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Solid State Chemistry of Proteins: I. Glass Transition Behavior in Freeze Dried Disaccharide Formulations of Human Growth Hormone (HGH)
Solid State Chemistry of Proteins: I. Glass Transition Behavior in Freeze Dried Disaccharide Formulations of Human Growth Hormone (hGH) MICHAEL J. PIKAL,1,2 D.R. RIGSBEE,2 M.L. ROY2 1
School of Pharmacy, University of Connecticut, Storrs, Connecticut
2
Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana
Received 1 November 2006; revised 26 January 2007; accepted 1 February 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20960
ABSTRACT: Although freeze dried formulations are commonly characterized using differential scanning calorimetry (DSC), a protein-rich system behaves as a ‘‘strong glass’’, and the glass transition temperature, Tg, cannot be directly determined by DSC. A strong glass means a small heat capacity change at Tg, DCp, and a very broad glass transition region, or a large DTg. However, direct experimental evidence for a small DCp and a large DTg have been lacking. Here, we utilize extrapolation of thermal analysis data in protein:disaccharide mixtures to evaluate Tg, DTg, and DCp for ‘‘pure’’ human growth hormone (hGH) from low to moderate residual water. We find that DTg is indeed large and DCp is very small. Also, the Tg for pure hGH decreases from a value of about 1368C when dry to around 258C at 12% water. This glass transition is not the onset of mobility within the protein molecule but rather signals onset of whole molecule rotation and translation. We also observe complex pre-Tg thermal events in the DSC data, which are interpreted as consequences of relaxation events, largely due to the disaccharide, and are characteristic of freeze dried systems having a broad distribution of relaxing substates. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2765–2776, 2007
Keywords: amorphous; biotechnology; calorimetry (DSC); freeze drying/lyophilization; glass transition; proteins; relaxation time; protein formulation
INTRODUCTION Proteins commonly undergo excessive degradation when stored in the aqueous solution state for long periods of time, and so therapeutic proteins are commonly dried to provide satisfactory storage stability. Freeze drying is, by far, the most popular drying technology for biotechnology products, but other drying techniques can also be employed.1 In essentially all cases, the solution being dried is aqueous and contains small Correspondence to: Michael J. Pikal (Telephone: 860-4863202; Fax: 860-486-4998; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2765–2776 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
amounts of a buffer as well as one or more ‘‘stabilizers’’ and results in a solid that is at least partially amorphous after drying. That is, the therapeutic protein, buffer system, and stabilizer remain amorphous and for optimal stability, should form a single amorphous phase.1 Just as physical characterization of a protein in the aqueous solution state is important to the prediction of the pharmaceutical stability of a solution formulation, so is characterization of the protein in the amorphous solid state important to the understanding and prediction of stability in the solid state. In principle, protein structure2 thermal denaturation3–6 and glass transition temperature1,7 are properties critical to stability behavior. In practice, thermal denaturation in the
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solid state occurs only at very high temperatures3–6 and therefore has no obvious direct relationship to practical stability behavior. However, protein structure by FTIR and the glass transition temperature in protein formulations are commonly measured in an attempt to predict stability behavior.1,2,8 While the glass transition temperature, Tg0 , in a frozen ‘‘pure’’ protein solution can be measured with differential scanning calorimetry (DSC), and is usually in the range of 108C to 158C9 glass transitions in ‘‘pure’’ dry proteins are problematic. That is, the sharp increase in heat capacity during a DSC scan, characteristic of the glass transition, typically cannot be observed for protein-rich systems.4–6,10–14 We note that some soy proteins do show the classical DSC signature of a glass transition, and a fully denatured protein often shows a strong increase in heat capacity, signaling a glass transition.10–13 However, most dry proteins appear to behave as ‘‘strong glasses’’12,13 meaning that the ‘‘strength parameter’’, D, is large (i.e., fragility is small). With a strong glass, the glass transition is spread over a very large temperature range, that is, DTg is large15,16 and the change in heat capacity at the glass transition, DCp is small. While the ‘‘strong glass’’ interpretation of dry amorphous protein behavior is generally accepted in pharmaceutical science, it is curious that there is no direct evidence for either a small DCp or large DTg in dry protein systems. The prime objective of this work is to demonstrate by quantitative extrapolation of DSC data on mixtures of disaccharides and human growth hormone (hGH) that ‘‘pure’’ hGH is indeed characterized by a very small DCp and large DTg, giving a large strength parameter, D. From the extrapolated Tg data, we characterize the effect of water content on the glass transition of the disaccharide-free protein system for water contents in the very low to moderate range.
EXPERIMENTAL Materials All lots formulated with sugars were prepared from the same lot of bulk hGH obtained from Eli Lilly (Indianapolis, IN). Sucrose was analytical reagent grade obtained from Baker (Phillipsburg, NJ), and sodium phosphate was analytical reagent grade obtained from Mallinckrodt (Phillipsburg, NJ). Trehalose was obtained from
Sigma (St. Louis, MO) and was ‘‘reduced metal ion content’’ material. All solutions were 5 mg/mL hGH buffered to pH 7.4 with sodium phosphate (0.75 mg/mL). Sucrose and trehalose were added at weight ratios, relative to hGH, of 1:1, 3:1, and 6:1 (i.e., sugar concentrations of 5 mg/mL, 15 mg/ mL, and 30 mg/mL, respectively). Freeze dried samples at elevated water content were prepared from the corresponding ‘‘dry’’ samples by equilibrating overnight with 11%, 22%, and 33% relative humidity in vacuum desiccators. All samples were confirmed 100% amorphous by noting the absence of birefringence by polarized light microscopy. Note that all samples contained sodium phosphate buffer at 15% (w/w) of the protein content, which remained amorphous and therefore could have a small impact on the DSC data reported.
Freeze Drying Samples were freeze dried in a Virtis 25-SRC-X (freeze dryer equipped with a MKS Baratron pressure measurement system and an electronic moisture sensor; Ondyne, Endress and Hauser, Greenwood, IN) to measure the partial pressure of water vapor in the drying chamber. Product and shelf surface temperatures were measured via 30 gauge copper-constantan thermocouples. Product temperature sensors were placed in the bottom center of a vial while the shelf surface sensors consisted of the thermocouple junctions silver soldered into a brass disk about 7 mm in diameter and about 1.5 mm thick. The disk was ‘‘glued’’ to the shelf with vacuum grease. Shelf surface temperature was measured on the shelf surface near the shelf heat transfer fluid entry and near the fluid exit. All hGH formulations were freeze dried at a shelf temperature control setting of 208C in primary drying and a chamber pressure setting of 0.10 Torr throughout the drying process. Each sample contained 5 mg hGH in 1 mL fill volume. Nominal 5 mL tubing vials were used along with 13 mm finish West gray butyl rubber stoppers (#1816 rubber stock) or with Daikyo Florotec stoppers (low moisture release stoppers), as indicated. All formulations were freeze dried at least 38C below their collapse temperatures, and visual inspection confirmed the absence of collapse. The maximum product temperatures during primary drying varied with formulation: 1:1 hGH:trehalose ¼ 35.28C; 1:3 hGH: trehalose ¼
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33.78C; 1:6 hGH: trehalose ¼31.08C; 1:1 hGH:sucrose ¼34.48C; 1:3 hGH:sucrose¼ 33.68C; 1:6 hGH:sucrose ¼ 33.48C. For secondary drying, the shelf temperature was increased from 208C to þ338C in four steps over 3 h and then the shelf temperature was held at 338C for 2 h. Water content varied from 1.0% to 0.4%, depending upon formulation. All samples were stored at 208C until use. Water Content Water contents were determined by Coulometric Karl Fischer procedures, using a ‘‘weigh and dump’’ technique which minimizes exposure of the sample to ambient humidity, and also by the Lilly Analytical Division using a variation of Coulometric Karl Fischer procedures which involves dissolving the sample in the original sample container. The difference in water contents determined by the two procedures averaged only 0.2%. Reported water contents represent the mean of the two sets of results. Differential Scanning Calorimetry DSC studies were carried out with a Perkin-Elmer DSC-7 using sealed aluminum pans. A scan rate of 58C/min was used for the study of frozen solutions (i.e., determination of the glass transition temperature of the freeze concentrate, Tg0 ). Freeze dried powders (usual sample size ¼ 5–10 mg) were compacted into disks 2–3 mm in thickness before sealing in the sample pans. All sample handling was carried out in a dry bag continuously purged with dry air (< 2% relative humidity). Powders were generally scanned at 78C/min. Second scans were made after cooling from above the glass transition temperature, Tg, to below 08C at 308C/min. Glass transition temperatures reported represent the mid-point of the heat capacity transition. Calibration was performed using indium, as conventional, and also by using pure water. In all thermograms, an endotherm is ‘‘up’’.
RESULTS Features of the DSC Thermograms Examples of the diverse thermograms obtained are given by Figures 1–4. Figure 1 shows the effect of aging on DSC response for 3:1 trehalose:hGH (weight ratio) formulations, where (a) gives the DOI 10.1002/jps
Figure 1. Representative DSC thermograms for hGH formulations: Effect of aging on 3:1 (weight ratio) trehalose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; ________ ¼ second scan (complete) following first (partial) scan. (a) Baseline as noted, and initial sample, 6.4 mg, % H2O ¼ 0.6%; (b) aged 3 months 258C, 8.6 mg, % H2O ¼ 1.6%; (c) Aged 3 months 408C, 7.8 mg, % H2O ¼ 2.1%. Increase in moisture on aging is due to stopper to product moisture transfer.
data for an ‘‘initial’’ sample, sample (b) was aged 3 months at 258C, and sample (c) was aged 3 months at 408C. In each figure, results of a partial scan up to and through the glass transition at about 908C–1158C are shown as a broken line. Following this partial heating scan, the sample was quenched to below 08C and then scanned up to about 1808C, shown as a solid line. The glass transition is easily identified from the second scan
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Figure 2. Representative DSC thermograms for hGH formulations: Effect of relative humidity on 1:1 (weight ratio) trehalose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; __________ ¼ second scan (complete) following first (partial) scan. (a)’’Dry’’ sample, 4.5 mg; (b) equilibrated with 11% relative humidity, 7.6 mg, % H2O ¼ 3.1%; (c) equilibrated with 22% relative humidity, 8.0 mg, % H2O ¼ 5.0%.
Figure 3. Representative DSC thermograms for hGH formulations: Effect of aging on 1:1 (weight ratio) sucrose:hGH Systems: Symbols: _____ _____ ¼ First (partial) scan; _________ ¼ second scan (complete) following first (partial) scan. (a) Baseline as noted, and initial sample, 3.0 mg, % H2O ¼ 0.8%; (b) aged 3 months 258C, 7.4 mg, % H2O ¼ 2.2%; (c) aged 3 months 408C, 9.3 mg, % H2O ¼ 2.5%. Increase in moisture on aging is due to stopper to product moisture transfer.
as the large increase in heat capacity (i.e., endothermic baseline shift) which occurs in the range of about 908C–1158C. The variation is due to variable water content, which in turn is a result of moisture transfer from stopper to product with the grey butyl stoppers used here. The large endotherm around 1508C–1608C is the thermal
denaturation endotherm for the protein in the ‘‘solid’’ state. Note, however, that denaturation occurs well above the glass transition temperature, so strictly speaking, the denaturation occurs in a viscous system of molten trehalose and protein. Denaturation is irreversible in the sense that if a sample is scanned through
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Figure 4. Representative DSC thermograms for hGH formulations: Effect of relative humidity on 1:1 (weight ratio) sucrose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; _________ ¼ second scan (complete) following first (partial) scan. (a) Equilibrated with 11% relative humidity, 8 mg, % H2O ¼ 2.5%; (b) equilibrated with 22% relative humidity, 9.5 mg, % H2O ¼ 3.9%.
the denaturation endotherm event, cooled, and then re-scanned, no denaturation endotherm is observed. The most curious features occur only in the first scan before and around the glass transition; thus, these features correspond to irreversible phenomena. With the ‘‘initial sample’’, we observe a slight endothermic shift (i.e., ‘‘up’’) at around 608C, followed by a sharp exothermic shift, giving the appearance of a negative heat capacity shift around 708C. With aging and perhaps also because of the increasing moisture content during aging, the endothermic shift and subsequent exothermic shift become more distinct and move to higher temperature, occurring essentially at the glass transition for the sample aged at 408C (Fig. 1c). Figure 2 illustrates behavior of 1:1 trehalose:hGH for ‘‘initial’’ samples where the water content of very dry material (Fig. 2a) was deliberately increased by equilibration with relative humidities of 11% (Fig. 2b) and 22% (Fig. 2c). DOI 10.1002/jps
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Again, the glass transitions are clearly observed in the second scans from about 1208C (dry) to about 508C (22% Relative humidity), and the (partial) first scan shows complex behavior at and below the glass transition region. The very dry sample shows a distinct negative heat capacity shift, which transforms into an endotherm followed by an exotherm with higher moisture content samples. With the 22% relative humidity sample, the irreversible phenomena appear much like a classical enthalpy recovery endotherm17 superimposed on the positive heat capacity shift due to the glass transition. The thermal behavior of 1:1 sucrose:hGH formulations is illustrated by Figures 3 and 4, where Figure 3 shows the effect of aging and Figure 4 shows the effect of exposure to 11% and 22% relative humidity. Figure 3a gives only a first scan to 1808C and the baseline, showing the pre-Tg event which is qualitatively the same as for the 258C aged 3:1 trehalose:hGH formulation (Fig. 1b); that is, we see a slight endotherm, followed by an exothermic shift and then the glass transition. Aging at 258C makes the ‘‘endo– exo’’ event more distinct, and aging at 408C leads to a strong endotherm superimposed on the glass transition, much like a classical enthalpy recovery event. Note the strong denaturation endotherm followed by a strong exothermic shift. The exothermic event corresponds to cold crystallization of the sucrose (i.e., crystallization from the amorphous state below the melting point). Due to interference from sucrose crystallization, thermal denaturation cannot be observed with the higher sucrose content formulations. Figure 4 shows the effect of elevated moisture content on the thermal behavior of the 1:1 sucrose:hGH formulation. The ‘‘dry’’ sample is shown in Figure 3a. Note that the effect of higher moisture content is qualitatively much the same as observed for the 1:1 trehalose:hGH system; as moisture increases, the ‘‘endo–exo’’ thermal event becomes more distinct and moves up to the Tg region, with the thermogram for 22% relative humidity material appearing as a classical enthalpy recovery endotherm superimposed on the glass transition.
DTg and DCp Results Table 1 summarizes the widths of the glass transition event and the change in heat capacity at the glass transition for the various hGH
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Table 1. Relative Width of the Glass Transition and Change in Heat Capacity at the Glass Transition
Disaccharide Sucrose Sucrose Sucrose Sucrose Trehalose Trehalose Trehalose Trehalose
Saccharaide:hGH
DCp(J g1 K1)
DTg/Tg
Pure sucrose 6 3 1 Pure trehalose 6 3 1
0.596 0.543 0.019 0.498 0.018 0.332 0.027 0.604 0.534 0.009 0.502 0.012 0.271 0.017
0.0187 0.0253 0.0013 0.0278 0.0013 0.0366 0.0019 0.0171 0.0191 0.0003 0.0244 0.0007 0.0410 0.0037
The DTg and DCp values were obtained by linear extrapolation of raw data versus % water data to zero water content. Uncertainties are the standard deviation in the intercept for extrapolation of the data to zero water content. Pure sucrose and pure trehalose data were means of duplicates taken on 0 water content samples.
formulations. The width of the glass transition has possible application in estimating the fragility (or strength) of the glass as well as providing an estimate of the structural relaxation time at temperatures below Tg,16 and values of DCp are useful in interpreting fragility results and in the determination of calorimetric structural relaxation times.18 All data reported were taken from second scan results to avoid ambiguities and inaccuracies resulting from the complex irreversible pre-Tg events described above. However, it should be noted that the values of DTg obtained from first scan results, when possible, are essentially the same as obtained from second scan; the ratio of first scan to second scan averages 0.96 0.025 (std error). The width of the glass transition temperature region was evaluated using the ‘‘E ¼ 0 method’’16 since, particularly for second scan data, ‘‘overshoot’’ endotherms at Tg were largely absent. Note that the glass transition is a weak transition (i.e., small DCp and large DTg/Tg) at high protein (1:1) and as noted earlier, cannot be seen with most dry pure protein systems,4–6,10–14 suggesting that a pure protein system is a ‘‘strong glass’’.12,13
Thermal Data for hGH:Saccharide Systems Equilibrated With Various Humidities Thermal data obtained on systems of varying water content are provided by Table 2. For a given formulation, all samples were generated from a given batch by equilibration with fixed relative humidities. All data represent ‘‘initial’’ values (i.e., no deliberate aging). The formulation, relative humidity (RH), mid point of the glass
transition in 8C (Tg), both the extrapolated onset and mid point of the denaturation endotherm, and the corresponding heat of denaturation (i.e., from the area of the denaturation endotherm) are listed. We note that the endotherms attributed to denaturation do indeed represent highly cooperative unfolding of the protein, as viewed by FTIR. This topic will be developed in detail in a later publication. The change in heat capacity upon denaturation could not be reliably measured since the DSC techniques used here did not allow separation of irreversible events from heat capacity changes. This topic will be explored with modulated DSC in a later publication. As expected, increasing the water content decreases Tg significantly, and increasing the saccharide content also decreases Tg, but only slightly for trehalose systems, suggesting that the Tg of pure protein is near that of trehalose. The thermal denaturation temperature also decreases with increasing water content and increasing saccharide content. Note that the thermal denaturation temperature for pure hGH is about 1808C when dry.4,5 In this sense, the saccharide ‘‘interacts’’ with the protein as does water, consistent with the concept of ‘‘water substitution’’ by the saccharide.3,19 In some of the sucrose-rich systems, denaturation could not be observed due to interference from cold crystallization of sucrose. With 1:1 saccharide:hGH, the heat of denaturation decreases as water content increases, and at least for trehalose where data are available at various saccharide levels, the heat of denaturation decreases with increasing trehalose level except at the highest water content studied. Thus, trends in the heat of denaturation with saccharide content mimic the trends with
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Table 2. Thermal Data For hGH:Saccharide systems Equilibrated With Selected Relative Humidities
Saccharide
Saccharide:hGH
%RH
%H2O
Tg, 8C Mid pt
Pure sugar 6 3 1 6 3 1 6 3 1 3 1 Pure sugar 6 3 1 6 3 1 6 3 1 6 3 1
0 0 0 0 11 11 11 22 22 22 33 33 0 0 0 0 11 11 11 22 22 22 33 33 33
0 0 0 0 2.15 2.3 2.5 4.1 4.0 3.9 5.8 5.3 0 0 0 0 3.3 3.2 3.1 5.4 5.2 5.0 7.1 6.9 6.6
77.0 80.7 89.0 103.9 49.0 50.4 58.3 36.4 38.4 42.4 24.9 30.5 118.0 121.l 122.9 126.8 69.2 67.8 70.1 49.9 49.3 42.4 38.1 36.9 37.2
Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose
water content. However, the trends in heat of denaturation do not predict trends of increasing ‘‘native structure’’ as increasing levels of saccharide increase native structure1,2,8,19 and would be expected to increase heat of denaturation, not cause a decrease!
DISCUSSION Strength of Protein-Rich Glasses Figure 5 shows DCp at Tg as a function of disaccharide level. Note that while the range of extrapolation is large and linearity is not assured over the entire composition range, there is no question that DCp is quite small for protein-rich systems and may decrease to nearly zero for pure protein systems. With a small DCp, one expects a large value of the Angell strength parameter, D 20; that is, one expects a strong glass. The Angell strength parameter may be estimated from the width of the glass transition region16 using the following procedure. First, we note that the activation energy for structural relaxation at Tg, DH , may be estimated from DOI 10.1002/jps
TD, 8C Onset
TD, 8C Mid pt
DHD J/ghGH
139.7 147.0
152.4
17.2
131.0
141.2
18.8
124.6
135.6
13.5
120.6
131.2
12.3
153.4 153.4 158.4 133.4 131.6 131.4 125.4 124.8 124.5 122.0 121.0 118.5
161.7 161.2 165.2 141.9 143.0 138.4 135.8 136.8 133.4 130.6 130.8 129.0
11.8 12.3 21.0 11.4 14.8 19.3 13.4 13.3 15.4 15.6 12.5 13.0
the width of the glass transition from the approximation, b
DH C ¼ ; RTg DTg =Tg
(1)
where C is a constant depending on DSC protocol (i.e., thermal history) and the method for determination of DTg. The symbol, b, is the ‘‘stretched exponent’’ of the Kohlrausch–Williams–Watts (KWW) equation for expressing the kinetics of enthalpy relaxation. For DSC protocols where the cooling and heating rates are 308/min and 78/min, respectively, and the ‘‘E ¼ 0 method’’1 is used to determine DTg, the value of C ¼ 3.2.16 The activa tion energy for structural relaxation at Tg, DH , is related to the Angell strength parameter, D, the ‘‘zero mobility’’ temperature, T0, and Tg by DH DðTg =T0 Þ ¼ RTg ðTg =T0 1Þ
(2)
1 The ‘‘E ¼ 0 method’’ refers to determination of the end of the glass transition region by using the intercept of the tangent drawn to the mid-point of the glass transition with the tangent drawn to the heat capacity curve of the equilibrium liquid, post Tg transition.
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Figure 5. The effect of composition on the change in heat capacity at the glass transition, DCp, for disaccharide:hGH systems. The values of DCp refer to data at 0% water and were taken from Table 1. key: open squares ¼ trehalose; filled triangles ¼ sucrose.
Figure 6. The effect of composition on the Angell strength parameter, D, for disaccharide:hGH systems. The values of D refer to systems at 0% water and were calculated using DTg data from Table 1 using equations (1) and (2). Symbols key: open squares ¼ trehalose; filled triangles ¼ sucrose.
and Angell20 also argues that the ratio Tg/T0 is directly related to the strength parameter by
expected from the estimated small DCp for protein-rich systems, the values of D for such systems are large, extrapolating to a D value for the pure protein which is about a factor of 4 higher than found for the pure disaccharide systems. Thus, consistent with the speculation provided more than a decade ago,12,13 to the extent that hGH is representative, proteins indeed behave as strong glasses. While it is clear that there are significant differences between a ‘‘native’’ protein and a typical small molecule (or synthetic polymer) in the changes in dynamics that occur at Tg, our view is that both reflect an increase in global mobility and the onset of viscous flow. The difference is that, in protein-rich systems, the change in heat capacity over the transition range is small, and thus the material at least behaves as a ‘‘strong glass’’, although it is obvious that such an observation does not mean the structure is similar to a ‘‘network glass’’. The reason for the small DCp in the glass transition range is likely a decoupling of the protein internal motions22–26 from the motions that characterize whole molecule translational/rotational diffusion and viscous flow. Either the internal motions are mostly actuated at a lower temperature (i.e., at the protein dynamical transition temperature, Td)22 and/or the internal degrees of freedom are gradually accessed over a large temperature range prior to the DSC Tg. Either way, at Tg, it is then only the relatively few remaining modes of motion of the
Tg ¼ 1 þ 0:0271 D T0
(3)
Thus, knowing the glass transition temperature, Tg and the width of the glass transition region, DTg, one can evaluate D using equations (1)–(3), provided an estimate of b is available. We take b ¼ 0.4 for all samples. While this value is somewhat arbitrary, the true value cannot be far different. In this regard, we note that the very small overshoot endotherms on the thermograms used to evaluate the widths of the glass transition indicate that b is very small, 0.4.16 Further, simulations suggest that values of 0.4 seem consistent with calorimetric relaxation data on saccharide-rich systems and therefore are appropriate for our current application. We note that while values of b are determined by a fit of the KWW equation to calorimetric relaxation data, such determinations are subject to systematic error in both b and t, but not tb.21 Actually, as long as b is not highly variable, the exact value is not critical for the qualitative conclusions we draw. The estimated values of the strength parameter, D, are plotted as a function of saccharide content in Figure 6. Note that the trends in D evaluated from either sucrose or trehalose systems are similar and suggest a relatively high value of D for the pure protein. As would be
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molecule ‘‘as a whole’’ that are cooperatively actuated, and the DCp is then small. The fact that both the glass transition temperature we measure and the transition width vary with composition indicates that both components determine the properties of the resulting glass, but this observation alone does not necessarily mean the protein internal motions are coupled to the disaccharide dynamics.
Estimates of the Glass Transition of ‘‘Pure Protein’’ Note that, ‘‘pure protein’’ in this context means protein without saccharide but containing 15% by weight sodium phosphate buffer. In principle, the value of the glass transition temperature for the pure protein may be obtained by extrapolation of the Tg’s of disaccharide:protein mixtures to zero saccharide content using, for example, the Fox equation, which assumes linearity of the reciprocal of glass transition temperature in weight fraction of the components, provided the extrapolation function is consistent with the data. In fact, the Fox equation is fully consistent with the data in that excellent linearity is found and moreover, essentially the same value of the glass transition temperature of pure protein is found using both the sucrose and the trehalose-based formulations. An illustration of the extrapolation of Tg data for disaccharide mixtures to zero saccharide level to obtain Tg of the pure protein is given by Figure 7 where ‘‘dry’’ Tg values from Table 2 are used. Here, both sucrose and trehalose data give exactly the same value for the Tg of the pure protein, 1368C. Data corresponding to other relative humidities give similar agreement (i.e., differences of less than 18C) except for the data at 33% relative humidity where the difference is 68C. Thus, with the possible exception of the data at 33% relative humidity, the excellent agreement between the sucrose and trehalose values, provides credibility for the estimates of Tg for pure protein. For dry protein, the Tg is very high, consistent with previous DSC observations.11,13 In addition, the Tg data obtained for the dry ‘‘pure protein’’ by extrapolation of the DSC data do agree with the results from the global thermally stimulated current measurements,27 the latter reflecting global motion but not depending on a heat capacity change at Tg for detection. Thus, the Tg data obtained do not correspond to the thermal events noted for hGH5 and insulin28 around 608C. These low temperature thermal events appear to DOI 10.1002/jps
Figure 7. Illustration of the extrapolation of glass transition data to ‘‘pure protein’’ using the Fox equation. The extrapolated values are 136.08C from both the sucrose and trehalose data. Similar extrapolations for samples equilibrated at selected relative humidities showed agreement between sucrose and trehalose data within 18C, except for the data at 33% relative humidity where the difference was 68C. Symbols key: open squares ¼ trehalose; filled triangles ¼ sucrose.
be enthalpy recovery events that are associated with a glass transition, but clearly such a glass transition is not the one we are measuring in Figure 7. The Tg data in Figure 7, obtained by DSC and by extrapolation to zero saccharide level, refer to the onset of global motion that is coupled with system viscosity, which is whole molecule translation and rotation. The low temperature thermal events, if they are associated with a glass transition, may be associated with Tg-like transitions within the protein molecule itself often denoted ‘‘protein dynamical temperature, Td’’, which have been a subject of investigation for some time22–26 and recently were discussed in terms of pharmaceutical stability applications.22 It is argued that the mobility responsible for this transition needs to be strongly coupled to the mobility of the matrix (i.e., the stabilizer) to dampen motion within the protein and reduce reactivity.22 This is an interesting concept that is plausible but unfortunately lacks direct experimental validation at this time. From our perspective, the question of whether or not it is ‘‘whole molecule’’ global motion reflected by Tg or protein internal motion that is most relevant to stability of dried protein products remains an open question. Some opinions and
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observations suggest that both are important. The concept that pharmaceutical stability is dependent on the degree of coupling between protein internal motion and matrix motion22 is in itself an acknowledgement of the potential importance of both the matrix motion (i.e., of Tg and the global dynamics) and internal protein dynamics. The observation that proteins do not unfold in the ‘‘dry’’ state until the glass transition temperature is exceeded (see, for example Tab. 2) suggests that Tg is a relevant parameter for at least large-scale conformational change. Further, as noted in the introduction, Tg and global dynamics can be a useful, though imperfect, indicator of pharmaceutical stability (i.e., chemical degradation, protein aggregation, and crystallization from the amorphous state) for both small molecule and protein systems. However, recent data29,30 suggest better correlations between pharmaceutical stability and ‘‘Fast Dynamics’’ (i.e., dynamics measured on the nanosecond time scale by neutron scattering) in cases where stability is studied well below Tg. However, at least in most cases, these ‘‘Fast Dynamics’’ refer to the carbohydrate matrix, not the internal motion of the protein, and so we still seek direct evidence that protein internal dynamics are critical to pharmaceutical stability. Values of Tg of pure hGH, obtained from extrapolations such as illustrated by Figure 7, are given as a function of water content by Figure 8. Note that although Tg of a dry hGH sample is very high, well above 1008C, at a water content of about 12%, the Tg is 258C. Thus, for water contents in excess of 12%, a pure protein sample is above its Tg at room temperature and might be expected to exhibit characteristics of high mobility (i.e., structural collapse during storage and poor stability).
Origin of Nonreversible Thermal Events Below Tg All the disaccharide:hGH formulations studied exhibit sub-Tg thermal events under at least some conditions (Figs. 1–4). As discussed above, pure protein systems also exhibit irreversible DSC thermal events at moderate temperatures,5,28 and given that the system glass transitions in pure proteins occur well above 1008C, these irreversible endotherms occurring around 608C in pure solid proteins5,28 may also be classified as subTg thermal events. However, while we have suggested that the pre-Tg endotherms in the pure
Figure 8. Estimated glass transitions of pure protein as a function of water content. The glass transition temperatures were taken from extrapolations as illustrated in Figure 7, and the water contents corresponding to the given relative humidities were taken from the water sorption isotherm of pure hGH. The values of ‘‘% relative humidity, % water’’ are: 11% RH, 4.2%; 22% RH, 6.6%; 33% RH, 9.5%.
protein systems may be associated with internal motions in the protein molecule, it seems clear that the pre-Tg events displayed in the saccharide:protein systems have, for the most part, a different origin since the magnitude of these transitions is not proportional to the percentage of protein in the sample. Also, similar sub-Tg transitions are observed in disaccharide systems without proteins (unpublished observations). We note that sub-Tg endotherms can be a result of a broad distribution of relaxing states. Modeling the DSC thermogram using a KWW stretched exponential function to describe relaxation kinetics, with small beta values indicating a very broad distribution of relaxation times, and using the Tool-Narayanaswamy-Moynihan (TNM) equation to represent nonlinearity, one can reproduce sub-Tg endotherms.17 It has also been recently suggested that the pre-Tg endotherms arise from ‘‘b-relaxations’’.31 However, the apparent negative heat capacity shifts and the combination of exotherm and endotherm, as observed in the present work, apparently cannot be reproduced by the theoretical analysis described above. Sub-Tg thermal events similar to those illustrated in Figures 1–4 were observed a number of years ago in polymer systems prepared by freeze-drying
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from naphthalene.32 While an unambiguous explanation for the observations was not provided, it was suggested that the complex thermal events below Tg were likely a result of the unique type of glass created by freeze drying-an ‘‘open structure’’ with a very broad distribution of relaxation times.
REFERENCES 1. Pikal M. 2002. Lyophilization In: Swarbrick J, Boylan JC, editors. chapter in Encyclopedia of pharmaceutical technology. New York: Marcel Dekker. pp 1299–1326. 2. Carpenter JF, Prestrelski SJ, Dong A. 1998. Application of infrared spectroscopy to development of stable lyophilized protein formulations. Eur J Pharm Biopharm 45:231–238. 3. Johnson DS, Castelli F. 1989. The influence of sugars on the properties of freeze-dried lysozyme and haemoglobin. Thermochim Acta 144:195–208. 4. Pikal MJ, Dellerman KM, Roy ML, Riggin RM. 1991. The effects of formulation variables on the stability of freeze dried human growth hormone. Pharm Res 8:427–436. 5. Pikal MJ, Dellerman KM, Roy ML. 1991. Formulation and stability of freeze-dried proteins: Effects of moisture and oxygen on the stability of freeze-dried formulations of human growth hormone. Develop Biol Standard 74:21–38. 6. Bell LN, Hageman MJ, Muraoka LM. 1995. Thermally induced denaturation of lyophilized bovine somatotroppin and lysozyme as impacted by moisture and excipients. J Pharm Sci 84:707–712. 7. Pikal MJ. 2004. Mechanisms of protein stabilization during freeze-drying and storage: The relative importance of thermodynamic stabilization and glassy state relaxation dynamics, In: Rey Louis, May Joan, editors. Chapter 3 in book, Freeze-drying/lyophilization of pharmaceutical and biological products. New York: Marcel Dekker. 63–107. 8. Carpenter JF, Pikal MJ, Chang BS, Randolph TW. 1997. Rational design of stable lyophilized protein formulations: Some practical advice. Pharm Res 14: 969–975. 9. Chang BS, Randall CS. 1992. Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology 29:632–656. 10. Sochava IV, Tsereteli GI, Smirnova OI. 1991. The denaturing specific heat increment in globular proteins and its relation with glass transition. Biofizika 36:1–6 (translated from Russian). 11. Sochava IV, Smirnova OI. 1993. Heat capacity of hydrated and dehydrated globular proteins. Denaturation increment of heat capacity. Food Hydrocolloids 6:513–524.
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12. Fan J, Cooper EI, Angell CA. 1994. Glasses with strong calorimetric b-glass transitions and the relation to the protein glass transition problem. J Phys Chem 98:9345–9349. 13. Green JL, Fan J, Angell CA. 1994. The proteinglass analogy: Some insights from homopeptide comparisons. J Phys Chem 98:13780–13790. 14. Chang L, Shepherd D, Sun J, Ouellette D, Grant K, Tang X, Pikal MJ. 2005. Mechanism of protein stabilization by sugars during freeze drying and storage: Native structure preservation, specific Interaction and/or immobilization in a glassy matrix? J Pharm Sci 94:1427–1444. 15. Moynihan CT1993. Correlation between the width of the glass transition region and the temperature dependence of the viscosity of high-Tg glasses. J Am Ceram Soc 76:1081–1087. 16. Pikal MJ, Chang L, Tang X. 2004. Evaluation of glassy-state dynamics from the width of the glass transition: Results from theoretical simulation of differential scanning calorimetry and comparisons with experiment. J Pharm Sci 93: 981–994. 17. Hodge IM. 1994. Enthalpy relaxation and recovery in amorphous materials. J Non-Cryst Solids 169: 211–266. 18. Liu J, Rigsbee D, Stotz C, Pikal MJ. 2002. Dynamics of pharmaceutical amorphous solids: The study of enthalpy relaxation by isothermal microcalorimetry. J Pharm Sci 91:1853–1862. 19. Carpenter JF, Crowe JH. 1989. An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry 28:3916–3922. 20. Angell CA. 1995. Formation of glasses from liquids and biopolymers. Science 267:1924–1935. 21. Kawakami K, Pikal MJ. 2005. Calorimetric investigation of the structural relaxation of amorphous materials: Evaluating validity of the methodologies. J Pharm Sci 94:948–965. 22. Hill JJ, Shalaev EY, Zografi G. 2005. Thermodynamic and dynamic factors involved in the stability of native protein structure in amorphous solids in relation to levels of hydration. J Pharm Sci 94: 1636–1667. 23. Dirama TE, Curtis JE, Carri GA, Sokolov AP. 2006. Coupling between lysozyme and trehalose dynamics: Microscopic insights from molecular dynamics simulations. J Chem Phys 124: (043901 download). 24. Fenimore PW, Frauenfelder H, McMahan BH, Young RD. 2004. Bulk-solvent and hydration-shell fluctuations, similar to a–and b–fluctuations in glasses, control protein motions and functions. Proc Natl Acad Sci 101:14408–14413. 25. Iben IET, Braunstein D, Doster W, Frauenfelder H, Hong MK, Johnson JB, Luck S, Ormos P, Schulte A, Steinbach PJ, Xie AH, Young RD. 1989. Phys Rev Lett 62:1916–1919.
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26. Giuffrida S, Cottone G, Cordone L. 2006. Role of solvent on protein-matrix coupling in MbCO embedded in water-saccharide systems: A Fourier infrared spectroscopy study. Biophys J 91:968–980. 27. Shalaev E, Chang L, Reddy R, Collins G, Luthra S, Pikal M. 2003. Molecular mobility and the glass transition in globular lyophilized proteins. AAPS Pharm Sci 5: Abstract T2300. 28. Pikal MJ, Rigsbee DR. 1997. The stability of insulin in crystalline and amorphous solids: Observation of greater stability for the amorphous form. Pharm Res 14:1379–1387. 29. Cicerone M, Soles CL, Chowdhuri C, Pikal MJ, Chang L. 2005. Fast dynamics as a diagnostic for
excipients in preservation of dried proteins. Am Pharm Rev 8:22–27. 30. Chang L, Shepherd D, Sun J, Tang X, Pikal MJ. 2005. Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state. J Pharm Sci 94:1445– 1455. 31. Vyazovkin S, Dranca I. 2006. Probing beta relaxation in pharmaceutically relevant glasses by using DSC. Pharm Res 23:422–428. 32. Shultz AR, Young AL. 1980. DSC on freeze-dried poly(methyl methacrylate)-polystyrene blends. Macromolecules 13:663–668.
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School of Pharmacy, University of Connecticut, Storrs, Connecticut
2
Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana
Received 1 November 2006; revised 26 January 2007; accepted 1 February 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20960
ABSTRACT: Although freeze dried formulations are commonly characterized using differential scanning calorimetry (DSC), a protein-rich system behaves as a ‘‘strong glass’’, and the glass transition temperature, Tg, cannot be directly determined by DSC. A strong glass means a small heat capacity change at Tg, DCp, and a very broad glass transition region, or a large DTg. However, direct experimental evidence for a small DCp and a large DTg have been lacking. Here, we utilize extrapolation of thermal analysis data in protein:disaccharide mixtures to evaluate Tg, DTg, and DCp for ‘‘pure’’ human growth hormone (hGH) from low to moderate residual water. We find that DTg is indeed large and DCp is very small. Also, the Tg for pure hGH decreases from a value of about 1368C when dry to around 258C at 12% water. This glass transition is not the onset of mobility within the protein molecule but rather signals onset of whole molecule rotation and translation. We also observe complex pre-Tg thermal events in the DSC data, which are interpreted as consequences of relaxation events, largely due to the disaccharide, and are characteristic of freeze dried systems having a broad distribution of relaxing substates. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2765–2776, 2007
Keywords: amorphous; biotechnology; calorimetry (DSC); freeze drying/lyophilization; glass transition; proteins; relaxation time; protein formulation
INTRODUCTION Proteins commonly undergo excessive degradation when stored in the aqueous solution state for long periods of time, and so therapeutic proteins are commonly dried to provide satisfactory storage stability. Freeze drying is, by far, the most popular drying technology for biotechnology products, but other drying techniques can also be employed.1 In essentially all cases, the solution being dried is aqueous and contains small Correspondence to: Michael J. Pikal (Telephone: 860-4863202; Fax: 860-486-4998; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2765–2776 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
amounts of a buffer as well as one or more ‘‘stabilizers’’ and results in a solid that is at least partially amorphous after drying. That is, the therapeutic protein, buffer system, and stabilizer remain amorphous and for optimal stability, should form a single amorphous phase.1 Just as physical characterization of a protein in the aqueous solution state is important to the prediction of the pharmaceutical stability of a solution formulation, so is characterization of the protein in the amorphous solid state important to the understanding and prediction of stability in the solid state. In principle, protein structure2 thermal denaturation3–6 and glass transition temperature1,7 are properties critical to stability behavior. In practice, thermal denaturation in the
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solid state occurs only at very high temperatures3–6 and therefore has no obvious direct relationship to practical stability behavior. However, protein structure by FTIR and the glass transition temperature in protein formulations are commonly measured in an attempt to predict stability behavior.1,2,8 While the glass transition temperature, Tg0 , in a frozen ‘‘pure’’ protein solution can be measured with differential scanning calorimetry (DSC), and is usually in the range of 108C to 158C9 glass transitions in ‘‘pure’’ dry proteins are problematic. That is, the sharp increase in heat capacity during a DSC scan, characteristic of the glass transition, typically cannot be observed for protein-rich systems.4–6,10–14 We note that some soy proteins do show the classical DSC signature of a glass transition, and a fully denatured protein often shows a strong increase in heat capacity, signaling a glass transition.10–13 However, most dry proteins appear to behave as ‘‘strong glasses’’12,13 meaning that the ‘‘strength parameter’’, D, is large (i.e., fragility is small). With a strong glass, the glass transition is spread over a very large temperature range, that is, DTg is large15,16 and the change in heat capacity at the glass transition, DCp is small. While the ‘‘strong glass’’ interpretation of dry amorphous protein behavior is generally accepted in pharmaceutical science, it is curious that there is no direct evidence for either a small DCp or large DTg in dry protein systems. The prime objective of this work is to demonstrate by quantitative extrapolation of DSC data on mixtures of disaccharides and human growth hormone (hGH) that ‘‘pure’’ hGH is indeed characterized by a very small DCp and large DTg, giving a large strength parameter, D. From the extrapolated Tg data, we characterize the effect of water content on the glass transition of the disaccharide-free protein system for water contents in the very low to moderate range.
EXPERIMENTAL Materials All lots formulated with sugars were prepared from the same lot of bulk hGH obtained from Eli Lilly (Indianapolis, IN). Sucrose was analytical reagent grade obtained from Baker (Phillipsburg, NJ), and sodium phosphate was analytical reagent grade obtained from Mallinckrodt (Phillipsburg, NJ). Trehalose was obtained from
Sigma (St. Louis, MO) and was ‘‘reduced metal ion content’’ material. All solutions were 5 mg/mL hGH buffered to pH 7.4 with sodium phosphate (0.75 mg/mL). Sucrose and trehalose were added at weight ratios, relative to hGH, of 1:1, 3:1, and 6:1 (i.e., sugar concentrations of 5 mg/mL, 15 mg/ mL, and 30 mg/mL, respectively). Freeze dried samples at elevated water content were prepared from the corresponding ‘‘dry’’ samples by equilibrating overnight with 11%, 22%, and 33% relative humidity in vacuum desiccators. All samples were confirmed 100% amorphous by noting the absence of birefringence by polarized light microscopy. Note that all samples contained sodium phosphate buffer at 15% (w/w) of the protein content, which remained amorphous and therefore could have a small impact on the DSC data reported.
Freeze Drying Samples were freeze dried in a Virtis 25-SRC-X (freeze dryer equipped with a MKS Baratron pressure measurement system and an electronic moisture sensor; Ondyne, Endress and Hauser, Greenwood, IN) to measure the partial pressure of water vapor in the drying chamber. Product and shelf surface temperatures were measured via 30 gauge copper-constantan thermocouples. Product temperature sensors were placed in the bottom center of a vial while the shelf surface sensors consisted of the thermocouple junctions silver soldered into a brass disk about 7 mm in diameter and about 1.5 mm thick. The disk was ‘‘glued’’ to the shelf with vacuum grease. Shelf surface temperature was measured on the shelf surface near the shelf heat transfer fluid entry and near the fluid exit. All hGH formulations were freeze dried at a shelf temperature control setting of 208C in primary drying and a chamber pressure setting of 0.10 Torr throughout the drying process. Each sample contained 5 mg hGH in 1 mL fill volume. Nominal 5 mL tubing vials were used along with 13 mm finish West gray butyl rubber stoppers (#1816 rubber stock) or with Daikyo Florotec stoppers (low moisture release stoppers), as indicated. All formulations were freeze dried at least 38C below their collapse temperatures, and visual inspection confirmed the absence of collapse. The maximum product temperatures during primary drying varied with formulation: 1:1 hGH:trehalose ¼ 35.28C; 1:3 hGH: trehalose ¼
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33.78C; 1:6 hGH: trehalose ¼31.08C; 1:1 hGH:sucrose ¼34.48C; 1:3 hGH:sucrose¼ 33.68C; 1:6 hGH:sucrose ¼ 33.48C. For secondary drying, the shelf temperature was increased from 208C to þ338C in four steps over 3 h and then the shelf temperature was held at 338C for 2 h. Water content varied from 1.0% to 0.4%, depending upon formulation. All samples were stored at 208C until use. Water Content Water contents were determined by Coulometric Karl Fischer procedures, using a ‘‘weigh and dump’’ technique which minimizes exposure of the sample to ambient humidity, and also by the Lilly Analytical Division using a variation of Coulometric Karl Fischer procedures which involves dissolving the sample in the original sample container. The difference in water contents determined by the two procedures averaged only 0.2%. Reported water contents represent the mean of the two sets of results. Differential Scanning Calorimetry DSC studies were carried out with a Perkin-Elmer DSC-7 using sealed aluminum pans. A scan rate of 58C/min was used for the study of frozen solutions (i.e., determination of the glass transition temperature of the freeze concentrate, Tg0 ). Freeze dried powders (usual sample size ¼ 5–10 mg) were compacted into disks 2–3 mm in thickness before sealing in the sample pans. All sample handling was carried out in a dry bag continuously purged with dry air (< 2% relative humidity). Powders were generally scanned at 78C/min. Second scans were made after cooling from above the glass transition temperature, Tg, to below 08C at 308C/min. Glass transition temperatures reported represent the mid-point of the heat capacity transition. Calibration was performed using indium, as conventional, and also by using pure water. In all thermograms, an endotherm is ‘‘up’’.
RESULTS Features of the DSC Thermograms Examples of the diverse thermograms obtained are given by Figures 1–4. Figure 1 shows the effect of aging on DSC response for 3:1 trehalose:hGH (weight ratio) formulations, where (a) gives the DOI 10.1002/jps
Figure 1. Representative DSC thermograms for hGH formulations: Effect of aging on 3:1 (weight ratio) trehalose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; ________ ¼ second scan (complete) following first (partial) scan. (a) Baseline as noted, and initial sample, 6.4 mg, % H2O ¼ 0.6%; (b) aged 3 months 258C, 8.6 mg, % H2O ¼ 1.6%; (c) Aged 3 months 408C, 7.8 mg, % H2O ¼ 2.1%. Increase in moisture on aging is due to stopper to product moisture transfer.
data for an ‘‘initial’’ sample, sample (b) was aged 3 months at 258C, and sample (c) was aged 3 months at 408C. In each figure, results of a partial scan up to and through the glass transition at about 908C–1158C are shown as a broken line. Following this partial heating scan, the sample was quenched to below 08C and then scanned up to about 1808C, shown as a solid line. The glass transition is easily identified from the second scan
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Figure 2. Representative DSC thermograms for hGH formulations: Effect of relative humidity on 1:1 (weight ratio) trehalose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; __________ ¼ second scan (complete) following first (partial) scan. (a)’’Dry’’ sample, 4.5 mg; (b) equilibrated with 11% relative humidity, 7.6 mg, % H2O ¼ 3.1%; (c) equilibrated with 22% relative humidity, 8.0 mg, % H2O ¼ 5.0%.
Figure 3. Representative DSC thermograms for hGH formulations: Effect of aging on 1:1 (weight ratio) sucrose:hGH Systems: Symbols: _____ _____ ¼ First (partial) scan; _________ ¼ second scan (complete) following first (partial) scan. (a) Baseline as noted, and initial sample, 3.0 mg, % H2O ¼ 0.8%; (b) aged 3 months 258C, 7.4 mg, % H2O ¼ 2.2%; (c) aged 3 months 408C, 9.3 mg, % H2O ¼ 2.5%. Increase in moisture on aging is due to stopper to product moisture transfer.
as the large increase in heat capacity (i.e., endothermic baseline shift) which occurs in the range of about 908C–1158C. The variation is due to variable water content, which in turn is a result of moisture transfer from stopper to product with the grey butyl stoppers used here. The large endotherm around 1508C–1608C is the thermal
denaturation endotherm for the protein in the ‘‘solid’’ state. Note, however, that denaturation occurs well above the glass transition temperature, so strictly speaking, the denaturation occurs in a viscous system of molten trehalose and protein. Denaturation is irreversible in the sense that if a sample is scanned through
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Figure 4. Representative DSC thermograms for hGH formulations: Effect of relative humidity on 1:1 (weight ratio) sucrose:hGH Systems. Symbols: _____ _____ ¼ First (partial) scan; _________ ¼ second scan (complete) following first (partial) scan. (a) Equilibrated with 11% relative humidity, 8 mg, % H2O ¼ 2.5%; (b) equilibrated with 22% relative humidity, 9.5 mg, % H2O ¼ 3.9%.
the denaturation endotherm event, cooled, and then re-scanned, no denaturation endotherm is observed. The most curious features occur only in the first scan before and around the glass transition; thus, these features correspond to irreversible phenomena. With the ‘‘initial sample’’, we observe a slight endothermic shift (i.e., ‘‘up’’) at around 608C, followed by a sharp exothermic shift, giving the appearance of a negative heat capacity shift around 708C. With aging and perhaps also because of the increasing moisture content during aging, the endothermic shift and subsequent exothermic shift become more distinct and move to higher temperature, occurring essentially at the glass transition for the sample aged at 408C (Fig. 1c). Figure 2 illustrates behavior of 1:1 trehalose:hGH for ‘‘initial’’ samples where the water content of very dry material (Fig. 2a) was deliberately increased by equilibration with relative humidities of 11% (Fig. 2b) and 22% (Fig. 2c). DOI 10.1002/jps
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Again, the glass transitions are clearly observed in the second scans from about 1208C (dry) to about 508C (22% Relative humidity), and the (partial) first scan shows complex behavior at and below the glass transition region. The very dry sample shows a distinct negative heat capacity shift, which transforms into an endotherm followed by an exotherm with higher moisture content samples. With the 22% relative humidity sample, the irreversible phenomena appear much like a classical enthalpy recovery endotherm17 superimposed on the positive heat capacity shift due to the glass transition. The thermal behavior of 1:1 sucrose:hGH formulations is illustrated by Figures 3 and 4, where Figure 3 shows the effect of aging and Figure 4 shows the effect of exposure to 11% and 22% relative humidity. Figure 3a gives only a first scan to 1808C and the baseline, showing the pre-Tg event which is qualitatively the same as for the 258C aged 3:1 trehalose:hGH formulation (Fig. 1b); that is, we see a slight endotherm, followed by an exothermic shift and then the glass transition. Aging at 258C makes the ‘‘endo– exo’’ event more distinct, and aging at 408C leads to a strong endotherm superimposed on the glass transition, much like a classical enthalpy recovery event. Note the strong denaturation endotherm followed by a strong exothermic shift. The exothermic event corresponds to cold crystallization of the sucrose (i.e., crystallization from the amorphous state below the melting point). Due to interference from sucrose crystallization, thermal denaturation cannot be observed with the higher sucrose content formulations. Figure 4 shows the effect of elevated moisture content on the thermal behavior of the 1:1 sucrose:hGH formulation. The ‘‘dry’’ sample is shown in Figure 3a. Note that the effect of higher moisture content is qualitatively much the same as observed for the 1:1 trehalose:hGH system; as moisture increases, the ‘‘endo–exo’’ thermal event becomes more distinct and moves up to the Tg region, with the thermogram for 22% relative humidity material appearing as a classical enthalpy recovery endotherm superimposed on the glass transition.
DTg and DCp Results Table 1 summarizes the widths of the glass transition event and the change in heat capacity at the glass transition for the various hGH
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Table 1. Relative Width of the Glass Transition and Change in Heat Capacity at the Glass Transition
Disaccharide Sucrose Sucrose Sucrose Sucrose Trehalose Trehalose Trehalose Trehalose
Saccharaide:hGH
DCp(J g1 K1)
DTg/Tg
Pure sucrose 6 3 1 Pure trehalose 6 3 1
0.596 0.543 0.019 0.498 0.018 0.332 0.027 0.604 0.534 0.009 0.502 0.012 0.271 0.017
0.0187 0.0253 0.0013 0.0278 0.0013 0.0366 0.0019 0.0171 0.0191 0.0003 0.0244 0.0007 0.0410 0.0037
The DTg and DCp values were obtained by linear extrapolation of raw data versus % water data to zero water content. Uncertainties are the standard deviation in the intercept for extrapolation of the data to zero water content. Pure sucrose and pure trehalose data were means of duplicates taken on 0 water content samples.
formulations. The width of the glass transition has possible application in estimating the fragility (or strength) of the glass as well as providing an estimate of the structural relaxation time at temperatures below Tg,16 and values of DCp are useful in interpreting fragility results and in the determination of calorimetric structural relaxation times.18 All data reported were taken from second scan results to avoid ambiguities and inaccuracies resulting from the complex irreversible pre-Tg events described above. However, it should be noted that the values of DTg obtained from first scan results, when possible, are essentially the same as obtained from second scan; the ratio of first scan to second scan averages 0.96 0.025 (std error). The width of the glass transition temperature region was evaluated using the ‘‘E ¼ 0 method’’16 since, particularly for second scan data, ‘‘overshoot’’ endotherms at Tg were largely absent. Note that the glass transition is a weak transition (i.e., small DCp and large DTg/Tg) at high protein (1:1) and as noted earlier, cannot be seen with most dry pure protein systems,4–6,10–14 suggesting that a pure protein system is a ‘‘strong glass’’.12,13
Thermal Data for hGH:Saccharide Systems Equilibrated With Various Humidities Thermal data obtained on systems of varying water content are provided by Table 2. For a given formulation, all samples were generated from a given batch by equilibration with fixed relative humidities. All data represent ‘‘initial’’ values (i.e., no deliberate aging). The formulation, relative humidity (RH), mid point of the glass
transition in 8C (Tg), both the extrapolated onset and mid point of the denaturation endotherm, and the corresponding heat of denaturation (i.e., from the area of the denaturation endotherm) are listed. We note that the endotherms attributed to denaturation do indeed represent highly cooperative unfolding of the protein, as viewed by FTIR. This topic will be developed in detail in a later publication. The change in heat capacity upon denaturation could not be reliably measured since the DSC techniques used here did not allow separation of irreversible events from heat capacity changes. This topic will be explored with modulated DSC in a later publication. As expected, increasing the water content decreases Tg significantly, and increasing the saccharide content also decreases Tg, but only slightly for trehalose systems, suggesting that the Tg of pure protein is near that of trehalose. The thermal denaturation temperature also decreases with increasing water content and increasing saccharide content. Note that the thermal denaturation temperature for pure hGH is about 1808C when dry.4,5 In this sense, the saccharide ‘‘interacts’’ with the protein as does water, consistent with the concept of ‘‘water substitution’’ by the saccharide.3,19 In some of the sucrose-rich systems, denaturation could not be observed due to interference from cold crystallization of sucrose. With 1:1 saccharide:hGH, the heat of denaturation decreases as water content increases, and at least for trehalose where data are available at various saccharide levels, the heat of denaturation decreases with increasing trehalose level except at the highest water content studied. Thus, trends in the heat of denaturation with saccharide content mimic the trends with
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Table 2. Thermal Data For hGH:Saccharide systems Equilibrated With Selected Relative Humidities
Saccharide
Saccharide:hGH
%RH
%H2O
Tg, 8C Mid pt
Pure sugar 6 3 1 6 3 1 6 3 1 3 1 Pure sugar 6 3 1 6 3 1 6 3 1 6 3 1
0 0 0 0 11 11 11 22 22 22 33 33 0 0 0 0 11 11 11 22 22 22 33 33 33
0 0 0 0 2.15 2.3 2.5 4.1 4.0 3.9 5.8 5.3 0 0 0 0 3.3 3.2 3.1 5.4 5.2 5.0 7.1 6.9 6.6
77.0 80.7 89.0 103.9 49.0 50.4 58.3 36.4 38.4 42.4 24.9 30.5 118.0 121.l 122.9 126.8 69.2 67.8 70.1 49.9 49.3 42.4 38.1 36.9 37.2
Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose Trehalose
water content. However, the trends in heat of denaturation do not predict trends of increasing ‘‘native structure’’ as increasing levels of saccharide increase native structure1,2,8,19 and would be expected to increase heat of denaturation, not cause a decrease!
DISCUSSION Strength of Protein-Rich Glasses Figure 5 shows DCp at Tg as a function of disaccharide level. Note that while the range of extrapolation is large and linearity is not assured over the entire composition range, there is no question that DCp is quite small for protein-rich systems and may decrease to nearly zero for pure protein systems. With a small DCp, one expects a large value of the Angell strength parameter, D 20; that is, one expects a strong glass. The Angell strength parameter may be estimated from the width of the glass transition region16 using the following procedure. First, we note that the activation energy for structural relaxation at Tg, DH , may be estimated from DOI 10.1002/jps
TD, 8C Onset
TD, 8C Mid pt
DHD J/ghGH
139.7 147.0
152.4
17.2
131.0
141.2
18.8
124.6
135.6
13.5
120.6
131.2
12.3
153.4 153.4 158.4 133.4 131.6 131.4 125.4 124.8 124.5 122.0 121.0 118.5
161.7 161.2 165.2 141.9 143.0 138.4 135.8 136.8 133.4 130.6 130.8 129.0
11.8 12.3 21.0 11.4 14.8 19.3 13.4 13.3 15.4 15.6 12.5 13.0
the width of the glass transition from the approximation, b
DH C ¼ ; RTg DTg =Tg
(1)
where C is a constant depending on DSC protocol (i.e., thermal history) and the method for determination of DTg. The symbol, b, is the ‘‘stretched exponent’’ of the Kohlrausch–Williams–Watts (KWW) equation for expressing the kinetics of enthalpy relaxation. For DSC protocols where the cooling and heating rates are 308/min and 78/min, respectively, and the ‘‘E ¼ 0 method’’1 is used to determine DTg, the value of C ¼ 3.2.16 The activa tion energy for structural relaxation at Tg, DH , is related to the Angell strength parameter, D, the ‘‘zero mobility’’ temperature, T0, and Tg by DH DðTg =T0 Þ ¼ RTg ðTg =T0 1Þ
(2)
1 The ‘‘E ¼ 0 method’’ refers to determination of the end of the glass transition region by using the intercept of the tangent drawn to the mid-point of the glass transition with the tangent drawn to the heat capacity curve of the equilibrium liquid, post Tg transition.
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Figure 5. The effect of composition on the change in heat capacity at the glass transition, DCp, for disaccharide:hGH systems. The values of DCp refer to data at 0% water and were taken from Table 1. key: open squares ¼ trehalose; filled triangles ¼ sucrose.
Figure 6. The effect of composition on the Angell strength parameter, D, for disaccharide:hGH systems. The values of D refer to systems at 0% water and were calculated using DTg data from Table 1 using equations (1) and (2). Symbols key: open squares ¼ trehalose; filled triangles ¼ sucrose.
and Angell20 also argues that the ratio Tg/T0 is directly related to the strength parameter by
expected from the estimated small DCp for protein-rich systems, the values of D for such systems are large, extrapolating to a D value for the pure protein which is about a factor of 4 higher than found for the pure disaccharide systems. Thus, consistent with the speculation provided more than a decade ago,12,13 to the extent that hGH is representative, proteins indeed behave as strong glasses. While it is clear that there are significant differences between a ‘‘native’’ protein and a typical small molecule (or synthetic polymer) in the changes in dynamics that occur at Tg, our view is that both reflect an increase in global mobility and the onset of viscous flow. The difference is that, in protein-rich systems, the change in heat capacity over the transition range is small, and thus the material at least behaves as a ‘‘strong glass’’, although it is obvious that such an observation does not mean the structure is similar to a ‘‘network glass’’. The reason for the small DCp in the glass transition range is likely a decoupling of the protein internal motions22–26 from the motions that characterize whole molecule translational/rotational diffusion and viscous flow. Either the internal motions are mostly actuated at a lower temperature (i.e., at the protein dynamical transition temperature, Td)22 and/or the internal degrees of freedom are gradually accessed over a large temperature range prior to the DSC Tg. Either way, at Tg, it is then only the relatively few remaining modes of motion of the
Tg ¼ 1 þ 0:0271 D T0
(3)
Thus, knowing the glass transition temperature, Tg and the width of the glass transition region, DTg, one can evaluate D using equations (1)–(3), provided an estimate of b is available. We take b ¼ 0.4 for all samples. While this value is somewhat arbitrary, the true value cannot be far different. In this regard, we note that the very small overshoot endotherms on the thermograms used to evaluate the widths of the glass transition indicate that b is very small, 0.4.16 Further, simulations suggest that values of 0.4 seem consistent with calorimetric relaxation data on saccharide-rich systems and therefore are appropriate for our current application. We note that while values of b are determined by a fit of the KWW equation to calorimetric relaxation data, such determinations are subject to systematic error in both b and t, but not tb.21 Actually, as long as b is not highly variable, the exact value is not critical for the qualitative conclusions we draw. The estimated values of the strength parameter, D, are plotted as a function of saccharide content in Figure 6. Note that the trends in D evaluated from either sucrose or trehalose systems are similar and suggest a relatively high value of D for the pure protein. As would be
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molecule ‘‘as a whole’’ that are cooperatively actuated, and the DCp is then small. The fact that both the glass transition temperature we measure and the transition width vary with composition indicates that both components determine the properties of the resulting glass, but this observation alone does not necessarily mean the protein internal motions are coupled to the disaccharide dynamics.
Estimates of the Glass Transition of ‘‘Pure Protein’’ Note that, ‘‘pure protein’’ in this context means protein without saccharide but containing 15% by weight sodium phosphate buffer. In principle, the value of the glass transition temperature for the pure protein may be obtained by extrapolation of the Tg’s of disaccharide:protein mixtures to zero saccharide content using, for example, the Fox equation, which assumes linearity of the reciprocal of glass transition temperature in weight fraction of the components, provided the extrapolation function is consistent with the data. In fact, the Fox equation is fully consistent with the data in that excellent linearity is found and moreover, essentially the same value of the glass transition temperature of pure protein is found using both the sucrose and the trehalose-based formulations. An illustration of the extrapolation of Tg data for disaccharide mixtures to zero saccharide level to obtain Tg of the pure protein is given by Figure 7 where ‘‘dry’’ Tg values from Table 2 are used. Here, both sucrose and trehalose data give exactly the same value for the Tg of the pure protein, 1368C. Data corresponding to other relative humidities give similar agreement (i.e., differences of less than 18C) except for the data at 33% relative humidity where the difference is 68C. Thus, with the possible exception of the data at 33% relative humidity, the excellent agreement between the sucrose and trehalose values, provides credibility for the estimates of Tg for pure protein. For dry protein, the Tg is very high, consistent with previous DSC observations.11,13 In addition, the Tg data obtained for the dry ‘‘pure protein’’ by extrapolation of the DSC data do agree with the results from the global thermally stimulated current measurements,27 the latter reflecting global motion but not depending on a heat capacity change at Tg for detection. Thus, the Tg data obtained do not correspond to the thermal events noted for hGH5 and insulin28 around 608C. These low temperature thermal events appear to DOI 10.1002/jps
Figure 7. Illustration of the extrapolation of glass transition data to ‘‘pure protein’’ using the Fox equation. The extrapolated values are 136.08C from both the sucrose and trehalose data. Similar extrapolations for samples equilibrated at selected relative humidities showed agreement between sucrose and trehalose data within 18C, except for the data at 33% relative humidity where the difference was 68C. Symbols key: open squares ¼ trehalose; filled triangles ¼ sucrose.
be enthalpy recovery events that are associated with a glass transition, but clearly such a glass transition is not the one we are measuring in Figure 7. The Tg data in Figure 7, obtained by DSC and by extrapolation to zero saccharide level, refer to the onset of global motion that is coupled with system viscosity, which is whole molecule translation and rotation. The low temperature thermal events, if they are associated with a glass transition, may be associated with Tg-like transitions within the protein molecule itself often denoted ‘‘protein dynamical temperature, Td’’, which have been a subject of investigation for some time22–26 and recently were discussed in terms of pharmaceutical stability applications.22 It is argued that the mobility responsible for this transition needs to be strongly coupled to the mobility of the matrix (i.e., the stabilizer) to dampen motion within the protein and reduce reactivity.22 This is an interesting concept that is plausible but unfortunately lacks direct experimental validation at this time. From our perspective, the question of whether or not it is ‘‘whole molecule’’ global motion reflected by Tg or protein internal motion that is most relevant to stability of dried protein products remains an open question. Some opinions and
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observations suggest that both are important. The concept that pharmaceutical stability is dependent on the degree of coupling between protein internal motion and matrix motion22 is in itself an acknowledgement of the potential importance of both the matrix motion (i.e., of Tg and the global dynamics) and internal protein dynamics. The observation that proteins do not unfold in the ‘‘dry’’ state until the glass transition temperature is exceeded (see, for example Tab. 2) suggests that Tg is a relevant parameter for at least large-scale conformational change. Further, as noted in the introduction, Tg and global dynamics can be a useful, though imperfect, indicator of pharmaceutical stability (i.e., chemical degradation, protein aggregation, and crystallization from the amorphous state) for both small molecule and protein systems. However, recent data29,30 suggest better correlations between pharmaceutical stability and ‘‘Fast Dynamics’’ (i.e., dynamics measured on the nanosecond time scale by neutron scattering) in cases where stability is studied well below Tg. However, at least in most cases, these ‘‘Fast Dynamics’’ refer to the carbohydrate matrix, not the internal motion of the protein, and so we still seek direct evidence that protein internal dynamics are critical to pharmaceutical stability. Values of Tg of pure hGH, obtained from extrapolations such as illustrated by Figure 7, are given as a function of water content by Figure 8. Note that although Tg of a dry hGH sample is very high, well above 1008C, at a water content of about 12%, the Tg is 258C. Thus, for water contents in excess of 12%, a pure protein sample is above its Tg at room temperature and might be expected to exhibit characteristics of high mobility (i.e., structural collapse during storage and poor stability).
Origin of Nonreversible Thermal Events Below Tg All the disaccharide:hGH formulations studied exhibit sub-Tg thermal events under at least some conditions (Figs. 1–4). As discussed above, pure protein systems also exhibit irreversible DSC thermal events at moderate temperatures,5,28 and given that the system glass transitions in pure proteins occur well above 1008C, these irreversible endotherms occurring around 608C in pure solid proteins5,28 may also be classified as subTg thermal events. However, while we have suggested that the pre-Tg endotherms in the pure
Figure 8. Estimated glass transitions of pure protein as a function of water content. The glass transition temperatures were taken from extrapolations as illustrated in Figure 7, and the water contents corresponding to the given relative humidities were taken from the water sorption isotherm of pure hGH. The values of ‘‘% relative humidity, % water’’ are: 11% RH, 4.2%; 22% RH, 6.6%; 33% RH, 9.5%.
protein systems may be associated with internal motions in the protein molecule, it seems clear that the pre-Tg events displayed in the saccharide:protein systems have, for the most part, a different origin since the magnitude of these transitions is not proportional to the percentage of protein in the sample. Also, similar sub-Tg transitions are observed in disaccharide systems without proteins (unpublished observations). We note that sub-Tg endotherms can be a result of a broad distribution of relaxing states. Modeling the DSC thermogram using a KWW stretched exponential function to describe relaxation kinetics, with small beta values indicating a very broad distribution of relaxation times, and using the Tool-Narayanaswamy-Moynihan (TNM) equation to represent nonlinearity, one can reproduce sub-Tg endotherms.17 It has also been recently suggested that the pre-Tg endotherms arise from ‘‘b-relaxations’’.31 However, the apparent negative heat capacity shifts and the combination of exotherm and endotherm, as observed in the present work, apparently cannot be reproduced by the theoretical analysis described above. Sub-Tg thermal events similar to those illustrated in Figures 1–4 were observed a number of years ago in polymer systems prepared by freeze-drying
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from naphthalene.32 While an unambiguous explanation for the observations was not provided, it was suggested that the complex thermal events below Tg were likely a result of the unique type of glass created by freeze drying-an ‘‘open structure’’ with a very broad distribution of relaxation times.
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