Mark C. Manning

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.

Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.

Inhibition of stress-induced aggregation of protein therapeutics

Publisher Summary This chapter describes the types of stresses and conditions that are routinely found to cause aggregation of purified therapeutic proteins. Stresses encountered during processing include short-term exposure to high temperatures during pasteurization of aqueous solutions, freeze-thawing, freeze-drying, and exposure to denaturing interfaces because of agitation, filtration, air bubble entrainment during filling, and so on. It also considers the effects of long-term storage on protein stability in aqueous solutions and dried solids. Each section describes how the rational choice of stabilizing additives can be used to inhibit protein aggregation. These choices are based on a clear understanding of the mechanisms by which different additives succeed or fail as protein stabilizers under different conditions. The mechanisms are considered in detail and are illustrated in the chapter by selected examples. Finally, this chapter briefly describes a model that allows the calculation of the degree of expansion of the native state needed to form an aggregate-fostering species in aqueous solution and the utility of infrared spectroscopy to characterize the structure of proteins in precipitates.

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.

Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native‐state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native‐state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far‐ and near‐UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native‐state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl‐induced unfolding, increased in proportion to sucrose concentration. Native‐state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange‐resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large‐scale than on small‐scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native‐state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.

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.

The stability factor: importance in formulation development.

Efficient development of stable formulations of protein pharmaceuticals requires an intimate knowledge of the protein and its chemical and physical properties. In particular, understanding the mechanisms by which a protein could degrade is critical for designing and testing formulations. This review describes the major pathways by which proteins can degrade, including denaturation, aggregation, oxidation, and interfacial damage. The methods to detect the degradation are covered, along with generalized strategies to retard or prevent each type of decomposition. Without an appreciation of the current best practices for devising stable formulations, the formulation process will be neither efficient nor optimal.

Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution.

There has been an increasing awareness that proteins, like other biopolymers, are large enough to exhibit colloidal behavior in aqueous solution. Net attractive or repulsive forces have been found to govern important physical properties, such as solubility and aggregation. The extent of intermolecular interactions, usually expressed in terms of the osmotic second virial coefficient, B, is most often measured using static light scattering. More recently, self-interaction chromatography (SIC) has emerged as a method for rapid determination of B in actual formulations, as it uses much less protein and has higher throughput. This review will summarize the relationship of B to crystallization, solubility, and aggregation of proteins in aqueous solution. Moreover, the capability of SIC to obtain B values in a rapid and reproducible fashion will be described in detail. Finally, the use of miniaturized devices to measure B is presented.

Thermal stability of low molecular weight urokinase during heat treatment. II. Effect of polymeric additives

Turbidimetric or light scattering assays can be used to determine the extent of aggregation in protein formulations. Using low molecular weight urokinase (LMW-UK) as a model protein, the effect of polymeric additives on heat-induced aggregation was evaluated. Previous work has shown that under 60°C heat treatment, LMW-UK initially denatures and the unfolded protein associates to form soluble aggregates. Eventually, these aggregates associate to form a precipitate. The effects of polymers on the initial aggregation phase was examined. Hydroxyethyl (heta) starch, polyethylene glycol 4000, and gelatin were found to be effective, concentration-dependent inhibitors of aggregation, whereas polyvinylpyrrolidone (PVP) and polyethylene glycol 300 were ineffective. Overall, the effect of polymeric additives on the stability of thermally-stressed LMW-UK can be accounted for by preferential exclusion of the solute from the surface of the 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.