A new mechanism for decreasing aggregation of recombinant human interferon‐γ by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20

A New Mechanism for Decreasing Aggregation of Recombinant Human Interferon-g by a Surfactant: Slowed Dissolution of Lyophilized Formulations in a Solution Containing 0.03% Polysorbate 20 SERENA D. WEBB,1 JEFFREY L. CLELAND,2 JOHN F. CARPENTER,3 THEODORE W. RANDOLPH1 1

Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Engineering Center, Room ECCH 111, Boulder, Colorado 80309-0424 2

Genentech, Inc., 460 Pt. San Bruno Boulevard, South San Francisco, California 94080

3

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received 16 March 2001; revised 19 July 2001; accepted 28 August 2001

ABSTRACT: To study the mechanisms by which Tween 20 (polysorbate 20) used in a reconstitution solution affects the aggregation of lyophilized recombinant human interferon-g (rhIFN-g), we used four types of buffered formulations containing 0.4± 5 mg/mL rhIFN-g in either 10 mM potassium phosphate or phosphate buffered saline: (1) without excipients, (2) with 5% sucrose, (3) with 0.03% polysorbate 20, or (4) with the combination of 5% sucrose and 0.03% polysorbate 20. After lyophilization, infrared spectroscopy was used to analyze the secondary structure of the protein in the freezedried solid. Each solid showed structural perturbation of the protein. Each formulation was reconstituted with water or a 0.03% polysorbate 20 solution. Aggregation of rhIFNg after reconstitution was measured by optical density at A350, and recovery of soluble protein was determined by high-performance liquid chromatography and ultraviolet spectroscopy. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. These results revealed the potential for recovery of native protein using the appropriate reconstitution conditions, even though the protein is non-native in the lyophilized state. Urea-induced unfolding with and without polysorbate 20 as measured by second-derivative ultraviolet spectroscopy indicated that a concentration of 0.03% polysorbate 20 lowered the free energy of unfolding for rhIFN-g (destabilizing). Polysorbate 20 also retarded refolding from urea solutions and increased aggregation. At a level of 0.03%, polysorbate 20 did not protect the protein against surface-induced aggregation during agitation. Dissolution times in water versus a 0.03% polysorbate 20 solution were measured using a rotating disk electrode for lyophilized formulations containing an electrochemically reactive species. The presence of 0.03% polysorbate 20 in the reconstitution solution nearly doubled the time required for dissolution of the phosphate buffered saline formulation, and the sucrose formulations dissolved 33±57% more slowly. Slowing the dissolution rates of lyophilized powders allows more time for

Correspondence to: Theodore W. Randolph (Telephone: 303492-4776; Fax: 303-492-4341; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 543±558 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

543

544

WEBB ET AL.

the protein to refold while it decreases the maximum concentration of the protein at the dissolution interface, thus reducing the total amount of aggregation. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:543±558, 2002

Keywords: rehydration; protein stabilization; surfactant; protein formulation; dissolution rates; dissolution model

INTRODUCTION Most native proteins are only marginally stable, with a free energy of stabilization (DGN ˆ > D ˆ Gnative Gdenatured) of only about 50  15 kJ/mol.1 A protein formulated in aqueous solution is particularly susceptible to conformational changes in its native structure, often leading to physical or chemical degradation. Therefore, proteins are commonly lyophilized to achieve long-term stability.2,3 Lyophilization produces rapidly dissolving powders, allows storage at higher temperatures, and reduces conformational mobility due to slowed molecular motion. The process of lyophilization often damages proteins. The addition of excipients and carefully designed lyophilization cycles are generally required to yield high recoveries of native protein after lyophilization. The majority of protein lyophilization research has been directed toward protecting the protein during the freezing and drying steps.3 Minimal effort has been directed toward the reconstitution step.4,5 Reconstitution occurs under conditions of temperature and pressure clearly different from reversing the path of the freeze dryer. Therefore, different methods for stabilizing the protein may be required. By adding excipients, such as amino acids, heparin, dextran sulfate, and surfactants to the reconstitution solutions for lyophilized proteins, Zhang et al.4,5 showed that recovered activities could be increased and levels of aggregate formation decreased compared with using water alone. However, the mechanism(s) by which these additives reduced protein aggregation during reconstitution was not clear. The purposes of the current study were to (a) determine whether the aggregation of rhIFN-g is decreased by the use of polysorbate 20 in a reconstitution medium; (b) identify the mechanism(s) by which polysorbate 20 affects the aggregation level; and (c) compare the effect of polysorbate 20 included in the lyophilization formulation versus its addition during reconstitution. The protein selected for this study, recombinant human interferon-g (rhIFN-g, has been shown to aggregate after acid denaturation,6 after attempted refolding,7±9 and above pH 5 during JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

thermal unfolding.7±12 It also aggregates during refolding from guanidine hydrochloride13 or urea.10 Phosphate buffer systems were selected because they cause signi®cant protein structural perturbation during lyophilization, resulting in aggregation upon reconstitution. Phosphate salts can crystallize during freezing,14 creating pH changes that could perturb rhIFN-g structure. Phosphate buffered saline (PBS) was used because of its propensity for large pH changes during freezing, whereas potassium phosphate buffer undergoes very little change. Sucrose was added in some of the formulations to partially stabilize the protein during lyophilization.3,15±18 Polysorbate 20 was selected speci®cally because its addition to a reconstitution medium had been shown to reduce aggregation of keratinocyte growth factor and interleukin-2 after reconstitution.4,5

MATERIALS AND METHODS Protein and Reagents Pharmaceutical-quality rhIFN-g expressed in Escherichia coli was produced and puri®ed at Genentech, Inc., South San Francisco, CA. Snakeskinß pleated dialysis tubing (7000 MWCO) and 10% polysorbate 20 (Tween 20 in Surfact-Ampsß 20) were purchased from Pierce, Rockford, IL. High-purity sucrose was purchased from Pfanstiel, Waukegan, IL. Sodium chloride, potassium chloride, sodium phosphate (dibasic), and potassium phosphate (monobasic and dibasic salts) were purchased from Fisher Scienti®c, Atlanta, GA. Potassium ferrocyanide trihydrate was purchased from Sigma, St. Louis, MO. The NonInterfering Protein Assayß was purchased from Bioworld Laboratory Essentials, Dublin, OH, and used according to the manufacturer's Protocol I instructions. All reagents were ACS reagent grade or higher quality. Millipore water was used in the preparation of solutions. Formulation solutions containing protein were ®ltered with low protein-binding ®lters (polyvinylidine di¯uoride) from Whatman, Clifton, NJ, with pore sizes of 0.22 mm. Buffer solutions were ®ltered using a 0.22 mm ®lter from Millipore, Bedford, MA.

DECREASING AGGREGATION OF rhIFN-g

Formulation Buffers and Filling Solutions Two buffers were used to prepare the formulations: 10 mM potassium phosphate, pH 7.5 or PBS containing 10 mM sodium phosphate (dibasic), 2 mM potassium phosphate (monobasic), 137 mM sodium chloride, and 3 mM potassium chloride, pH 7.0. rhIFN-g was dialyzed into each buffer, and vials were prepared with 0.4 mg/mL rhIFN-g in one of four buffered solutions: (1) no excipients, (2) 5% sucrose, (3) 0.03% polysorbate 20, or (4) the combination of 5% sucrose and 0.03% polysorbate 20. The concentration of polysorbate 20 was selected because it is signi®cantly above the critical micelle concentration (0.007%) in water. Protein concentrations of 0.4, 1, and 5 mg/mL rhIFN-g in each buffer only were also lyophilized. All concentrations identi®ed as percentages (i.e., sucrose and polysorbate 20) were prepared as w/v solutions. Lyophilization Procedure Lyophilization vials of 3 mL-volume (West Company no. 6800-0316, ¯int glass, Lionville, PA) were ®lled with 1 mL solution and loaded into an FTS Durastop freeze dryer, Stone Ridge, NY. Thermocouples were placed in two vials. Vials were loaded randomly into the freeze dryer and equilibrated at 18C for 30 min. The shelf temperature was decreased by 2.58C/min to 458C. When the temperature of the samples reached 308C, the shelf temperature was held at 458C for an additional 2 h. Primary drying was performed with a shelf temperature of 358C and a chamber pressure < 100 mTorr for approximately 40 h. The shelf temperature was increased during secondary drying by 18C/min and chamber pressure was increased to 200 mTorr. Shelf temperature was held at 20, 0, and 208C for 4 h each, and increased to 30 and 408C for 1 h each to complete the cycle. Moisture Determination Random lyophilized samples from formulations in both buffers were prepared in a dry-nitrogenpurged glove box and analyzed for moisture content using the Karl Fisher method.19 A Mettler DL37 coulometric moisture analyzer (Hightstown, NJ) was used with Hydranal reagents (Reidel de Haen, Seelze, Germany). Protein Secondary Structure by Infrared Spectroscopy Lyophilized samples containing 0.2 mg of rhIFN-g were mixed with 300 mg of potassium bromide

545

and pressed at 30 mPa into pellets for secondary structure analysis. Calcium ¯uoride windows separated by a 6 mm spacer were used for liquid samples. Spectra were collected at 258C with a Nicolet Magna-IRß 750 Series II spectrometer, equipped with a DTGS detector as described previously.20 For each spectrum, a 256-scan interferogram was collected in single beam mode, with a 4 cm 1 resolution using Omnicß (v. 2.1) software from Nicolet. The optical bench and sample chamber were continuously purged with dry air supplied from a Whatman model 75-52 IR purge gas generator (Haverhill, MA). Background liquid and gaseous water spectra were subtracted from the protein spectra according to previously established criteria.21 Second-derivative spectra were calculated using Nicolet Omnic software. The ®nal protein spectra were smoothed with a 7point function to remove white noise. All secondderivative spectra were baseline corrected using Galactic's GRAMS 386 software based on a previously described method,21 and were area normalized under the second-derivative amide I region, 1600±1700 cm 1.22 The native control sample consisted of 20 mg/mL rhIFN-g in 5 mM sodium succinate, pH 5.0. Reconstitution Procedure Reconstitution was performed at room temperature (238C). Stoppers were removed and samples were reconstituted in a random order with 1 mL of water or an aqueous 0.03% polysorbate 20 solution added within 2 s to the center of the lyophilized plug by a micropipet. Vials were placed on a Barnstead/Thermolyne Labquake red blood cell suspender and mixed at 8 rpm for 4 min. To verify that this mixing process does not cause aggregation, nonlyophilized controls for all four formulations in each buffer containing 0.4, 1, and 5 mg/ mL rhIFN-g were mixed under identical conditions. Absorbances were read at A350 to evaluate optical densities before and after the mixing process was completed. After 4 min of mixing, aggregates were not detected in any of the control samples. Optical Density and Protein Concentration Measurements Using Ultraviolet (UV) Spectroscopy A Perkin-Elmer Lambda 3B UV/VIS spectrophotometer was used to measure absorbances at 280 nm (A280) and 350 nm (A350). A280 was used to determine protein concentration, and A350/mg JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

546

WEBB ET AL.

protein was used to identify the presence of protein aggregates.23,24 A350 was recorded immediately upon reconstitution, and A280 was recorded after centrifugation to remove any insoluble aggregates. A350 was also recorded after centrifugation to verify the absence of signi®cant optical density. Protein concentration was calculated using 2 ˆ 0.75 mL mg 1cm 1 for rhIFN-g at 280 nm.25 Soluble Aggregate Detection and Protein Concentration Using High-Performance Liquid Chromatography±Size Exclusion Chromatography (HPLC±SEC) A Dionex DX500 chromatography system with a GP40 gradient pump, HP1050 series auto sampler and an AD20 absorbance detector at 214 nm was used with a Tosohaas TSK-GEL G2000SWXL stainless steel column. A 1.2 M KCl mobile phase was used at a ¯ow rate of 0.2 mL/min. The native dimeric form of rhIFN-g eluted at 41 min, and soluble aggregates appeared between 33 and 35 min. The column was calibrated with cytochrome C, carbonic anhydrase, albumen, and alcohol dehydrogenase. Native rhIFN-g controls, run at the beginning and end of each HPLC sample run, were used to calculate the concentration of protein in the samples using peak areas. Measurement of pH in Freeze-Concentrated Formulations pH in frozen solutions was measured using the method of Anchordoquy and Carpenter.26 An Ingold pH electrode containing a low-temperature electrolyte, Friscolyte ``B,'' was calibrated by measuring pH and conductivity (mV) at 25, 10, and 08C, using three standard buffer solutions. The pH electrode was placed into a 15-mL falcon tube containing each solution to be measured. Each tube was placed into an ethylene glycol bath maintained at 158C or below. A type T thermocouple was placed within 3 mm of the probe tip to monitor sample temperature. The pH was monitored until the value remained constant (at least 30 min after ice formation in the sample). Protein Unfolding in Urea Followed by Second-Derivative UV Spectroscopy A stock solution of urea in PBS, pH 7.0, was prepared as per the method of Pace et al.27 Solutions of 1 mg/mL rhIFN-g in urea with and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

without 0.03% polysorbate 20 were prepared, held overnight at 2±88C, and analyzed the next morning. Triplicate samples at each solution condition were analyzed. UV scans were measured from 310 to 250 nm with a scan rate of 15 nm per minute using a Perkin-Elmer Lambda 3B dual beam spectrophotometer. Data acquisition was made via a National Instruments (Austin, TX) model AT-MIO-16E-10 data acquisition board at a rate of 5 samples per second. National Instruments LabViewß software was used to control data acquisition and Microsoft Excelß to convert the wavelength and absorption data from volts to nanometers and absorbance units, respectively. Background spectra from samples containing all solution components except protein were subtracted for all samples. The second derivatives of the absorption spectra (d2A/dl2) were calculated in GRAMS/386 (v. 3.02) software (Galactic Industries) using the Savitzky-Golay method with a second-order polynomial smoothed over  2 nm. Unfolding of rhIFN-g was followed by change in extremum depth near 286 nm in the secondderivative absorption spectra.28 The native protein spectrum has a minimum near 286 nm re¯ective of the microenvironments of tryptophan and tyrosine residues.28±31 The depth of this minimum is reduced, eventually becoming a maximum as the concentration of urea is increased. The data are converted to the fraction of native protein (fN) as a function of urea by using a baseline correction for the pre- and post-transition regions as described by the method of Pace et al.27 The free energy of unfolding, DG, at each urea concentration was calculated as follows:
G ˆ

RT ln K

…1†

Temperature (T) is reported in Kelvin and R is the gas constant. The calculation for the equilibrium constant, K, assumes dissociation of the native dimer (N) into two monomers (D)28: N $ 2D K ˆ 4No ‰fN ‡ 1=fN

…2† 2Š

…3†

where No is the initial concentration of the native protein, and fN is the fraction of native protein. Note that because of the concentration term in the calculation for K, units are introduced. For the calculation of DG, a reference state of 1 M rhIFN-g at 238C was assumed,28 making K dimensionless. DG at 0 M urea was calculated by linear extrapolation of DG as a function of urea

DECREASING AGGREGATION OF rhIFN-g

concentration.27 Reported error for DG at 0 M urea is based on 95% con®dence limits on the linear extrapolation. Refolding of rhIFN-g in Urea One or three milligrams/milliliter rhIFN-g was equilibrated for 4 h at 238C in 3.5 or 5 M urea in PBS, pH 7.0. Solutions were rapidly diluted to a urea concentration of 1 M with PBS, pH 7.0, or polysorbate 20 in PBS, pH 7.0. The level of polysorbate 20 was adjusted such that the ®nal concentration upon dilution was 100 mM (0.012%). Optical densities of the solutions were recorded immediately at A350.23,24 Solutions were centrifuged and protein concentration in the supernatant was determined by the Non-Interfering Protein Assay. Agitation Procedure for Surface-Induced Aggregation Solutions of 1-mL sample size containing 1 or 5 mg rhIFN-g in PBS, pH 7.0, with and without polysorbate 20 (0.03 or 0.1%) were added to 1.7-mL Eppendorf tubes and rotated at 8 rpm and 238C for 0.25, 1, 1.5, 3, and 15 h. For some samples, PBS buffer was diluted to 1/15 its initial strength to determine ionic strength effects (from 152 to 10 mM). Protein aggregation was monitored by the ratio of A350 to protein concentration. Samples were centrifuged and protein recovery in the supernatant was analyzed using the Non-Interfering Protein Assay. Dissolution Rate Determination We determined dissolution times in water versus a 0.03% Tween 20 solution electrochemically using a rotating disk electrode at 238C. The method is brie¯y described herein, whileas the details may be found elsewhere.32 Potassium ferrocyanide was lyophilized with the formulations that had produced aggregation after reconstitution (all formulations except 10 mM potassium phosphate, 5% sucrose/0.03% Tween 20 in potassium phosphate and in PBS). Each lyophilized vial contained suf®cient potassium ferrocyanide to result in a concentration of 0.8 mM when reconstituted with 30 mL of solution. The platinum rotating disk electrode was submerged slightly below the surface of the reconstitution solution in a 50-mL beaker. A platinum counter electrode and Ag/AgCl/KCl (saturated) reference

547

electrode were af®xed together near the inside edge of the beaker. An Analytical Rotator (model AFASRP) from the Pine Instrument Company was used to control the rotating disk speed. A Pine Instrument Company Bi-Potentiostat (model AFRDE5) was used to apply a constant potential (600 mV) while a lyophilized sample was added to the reconstitution solution. The solution also contained a background electrolyte, 0.15 M NaCl, which was necessary to prevent the migration of ionic species in a ®eld and therefore enable measurement of diffusive processes. Data acquisition was made using a National Instruments (Austin, TX) model AT-MIO-16E-10 data acquisition board at a rate of 50 samples per second, and via a Kipp and Zonen X-Y Recorder (type BD91) concurrently. National Instruments LabView software was used to control the electronic data acquisition. Rates of dissolution were measured in triplicate. As the lyophilized material dissolves, the ferrocyanide becomes solvated and begins to undergo the following oxidation reaction: Fe…CN†46

Pt

! Fe…CN†36 ‡ e

NaCl

…4†

The Pt concentration of ferrocyanide in solution is directly proportional to the current, and both increase until dissolution is complete. Dissolution pro®les were found to ®t a ®rst order response, and a ®rst order time constant (t) was calculated for each sample type.

RESULTS Lyophilization and Reconstitution of rhIFN-g in Phosphate Buffers Characterization of the Lyophilized Formulations Residual moisture in lyophilized protein formulations may impact the overall stability as well as the reconstitution process. The water content remaining in the samples after lyophilization ranged from 0.23 to 0.77  0.1% (two standard deviations). Random samples were tested from both the potassium phosphate and PBS buffer formulations, and vials containing 0.4±5 mg rhIFN-g were included. No apparent differences in water content were obvious between sample types, and therefore differences in formulations cannot be attributed to variations in water content in the lyophilized samples. Previous studies have shown that phosphate buffers may induce signi®cant damage to proteins during freeze-drying as the result of pH changes JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

548

WEBB ET AL.

upon freezing.33±36 To assess this effect on rhIFNg, the secondary structure of the protein in the lyophilized formulations was characterized by infrared (IR) spectroscopy. The second-derivative IR spectra of the amide I region for lyophilized 0.4 mg/mL rhIFN-g in 10 mM potassium phosphate, pH 7.5, are shown in Figure 1A. Bands in the second-derivative IR spectrum of native rhIFN-g have been assigned previously.6 The band at 1682 cm 1 is a combination of two bands at 1677 and 1684 cm 1. The band at 1677 cm 1 is assigned to extended chain, whereas the band at 1684 cm 1 is assigned to turn structure. The band appearing at 1633 cm 1 is also composed of two bands at 1630 and 1635 cm 1, both of which are

Figure 1. (A) Second-derivative IR spectra of 0.4 mg/ mL rhIFN-g lyophilized in 10 mM potassium phosphate, pH 7.5. Formulations: 10 mM potassium phosphate (- - - - - -); 5% sucrose (± ± ±); 0.03% polysorbate 20 (Ð Ð); 5% sucrose and 0.03% polysorbate 20 (- - -); native control (Ð Ð, bold). (B) Second-derivative IR spectra of 0.4±5 mg/mL rhIFN-g lyophilized in 10 mM potassium phosphate, pH 7.5. Symbols: 0.4 mg/ mL (Ð Ð); 1mg/mL (± ± ±); 5 mg/mL (- - - - - -); native control (- - -, bold). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

assigned to extended chain. Finally, the dominant band at 1656 cm 1 corresponds to a-helix. Less-intense a-helix bands were seen in sucrose-free formulations with and without polysorbate 20. These losses were compensated for by an increase in turn structure (at 1684 cm 1), and formation of b-sheet structure at 1695 cm 1,37,38 particularly in the excipient-free formulation. The general band broadening of the amide I region in these same formulations is indicative of heterogeneity in the secondary structure of rhIFN-g after lyophilization.6,38,39 When sucrose is present in the 10-mM potassium phosphate formulations, native bands are retained to a greater extent, though there is still substantial perturbation of secondary structure in the dried solid. The IR spectra for 0.4±5 mg/mL rhIFN-g lyophilized in 10 mM potassium phosphate, pH 7.5, are shown in Figure 1B. All samples show signi®cant perturbation of native protein structure, as indicated by the dramatic reduction in helix band intensity in the IR spectra. As protein concentration increases, this perturbation increases slightly but remains similar (within error) among the different concentrations. The second-derivative IR spectra of the amide I region for 0.4 mg/mL rhIFN-g lyophilized in PBS, pH 7.0, are shown in Figure 2A. In contrast to results shown in Figure 1A for samples lyophilized in 10 mM potassium phosphate, sucrosecontaining formulations in PBS exhibit little to no improvement in retention of native a-helix structure at 1656 cm 1, although they do retain more extended chain structure at 1677 and 1635 cm 1. The IR spectra for 0.4±5 mg/mL rhIFN-g in PBS, pH 7.0, are shown in Figure 2B. Clearly, the secondary structure after lyophilization is not dependent on protein concentration. Overall, the samples lyophilized in PBS (Fig. 2B) retained more a-helical content than the samples lyophilized in 10 mM potassium phosphate (Fig. 1B). To determine whether damage during lyophilization was possible due to pH changes occurring during freezing, we measured apparent pH values reached during freezing of the formulations. The change from the initial pH 7.5 in potassium phosphate was greatest when polysorbate 20 was present, in which it increased to 8.24 (see Table 1). The presence of sucrose caused only a slight decrease in pH in the phosphate buffer. Overall, the changes in pH were small in the potassium phosphate buffer as compared with PBS. In PBS, the largest change in pH during freezing, a decrease of nearly 3 pH units, again occurred in

DECREASING AGGREGATION OF rhIFN-g

549

Figure 3. Second-derivative IR spectra of rhIFN-g at the apparent pH of freeze concentration in potassium phosphate and PBS buffers. Legend: native control (Ð Ð, bold); 10 mM potassium phosphate, pH 7.5 (Ð Ð); potassium phosphate, pH 8.2 (± ± ±); PBS, pH 7.0 (- - Ð); PBS, pH 5.2 (- - - - - -).

Figure 2. (A) Second-derivative IR spectra of 0.4 mg/ mL rhIFN-g lyophilized in PBS, pH 7. Formulations: PBS, pH 7 (- - - - - -); 5% sucrose in buffer (± ± ±); 0.03% polysorbate 20 in buffer (Ð Ð); 5% sucrose and 0.03% polysorbate 20 in buffer (- - - Ð); native control (Ð Ð, bold). (B) Second-derivative IR spectra of 0.4±5 mg/mL rhIFN-g lyophilized in PBS, pH 7.0. Symbols: 0.4 mg/ mL (Ð Ð); 1mg/mL (± ± ±); 5 mg/mL (- - - - - -); native control (Ð Ð, bold).

the presence of polysorbate 20. In contrast, in buffer alone, the pH decreased about 2 units. To assess potential damage to the protein induced by pH changes of the buffers during freezing, the pH of solutions of rhIFN-g in each buffer alone were adjusted to those measured in the freeze-concentration experiment. The secondderivative IR spectra of rhIFN-g in these solutions are shown in Figure 3. Alkalinization of potassium phosphate buffer from pH 7.5 to 8.2 caused a slight reduction in the intensity of the a-helix band at 1656 cm 1. In contrast, acidi®cation of PBS from pH 7.0 to 5.2 caused a dramatic reduction in the intensity of the a-helix band concomitant with appearance of new bands at 1620 cm 1 and 1692 cm 1. These changes were unexpected because the protein is stable in 5 mM sodium succinate, pH 5.0, and maintains native

Table 1. Determination of Apparent Frozen pH for Both Potassium Phosphate and PBS Buffer Formulations Formulation No excipients 5% Surcrose 0.03% Polysorbate 20 5% Sucrose/0.03% Polysorbate 20

10 mM Potassium Phosphate, pH 7.5 8.17 7.23 8.24 7.18

PBS, pH 7.0 5.21 5.70 4.33 5.77

Based on calibration of pH standards 4, 7, and 10 at 0, 10, and 258C, the error in measurement was < 0.1% for 95% con®dence. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

550

WEBB ET AL.

structure under these conditions.25,40 It is likely that the combination of high salt (152 mM) and low pH disrupt the helix dipoles resulting in protein denaturation. These bands at 1620 cm 1 and 1692 cm 1 are indicative of intermolecular b-sheet associated with formation of protein aggregates,37,38 and have been used to monitor aggregation of rhIFN-g in the liquid state.6 Reconstitution of the Lyophilized Formulations Formulations lyophilized in potassium phosphate and reconstituted in water showed either very little or no aggregation (Table 2). In samples in which aggregates were detected, inclusion of 0.03% polysorbate 20 in the reconstitution solution reduced aggregate levels. An exception occurred when rhIFN-g was lyophilized in the presence of 0.03% polysorbate 20. In this case, aggregation levels were unaffected by the further addition of polysorbate 20 to the reconstitution medium. When lyophilized samples of rhIFN-g were reconstituted with water alone, aggregates were present in all PBS formulations except the formulation containing both sucrose and polysorbate 20, based on A350 and SEC results (Table 2). The greatest levels of aggregation were noted in samples lyophilized in PBS alone. The addition of sucrose to the PBS formulation reduced but did not eliminate aggregation upon reconstitution. In contrast, PBS formulations reconstituted with 0.03% polysorbate 20 showed statistically insigni®cant levels of aggregation by protein recovery from SEC, and reduced light scattering at 350 nm. Mechanism of Stabilization by Polysorbate 20 To determine the mechanism of stabilization by polysorbate 20, we investigated several possibilities. Polysorbate 20 may stabilize the native state of the protein by increasing its free energy of unfolding, or it may facilitate refolding to the native state. Alternatively, the surfactant may inhibit surface-induced denaturation during reconstitution. Another potential mechanism is the in¯uence of polysorbate 20 on the rate of reconstitution of the lyophilized protein. Each of these mechanisms was assessed in detail. Unfolding rhIFN-c in Urea The urea-unfolding curve for rhIFN-g was shifted to lower concentrations of urea in the presence of polysorbate 20 (Fig. 4). In both cases, the curves JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

showed sigmoidal, highly cooperative transitions typical of two-state processes. No precipitation of the protein occurred. A two-state, equilibrium model was assumed for thermodynamic calculations. For the unfolding of rhIFN-g in PBS, DG ˆ 60.6  2.0 kJ/mol was calculated versus 53.5  3.2 kJ/mol in the presence of polysorbate 20. These results suggested that polysorbate 20 destabilized the protein under these conditions. Refolding rhIFN-c After Rapid Dilutions From Urea Solutions rhIFN-g was equilibrated in solutions of urea at concentrations of 3.5 and 5 M. These chaotrope concentrations correspond to fn & 0.5 and fn & 0, respectively, as shown by the unfolding curve in Figure 4. During rapid dilutions to 1 M urea, the introduction of polysorbate 20 increased A350/mg and decreased soluble protein remaining after the dilutions were completed (see Table 3). Apparently, polysorbate 20 did not facilitate refolding and instead fostered aggregation during refolding. In addition, higher concentrations of protein resulted in larger losses because of aggregation (Table 3). Surface-Induced Denaturation of rhIFN-c Resulting From Mild Agitation We hypothesized that polysorbate 20 might inhibit rhIFN-g aggregation during reconstitution by competing with protein for access to air± water interfaces created during injection of reconstitution solution into vials. To test whether polysorbate 20 is effective at reducing potential denaturation at air/water interfaces, we exposed rhIFN-g to air±water interfaces by agitating solutions of rhIFN-g for 15 h. The percent of soluble protein recovered after agitation was invariant at both protein concentrations tested, in the presence or absence of 0.03% polysorbate 20, and in solutions containing PBS or 15-fold diluted PBS (Fig. 5, inset). However, the inclusion of 0.1% polysorbate 20 yielded full recovery of the initial starting concentration of rhIFN-g after 15 h of agitation. After 15 h of agitation, A350/mg protein was higher in solutions containing 0.03% polysorbate 20 than in solutions without polysorbate 20 (Fig. 5). A350/mg protein was lower in solutions with lower ionic strength, i.e., 15-fold diluted PBS (Fig. 5), as well as in the solution containing 0.1% polysorbate 20. Therefore, the polysorbate 20 concentration used in the reconstitution studies (0.03%) did not stabilize the

0.4 0.4 0.4 0.4 0.4 0.4

B

A

B

A

B

5

1

0.4

5

1

0.4

[rhIFN-g] in Sample (mg/mL)

A

B

A

Buffer

0.325 0.278 0.260 0.085 0.025 0.023 0.018 0.015 20

20

20

20

Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate

0.033 0.038 0.058 0.043 0.017 0.019 0.525 0.175 0.184 0.041 0.109 0.058 0.063 0.020 0.038 0.013

20

20

20

20

20

20

A350/mg

Water 0.03% Polysorbate 20 Water 0.03% Polysorbate 20

Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate Water 0.03% Polysorbate

Reconstitution Solution

0.018 0.140 0.153 0.098 0.028 0.030 0.005 0.005

0.025 0.025 0.010 0.013

0.008 0.025 0.013 0.029 0.004 0.013 0.038 0.058 0.013 0.013 0.008 0.019



95.6 95.7 99.4 100 96.9 99.2 97.0 98.1

95.2 99.0 96.3 100

93.1 99.7 98.0 99.7 98.7 98.8 91.5 100 93.0 100 85.6 97.1

% recovereda

6.6 4.1 3.0 4.9 6.2 1.0 4.1 1.6

7.9 2.9 1.7 6.0

6.0 6.0 1.8 1.8 1.7 1.7 5.1 2.3 5.1 3.1 5.1 5.1



98.6 98.3 Ð Ð 99.8 99.0 Ð Ð

100 99.6 Ð Ð

99.9 99.9 99.2 99.3 100 99.9 100 99.9 99.7 100 99.9 99.8

% Dimer

1.4 1.7 Ð Ð 0.2 1.0 Ð Ð

0.0 0.4 Ð Ð

0.1 0.1 0.8 0.7 0.0 0.1 0.0 0.1 0.3 0.0 0.1 0.2

% Soluble Aggregates

Total rhIFN-g eluted

a Buffer types: A ˆ 10 mM potassium phosphate, pH 7.5, and B ˆ PBS, pH 7.0. For samples without SEC results, protein recoveries are calculated from A280 measurements recorded after centrifugation. Remaining recoveries are based on SEC results. Errors are  2 standard deviations.

5% Sucrose and 0.03% Polysorbate 20

0.03% Polysorbate 20

5% Sucrose

No excipients

Formulation

Table 2. Optical Density, Protein Recovery, and SEC Results

DECREASING AGGREGATION OF rhIFN-g

551

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

552

WEBB ET AL.

stituted in a 0.03% polysorbate 20 solution required nearly double the time for dissolution than when water was used to reconstitute (see Table 4). A similar trend was seen for dissolution of the 5% sucrose formulations, which required 33±57% more time when polysorbate 20 was present in the reconstitution solution. Dissolution of the 0.03% polysorbate 20 formulations required about half the time to dissolve in the 0.03% polysorbate 20 solution as compared with dissolution in water.

DISCUSSION Figure 4. The unfolding and dissociation by secondderivative UV spectroscopy for 1 mg/mL rhIFN-g in urea. The unfolding transition is shifted to lower concentrations of urea in the presence of polysorbate 20. Key: (^) PBS, pH 7.0 and (&) 0.03% polysorbate 20 in PBS, pH 7.0.

protein against denaturation at air±liquid interfaces. Dissolution Rates for Lyophilized Powders The total apparent dissolution time (5 t; 99.3% complete) of each lyophilized sample, tdis, is reported in Table 4. The PBS formulation recon-

It is well known that the lyophilization process often denatures proteins. This can be the result of many factors, including the concentration of solutes during freezing, pH changes due to salt crystallization, introduction of new ice/water and solid/air interfaces, the imposition of mechanical stresses due to shrinking of the cake during drying, and dehydration itself. Not surprisingly, the secondary structure of rhIFN-g was perturbed after lyophilization in all formulations tested. The changes observed in the amide I IR spectra after lyophilization were not uniform among the formulations, indicating that the ``starting points'' for the protein population during reconstitution differed in terms of native protein content. Further, the degree of lyophilization-induced

Table 3. Refolding of rhIFN-g From Concentrated Urea Solutions Post-Dilution [Protein] mg/mL

A350/mg



mg/mL



% Soluble Protein ( 5%)

in

3 3

0.6 0.6

3.667 4.133

1.083 1.233

0.15 0.11

0.01 0.01

25% 18%

in

1 1

0.2 0.2

0.920 3.825

0.270 1.135

0.10 0.07

0.01 0.003

50% 35%

in

3 3

0.86 0.86

1.477 1.907

0.442 0.570

0.70 0.64

0.04 0.03

81% 74%

in

1 1

0.29 0.29

0.021 0.676

0.007 0.200

0.26 0.23

0.01 0.01

90% 79%

Diluent A PBS, pH 7 Polysorbate 20 PBS, pH 7 PBS, pH 7 Polysorbate 20 PBS, pH 7 B PBS, pH 7 Polysorbate 20 PBS, pH 7 PBS, pH 7 Polysorbate 20 PBS, pH 7

Post-Dilution Soluble [Protein]

Initial [Protein] mg/mL

Post-Dilution O.D.

The refolding of rhIFN-g in urea and PBS, pH 7.0, at 238C rapidly diluted with: (1) PBS, pH 7.0, or (2) a polysorbate 20 solution to make a ®nal concentration of 100 mM (0.012%) polysorbate 20 after dilution. A: Urea dilution from 5 M to 1 M urea, and (B) 3.5 M to 1 M urea. Errors are  2 standard deviations. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

DECREASING AGGREGATION OF rhIFN-g

553

Figure 5. Effect of polysorbate 20 on the optical density at 350 nm of agitated rhIFN-g solutions in PBS, pH 7.0, adjusted by protein concentration. Samples were rotated at 8 rpm at 238C. Error bars re¯ect  2 standard deviations. Legend: 1 mg/mL rhIFN-g (^); 1 mg/mL rhIFN-g in 0.03% polysorbate 20 (&); 5 mg/mL rhIFN-g (~); 5 mg/mL rhIFN-g in

0.03% polysorbate 20 (); 5 mg/mL rhIFN-g in 0.1% polysorbate 20 (Ð); 1 mg/mL rhIFN-g in 1/15 PBS, pH 7.0 ( ); and 1 mg/mL rhIFN-g in 0.03% and 1/15 PBS, pH 7.0 (*). Recovery of protein refers to soluble protein remaining after solutions were centrifuged, as determined by Non-Interfering Protein Assay. Error re¯ects  2 standard deviations.

unfolding also did not always correlate to the degree of aggregation after reconstitution. For example, the most non-native protein structure overall was observed in the 10 mM potassium phosphate formulation which yielded nearly complete recovery of soluble protein upon reconstitution. Perturbations in the secondary structure of rhIFN-g observed in the dried solid observed by IR spectroscopy could not be explained by pH changes due to freezing. It has been shown

previously that rhIFN-g undergoes aggregation in buffers undergoing acidi®cation during lyophilization.40 rhIFN-g dissociates into monomers at low pH,7,12 and the monomeric species has been linked to aggregation of the protein.28,41,42 However, the magnitudes of the pH changes in this study did not correlate to aggregation levels after reconstitution. In fact, the commercial product is formulated at pH 5.0, though a buffer with low ionic strength (5 mM sodium succinate) is used.

Table 4. Dissolution Rate Results Dissolution in Water ÐÐÐÐÐÐÐ tdis 

Formulation PBS 5% Sucrose in PBS 5% Sucrose in 10 mM potassium phosphate 0.03% Tween 20 in PBS 0.03% Tween 20 in 10 mM potassium phosphate

1.2 1.5 1.4 3.4 3.2

0.15 0.31 0.25 0.61 0.89

0.03% Tween 20 ÐÐÐÐÐÐÐÐ tdis  2.3 2.0 2.2 1.6 1.7

0.66 0.15 0.50 0.44 0.44

Dissolution rates found by the rotating disk electrode method. Error represents  2 standard deviations. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

554

WEBB ET AL.

The presence of polysorbate 20 in the reconstitution solution decreased the total amount of aggregated rhIFN-g after reconstitution of formulations that did not contain polysorbate 20 in the lyophilized cake. We investigated several possible mechanisms that might explain this effect. Assuming that non-native IR spectra were indicative of the presence of a substantial fraction of unfolded protein molecules, then during rehydration there may be a kinetic competition between aggregation and refolding.43 Refolding of proteins may sometimes be assisted by surfactants.44±47 However, when solutions of unfolded or partially unfolded rhIFN-g in urea were diluted to foster refolding, the presence of polysorbate 20 actually increased aggregation. Therefore, polysorbate 20 probably does not reduce aggregation during reconstitution by enhancing refolding. Polysorbate 40 has been observed to bind to the native structure of rhIFN-g48 and thus, it might have been plausible that polysorbate 20 could thermodynamically stabilize the native conformation of rhIFN-g. However, the free energy necessary to unfold rhIFN-g actually decreased by about 7 kJ/mol in the presence of 0.03% polysorbate 20. Therefore, polysorbate 20-induced thermodynamic stabilization of rhIFN-g does not occur and was not the mechanism for the decreased aggregation during reconstitution. Polysorbate 20 must therefore bind more to nonnative forms of rhIFN-g than to the native form.49 Surfactants may also protect proteins from surface-induced denaturation.50±54 Upon the addition of a reconstitution medium to a lyophilized powder, a large number of small air bubbles may be formed. To determine whether polysorbate 20 protects rhIFN-g against denaturation at these air±water interfaces, we agitated polysorbate 20 solutions of rhIFN-g. Surprisingly, 0.03% polysorbate 20 failed to increase the recovery of rhIFN-g. Thus, prevention of denaturation at air± liquid interfaces was not the mechanism for increased recovery of native protein upon reconstitution with 0.03% polysorbate 20. However, in agitation studies, 0.1% polysorbate 20 was effective at preventing surface-induced denaturation of rhIFN-g. Higher concentrations of Tween 20 ( >> 0.03%) would be attained upon freeze concentration of formulations containing the surfactant. During dissolution of these formulations, the higher initial concentration of Tween 20 may attenuate surface-induced denaturation of rhIFN-g. Polysorbate 20, 0.03%, provides inadequate protection to stabilize rhIFN-g by preventing JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

surface-induced damage, by thermodynamically stabilizing the protein's conformation, or by catalyzing refolding. However, formulations that displayed decreased aggregation levels after reconstitution with polysorbate 20 also dissolved more slowly in the presence of the surfactant. Because polysorbate 20 is surface active, equilibrium concentrations at a solid±liquid interface should be in excess. As a general rule, adsorption onto polar solid surfaces occurs with the surfactant's polar groups in close proximity to the solid and the nonpolar groups oriented toward the aqueous solution.55 This creates a hydrophobic barrier that impedes wetting and dissolution. Why might the slower dissolution rates decrease protein aggregation? As the powder begins to wet and dissolve, protein molecules, many of which are at least partially unfolded, will be found at very high concentration in a stagnant thin ®lm of solution of thickness d near the solid/ water interface. Convection and diffusion serve to reduce this concentration by transporting protein away from the stagnant layer. If dissolution occurs quickly, there will be more protein molecules (as well as other dissolving solutes) at the interface than if the powder were to dissolve more slowly. Assuming a pseudo-steady-state dissolution rate, the rate at which protein molecules enter the stagnant ®lm at the interface is equal to the rate at which it is transported out of the ®lm to the bulk by diffusion. This in turn requires a higher concentration gradient for diffusion across the stagnant ®lm for higher dissolution rates, and a concomitantly higher protein concentration at the solid±liquid interface (see Appendix). As the powder dissolves, it releases partially or completely unfolded protein. This protein may refold into the native conformation, or aggregate irreversibly. The experiments wherein we monitored rhIFN-g aggregation after dilution from urea solutions suggest that, at a given rhIFN-g concentration, the presence of polysorbate 20 does not favor refolding over aggregation. Protein aggregation kinetics are usually second order or higher.11,56±58 For purposes of discussion, we assume that the aggregation kinetics are second order in the concentration of structurally perturbed protein: r ˆ d‰AŠ=dt ˆ k‰CA Š2

…5†

where r is the rate of aggregation, [A] is the concentration of aggregated protein, t is time, k is the rate constant, and [CA] is the concentration of

DECREASING AGGREGATION OF rhIFN-g

aggregation-competent protein within the stagnant thin ®lm surrounding a dissolving particle. [CA] varies linearly across the boundary layer from [CA,max]at the interface to approximately 0 at the outer edge of the layer (z ˆ d). [CA(z)] is constant with respect to time during the pseudosteady-state dissolution of a sample, but will increase with increasing dissolution rates. Integration of the rate of aggregation across the boundary layer and over the total time of dissolution (tD) gives the total amount of aggregated protein Atot in the stagnant thin ®lm: ZtD Z  Atot ˆ Np p 0

ZtD ˆ N p p

k‰CA Š2 dzdt

0

k‰CA;max Š2 =3dt

…6†

0

ˆ Np p k‰CA;max Š2 tD =3 where Np represents the number of particles dissolving, and sp is the surface area of each particle. [CA,max] is proportional to the dissolution rate, dr/dt (see Appendix), so the total amount of aggregate, Atot, is then inversely proportional to the dissolution time, tD. Therefore, a slower dissolution rate results in less total aggregation. Further, recovery of native protein is decreased in PBS buffer relative to 10 mM potassium phosphate buffer (Table 2), suggesting that the rate constant for rhIFN-g aggregation, k, may increase with ionic strength, perhaps because of charge shielding at higher ionic strengths.8 High ionic strength has been shown to promote aggregation of rhIFN-gin aqueous solution.8,59 More rapid dissolution of powders containing protein and salts not only results in higher concentrations of protein at the solid±water interface, but also in concomitantly higher ionic strength, thus potentially further augmenting the aggregation rate through increases in k. An exception to the relationship between dissolution rate and protein aggregation occurs when polysorbate 20 formulations are reconstituted. In this case, reconstitution with polysorbate 20 solutions results in faster dissolution rates versus reconstitution with water alone, yet there is no increase in protein aggregation. Perhaps the higher ®nal concentration of polysorbate 20 (0.06% overall) and the even higher concentration of polysorbate 20 in the stagnant boundary layer during dissolution are suf®cient

555

to prevent protein aggregation, as was seen when higher (0.1%) concentrations of polysorbate 20 were used in the agitation studies. One ®nal observation was that the use of A350 to determine the extent of aggregation present in samples is inappropriate. Caution must be used when interpreting A350 measurements, because although the data do re¯ect the concentration (``optical density'') of scattering species, the measurement is very sensitive to the sample's particle-size distribution. From Table 2, it is evident that during reconstitution, the presence of polysorbate 20 in the reconstitution solution often decreased A350 without affecting the amount of soluble protein recovered, indicating a shift in the particle-size distribution toward smaller aggregates. When rhIFN-g was agitated in high ionic strength and/or in the presence of 0.03% polysorbate 20, the increase in A350 suggests that larger aggregates were formed, because protein recoveries were statistically similar for all samples (Fig. 5). Therefore, A350 measurements can be used with con®dence only as a qualitative measure for the presence or absence of scattering species.

CONCLUSIONS Although non-ionic surfactants are nearly ubiquitous in protein formulations, both the effect of surfactant addition is unpredictable and the mechanism(s) by which these effects occur is poorly understood. In the current study, none of the commonly cited mechanisms for surfactantinduced inhibition of protein aggregation was suf®cient to explain the reduction in aggregation seen when polysorbate 20 was added to reconstitution media. In this case, the indirect effects of surfactant on the dissolution rate of lyophilized powders appear to dominate the observed recoveries of native protein.

ACKNOWLEDGMENTS We gratefully acknowledge Genentech, Inc., for providing rhIFN-g for these studies. Financial support was provided by NSF-BES9816975 and predoctoral fellowships for SDW from the National Institutes of Health and the National Science Foundation. We thank Drs. Catherine Randolph, Carl Koval, and Heather Shafer for their ideas and assistance regarding the electrochemistry/dissolution experiments. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

556

WEBB ET AL.

APPENDIX The goals of this appendix were to describe the application of the thin ®lm model to the dissolution process, and to outline the representative equations that explain the importance of the dissolution rates for the lyophilized powders. Dissolution of the lyophilized powder can be characterized in terms of a mass balance between dissolution at the solid/liquid interface and the subsequent mass transfer of the solutes into the bulk solution. A stagnant thin ®lm of thickness d is the simplest model of an interfacial region.60 Because the ®lm will be very thin in a wellmixed system such as that seen when reconstitution medium is rapidly injected into a vial containing powder, mass transfer can be approximated as one-dimensional across the ®lm. We assume a pseudo-steady-state ¯ux of material across the thin ®lm. This results in a linear concentration pro®le for a species in the thin ®lm: d2 ‰CŠ=dz2 ˆ 0

…A1†

where z varies from 0 to d across the ®lm thickness, and [C] represents the concentration of a species in the ®lm, e.g., potassium ferrocyanide or rhIFN-g. Flux of a species across the thin ®lm can be represented by: D  A  d‰CŠ=dz ˆ fd=dt…V†g  ‰Csolid Š

…A2†

where D is the diffusion coef®cient, A is a representative cross-sectional area of the thin ®lm, and V is the volume of material in the solid, decreasing over time during dissolution. The concentration of the species in the solid, [Csolid], is a constant. For simpli®cation, we assume a spherical shape for our lyophilized particles, allowing us to transform eq. A2 to: d‰CŠ=dz ˆ d=dt…4=3    r3 †g  ‰Csolid Š=… D  4    r2 †

…A3†

Taking the derivative of the volume term and simplifying: d‰CŠ=dz ˆ

dr=dt  ‰Csolid Š=D

…A4†

At z ˆ d, [C] ˆ [Cbulk] and the following expression for concentrations within the ®lm results after integration: ‰CŠ ˆ

…dr=dt†  ‰Csolid Š  …

z†=D ‡ ‰Cbulk Š …A5†

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

Evaluation of eq. A5 at z ˆ 0 gives the maximum concentration, [Cmax], which is directly proportional to the dissolution rate, dr/dt.

REFERENCES 1. Jaenicke R. 1991. Protein folding: Local structures, domains, subunits, and assemblies. Biochemistry 30:3147±3161. 2. Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF. 1993. Factors affecting short-term and longterm stabilities of proteins. Adv Drug Deliv Rev 10:1±28. 3. Carpenter JF, Pikal MJ, Chang BS, Randolph TW. 1997. Rational design of stable lyophilized protein formulations: Some practical advice. Pharm Res 14:969±975. 4. Zhang MZ, Wen J, Arakawa T, Prestrelski SJ. 1995. A new strategy for enhancing the stability of lyophilized protein: The effect of the reconstitution medium on keratinocyte growth-factor. Pharm Res 12:1447±1452. 5. Zhang MZ, Pikal K, Nguyen T, Arakawa T, Prestrelski SJ. 1996. The effect of the reconstitution medium on aggregation of lyophilized recombinant interleukin-2 and ribonuclease A. Pharm Res 13:643±646. 6. 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. 7. Arakawa T, Hsu YR, Yphantis DA. 1987. Acid unfolding and self-association of recombinant Escherichia coli derived human interferon-gamma. Biochemistry 26:5428±5432. 8. Hsu YR, Arakawa T. 1985. Structural studies on acid unfolding and refolding of recombinant human interferon-gamma. Biochemistry 24:7959±7963. 9. Arakawa T, Hsu YR, Narachi MA, Herrera C, Rohde MF, Hennigan P. 1990. Reversibility of acid denaturation of recombinant interferon-gamma. Biopolymers 29:1065±1068. 10. Arakawa T, Alton NK, Hsu YR. 1985. Preparation and characterization of recombinant DNA-derived human interferon-gamma. J Biol Chem 260:4435± 4439. 11. Mulkerrin MG, Wetzel R. 1989. pH dependence of the reversible and irreversible thermal denaturation of gamma interferons. Biochemistry 28:6556± 6561. 12. Yphantis DA, Arakawa T. 1987. Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma. Biochemistry 26:5422±5427. 13. Cleland JL, Builder SE, Swartz JR, Winkler M, Chang JY, Wang DIC. 1992. Polyethylene glycol

DECREASING AGGREGATION OF rhIFN-g

14. 15.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25. 26.

enhanced protein refolding. Biotechnology 10: 1013±1019. van den Berg L. 1966. pH changes in buffers and foods during freezing and subsequent storage. Cryobiology 3:236±242. Remmele RL, Stushnoff C, Carpenter JF. 1997. Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: Dehydration stress in the presence of sucrose. Pharm Res 14:1548±1555. Pikal MJ. 1994. Freeze-drying of proteins: Process, formulation, and stability. In: Cleland JL, Langer R, editors. Formulation and delivery of proteins and peptides; Washington DC: American Chemical Society, pp 120±133. Carpenter JF, Chang BS. 1996. Lyophilization of protein pharmaceuticals. In: Avis KE, editor. Biotechnology and biopharmaceutical manufacturing, processing, and preservation; Buffalo Grove, IL: Interpharm Press, pp 199±264. Prestrelski SJ, Tedeschi N, Arakawa T, Carpenter JF. 1993. Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. Biophys J 65:661±671. May JC, Grim E, Wheeler RM, West J. 1982. Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer gravimetric and thermogravimetric methodologies. J Biol Stand 10: 249±259. Dong A, Matsuura J, Allison SD, Chrisman E, Manning MC, Carpenter JF. 1996. Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B. Biochemistry 35:1450±1457. Dong AC, Caughey WS. 1994. Infrared methods for study of hemoglobin reactions and structures. In: Everse J, Vandegriff KD, Winslow RM, editors. Hemoglobins part C; San Diego: Academic Press, pp 139±175. Kendrick BS, Dong AC, Allison SD, Manning MC, Carpenter JF. 1996. Quantitation of the area of overlap between second-derivative amide I infrared spectra to determine the structural similarity of a protein in different states. J Pharm Sci 85:155± 158. DeFelippis MR, Alter LA, Pekar AH, Havel HA, Brems DN. 1993. Evidence for a self-associating equilibrium intermediate during folding of human growth hormone. Biochemistry 32:1555± 1562. Eckhardt BM, Oeswein JQ, Bewley TA. 1991. Effect of freezing on aggregation of human growth hormone. Pharm Res 8:1360±1364. Lam XM, Patapoff TW, Nguyen TH. 1997. The effect of benzyl alcohol on recombinant human interferon gamma. Pharm Res 14:725±729. Anchordoquy TJ, Carpenter JF. 1996. Polymers protect lactate dehydrogenase during freeze-drying

27.

28.

29.

30.

31.

32.

33.

34. 35.

36.

37.

38.

557

by inhibiting dissociation in the frozen state. Arch Biochem Biophys 332:231±238. Pace CN, Shirley BA, Thomson JA. 1989. Measuring the conformational stability of a protein. In: Creighton TE, editor. Protein structure: A practical approach; New York: Oxford University Press, pp 311±330. Webb JN, Webb SD, Cleland JL, Carpenter JF, Randolph TW. 2001. Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and co-solute studies on recombinant human interferon-gamma. Proc Natl Acad Sci USA 98:7259±7264. Ragone R, Colonna G, Balestrieri C, Servillo L, Irace G. 1984. Determination of tyrosine exposure in proteins by second-derivative spectroscopy. Biochemistry 23:1871±1875. Servillo L, Colonna G, Balestrieri C, Ragone R, Irace G. 1982. Simultaneous determination of tyrosine and tryptophan residues in proteins by second-derivative spectroscopy. Anal Biochem 126:251±257. Balestrieri C, Colonna G, Giovane A, Irace G, Servillo L. 1978. Second-derivative spectroscopy of proteins: Method for quantitative determination of aromatic amino acids in proteins. Eur J Biochem 90:433±440. Webb SD, Koval CA, Randolph CM, Randolph TW. 2001. Electrochemical determination of dissolution rates of lyophilized pharmaceutical preparations. Anal Chem 73:5296±5301. Szkudlarek BA, Anchordoquy TJ, Garcia GA, Pikal MJ, Carpenter JF, Rodriguez Hornedo N. 1996. pH changes of phosphate buffer solutions during freezing and their in¯uence on the stability of a model protein, lactate dehydrogenase. Abstr Pap Am Chem Soc 211:138-BIOT. Townsend MW, Deluca PP. 1991. Nature of aggregates formed during storage of freeze-dried ribonuclease-a. J Pharm Sci 80:63±66. Tarelli E, Wheeler SF. 1994. Drying from phosphate-buffered solutions can result in the phosphorylation of primary and secondary alcohol groups of saccharides, hydroxylated amino acids, proteins, and glycoproteins. Anal Biochem 222:196±201. Chang BS, Reeder G, Carpenter JF. 1996. Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist. Pharm Res 13:243±249. Dong AC, Prestrelski SJ, Allison SD, Carpenter JF. 1995. Infrared spectroscopic studies of lyophilization-induced and temperature-induced protein aggregation. J Pharm Sci 84:415±424. Dong AC, Kendrick B, Kreilgard L, Matsuura J, Manning MC, Carpenter JF. 1997. Spectroscopic study of secondary structure and thermal

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

558

39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

WEBB ET AL.

denaturation of recombinant human factor XIII in aqueous solution. Arch Biochem Biophys 347:213± 220. Byler DM, Susi H. 1986. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 25:469±487. Lam XM, Costantino HR, Overcashier DE, Nguyen TH, Hsu CC. 1996. Replacing succinate with glycolate buffer improves the stability of lyophilized interferon-gamma. Int J Pharm 142:85±95. Boteva R, Zlateva T, Dorovska Taran V, Visser A, Tsanev R, Salvato B. 1996. Dissociation equilibrium of human recombinant interferon gamma. Biochemistry 35:14825±14830. Zlateva T, Boteva R, Salvato B, Tsanev R. 1999. Factors affecting the dissociation and aggregation of human interferon gamma. Int J Biol Macromol 26:357±362. Prestrelski SJ, Arakawa T, Carpenter JF. 1993. Separation of freezing-induced and drying-induced denaturation of lyophilized proteins using stressspeci®c stabilization. 2. Structural studies using infrared-spectroscopy. Arch Biochem Biophys 303: 465±473. Wetlaufer DB, Xie Y. 1995. Control of aggregation in protein refolding: A variety of surfactants promote renaturation of carbonic anhydrase II. Protein Sci 4:1535±1543. Bhattacharyya J, Das KP. 1999. Effect of surfactants on the prevention of protein aggregation during unfolding and refolding processes: Comparison with molecular chaperone alpha-crystallin. J Dispersion Sci Technol 20:1163±1178. Jones LS, Bam NB, Randolph TW. 1997. Surfactant-stabilized protein formulations: A review of protein-surfactants interactions and novel analytical methodologies. In: Shahrokh Z, Sluzky V, Cleland JL, editors. Therapeutic protein and peptide formulation and delivery; Washington DC: American Chemical Society, pp 206±222. Bam NB, Cleland JL, Randolph TW. 1996. Molten globule intermediate of recombinant human growth hormone: Stabilization with surfactants. Biotechnol Prog 12:801±809. Bam NB, Randolph TW, Cleland JL. 1995. Stability of protein formulations: Investigation of surfactant effects by a novel EPR spectroscopic technique. Pharm Res 12:2±11.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002

49. Timasheff SN. 1998. Control of protein stability and reactions by weakly interacting cosolvents: The simplicity of the complicated. In: Richards FM, Eisenberg DS, Kim PS, editors. Advances in protein chemistry, Vol. 51. San Diego: Academic Press. pp 355±432. 50. 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. 51. Duncan MR, Lee JM, Warchol MP. 1995. In¯uence of surfactants upon protein peptide adsorption to glass and polypropylene. Int J Pharm 120:179±188. 52. Miller R, Fainerman VB, Makievski AV, Kragel J, Wustneck R. 2000. Adsorption characteristics of mixed monolayers of a globular protein and a nonionic surfactant. Colloid Surf A Physicochem Eng Asp 161:151±157. 53. Fatouros A, Sjostrom B. 2000. Recombinant factor VIIISQ: The in¯uence of formulation parameters on structure and surface adsorption. Int J Pharm 194:69±79. 54. Sluzky V, Klibanov AM, Langer R. 1992. Mechanism of insulin aggregation and stabilization in agitated aqueous solutions. Biotechnol Bioeng 40:895±903. 55. Zogra® G. 1985. Interfacial phenomena. In: Gennaro AR, editor. Remington's pharmaceutical sciences; Easton, PA: Mack Publishing, pp 258± 270. 56. Lumry R, Eyring H. 1954. Conformational changes of protein. J Phys Chem 58:110±120. 57. Minton KW, Karmin P, Hahn GM, Minton AP. 1982. Nonspeci®c stabilization of stress-susceptible proteins by stress-resistant proteins: A model for the biological role of heat-shock proteins. Proc Natl Acad Sci USA 79:7107±7111. 58. Georgiou G, Valax P, Ostermeier M, Horowitz PM. 1994. Folding and aggregation of TEM betalactamase: Analogies with the formation of inclusion bodies in Escherichia coli. Protein Sci 3:1953± 1960. 59. Beldarrain A, Lopez-Lacomba JL, Furrazola G, Barberia D, Cortijo M. 1999. Thermal denaturation of human gamma-interferon: A calorimetric and spectroscopic study. Biochemistry 38:7865±7873. 60. Cussler EL. 1984. Diffusion: Mass transfer in ¯uid systems. New York: Cambridge University Press.