Effects of annealing lyophilized and spray‐lyophilized formulations of recombinant human interferon‐γ

Effects of Annealing Lyophilized and Spray-Lyophilized Formulations of Recombinant Human Interferon-g SERENA D. WEBB,1 JEFFREY L. CLELAND,2 JOHN F. CARPENTER,3 THEODORE W. RANDOLPH1 1

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

Genentech, Inc., South San Francisco, California 94080

3

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received 29 April 2002; revised 13 September 2002; accepted 22 October 2002

ABSTRACT: The purpose of this study was to examine the effects of adsorption of recombinant human interferon-g (rhIFN-g) on ice surfaces and subsequent drying during processing by spray-lyophilization and lyophilization. Ice/liquid interfacial areas were manipulated by the freezing method as well as by the addition of an annealing step during lyophilization; that is, rhIFN-g adsorption was modified by the addition of nonionic surfactants. rhIFN-g was lyophilized or spray-lyophilized at a concentration of 1 mg/mL in 5% sucrose, 5% hydroxyethyl starch (HES)
0.03% polysorbate 20 in 140 mM KCl, and 10 mM potassium phosphate, pH 7.5. After the samples were frozen, half were annealed on the lyophilizer shelf. Recovery of soluble protein was measured at intermediate points during processing. On drying, the secondary structure of rhIFN-g was determined by second-derivative infrared (IR) spectroscopy, specific surface areas (SSAs) were measured, scanning electron micrographs (SEM) were taken, and dissolution times were recorded. Adsorption of rhIFN-g to ice/liquid interfaces alone was not responsible for aggregation. Rather, drying was necessary to cause aggregation in lyophilized sucrose formulations. Addition of an annealing step to the lyophilization cycle resulted in more native-like secondary protein structure in the dried solid, eliminated cracking of the dried cakes, and suppressed both the formation of air/liquid interfaces and rhIFN-g aggregation on reconstitution. ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:715–729, 2003

Keywords: rhIFN-g; surfactant, Tween 20; polysorbate 20; lyophilization; freeze drying; spray-lyophilization; annealing; ice/liquid interface; air/liquid interface

INTRODUCTION A large ice/liquid interfacial area (0.1–2 m2/mL) forms during the freezing step of a lyophilization or spray-lyophilization process. This interfacial area has been implicated in aggregation of proteins,1,2 which may adsorb to the ice/liquid

Correspondence to: Theodore W. Randolph (Telephone: 303492-4776; Fax: 303-492-4341; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 715–729 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association

interface and undergo subsequent unfolding and aggregation. In previous studies of interfacial adsorption of protein at the ice/liquid interface, fast or slow cooling protocols have been used to manipulate the size of the interface.1–5 Chang et al.2 noted that rapid cooling in liquid nitrogen caused nearly the same increase in turbidity in solutions containing one of a variety of proteins as 11 slow freeze–thaw cycles. This result was explained in terms of the larger ice/liquid interfacial surface area in the rapidly frozen samples. Presumably, however, the cumulative total surface area to which the

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proteins were exposed was greater in the case of the 11 repetitive freeze–thaw cycles, and damage arising during a given freezing step could be at least partially reversed during thawing. Strambini and Gabellieri1 noted dramatic decreases in phosphorescence lifetimes for proteins in the presence of ice/liquid interfaces, and ascribed this effect to unfolding of the proteins at the ice/liquid interface. However, a number of variables that might affect protein stability are coupled to the cooling rate. For instance, during freezing, the crystallization of pure ice results in freeze-concentration (often 10–15-fold) of the remainder of the formulation. If this concentration exceeds the solubility limit of solutes, precipitation can occur. In the specific case of buffer salts, drastic changes in pH can result from differential precipitation of buffer salts.6 The extent to which such differential precipitation occurs is linked to cooling rates.7 Crystallization of other excipients is also affected by the cooling rate,8 which can in turn affect protein stability via introduction of new solid/ liquid surfaces or by reducing protective protein– excipient interactions during subsequent drying. The cooling rate may also affect free volume of glassy phases formed on cooling below the glass transition temperature of the maximally freezeconcentrated solid (Tg0 ).4,9 Faster cooling leads to greater excess free volume, which in turn results in higher levels of residual stress in the solid. Presumably, residual stress in the dried solid could also have an adverse effect of protein stability during storage. Our previous results10 on lyophilization and spray-lyophilization of rhIFN-g indicated that rhIFN-g adsorbs to both air/liquid and ice/liquid interfaces. Significant protein aggregation was evident after air/liquid interfacial adsorption, but aggregation resulting from adsorption to the ice/ liquid interface was insignificant. In contrast, drying of frozen solutions caused significant aggregation. We propose that an alternative explanation to surface-induced aggregation at the ice/ liquid interface is provided by the residual stress in the glasses formed on freezing. Shrinkage of lyophilized cakes occurs during primary drying.11,12 If protein adsorbs to ice surfaces, differential stresses exacerbated by sublimation of ice may be responsible for loss of soluble protein on reconstitution. Another possibility is that protein adsorption at ice interfaces is unimportant, and that the mechanical stress in the amorphous phase affects the protein throughout the glass JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

independent of ice/glass surfaces. Additionally, we propose that exposure to air/liquid interfaces formed during reconstitution results in aggregation of rhIFN-g. We test these hypotheses by adding an annealing step to alleviate residual stress in frozen solids, altering the degree of surface adsorption by the addition of polysorbate 20, and changing cooling rates and amounts of air/liquid interface formed during reconstitution by lyophilization or spraylyophilization techniques.

MATERIALS AND METHODS Protein and Reagents Pharmaceutical quality rhIFN-g expressed in E. coli was produced and purified at Genentech, Inc., and supplied in 5 mM sodium succinate, pH 5. Snakeskinß pleated dialysis tubing (7000 MWCO) and 10% polysorbate 20 (Surfact-Ampsß 20, or Tween 20) were purchased from Pierce. High purity sucrose (low metal content) was purchased from Pfanstiel. Hydroxyethyl starch (HES) was purchased as Viastarchß for injection from Fresenius. Potassium phosphate monobasic and dibasic salts were purchased from Fisher Scientific. Potassium ferrocyanide trihydrate was purchased from Sigma. The Non-Interfering Protein Assayß was purchased from Bioworld Laboratory Essentials and used according to the manufacturer’s Protocol I instructions. All reagents were A.C.S. reagent grade or higher quality. Millipore water was used in the preparation of solutions. Solutions containing protein were filtered with a low protein-binding filter (polyethersulfone) from Whatman with a pore size of 0.2 mm. Buffer solutions were filtered using a 0.22-mm filter from Millipore. Formulation Buffer and Solutions rhIFN-g was dialyzed into 10 mM potassium phosphate, pH 7.5. Formulations were prepared in 10 mM potassium phosphate, pH 7.5, with 1 mg/ mL rhIFN-g, 140 mM KCl, and (1) 5% sucrose and 5% HES, (2) 5% sucrose, 5% HES, and 0.03% polysorbate 20, or (3) 10% HES. All concentrations identified as percentages were prepared as w/v solutions. The three formulations were lyophilized or spray-lyophilized (vide infra). Half of the samples were annealed for 4 h at 58C, and the other half were lyophilized per the normal cycle as

ANNEALING EFFECTS ON LYOPHILIZED RECOMBINANT HUMAN INTERFERON-g

described later in the lyophilization section. Formulations that dissolved rapidly (non-annealed lyophilized sucrose formulations) were prepared in an identical manner for electrochemical dissolution analyses, except potassium ferrocyanide trihydrate was added to each formulation prior to lyophilization, as described previously.13 Differential Scanning Calorimetry (DSC) Glass transition temperatures of the maximally concentrated amorphous phase (Tg0 ) were determined for the formulations with a Perkin Elmer DSC-7 calorimeter, controlled by a Perkin Elmer Thermal Analysis Controller TAC 7/DX. The DSC was calibrated with both indium and water, and a baseline was obtained with an empty sample pan prior to Tg0 determinations. For Tg0 measurements, 20 mL of solution were placed in aluminum sample pans with a floating lid. Samples were loaded at 258C, cooled to 1008C at a rate of 508/min, and scanned from 100 to 108C at a rate of 108/min. Tg0 was taken as the midpoint of the transition region. Spray-Lyophilization Liquid formulations were sprayed through an atomizing nozzle as described previously.14 An ISCO Series D pump controller and model 260D syringe pump maintained the liquid feed rate at 1.5 mL/min. A mass flow controller (Sierra Instruments, model 810C-DR-13) produced a gas flow rate of 0.42 L/min to sustain a pressure drop across the nozzle of 2.6  104 Pa. Ultra-high purity nitrogen was used as the carrier gas. A small amount (2 mL) of each formulation was atomized through the nozzle and collected in a beaker without freezing to monitor aggregation to the protein due to the atomization process. These samples were centrifuged to remove precipitated protein, and the amount of soluble protein in the supernatant was determined with the Non-Interfering Protein Assayß. Other samples were sprayed into a stainless steel bowl containing liquid nitrogen, and the bowl was transferred into a cooler filled with dry ice. The liquid nitrogen was allowed to boil off prior to filling the frozen powder into pre-cooled vials (5-mL tubing vial, West Company 6800-0344) using a pre-cooled plastic spoon and tongs. Lyophilization stoppers were partially inserted into filled vials (20 mm, West Company 1014-4899). A thermocouple was placed just above the vial filling location, and the

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temperature was maintained below 408C at all times during the storage and filling of the material prior to transfer to the lyophilizer shelf. A subset of the filled vials were stoppered, and the samples were allowed to thaw at room temperature to assess spray freeze-thaw aggregation using the Non-Interfering Protein Assayß. The remaining filled vials were loaded onto lyophilizer shelves maintained at 408C, and lyophilized according to the cycle presented next. Lyophilized Sample Preparation and Lyophilization Cycle Lyophilization vials of 5 mL volume (West Company 6800-0344), flint glass) were filled with 2 mL of formulation solution. Thermocouples were placed in two representative vials. Samples were frozen by submersion in liquid nitrogen for at least 1 min. Vials were removed from the liquid nitrogen and directly loaded into a Thermal Kinetics Systems LyoStar1 lyophilizer, with the shelf temperature maintained at 408C. Three vials from each formulation were removed from the lyophilizer, and their contents were allowed to thaw at room temperature to assess protein aggregation at this step. The vials containing the spray-frozen samples were added to the lyophilizer and the door was sealed. All samples were equilibrated with a shelf at 408C and maintained at this temperature for 2 h. Half of the samples from each formulation were lyophilized using a cycle containing an annealing step: the shelf temperature was increased by 18C/ min to 58C, maintained at this temperature for 4 h, and then decreased by 18C/min to 408C. The remainder of the cycle was used for both annealed and non-annealed sample sets. Chamber pressure was reduced to <100 mTorr, and the shelf temperature was increased by 18C/min to 258C, and maintained at this temperature for 35 h. The chamber pressure was increased to 200 mTorr, and the shelf temperature was increased by 0.038/ min to 308C and maintained at that temperature for 2 h. The vials were stoppered under vacuum prior to unloading from the freeze dryer. Primary Drying Endpoint Determination All vials and stoppers were numbered to maintain pairings. Vials and stoppers were washed with mild detergent and rinsed with distilled, deionized water, and then dried on the lyophilizer shelves at 408C (pressure <100 mTorr) to a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

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constant final weight (3–4 h). Vials for lyophilized samples were filled at room temperature (238C) with 2 mL of formulation solution, and vials for spray-lyophilization were filled approximately half full with spray-frozen powder while in the dry ice cooler, as already reported. Thermocouples were inserted in all the lyophilization vials prior to immersion in liquid nitrogen. Prechilled thermocouples were inserted into vials of spray-lyophilized powder within the dry ice cooler. After freezing the samples to be lyophilized by immersing the vials in liquid nitrogen for at least 1 min, the samples were added to the dry ice cooler. Spray-lyophilized and lyophilized sample vials were grouped together and surrounded by a ring of dummy vials filled with ice and then encircled in a radiation shield.15 The group of vials was rapidly transferred from the cooler onto the 408C lyophilizer shelf. All samples were equilibrated at 408C and maintained at this temperature for 2 h. Chamber pressure was reduced to <100 mTorr, and the shelf temperature was increased by 18C/min to 258C and held until all vials reached a shelf temperature of 258C or greater. For each vial, the time from initiation of drying (pressure <100 mTorr) until the thermocouple reached 258C was recorded as the total primary drying time. All 12 sample types were included in the run, with two types run in triplicate to provide an estimation of error. Interrupted Drying Protocol All vials and stoppers were numbered, washed, dried, and weighed as in the endpoint determination method just described. Vials for lyophilized samples were filled at room temperature (238C) with 2 mL of formulation solution, stoppers were inserted, and vials were weighed again prior to immersion in liquid nitrogen. Similar to the previous treatment, all samples were grouped together in the dry ice cooler, surrounded by frozen dummy vials, and wrapped in a radiation shield. The group was rapidly transferred from the cooler and onto the 408C lyophilizer shelf. All samples were equilibrated with a shelf at 408C and held at that temperature for 2 h. Primary drying was initiated, and the cycle protocol was followed (vide supra). The drying was aborted during three separate runs by stoppering the vials while under vacuum. The first two runs were aborted prior to thermocouples reaching 258C, whereas the third run was aborted after the thermocouples reached 258C. The vials were JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

removed and weighed. The stoppers were placed in the upright (open) position, reloaded into the lyophilizer, and dried to a constant weight as reported previously.10 Using the total solids mass determined after baking the lyophilized samples to a constant mass, the grams of water sublimed were calculated. For samples undergoing lyophilization: Water sublimed ðgÞ ¼ Initial mass ðgÞ  Mass after primary drying ðgÞ

ð1Þ

The amount of water sublimed was added back to each sample. The rehydrated samples were analyzed for protein aggregates. Protein Secondary Structure by Infrared Spectroscopy Lyophilized samples containing 0.2 mg rhIFN-g were mixed with 300 mg of potassium bromide and pressed at 30 MPa into pellets for secondary structure analysis. Calcium fluoride 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.16 For each spectrum, a 256-scan interferogram was collected in single beam mode, with a 4 cm1 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.17 HES absorbs weakly in the amide I region, and a subtraction procedure was developed. A spectrum of lyophilized pure HES was subtracted from sample spectra by minimizing peak absorbances near 730, 815, 3024, and the region surrounding 2000 cm1. Second-derivative protein spectra were calculated using Nicolet Omnicß software. The final protein spectra were smoothed with a 7-point function to remove white noise. All second-derivative spectra were baseline corrected using Galactic’s GRAMS 386 software based on a previously described method,17 and were area normalized in the amide I region 1600– 1700 cm1.18 Spray Freeze-Dried Particle Size Determination The aerodynamic diameters of spray-lyophilized particles were determined using an Aerosizer1

ANNEALING EFFECTS ON LYOPHILIZED RECOMBINANT HUMAN INTERFERON-g

DSP (Model 3225) equipped with an AeroDisperser1 (Model 3230). The dispersion pin was calibrated and a diagnostic test was performed prior to powder addition and sample measurements. More than 200,000 particles were used for each measurement. Scanning Electron Microscopy (SEM) Scanning electron micrographs (SEMs) were taken of the lyophilized and spray-lyophilized 10% HES formulations (both annealed and nonannealed samples) using an ISI-SX-30 SEM (International Scientific Instruments) after sputter-coating the samples with gold using a Polaron SEM coating system. Surface Area Measurement A Quantachrome Autosorb-1C (Boynton Beach, FL) was used to measure the surface areas of the lyophilized and spray-lyophilized (both annealed and non-annealed) samples at 77 K. Nitrogen was used as the adsorbate for the non-annealed spraylyophilized samples, but because of sample size limitations, it was necessary to use krypton for annealed and lyophilized samples. The Brunauer, Emmett, and Teller19 (BET) adsorption theory equation was used to calculate the specific surface areas (SSAs), using a 0.05–0.30 relative pressure range. Moisture Determination Random lyophilized samples were prepared in a dry-nitrogen-purged glove box and analyzed for moisture content using the Karl Fischer method.20 A Mettler DL37 coulometric moisture analyzer (Hightstown, NJ) was used with Photovolt reagents (Indianapolis, Indiana). Reconstitution To reconstitute a lyophilized sample, the stopper was removed from the sample vial and 2 mL of water or a solution of 0.03% polysorbate 20 was pipetted quickly to the center of the cake. For spray-lyophilized samples, 226 mg of powder was weighed into a separate clean vial. The volume of water needed to reconstitute to the original concentration was calculated (2 mL) and added in the same fashion as for the lyophilized samples. Stoppers were replaced, and the vials remained stationary until the powders were

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fully dissolved and any bubbles had cleared. A digital camera was used to photograph representative vials during the dissolution process immediately following addition of water and at 1 and 3 min after reconstitution. Samples were reconstituted in triplicate and analyzed for the presence of aggregates by optical density at 350 nm (OD350). Samples were centrifuged, and the supernatant was analyzed by the Non-Interfering Protein Assayß to determine soluble protein remaining. Error from triplicate samples was used to calculate a pooled error. Dissolution Rate Determination We determined dissolution times for lyophilized and spray-lyophilized powders visually and dissolution times for the relatively fast-dissolving lyophilized samples electrochemically using a rotating disk electrode (RDE) at 238C as described previously.13 Storage Stability Testing Samples were placed in an incubator maintained at 45
18C for 2 weeks. On removal, the samples were reconstituted with water and checked for the presence of aggregates using OD350, centrifuged, and analyzed for soluble protein in the supernatant via the Non-Interfering Protein Assayß.

RESULTS Selection of Annealing and Drying Conditions The Tg0 measurements were used to define the lyophilization temperatures for primary drying and annealing. Tg0 values measured by DSC were 28.0, 29.0, and 18.78C for the 5% HES/5% sucrose, 5% HES/5% sucrose/0.03% polysorbate 20 and 10% HES frozen solutions, respectively. Tg0 values were similar to those measured previously21 for comparable mixtures of HES and sucrose. Based on these temperatures and previous data21 from successful annealing studies, a temperature of 58C was selected for the annealing of these samples. Further, because primary drying must be carried out below Tg0 to avoid collapse, a shelf temperature of 258C was selected. Sublimation of ice cools the samples during drying, and sample temperatures were typically below 408C during the primary drying step. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

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Secondary Structure of rhIFN-g after Lyophilization The secondary structure of rhIFN-g was measured in lyophilized powders by IR spectroscopy. The dominant band in the conformationally sensitive amide I region is at 1656 cm1 and corresponds to the a-helix,22 which comprises 62% of the native structure of rhIFN-g.23 The band at 1682 cm1 is a combination of two bands at 1677 cm1 (extended chain) and at 1684 cm1 (turn structure), and likewise, the band at 1633 cm1 is composed of bands at 1630 and 1635 cm1 (both extended chain).22 In nonannealed lyophilized samples, intensities of the extended chain and turn structure bands (1677 and 1684 cm1) and the a-helix band (1656 cm1) decreased relative to those in the spectrum for the native control protein (see Figure 1A–C). The general band broadening of the amide I region is indicative of heterogeneity in the secondary structure of rhIFN-g after lyophilization.22,24,25 The 10% HES formulation exhibited the least native-like structure, with an increased intensity of turn structure in the bands at 1630 and 1635 cm1 in addition to large broadening of the a-helix band. Overall, the most native-like structure in the non-annealed lyophilized samples was present in the 5% HES/5% sucrose/0.03% polysorbate 20 formulation. The secondary protein structure became more native-like on annealing, with significantly increased intensity and narrowing of the a-helix band for all lyophilized formulations. After annealing, the 5% HES/5% sucrose/ 0.03% polysorbate 20 formulation again appeared the most native-like, whereas the 10% HES formulation was the least native-like.

Secondary Structure of rhIFN-g after Spray-Lyophilization

Figure 1. Secondary structure of rhIFN-g by secondderivative IR spectroscopy, after lyophilization of 1 mg/ mL in: (A) 5% sucrose, 5% HES, 140 mM KCl, and 10 mM potassium phosphate, pH 7.5; (B) 5% sucrose, 5% HES, 0.03% polysorbate 20, 140 mM KCl, and 10 mM potassium phosphate, pH 7.5; and (C) 10% HES in 140 mM KCl and 10 mM potassium phosphate, pH 7.5. Key: (---) non-annealed; (—) annealed; (—, bold) compared with native control.

Second-derivative IR spectra of rhIFN-g in the dried powders after spray-lyophilization are shown in Figure 2A–C. In the non-annealed sucrose formulations, there was a greater decrease in intensity of the bands at 1677 (extended chain), 1684 (turn), and 1656 cm1 (a-helix) than that observed in the non-annealed lyophilized samples. The bands at 1630 and 1635 cm1 (extended chain) increased, and a new band appeared near 1638 cm1, a wave number commonly assigned to b-sheet.24 Again, general band broadening of the amide I region was present in all nonannealed spray-lyophilized samples. There was

very little native secondary structure remaining after spray-lyophilization in the 10% HES formulation. Annealing of the spray-lyophilized sucrose formulations increased the intensity and decreased the width of the bands at 1656 cm1 (ahelix), 1677 cm1 (extended chain), and 1684 cm1 (turn), whereas it again decreased the intensity of the bands near 1633 cm1 (extended chain). These changes were larger than those observed after annealing and lyophilizing sucrose formulations, although the final spectra were similar. Conversely, annealing resulted in minor changes in the

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Figure 3. Picture of lyophilized vials. From left to right: 5% HES/5% sucrose non-annealed, annealed; 5% HES/5% sucrose/0.03% polysorbate 20 non-annealed, annealed; 10% HES non-annealed, annealed.

Figure 2. Secondary structure of rhIFN-g by secondderivative IR spectroscopy, after spray-lyophilization of 1 mg/mL in: (A) 5% sucrose, 5% HES, 140 mM KCl, and 10 mM potassium phosphate, pH 7.5; (B) 5% sucrose, 5% HES, 0.03% polysorbate 20, 140 mM KCl, and 10 mM potassium phosphate, pH 7.5; and (C) 10% HES in 140 mM KCl and 10 mM potassium phosphate, pH 7.5. Key: (---) non-annealed; (—) annealed; (—, bold) compared with native control.

spectrum for the spray-lyophilized 10% HES formulation. For both annealed and non-annealed spray-lyophilized samples, the 5% HES/5% sucrose/0.03% polysorbate 20 formulation was the most native-like and the 10% HES formulation was the least native-like, similar to the results of the lyophilized samples. Physical Appearance and Reconstitution of the Dried Cakes and Powders Not only were there distinct improvements in the secondary structure of rhIFN-g in every lyophilized sample after annealing, the lyophilized cakes themselves appeared more pharmaceuti-

cally elegant (see Figure 3). All of the nonannealed 5% HES/5% sucrose samples were severely cracked, about half of the 5% HES/5% sucrose/0.03% polysorbate 20 samples were cracked, and all but a very small percentage of the 10% HES samples were cracked. In contrast, none of the annealed formulations exhibited stress cracks in the cakes. Physical changes after annealing the spray-lyophilized formulations could not be detected by visual observation. Annealing also had a large impact on bubble formation during reconstitution of all the lyophilized samples. Time-lapse photographs of reconstitution of the 5% HES/5% sucrose formulation (non-annealed versus annealed) is shown in Figure 4A–C. All three formulations exhibited similar behavior (data not shown). Reconstitution of the non-annealed samples was a very vigorous process, with extensive bubble formation and even some splattering of the solution onto the sides of the vials. Reconstitution of the annealed samples was much less energetic, with fewer bubbles formed and no splattering. As long as 3 min after reconstitution (Figure 4C), significantly more bubbles remained in the solutions of the nonannealed formulations. Aggregation of rhIFN-g During Lyophilization and Spray-Lyophilization Lyophilized samples all showed substantial aggregation after reconstitution (Table 1). Annealing resulted in decreases in rhIFN-g aggregation JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

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student’s t-test (p ¼ 0.001). Pooled error was
0.03 mg/mL (3%) for one standard deviation. Recoveries of soluble protein from non-annealed spray-lyophilized samples after reconstitution were lower than from non-annealed lyophilized samples. Annealing increased the recovery of soluble protein from spray-lyophilized samples (Table 1). The presence of aggregates in each reconstituted solution was also detected by optical density of the solution at OD350 (see Table 2). For the same formulation reconstituted with water, spray-lyophilized samples had much greater optical densities than lyophilized samples. Annealing of samples in formulations without polysorbate 20 greatly reduced OD350. Additionally, the presence of 0.03% polysorbate in the formulation greatly decreased the optical densities of the reconstituted solutions. However, optical densities did not correspond with amount of insoluble aggregates that were detected by protein analysis. For example, samples lyophilized in the sucrose/ HES/polysorbate 20 formulation showed an OD350 value 300 times smaller than that for the sucrose/ HES formulation, even though the difference in aggregate levels was small (4%). Because light scattering depends both on particle size and concentration, these results suggest that the particle size distributions of aggregates are formulation- and process-dependent. It is important to note that size-exclusion HPLC analysis documented that soluble aggregates of rhIFN-g could not be detected after reconstitution of lyophilized or spray-lyophilized samples (data not shown). Aggregation during Intermediate Processing Points of Lyophilization

Figure 4. Reconstitution of contents of lyophilized vials (A) immediately after water addition, (B) 1 min after water addition, and (C) 3 min after water addition. For each panel, 2 mL of water was added to the lyophilized 5% HES/5% sucrose formulation. The nonannealed vial is on the left and the annealed vial is on the right.

levels for all formulations on reconstitution (see Table 1). The effect of annealing was significant as determined by a difference in the means of formulation pairs (i.e., annealed versus non-annealed, same formulation and processing method) using a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

Because significant losses of soluble protein to aggregation were detected after reconstitution of lyophilized samples, samples were tested at intermediate processing points to determine the extent of protein losses. Freezing of the contents of a vial by immersion in liquid nitrogen, with subsequent thawing of the contents, was responsible for a 22% decrease in recovery of soluble protein from the 10% HES formulation (Figure 5). Freeze–thawing resulted in no significant aggregation in the formulations containing sucrose (Figure 5). Furthermore, samples frozen and partially dried by removing them from the lyophilizer during the primary drying step (82 and 55% of initial water remaining) melted with very little bubble formation and showed no loss of soluble rhIFN-g. Samples retaining only 1% of the

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Table 1. Recovery of Lyophilized or Spray-Lyophilized rhIFN-g (1 mg/mL)a Lyophilized Non-Annealed

Spray-Lyophilized Annealed

Non-Annealed

Reconstituted with:

Formulation 5% Sucrose, 5% HES 5% Sucrose, 5% HES, and 0.03% polysorbate 20 10% HES

Annealed

Reconstituted with:

0.03% 0.03% 0.03% 0.03% Water Polysorbate 20 Water Polysorbate 20 Water Polysorbate 20 Water Polysorbate 20 0.79 0.83

0.85 0.82

0.86 0.90

0.89 0.87

0.70 0.80

0.75 0.79

0.76 0.80

0.79 0.83

0.75

0.78

0.81

0.88

0.66

0.71

0.66

0.73

a Recoveries in 140 mM KCl and 10 mM potassium phosphate, pH 7.5, after reconstitution with water or water containing 0.03% polysorbate 20. Protein recoveries are reported in units of mg/mL. The concentration of the initial protein control for each of the three formulations was 1.0 mg/mL. The pooled error is þ/0.03 mg/mL for a standard deviation.

initial water (9 mg of water, or about 50% residual water on a sample mass basis) were removed after primary drying but before secondary drying was complete. Visual inspection of the samples indicated that a degree of cracking had already occurred in the cakes, and some air bubbles formed on reconstitution with water. However, the 5% HES/5% sucrose formulation retained 96% soluble protein, whereas the 5% HES/5% sucrose/0.03% polysorbate 20 formulation yielded 100% recovery. Comparison of these samples with results measured after secondary drying was complete (Table 1) suggests that the processes leading to aggregation must occur in the latter stages of secondary drying. Aggregation of rhIFN-g during Atomization Because spray lyophilization forms large amounts of air/liquid interfacial area during the initial

atomization step, it is important to assess the effects of this step on protein stability. Atomization of the 5% HES/5% sucrose formulation resulted in aggregation of 13% of the rhIFN-g (Figure 5). A slightly greater aggregation level of 16% was observed on atomization of the 10% HES formulation. As expected, the presence of 0.03% polysorbate 20 in the sucrose/HES formulation reduced aggregation to 6%. Aggregation of rhIFNg on freezing the atomized sprays and thawing was not statistically different from that seen after atomization alone (Figure 5).

Residual Water Analyses As shown in Table 3, all formulations have very low final water contents on completion of the lyophilization cycle. Non-annealed spray-lyophilized samples (all formulations) and all samples

Table 2. Optical Density of the Formulations after Reconstitution with Water or 0.03% Polysorbate 20 as Measured by Absorbance at 350 nm Lyophilized Non-Annealed

Spray-Lyophilized Annealed

Non-Annealed

Reconstituted with:

Formulation

Annealed

Reconstituted with:

0.03% 0.03% 0.03% 0.03% Water Polysorbate 20 Water Polysorbate 20 Water Polysorbate 20 Water Polysorbate 20

5% Sucrose, 5% HES 0.459 5% Sucrose, 5% HES, 0.014 and 0.03% polysorbate 20 10% HES 0.539

0.254 0.016

0.121 0.029

0.102 0.031

0.863 0.408

0.884 0.433

0.389 0.332

0.449 0.335

0.400

0.149

0.144

1.60

1.68

0.583

0.491

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and OD350 (Table 2). Reconstitution of lyophilized formulations with 0.03% polysorbate 20 resulted in no effect or slightly improved recoveries for most samples (see Table 1). However, reconstitution with 0.03% polysorbate 20 resulted in lower OD350 values for formulations without surfactant initially present, presumably because of reduced aggregate particle size, rather than lower aggregate concentration. We found no difference in dissolution rates when reconstituting with 0.03% polysorbate 20 versus water (data not shown). Dissolution rates did depend on the processing method, and for the same processing method, the rates were affected significantly by annealing. Generally, lyophilized samples dissolved much more rapidly than spray-lyophilized samples (see Table 4). All annealed lyophilized samples dissolved more slowly than their non-annealed counterparts, whereas the opposite was observed for spraylyophilized samples.

Figure 5. Recovery of soluble rhIFN-g at intermediate processing points during lyophilization and spraylyophilization in 140 mM KCl and 10 mM potassium phosphate, pH 7.5. X-axis labels represent the following formulations: (1) 5% HES/5% sucrose, (2) 5% HES/5% sucrose/0.03% polysorbate 20, and (3) 10% HES. Recovery of rhIFN-g after immersion of vial in liquid nitrogen and thawing of contents slowly at room temperature (solid); after spraying (diagonal bars); andafter sprayfreezing and thawing slowly at room temperature (checkerboard).

Surface Area Measurements and Morphology of Spray-Lyophilized and Lyophilized Powders To quantify the effects of processing method and annealing on dried sample surface areas, we used the BET gas adsorption technique (see Table 5). Non-annealed spray-lyophilized samples had much greater SSAs than the respective lyophilized formulations. The inclusion of polysorbate 20 caused a significant reduction in SSA in the non-annealed spray-lyophilized powder. For lyophilized samples, annealing decreased the SSAs of the formulations 4-fold. The reductions in surface area were larger for the spray-lyophilized samples after annealing, with SSAs reduced 20fold. The final SSAs of the annealed spraylyophilized powders were very similar to those of the annealed lyophilized samples. The effect of annealing on the morphology of the samples is shown in scanning electron micro-

containing 10% HES (non-annealed or annealed, lyophilized, or spray-lyophilized) displaying the lowest water contents (0.04–0.15%, g H2O/100 g solid). Interestingly, when lyophilized and spraylyophilized sucrose formulations were annealed, they retained identical amounts of water (0.40%). Effect of Reconstitution Solutions Previously, we reported increased recovery of soluble rhIFN-g in formulations after reconstitution with 0.03% polysorbate 20 compared with the same formulations reconstituted with water alone.26 Therefore, we reconstituted the current formulations with 0.03% polysorbate 20, and measured soluble protein recoveries (see Table 1)

Table 3. Results from Karl Fischer Analysis of Lyophilized and Spray-Lyophilized Samplesa Non-annealed Formulation 5% Sucrose and 5% HES 5% Sucrose, 5% HES and 0.03% Tween 20 10% HES a

Annealed

Lyophilized

Spray-Lyophilized

Lyophilized

Spray-Lyophilized

0.22 0.28

0.15 0.04

0.40 0.40

0.38 0.42

0.14

0.12

0.14

0.11

Results are reported in % water. Two standard deviations (pooled error) are þ/0.03 for each individual sample.

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ANNEALING EFFECTS ON LYOPHILIZED RECOMBINANT HUMAN INTERFERON-g

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Table 4. Dissolution Rates of Lyophilized and Spray-Lyophilized rhIFN-g (1 mg/mL) in 140 mM KCl and 10 mM Potassium Phosphate (pH 7.5) in Watera Lyophilized Formulation

Spray-Lyophilized

Non-annealed

Annealed

Non-annealed

Annealed

29 30

23 7.6

0.53 0.69

0.91 3.4

23

1.3

0.15

0.47

5% Sucrose and 5% HES 5% Sucrose, 5% HES and 0.03% polysorbate 20 10% HES a

Values are reported in mg solid/s; error is þ/ 11% for one standard deviation.

graphs (Figure 6A–D). Freezing by immersion of vials in liquid nitrogen causes directional solidification with a resulting fine structure,15,21 as shown in Figure 6A. The view shown is looking down at a cross-section in the vial, with the top surface of the cake removed. Annealing destroyed this fine structure21 (Figure 6B). Spray lyophilization creates spherical, highly porous particles (Figure 6C), independent of formulation.14 The average diameter of the spray-lyophilized particles shown is 90 mm (
1.6 mm, one standard deviation as measured by the Aerosizer1 DSP). Macroscopically, the volume of particles in the vials shrank slightly on annealing spray-lyophilized powders. However, massive changes were evident on the microscopic scale (see Figure 6D). Individual particulate structures were lost on annealing, and the microstructure of the annealed spraylyophilized powder resembled that of the annealed lyophilized sample. Effect of Annealing on Primary Drying Times The morphological changes occurring during annealing impacted primary drying times significantly. On average, annealing decreased the primary drying time for the lyophilized and spraylyophilized samples by 9.6 and 21 h, respectively.

Annealing reduces drying rate heterogeneity.21 Standard deviations were 3–4 times larger for primary drying endpoints of non-annealed versus those of annealed lyophilized samples (e.g., 4.2 versus. 1.0 h). Storage Stability Studies Samples were stored at 458C for 2 weeks to determine if any differences in recovery of soluble rhIFN-g between annealed and non-annealed samples might become magnified. However, on reconstitution, all samples exhibited recoveries and optical densities similar to those reported prior to storage (data not shown).

DISCUSSION Protein Damage during Spray-Lyophilization Previously, we showed that adsorption on air/ liquid interfaces could quantitatively account for aggregation of rhIFN-g observed during atomization into liquid nitrogen.10 In the present study, 6–16% of the protein aggregated during atomization, and no further damage was noted during subsequent freezing and thawing. In contrast, complete spray-lyophilization and rehydration

Table 5. Specific Surface Areas (SSAs) of Lyophilized or Spray-Lyophilized rhIFN-g (1 mg/mL) in 140 mM KCl and 10 mM Potassium Phosphate (pH 7.5)a Lyophilized Formulation 5% Sucrose and 5% HES 5% Sucrose, 5% HES and 0.03% polysorbate 20 10% HES a

Spray-Lyophilized

Non-annealed

Annealed

Non-annealed

Annealed

2.1 2.0

0.6 0.6

13 6.8

1.0 0.3

2.9

0.6

15

0.5

2

SSAs were measured by BET gas adsorption, and are reported in m /g. Error is þ/ 4% for 1 standard deviation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

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Figure 6. Scanning electron micrograph images of 1 mg/mL rhIFN-g in 10% HES, 140 mM KCl, and 10 mM potassium phosphate, pH 7.5: (A) lyophilized, non-annealed; (B) lyophilized, annealed; (C) spray-lyophilized, non-annealed; and (D) spray-lyophilized, annealed.

induced an additional 15% increase in aggregation, irrespective of formulation. Adsorption of protein to ice/liquid interfaces has been implicated in protein damage during freezethawing.1–5 During spray-lyophilization, adsorption of protein to ice/liquid interfaces does not occur because of the rapidity of the freezing step.10,27,28 Previously, it has been estimated that the time required for a 10-mm droplet to freeze on submersion in liquid nitrogen is of the order of 1 ms.28 The freezing front moves extremely fast (1 cm/s) through the droplet, and would completely pass through the diameter of a rhIFN-g molecule within 0.4 ms. Because protein does not have enough time to adsorb to the ice/water interfaces, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

neither the conformational changes nor the additional aggregation seen in the final dried powders can be the result of damage at the ice/liquid interface. Moreover, the three formulations tested in the current study contained excipients that are effective at protecting proteins during freeze– thawing. Protein Damage during Lyophilization When vials are immersed in liquid nitrogen, the vial contents solidify and the temperature is reduced below Tg0 within seconds, which is orders of magnitude slower than the time required to cool below Tg0 during spray-freezing. Thus, the protein

ANNEALING EFFECTS ON LYOPHILIZED RECOMBINANT HUMAN INTERFERON-g

has more time to adsorb to ice surfaces during freezing and cooling in a vial, but the extent of adsorption is also related to the area of ice/liquid interface. Surface areas measured for the lyophilized powders indicated that 0.2–0.3 m2 of ice/ liquid surface area formed per milliliter of solution. From our previous work,10 surface concentrations of 1.3 mg/m2 rhIFN-g on ice surfaces were measured by electron spectroscopy for chemical analysis for lyophilized trehalose formulations. Assuming the same concentrations on the ice surfaces of the formulations in the current work, 26–39% of rhIFN-g could be adsorbed to ice surfaces. However, there was no aggregation of rhIFN-g on thawing the frozen sucrose formulations, presumably because of stabilization by sucrose. Twenty percent aggregation occurred in the HES formulation, which could be due to adsorption to ice as well as other stresses, such as cold denaturation and freeze-concentration. In our previous work,10 we showed that addition of polysorbate 20 reduced rhIFN-g concentrations at ice/liquid interfaces by a factor of 3, yet addition of polysorbate 20 did not result in significant additional recovery of soluble protein after reconstitution. These data suggest that even when adsorption to ice surfaces occurred in sucrosecontaining formulations, it was not responsible for aggregation of the protein. Costantino et al.3 also reported that ice/liquid interfaces formed during freezing were not responsible for loss of protein. In our formulations containing sucrose, neither freeze–thawing nor partial drying caused aggregation. Thus, we attribute aggregation of rhIFN-g in these solutions to damage arising during the terminal stages of drying. Residual Stress in Glasses and Protein Damage Glasses are nonequilibrium states, and more rapid cooling during glass formation results in greater deviations from equilibrium, corresponding to greater excess free volume of the glassy matrix containing the protein.9 When water is removed from the glass during secondary drying, the excess free volume is increased further. In turn, this excess free volume corresponds to greater internal stress in the glass. Stress in the glassy phase often causes shrinkage of the glass during drying.11,12 In the current study, stress is macroscopically evident by the cracks formed during primary drying. We speculate that the same mechanical forces that (on a macroscopic scale) cause cracking of the cakes may also be

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responsible (on a microscopic scale) for causing the observed perturbations of protein secondary structure in the dried solids and, ultimately, aggregation on reconstitution. Experimentally, crack formation was completely absent in the cakes of all annealed samples. Interestingly, cracks were present in only about half of the samples containing polysorbate 20. This result suggests that mechanical stresses encountered during lyophilization are reduced in the presence of the surfactant, which may at least partially explain why the secondary structure of rhIFN-g in the dried samples was most nativelike in formulations containing polysorbate 20. Another manner by which to reduce the level of cracking is to anneal. Annealing above Tg0 allows the liquid to relax and results in a glass with lower excess free volume and decreased internal stress.29 We speculate that this reduced stress is responsible for the more native-like protein secondary structures observed when an annealing step precedes lyophilization or spray-lyophilization. Such an effect may also explain in part the differences between the HES-only formulation and those containing sucrose. Because of its higher molecular weight, the freeze-concentrated liquid phase present during the annealing of HES solutions will relax more slowly than liquids containing sucrose, and will thus retain more residual stress on drying. As a result, protein in dried HES formulations showed the greatest perturbations from native secondary structure, and the highest levels of aggregates after reconstitution. Another important effect of annealing was to reduce the amounts of air bubbles formed during reconstitution. Annealing increases the average pore diameter in the lyophilized solid through an Ostwald ripening process.,21 which in turn results in larger, and hence fewer, bubbles on reconstitution.13 Protein adsorption at air/liquid interfaces during processing can cause aggregation,10 perhaps explaining the reduction in aggregation observed for annealed samples. Effects of Dissolution Rates Slow wetting and the low solubility of HES limited the dissolution rates of the dried powders, and consequently obscured any effect on dissolution rate by the inclusion of a small amount of surfactant in the reconstitution solution. In contrast, our previous studies10 showed that reconstitution of trehalose formulations with a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

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polysorbate 20 solution improved recovery of soluble rhIFN-g by slowing the rapid dissolution rates. However, the HES formulations in this study already dissolved less rapidly than the most slowly dissolving trehalose formulations of the previous study, reducing the impact of any further decrease in dissolution rate. The mechanism by which annealing slowed the dissolution rates of lyophilized samples was through a reduction in the surface area of the cake available for wetting, resulting in as much as an 18-fold reduction in the dissolution rate. However, a different mechanism is operative for spraylyophilized particles. Spray-lyophilization creates spherical particles with 90% of the surface area existing internally.10 These porous particles float on the surface of the reconstitution solution, wetting very slowly. Annealing caused collapse of the internal surface area, increasing the density of the powder, resulting in more rapid submersion of the powders and faster dissolution rates. Collapse of the internal porous structure of spray-lyophilized particles was also assisted by the presence of polysorbate 20 in the formulation, causing a reduction in the SSAs measured for the non-annealed spray lyophilized powders. Annealing Effects on Primary Drying Time Lyophilization of both pharmaceutical30,31 and biopharmaceutical32–37 formulations is commonplace, even though lyophilization is both timeconsuming and expensive.30,38 Therefore, opportunities to streamline lyophilization cycles are of paramount importance. Recently, it has been shown that the addition of an annealing step can significantly decrease the time needed for primary drying,21 which is normally the longest phase of a typical lyophilization cycle. In the present study, even with the annealing time included, the overall length of the lyophilization cycle was decreased. As has been previously reported,21 annealing produced more consistent drying among vials in a lyophilized batch, and the reduced variation in drying times for annealed samples in this study supports this conclusion.

CONCLUSIONS Excess free volume in glassy formulations results in residual stress in the dried solid. Conditions that relieved this residual stress (annealing, low molecular weight excipients, addition of a plastiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003

cizing agent, polysorbate 20) correlated with reduced protein unfolding in the dried solids and less aggregation after reconstitution. Furthermore, annealed samples form fewer bubbles during reconstitution, which may act to avoid damage at air/liquid interfaces. Finally, annealing improved the pharmaceutical elegance of the final product, shortened the primary drying time, and reduced drying rate heterogeneity between samples.

ACKNOWLEDGMENTS We thank Ken Abbott, of CU Photography, for the digital pictures of the vials. We also thank Genentech, Inc. for its donation of rhIFN-g. Funding was provided by NSF-BES9816975.

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