Therapeutic Protein Formulations Research Articles

Effect of the spraying conditions and nozzle design on the shape and size distribution of particles obtained with supercritical fluid drying

In the perspective of production of dry therapeutic protein formulations, spray drying of lysozyme (as a model protein) into supercritical carbon dioxide was studied. The effects of the nozzle (i.e., co-current coaxial converging and converging–diverging, and T-mixer impinging) and process conditions (i.e., flow rates, pressure) on the drying of the lysozyme prepared in aqueous solution dried with supercritical carbon dioxide enriched with ethanol were investigated. The particle size distribution, width of particle size distribution and morphology were used to determine the effect of the various parameters assessed. Particles with a median size of ∼1.5, ∼5 or ∼25 μm were produced depending of the nozzle selected. A basic comparative study of the nozzle was done by computational fluid dynamics, but the differences in particle size could not be depicted by these computations. The proportional increase of the flow rates (up to fivefold) caused a decrease in particle size (7- to 12-fold), and doubling the pressure caused a moderate decrease of the size (5–20%). The individual effect of the supercritical carbon dioxide, ethanol and solution streams was explained with a mass transfer model. Changing the ratio between flow rates slightly affected the particle size in various ways because of the swelling and shrinking stages of the drying droplet in supercritical carbon dioxide enriched with ethanol.

Preparation of protein nanocrystals and their characterization by solid state NMR

Preparation of proteins in their crystalline state has been found to be important in producing stable therapeutic protein formulations, cross-linked enzyme crystals for application in industrial processes, generating novel porous media for separations, and of course in structure elucidation. Of these applications only X-ray crystallography requires large crystals, defined here as being crystals 100s of microns or greater in size. Smaller crystals have attractive attributes in many instances, and are just as useful in structure determination by solid state NMR (ssNMR) as are large crystals. In this paper we outline a simple set of procedures for preparing nanocrystalline protein samples for ssNMR or other applications and describe the characterization of their crystallinity by ssNMR and X-ray powder diffraction. The approach is demonstrated in application to five different proteins: ubiquitin, lysozyme, ribonuclease A, streptavidin, and cytochrome c. In all instances the nanocrystals produced are found to be highly crystalline as judged by natural abundance 13C ssNMR and optical and electron microscopy. We show for ubiquitin that nanocrystals prepared by rapid batch crystallization yield equivalent 13C ssNMR spectra to those of larger X-ray diffraction quality crystals. Single crystal and powder X-ray diffraction measurements are made to compare the degree of order present in polycrystalline, nanocrystalline, and lyophilized ubiquitin. Solid state 13C NMR is also used to show that ubiquitin nanocrystals are thermally robust, giving no indication of loss of local order after repeated temperature cycling between liquid nitrogen and room temperature. The methods developed are rapid and should scale well from the tenths of milligram to multi-gram scales, and as such should find wide utility in the preparation of protein nanocrystals for applications in catalysis, separations, and especially in sample preparation for structural studies using ssNMR.

Effect of secondary structure on protein aggregation: A replica exchange simulation study

The ability to control or reverse protein aggregation is vital to the production and formulation of therapeutic proteins and may be the key to the prevention of a number of neurodegenerative diseases. In recent years, laboratory studies of the phenomenon have been accompanied by a growing number of computational treatments aimed at elucidating the molecular mechanisms of aggregation. The present article is a continuation of our simulation studies of coarse-grained model oligopeptides that mimic aggregating proteins. The potential function of a multichain system is expressed in terms of a generalized Go model for a set of sequences with varying contents of secondary-structural motifs akin to α-helices and β-sheets. Conformational evolution is considered by conventional Monte Carlo simulation, and by a variation of the Replica Monte Carlo technique that facilitates barrier-crossing in glasslike aggregated systems. The foldability and aggregation propensity are monitored as functions of the extent of different secondary structures and the length of the chains. Our results indicate that an increased proportion of sheetlike structures facilitates folding of isolated chains, but strongly favors the formation of misfolded aggregates in multichain systems, in agreement with experimental observations. This behavior is interpreted in terms of cooperativity effects associated with the formation of multiple residue–residue bonds involving adjacent monomers in interacting segments, which enhance both intramolecular binding and interprotein association.

Thermal Stability: A Means to Assure Tertiary Structure in Therapeutic Proteins

To be both safe and effective, a therapeutic product must have the correct chemical structure and be free of harmful contaminants. Structure in protein therapeutic products, however, implies not only the correct sequence of amino acids (primary structure) but also the proper folding of that amino acid chain in three-dimensional space (tertiary structure). This work is part of a general strategy to develop a battery of physico-chemical methods that could give assurances of structure (and hence function) in formulated therapeutic proteins in the absence of in vivo data. It focuses on recombinant human growth hormone (rhGH), a well-characterized therapeutic protein, and examines the utility of thermodynamic parameters in assessing its tertiary structure. Resistance of solutions of formulated rhGH to thermal denaturation was followed using Fourier Transform Infrared Spectroscopy (FTIR) by observing decreases in total helicity and increases in intermolecular beta-sheet formation. Under conditions known to induce changes in the intra-molecular ionic and H-bonding patterns stabilizing the tertiary structure but not affecting the protein's secondary structure or global fold, we have observed upwards of a 12°C shift in the melting temperature of the protein. Furthermore, our results indicated that the T m of unfolding of rhGH was sensitive to much more subtle changes in the protein structure. Thus, resistance to thermal denaturation may well be a useful means to measure structure in formulations of well-characterized therapeutic proteins. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI–PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI–PEGs retained ∼50% of enzyme activity when PEG was covalently attached to ∼40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI–PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.

Practical approaches to protein formulation development.

As is the case with other pharmaceuticals, formulation development is one of the critical steps in developing a protein as a therapeutic product. Development of stable protein formulations may require even more resources and effort than conventional small molecule pharmaceuticals. Proteins typically have more stability issues as a result of their complexity and delicate structural stability. Fortunately, a great deal of research regarding protein stability has been conducted and this information is readily available in the literature (reviewed by Manning et al., 1989; Chen, 1992; Ahern and Manning, 1992a; Ahern and Manning, 1992b; Arakawa et al., 1993; Cleland et al., 1993; Wang and Pearlman, 1993; Pearlman and Wang, 1996; Volkin and Middaugh, 1997). Ultimately, it would be ideal to be able to develop a pure pharmaceutical containing only the native protein. However, it is not practical to have only the native form of a protein in the formulation because the protein must be purified from a complex biological mixture containing a pool of other proteins which includes misfolded, denatured, and degraded forms of the same protein. Furthermore, a major challenge is to maintain the integrity of the purified protein during routine pharmaceutical processing, storage, handling, and delivery to the patient. One could envision achieving this goal by developing a formulation with perfect stability, i.e., no physical and chemical change in the protein. Becauise proteins are complex molecules composed of numerous reactive chemical groups and delicate three-dimensional structures, identifying a set of conditions to keep all components stable is virtually impossible. In general, commercial therapeutic protein formulations are developed under the assumption that some degree of physicochemical changes will occur during storage and handling.

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

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).

Unfolding and conformational distributions during protein precipitation.

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.

DepoFoam™ technology: a vehicle for controlled delivery of protein and peptide drugs

A major challenge in the development of sustained-release formulations for protein and peptide drugs is to achieve high drug loading sufficient for prolonged therapeutic effect coupled with a high recovery of the protein/peptide. This challenge has been successfully met in the formulation of several peptide and protein drugs using the DepoFoam™, multivesicular lipid-based drug delivery system. DepoFoam technology consists of novel multivesicular liposomes characterized by their unique structure of multiple non-concentric aqueous chambers surrounded by a network of lipid membranes. The objective of this paper is to demonstrate that DepoFoam technology can be used to develop sustained-release formulations of therapeutic proteins and peptides with high loading. DepoFoam formulations of a protein such as insulin, and peptides such as leuprolide, enkephalin and octreotide have been developed and characterized. The data show that these formulations have high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the drug caused by the formulation process, narrow particle size distribution, and spherical particle morphology. Drug release assays conducted in vitro in biological suspending media such as human plasma indicate that these formulations provide sustained release of encapsulated drug over a period from a few days to several weeks, and that the rate of release can be modulated. In vivo pharmacodynamic studies in rats also show a sustained therapeutic effect over a prolonged period. These results demonstrate that the DepoFoam system is capable of efficiently encapsulating therapeutic proteins and peptides and effectively providing controlled delivery of these biologically active macromolecules.

In vitro chromosome aberration assay of formulated recombinant human granulocyte-macrophage colony-stimulating factor in human peripheral blood lymphocytes

The clastogenic potential of a recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) therapeutic protein formulation was assessed in human peripheral blood lymphocyte (HPBL) cultures. Final formulation, rather than pure protein, was evaluated to simulate exposure conditions encountered in man. Because the formulation excipients (citric acid, sodium phosphate, mannitol, human serum albumin and polyethylene glycol) were found to alter cell-cycle kinetics and interfere with S-9 metabolic activation in vitro, a novel testing sequence and protocol were used to distinguish between rhGM-CSF and excipient effects and to guard against false negative results. Initial trials were performed to establish the volume of formulation alone (with and without rhGM-CSF) that would not cause severe cell-cycle delay, interfere with S-9 metabolic activation or inhibit positive control responses. This maximum tolerated volume of formulation was then incorporated in all dosed and control groups of each respective assay phase to give the same extent of cell-cycle delay. In the main assay, 24-hr HPBL cultures were exposed to dose levels of 0, 50, 100, 200 and 350 μg rhGM-CSF/ml for 24 hr in −S-9 phases and 0, 200, 350, 500 and 650 μg/ml for 2 hr in +S-9 phases, and harvested 24 hr later. No significant increases in the percentage of cells with structural chromosomal aberrations were found in either test phase. These results show that rhGM-CSF is not clastogenic in HPBL at greater than 17,000 times human exposure levels, and demonstrate that valid cytogenetic assay data with formulations containing cytotoxic excipient can be obtained in vitro.