Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development

The purpose of this review is to demonstrate the critical importance of understanding protein-excipient interactions as a key step in the rational design of formulations to stabilize and deliver protein-based therapeutic drugs and vaccines. Biophysical methods used to examine various molecular interactions between solutes and protein molecules are discussed with an emphasis on applications to pharmaceutical excipients in terms of their effects on protein stability. Key mechanisms of protein-excipient interactions such as electrostatic and cation-pi interactions, preferential hydration, dispersive forces, and hydrogen bonding are presented in the context of different physical states of the formulation such as frozen liquids, solutions, gels, freeze-dried solids and interfacial phenomenon. An overview of the different classes of pharmaceutical excipients used to formulate and stabilize protein therapeutic drugs is also presented along with the rationale for use in different dosage forms including practical pharmaceutical considerations. The utility of high throughput analytical methodologies to examine protein-excipient interactions is presented in terms of expanding formulation design space and accelerating experimental timelines.     

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

Purpose

The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported.

Methods

The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively.

Results

Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D₂O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing.

Conclusions

The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.

     

Interference from Proteins and Surfactants on Particle Size Distributions Measured by Nanoparticle Tracking Analysis (NTA)

Purpose

Characterization of submicron protein particles continues to be challenging despite active developments in the field. NTA is a submicron particle enumeration technique, which optically tracks the light scattering signal from suspended particles undergoing Brownian motion. The submicron particle size range NTA can monitor in common protein formulations is not well established. We conducted a comprehensive investigation with several protein formulations along with corresponding placebos using NTA to determine submicron particle size distributions and shed light on potential non-particle origin of size distribution in the range of approximately 50–300 nm.

Methods

NTA and DLS are performed on polystyrene size standards as well as protein and placebo formulations.

Results

Protein formulations filtered through a 20 nm filter, with and without polysorbate-80, show NTA particle counts. As such, particle counts above 20 nm are not expected in these solutions. Several other systems including positive and negative controls were studied using NTA and DLS.

Conclusions

These apparent particles measured by NTA are not observed in DLS measurements and may not correspond to real particles. The intent of this article is to raise awareness about the need to interpret particle counts and size distribution from NTA with caution.

     

Interactions of formulation excipients with proteins in solution and in the dried state

A variety of excipients are used to stabilize proteins, suppress protein aggregation, reduce surface adsorption, or to simply provide physiological osmolality. The stabilizers encompass a wide variety of molecules including sugars, salts, polymers, surfactants, and amino acids, in particular arginine. The effects of these excipients on protein stability in solution are mainly caused by their interaction with the protein and the container surface, and most importantly with water. Some excipients stabilize proteins in solution by direct binding, while others use a number of fundamentally different mechanisms that involve indirect interactions. In the dry state, any effects that the excipients confer to proteins through their interactions with water are irrelevant, as water is no longer present. Rather, the excipients stabilize proteins through direct binding and their effects on the physical properties of the dried powder. This review will describe a number of mechanisms by which the excipients interact with proteins in solution and with various interfaces, and their effects on the physical properties of the dried protein structure, and explain how the various interaction forces are related to their observed effects on protein stability.     

Manipulating Aggregation Behavior of the Uncharged Peptide Carbetocin

Peptides are usually administered through subcutaneous injection. For low potency drugs, this may require high concentration formulations increasing the risk of peptide aggregation, especially for compounds without any intrinsic chargeable groups. Carbetocin was used as a model to study the behavior of uncharged peptides at high concentrations. Manipulation of the aggregation behavior of 70 mg/mL carbetocin was attempted by selecting excipients which interact with hydrophobic groups in carbetocin, and cover hydrophobic surfaces and interfaces. Peptide aggregation was induced by shaking stress and followed over time. Carbetocin solutions showed significant visible particle formation already after 4 h of shaking stress. This particle formation was not due to supersaturation or phase separation but suggested a nucleated aggregation process. None of the excipients prevented carbetocin aggregation, though altered aggregation behavior was observed, such as induction of fibril formation for most, but not all, charged excipients. Sodium dodecyl sulfate was found to accelerate peptide aggregation both below and above the critical micelle concentration in half-filled vials. However, in the absence of an air headspace, sodium dodecyl sulfate above the critical micelle concentration was capable of preventing shaking-induced carbetocin aggregation. Our study highlights the complexity in rational excipient selection to stabilize uncharged peptides at high concentration.     

纳米颗粒跟踪分析和纳米流式细胞仪对脂肪间充质干细胞的细胞外囊泡检测结果评价

目的 探讨纳米颗粒跟踪分析（nanoparticle tracking analysis,NTA）和纳米流式细胞仪（nano-flow cytometry,nanoFCM）对对照品微球和脂肪间充质干细胞（adipose mesenchymal stem cells,AdMSCs）的细胞外囊泡（extracellular vesicles,EVs）的检测能力,为EVs的鉴定和质量控制提供依据。方法 首先使用对照品微球来验证NTA和nanoFCM的检测能力。随后,对于聚乙二醇6000沉淀-超速离心法制备的AdMSCs-EVs,通过Western blotting和电子透射显微镜进行一般特征鉴定后,使用NTA和nanoFCM来检测其粒径分布和PKH67染色后的荧光颗粒占比。结果 NTA和nanoFCM在颗粒浓度方面的检测能力相近,但是nanoFCM的精度更高,粒径区分度更好。用NTA检测EVs的检测结果显示NTA粒径分布较广,nanoFCM粒径分布较窄;NTA的PKH67的荧光检测阳性率显著低于nanoFCM。结论 NanoFCM拥有较高的精度,但也存在着一定应用局限性。NTA的粒径区分度相对较差,但是可检测的粒径范围更广。因此,NTA可以满足EVs的粒径分布检测,对荧光检测具有较高灵敏度的nanoFCM更适合分析EVs的表面标志物比例。     

Physical characterization of HPMC and HEC and investigation of their use as pelletization aids

Pelletization of drugs with suitable excipients by extrusion/spheronization is one of the popular approaches in the development of solid dosage forms, especially for extended-release formulations. However, the choice of pelletization aids is limited to the use of microcrystalline cellulose (MCC), which is generally wet massed using water. In the formulation of water-sensitive drugs, however, pelletization excipients which may be wet massed using an organic liquid are desired. In the present study therefore, two cellulose ethers, hydroxypropyl methylcellulose (HPMC) and hydroxyethyl cellulose (HEC), were characterized for their physical properties, such as moisture content, bulk and tapped densities, median particle size and size distribution, surface area and surface morphology, in an attempt to evaluate the feasibility of their use as pelletization aids. Since HPMC and HEC are water-soluble, but insoluble in isopropyl alcohol, the latter was used as the wet massing liquid for pelletization. Microcrystalline cellulose, because of its established uniqueness as a pellet former, was used as the reference material and was also wet massed using isopropyl alcohol for consistency and comparison. Placebo pellets of the three cellulosic excipients, HPMC, HEC and MCC, were prepared by extrusion/spheronization. The placebo pellets were evaluated for their size and size distribution, hardness, friability, bulk and tapped densities and sphericity. Of the three cellulosic excipients, HPMC and MCC produced pellets with the most desirable attributes. In water as the dissolution medium, the HPMC pellets absorbed water and formed a single viscous gel matrix that slowly dissolved. HEC pellets were swollen but intact and slowly eroded, whereas MCC pellets stayed intact without dissolution or erosion. These findings indicate that HPMC will find application in pellet formulations of water-sensitive drugs, as well as in those formulations where water may not be used as wet massing liquid and an organic liquid must be used. It will also be a good choice as a pelletization excipient when complete water-solubility of all the formulation excipients is desired. It may be anticipated that modification of drug release from pelletized formulations may also be achieved by using cellulose ethers of varying viscosity grades and hydration rates.     

Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors

Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. This review provides a comprehensive analysis of upstream, downstream and formulation unit operation challenges encountered during AAV vector manufacturing, and discusses how desired product quality attributes can be maintained throughout product shelf-life by understanding the degradation mechanisms and formulation strategies. The mechanisms of various physical and chemical instabilities that the viral vector may encounter during its production and shelf-life because of various stressed conditions such as thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH, excipients, and impurities on the stability of viral vectors is also discussed. As such, the aim of this review is to outline the tools and a potential roadmap for improving the quality of AAV-based drug products by stressing the need for a mechanistic understanding of the involved processes.     

Use of microcalorimetry and its correlation with size exclusion chromatography for rapid screening of the physical stability of large pharmaceutical proteins in solution

The utility of microcalorimetry as a rapid screening tool for assessing the solution stability of high molecular weight pharmaceutical proteins was evaluated by using model recombinant antibodies, Protein I and Protein II. Changes in the transition midpoint, T(m), were monitored as a function of pH and/or in the presence of excipients, and results were compared with traditional accelerated stability data from samples that were analyzed by size exclusion chromatography (SEC). The data from microcalorimetry were well correlated with those from SEC for predicting both optimal solution pH as well as excipient effects on solution stability. These results indicate that microcalorimetry can be an efficient screening tool useful in identifying optimal pH conditions and excipients to stabilize pharmaceutical proteins in solution formulations.     

药物制剂中药物与辅料相互作用的研究进展

药物制剂中药物与辅料间的相互作用对药物的溶解度、稳定性、生物利用度等方面具有正向或负向的影响.因此,在药物制剂研发过程中,对药物与辅料间、辅料与辅料间相互作用的考察不可缺少.本文就近年来国内外固体制剂、液体制剂和载体给药系统中的原辅料相互作用以及用于相容性研究的分析方法等研究进展进行综述,为药物制剂研究中的处方筛选及剂...     

Caffeine as a Viscosity Reducer for Highly Concentrated Monoclonal Antibody Solutions

Many monoclonal antibody (mAb) solutions exhibit high viscosity at elevated concentrations, which prevents manufacturing and injecting of concentrated mAb drug products at the small volumes needed for subcutaneous (SC) administration. Addition of excipients that interrupt intermolecular interactions is a common approach to reduce viscosity of high concentration mAb formulations. However, in some cases widely used excipients can fail to lower viscosity. Here, using infliximab and ipilimumab as model proteins, we show that caffeine effectively lowers the viscosity of both mAb formulations, whereas other common viscosity-reducing excipients, sodium chloride and arginine, do not. Furthermore, stability studies under accelerated conditions show that caffeine has no impact on stability of lyophilized infliximab or liquid ipilimumab formulations. In addition, presence of caffeine in the formulations does not affect in vitro bioactivities of infliximab or ipilimumab. Results from this study suggest that caffeine could be a useful viscosity reducing agent that complements other traditional excipients and provides viscosity reduction to a wider range of mAb drug products.     

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ～70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ～50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from ?80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at ?20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.     

Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.     

Mechanistic studies of glass vial breakage for frozen formulations. I. Vial breakage caused by crystallizable excipient mannitol

The process of freeze-thaw not only subjects bioproducts to potentially destabilizing stress, but also imposes challenges to retain container integrity. Shipment and storage of frozen products in glass vials and thawing of the vials prior to use at clinics is a common situation. Vial integrity failure during freeze-thaw results in product loss and safety issues. Formulations of biomolecules often include crystallizable excipients, which can cause glass vial breakage during freeze-thaw operations. In this study, mannitol formulations served as models for mechanistic investigation of root causes for vial breakage. Several parameters and their impacts on vial breakage were investigated, including mannitol concentration (5% and 15%), different freeze-thaw conditions (fast, slow, and staging), fill configurations (varying fill volume/vial size ratio), and vial tray materials (plastic, stainless steel, corrugated cardboard, aluminum, and polyurethane foam). The results in this study were subjected to a statistical proportion test. The data showed that large fill volumes strongly correlated with higher percentage of vial cracks. Furthermore, the 15% mannitol was found to cause more breakage than 5% mannitol, especially with fast temperature gradient. Significantly more thawing vial breakage occurred in the fast compared to slow freeze-thaw with all types of vial trays. The freezing breakage was substantially lower than the thawing breakage using the fast temperature gradient, and the trend was reversed with the slow temperature gradient. An intermediate hold at -30 degrees C prior to further decrease in temperature proved to be a practical approach to minimize mannitol-induced vial breakage. Thermal mechanical analysis (TMA) and strain gage techniques were employed to gain mechanistic insights, and it was found that the primary causes for mannitol-induced vial breakage were partial crystallization during freezing and "secondary" crystallization of non-crystallized fraction during thawing. The strain on the vial's axial direction was significantly higher than the hoop direction, typically resulting in bottom lens of the vial coming off. Without a -30 degrees C hold, rapid volume expansions due to initial crystallization and secondary crystallization of mannitol were observed in TMA profiles, and these expansions were more apparent in 15% mannitol compared to 5% mannitol. With the introduction of a -30 degrees C hold step, abrupt expansions diminished in TMA profiles, suggesting that most of the mannitol crystallization occurred concurrently with ice solidification during the -30 degrees C holding step and, thus, secondary crystallization during thawing was minimal and the sudden expansion event was eliminated. Therefore, vial breakage during both freezing and thawing was reduced.     

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches

Purpose

Characterizing submicron protein particles (approximately 0.1–1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).

Methods

Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment.

Results

A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4?×?10⁷-1?×?10¹¹ particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering.

Conclusion

RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

     

Mechanistic studies of glass vial breakage for frozen formulations. II. Vial breakage caused by amorphous protein formulations

In an accompanying article we have described parameters that influence vial breakage in freeze-thaw operations when using crystalizable mannitol formulations, and further provided a practical approach to minimize the breakage in manufacturing settings. Using two diagnostic tools-thermal mechanical analysis (TMA) and strain gage, we investigated the mechanism of mannitol vial breakage and concluded that the breakage is related to sudden volume expansions in the frozen plug due to crystallization events. Glass vial breakage has also been observed with a number of frozen protein formulations consisting of only amorphous ingredients. Therefore, in this study, we applied the methodologies and learnings from the prior investigation to further explore the mechanism of vial breakage during freeze-thaw of amorphous protein products. It was found that temperature is a critical factor, as breakage typically occurred when the products were frozen to -70 degrees C, while freezing only to -30 degrees C resulted in negligible breakage. When freezing to -70 degrees C, increased protein concentration and higher fill volume induced more vial breakage, and the breakage occurred mostly during freezing. In contrast to the previous findings for crystallizable formulations, an intermediate staging step at -30 degrees C did not reduce breakage for amorphous protein formulations, and even slightly increased the breakage rate. The TMA profiles revealed substantially higher thermal contraction of frozen protein formulations when freezing below -30 degrees C, as compared to glass. Such thermal contraction of frozen protein formulations caused inward deformation of glass and subsequent rapid movement of glass when the frozen plug separates from the vial. Increasing protein concentration caused more significant inward glass deformation, and therefore a higher level of potential energy was released during the separation between the glass and frozen formulation, causing higher breakage rates. The thermal expansion during thawing generated moderate positive strain on glass and explained the thaw breakage occasionally observed. The mechanism of vial breakage during freeze-thaw of amorphous protein formulations is different compared to crystallizable formulations, and accordingly requires different approaches to reduce vial breakage in manufacturing. Storing and shipping at no lower than -30 degrees C effectively prevents breakage of amorphous protein solutions. If lower temperature such as -70 degrees C is unavoidable, the risk of breakage can be reduced by lowering fill volume.     

Arginine as an Excipient for Protein Freeze-Drying: A Mini Review

Successful development of marketable freeze-dried protein formulations requires adequate stabilization of the active biopharmaceutical ingredient. The choice of a stabilizer must therefore be based on sound knowledge of the physical and chemical properties of the excipients and specific needs of the protein component. Amino acids, such as arginine, have exhibit cryo- and lyoprotective effects similar to those of sugars and polymers and may therefore be considered to be an alternative approach to these established formulation strategies. The chemical structure and physicochemical characteristics of arginine are unique among amino acids and can provide additional benefits to freeze-dried protein formulations with regard to liquid and solid-state stability. This mini review provides a brief summary of research focused on the application of arginine in freeze-dried protein pharmaceuticals, including a discussion of its basic physical and chemical attributes as well as thermal behavior in the frozen and solid states. Mechanisms contributing to solid-state stabilization by arginine are discussed in the context of available stability studies on arginine-containing protein formulations. This mini review seeks to deepen the understanding of the opportunities and challenges associated with arginine-based preparations for freeze-dried protein pharmaceuticals.     

Influence of Histidine on the Stability and Physical Properties of a Fully Human Antibody in Aqueous and Solid Forms

PURPOSE: The aim of the study was to investigate the effect of histidine on the stability and physical properties of a fully human anti-IL8 monoclonal antibody (ABX-IL8) in aqueous and solid forms. METHODS: Using a fractional factorial design, we tested many excipients, including histidine, sucrose, and other commonly used excipients, on the stability and physical properties of the antibody in both liquid and lyophilized forms. Antibody stability and physical properties were evaluated using size-exclusion high-performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a viscometer. Residual moisture content was determined by coulometric Karl Fischer titrator. Differential scanning calorimetry (DSC) was used to detect the glass transition temperatures (Tg) of the solid cakes and melting temperatures (Tm) of the antibody in liquid formulations. Fourier-transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure. RESULTS: Increasing the histidine concentration in the bulk solution inhibited the increases of high-molecular-weight (HMW) species and aggregates upon lyophilization and storage. In addition, histidine bulk enhanced solution stability of the antibody under freezing and thermal stress conditions, as evidenced by the lower levels of aggregates. Furthermore, histidine reduced viscosity of the antibody solution, which is desirable for the manufacture of the dosage form. However, high concentrations of histidine in liquid formulations led to coloration of the solution and high levels of aggregates on storage at elevated temperature (40 degrees C) after the formulations were exposed to stainless steel containers during bulk freezing-thawing. CONCLUSIONS: Histidine enhanced the stability of ABX-IL8 in both aqueous and lyophilized forms. Histidine also improved the physical properties such as reducing the solution viscosity. Liquid formulations containing high concentrations of histidine should not be stored in stainless steel tanks at elevated temperatures.     

Stable Formulations of Recombinant Human Growth Hormone and Interferon-γ for Microencapsulation in Biodegradable Mircospheres

Purpose. The successful development of controlled release formulations for proteins requires that the protein not be denatured during the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon- (rhIFN-), at high protein concentrations (>100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate. Methods. Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized, milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in poly-lactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. Results. The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA micro-spheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN- (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN- from PLGA microspheres. Conclusions. The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface. Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.