al., 1991; Crowe et al., 1998). intimacy of hydrogen bond formation. Since an amorphous state of proteins and stabi- In addition to glass formation, many excipients,lizers allows maximal H-bonding between protein especially polymers, can stabilize proteins by in-and stabilizer molecules, crystallization of any creasing Tg of protein formulations, since higheramorphous protein stabilizers during lyophiliza- Tgs generally result in more stable protein formula-tion often causes protein destabilization due to tions during lyophilization. For example,inefficient hydrogen bonding. Mannitol can easily Costantino et al. (1998b) examined six stabilizersbe crystallized and its crystallization is apparently (lactose, trehalose, cellobiose, mannitol, sorbitol,responsible for the destabilization of some proteins and methyl a-D-mannopyranoside) duringduring lyophilization. The aggregation of IL-6 lyophilization of rhGH and found that the higherduring lyophilization could not be inhibited effec- the Tg of the stabilized formulation, the greater thetively in a formulation containing only mannitol degree of structural (such as a-helix) preservation(Lueckel et al., 1998b). In the presence of 1% PEG, in the co-lyophilizate with less protein aggregation.increasing the mannitol concentration above 10 In general, larger carbohydrates form a glass moremM reduced the activity of LDH and PFK, possi- readily with a higher Tg than smaller ones, but havebly due to crystallization of mannitol (Carpenter et more steric hindrance interfering with intimateal., 1993). Mannitol at 300 mM destabilized Humi- hydrogen bonding with a dried protein (Crowe etcola lanuginosa lipase during lyophilization and al., 1993b). Therefore, selection of such an excipientDSC analysis indicated that 85% mannitol was needs balancing both the formation of a glass withcrystallized during lyophilization (Kreilgaard et al.,1999). a high Tg and intimacy of hydrogen bonding. Although it was debatable whether or not hydro- Other mechanisms of protein stabilization alsogen bond was indeed formed between trehalose and seem operable. Sugars may stabilize proteins bylysozyme upon lyophilization (Belton and Gil, inhibiting crystallization of other excipients such as1994), many studies have confirmed hydrogen PEGs during lyophilization (Izutsu et al., 1995), bybonding by IR spectroscopy between carbohy- inhibiting acute lyophilization-induced protein un-drates and freeze-dried proteins, such as lysozyme, folding such as rhIL-1ra (Chang et al., 1996a), orBSA, and PFK (Carpenter and Crowe, 1989; by preserving a protein’s internal mobility such asCrowe et al., 1993b; Remmele et al., 1997; Allison sperm whale Mb (Sastry and Agmon, 1997).et al. 1999) and bFGF, g-IFN, recombinant G- Polyelectrolytes can stabilize a protein duringCSF, bovine a-lactalbumin, and bovine a-casein lyophilization by forming multiple electrostatic(Prestrelski et al., 1993b). The degree of structural interactions with the protein (Gibson, 1996).protection of lysozyme by sucrose and trehalosespectra was shown to correlate with the extent ofhydrogen bonding between the sugars and the 4. Design of a robust lyophilization cycle — aprotein (Allison et al., 1999). Hydrogen bonding step-by-step analysishas also been demonstrated between sucrose andother non-protein polymers, such as poly-L-lysine The purpose of designing a robust lyophiliza-(Wolkers et al., 1998b), and PVP (Taylor and tion cycle for protein pharmaceuticals is to obtainZografi, 1998b). Different excipients may form a consistent, stable, and esthetically acceptablehydrogen bonds with proteins to different extents product. To achieve this goal, a number of
  17. 17. W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60 17parameters that directly determine or characterize eventually changes the solution from a viscousa lyophilization cycle need to be determined or liquid to a brittle glass, which contains aboutdefined. These parameters should include glass 20–50% water (Pikal, 1990b; Hatley et al., 1996).transition temperature (T % )/collapse temperature g The temperature of this reversible transition for(Tcol), cooling rate, drying rate, and residual mois- the maximally freeze-concentrated solution isture content. termed glass transition temperature, T % . This tem- g perature is also called the temperature of vitreous4.1. Characterization of protein formulations prior transformation (Rey, 1999). T % is used to differen- gto lyophilization tiate this transition from the softening point of a true glass transition, Tg of a pure polymer. T % is g In addition to glass transition temperature (T % )/ g one of the most important parameters for opti-collapse temperature (Tcol), several other critical mization of a lyophilization process (Franks,temperatures, including crystallization tempera- 1990).ture (Tcry), eutectic temperature (Teut), and devit- The collapse temperature (Tcol) is the tempera-rification temperature (Tdev), should be ture at which the interstitial water in the frozendetermined in order to design a robust lyophiliza- matrix becomes significantly mobile (Jennings,tion cycle. These temperatures are mostly deter- 1999). Tcol is closely related to T % . In fact, Tcol has gmined by thermal analysis such as DSC, electrical been considered to be equivalent to Tg of anresistance measurements, and direct microscopic amorphous system or to the eutectic melting tem-observation. perature of a crystalline system (Slade et al., 1989; Pikal, 1990a,b). Recent literature indicates that4.1.1. Glass transition temperature (T % ) and g the Tcol of many small carbohydrates is consis-collapse temperature (Tcol) tently higher than their T % by about 12 K (Sun, g Ice formation during cooling of a protein solu- 1997). The discrepancy between T % and Tcol for gtion concentrates all solutes. Solute concentration polymers seems even larger (Roos and Karel, 1991). This is because the decrease in viscosity at T % may not be sufficient enough to cause struc- g tural collapse (Bindschaedler, 1999). For refer- ence, Table 1 lists T % s and Tcols of some g commonly used excipients and buffers. 4.1.2. Crystallization temperature (Tcry) When the temperature of an aqueous protein formulation drops below 0°C, water usually crys- tallizes out first. Then, the crystalline component, which usually has the least solubility in the formu- lation, may crystallize out. This temperature is termed crystallization temperature. 4.1.3. Eutectic crystallization/melting temperature (Teut) When the temperature of an aqueous protein formulation further decreases after crystallization of the least soluble component, this component and water crystallize out at the same time as aFig. 1. A theoretical phase diagram showing ice formation, mixture. This temperature is termed eutectic crys-solute crystallization, eutectic point, and glass transition dur- tallization/melting temperature. The relationshiping freezing. between Teut and T % is shown in Fig. 1. Due to g
  18. 18. 18Table 1Glass transition and collapse temperatures (°C) of buffers, excipients and proteinsCompounds T% g Tcol Tg ReferencesBuffering agents aCitric acid −54a, −53b 11b Chang and Randall, 1992; bLu and Zografi, 1997Hepes −63 Chang and Randall, 1992 aHistidine −33a, −32b Chang and Randall, 1992; bOsterberg and Wadsten, 1999 8Potassium −76 Franks, 1993acetatePostassium −62 Franks, 1993citratePotassium −55 Chang and Randall, 1992phosphate(KH2PO4)Sodium acetate −64 Chang and Randall, 1992Sodium −52 Franks, 1993bicarbonateSodium citrate −41 Chang and Randall, 1992Sodium −45 Chang and Randall, 1992phosphate(NaH2PO4)Tris base −55 Franks, 1993Tris–HCl −65 Chang and Randall, 1992Excipients, low MWb-Alanine −65 Franks, 1993Arabinose 3 Roos, 1993Arginine 42 Mattern et al., 1999Cellobiose 77 Franks, 1990; Costantino et al., 1998b aFructose −42b 10a, 13b Roos, 1993; bWisniewski, 1998Fucose 31 Roos, 1993 aGalactose −41a 31b, c38 Franks, 1990; bLevine and Slade, 1992; cRoos, 1993 a W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60Glucose −43a −41d 4c, 32b, 39a Franks, 1990; bLevine and Slade, 1992; cPrestrelski et al., 1995; dAdams and Ramsay, 1996Glutamic acid −17 Chang and Randall, 1992 aGlycerol −65a −92b, −93a Franks, 1990; bBell et al., 1995Glycine −37 Chang and Fischer, 1995 b a aHistidine −32 37 Mattern et al., 1999; bOsterberg and Wadsten, 1999 8 b aLactose −30.5 101a, 114c Levine and Slade, 1992; bAdams and Ramsay, 1996; cTaylor and Zografi, 1998bLysine 68 Mattern et al., 1999 aMaltose −30a 43a, 82b, Franks, 1990; bLevine and Slade, 1992; cTaylor and Zografi, 1998b 100cMaltotriose −23 Hatley and Franks, 1991 aMannitol −33a,−27b 13c Meredith et al., 1996; bLueckel et al., 1998a; cKim et al., 1998
  19. 19. Table 1 (Continued)Compounds T% g Tcol Tg References aMannose −41a 30a, 33b Franks, 1990; bLevine and Slade, 1992Melibiose 91 Roos, 1993Octulose 21 Wolkers et al., 1998a aRaffinose −26c 108b, 114a Taylor and Zografi, 1998b; bWolkers et al., 1998a; cWillemer, 1999 aRibose −47b −10b, −13a Roos, 1993; bWisniewski, 1998Sodium chloride B−60 Franks, 1993 aSorbitol (glucitol) −44a,b −54c −3a Franks, 1990; bKerr et al., 1993; cAdams and Ramsay, 1996Sucrose −32f −31d, −40h 31c, 61a, 72e, aLevine and Slade, 1992; bHancock et al., 1995; cPrestrelski et al., 1995; dAdams and 77b, 75g Ramsay, 1996; eCostantino et al., 1998c; fKasraian et al., 1998; fLueckel et al., 1998a; g Taylor and Zografi, 1998b; hOvercashier et al., 1999Trehalose −29c, −34h 78a, 46b, 117d, aLevine and Slade, 1992; bPrestrelski et al., 1995; cAdams and Ramsay, 1996; dDuddu and 115c, 105f, 118g Dal Monte, 1997; eMiller et al., 1997; fLueckel et al., 1998a; gTaylor and Zografi, 1998b; h Overcashier et al., 1999 aWater −133a, −137b Bell et al., 1995; bMiller et al., 1997 bXylitol −47b −23a, −39b Franks, 1990; aRoos, 1993 aXylose −48a 9a, 14b Franks, 1990; bRoos, 1993Excipients, high MWCellulose 227 Hancock and Zografi, 1994b-Cyclodextrin 108 Prestrelski et al., 1995 aDextran (10 kD) −10b 91a Prestrelski et al., 1995; bWillemer, 1999 aDextran (40 kD) 94a, 101b te Booy et al., 1992; bPrestrelski et al., 1995Dextran (70 kD) −11 Adams and Ramsay, 1996Ficoll −20 Pikal, 1990b; Izutsu et al., 1996Gelatin −8 Pikal, 1990bHydroxypropylmeth 155 Hancock and Zografi, 1994yl-cellulose aHydroxyethyl −12a −5b 110c Crowe et al., 1993b; bPikal, 1990b; cCrowe et al., 1998starchMaltodextrin 860 169 Taylor and Zografi, 1998bMethocel −9 Willemer, 1999 W. Wang / International Journal of Pharmaceutics 203 (2000) 1–60PEG (6 kD) −13 Willemer, 1999Polydextrose −27 Kerr et al., 1993 aPVP K15 (10 kD) −27b 100a Bell et al., 1995; bIzutsu et al., 1996PVP (40 kD) −24 Adams and Ramsay, 1996PVP K30 (40 kD) 180 Bell et al., 1995 aPVP K90 (1000 185a, 176b Hancock et al., 1995; bTaylor and Zografi, 1998bkD)Sephadex G 200 −10 Willemer, 1999Starch 227 Hancock and Zografi, 1994Proteins a 19BSA −13a, −11b Slade et al., 1989; bChang and Randall, 1992