Showing papers by "Theodore W. Randolph published in 2002"

Rational design of stable lyophilized protein formulations: theory and practice.

Abstract: For ease of preparation and cost containment by the manufacturer, and ease of handling by the end user, an aqueous therapeutic protein formulation usually is preferred. However, with many proteins it is not possible—especially considering the time constraints for product development—to develop sufficiently stable aqueous formulations. Unacceptable denaturation and aggregation can be induced readily by the numerous stresses to which a protein in aqueous solution is sensitive; e.g., heating, agitation, freezing, pH changes, and exposure to interfaces or denaturants (Arakawa et al., 1993; Cleland et al., 1993; Brange, 2000; Bummer and Koppenol, 2000). Furthermore, even under conditions that thermodynamically greatly favor the native state of proteins, aggregation can arise during months of storage in aqueous solution (e.g., Gu et al., 1991; Arakawa et al., 1993; Chen et al., 1994; Chen et al., 1994; Chang et al., 1996a). In addition, several chemical degradation pathways (e.g., hydrolysis and deamidation) are mediated by water. In aqueous formulations, the rates of these and other (e.g., oxidation) chemical degradation reactions can be unacceptably rapid on the time scale of storage (e.g., 18–24 months) for pharmaceutical products (Manning et al., 1989; Cleland et al., 1993; Goolcharran et al., 2000; Bummer and Koppenol, 2000).

Surfactant-protein interactions.

Abstract: To retain biological activity, proteins generally must be maintained in a specific, three-dimensional conformation. This conformation is only marginally stable, and thus relatively minor perturbing forces can disrupt protein structure, causing loss of biological activity, as well as formation of non-native protein aggregates. Such perturbations are commonly encountered as proteins are produced, stored, transported, and delivered to patients. For example, it is well known that during common industrial processes such as filtering (Maa and Hsu, 1998), storage (Mcleod et al., 2000), agitation (Thurow and Geisen, 1984; Maa and Hsu, 1997) freeze/thawing (Eckhardt, Oeswein et al., 1991; Nema and Avis, 1993; Izutsu et al., 1994), lyophilization (Carpenter and Chang, 1996; Carpenter et al., 1997), nebulization (Ip et al., 1995) and spray-drying (Broadhead et al., 1994; Mumenthaler et al., 1994; Maa et al., 1998; Adler and Lee, 1999; Millqvist-Fureby, Malmsten et al., 1999; Tzannis and Prestrelski, 1999) proteins can suffer damage to their native conformation. Further, delivery of protein pharmaceuticals to patients may also provoke losses of conformational integrity via unfavorable interactions of proteins with surfaces (e.g., inner walls of catheter tubing or syringes (Tzannis et al., 1996)).

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.

Abstract: We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 °C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen−deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protei...

Long-term storage of proteins.

Abstract: This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins. It briefly explains the stresses that induce protein aggregation the major causes for chemical degradation. It also discusses how to use various storage strategies to increase the long-term stability of proteins. When appropriate it points out critical mistakes to avoid. This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins.

High-Pressure Refolding of Disulfide-Cross-Linked Lysozyme Aggregates: Thermodynamics and Optimization

Abstract: Previous exploratory work revealed that high pressure (200 MPa), in combination with oxido-shuffling agents such as glutathione, effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules, at concentrations up to 2 mg/mL (1). To understand further and optimize this process, in the current study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH). Yields decreased dramatically at more oxidizing conditions ([GSSG] > [GSH]). Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions. At GSSG:GSH ratios of 4:1, 1:1, and 1:16, lysozyme dissolved and refolded with time constants of 62, 20, and 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.

Preparation and use of photopolymerized microparticles

Abstract: Methods of forming crosslinked polymer particles in situ from polymer precursors such as monomers or oligomers, comprising exposing a composition comprising at least one polymer precursor, a solvent or solvent mixture, and an antisolvent or antisolvent mixture to photoradiation under conditions whereby particles are formed are provided. The polymer precursor may be photosensitive, or a separate polymerization initiator may be used. In a preferred embodiment, the polymer precursor is insoluble in the antisolvent or antisolvent mixture and the solvent or solvent mixture is soluble in the antisolvent or antisolvent mixture at the concentrations used. Crosslinked polymer particles and crosslinked polymer particles comprising a polymer and a bioactive material are also provided. The polymer may be erodable, and the polymer particles formed may be used in a variety of applications, including controlled release of bioactive materials such as drugs. Polymer particles formed using the methods of the invention have low residual solvent levels and high additive encapsulation efficiencies. The processes of the invention allow control of particle size and morphology, use low operating temperatures and are useful for efficient bulk production.

Dissolution and partitioning behavior of hydrophobic ion-paired compounds.

Abstract: Purpose. This study was conducted to determine the effects of counterion hydrophobicity on organic/aqueous partition coefficients for hydrophobic ion paired (HIP) complexes. Furthermore, the coupled dissolution and reverse ion-exchange kinetics for dissolution of HIP complexes into aqueous electrolyte solutions were measured and mathematically modeled.

Devices and methods for the production of particles

Abstract: The present invention provides methods and devices for producing particles with an average diameter less than about 15νm using the precipitation with compressed fluid-antisolvent (PCA) process and the carbon-dioxide assisted nebulization with a bubble dryer (CAN-BD) process. In the methods and nozzles of the invention, at least one jet of supercritical or near-supercritical fluid and at least one jet of solution interact to mix the supercritical or near-supercritical fluid and the solution within a chamber. The solution contains at least one solvent and at least one solute. At least one of the jets is a swirling jet. To form particles, the solvent and supercritical or near-supercritical fluid are then injected into a PCA or a CAN-BD process chamber. The degree of mixing depends in part on the power input into the mixing chamber. Power inputs of about 6.5xl09W/m3 enhance the degree of mixing and allow production of nanoscale particles with the PCA process. The nanoscale particles have a size distribution so that polydispersity is less than about 1.75.

Encapsulating DNA within biodegradable polymeric microparticles.

Abstract: In order for genetic medicines to become viable commercial products, the active form of the drug (e.g., DNA) must be able to reach the site of action and remain there long enough to accomplish its intended function. Encapsulation of plasmid DNA into biodegradable microspheres is one approach towards solving this challenge. This review describes the primary methods for satisfactorily entrapping intact DNA into biodegradable polymeric matrices. In particular, the materials, processes, and equipment required for each encapsulation method are described in detail. The resulting microspheres could be used for parenteral, oral, and inhalation therapy.

Compressed Antisolvent Precipitation and Photopolymerization To Form Highly Cross-Linked Polymer Particles

Abstract: We present a novel antisolvent processing technique by simultaneous compressed antisolvent precipitation and photopolymerization for forming cross-linked polymer microparticles. In this process, an organic solvent dissolves monomer and polymerization photoinitiators to form a homogeneous solution. Photopolymerization and microparticle formation occur when the homogeneous solution is sprayed into a compressed antisolvent while being simultaneously exposed to initiating light. High miscibility of the solvent in the supercritical antisolvent allows for its quick extraction from the polymerizing phase, leaving progressively higher concentrations of monomer. The high monomer concentration combined with photoinitiated polymerization facilitates rapid reaction rates and ultimately results in polymer precipitation. Particle size and morphology are adjustable by changing the processing conditions, as simultaneous polymerization and solvent extraction result in microparticles with a wide range of diameters.