The Impact of Post-Processing Temperature on PLGA Microparticle Properties.

PURPOSE: Biodegradable poly(lactide-co-glycolide) (PLGA) microparticles loaded with either risperidone or naltrexone were prepared from an emulsification homogenization process. The objective of this study was to determine the impact the post-treatment temperature has on the properties and subsequent performance of the microparticles. METHODS: The post-treatment temperature of an ethanolic solution was characterized from 10 ~ 35ºC for the naltrexone and risperidone micropartilces. RESULTS: The wash temperature resulted in a typical triphasic in vitro release pattern at low wash temperatures or a biphasic pattern consisting of an elevated release rate at higher post-treatment temperatures. The post-treatment temperature largely influences the particle morphology, residual solvent levels, glass transition temperature, and drug loading and is molecule dependent, whereby these characteristics subsequently influence the drug release rate. CONCLUSION: The study highlights the importance of both the post-treatment process and control during manufacturing to obtain a formulation within the desired product profile.

Scanning Analysis of Sequential Semisolvent Vapor Impact To Study Naltrexone Release from Poly(lactide-co-glycolide) Microparticles.

Poly(lactide-co-glycolide) (PLGA)-based microparticle formulations have been a mainstay of long-acting injectable drug delivery applications for decades. Despite a long history of use, tools and techniques to analyze and understand these formulations are still under development. Recently, a new characterization method was introduced known as the surface analysis after sequential semisolvent impact using sequential semisolvent vapors. The vapor-based technique is named, for convenience, surface analysis of (semisolvent) vapor impact (SAVI). In the SAVI method, discretely controlled quantities of selected organic semisolvents in the vapor phase were applied to PLGA microparticles to track particle morphological changes by laser scanning confocal microscopy. Subsequently, the morphological images were analyzed to calculate mean peak height (Sa), core height (Sk), kurtosis (Sku), dale void volume (Vvv), the density of peaks (Spd), maximum height (Hm), and the shape ratio (Rs). Here, the SAVI method was applied to naltrexone-loaded microparticles manufactured internally and Vivitrol, a commercial formulation. SAVI analysis of these microparticles indicated that the two primary mechanisms controlling the naltrexone release were the formation of discrete, self-crystallized portions of naltrexone within the PLGA structure and the degradation of PLGA chains through nucleophilic substitution. The relatively higher amounts of naltrexone crystals resulted in prolonged release than lower amounts of crystals. Data from gel permeation chromatography, differential scanning calorimetry, and in vitro release measurements all point to the importance of naltrexone crystal formation. This study highlights the utility of SAVI for gaining further insights into the microstructure of PLGA formulations and using SAVI data to support research, product development, and quality control applications for microparticle formulations of pharmaceuticals.

Implications of particle size on the respective solid-state properties of naltrexone in PLGA microparticles.

A thorough understanding of the complexities in formulating and manufacturing polymeric microspheres is required for new and generic drug applications. Specifically, for an ANDA application for polymeric microsphere-based products, the applicant must meet Q1 (qualitative) and Q2 (quantitative) sameness, and in some cases, Q3 (e.g., microstructural) sameness. Herein, we report the naltrexone crystallinity in a PLGA microparticle system prepared from a dichloromethane-benzyl alcohol solvent system results in a crystallinity dependence as a function of microparticle size from the same batch - illustrating intrabatch microstructural variability. As the particle size increases, the crystallinity increases, with additional polymorphic forms more readily noted at the large particle sizes. Furthermore, during dissolution, a polymorphic transition and/or crystallization occurs at larger size fractions. This study highlights the importance of controlling the manufacturing parameters during microparticle formation, specifically solvent extraction and particle size control. Furthermore, with the approval of generic microparticles formulations on the horizon, this study highlights the importance of Q3, the same components in the same concentration with the same arrangement of matter, whereby microparticles can have varying microstructural properties across particle sizes from the same batch.

Transitioning from a lab-scale PLGA microparticle formulation to pilot-scale manufacturing.

The complexity of scale-up manufacturing of PLGA microparticles creates a significant challenge when transitioning from benchtop-scale formulation development into larger clinical scale batches. Minor changes in the initial formulation composition (e.g., PLGA molecular weight, solvent type, and drug concentration) and processing parameters (e.g., extraction kinetics and drying condition) during scale-up production can result in significantly different performance of the prepared microparticles. The objectives of the present study were to highlight the in vitro and in vivo performance of a candidate benchtop-scale batch created with a rotor-stator mixer, transitioned into an in-line manufacturing process at ~15× scale of a long-acting naltrexone formulation. Physicochemical properties (such as drug loading, residual benzyl alcohol content, and morphology) as well as the in vitro release characteristics of the prepared naltrexone microparticles between the benchtop-scale and in-line process pilot-scale were determined. The pharmacokinetics of the naltrexone microspheres were investigated using the rat model. The results demonstrate that while the morphologies of the particles were different from a visual assessment and slight differences were observed in the in vitro release profiles, the in vivo pharmacokinetics illustrate similar kinetics. Our study shows that scale-up production having the same drug release kinetics can be made by controlling the formulation and processing parameters.

Evolution of drug delivery systems: From 1950 to 2020 and beyond.

Modern drug delivery technology began in 1952 with the advent of the Spansule® sustained-release capsule technology, which can deliver a drug for 12 h after oral administration through an initial immediate dose followed by the remaining released gradually. Until the 1980s, oral and transdermal formulations providing therapeutic durations up to 24 h for small molecules dominated the drug delivery field and the market. The introduction of Lupron Depot® in 1989 opened the door for long-acting injectables and implantables, extending the drug delivery duration from days to months and occasionally years. Notably, the new technologies allowed long-term delivery of peptide and protein drugs, although limited to parenteral administration. The introduction of the first PEGylated protein, Adagen®, in 1990 marked the new era of PEGylation, resulting in Doxil® (doxorubicin in PEGylated liposome) in 1995, Movantik® (PEGylated naloxone - naloxegol) in 2014, and Onpattro® (Patisiran - siRNA in PEGylated lipid nanoparticle) in 2018. Drug-polymer complexes were introduced, e.g., InFed® (iron-dextran complex injection) in 1974 and Abraxane® (paclitaxel-albumin complex) in 2005. In 2000, both Mylotarg™ (antibody-drug conjugate - gemtuzumab ozogamicin) and Rapamune® (sirolimus nanocrystal formulation) were introduced. The year 2000 also marked the launching of the National Nanotechnology Initiative by the U.S. government, which was soon followed by the rest of the world. Extensive work on nanomedicine, particularly formulations designed to escape from endosomes after being taken by tumor cells, along with PEGylation technology, ultimately resulted in the timely development of lipid nanoparticle formulations for COVID-19 vaccine delivery in 2020. While the advances in drug delivery technologies for the last seven decades are breathtaking, they are only the tip of an iceberg of technologies that have yet to be utilized in an approved formulation or even to be discovered. As life expectancy continues to increase, more people require long-term care for various diseases. Filling the current and future unmet needs requires innovative drug delivery technologies to overcome age-old familiar hurdles, e.g., improving water-solubility of poorly soluble drugs, overcoming biological barriers, and developing more efficient long-acting depot formulations. The lessons learned from the past are essential assets for developing future drug delivery technologies implemented into products. As the development of COVID-19 vaccines demonstrated, meeting the unforeseen crisis of the uncertain future requires continuous cumulation of failures (as learning experiences), knowledge, and technologies. Conscious efforts of supporting diversified research topics in the drug delivery field are urgently needed more than ever.

Initial Formation of the Skin Layer of PLGA Microparticles.

Poly(lactide-co-glycolide) (PLGA) has been extensively used in making long-acting injectable formulations. The critical factors affecting the PLGA formulation properties have been adjusted to control the drug release kinetics and obtain desirable properties of PLGA-based drug delivery systems. The PLGA microparticle formation begins as soon as the drug/PLGA-dissolved in the organic solvent phase (oil phase) is exposed to the water phase. The initial skin (or shell) formation on the oil droplets occurs very quickly, sometimes in the matter of milliseconds, and studying the process has been difficult. The skin formation on the PLGA emulsion droplet surface that can affect the subsequent hardening steps is examined. PLGA droplets with different compositions are prepared. Using collimated light and a high-speed camera made it possible to detect the diffusion of acetonitrile from the oil phase into the water phase during the oil droplet formation. Although the skin formation is not visible on the surface of the oil phase droplet with the current setup, the droplet shapes, solid strand formation, and the difference in the spreading time suggest that the initial contact time between the oil and water phases in the range of a few seconds is critical to the properties of the skin.

Engineering Quick- and Long-acting Naloxone Delivery Systems for Treating Opioid Overdose.

PURPOSE: Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick- and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. METHODS: The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 µm and 50 µm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with ß-arrestin recruitment assays. RESULTS: The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 µm to 23 µm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 µm to 23 µm helped eliminate the lag phase and obtain the steady-state drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a µ-opioid receptor, to recruit ß-arrestin 2. CONCLUSIONS: Naloxone released from the three selected formulations could inhibit DAMGO-induced ß-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration.

Coupling the in vivo performance to the in vitro characterization of PLGA microparticles.

The main objective of the study was to determine if rodent housing conditions, specifically housing climate, could impact the in vivo performance of poly(lactide-co-glycolide) (PLGA) microspheres through temperature modification of the subcutaneous space. Vivitrol®, a once monthly naltrexone injectable suspension, was chosen as a model PLGA microparticle formulation for this study. Two lots of Vivitrol were used to ascertain any potential differences that may exist between the batches and if in vitro characterization techniques could delineate any variation(s). The pharmacokinetics of the naltrexone-PLGA microparticles were determined in the rodent model under two different housing climates (20 vs. 25 °C). The results demonstrate that such difference in housing temperature resulted in a change in subcutaneous temperature but actually within a narrow range (36.31-36.77 °C) and thus minimally influenced the in vivo performance of subcutaneously injected microparticles. The shake-flask method was used to characterize the in vitro release at 35, 36, and 37 °C and demonstrated significant differences in the in vitro release profiles across this range of temperatures. Minimal differences in the in vitro characterization of the two lots were found. While these results did not provide statistical significance, the local in vivo temperature may be a parameter that should be considered when evaluating microparticle performance. The IVIVCs demonstrate that in vitro release at 37 °C may not accurately represent the in vivo conditions (i.e., subcutaneous space in rodents), and in certain instances lower in vitro release temperatures may more accurately represent the in vivo microenvironment and provide better correlations. Future studies will determine the extent temperature and specifically co-housing, may have on the relative impact of the in vivo performance of injectable polymeric microparticles based upon the significant differences observed in the in vitro release profiles across the range of 35-37 °C.

Potential Roles of the Glass Transition Temperature of PLGA Microparticles in Drug Release Kinetics.

Poly(lactic-co-glycolic acid) (PLGA) has been used for long-acting injectable drug delivery systems for more than 30 years. The factors affecting the properties of PLGA formulations are still not clearly understood. The drug release kinetics of PLGA microparticles are influenced by many parameters associated with the formulation composition, manufacturing process, and post-treatments. Since the drug release kinetics have not been explainable using the measurable properties, formulating PLGA microparticles with desired drug release kinetics has been extremely difficult. Of the various properties, the glass transition temperature, Tg, of PLGA formulations is able to explain various aspects of drug release kinetics. This allows examination of parameters that affect the Tg of PLGA formulations, and thus, affecting the drug release kinetics. The impacts of the terminal sterilization on the Tg and drug release kinetics were also examined. The analysis of drug release kinetics in relation to the Tg of PLGA formulations provides a basis for further understanding of the factors controlling drug release.

Formulation composition, manufacturing process, and characterization of poly(lactide-co-glycolide) microparticles.

Injectable long-acting formulations, specifically poly(lactide-co-glycolide) (PLGA) based systems, have been used to deliver drugs systemically for up to 6 months. Despite the benefits of using this type of long-acting formulations, the development of clinical products and the generic versions of existing formulations has been slow. Only about two dozen formulations have been approved by the U.S. Food and Drug Administration during the last 30 years. Furthermore, less than a dozen small molecules have been incorporated and approved for clinical use in PLGA-based formulations. The limited number of clinically used products is mainly due to the incomplete understanding of PLGA polymers and the various variables involved in the composition and manufacturing process. Numerous process parameters affect the formulation properties, and their intricate interactions have been difficult to decipher. Thus, it is necessary to identify all the factors affecting the final formulation properties and determine the main contributors to enable control of each factor independently. The composition of the formulation and the manufacturing processes determine the essential property of each formulation, i.e., in vivo drug release kinetics leading to their respective pharmacokinetic profiles. Since the pharmacokinetic profiles can be correlated with in vitro release kinetics, proper in vitro characterization is critical for both batch-to-batch quality control and scale-up production. In addition to in vitro release kinetics, other in vitro characterization is essential for ensuring that the desired formulation is produced, resulting in an expected pharmacokinetic profile. This article reviews the effects of a selected number of parameters in the formulation composition, manufacturing process, and characterization of microparticle systems. In particular, the emphasis is focused on the characterization of surface morphology of PLGA microparticles, as it is a manifestation of the formulation composition and the manufacturing process. Also, the implication of the surface morphology on the drug release kinetics is examined. The information described here can also be applied to in situ forming implants and solid implants.

Interfacial tension effects on the properties of PLGA microparticles.

Many types of long-acting injectables, including in situ forming implants, preformed implants, and polymeric microparticles, have been developed and ultimately benefited numerous patients. The advantages of using long-acting injectables include greater patient compliance and more steady state drug plasma levels for weeks and months. However, the development of long-acting polymeric microparticles has been hampered by the lack of understanding of the microparticle formation process, and thus, control of the process. Of the many parameters critical to the reproducible preparation of microparticles, the interfacial tension (IFT) effect is an important factor throughout the process. It may influence the droplet formation, solvent extraction, and drug distribution in the polymer matrix, and ultimately drug release kinetics from the microparticles. This mini-review is focused on the IFT effects on drug-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles.

Continuous in-line homogenization process for scale-up production of naltrexone-loaded PLGA microparticles.

Injectable, long-acting drug delivery systems provide effective drug concentrations in the blood for up to 6 months. Naltrexone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared using an in-line homogenization method. It allows the transition from a laboratory scale to scale-up production. This research was designed to understand how the processing parameters affect the properties of the microparticles, such as microparticle size distributions, surface and internal morphologies, drug loadings, and drug release kinetics, and thus, to control them. The in-line homogenization system was used at high flow rates for the oil- and water-phases, e.g., 100 mL/min and 400 mL/min, respectively, to continuously generate microparticles. A high molecular weight (148 kDa) PLGA at various concentrations was used to generate oil-phases with a range of viscosities and also to compare with a 64 and 79 kDa at a single, high concentration. The uniformity of the microparticles was found to be related to the viscosity of the oil-phase. As the viscosity of the oil-phase increased from 52.6 mPa∙s to 4046 mPa∙s, the span value (a measure of uniformity) increased from 1.24 to 3.1 for the microparticles generated at the homogenization speed of 2000 RPM. Increasing the PLGA concentration from 5.58% to 16.85% showed a corresponding rise in the encapsulation efficiency from 74.0% to 85.8% and drug loading (DL) from 27.4% to 31.7% for the microparticles made with the homogenization speed of 2000 RPM. These increases may be due to a faster shell formulation, enabling PLGA microparticles to entrap more naltrexone into the structure. A higher DL, however, shortened the drug release duration from 56 to 42 days. The changes in morphology of the microparticles during different phases of the in vitro release study were also studied for three types of microparticles made with different PLGA concentrations and molecular weights. As PLGA microparticles went through structural changes, the surface showed raisin-like wrinkled morphologies within the first 10 days. Then, the microparticles swelled to form smooth surfaces. The in-line approach produced PLGA microparticles with a highly reproducible size distribution, DL, and naltrexone release rate.

Injectable, long-acting PLGA formulations: Analyzing PLGA and understanding microparticle formation.

Injectable, long-acting depot formulations based on poly(lactide-co-glycolide) (PLGA) have been used clinically since 1989. Despite 30 years of development, however, there are only 19 different drugs in PLGA formulations approved by the U.S. Food and Drug Administration (FDA). The difficulty in developing depot formulations stems in large part from the lack of a clear molecular understanding of PLGA polymers and a mechanistic understanding of PLGA microparticles formation. The difficulty is readily apparent by the absence of approved PLGA-based generic products, limiting access to affordable medicines to all patients. PLGA has been traditionally characterized by its molecular weight, lactide:glycolide (L:G) ratio, and end group. Characterization of non-linear PLGA, such as star-shaped glucose-PLGA, has been difficult due to the shortcomings in analytical methods typically used for PLGA. In addition, separation of a mixture of different PLGAs has not been previously identified, especially when only their L:G ratios are different while the molecular weights are the same. New analytical methods were developed to determine the branch number of star-shaped PLGAs, and to separate PLGAs based on L:G ratios regardless of the molecular weight. A deeper understanding of complex PLGA formulations can be achieved with these new characterization methods. Such methods are important for further development of not only PLGA depot formulations with controllable drug release kinetics, but also generic formulations of current brand-name products.

Prevention of Opioid Abuse and Treatment of Opioid Addiction: Current Status and Future Possibilities.

Prescription opioid medications have seen a dramatic rise in misuse and abuse, leading regulators and scientists to develop policies and abuse-deterrent technologies to combat the current opioid epidemic. These abuse-deterrent formulations (ADFs) are intended to deter physical and chemical tampering of opioid-based products, while still providing safe and effective delivery for therapeutic purposes. Even though formulations with varying abuse-deterrent technologies have been approved, questions remain about their effectiveness. While these formulations provide a single means to combat the epidemic, a greater emphasis should be placed on formulations for treatment of addiction and overdose to help those struggling with opioid dependence. This article analyzes various ADFs currently in clinical use and explores potential novel systems for treatment of addiction and prevention of overdose.

Liquid crystalline drug delivery vehicles for oral and IV/subcutaneous administration of poorly soluble (and soluble) drugs.

Poorly soluble drug molecules often have low bioavailability issues and absorption problems in the clinical setting. As the number of poorly soluble drugs increases from discovery, developing technologies to enhance their solubility, while also controlling their release is one of the many challenges facing the pharmaceutical industry today. Liquid crystalline systems, nanoparticulate or macro-matrix, self-assemble in the presence of an aqueous environment and can provide a solubility enhancement, while also controlling the drug release rate. This review examines the fundamentals of liquid crystalline systems through the representative literature, concluding with examples of liquid crystalline systems in clinical trials development. The review focus is on the potential of utilizing liquid crystalline systems for poorly soluble drugs, in the areas of oral delivery and IV/subcutaneous, followed by water soluble molecules. Key considerations in utilizing liquid crystalline systems advantages while also discussing potential areas of key research that may be needed will be highlighted.

The in vivo transformation and pharmacokinetic properties of a liquid crystalline drug delivery system.

A liquid crystalline (LC) system, composed of phosphatidylcholine, sorbitan monoleate, and tocopherol acetate, was investigated to understand the in vivo transformation after subcutaneous injection, coupled with the physicochemical and pharmacokinetic properties of the formulation. The rat model was utilized to monitor a pseudo-time course transformation from a precursor LC formulation to the LC matrix, coupled with the blood concentration profiles of the formulations containing leuprolide acetate. Three formulations that result in the HII phase, demonstrating dissimilar in vitro release profiles, were used. The formulation showing the highest AUC, Cmax and Tmax, also displayed the greatest release rate in vitro, the lowest viscosity (LC matrix), and an earlier transformation (LC precursor to matrix) in vivo. A potential link between viscosity, phase transformation, and drug release properties of a liquid crystalline system is described.

Formulation and characterization of a liquid crystalline hexagonal mesophase region of phosphatidylcholine, sorbitan monooleate, and tocopherol acetate for sustained delivery of leuprolide acetate.

Although liquid crystal (LC) systems have been studied before, their utility in drug delivery applications has not been explored in depth. This study examined the development of a 1-month sustained release formulation of leuprolide acetate using an in situ-forming LC matrix. The phase progression upon water absorption was tested through construction of ternary phase diagrams of phosphatidylcholine, sorbitan monooleate, and tocopherol acetate (TA) at increasing water content. Small angle X-ray scattering revealed the presence of lamellar and hexagonal mesophases. The physicochemical characteristics and in vitro drug release were evaluated as a function of the ternary component ratio and its resultant phase behavior. Formulations with increased water uptake capacity displayed greater drug release and enhanced erodability. Removal of TA resulted in increased water uptake capacity and drug release, where 8% (w/w) TA was determined as the critical concentration threshold for divergence of release profiles. In conclusion, characterization of the resultant HII mesophase region provided information of the impact the individual components have on the physicochemical properties and potential drug release mechanisms. This high mitigating impact of TA on drug release indicates the use of TA as a tailoring agent, broadening the therapeutic applications of this LC system.

A Fast and Sensitive Method for the Detection of Leuprolide Acetate: A High-Throughput Approach for the In Vitro Evaluation of Liquid Crystal Formulations.

The suitability of using fluorescence spectroscopy to rapidly assay drug release by quantifying the time-dependent increase in total intrinsic protein fluorescence was assessed. Leuprolide acetate, a synthetic nonapeptide analogue of gonadotropin-releasing hormone (GnRH or LHRH), is the active pharmaceutical ingredient used to treat a wide range of sex hormone-related disorders, including advanced prostatic cancer, endometriosis, and precocious puberty. During the in vitro evaluation of drug delivery technologies for leuprolide acetate, one of the most time-consuming steps is the detection and accurate quantification of leuprolide release from formulation candidates. Thus far, the dominant means for leuprolide detection involves conventional multistep high-performance liquid chromatography (HPLC) methods, requiring sampling, dilutions, sample filtration, and chromatography, which can take up to 40 min for each sample. With the increasing demand for assay adaptation to high-throughput format, here we sought to exploit fluorescence spectroscopy as a tool to develop a novel method to rapidly assay the in vitro release of leuprolide acetate. By utilizing the intrinsic fluorescence of the tryptophan (Trp) and tyrosine (Tyr) amino acid residues present in the leuprolide nonapeptide, the in vitro release from liquid crystal formulations was accurately quantified as a function of fluorescence intensity. Here, we demonstrate that assaying leuprolide release using intrinsic protein fluorescence in a 96-well format requiring volumes as low as 100 µL is a cost-effective, rapid, and highly sensitive alternative to conventional HPLC methods. Furthermore, the high signal-to-noise ratios and robust Z'-factors of >0.8 indicate high sensitivity, precision, and feasibility for miniaturization, high-throughput format adaptation, and automation.

Investigation of Film with ß-Galactosidase Designed for Stabilization and Handling in Dry Configuration.

Gelatin-based films with an immobilized enzyme designed for extending the stability of the protein in dry, non-powder configuration with precise dosing attributes were subjected to stress conditions of temperature and relative humidity. ß-galactosidase was used as model functional protein. The film configuration preserved the activity of the enzyme under the different storage conditions investigated, which include room temperature under low (ambient) and high (75%) relative humidity, and 36 °C under low (oven) and high relative humidity conditions for a period of 46 days. The influence of the enzyme and plasticizer (glycerol) on the physical and mechanical properties of the films was investigated using DMA (dynamic mechanical analysis). Films containing 5% ß-galactosisdase and glycerol concentrations of 14% or greater exhibited greater tensile strength, Young's modulus, and elongation at break than films with equal concentrations of plasticizer but devoid of any enzyme. The surface texture of the films was analyzed using scanning electron microscopy (SEM). ß-galactosidase and glycerol have opposite effects on the surface morphology of the films. Increasing concentrations of the enzyme result in rougher film surface, whereas increasing the concentration of glycerol leads to films with denser and smoother surface. The results obtained suggest that the dry film configuration approach can help in facilitating the stabilization, handling, storage, and transportation of functional proteins in a cost effective manner.