Jeffrey D. Meyer

Controlled release of ionic compounds from poly (l-lactide) microspheres produced by precipitation with a compressed antisolvent

Abstract Poly ( l -lactide) microspheres containing low molecular weight pharmaceutical agents were prepared using the precipitation with a compressed antisolvent (PCA) process with supercritical carbon dioxide as the antisolvent. Gentamycin, naloxone, and naltrexone were solubilized in methylene chloride using hydrophobic ion pairing (HIP) to stoichiometrically replace polar counter ions with an anionic detergent, aerosol OT (AOT, sodium bis-2-ethylhexyl sulfosuccinate). Through HIP complexation, solubilities in excess of 1 mg/ml were attainable in methylene chloride, allowing levels of direct incorporation that are not possible with other PCA approaches. The drug/polymer particles were spherical in shape and between 0.2 and 1.0 μm in diameter, as determined by scanning electron microscopy. Drug incorporation efficiencies were determined and in vitro release profiles measured. At 37°C, the release of the ion-paired drugs into phosphate-buffered saline displays minimal burst effects and exhibits release kinetics that are approximately linear with the square root of time, indicating matrix diffusion control of drug release. For gentamycin, linear release from the poly ( l -lactide) microspheres was observed for more than 7 weeks, even at a drug loading of near 25% (w/w). Naltrexone exhibits similar release characteristics, although more drug was found on the surface of the microspheres. Conversely, rifampin, which was not ion-paired, was poorly encapsulated.

Hydrophobic Ion Pairing: Altering the Solubility Properties of Biomolecules

The high aqueous solubility of ionic compounds can be attributed to the ease of solvation of the counter ions. Replacement of the counter ions with ionic detergents dramatically alters the solubility properties of the molecule. Not only does the aqueous solubility drop precipitously, but the solubility in organic phases increases as well. Consequently, the partition coefficient changes by orders of magnitude. This ion pairing phenomenon, which we term hydrophobic ion pairing (HIP), has been extended to polyelectrolytes, such as proteins and polynucleotides. These materials form HIP complexes that dissolve in a range of organic solvents, often with retention of native structure and enzymatic activity. The HIP process has been used to purify protein mixtures, conduct enzymatic reactions in nonaqueous environments, increase structural stability, enhance bioavailability, and prepare new dosage forms.

Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor

Metal-induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc-induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc-induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation.

Stability of human serum albumin during bioprocessing: denaturation and aggregation during processing of albumin paste.

Purpose. To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin (HSA) obtainedfrom pooled human plasma.Methods. After initial precipitation of HSA from plasma, the resultantpaste is either (a) lyophilized or (b) washed with acetone and thenair-dried in order to obtain a dry powder. The structure of HSA wasexamined using Fourier transform infrared (IR) spectroscopy. Theextent of aggregation of redissolved HSA was measured using bothdynamic light scattering and SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Results. Both lyophilization and air-drying perturb the secondarystructural composition of HSA, as detected by infrared (IR) spectroscopy.Upon dissolution of dried paste, most of the protein refolds to anative-like conformation. However, a small fraction of the protein moleculesform soluble aggregates that can be detected by both dynamic lightscattering and SDS-PAGE. The level of aggregation is so low that itcould not be detected in the bulk by either circular dichroism orIR spectroscopy. The lyophilized protein, which appears to be moreunfolded in the solid state than the acetone washed/air-dried material,exhibits a higher level of aggregation upon dissolution.Conclusions. There is a direct correlation between the extent ofunfolding in the solid state and the amount of soluble aggregate presentafter dissolution. Moreover, the presence of the aggregates persiststhroughout the remainder of the purification process, which includesdissolution, chromatography, sterile filtration and viral inactivationsteps. Analytical methods used to monitor the stability ofbiopharmaceuticals in the final product can be used to assess damage inflictedduring processing of protein pharmaceuticals.

Impact of bulking agents on the stability of a lyophilized monoclonal antibody

The impact of the bulking agents on the stability of lyophilized protein pharmaceuticals is not typically considered, as they usually crystallize, preventing them from stabilizing the protein. In this study, combinations of sucrose with mannitol or glycine were evaluated for their effects on antibody stability (deamidation and aggregation) and solid-state properties (water content, residual antibody structure, and T(g) values). While sucrose remains the primary stabilizing agent for the antibody in the solid state, inclusion of some amorphous glycine leads to a significant increase in stability. Mannitol displays little, if any, stabilizing ability in this system. In formulations where infrared spectroscopy could be applied, maintenance of secondary structure is an important factor in predicting storage stability. This study demonstrates that bulking agents can impact protein stability in the solid state. By remaining partially amorphous, bulking agents can sometimes lower degradation rates, while at the same time, providing a crystalline matrix producing elegant cakes. It appears that bulking agents may belong to a larger class of low molecular weight plasticizers, which appears to be able to increase solid-state stability of proteins despite lowering the glass transition temperature.

In Vitro Release Kinetics of Gentamycin from a Sodium Hyaluronate Gel Delivery System Suitable for the Treatment of Peripheral Vestibular Disease

For certain patients who experience intense vertigo arising from unilateral vestibular lesions, the primary therapy is a vestibular nerve section, an intracranial surgical procedure. One alternative to this treatment is therapeutic ablation of vestibular function on the unaffected side using an ototoxic agent. We prepared a biodegradable sustained-release gel delivery system using sodium hyaluronate that can be administered into the middle ear using only a local anesthetic. The gel contains gentamycin sulfate, the ototoxic agent of choice for treatment of unilateral vestibulopathy, and it exhibits diffusion-controlled release of the drug over a period of hours. The released gentamycin could then diffuse into the inner ear through the round membrane. This represents an important advance over previous formulations, which used only gentamycin sulfate solutions, in that it should allow more careful control of the dose, it should reduce loss of the drug from the middle ear site, and it should maintain intimate contact with the round membrane. By carefully controlling the dose, it should be possible to inhibit vestibular function while minimizing hearing loss. Herein we describe the in vitro release kinetics of gentamycin sulfate from sodium hyaluronate gels and find that the system obeys Fickian behavior.

Effects of Conformation on the Chemical Stability of Pharmaceutically Relevant Polypeptides

Control of chemical instability in protein pharmaceuticals continues to be a critical issue in developing stable formulations. While the effects of pH, buffer composition, ionic strength and temperature remain the most effective methods for controlling hydrolysis and oxidation reactions, it appears that conformational control may also be important. Addition of excipients to maintain native structure and reduce the propensity of the protein to denature and/or aggregate is already a central theme in stabilizing proteins (Arakawa et al., 1993). The same additives have now been found to slow both deamidation and oxidation, whether in solution or in the solid state. What is emerging is an additional approach for producing protein pharmaceuticals that maintain native structure and activity during long-term storage.

Lipid unsaturation determines the interaction of AFP type I with model membranes during thermotropic phase transitions

We have previously shown that antifreeze protein (AFP) type I from winter flounder interacts with the acyl chains of lipids in model membranes containing a mixture of dimyristoylphosphatidylcholine (DMPC) and the plant thylakoid lipid digalactosyldiacylglycerol (DGDG), most likely through hydrophobic interactions. By contrast, in studies with pure phospholipid membranes, no such interaction was seen. DGDG is a highly unsaturated lipid, which renders these studies quite different from the previous studies of AFP-membrane interaction where the lipids were saturated or trans-unsaturated. Therefore, it seemed possible that either the digalactose headgroups or the unsaturated DGDG acyl chains, or both, may be important for interactions of membranes with AFP type I. To distinguish between these possibilities, we catalytically hydrogenated the DGDG to obtain a galactolipid with completely saturated fatty acyl chains. The results with the hydrogenated DGDG were strikingly different from those obtained previously with the unsaturated DGDG; the clear binding of AFPs to the bilayer appeared to be lost. Nevertheless, the temperature-dependent folding of AFP type I was inhibited in the presence of liposomes containing either the unsaturated or the hydrogenated DGDG. The results indicate that the liposomes and protein still interact, even following hydrogenation of the acyl chains, perhaps at the membrane-solution interface.

Thermal stability of low molecular weight urokinase during heat treatment. III. Effect of salts, sugars and Tween 80

A turbidimetric assay was used to determine the extent of thermally-induced aggregation in low molecular weight urokinase (LMW-UK). Previous work has shown that, under 60°C heat treatment, LMW-UK denatures and the unfolded protein proceeds to form soluble aggregates. The effects of excipients on the extent of aggregation were examined. Both salts (ammonium sulfate and magnesium chloride) and sugars (sucrose, glucose, trehalose, raffinose) were found to be effective, concentration-dependent inhibitors of aggregation, although excessive salt concentrations did lead to salting out of the protein. Addition of Tween 80, a nonionic detergent, was ineffective. Overall, the effect of these additives on the stability of thermally-stressed LMW-UK can be understood in terms of preferential exclusion of the solute from the surface of the protein. These interactions affect the extent of denaturation, or unfolding, of LMW-UK at 60°C, thereby controlling the degree of aggregation. Purification and incubation experiments indicate that a thermally-unstable subpopulation of LMW-UK exists and is responsible for the majority of the aggregation observed.