Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein.

Encapsulation of the model protein bovine serum albumin (BSA) into poly(D,L lactide-co-glycolide) (PLG) microspheres was performed by a non-aqueous oil-in-oil (o/o) methodology. Powder formulations of BSA obtained by spray-freeze drying were first suspended in methylene chloride containing PLG followed by coacervation by adding silicon oil and microsphere hardening in heptane. The secondary structure of BSA was determined at relevant steps of the encapsulation procedure by employing Fourier-transform infrared (FTIR) spectroscopy. This fast and non-invasive method demonstrated the potential to rapidly screen pharmaceutically relevant protein delivery systems for their suitability. Structural perturbations in BSA were reduced during the spray-freeze drying step by employing the excipient trehalose. The protein was then encapsulated into PLG microspheres under various conditions without inducing significant structural perturbations. BSA released from these microspheres had a similar monomer content as unencapsulated BSA and also the same secondary structure. Upon blending of a poloxamer (Pluronic F-68) with the polymer phase, in vitro release was characterized by a small initial release and a prolonged and continuous sustained phase. In conclusion, the developed o/o methodology coupled with FTIR spectroscopic monitoring of protein structure is a powerful approach for the development of sustained release microspheres.

Protein spray-freeze drying. Effect of atomization conditions on particle size and stability.

PURPOSE: To investigate the effect of atomization conditions on particle size and stability of spray-freeze dried protein. METHODS: Atomization variables were explored for excipient-free (no zinc added) and zinc-complexed bovine serum albumin (BSA). Particle size was measured by laser diffraction light scattering following sonication in organic solvent containing poly(lactide-co-glycolide) (PLG). Powder surface area was determined from the N2 vapor sorption isotherm. Size-exclusion chromatography (SEC) was used to assess decrease in percent protein monomer. Fourier-transform infrared (FTIR) spectroscopy was employed to estimate protein secondary structure. PLG microspheres were made using a non-aqueous, cryogenic process and release of spray-freeze dried BSA was assessed in vitro. RESULTS: The most significant atomization parameter affecting particle size was the mass flow ratio (mass of atomization N2 relative to that for liquid feed). Particle size was inversely related to specific surface area and the amount of protein aggregates formed. Zinc-complexation reduced the specific surface area and stabilized the protein against aggregation. FTIR data indicated perturbations in secondary structure upon spray-freeze drying for both excipient-free and zinc-complexed protein. CONCLUSIONS: Upon sonication, spray-freeze dried protein powders exhibited friability, or susceptibility towards disintegration. For excipient-free protein, conditions where the mass flow ratio was > -0.3 yielded sub-micron powders with relatively large specific surface areas. Reduced particle size was also linked to a decrease in the percentage of protein monomer upon drying. This effect was ameliorated by zinc-complexation, via a mechanism involving reduction in specific surface area of the powder rather than stabilization of secondary structure. Reduction of protein particle size was beneficial in reducing the initial release (burst) of the protein encapsulated in PLG microspheres.

The effect of formulation excipients on protein stability and aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody.

PURPOSE: To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. METHODS: Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. RESULTS: Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300: 1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. CONCLUSIONS: Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.

Influence of calcium ions on the structure and stability of recombinant human deoxyribonuclease I in the aqueous and lyophilized states.

The effect of calcium ions on the structure and stability of recombinant human DNase I (rhDNase) in the aqueous and solid (lyophilized) states was investigated. Fourier transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure, while chemical stability was monitored in terms of deamidation and soluble aggregate formation at 40 degrees C. The exogenous calcium was removed by EGTA. This process can remove all but approximately one calcium ion per protein molecule. Analysis of the FTIR spectra in the amide III region in either the aqueous or lyophilized state demonstrated that removal of exogenous Ca2+ by EGTA-treatment had little effect on the secondary structure (and lyophilization-induced rearrangement thereof). For the aqueous solution, circular dichroism was used as an independent technique and confirmed that there was no large overall change in the secondary or tertiary structure upon the removal of calcium. The primary degradation route for the aqueous protein was deamidation. For the EGTA-treated protein, there was also severe covalent aggregation, e.g., formation of intermolecular disulfides facilitated by the cleavage of Cys173-Cys209. The aggregates exhibited a markedly different secondary structure compared to the native protein. For instance, the beta-sheet band observed at ca. 1620 cm-1 wavenumber in the amide I second derivative spectra was increased. Enzymatic activity was completely lost upon aggregation, consistent with the cleavage of the aforementioned native disulfide. For the protein lyophilized in the presence of Ca2+, there was no increase in deamidated species during solid-state storage; however, some aggregation was observed. For the lyophilized EGTA-treated protein, aggregation was even more pronounced, and there was some loss in enzymatic activity upon reconstitution. Thus, the removal of calcium ions by EGTA-treatment decreased the stability of rhDNase in both the aqueous and solid states even though no large overall calcium-induced structural changes could be observed by the techniques used in this study.

On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer.

In this work we describe the structural investigation of the model protein recombinant human growth hormone (rhGH) under conditions relevant to polymeric sustained-delivery depots, including the dried protein entrapped in a film of poly(DL-lactic-co-glycolic)acid. At each step of the procedure, dehydration of rhGH by lyophilization, suspension in methylene chloride, and drying from that suspension, the structure of rhGH was probed noninvasively using Fourier transform infrared (FTIR) spectroscopy. We found that the structure of rhGH was significantly changed by the dehydration process as indicated by a marked drop in the alpha-helix content and increase in the beta-sheet content. Subsequent suspension of this powder in methylene chloride, drying from that suspension, and drying from a methylene chloride/PLGA solution introduced only minor additional structural changes when using appropriate conditions. This result is likely due to the limited molecular mobility of proteins in nonprotein-dissolving organic solvents. Finally, when rhGH was co-lyophilized with the lyoprotectant trehalose, which preserves the secondary structure, the rhGH entrapped in the PLGA matrix also had a nativelike secondary structure.

Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody.

We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.

Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone.

We have investigated the effect of mannitol, sorbitol, methyl alpha-D-mannopyranoside, lactose, trehalose, and cellobiose on the stability and structure of the pharmaceutical protein recombinant human growth hormone (rhGH) in the lyophilized state. All excipients afforded significant protection of the protein against aggregation, particularly at levels to potentially satisfy water-binding sites on the protein in the dried state (i.e., 131:1 excipient-to-protein molar ratio). At higher excipient-to-protein ratios, X-ray diffraction studies showed that mannitol and sorbitol were prone to crystallization and afforded somewhat less stabilization than at lower ratios where the excipient remained in the amorphous, protein-containing phase. The secondary structure of rhGH was determined using Fourier transform infrared (FTIR) spectroscopy. rhGH exhibited a decrease in alpha-helix and increase in beta-sheet structures upon drying. Addition of excipient stabilized the secondary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conservation, as indicated by the alpha-helix content, generally exhibited reduced aggregation. In addition, prevention of protein-protein interactions (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving the protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-protein distance and thus prevent aggregation.

Deterioration of lyophilized pharmaceutical proteins.

The successful use of proteins in pharmaceutical and other commercial applications requires close examination of their relative fragility. Because of the resultant enhanced stability, proteins are often formulated in the solid state, even though dehydration tends to alter their structure. Even in the solid form, however, proteins may become inactivated due to various deleterious processes, e.g., aggregation. This review focuses on such mechanisms, with an emphasis on case studies conducted in our laboratory. Proteins which have both disulfide bonds and free thiols may aggregate via thiol-disulfide exchange, and this process may be facilitated by lyophilization-induced structural perturbations. For proteins possessing disulfides but not free thiols, aggregation also may occur when native disulfides are beta-eliminated, thus giving rise to thiol species which can catalyze disulfide scrambling. Other deleterious processes have also been uncovered, including a formaldehyde-mediated aggregation of formalinized vaccines. It is illustrated how knowledge of such deterioration pathways makes possible the rational development of stable solid protein formulations.

The effect of operating and formulation variables on the morphology of spray-dried protein particles.

The purpose of this research was to investigate the shape and morphology of various spray-dried protein powders as a function of spray-drying conditions and protein formulations. A benchtop spray dryer was used to spray dry three model proteins in formulation with a sugar or a surfactant. Physical characterizations of the powder included morphology (scanning electron microscopy), particle size, residual moisture, and X-ray powder diffraction analyses. A significant change in particle shape from irregular (e.g., "donut") to spherical was observed as the outlet temperature of the dryer was decreased. The drying air outlet temperature was shown to depend on various operating parameters and was found to correlate with the drying rate of atomized droplets in the drying chamber. The morphology of spray-dried protein particles was also affected by formulation. In protein:sugar formulations, spray-dried particles exhibited a smooth surface regardless of the protein-to-lactose ratio, whereas roughness was observed when mannitol was present at > 30% of total solids, due to recrystallization. Protein particles containing trehalose at concentrations > 50% were highly agglomerated. The presence of surfactant resulted in noticeably smoother, more spherical particles. The shape and the morphology of spray-dried powders are affected by spray drying conditions and protein formulation. This study provides information useful for development of dry proteins for fine powder (e.g., aerosol) applications.

Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.

Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control.

On the pH memory of lyophilized compounds containing protein functional groups.

Lyophilized proteins exhibit "pH memory," i.e., their behavior in the solid form corresponds to the pH of the aqueous solution from which they were freeze dried. Herein, we directly tested whether the ionization state is "remembered" by model organic compounds containing various protein functional groups (amino, carboxyl, and phenolic). The fraction of ionized species was quantitated from the infrared spectra of both the aqueous and lyophilized states. The pK(a) values in the aqueous and lyophilized forms for each compound were found to be quite similar, within 0.3 units from each other.

Determining the water sorption monolayer of lyophilized pharmaceutical proteins.

The concept of monolayer water coverage is useful in the development of lyophilized protein formulations. Herein, we have explored three different methodologies to determine the water monolayer for pharmaceutical proteins: (1) theoretical prediction based on the amino acid composition and their relative propensities to sorb water; (2) a traditional adsorption isotherm measurement by Karl Fischer water titration of samples held at various relative humidities (created by saturated salt solutions); and (3) an adsorption isotherm measurement with a gravimetric sorption analyzer (GSA), which consists of a microbalance within a computer-controlled humidified environment. Data from the latter two methods were analyzed with the Brunauer-Emmett-Teller (BET) gas adsorption equation to yield experimental monolayers. In our study, we examined six different therapeutic proteins and found that for each case all three approaches yielded similar results for the water monolayer. We also attempted to use the BET equation to determine the water monolayer for a model sugar (trehalose) and polyol (mannitol), which are potential excipients in pharmaceutical protein formulations. We found that calculations from the data obtained by the traditional and GSA methods yielded consistent results for trehalose, which remained amorphous upon lyophilization. Mannitol tended to form anhydrous crystals upon freezedrying, and was thus not amenable to analysis. The utility of both traditional and GSA methods for determining the water monolayer was extended to colyophilized protein:sugar systems as well.

The secondary structure and aggregation of lyophilized tetanus toxoid.

Tetanus toxoid (TT), the vaccine for tetanus, is an important protein antigen and candidate for sustained release from polymeric matrices. During administration from the latter, the solid (e.g., lyophilized) protein will be exposed to elevated levels of temperature and moisture, conditions which trigger its aggregation. To examine the connection between this aggregation and the structure of the TT molecule in the solid state, Fourier-transform infrared (FTIR) spectroscopy was employed to determine the secondary structure of TT in the presence of various excipients. We found that excipient-free TT undergoes a significant alteration (mostly reversible) in the secondary structure during lyophilization. Specifically, more than half the total alpha-helix content was lost with a concomitant increase in beta-sheet structure. The extent of structural alterations in the presence of 1:5 (g:g protein) NaCl, sorbitol, or poly-(ethylene glycol), did not correlate with stability conferred towards moisture-induced aggregation. These results suggest that the degree of retention of the native protein structure in the dry state is not a general predictor of stability for the "wetted" solid within polymer controlled-release vehicles.

Strategies for stabilising tetanus toxoid towards the development of a single-dose tetanus vaccine.

The stability of tetanus toxoid (TT) has been investigated for the purpose of enhancing its immunogenicity when encapsulated in TT-polylactide-co-glycolide (PLGA) microspheres. In this paper we describe our simulations of various potential inactivating events during microsphere processing and release of the antigen. These include: i) processing aqueous TT solutions in the presence of organic solvents, ii) exposing lyophilized TT to moisture, and iii) incubating vaccine with the degrading PLGA. At 37 degrees C, TT began to aggregate in solution a few hours after the addition of either methylene chloride or ethyl acetate to form a single oil-water interface. Similarly, exposure of the lyophilized vaccine to elevated humidity caused the antigen to lose solubility. Previous analysis of moisture-induced aggregates has revealed that formaldehyde, which is stored in labile linkages in the vaccine following its detoxification, is the precursor to the reactive species in the principal aggregation pathway [1]. Methods to inhibit this mechanism, such as blocking nucleophilic amino groups of TT with succinic anhydride, were verified. Succinylation of TT resulted in the incorporation of 10-fold greater antigenically active vaccine in PLGA microspheres relative to the unmodified TT following processing by double-emulsion/solvent evaporation with ethyl acetate, strongly suggesting the formaldehyde-mediated aggregation pathway also occurs during the deleterious conditions of microsphere processing. Incubation of solutions of TT in the presence of excess blank (unloaded) poly (D,L) lactide (mol. wt. 2000) microspheres led to a dramatic reduction in pH (approximately 3.2 units after one day at 45 degrees C) and simultaneous precipitation of TT. Stabilisation in the presence of the degrading polymer is likely to be the final obstacle before controlled-release preparations can be formulated to release antigenically active TT over extended time periods. Hence, mechanistic analyses as described here may be crucial for the development of effective single-dose vaccines.

Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form.

Upon the removal of water, proteins undergo a major, reversible rearrangement of their secondary structure, as revealed by FTIR spectroscopy. We have found herein that for recombinant human albumin (rHA) the extent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (protein concentration, pH, and the presence of excipients such as dextran or NaCl) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rHA's solid-state stability against moisture-induced aggregation. In all cases, the alpha-helix content of rHA drops from 58% in solution to 25-35% in the dehydrated state, the beta-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and confirmed that it too undergoes a significant alteration of the secondary structure upon lyophilization. The extent of this structural reorganization has been found to be insensitive to the pH of the aqueous solution prior to lyophilization from pH 1.9 to 5.1, even though the thermal transition temperature (Tm) in aqueous solution over this range varies by 30 degrees C.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

Aggregation of a lyophilized pharmaceutical protein, recombinant human albumin: effect of moisture and stabilization by excipients.

In the presence of water vapor at 37 degrees C, lyophilized recombinant human albumin (rHA) undergoes intermolecular thiol-disulfide interchange, eventually forming high-molecular-weight, water-insoluble aggregates. The relationship between the extent of aggregation and the water content of the lyophilized protein was bell-shaped, with maximum aggregation (over 80% after one day) at approximately 50 g water per 100 g dry protein, corresponding to incubation at 96% relative humidity. Nineteen different excipients were co-lyophilized with rHA to test their ability to inhibit aggregation under these conditions. These compounds included low- and high-molecular-weight sugars, as well as various organic acids (amino, hydroxy, and aliphatic), and the simple inorganic salt sodium chloride. Seven of them afforded complete stabilization of rHA against moisture-induced aggregation. The stabilizing potency of the excipients correlated with their water-sorbing capability, presumably due to increasing the moisture level in the vicinity of rHA.

Solid-phase aggregation of proteins under pharmaceutically relevant conditions.

In order to successfully employ proteins as pharmaceuticals, it is essential to understand mechanistically the stability issues relevant to their formulation and delivery. Various deleterious processes may occur in protein formulations, thereby diminishing their therapeutic value. This review focuses upon one aspect of this problem, namely aggregation of solid proteins under pharmaceutically relevant conditions (elevated temperature and water activity). Strategies to pursue such studies are presented with an emphasis on a mechanistic analysis of aggregate formation. Both covalent and noncovalent aggregation pathways have been elucidated. Proteins that contain disulfide bonds as well as free thiol residues may aggregate via thiol-disulfide interchange. For proteins which contain disulfides but not free thiol residues, intermolecular disulfide bonding may still occur when intact disulfides undergo beta-elimination, yielding free thiols which can catalyze disulfide scrambling. Finally, proteins containing no cysteine/cystine residues may aggregate by other covalent pathways or by noncovalent routes. On the basis of these pathways, some rational stabilization strategies have been proposed and verified. Ultimately, application of this knowledge should lead to more stable and effective pharmaceutical protein formulations.

Moisture-induced aggregation of lyophilized insulin.

A critical problem in the storage and delivery of pharmaceutical proteins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies of protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, we have found that, when exposed to an environment simulating these conditions, lyophilized insulin undergoes both covalent and noncovalent aggregation. The covalent process has been elucidated to be intermolecular thiol-catalyzed disulfide interchange following beta-elimination of an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the insulin powder or by performing lyophilization and/or dissolution of insulin in alkaline media. The aggregation can be ameliorated by the presence of Cu2+, which presumably catalyzes the oxidization of free thiols. The water sorption isotherm for insulin reveals that the extent of aggregation directly correlates with the water uptake by the lyophilized insulin powder, thus pointing to the critical role of protein conformational mobility in the aggregation process.

Characterization of Amylolytic Enzyme Activities Associated with the Hyperthermophilic Archaebacterium Pyrococcus furiosus.

The hyperthermophilic archaebacterium Pyrococcus furiosus produces several amylolytic enzymes in response to the presence of complex carbohydrates in the growth medium. These enzyme activities, alpha-glucosidase, pullulanase, and alpha-amylase, were detected in both cell extracts and culture supernatants. All activities were characterized by temperature optima of at least 100 degrees C as well as a high degree of thermostability. The existence of this collection of activities in P. furiosus suggests that polysaccharide availability in its growth environment is a significant aspect of the niche from which it was isolated.