Amino acids as stabilizers for lysozyme during the spray-drying process and storage.

Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms by which they can stabilize a protein have not been fully elucidated yet. The purpose of this study was to investigate the effect of AAs with distinct physicochemical properties on the stability of a model protein (lysozyme, LZM) during the spray-drying process and subsequent storage. Molecular descriptor based multivariate data analysis was used to select distinct AAs from the group of 20 natural AAs. Then, LZM and the five selected AAs (1:1 wt ratio) were spray-dried (SD). The solid form, residual moisture content (RMC), hygroscopicity, morphology, secondary/tertiary structure and enzymatic activity of LZM were evaluated before and after storage under 40 °C/75 % RH for 30 days. Arginine (Arg), leucine (Leu), glycine (Gly), tryptophan (Trp), aspartic acid (Asp) were selected because of their distinct properties by using principal component analysis (PCA). The SD LZM powders containing Arg, Trp, or Asp were amorphous, while SD LZM powders containing Leu or Gly were crystalline. Recrystallization of Arg, Trp, Asp and polymorph transition of Gly were observed after the storage under accelerated conditions. The morphologies of the SD particles vary upon the different AAs formulated with LZM, implying different drying kinetics of the five model systems. A tertiary structural change of LZM was observed in the SD powder containing Arg, while a decrease in the enzymatic activity of LZM was observed in the powders containing Arg or Asp after the storage. This can be attributed to the extremely basic and acidic conditions that Arg and Asp create, respectively. This study suggests that when AAs are used as stabilizers instead of traditional disaccharides, not only do classic vitrification theory and water replacement theory play a role, but the microenvironmental pH conditions created by basic or acidic AAs in the starting solution or during the storage of solid matter are also crucial for the stability of SD protein products.

Formulation factors affecting foam properties during vacuum foam-drying.

This paper explores how vacuum foam-drying of a protein is influenced by formulation parameters by investigating the foam structure, physical properties of the foam, and the stability of the protein. Recombinant human bile salt-stimulated lipase was used as a model of a protein drug. The stability of the lipase was evaluated through activity measurements. Two disaccharides (sucrose and trehalose), strongly tending to an amorphous form, were used as matrix formers, and the physical properties were assessed through residual water content, glass transition temperature, and crystalline state. Moreover, some formulations included surfactants with different sizes and structures of the head group. The alkyl chain length was kept constant to only investigate the impact of the surfactant head group, in the presence of the lipase, on the foamability and surface coverage of the lipase. The study demonstrated that the lipase allowed for a dry, solid foam with a foam overrun of up to 2600 %. The wall thickness of the dry, solid foam was estimated to be 20-50 µm. Clear differences between sucrose and trehalose as matrix former were identified. The lipase showed no tendency to lose activity because of the drying and rehydration, despite a proportion of the lipase covering the surfaces of the dry material.

Co-formulations of adalimumab with hyaluronic acid/polyvinylpyrrolidone to combine intraarticular drug delivery and viscosupplementation.

Polymer-based formulations present an attractive strategy in intraarticular drug-delivery to refrain biologicals from early leakage from the joint. In this study, co-formulations of hyaluronic acid and polyvinylpyrrolidone were investigated for their potential as viscosupplements and their influence on the transsynovial loss of adalimumab. For this purpose, polymer mixtures were evaluated for their viscosity and elasticity behavior while their influence on the permeation of adalimumab across a porcine ex-vivo synovial membrane was determined. Hyaluronic acid showed strong shear thinning behavior and exhibited high viscosity and elasticity at low motions, while combinations with polyvinylpyrrolidone provided absorption and stiffness at high mechanical stress, so that they can potentially restore the rheological properties of the synovial fluid over the range of joint motion. In addition, the formulations showed significant influence on transsynovial permeation kinetics of adalimumab and hyaluronic acid, which could be decelerated up to 5- and 3-fold, respectively. Besides viscosity effects, adalimumab was retained primarily by an electrostatic interaction with hyaluronic acid, as detected by isothermal calibration calorimetry. Furthermore, polymer-mediated stabilization of the antibody activity was detected. In summary, hyaluronic acid - polyvinylpyrrolidone combinations can be efficiently used to prolong the residence of adalimumab in the joint cavity while simultaneously supplying viscosupplementation.

Assessment of joint pharmacokinetics and consequences for the intraarticular delivery of biologics.

Intraarticular (IA) injections provide the opportunity to deliver biologics directly to their site of action for a local and efficient treatment of osteoarthritis. However, the synovial joint is a challenging site of administration since the drug is rapidly eliminated across the synovial membrane and has limited distribution into cartilage, resulting in unsatisfactory therapeutic efficacy. In order to rationally develop appropriate drug delivery systems, it is essential to thoroughly understand the unique biopharmaceutical environments and kinetics in the joint to adequately simulate them in relevant experimental models. This review presents a detailed view on articular kinetics and drug-tissue interplay of IA administered drugs and summarizes how these can be translated into reasonable formulation strategies by identification of key factors through which the joint residence time can be prolonged and specific structures can be targeted. In this way, pros and cons of the delivery approaches for biologics will be evaluated and the extent to which biorelevant models are applicable to gain mechanistic insights and ameliorate formulation design is discussed.

Sustained In-Vivo Release of Triptorelin Acetate from a Biodegradable Silica Depot: Comparison to Pamorelin® LA.

Triptorelin acetate was encapsulated into silica microparticles by spray-drying a mixture of colloidal silica sol and triptorelin acetate solution. The resulting microparticles were then combined with another silica sol containing silica nanoparticles, which together formed an injectable silica-triptorelin acetate depot. The particle size and surface morphology of the silica-triptorelin acetate microparticles were characterized together with the in vitro release of triptorelin, injectability and rheology of the final injectable silica-triptorelin acetate depot. In vivo pharmacokinetics and pharmacodynamics of the silica-triptorelin acetate depot and Pamorelin® were evaluated and compared in Sprague-Dawley male rats after subcutaneous administration. Serum samples up to 91 days were collected and the plasma concentrations of triptorelin and testosterone were analyzed with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In vivo pharmacokinetics showed that injections of the silica-triptorelin acetate depot gave 5-fold lower Cmax values than the corresponding Pamorelin® injections. The depot also showed a comparable sustained triptorelin release and equivalent pharmacodynamic effect as the Pamorelin® injections. Detectable triptorelin plasma concentrations were seen with the depot after the 91-day study period and testosterone plasma concentrations remained below the human castration limit for the same period.

A cost-efficient low-weight autonomous profiler for measurements in polar coastal waters and other regions with strong density gradients.

The rapid warming of our planet has resulted in accelerated melting of ice in polar regions. Currently we have limited knowledge on how, where and when the surface meltwater layer is mixed with the underlying ocean due to lack of observations in these remote areas. We present a lightweight (17 kg) and low-cost (6000€) instrument for autonomous profiling across the strongly stratified upper layer in Arctic coastal waters, freshened by the riverine input and meltwater from glaciers, icebergs, and sea ice. The profiler uses a specially designed plunger buoyancy engine to displace up to 700 cm3 of water and allows for autonomous dives to 200 m depth. It can carry different sensor packages and convey its location by satellite communication. Two modes are available: (a) a free-floating mode and (b) a moored mode, where the instrument is anchored to the seafloor. In both modes, the profiler controls its velocity of 12 ± 0.3 cm/s resulting in 510 ± 22 data points per 100 m depth. Equipped with several sensors, e.g. conductivity, temperature, oxygen, and pressure, the autonomous profiler was successfully tested in a remote Northeast Greenlandic fjord. Data has been compared to traditional CTD instrument casts performed nearby.

Transcellular stomach absorption of a derivatized glucagon-like peptide-1 receptor agonist.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may decrease the permeability. In conclusion, acylated sCT acts as its own in vitro intestinal permeation enhancer, with reversible effects on Caco-2 cells, indicating that acylation of sCT may represent a promising tool to increase intestinal permeability without adding oral permeation enhancers.

Acylation of Glucagon-like peptide-2: interaction with lipid membranes and in vitro intestinal permeability.

BACKGROUND: Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation as well as increasing enzymatic stability without disrupting biological potency. Acylation has furthermore been shown to increase interactions with the lipid membranes of mammalian cells. The extent to which such interactions hinder or benefit delivery of acylated peptide drugs across cellular barriers such as the intestinal epithelia is currently unknown. The present study investigates the effect of acylating peptide drugs from a drug delivery perspective. PURPOSE: We hypothesize that the membrane interaction is an important parameter for intestinal translocation, which may be used to optimize the acylation chain length for intestinal permeation. This work aims to characterize acylated analogues of the intestinotrophic Glucagon-like peptide-2 by systematically increasing acyl chain length, in order to elucidate its influence on membrane interaction and intestinal cell translocation in vitro. RESULTS: Peptide self-association and binding to both model lipid and cell membranes was found to increase gradually with acyl chain length, whereas translocation across Caco-2 cells depended non-linearly on chain length. Short and medium acyl chains increased translocation compared to the native peptide, but long chain acylation displayed no improvement in translocation. Co-administration of a paracellular absorption enhancer was found to increase translocation irrespective of acyl chain length, whereas a transcellular enhancer displayed increased synergy with the long chain acylation. CONCLUSIONS: These results show that membrane interactions play a prominent role during intestinal translocation of an acylated peptide. Acylation benefits permeation for shorter and medium chains due to increased membrane interactions, however, for longer chains insertion in the membrane becomes dominant and hinders translocation, i.e. the peptides get 'stuck' in the cell membrane. Applying a transcellular absorption enhancer increases the dynamics of membrane insertion and detachment by fluidizing the membrane, thus facilitating its effects primarily on membrane associated peptides.

Modulating protein release profiles by incorporating hyaluronic acid into PLGA microparticles Via a spray dryer equipped with a 3-fluid nozzle.

PURPOSE: The purpose of this study was to modulate the release profiles of the model protein drug from spray dried poly(DL-lactic-co-glycolic acid) (PLGA) microparticles by incorporating hyaluronic acid (HA) in the formulation. METHODS: Bovine serum albumin (BSA)-loaded PLGA microparticles with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron microscopy (SEM), laser diffraction (LD), confocal laser scanning microscopy (CLSM) and X-ray photoelectron spectroscopy (XPS). Circular dischoism (CD) was used to characterize conformational integrity of BSA released from the microparticles. RESULTS: Spherical microparticles with D50 of 5-10 µm were obtained. Addition of HA in inner feed solutions increased the feed viscosity, but with no influence on the surface tension. All inner feed solutions showed non-Newtonian shear thinning behavior and the rheological properties were not time dependent. The CLSM and XPS analyses suggested a core-shell like structure of the microparticles when HA was incorporated. The release profiles of BSA were extended and the initial burst releases were suppressed with an increase in HA in the microparticle formulations. In addition, HA seemed to protect BSA from degradation upon the spray-drying process. CONCLUSIONS: The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle.

One-step production of protein-loaded PLGA microparticles via spray drying using 3-fluid nozzle.

PURPOSE: The aim of this study was to investigate the potential of using a spray-dryer equipped with a 3-fluid nozzle to microencapsulate protein drugs into polymeric microparticles. METHODS: Lysozyme and PLGA were used as a model protein and a model polymer, respectively. The effects of process and formulation variables, such as i) the type of organic solvent, ii) the feeding rate ratio of the outer PLGA-containing feed solution to inner lysozyme-containing feed solution, and iii) the mass ratio of PLGA to protein, on the properties (morphology, internal structure, protein surface enrichment and release profiles) of the spray dried microparticles were investigated to understand protein microencapsulation and particle formation mechanisms. RESULTS: The spherical, condensed microparticles were obtained with D50 of 1.07-1.60 µm and Span in the range of 0.82-1.23. The lysozyme surface content decreased upon different organic solvents used as follows: acetonitrile > acetone > dichloromethane. Additionally, the lysozyme surface enrichment decreased slightly when increasing the feeding rate ratio of the outer feed solution to the inner feed solution from 4:1 to 10:1. Furthermore, it was observed that there was a correlation between the degree of burst release and the lysozyme surface enrichment, whereas the lysozyme loading content had no substantial impact on the release kinetics. CONCLUSIONS: The present work demonstrates the potential of spray dryer equipped with a 3-fluid nozzle in microencapsulation of proteins into PLGA matrices with different characteristics by varying process and formulation parameters.

Critical solvent properties affecting the particle formation process and characteristics of celecoxib-loaded plga microparticles via spray-drying.

PURPOSE: It is imperative to understand the particle formation mechanisms when designing advanced nano/microparticulate drug delivery systems. We investigated how the solvent power and volatility influence the texture and surface chemistry of celecoxib-loaded poly (lactic-co-glycolic acid) (PLGA) microparticles prepared by spray-drying. METHODS: Binary mixtures of acetone and methanol at different molar ratios were applied to dissolve celecoxib and PLGA prior to spray-drying. The resulting microparticles were characterized with respect to morphology, texture, surface chemistry, solid state properties and drug release profile. The evaporation profiles of the feed solutions were investigated using thermogravimetric analysis (TGA). RESULTS: Spherical PLGA microparticles were obtained, irrespectively of the solvent composition. The particle size and surface chemistry were highly dependent on the solvent power of the feed solution. An obvious burst release was observed for the microparticles prepared by the feed solutions with the highest amount of poor solvent for PLGA. TGA analysis revealed distinct drying kinetics for the binary mixtures. CONCLUSIONS: The particle formation process is mainly governed by the PLGA precipitation rate, which is solvent-dependent, and the migration rate of celecoxib molecules during drying. The texture and surface chemistry of the spray-dried PLGA microparticles can therefore be tailored by adjusting the solvent composition.

Multivariate analysis of phenol in freeze-dried and spray-dried insulin formulations by NIR and FTIR.

Dehydration is a commonly used method to stabilise protein formulations. Upon dehydration, there is a significant risk the composition of the formulation will change especially if the protein formulation contains volatile compounds. Phenol is often used as excipient in insulin formulations, stabilising the insulin hexamer by changing the secondary structure. We have previously shown that it is possible to maintain this structural change after drying. The aim of this study was to evaluate the residual phenol content in spray-dried and freeze-dried insulin formulations by Fourier transform infrared (FTIR) spectroscopy and near infrared (NIR) spectroscopy using multivariate data analysis. A principal component analysis (PCA) and partial least squares (PLS) projections were used to analyse spectral data. After drying, there was a difference between the two drying methods in the phenol/insulin ratio and the water content of the dried samples. The spray-dried samples contained more water and less phenol compared with the freeze-dried samples. For the FTIR spectra, the best model used one PLS component to describe the phenol/insulin ratio in the powders, and was based on the second derivative pre-treated spectra in the 850-650 cm(-1) region. The best PLS model based on the NIR spectra utilised three PLS components to describe the phenol/insulin ratio and was based on the standard normal variate transformed spectra in the 6,200-5,800 cm(-1) region. The root mean square error of cross validation was 0.69% and 0.60% (w/w) for the models based on the FTIR and NIR spectra, respectively. In general, both methods were suitable for phenol quantification in dried phenol/insulin samples.

Analysis of insulin allostery in solution and solid state with FTIR.

The insulin hexamer acts as an allosteric unit mediated by homotropic and heterotropic effects shifting the equilibrium between three distinct conformational states (T(6), R(3)T(3) and R(6)). The homotropic ligand phenol stabilises the R(6) state by binding to hydrophobic pockets only present in the R(6) state and shifts the equilibrium towards the R(6) state. The structural difference between the T(6) and R(6) state is primarily a change in the B1-B8 residues from extended conformation (T(6)) to alpha-helix (R(6)). The aim of this study was to investigate FTIR as an alternative method to monitor the T-R transition in the insulin hexamer upon phenol binding, and in addition to explore the advantage of infrared spectroscopy to measure solid state samples, and support the ability to maintain an allosteric state upon drying. The FTIR spectra of insulin in solution showed an increase in alpha-helix upon phenol binding and correlated well with the transition measured by CD yielding similar dissociation constants. Furthermore it was possible to maintain the increase in alpha-helix upon phenol binding after lyophilisation. The overall structure of the FTIR spectra changed upon lyophilisation but an increase in alpha-helix content was retained. Reconstitution of lyophilised insulin resulted in a change in structure resembling the structure of insulin prior to lyophilisation. Principal component analysis of all spectra was computed resulting in distinct clusters, and most variation in the data set could be explained by PC1 corresponding to a change in alpha-helix.

Quality by design - Spray drying of insulin intended for inhalation.

Quality by design (QBD) refers to a holistic approach towards drug development. Important parts of QBD include definition of final product performance and understanding of formulation and process parameters. Inhalation of proteins for systemic distribution requires specific product characteristics and a manufacturing process which produces the desired product. The objective of this study was to understand the spray drying process of insulin intended for pulmonary administration. In particular, the effects of process and formulation parameters on particle characteristics and insulin integrity were investigated. Design of experiments (DOE) and multivariate data analysis were used to identify important process parameters and correlations between particle characteristics. The independent parameters included the process parameters nozzle, feed, and drying air flow rate and drying air temperature along with the insulin concentration as a formulation parameter. The dependent variables included droplet size, geometric particle size, aerodynamic particle size, yield, density, tap density, moisture content, outlet temperature, morphology, and physical and chemical integrity. Principal component analysis was performed to find correlations between dependent and independent variables. Prediction equations were obtained for all dependent variables including both interaction and quadratic terms. Overall, the insulin concentration was found to be the most important parameter, followed by inlet drying air temperature and the nozzle gas flow rate. The insulin concentration mainly affected the particle size, yield and tap density, while the inlet drying air temperature mainly affected the moisture content. No change was observed in physical and chemical integrity of the insulin molecule.

Characterisation and physical stability of PEGylated glucagon.

Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay. Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon-PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular beta-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular beta-sheet was less apparent for glucagon-PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide-peptide interactions. Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for alpha-helix shifted to 1664 cm(-1), which could possibly be explained by formation of 3(10)-helix. Neither 3(10)-helix nor intermolecular beta-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra. In conclusion, glucagon-PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon.

Micropipette manipulation: a technique to evaluate the stability of water-in-oil emulsions containing proteins.

The interfacial properties and stability of water-in-oil emulsions containing protein were studied using micromanipulation. Micropipettes were used to produce individual water droplets in oil in a controlled manner on the micron scale. The pipettes were then used to bring two droplets into contact in order to observe fusion. The occurrence of fusion was investigated as a function of the compositions of both the continuous (oil) and dispersed (aqueous) phases. Various proteins, i.e., insulin, growth hormone, or serum albumin, were dissolved in the dispersed phase. When low concentrations of surfactants or no surfactant were present in the oil phase, a condensed protein film was formed at the surface of the droplets, which was revealed by the irregular topology of the droplet surface viewed with contrast microscopy. At higher surfactant concentrations, this topology was not observed nor was the stability apparently affected; emulsion droplets coalesce immediately upon contact with each other. There seems to be a limiting surfactant concentration, which stabilizes the droplets toward fusion and prevents formation of a condensed surface film, when the droplets contain protein. The technique exhibits potential for examination of the effects of various excipients on the coalescence stability of emulsion droplets.

Probing structural changes of proteins incorporated into water-in-oil emulsions.

The applicability of different techniques, that is, Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), and intrinsic tryptophan fluorescence, for probing the structural changes of proteins in the water-in-oil emulsions are investigated using nondefatted bovine (BSA) and human serum albumin (HSA) as model proteins. FTIR shows that the overall secondary structure of the proteins changes to some extent, 12% for BSA and 9% for HSA, when these are incorporated into the emulsion. There was no evidence of changes in the distribution of secondary structural elements apart from the changes in overall secondary structure. A blue shift of 12 to 14 nm in the fluorescence emission maximum was observed for proteins in the emulsion and 3 to 11 nm in the simulated interior of the aqueous phase, thus indicating structural changes around the tryptophan residues. DSC scans indicated that the domains in the proteins change because the shape of the transition peaks changes, when the proteins were incorporated into the emulsions. The total enthalpy decreases for BSA and HSA when these are incorporated into the emulsion, and some changes to the transition temperatures are observed. All the applied techniques supplement each other to give a more complete picture of the structural changes in proteins in intact water-in-oil emulsions.

Secondary structure alterations in insulin and growth hormone water-in-oil emulsions.

Water-in-oil (w/o) emulsions have shown a promising release profile of small drug molecules and proteins. However, the major concerns are the structural stability, the retention of the activity and to avoid unwanted immunological reactions caused by the changes in protein structure. In the present study, the secondary structure of insulin and growth hormone is investigated after manufacture of w/o emulsions, using Fourier transform infrared (FTIR) spectroscopy. Initial investigations indicate an altered distribution in the secondary structure elements, e.g. alpha-helix and beta-sheet, measured by area overlap calculations. The changes are more pronounced for growth hormone than for insulin. The overlapping area is 0.93 +/- 0.01 for the emulsion containing insulin manufactured at 0 degrees C and homogenised for 3 min, the corresponding value for growth hormone is 0.83 +/- 0.01. The droplet size changes from 0.27 +/- 0.04 microm in the blank w/o emulsion to 0.79 +/- 0.13 and 0.66 +/- 0.21 microm when insulin or growth hormone is incorporated into the w/o emulsions, respectively.