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Lyophilization‐induced protein denaturation in phosphate buffer systems: Monomeric and tetrameric β‐galactosidase
Lyophilization-Induced Protein Denaturation in Phosphate Buffer Systems: Monomeric and Tetrameric b-Galactosidase KATHERINE A. PIKAL-CLELAND,1,2 JOHN F. CARPENTER2 1
Inhale Therapeutic Systems, 150 Industrial Road, San Carlos, California 94070
2
University of Colorado Health Sciences Center, Denver, Colorado 80262
Received 28 August 2000; revised 1 February 2001; accepted 12 February 2001
ABSTRACT: During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate signi®cantly more inactivation and secondary structural perturbation of monomeric and tetrameric b-galactosidase (b-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the signi®cant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer.1 In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric b-gal in either NaP or KP buffer resulted in signi®cant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freezedrying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilizationinduced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active b-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freezedrying in phosphate buffers, even if there are large-scale changes in solution pH during freezing. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1255±1268, 2001
Keywords: b-galactosidase; pH changes; phosphate buffers; freeze-dry; protein denaturation; infrared spectroscopy
INTRODUCTION Correspondence to: K.A. Pikal-Cleland (Telephone: 650631-3564; Fax: 650-631-3150; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1255±1268 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
Freeze-drying is commonly used to manufacture protein pharmaceuticals, which are insuf®ciently stable for distribution and storage as aqueous formulations. However, proteins are often readily denatured during freezing and drying. Prestrelski
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Figure 1. The pathways of unfolding and refolding during freezing and drying of proteins was described previously.2 The native protein (Naq) in solution may unfold to form denatured or dissociated protein (Ufrozen). In the presence of stabilizing excipients that cause preferential hydration of the native protein, the native protein structure (Nfrozen) is maintained on freezing. During drying, the native frozen protein may either unfold (Udry) or remain in its native state (Ndry). To maintain the protein in its native state during drying, excipients that are able to hydrogen bond to the protein are often required (water replacement theory). Alternatively, if the protein is already unfolded in the frozen state, it is likely to undergo additional unfolding on drying (Udry). On reconstitution, the unfolded protein may either refold to the native form (Naq) or form an inactive species such as an aggregate (Uaq).
and colleagues2 previously described the protein conformational states during freezing, drying, and rehydration and the effects of excipients during each of these processes (Fig. 1). According to their scheme, a suitable cryoprotectant may prevent freezing-induced unfolding and thus the protein remains native in the frozen state (Nfrozen). A suitable cryoprotectant is a preferentially excluded solute, such as sucrose3 and poly(ethylene glycol) (PEG),4 that causes an increase in the chemical potential of the protein at the appropriate solute concentrations.5±7 The increase in chemical potential is greater for the denatured state than the native state, causing an increase in the free energy of unfolding. In the absence of a suitable cryoprotectant, a non-native protein conformation may result during freezing (Ufrozen). Even though an excipient may stabilize the native protein during freezing (i.e., a cryoproJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
tectant), it will not necessarily prevent dryinginduced denaturation. During drying, the hydration shell around the protein is removed, and thus the thermodynamics of the preferential exclusion mechanism do not apply. Stabilization of the native protein state during dehydration may be explained by the water substitute hypothesis. Excipients, such as sucrose and trehalose,8±11 which are able to hydrogen bond to the polar and charged groups of the protein in the place of lost water, have been shown to inhibit dehydration-induced protein unfolding (Ndry) as measured by infrared (IR) spectroscopy.8,12 Another theoretical explanation for solute-induced stabilization during dehydration is the vitri®cation hypothesis, which states that an effective stabilizer readily forms a glass, reducing any molecular motion relevant to stability.13,14 However, studies have documented that formation of a glass is not a suf®cient requirement for stabilization. For example, dextran, which forms a glass, does not inhibit protein unfolding during lyophilization9,11,15 ±18 and, because of steric hindrance, it does not hydrogen bond effectively to the dried protein.9,11,15±18 The ability of excipients to stabilize proteins against freezing and dehydration stresses is concentration dependent. Sucrose at concentrations as low as 10 mM may promote stability during drying, but higher sucrose concentrations (e.g., > 5% w/v) are often required for stabilization during freezing.6 PEG offers protection during freezing at much lower concentrations (1% w/v) than required for sugars,7 but it does not protect against drying-induced degradation. If during freezing the protein has already denatured (Ufrozen), subsequent drying in the presence of low sucrose levels (i.e., 10 mM sucrose) would prevent further dehydration-induced denaturation but still result in unfolded dried protein (Udry). In addition, if the excipient protects during freezing but not dehydration (i.e., PEG), the result after freeze-drying would also be a non-native conformation (Udry). To maintain the native structure throughout lyophilization, the protein must be protected against both the freezing- and drying-induced denaturation. Conformational stability upon rehydration must also be considered. A protein that maintains its native conformation throughout freezing and drying will most likely remain in its native state on rehydration as well (Naq). Even if the protein is in its unfolded state after freeze-drying (Udry), on reconstitution it may refold to its native
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
conformation (Naq). However, competing with this refolding pathway is the conversion of the unfolded protein in its dried state to an irreversibly denatured or aggregated species, thus resulting in some fraction (perhaps dominant) of the population of protein molecules being inactive and/or aggregated in the rehydrated samples. In this study, we investigated the impact of dehydration on the recovery of enzymatic activity and structural integrity of monomeric and tetrameric b-galactosidase (b-gal) formulated in phosphate buffers, to complement earlier freezethawing studies.1 Both monomeric and tetrameric b-gal displayed lower recoveries of activity and greater changes in secondary structure after freeze-thawing in the sodium phosphate (NaP) buffer system compared with the potassium phosphate (KP) system that was attributed to differences in pH of the freeze concentrate. During freezing in the NaP buffer system, precipitation of the less soluble buffer component (disodium salt) results in a signi®cant pH decrease to as low as 3.8. In contrast, the KP buffer does not experience a signi®cant pH change during freezing. Minimizing the residence time in the freeze-concentrated state by fast cooling and fast warming reduced exposure to concentrated buffer salts and acidic pH conditions (NaP buffer), yielding greater recovery of active protein. In addition, tetrameric b-gal was observed to be more freeze labile than monomeric b-gal due in part to cooling-induced and/or freezing-induced dissociation, which is commonly observed for multimeric proteins.19±21 With this understanding of the physical instability of b-gal during freeze-thawing, we investigated the stability during freeze-drying in the phosphate buffers to gain insight into the effects of the combined stresses of freezing, buffer acidi®cation, and dehydration on protein stability. Izutsu and colleagues22 have documented that monomeric b-gal is partially inactivated during freeze-drying in the absence of excipients. Because the degradation pathways for the model proteins chosen are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stress of drying alters the native structure of the protein, by infrared (IR) spectroscopy, and if preservation of the native conformation during freeze-drying is requisite for optimal recovery of activity. Finally, excipients of various classes just discussed were tested for their capacity to prevent lyophilizationinduced unfolding and to promote increased
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recovery of catalytic activity on reconstitution of the lyophilized sample.
MATERIALS AND METHODS Materials b-Galactosidase derived from A. oryzae (monomeric b-gal) was purchased from Toyobo Company (Osaka, Japan). b-Galactosidase derived from E. coli (tetrameric b-gal) was purchased from Worthington Biochemical Corporation. Sucrose was purchased from Pfanstiehl Laboratories. All other chemicals were purchased from Sigma Chemicals. All materials were reagent grade or higher quality. Freeze-Drying Conditions Samples (0.5 mL) at different protein concentrations (2, 25, 100, 1000 mg/mL) were transferred into 1-mL glass vials and freeze-dried in a FTS freeze-drier. Samples were frozen at a shelf temperature of ÿ508C for 2 h. Primary drying was achieved at a shelf temperature of ÿ408C for 30 h. Secondary drying was achieved by drying at an initial shelf temperature of ÿ108C for 4 h and then at 208C for an additional 4 h. A vacuum of 60 mTorr was maintained throughout the freeze-dry cycle. After completion of secondary drying, the vials were capped under vacuum. Enzyme Preparation and Activity Assay Monomeric and tetrameric b-gal were dialyzed against the appropriate phosphate buffer (pH 7.0) overnight at 48C before use. As described by Izutsu and colleagues,23 monomeric b-gal activity was determined by measuring the absorbance at 420 nm after a 10-min incubation at 258C using 2-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. Tetrameric b-gal activity was determined by monitoring the increase in absorbance at 405 nm at 258C resulting from the hydrolysis of ONPG as described by Cravens et al.24 Initial activity was calculated as a percentage of the activity in the control samples (for each formulation) that were not subjected to a freeze-thaw cycle or freeze-dry cycle. Freeze-thaw and freeze-dry samples were run in triplicate. The recoveries of activities are reported as a mean and standard deviation (SD) for three separate enzyme samples. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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Infrared Spectroscopy Secondary structural analysis of the protein freeze-dried at 1 mg/mL was conducted using a Nicolet Magna 550 IR spectrophotometer equipped with a dTGS detector. IR spectra (256 scans, 4 cmÿ1 resolution) were recorded at 258C. Freeze-dried samples (0.2±0.4 mg protein) were ground with 300 mg of KBr and pressed into pellets, as described previously.8 This procedure for preparing KBr pellets does not alter the structure of proteins in the dried state.8 Spectra of the unlyophilized aqueous proteins (native controls) were collected using an IR cell with CaF2 windows and a 6-mm spacer. The native controls were aqueous solutions at 18 mg/mL (tetrameric b-gal) or 27 mg/mL (monomeric b-gal).1 The protein spectra were processed as outlined previously.25,26 Brie¯y, the protein spectra were corrected with the appropriate buffer blank (for the aqueous native controls) and gaseous water, and the second-derivative amide I spectra were calculated with Nicolet Omnic software and area normalized.
RESULTS AND DISCUSSION Protein Concentration Dependence Because many proteins have displayed greater stability at higher protein concentrations,10,22,27,28 we ®rst investigated the impact of protein concentration on the stability of b-gal during freeze-drying and then determined the impact of the buffer salts at a ®xed protein concentration. Previously, we have shown that the percentage of activity recovered for both monomeric and tetrameric b-gal freeze-thawed in 100 mM KP buffer was not in¯uenced by protein concentration in the range of 25 to 1000 mg/ mL.1 The solution changes during freezing and the residence time of the protein in these destabilizing conditions are comparable to the freeze-thaw conditions (slow cooling/fast warming) that approximate the slow cooling and rapid reconstitution of samples in the current lyophilization study. Recoveries of activity on reconstitution of the freeze-dried monomeric and tetrameric b-gal formulated in 100 mM KP buffer at < 1 mg/mL (Fig. 2A) were signi®cantly lower than the recoveries of activity obtained in freezethawed samples (90%1). As the protein concentration decreased, the recovery of activity decreased after lyophilization and reconstitution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
Figure 2. The effect of protein concentration on the recovery of active protein after lyophilization and rehydration was determined for monomeric (solid bars) and tetrameric (open bars) b-gal in (A)100 mM KP and (B) 100 mM NaP buffers.
At the low protein concentrations, surface denaturation may play a greater role because potentially a larger fraction of protein would be denatured. Surface-induced denaturation during the drying process or on reconstitution of the freeze-dried cake may explain why the concentration dependence is apparent during freeze-drying but not freeze-thawing.29±33 Although surface denaturation can impact the stability of a tetrameric protein, concentration dependence during freezing is governed by its degree of association, which is described by the law of mass action. As the concentration of native tetramer increases, the ratio of monomer/dimer to tetramer decreases at equilibrium. Interestingly, when freeze-drying
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
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in KP buffer at the lower protein concentrations, the tetrameric protein is more stable than the monomeric protein, possibly because of the selfstabilization of the tetrameric subunits. Also, it is possible that during reconstitution, concentration gradients may occur because of the resolubilization of the protein, and the concentrated regions of protein can promote reassociation of the tetrameric subunits. Similar to the KP buffer system, the recovery of activity after freeze-drying monomeric and tetrameric b-gal in 100 mM NaP buffer decreased as a function of decreasing protein concentration (Fig. 2B). This behavior was also observed for freeze-thawed tetrameric b-gal but not monomeric b-gal.1 The recovery of activity after lyophilization and reconstitution was lower in 100 mM NaP than 100 mM KP, indicating that exposure to low pH (100 mM NaP, pH 3.8)1 during freezing fosters greater damage during lyophilization. Effect of Buffer Salts Activity Analysis To assess the impact of the concentration and type of buffer salts on the recovery of activity, we compared the recoveries of activity after freezethawing (slow cooling/fast warming) with freezedrying at one protein concentration (1 mg/mL). For the monomeric protein, the additional stress of dehydration did not further denature the protein except when formulated in 10 mM NaP buffer (Fig. 3A). Similar recoveries of activity after freeze-thawing and freeze-drying would indicate that b-gal inactivation occurs primarily during the freezing process because the freezing protocols are similar for the freeze-thawed and freeze-dried samples. During freezing of monomeric b-gal in the 10 mM NaP buffer,1 the pH decreases to 5.5 and this exposure results in a certain amount of unfolded protein (Ufrozen; Fig. 1), which is further denatured on drying. In contrast, freezing monomeric b-gal in the 100 mM NaP buffer unfolds the protein to the greatest extent (Ufrozen,irreversible), due to exposure to pH 3.8, yielding low recoveries of active protein during freezing.1 Drying after freezing in the 100 mM NaP buffer has little impact on the extensively denatured protein. Additional denaturation during dehydration did not occur for monomeric b-gal when formulated in KP buffer, as evident by the similar recoveries of activity after freeze-thawing
Figure 3. The recovery of activity during freezethawing (solid bars) and freeze-drying (open bars) was determined in each of the phosphate buffers for (A) monomeric and (B) tetrameric b-gal formulated at 1 mg/ mL protein. The freeze-thaw data were obtained in a previous study.1
(slow cooling/fast warming) and after freezedrying. Tetrameric b-gal was susceptible to additional denaturation during dehydration as evident by a greater loss of activity after freeze-drying compared with that after freeze-thawing in 100 mM NaP buffer or the KP buffers (Fig. 3B). Freezethawing b-gal in the 100 mM NaP buffer resulted in higher recoveries of activity compared with that in the 10 mM NaP buffer (Fig. 3B), but similar recoveries were observed in both buffers after freeze-drying. Infrared Spectroscopic Analysis During freezing, exposure of the protein to concentrated buffer salts and acidic pH (for NaP JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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buffer) may produce a certain amount of dissociated or unfolded protein (Ufrozen) that on subsequent exposure to dehydration stress may potentially form an aggregated or irreversibly denatured species (Udry). Because the KP buffer system does not experience pH changes during freezing, b-gal frozen in KP buffer will result in more native protein (Nfrozen), possibly increasing the amount of native protein in the dried state (Ndry). Higher recoveries after freeze-thawing compared with after freeze-drying (tetrameric b-gal in KP buffer, Fig. 3B) also indicates that additional protein inactivation may occur during the drying process. The cooling cycle during freeze-thawing is re¯ective of that during freezedrying; therefore, it can be hypothesized that the extent of protein unfolding occurring during the cooling portions of the different processes is similar. On drying, the protein undergoes additional structural perturbations and is exposed to a range of water contents during rehydration, causing aggregates or denatured protein to form in solution (Uaq, irreversible). Hence, some of the inactivation that occurs during freeze-drying is irreversible. To test this hypothesis, we used IR spectroscopy to study protein secondary structure in the dried solid. The native structure (aqueous control) of monomeric and tetrameric b-gal is dominated by beta sheet as indicated by a prominent band at 1635 cmÿ1 in the conformationally sensitive amide I region (Figs. 4 and 5). Monomeric b-gal has regions of random coil (1651 cmÿ1) but lacks helical structures,1 whereas tetrameric b-gal contains alpha helical structures (1656 cmÿ1) without random coil.1,34,35 b-Gal at 1 mg/mL was freeze-dried in either NaP or KP buffer. The stress of dehydration resulted in severe damage to the native secondary structure of monomeric b-gal as seen by the signi®cant loss of random coil (1651 cmÿ1) and turn structure (1668 and 1685 cmÿ1) on freeze-drying in either KP or NaP buffers, suggesting the formation of a non-native dried protein (Udry; Fig. 4). Similarly, tetrameric b-gal displayed signi®cant structural perturbation, as illustrated by a dramatic loss in alpha helix (1656 cmÿ1) and turn structure (1680 cmÿ1) after freeze-drying in the phosphate buffers, which also indicated the presence of non-native dried protein (Fig. 5). Furthermore, the broadening and shifting of component bands in the IR spectrum of the dried protein indicate lyophilization-induced unfolding, a common observation for freeze-dried proteins.8,18,27,36 The decreases JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
Figure 4. Second-derivative Fourier transform infrared (FT-IR) spectra of monomeric b-gal. The protein (1 mg/mL) was lyophilized in (A) KP and (B) NaP buffers. The native (aqueous solution, Ð), 10 mM buffer (- - -), and 100 mM buffer (± ±) samples are plotted for each buffer system.
in band absorbances representing random coil (monomeric b-gal), alpha helix (tetrameric b-gal), and turn structure (monomeric and tetrameric b-gal) are compensated by enhanced absorbance at 1692 cmÿ1 and some increase at 1618 cmÿ1 for tetrameric b-gal. These bands are characteristic of intermolecular beta sheet structure formed by unfolding and subsequent aggregation of the protein during freeze-drying.36,37 On freeze-drying either protein in the phosphate buffers, a shift in the position of the beta
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
Figure 5. Second-derivative Fourier transform infrared (FT-IR) spectra of tetrameric b-gal. The protein (1 mg/mL) was lyophilized in (A) KP and (B) NaP buffers. The native (aqueous solution, Ð), 10 mM buffer (- - -), and 100 mM buffer (± ±) samples are plotted for each buffer system.
sheet band was observed, with a slightly greater band shift of 1 cmÿ1 for the NaP formulation. Freezing monomeric or tetrameric b-gal in the KP buffer did not result in a shift in the beta sheet band,1 whereas, freeze-drying in this buffer caused a signi®cant shift in the beta sheet band, as well as losses in other secondary structures (Figs. 4A and 5A). These results support the hypothesis that the monomeric and tetrameric protein in KP buffer are native in the frozen state (Nfrozen) but become denatured during drying (Udry). In contrast, freezing monomeric b-gal in
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NaP buffer caused a shift in the beta sheet band,1 and subsequent drying induced additional perturbations in the secondary structure (Fig. 4B). Therefore, the monomeric protein is somewhat unfolded during freezing (Ufrozen), and additional unfolding occurs during drying (Udry). Unlike the monomeric protein, the tetrameric protein did not undergo substantial changes during freezing1 but the dehydrated protein had dramatically altered secondary structure (Fig. 5B), suggesting that the native frozen protein (Nfrozen) unfolds due to the dehydration stress (Udry). In addition to providing insight into the differences in structure of the frozen and dried protein, the change in the beta sheet band appears to correlate with a loss in activity for freeze-dried monomeric and tetrameric b-gal. The loss in activity is greater for proteins freeze-dried in the NaP buffer than the KP buffer (Fig. 3) and a greater shift and broadening in the beta sheet band is observed for the NaP buffer (Figs. 4 and 5). Furthermore, a twofold greater band shift was seen for b-gal freeze-dried in the 100 mM as compared to the 10 mM buffers (Figs. 4 and 5). This change in secondary structure did not manifest itself in a loss in activity with the exception of the tetrameric b-gal in the KP buffer, where a greater loss in activity was observed in 100 mM KP (Fig. 3). The loss in secondary structure in the KP buffers did not adversely affect recovery of the native monomeric protein. Therefore, the unfolded monomeric protein observed in the dried state (Udry) may substantially refold to the native active form (Naq) during reconstitution. However, overall, minimization of the secondary structural changes during freezing and drying led to greater recovery of active protein. Excipient Effects on b-Gal during Freeze-Drying To inhibit denaturation during freeze-drying, we assessed the ability of excipients to protect the protein by maintaining it in its native state during freezing (Nfrozen) and drying (Ndry). Even at relatively high protein concentrations (i.e., 1 mg/mL), both monomeric and tetrameric b-gal display instability after freeze-drying in buffer alone. A greater loss in activity and secondary structure was observed for protein freeze-dried in NaP buffer compared to KP buffer (Figs. 3, 4, and 5). We therefore chose b-gal formulated at 1 mg/mL in the NaP buffer system (10 mM) to examine excipient-induced stabilization during JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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freeze-drying. As previously discussed (Fig. 1), to optimize the retention of native conformation after freeze-drying (Ndry), the protein must be protected from both freezing and drying stresses.2 Sucrose, which protects during both freezing and drying, and PEG, which protects only during freezing, were used at different concentrations to assess their impact on stability. Dextran, a good glass former, was also investigated because it is known to promote stability in the solid state.9,15,16,18 Furthermore, both sucrose38 and dextran may inhibit crystallization of the disodium phosphate during freezing. Thus, the protein would not be exposed to the potentially destabilizing acidic conditions. During the freezing step of lyophilization, Tween may inhibit aggregation, possibly by competing for denaturing interfaces (i.e., ice interface or air±water interface) or by binding to protein states (native and folding intermediates).39,40 Tween usually does not appear to inhibit protein unfolding during the drying step of lyophilization, but can facilitate refolding during rehydration.17,32 Sucrose The IR spectra of the dried protein indicated greater retention of native secondary structure (Ndry) when freeze-drying monomeric (Fig. 6A) and tetrameric (Fig. 7A) b-gal prepared with 100 or 500 mM sucrose as compared with the formulation without excipient. The bands related to the secondary structural components in the spectra of samples lyophilized with sucrose suggest that the protein is more native-like (Ndry) in the sucrose samples than when lyophilized in the buffer alone. Unlike freeze-drying in the absence of excipients, we did not observe enhanced absorbance at 1692 cmÿ1 in the presence of sucrose. Furthermore, essentially complete recovery of activity resulted when freeze-drying monomeric and tetrameric b-gal in formulations containing sucrose alone at concentrations of 100 or 500 mM (Table 1). For monomeric b-gal, the 100 mM sucrose formulation yielded the highest retention of native structure, as indicated by nearly native beta-sheet structure and close to native random structure (Ndry). Monomeric b-gal in the 500 mM sucrose formulation had less native-like structure (Udry) than in the 100 mM sucrose formulation, as seen by the decrease in intensity of the beta-sheet band. This decrease in intensity was compensated for by an increase in intensity of the random JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
structure. In addition, for the monomeric protein in the 500 mM sucrose formulation, the freezedried cake separated into two layers, with the bottom layer appearing somewhat glassy. It is possible that phase separation occurred during freeze-drying at the higher sucrose concentration due to concentration gradients that formed during the freezing process. The small conformational difference between monomeric b-gal in 100 and 500 mM sucrose did not signi®cantly change the recovery of activity (Table 1). For the tetrameric protein, the difference in dried state conformation between the 100 and 500 mM sucrose formulations was not as apparent; however, the broadening of dominant peaks (alpha helix and beta sheet) was more pronounced in the 500 mM sucrose formulation. This general broadening of peaks is likely indicative of static peptide chain disordering.8 The > 100% recovery in the sucrose formulations (Table 1) may be caused by the formation of a more stable native structure (quaternary) increasing the activity of the protein. Overall, sucrose at the concentrations used in this study effectively stabilized the protein during both freezing and drying, which explains the complete recovery of active protein and the native-like conformation (Ndry) after freeze-drying. As previously mentioned, during freezing, sucrose is preferentially excluded from the surface of the protein, and thus the more compact native state is thermodynamically favored during freezing (Nfrozen). On subsequent dehydration, the thermodynamics of preferential exclusion no longer apply and sucrose acts as a water substitute and hydrogen bonds to the polar and charged groups of the protein, thereby retaining the native conformation in the dried state (Ndry). Furthermore, a previous study38 using different weight ratios of sucrose to sodium phosphate buffer indicated that a weight ratio of 6:1 (sucrose-tobuffer) was suf®cient to completely inhibit a pH decrease during freezing due to buffer salt crystallization. Therefore, the weight ratios of sucrose to buffer (3:1 and 6:1) used in this study may have partially or completely inhibited buffer crystallization and subsequent pH decreases during freezing, which have been shown to result in a loss of activity and secondary structure.1 Dextran 10,000 Unlike sucrose, dextran did not promote stability of monomeric b-gal during freeze-drying (Table 1),
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Figure 6. The effect of excipients on the secondderivative IR spectra of monomeric b-gal (1 mg/mL) after lyophilization. The second-derivative Fourier transform infrared (FT-IR) spectrum was plotted for each set of formulation conditions (formulation buffer: 10 mM NaP): (A) sucrose: buffer alone (Ð); 100 mM sucrose ( ); 500 mM sucrose (Ð Ð); native protein
(aqueous solution, Ð Ð); (B) dextran: buffer alone (Ð); 5% dextran ( ); 10% dextran (Ð Ð); native protein (aqueous solution, Ð Ð); (C) PEG: buffer alone (Ð); 100 mM sucrose ( ); 1% PEG (Ð Ð); 1% PEG and 100 mM sucrose (Ð Ð); and (D) Tween: buffer alone (Ð); 100 mM sucrose ( ); 0.1% Tween (Ð Ð); 0.1% Tween and 100 mM sucrose (Ð Ð).
but a slight increase in recovery of activity for tetrameric b-gal was observed (Table 1). The IR spectra of the dextran formulations revealed even greater protein unfolding than noted without excipients (Udry), as indicated by greater broadening and shifting of component bands in the IR spectra (Figs. 6B and 7B). Monomeric b-gal appeared to be least stable in the 10% dextran samples, as indicated by a more dominant shift in position of the beta-sheet band for the 10%
dextran sample. The secondary structure of tetrameric b-gal formulated in 10% dextran was also grossly perturbed, as seen by a dramatic broadening of the beta-sheet band. In support of the IR spectroscopic analysis, recoveries of activity when freeze-drying monomeric and tetrameric b-gal in the 10% dextran formulation were slightly lower than those obtained after freezedrying in the 5% dextran formulation (Table 1). Formulating tetrameric b-gal at the lower JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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Figure 7. The effect of excipients on the second-derivative IR spectra of tetrameric b-gal (1 mg/mL) after lyophilization. The second-derivative spectra were obtained under the same conditions as described in Figure 6, and the same symbols were used for each of the graphs (A, sucrose; B, dextran; C, PEG; and D, Tween).
dextran concentration (5%) slightly improved the recovery of activity compared with that of the buffer alone formulation, but still did not offer as much stabilization as the sucrose formulations. Ideally, an effective inhibitor of dehydrationinduced protein unfolding should be able to hydrogen bond to the protein during dehydration; however, with dextran, steric hindrance most likely prevents this interaction from occurring. Also, dextran has been shown to remain amorphous during drying and thus molecular mobility was restricted, but protein unfolding was still JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
observed.9,15,16,18 Because of the greater denaturation in the dried state (Udry), the protein in the 10% dextran may undergo more aggregation on reconstitution (Uaq, irreversible) than the protein in the 5% dextran. Greater unfolding during lyophilization could be due to phase separation of the protein and dextran.16,41 PEG 8000 Freeze-drying monomeric and tetrameric b-gal in the presence of 1% PEG resulted in a signi®cantly
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
Table 1. Recovery of Activity for Monomeric and Tetrameric b-gala
Formulation Buffer alone 5% Dextran 10% Dextran 1% PEG 8000 0.1% Tween 80 100 mM Sucrose 500 mM Sucrose 100 mM Sucrose 1% PEG 8000 100 mM Sucrose 0.1% Tween 80
% Activity Monomeric b-gal
% Activity Tetrameric b-gal
69.97.5 78.98.4 59.74.1 62.48.3 79.07.9 10010.2 90.82.7 88.45.7
53.39.3 83.48.2 71.22.5 49.82.6 56.67.9 143.19.3 119.86.9 91.33.5
98.78.5
89.43.2
a b-gal (1 mg/ml) was freeze dried in 10 mM NaP buffer with and without excipients.
perturbed native structure (Udry), as seen by the dried state IR spectrum resembling that of the buffer alone sample (Figs. 6C and 7C). For the monomeric protein, the most notable differences are seen in the perturbations to the native random structure (Fig. 6C). In addition, enhanced absorbance at 1692 cmÿ1 indicates formation of a non-native structure during dehydration. For the tetrameric protein, although retention of native alpha helix structure is better than the buffer alone formulation, a signi®cant amount of nonnative species (1692 cmÿ1) is formed during dehydration. PEG is an excellent cryoprotectant but does not protect against drying-induced denaturation because the crystallization of PEG during lyophilization prevents interaction with the protein.6 Therefore, PEG favors maintenance of the native conformation (Nfrozen) during freezing, but on subsequent dehydration signi®cant unfolding occurs (Udry). Along with the poor conformational stability in the dried state, recoveries of activity on reconstitution of the freezedried monomeric and tetrameric protein in 1% PEG were comparable to recoveries after freezedrying in buffer alone (Table 1). Thus, the presence of PEG did not protect during lyophilization or favor refolding on reconstitution, and the protein remains in its unfolded state (Uaq). Because PEG alone is not an effective stabilizer during freeze-drying, we examined a formulation that should offer protection against both the stresses of freezing and drying. The addition of sucrose (100 mM) to the PEG formulation resulted in native-like conformation in the dried
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state (Ndry) for both monomeric and tetrameric bgal (Figs. 6C and 7C). In addition, when freezedrying in the presence of sucrose and PEG, high recoveries of active monomeric and tetrameric protein were observed as compared with those for protein freeze-dried in PEG alone (Table 1). Furthermore, for the monomeric protein, the PEG/sucrose formulation resulted in a more native-like conformation than the sucrose alone formulation, as indicated by an increase in intensity of the random structure band (Fig. 6C); however, recoveries of activity were similar between the sucrose and PEG/sucrose formulation (Table 1), suggesting that the slight differences in the secondary structure did not affect recovery of the native protein after reconstitution. Also, freeze-drying monomeric and tetrameric b-gal in sucrose alone at 100 mM or in the presence of sucrose and PEG minimized theformation of non-native species in the dried state. Even though PEG will promote the native conformation during freezing, it is apparent that sucrose alone at 100 mM effectively protects b-gal against both freezing- and drying-induced denaturation, as indicated by the native-like conformation (Ndry) in the dried state and the complete recovery of active protein (Table 1). Tween 80 Tween has been shown to promote stability during freeze-thawing by competing with the protein for denaturing interfaces, such as the vial± water or ice±water interfaces.17,32,39,40 Although the presence of 0.1% Tween 80 might prevent surface-induced denaturation of b-gal during freezing, freeze-drying in Tween alone did not prevent protein unfolding. Similar to PEG, Tween 80 did not offer protection against protein denaturation during freeze-drying of monomeric and tetrameric b-gal, as evident by the similarities in secondary structure in the dried state between the buffer alone and Tween formulations (Figs. 6D and 7D). However, for tetrameric b-gal, it is apparent that a small reduction in structural perturbation is conferred by Tween as indicated by the slightly improved resolution of the alphahelix band at 1656 cmÿ1 (Fig. 7D). For both proteins, enhanced absorbance at 1692 cmÿ1 indicates formation of a non-native species for the Tween and buffer alone samples (Udry). Furthermore, Tween did not appear to facilitate refolding during reconstitution, as noted by low recoveries of active monomeric and tetrameric JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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protein indicating signi®cant unfolded protein upon reconstitution (Uaq) (Table 1). However, the addition of sucrose (100 mM) to the Tween formulation resulted in a high retention of native structure after freeze-drying (Ndry), as well as high recoveries of active protein on reconstitution (Naq). Furthermore, similar to the PEG/sucrose data, monomeric b-gal freeze-dried in the Tween/ sucrose formulation resulted in a more native-like conformation than the sucrose alone formulation. However, the addition of Tween to the sucrose formulation did not improve the recovery of activity after freeze-drying, as evident by the complete recovery of active protein in the presence of 100 mM sucrose alone.
SUMMARY Freeze drying of b-gal was performed to assess the effects of dehydration stress on protein stability and compare this stress with the freezing stress previously reported for b-gal.1 As the protein concentration decreased, a lower fraction of active protein was obtained on reconstitution of monomeric and tetrameric b-gal freeze-dried in phosphate buffers. This concentration dependence was not observed for the monomeric b-gal during freeze-thawing. For monomeric b-gal, surfaceinduced denaturation during the drying process or on reconstitution of the freeze-dried protein may be the cause for signi®cant activity loss at the lower protein concentrations. Similar to the freeze-thaw results, the recoveries of activity were lower after freeze-drying in the NaP buffer than the recoveries achieved after freeze-drying in the KP buffer. These results may be explained by the exposure of the protein to acidic pH conditions during freezing. The protein is maintained in its native state during freezing (Nfrozen) in KP buffer. In contrast, the protein is substantially unfolded on freezing (Ufrozen) in the NaP buffers.1 In both buffers, the proteins were further denatured on drying (Udry); however, the NaP samples revealed more secondary structural perturbation than the KP samples (Figs. 4 and 5). On reconstitution, the protein in the NaP buffer did not regain full activity, indicating the presence of inactive species (Uaq, irreversible). In addition, recoveries of active monomeric protein were similar after freeze-thawing and freezedrying in the 100 mM NaP or either of the KP buffers, indicating that although some denaturation occurs during the drying process (Udry), most JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
of this denaturation is reversible on reconstitution (Naq). In contrast, lower recoveries of active tetrameric protein after freeze-drying compared with freeze-thawing in the 100 mM NaP buffer and KP buffers indicate signi®cant denaturation during the drying process (Udry) that is not reversible on reconstitution (Uaq). To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. We investigated the ability of different excipients to protect monomeric and tetrameric b-gal during freeze-drying in 10 mM sodium phosphate buffer. Freeze-dried monomeric and tetrameric b-gal displayed optimal recoveries of activity when the formulation contained sucrose (100 and 500 mM sucrose, PEG/sucrose, Tween/sucrose; Table 1). Dried-state conformational analysis by IR spectroscopy also revealed native-like secondary structure for the freeze-dried sucrose formulations. The presence of sucrose at 100 mM alone effectively protects monomeric and tetrameric bgal from both freezing- and drying-induced denaturation. Unlike sucrose, dextran, PEG, and Tween did not promote stability of monomeric and tetrameric b-gal, as indicated by the lower recovery of active protein (Table 1) and less retention of native protein structure (Figs. 6 and 7). Excipients that provide protection during freezing by the preferential hydration mechanism and replace water on the protein surface (water replacement hypothesis) as the protein dries are necessary to assure optimal recovery of these model proteins.
ACKNOWLEDGMENTS We gratefully acknowledge the following organizations for their ®nancial support: Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation and National Science Foundation (Grant # BES9816975).
REFERENCES 1. Pikal-Cleland KA, RodrõÂguez-Hornedo N, Amidon GL, Carpenter JF. 2000. Protein denaturation during freezing and thawing in phosphate buffer systems: Monomeric and tetrameric b-galactosidase. Arch Biochem Biophys 384:398±406. 2. Prestrelski SJ, Arakawa T, Carpenter JF. 1993. Separation of freezing- and drying-induced
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3. 4. 5.
6.
7.
8.
9.
10. 11.
12. 13. 14.
15.
16.
denaturation of lyophilized proteins using stressspeci®c stabilization. II. Structural studies using infrared spectroscopy. Arch Biochem Biophys 303:465±473. Lee JC, Timasheff SN. 1981. The stabilization of proteins by sucrose. J Biol Chem 256:7193±7201. Arakawa T, Timasheff SN. 1985. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry 24:6756±6762. Timasheff SN, Arakawa T. 1989. Stabilization of protein structure by solvents. In: Creighton TE, editor. Protein structure, a practical approach. New York: IRL Press. pp. 331±345. Carpenter JF, Prestrelski SJ, Arakawa T. 1993. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-speci®c stabilization. I. Enzyme activity and calorimetric studies. Arch Biochem Biophys 303:456±464. Carpenter JF, Prestrelski SJ, Anchordoquy TJ, Arakawa T. 1994. Interactions of stabilizers with proteins during freezing and drying. In: Cleland JL, Langer R, editors. Formulation and delivery of proteins and peptides. Washington, D.C.: American Chemical Society. pp. 134±147. Prestrelski SJ, Tedeschi N, Arakawa T, Carpenter JF. 1993. Dehydration-induced conformation transitions in proteins and their inhibition by stabilizers. Biophys J 65:661±671. Tanaka K, Takeda T, Miyajima K. 1991. Cryoprotective effects of saccharides on denaturation of catalase by freeze-drying. Chem Pharm Bull 35:1091±1094. Carpenter JF, Crowe JH. 1988. The mechanism of cryoprotection of proteins by solutes. Cryobiology 25:244±255. Allison SD, Chang B, Randolph TW, Carpenter JF. 1999. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydrationinduced protein unfolding. Arch Biochem Biophys 365:289±298. Carpenter JF, Crowe JH. 1989. An infrared spectroscopic study of interactions of carbohydrates with dried proteins. Biochemistry 28:3916±3922. Franks F. 1994. Long-term stabilization of biologicals. Biotechnology 12:253±256. Levine H, Slade L. 1992. Glass transitions in foods. In: Schwartzberg HG, Hartell RW, editors. Physical chemistry of foods. New York: Marcell Dekker. pp. 83±221. Allison SD, Randolph TW, Manning MC, Middleton K, Davis A, Carpenter JF. 1998. Effects of drying methods and additives on structure and function of actin: Mechanisms of dehydration-induced damage and its inhibition. Arch Biochem Biophys 358:171± 181. Kreilgaard L, Frokjaer S, Flink JM, Randolph TW, Carpenter JF. 1998. Effects of additives on the stability of recombinant human factor XIII during
17.
18.
19.
20. 21. 22. 23.
24.
25.
26.
27.
28. 29.
30.
1267
freeze-drying and storage in the dried solid. Arch Biochem Biophys 360:121±134. Kreilgaard L, Jones L, Randolph TW, Frokjaer S, Flink JM, Manning MC, Carpenter JF. 1998. Effects of Tween 20 on agitation- and freezethawing-induced aggregation of recombinant human factor XIII. J Pharm Sci 87:1597±1603. Prestrelski SJ, Pikal KA, Arakawa T. 1995. Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy. Pharm Res 12:1250±1259. Anchordoquy TJ, Carpenter JF. 1996. Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state. Arch Biochem Biophys 332:231±238. Jaenicke R. 1981. Enzymes under extremes of physical conditions. Ann Rev Biophys Bioeng 10:1±67. Privalov PL. 1990. Cold denaturation of proteins. Crit Rev Biochem Molec Biol 25:281±305. Izutsu K, Yoshioka S, Terao T. 1993. Stabilization of b-galactosidase by amphiphilic additives during freeze-drying. Int J Pharm 90:187±194. Izutsu K, Yoshioka S, Takeda Y. 1990. Protein denaturation in dosage forms measured by differential scanning calorimetry. Chem Pharm Bull 38:800±803. Craven GR, Steers E Jr, An®nsen CB. 1965. Puri®cation, composition and molecular weight of the b-galactosidase of Escherichia coli K12. J Biol Chem 240:2468±2473. Dong A, Huang P, Caughey WS. 1990. Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29: 3303±3308. Kendrick BS, Cleland JL, Lam X, Nguyen T, Randolph TW, Manning MC, Carpenter JF. 1998. Aggregation of recombinant human interferon gamma: Kinetics and structural transitions. J Pharm Sci 87:1069±1076. Allison SD, Dong A, Carpenter JF. 1996. Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration. Biophys J 71:2022±2032. Bock PE, Frieden C. 1974. pH-induced cold liability of rabbit skeletal muscle phosphofructokinase. Biochemistry 13:4191±4199. Hsu CC, Nguyen HM, Yeung DA, Brooks DA, Koe GS, Bewley TA, Pearlman R. 1995. Surface denaturation at solid-void interface Ð A possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures. Pharm Res 12:69± 77. Pikal MJ. 1994. Freeze-drying of proteins: process, formulation, and stability. In: Cleland JL, Langer R, editors. Protein formulations and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
1268
31. 32.
33.
34.
35. 36.
PIKAL-CLELAND AND CARPENTER
delivery. Washington, D.C.: American Chemical Society. pp.120±133. Jiang S, Nail SL. 1998. Effect of process conditions on recovery of protein activity during freezing and freeze-drying. Eur J Pharm Biopharm 45:249±257. Chang BS, Kendrick BS, Carpenter JF. 1996. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci 85:1325±1330. Sarciaux JM, Mansour S, Hageman MJ, Nail SL. 1999. Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci 88:1354±1361. Edwards RA, Jacobson AL, Huber RE. 1990. Thermal denaturation of b-galactosidase and of two site speci®c mutants. Biochemistry 29:11001± 11008. Jacobson RH, Zhang XJ, DuBose RF, Matthews BW. 1994. Three-dimensional structure of b-galactosidase from E. coli. Nature 369:761±766. Dong A, Prestrelski SJ, Allison SD, Carpenter JF. 1995. Infrared spectroscopic studies of lyophiliza-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
37.
38.
39.
40.
41.
tion- and temperature-induced protein aggregation. J Pharm Sci 84:415±424. Clark AH, Saunderson DHP, Suggett A. 1981. Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int J Pept Protein Res 17:353±364. GoÂmez G. 1995. Crystallization-related pH changes during freezing of sodium phosphate buffer solutions, University of Michigan Ph.D. Dissertation: University Micro®lms Inc. Bam NB, Cleland JL, Randolph TW. 1996. Molten globule intermediate of recombinant human growth hormone: Stabilization with surfactants. Biotech Progress 12:801±809. Bam NB, Cleland JL, Manning MC, Carpenter JF, Kelley RF, Randolph TW. 1998. Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interaction. J Pharm Sci 87:1554±1559. Heller MC, Carpenter JF, Randolph TW. 1996. Effect of phase separating systems on lyophilized hemoglobin. J Pharm Sci 85:1358±1362.
Inhale Therapeutic Systems, 150 Industrial Road, San Carlos, California 94070
2
University of Colorado Health Sciences Center, Denver, Colorado 80262
Received 28 August 2000; revised 1 February 2001; accepted 12 February 2001
ABSTRACT: During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate signi®cantly more inactivation and secondary structural perturbation of monomeric and tetrameric b-galactosidase (b-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the signi®cant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer.1 In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric b-gal in either NaP or KP buffer resulted in signi®cant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freezedrying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilizationinduced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active b-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freezedrying in phosphate buffers, even if there are large-scale changes in solution pH during freezing. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1255±1268, 2001
Keywords: b-galactosidase; pH changes; phosphate buffers; freeze-dry; protein denaturation; infrared spectroscopy
INTRODUCTION Correspondence to: K.A. Pikal-Cleland (Telephone: 650631-3564; Fax: 650-631-3150; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1255±1268 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
Freeze-drying is commonly used to manufacture protein pharmaceuticals, which are insuf®ciently stable for distribution and storage as aqueous formulations. However, proteins are often readily denatured during freezing and drying. Prestrelski
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Figure 1. The pathways of unfolding and refolding during freezing and drying of proteins was described previously.2 The native protein (Naq) in solution may unfold to form denatured or dissociated protein (Ufrozen). In the presence of stabilizing excipients that cause preferential hydration of the native protein, the native protein structure (Nfrozen) is maintained on freezing. During drying, the native frozen protein may either unfold (Udry) or remain in its native state (Ndry). To maintain the protein in its native state during drying, excipients that are able to hydrogen bond to the protein are often required (water replacement theory). Alternatively, if the protein is already unfolded in the frozen state, it is likely to undergo additional unfolding on drying (Udry). On reconstitution, the unfolded protein may either refold to the native form (Naq) or form an inactive species such as an aggregate (Uaq).
and colleagues2 previously described the protein conformational states during freezing, drying, and rehydration and the effects of excipients during each of these processes (Fig. 1). According to their scheme, a suitable cryoprotectant may prevent freezing-induced unfolding and thus the protein remains native in the frozen state (Nfrozen). A suitable cryoprotectant is a preferentially excluded solute, such as sucrose3 and poly(ethylene glycol) (PEG),4 that causes an increase in the chemical potential of the protein at the appropriate solute concentrations.5±7 The increase in chemical potential is greater for the denatured state than the native state, causing an increase in the free energy of unfolding. In the absence of a suitable cryoprotectant, a non-native protein conformation may result during freezing (Ufrozen). Even though an excipient may stabilize the native protein during freezing (i.e., a cryoproJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
tectant), it will not necessarily prevent dryinginduced denaturation. During drying, the hydration shell around the protein is removed, and thus the thermodynamics of the preferential exclusion mechanism do not apply. Stabilization of the native protein state during dehydration may be explained by the water substitute hypothesis. Excipients, such as sucrose and trehalose,8±11 which are able to hydrogen bond to the polar and charged groups of the protein in the place of lost water, have been shown to inhibit dehydration-induced protein unfolding (Ndry) as measured by infrared (IR) spectroscopy.8,12 Another theoretical explanation for solute-induced stabilization during dehydration is the vitri®cation hypothesis, which states that an effective stabilizer readily forms a glass, reducing any molecular motion relevant to stability.13,14 However, studies have documented that formation of a glass is not a suf®cient requirement for stabilization. For example, dextran, which forms a glass, does not inhibit protein unfolding during lyophilization9,11,15 ±18 and, because of steric hindrance, it does not hydrogen bond effectively to the dried protein.9,11,15±18 The ability of excipients to stabilize proteins against freezing and dehydration stresses is concentration dependent. Sucrose at concentrations as low as 10 mM may promote stability during drying, but higher sucrose concentrations (e.g., > 5% w/v) are often required for stabilization during freezing.6 PEG offers protection during freezing at much lower concentrations (1% w/v) than required for sugars,7 but it does not protect against drying-induced degradation. If during freezing the protein has already denatured (Ufrozen), subsequent drying in the presence of low sucrose levels (i.e., 10 mM sucrose) would prevent further dehydration-induced denaturation but still result in unfolded dried protein (Udry). In addition, if the excipient protects during freezing but not dehydration (i.e., PEG), the result after freeze-drying would also be a non-native conformation (Udry). To maintain the native structure throughout lyophilization, the protein must be protected against both the freezing- and drying-induced denaturation. Conformational stability upon rehydration must also be considered. A protein that maintains its native conformation throughout freezing and drying will most likely remain in its native state on rehydration as well (Naq). Even if the protein is in its unfolded state after freeze-drying (Udry), on reconstitution it may refold to its native
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
conformation (Naq). However, competing with this refolding pathway is the conversion of the unfolded protein in its dried state to an irreversibly denatured or aggregated species, thus resulting in some fraction (perhaps dominant) of the population of protein molecules being inactive and/or aggregated in the rehydrated samples. In this study, we investigated the impact of dehydration on the recovery of enzymatic activity and structural integrity of monomeric and tetrameric b-galactosidase (b-gal) formulated in phosphate buffers, to complement earlier freezethawing studies.1 Both monomeric and tetrameric b-gal displayed lower recoveries of activity and greater changes in secondary structure after freeze-thawing in the sodium phosphate (NaP) buffer system compared with the potassium phosphate (KP) system that was attributed to differences in pH of the freeze concentrate. During freezing in the NaP buffer system, precipitation of the less soluble buffer component (disodium salt) results in a signi®cant pH decrease to as low as 3.8. In contrast, the KP buffer does not experience a signi®cant pH change during freezing. Minimizing the residence time in the freeze-concentrated state by fast cooling and fast warming reduced exposure to concentrated buffer salts and acidic pH conditions (NaP buffer), yielding greater recovery of active protein. In addition, tetrameric b-gal was observed to be more freeze labile than monomeric b-gal due in part to cooling-induced and/or freezing-induced dissociation, which is commonly observed for multimeric proteins.19±21 With this understanding of the physical instability of b-gal during freeze-thawing, we investigated the stability during freeze-drying in the phosphate buffers to gain insight into the effects of the combined stresses of freezing, buffer acidi®cation, and dehydration on protein stability. Izutsu and colleagues22 have documented that monomeric b-gal is partially inactivated during freeze-drying in the absence of excipients. Because the degradation pathways for the model proteins chosen are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stress of drying alters the native structure of the protein, by infrared (IR) spectroscopy, and if preservation of the native conformation during freeze-drying is requisite for optimal recovery of activity. Finally, excipients of various classes just discussed were tested for their capacity to prevent lyophilizationinduced unfolding and to promote increased
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recovery of catalytic activity on reconstitution of the lyophilized sample.
MATERIALS AND METHODS Materials b-Galactosidase derived from A. oryzae (monomeric b-gal) was purchased from Toyobo Company (Osaka, Japan). b-Galactosidase derived from E. coli (tetrameric b-gal) was purchased from Worthington Biochemical Corporation. Sucrose was purchased from Pfanstiehl Laboratories. All other chemicals were purchased from Sigma Chemicals. All materials were reagent grade or higher quality. Freeze-Drying Conditions Samples (0.5 mL) at different protein concentrations (2, 25, 100, 1000 mg/mL) were transferred into 1-mL glass vials and freeze-dried in a FTS freeze-drier. Samples were frozen at a shelf temperature of ÿ508C for 2 h. Primary drying was achieved at a shelf temperature of ÿ408C for 30 h. Secondary drying was achieved by drying at an initial shelf temperature of ÿ108C for 4 h and then at 208C for an additional 4 h. A vacuum of 60 mTorr was maintained throughout the freeze-dry cycle. After completion of secondary drying, the vials were capped under vacuum. Enzyme Preparation and Activity Assay Monomeric and tetrameric b-gal were dialyzed against the appropriate phosphate buffer (pH 7.0) overnight at 48C before use. As described by Izutsu and colleagues,23 monomeric b-gal activity was determined by measuring the absorbance at 420 nm after a 10-min incubation at 258C using 2-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. Tetrameric b-gal activity was determined by monitoring the increase in absorbance at 405 nm at 258C resulting from the hydrolysis of ONPG as described by Cravens et al.24 Initial activity was calculated as a percentage of the activity in the control samples (for each formulation) that were not subjected to a freeze-thaw cycle or freeze-dry cycle. Freeze-thaw and freeze-dry samples were run in triplicate. The recoveries of activities are reported as a mean and standard deviation (SD) for three separate enzyme samples. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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Infrared Spectroscopy Secondary structural analysis of the protein freeze-dried at 1 mg/mL was conducted using a Nicolet Magna 550 IR spectrophotometer equipped with a dTGS detector. IR spectra (256 scans, 4 cmÿ1 resolution) were recorded at 258C. Freeze-dried samples (0.2±0.4 mg protein) were ground with 300 mg of KBr and pressed into pellets, as described previously.8 This procedure for preparing KBr pellets does not alter the structure of proteins in the dried state.8 Spectra of the unlyophilized aqueous proteins (native controls) were collected using an IR cell with CaF2 windows and a 6-mm spacer. The native controls were aqueous solutions at 18 mg/mL (tetrameric b-gal) or 27 mg/mL (monomeric b-gal).1 The protein spectra were processed as outlined previously.25,26 Brie¯y, the protein spectra were corrected with the appropriate buffer blank (for the aqueous native controls) and gaseous water, and the second-derivative amide I spectra were calculated with Nicolet Omnic software and area normalized.
RESULTS AND DISCUSSION Protein Concentration Dependence Because many proteins have displayed greater stability at higher protein concentrations,10,22,27,28 we ®rst investigated the impact of protein concentration on the stability of b-gal during freeze-drying and then determined the impact of the buffer salts at a ®xed protein concentration. Previously, we have shown that the percentage of activity recovered for both monomeric and tetrameric b-gal freeze-thawed in 100 mM KP buffer was not in¯uenced by protein concentration in the range of 25 to 1000 mg/ mL.1 The solution changes during freezing and the residence time of the protein in these destabilizing conditions are comparable to the freeze-thaw conditions (slow cooling/fast warming) that approximate the slow cooling and rapid reconstitution of samples in the current lyophilization study. Recoveries of activity on reconstitution of the freeze-dried monomeric and tetrameric b-gal formulated in 100 mM KP buffer at < 1 mg/mL (Fig. 2A) were signi®cantly lower than the recoveries of activity obtained in freezethawed samples (90%1). As the protein concentration decreased, the recovery of activity decreased after lyophilization and reconstitution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
Figure 2. The effect of protein concentration on the recovery of active protein after lyophilization and rehydration was determined for monomeric (solid bars) and tetrameric (open bars) b-gal in (A)100 mM KP and (B) 100 mM NaP buffers.
At the low protein concentrations, surface denaturation may play a greater role because potentially a larger fraction of protein would be denatured. Surface-induced denaturation during the drying process or on reconstitution of the freeze-dried cake may explain why the concentration dependence is apparent during freeze-drying but not freeze-thawing.29±33 Although surface denaturation can impact the stability of a tetrameric protein, concentration dependence during freezing is governed by its degree of association, which is described by the law of mass action. As the concentration of native tetramer increases, the ratio of monomer/dimer to tetramer decreases at equilibrium. Interestingly, when freeze-drying
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
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in KP buffer at the lower protein concentrations, the tetrameric protein is more stable than the monomeric protein, possibly because of the selfstabilization of the tetrameric subunits. Also, it is possible that during reconstitution, concentration gradients may occur because of the resolubilization of the protein, and the concentrated regions of protein can promote reassociation of the tetrameric subunits. Similar to the KP buffer system, the recovery of activity after freeze-drying monomeric and tetrameric b-gal in 100 mM NaP buffer decreased as a function of decreasing protein concentration (Fig. 2B). This behavior was also observed for freeze-thawed tetrameric b-gal but not monomeric b-gal.1 The recovery of activity after lyophilization and reconstitution was lower in 100 mM NaP than 100 mM KP, indicating that exposure to low pH (100 mM NaP, pH 3.8)1 during freezing fosters greater damage during lyophilization. Effect of Buffer Salts Activity Analysis To assess the impact of the concentration and type of buffer salts on the recovery of activity, we compared the recoveries of activity after freezethawing (slow cooling/fast warming) with freezedrying at one protein concentration (1 mg/mL). For the monomeric protein, the additional stress of dehydration did not further denature the protein except when formulated in 10 mM NaP buffer (Fig. 3A). Similar recoveries of activity after freeze-thawing and freeze-drying would indicate that b-gal inactivation occurs primarily during the freezing process because the freezing protocols are similar for the freeze-thawed and freeze-dried samples. During freezing of monomeric b-gal in the 10 mM NaP buffer,1 the pH decreases to 5.5 and this exposure results in a certain amount of unfolded protein (Ufrozen; Fig. 1), which is further denatured on drying. In contrast, freezing monomeric b-gal in the 100 mM NaP buffer unfolds the protein to the greatest extent (Ufrozen,irreversible), due to exposure to pH 3.8, yielding low recoveries of active protein during freezing.1 Drying after freezing in the 100 mM NaP buffer has little impact on the extensively denatured protein. Additional denaturation during dehydration did not occur for monomeric b-gal when formulated in KP buffer, as evident by the similar recoveries of activity after freeze-thawing
Figure 3. The recovery of activity during freezethawing (solid bars) and freeze-drying (open bars) was determined in each of the phosphate buffers for (A) monomeric and (B) tetrameric b-gal formulated at 1 mg/ mL protein. The freeze-thaw data were obtained in a previous study.1
(slow cooling/fast warming) and after freezedrying. Tetrameric b-gal was susceptible to additional denaturation during dehydration as evident by a greater loss of activity after freeze-drying compared with that after freeze-thawing in 100 mM NaP buffer or the KP buffers (Fig. 3B). Freezethawing b-gal in the 100 mM NaP buffer resulted in higher recoveries of activity compared with that in the 10 mM NaP buffer (Fig. 3B), but similar recoveries were observed in both buffers after freeze-drying. Infrared Spectroscopic Analysis During freezing, exposure of the protein to concentrated buffer salts and acidic pH (for NaP JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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buffer) may produce a certain amount of dissociated or unfolded protein (Ufrozen) that on subsequent exposure to dehydration stress may potentially form an aggregated or irreversibly denatured species (Udry). Because the KP buffer system does not experience pH changes during freezing, b-gal frozen in KP buffer will result in more native protein (Nfrozen), possibly increasing the amount of native protein in the dried state (Ndry). Higher recoveries after freeze-thawing compared with after freeze-drying (tetrameric b-gal in KP buffer, Fig. 3B) also indicates that additional protein inactivation may occur during the drying process. The cooling cycle during freeze-thawing is re¯ective of that during freezedrying; therefore, it can be hypothesized that the extent of protein unfolding occurring during the cooling portions of the different processes is similar. On drying, the protein undergoes additional structural perturbations and is exposed to a range of water contents during rehydration, causing aggregates or denatured protein to form in solution (Uaq, irreversible). Hence, some of the inactivation that occurs during freeze-drying is irreversible. To test this hypothesis, we used IR spectroscopy to study protein secondary structure in the dried solid. The native structure (aqueous control) of monomeric and tetrameric b-gal is dominated by beta sheet as indicated by a prominent band at 1635 cmÿ1 in the conformationally sensitive amide I region (Figs. 4 and 5). Monomeric b-gal has regions of random coil (1651 cmÿ1) but lacks helical structures,1 whereas tetrameric b-gal contains alpha helical structures (1656 cmÿ1) without random coil.1,34,35 b-Gal at 1 mg/mL was freeze-dried in either NaP or KP buffer. The stress of dehydration resulted in severe damage to the native secondary structure of monomeric b-gal as seen by the signi®cant loss of random coil (1651 cmÿ1) and turn structure (1668 and 1685 cmÿ1) on freeze-drying in either KP or NaP buffers, suggesting the formation of a non-native dried protein (Udry; Fig. 4). Similarly, tetrameric b-gal displayed signi®cant structural perturbation, as illustrated by a dramatic loss in alpha helix (1656 cmÿ1) and turn structure (1680 cmÿ1) after freeze-drying in the phosphate buffers, which also indicated the presence of non-native dried protein (Fig. 5). Furthermore, the broadening and shifting of component bands in the IR spectrum of the dried protein indicate lyophilization-induced unfolding, a common observation for freeze-dried proteins.8,18,27,36 The decreases JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
Figure 4. Second-derivative Fourier transform infrared (FT-IR) spectra of monomeric b-gal. The protein (1 mg/mL) was lyophilized in (A) KP and (B) NaP buffers. The native (aqueous solution, Ð), 10 mM buffer (- - -), and 100 mM buffer (± ±) samples are plotted for each buffer system.
in band absorbances representing random coil (monomeric b-gal), alpha helix (tetrameric b-gal), and turn structure (monomeric and tetrameric b-gal) are compensated by enhanced absorbance at 1692 cmÿ1 and some increase at 1618 cmÿ1 for tetrameric b-gal. These bands are characteristic of intermolecular beta sheet structure formed by unfolding and subsequent aggregation of the protein during freeze-drying.36,37 On freeze-drying either protein in the phosphate buffers, a shift in the position of the beta
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
Figure 5. Second-derivative Fourier transform infrared (FT-IR) spectra of tetrameric b-gal. The protein (1 mg/mL) was lyophilized in (A) KP and (B) NaP buffers. The native (aqueous solution, Ð), 10 mM buffer (- - -), and 100 mM buffer (± ±) samples are plotted for each buffer system.
sheet band was observed, with a slightly greater band shift of 1 cmÿ1 for the NaP formulation. Freezing monomeric or tetrameric b-gal in the KP buffer did not result in a shift in the beta sheet band,1 whereas, freeze-drying in this buffer caused a signi®cant shift in the beta sheet band, as well as losses in other secondary structures (Figs. 4A and 5A). These results support the hypothesis that the monomeric and tetrameric protein in KP buffer are native in the frozen state (Nfrozen) but become denatured during drying (Udry). In contrast, freezing monomeric b-gal in
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NaP buffer caused a shift in the beta sheet band,1 and subsequent drying induced additional perturbations in the secondary structure (Fig. 4B). Therefore, the monomeric protein is somewhat unfolded during freezing (Ufrozen), and additional unfolding occurs during drying (Udry). Unlike the monomeric protein, the tetrameric protein did not undergo substantial changes during freezing1 but the dehydrated protein had dramatically altered secondary structure (Fig. 5B), suggesting that the native frozen protein (Nfrozen) unfolds due to the dehydration stress (Udry). In addition to providing insight into the differences in structure of the frozen and dried protein, the change in the beta sheet band appears to correlate with a loss in activity for freeze-dried monomeric and tetrameric b-gal. The loss in activity is greater for proteins freeze-dried in the NaP buffer than the KP buffer (Fig. 3) and a greater shift and broadening in the beta sheet band is observed for the NaP buffer (Figs. 4 and 5). Furthermore, a twofold greater band shift was seen for b-gal freeze-dried in the 100 mM as compared to the 10 mM buffers (Figs. 4 and 5). This change in secondary structure did not manifest itself in a loss in activity with the exception of the tetrameric b-gal in the KP buffer, where a greater loss in activity was observed in 100 mM KP (Fig. 3). The loss in secondary structure in the KP buffers did not adversely affect recovery of the native monomeric protein. Therefore, the unfolded monomeric protein observed in the dried state (Udry) may substantially refold to the native active form (Naq) during reconstitution. However, overall, minimization of the secondary structural changes during freezing and drying led to greater recovery of active protein. Excipient Effects on b-Gal during Freeze-Drying To inhibit denaturation during freeze-drying, we assessed the ability of excipients to protect the protein by maintaining it in its native state during freezing (Nfrozen) and drying (Ndry). Even at relatively high protein concentrations (i.e., 1 mg/mL), both monomeric and tetrameric b-gal display instability after freeze-drying in buffer alone. A greater loss in activity and secondary structure was observed for protein freeze-dried in NaP buffer compared to KP buffer (Figs. 3, 4, and 5). We therefore chose b-gal formulated at 1 mg/mL in the NaP buffer system (10 mM) to examine excipient-induced stabilization during JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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freeze-drying. As previously discussed (Fig. 1), to optimize the retention of native conformation after freeze-drying (Ndry), the protein must be protected from both freezing and drying stresses.2 Sucrose, which protects during both freezing and drying, and PEG, which protects only during freezing, were used at different concentrations to assess their impact on stability. Dextran, a good glass former, was also investigated because it is known to promote stability in the solid state.9,15,16,18 Furthermore, both sucrose38 and dextran may inhibit crystallization of the disodium phosphate during freezing. Thus, the protein would not be exposed to the potentially destabilizing acidic conditions. During the freezing step of lyophilization, Tween may inhibit aggregation, possibly by competing for denaturing interfaces (i.e., ice interface or air±water interface) or by binding to protein states (native and folding intermediates).39,40 Tween usually does not appear to inhibit protein unfolding during the drying step of lyophilization, but can facilitate refolding during rehydration.17,32 Sucrose The IR spectra of the dried protein indicated greater retention of native secondary structure (Ndry) when freeze-drying monomeric (Fig. 6A) and tetrameric (Fig. 7A) b-gal prepared with 100 or 500 mM sucrose as compared with the formulation without excipient. The bands related to the secondary structural components in the spectra of samples lyophilized with sucrose suggest that the protein is more native-like (Ndry) in the sucrose samples than when lyophilized in the buffer alone. Unlike freeze-drying in the absence of excipients, we did not observe enhanced absorbance at 1692 cmÿ1 in the presence of sucrose. Furthermore, essentially complete recovery of activity resulted when freeze-drying monomeric and tetrameric b-gal in formulations containing sucrose alone at concentrations of 100 or 500 mM (Table 1). For monomeric b-gal, the 100 mM sucrose formulation yielded the highest retention of native structure, as indicated by nearly native beta-sheet structure and close to native random structure (Ndry). Monomeric b-gal in the 500 mM sucrose formulation had less native-like structure (Udry) than in the 100 mM sucrose formulation, as seen by the decrease in intensity of the beta-sheet band. This decrease in intensity was compensated for by an increase in intensity of the random JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
structure. In addition, for the monomeric protein in the 500 mM sucrose formulation, the freezedried cake separated into two layers, with the bottom layer appearing somewhat glassy. It is possible that phase separation occurred during freeze-drying at the higher sucrose concentration due to concentration gradients that formed during the freezing process. The small conformational difference between monomeric b-gal in 100 and 500 mM sucrose did not signi®cantly change the recovery of activity (Table 1). For the tetrameric protein, the difference in dried state conformation between the 100 and 500 mM sucrose formulations was not as apparent; however, the broadening of dominant peaks (alpha helix and beta sheet) was more pronounced in the 500 mM sucrose formulation. This general broadening of peaks is likely indicative of static peptide chain disordering.8 The > 100% recovery in the sucrose formulations (Table 1) may be caused by the formation of a more stable native structure (quaternary) increasing the activity of the protein. Overall, sucrose at the concentrations used in this study effectively stabilized the protein during both freezing and drying, which explains the complete recovery of active protein and the native-like conformation (Ndry) after freeze-drying. As previously mentioned, during freezing, sucrose is preferentially excluded from the surface of the protein, and thus the more compact native state is thermodynamically favored during freezing (Nfrozen). On subsequent dehydration, the thermodynamics of preferential exclusion no longer apply and sucrose acts as a water substitute and hydrogen bonds to the polar and charged groups of the protein, thereby retaining the native conformation in the dried state (Ndry). Furthermore, a previous study38 using different weight ratios of sucrose to sodium phosphate buffer indicated that a weight ratio of 6:1 (sucrose-tobuffer) was suf®cient to completely inhibit a pH decrease during freezing due to buffer salt crystallization. Therefore, the weight ratios of sucrose to buffer (3:1 and 6:1) used in this study may have partially or completely inhibited buffer crystallization and subsequent pH decreases during freezing, which have been shown to result in a loss of activity and secondary structure.1 Dextran 10,000 Unlike sucrose, dextran did not promote stability of monomeric b-gal during freeze-drying (Table 1),
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
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Figure 6. The effect of excipients on the secondderivative IR spectra of monomeric b-gal (1 mg/mL) after lyophilization. The second-derivative Fourier transform infrared (FT-IR) spectrum was plotted for each set of formulation conditions (formulation buffer: 10 mM NaP): (A) sucrose: buffer alone (Ð); 100 mM sucrose ( ); 500 mM sucrose (Ð Ð); native protein
(aqueous solution, Ð Ð); (B) dextran: buffer alone (Ð); 5% dextran ( ); 10% dextran (Ð Ð); native protein (aqueous solution, Ð Ð); (C) PEG: buffer alone (Ð); 100 mM sucrose ( ); 1% PEG (Ð Ð); 1% PEG and 100 mM sucrose (Ð Ð); and (D) Tween: buffer alone (Ð); 100 mM sucrose ( ); 0.1% Tween (Ð Ð); 0.1% Tween and 100 mM sucrose (Ð Ð).
but a slight increase in recovery of activity for tetrameric b-gal was observed (Table 1). The IR spectra of the dextran formulations revealed even greater protein unfolding than noted without excipients (Udry), as indicated by greater broadening and shifting of component bands in the IR spectra (Figs. 6B and 7B). Monomeric b-gal appeared to be least stable in the 10% dextran samples, as indicated by a more dominant shift in position of the beta-sheet band for the 10%
dextran sample. The secondary structure of tetrameric b-gal formulated in 10% dextran was also grossly perturbed, as seen by a dramatic broadening of the beta-sheet band. In support of the IR spectroscopic analysis, recoveries of activity when freeze-drying monomeric and tetrameric b-gal in the 10% dextran formulation were slightly lower than those obtained after freezedrying in the 5% dextran formulation (Table 1). Formulating tetrameric b-gal at the lower JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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Figure 7. The effect of excipients on the second-derivative IR spectra of tetrameric b-gal (1 mg/mL) after lyophilization. The second-derivative spectra were obtained under the same conditions as described in Figure 6, and the same symbols were used for each of the graphs (A, sucrose; B, dextran; C, PEG; and D, Tween).
dextran concentration (5%) slightly improved the recovery of activity compared with that of the buffer alone formulation, but still did not offer as much stabilization as the sucrose formulations. Ideally, an effective inhibitor of dehydrationinduced protein unfolding should be able to hydrogen bond to the protein during dehydration; however, with dextran, steric hindrance most likely prevents this interaction from occurring. Also, dextran has been shown to remain amorphous during drying and thus molecular mobility was restricted, but protein unfolding was still JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
observed.9,15,16,18 Because of the greater denaturation in the dried state (Udry), the protein in the 10% dextran may undergo more aggregation on reconstitution (Uaq, irreversible) than the protein in the 5% dextran. Greater unfolding during lyophilization could be due to phase separation of the protein and dextran.16,41 PEG 8000 Freeze-drying monomeric and tetrameric b-gal in the presence of 1% PEG resulted in a signi®cantly
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
Table 1. Recovery of Activity for Monomeric and Tetrameric b-gala
Formulation Buffer alone 5% Dextran 10% Dextran 1% PEG 8000 0.1% Tween 80 100 mM Sucrose 500 mM Sucrose 100 mM Sucrose 1% PEG 8000 100 mM Sucrose 0.1% Tween 80
% Activity Monomeric b-gal
% Activity Tetrameric b-gal
69.97.5 78.98.4 59.74.1 62.48.3 79.07.9 10010.2 90.82.7 88.45.7
53.39.3 83.48.2 71.22.5 49.82.6 56.67.9 143.19.3 119.86.9 91.33.5
98.78.5
89.43.2
a b-gal (1 mg/ml) was freeze dried in 10 mM NaP buffer with and without excipients.
perturbed native structure (Udry), as seen by the dried state IR spectrum resembling that of the buffer alone sample (Figs. 6C and 7C). For the monomeric protein, the most notable differences are seen in the perturbations to the native random structure (Fig. 6C). In addition, enhanced absorbance at 1692 cmÿ1 indicates formation of a non-native structure during dehydration. For the tetrameric protein, although retention of native alpha helix structure is better than the buffer alone formulation, a signi®cant amount of nonnative species (1692 cmÿ1) is formed during dehydration. PEG is an excellent cryoprotectant but does not protect against drying-induced denaturation because the crystallization of PEG during lyophilization prevents interaction with the protein.6 Therefore, PEG favors maintenance of the native conformation (Nfrozen) during freezing, but on subsequent dehydration signi®cant unfolding occurs (Udry). Along with the poor conformational stability in the dried state, recoveries of activity on reconstitution of the freezedried monomeric and tetrameric protein in 1% PEG were comparable to recoveries after freezedrying in buffer alone (Table 1). Thus, the presence of PEG did not protect during lyophilization or favor refolding on reconstitution, and the protein remains in its unfolded state (Uaq). Because PEG alone is not an effective stabilizer during freeze-drying, we examined a formulation that should offer protection against both the stresses of freezing and drying. The addition of sucrose (100 mM) to the PEG formulation resulted in native-like conformation in the dried
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state (Ndry) for both monomeric and tetrameric bgal (Figs. 6C and 7C). In addition, when freezedrying in the presence of sucrose and PEG, high recoveries of active monomeric and tetrameric protein were observed as compared with those for protein freeze-dried in PEG alone (Table 1). Furthermore, for the monomeric protein, the PEG/sucrose formulation resulted in a more native-like conformation than the sucrose alone formulation, as indicated by an increase in intensity of the random structure band (Fig. 6C); however, recoveries of activity were similar between the sucrose and PEG/sucrose formulation (Table 1), suggesting that the slight differences in the secondary structure did not affect recovery of the native protein after reconstitution. Also, freeze-drying monomeric and tetrameric b-gal in sucrose alone at 100 mM or in the presence of sucrose and PEG minimized theformation of non-native species in the dried state. Even though PEG will promote the native conformation during freezing, it is apparent that sucrose alone at 100 mM effectively protects b-gal against both freezing- and drying-induced denaturation, as indicated by the native-like conformation (Ndry) in the dried state and the complete recovery of active protein (Table 1). Tween 80 Tween has been shown to promote stability during freeze-thawing by competing with the protein for denaturing interfaces, such as the vial± water or ice±water interfaces.17,32,39,40 Although the presence of 0.1% Tween 80 might prevent surface-induced denaturation of b-gal during freezing, freeze-drying in Tween alone did not prevent protein unfolding. Similar to PEG, Tween 80 did not offer protection against protein denaturation during freeze-drying of monomeric and tetrameric b-gal, as evident by the similarities in secondary structure in the dried state between the buffer alone and Tween formulations (Figs. 6D and 7D). However, for tetrameric b-gal, it is apparent that a small reduction in structural perturbation is conferred by Tween as indicated by the slightly improved resolution of the alphahelix band at 1656 cmÿ1 (Fig. 7D). For both proteins, enhanced absorbance at 1692 cmÿ1 indicates formation of a non-native species for the Tween and buffer alone samples (Udry). Furthermore, Tween did not appear to facilitate refolding during reconstitution, as noted by low recoveries of active monomeric and tetrameric JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
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protein indicating signi®cant unfolded protein upon reconstitution (Uaq) (Table 1). However, the addition of sucrose (100 mM) to the Tween formulation resulted in a high retention of native structure after freeze-drying (Ndry), as well as high recoveries of active protein on reconstitution (Naq). Furthermore, similar to the PEG/sucrose data, monomeric b-gal freeze-dried in the Tween/ sucrose formulation resulted in a more native-like conformation than the sucrose alone formulation. However, the addition of Tween to the sucrose formulation did not improve the recovery of activity after freeze-drying, as evident by the complete recovery of active protein in the presence of 100 mM sucrose alone.
SUMMARY Freeze drying of b-gal was performed to assess the effects of dehydration stress on protein stability and compare this stress with the freezing stress previously reported for b-gal.1 As the protein concentration decreased, a lower fraction of active protein was obtained on reconstitution of monomeric and tetrameric b-gal freeze-dried in phosphate buffers. This concentration dependence was not observed for the monomeric b-gal during freeze-thawing. For monomeric b-gal, surfaceinduced denaturation during the drying process or on reconstitution of the freeze-dried protein may be the cause for signi®cant activity loss at the lower protein concentrations. Similar to the freeze-thaw results, the recoveries of activity were lower after freeze-drying in the NaP buffer than the recoveries achieved after freeze-drying in the KP buffer. These results may be explained by the exposure of the protein to acidic pH conditions during freezing. The protein is maintained in its native state during freezing (Nfrozen) in KP buffer. In contrast, the protein is substantially unfolded on freezing (Ufrozen) in the NaP buffers.1 In both buffers, the proteins were further denatured on drying (Udry); however, the NaP samples revealed more secondary structural perturbation than the KP samples (Figs. 4 and 5). On reconstitution, the protein in the NaP buffer did not regain full activity, indicating the presence of inactive species (Uaq, irreversible). In addition, recoveries of active monomeric protein were similar after freeze-thawing and freezedrying in the 100 mM NaP or either of the KP buffers, indicating that although some denaturation occurs during the drying process (Udry), most JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
of this denaturation is reversible on reconstitution (Naq). In contrast, lower recoveries of active tetrameric protein after freeze-drying compared with freeze-thawing in the 100 mM NaP buffer and KP buffers indicate signi®cant denaturation during the drying process (Udry) that is not reversible on reconstitution (Uaq). To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. We investigated the ability of different excipients to protect monomeric and tetrameric b-gal during freeze-drying in 10 mM sodium phosphate buffer. Freeze-dried monomeric and tetrameric b-gal displayed optimal recoveries of activity when the formulation contained sucrose (100 and 500 mM sucrose, PEG/sucrose, Tween/sucrose; Table 1). Dried-state conformational analysis by IR spectroscopy also revealed native-like secondary structure for the freeze-dried sucrose formulations. The presence of sucrose at 100 mM alone effectively protects monomeric and tetrameric bgal from both freezing- and drying-induced denaturation. Unlike sucrose, dextran, PEG, and Tween did not promote stability of monomeric and tetrameric b-gal, as indicated by the lower recovery of active protein (Table 1) and less retention of native protein structure (Figs. 6 and 7). Excipients that provide protection during freezing by the preferential hydration mechanism and replace water on the protein surface (water replacement hypothesis) as the protein dries are necessary to assure optimal recovery of these model proteins.
ACKNOWLEDGMENTS We gratefully acknowledge the following organizations for their ®nancial support: Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation and National Science Foundation (Grant # BES9816975).
REFERENCES 1. Pikal-Cleland KA, RodrõÂguez-Hornedo N, Amidon GL, Carpenter JF. 2000. Protein denaturation during freezing and thawing in phosphate buffer systems: Monomeric and tetrameric b-galactosidase. Arch Biochem Biophys 384:398±406. 2. Prestrelski SJ, Arakawa T, Carpenter JF. 1993. Separation of freezing- and drying-induced
PROTEIN DENATURATION OF b-GALACTOSIDASE INDUCED BY LYOPHILIZATION
3. 4. 5.
6.
7.
8.
9.
10. 11.
12. 13. 14.
15.
16.
denaturation of lyophilized proteins using stressspeci®c stabilization. II. Structural studies using infrared spectroscopy. Arch Biochem Biophys 303:465±473. Lee JC, Timasheff SN. 1981. The stabilization of proteins by sucrose. J Biol Chem 256:7193±7201. Arakawa T, Timasheff SN. 1985. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry 24:6756±6762. Timasheff SN, Arakawa T. 1989. Stabilization of protein structure by solvents. In: Creighton TE, editor. Protein structure, a practical approach. New York: IRL Press. pp. 331±345. Carpenter JF, Prestrelski SJ, Arakawa T. 1993. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-speci®c stabilization. I. Enzyme activity and calorimetric studies. Arch Biochem Biophys 303:456±464. Carpenter JF, Prestrelski SJ, Anchordoquy TJ, Arakawa T. 1994. Interactions of stabilizers with proteins during freezing and drying. In: Cleland JL, Langer R, editors. Formulation and delivery of proteins and peptides. Washington, D.C.: American Chemical Society. pp. 134±147. Prestrelski SJ, Tedeschi N, Arakawa T, Carpenter JF. 1993. Dehydration-induced conformation transitions in proteins and their inhibition by stabilizers. Biophys J 65:661±671. Tanaka K, Takeda T, Miyajima K. 1991. Cryoprotective effects of saccharides on denaturation of catalase by freeze-drying. Chem Pharm Bull 35:1091±1094. Carpenter JF, Crowe JH. 1988. The mechanism of cryoprotection of proteins by solutes. Cryobiology 25:244±255. Allison SD, Chang B, Randolph TW, Carpenter JF. 1999. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydrationinduced protein unfolding. Arch Biochem Biophys 365:289±298. Carpenter JF, Crowe JH. 1989. An infrared spectroscopic study of interactions of carbohydrates with dried proteins. Biochemistry 28:3916±3922. Franks F. 1994. Long-term stabilization of biologicals. Biotechnology 12:253±256. Levine H, Slade L. 1992. Glass transitions in foods. In: Schwartzberg HG, Hartell RW, editors. Physical chemistry of foods. New York: Marcell Dekker. pp. 83±221. Allison SD, Randolph TW, Manning MC, Middleton K, Davis A, Carpenter JF. 1998. Effects of drying methods and additives on structure and function of actin: Mechanisms of dehydration-induced damage and its inhibition. Arch Biochem Biophys 358:171± 181. Kreilgaard L, Frokjaer S, Flink JM, Randolph TW, Carpenter JF. 1998. Effects of additives on the stability of recombinant human factor XIII during
17.
18.
19.
20. 21. 22. 23.
24.
25.
26.
27.
28. 29.
30.
1267
freeze-drying and storage in the dried solid. Arch Biochem Biophys 360:121±134. Kreilgaard L, Jones L, Randolph TW, Frokjaer S, Flink JM, Manning MC, Carpenter JF. 1998. Effects of Tween 20 on agitation- and freezethawing-induced aggregation of recombinant human factor XIII. J Pharm Sci 87:1597±1603. Prestrelski SJ, Pikal KA, Arakawa T. 1995. Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy. Pharm Res 12:1250±1259. Anchordoquy TJ, Carpenter JF. 1996. Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state. Arch Biochem Biophys 332:231±238. Jaenicke R. 1981. Enzymes under extremes of physical conditions. Ann Rev Biophys Bioeng 10:1±67. Privalov PL. 1990. Cold denaturation of proteins. Crit Rev Biochem Molec Biol 25:281±305. Izutsu K, Yoshioka S, Terao T. 1993. Stabilization of b-galactosidase by amphiphilic additives during freeze-drying. Int J Pharm 90:187±194. Izutsu K, Yoshioka S, Takeda Y. 1990. Protein denaturation in dosage forms measured by differential scanning calorimetry. Chem Pharm Bull 38:800±803. Craven GR, Steers E Jr, An®nsen CB. 1965. Puri®cation, composition and molecular weight of the b-galactosidase of Escherichia coli K12. J Biol Chem 240:2468±2473. Dong A, Huang P, Caughey WS. 1990. Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29: 3303±3308. Kendrick BS, Cleland JL, Lam X, Nguyen T, Randolph TW, Manning MC, Carpenter JF. 1998. Aggregation of recombinant human interferon gamma: Kinetics and structural transitions. J Pharm Sci 87:1069±1076. Allison SD, Dong A, Carpenter JF. 1996. Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration. Biophys J 71:2022±2032. Bock PE, Frieden C. 1974. pH-induced cold liability of rabbit skeletal muscle phosphofructokinase. Biochemistry 13:4191±4199. Hsu CC, Nguyen HM, Yeung DA, Brooks DA, Koe GS, Bewley TA, Pearlman R. 1995. Surface denaturation at solid-void interface Ð A possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures. Pharm Res 12:69± 77. Pikal MJ. 1994. Freeze-drying of proteins: process, formulation, and stability. In: Cleland JL, Langer R, editors. Protein formulations and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
1268
31. 32.
33.
34.
35. 36.
PIKAL-CLELAND AND CARPENTER
delivery. Washington, D.C.: American Chemical Society. pp.120±133. Jiang S, Nail SL. 1998. Effect of process conditions on recovery of protein activity during freezing and freeze-drying. Eur J Pharm Biopharm 45:249±257. Chang BS, Kendrick BS, Carpenter JF. 1996. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci 85:1325±1330. Sarciaux JM, Mansour S, Hageman MJ, Nail SL. 1999. Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci 88:1354±1361. Edwards RA, Jacobson AL, Huber RE. 1990. Thermal denaturation of b-galactosidase and of two site speci®c mutants. Biochemistry 29:11001± 11008. Jacobson RH, Zhang XJ, DuBose RF, Matthews BW. 1994. Three-dimensional structure of b-galactosidase from E. coli. Nature 369:761±766. Dong A, Prestrelski SJ, Allison SD, Carpenter JF. 1995. Infrared spectroscopic studies of lyophiliza-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 9, SEPTEMBER 2001
37.
38.
39.
40.
41.
tion- and temperature-induced protein aggregation. J Pharm Sci 84:415±424. Clark AH, Saunderson DHP, Suggett A. 1981. Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int J Pept Protein Res 17:353±364. GoÂmez G. 1995. Crystallization-related pH changes during freezing of sodium phosphate buffer solutions, University of Michigan Ph.D. Dissertation: University Micro®lms Inc. Bam NB, Cleland JL, Randolph TW. 1996. Molten globule intermediate of recombinant human growth hormone: Stabilization with surfactants. Biotech Progress 12:801±809. Bam NB, Cleland JL, Manning MC, Carpenter JF, Kelley RF, Randolph TW. 1998. Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interaction. J Pharm Sci 87:1554±1559. Heller MC, Carpenter JF, Randolph TW. 1996. Effect of phase separating systems on lyophilized hemoglobin. J Pharm Sci 85:1358±1362.