Paul M. Bummer

Purification of Proteins Using Foam Fractionation

Purification is an important step in the production of pharmaceuticals from recombinant proteins. The characteristics of industrial-scale purification schemes, such as conventional chromatography, have a significant impact on the cost of production. Foam fractionation, a novel separation technique based upon the differences in affinities of components for the gas/aqueous interface of a foam, has the potential to be a cost-effective component in a purification scheme. This review covers some of the more recent studies in understanding the process and applications of foam fractionation in protein-containing systems with special attention to the requirements of pharmaceutical products.

Behavior of partially fluorinated carboxylic acids at the air-water interface

Abstract Langmuir isotherms were recorded for 1-(perfluorobutyl)undecanoic acid (F 3 C(CF 2 ) 3 (CH 2 ) 10 CO 2 H), 1-(perfluorohexyl)undecanoic acid (F 3 C(CF 2 ) 5 (CH 2 ) 10 CO 2 H) and 1-(perfluorooctyl)undecanoic acid (F 3 C(CF 2 ) 7 (CH 2 ) 10 CO 2 H), their hydrocarbon analog, perfluorododecanoic and perfluorotetradecanoic acid after spreading onto hydrochloric acid (pH=1.9–2.0) at 32°C. All acids formed stable monolayers at the air–water interface. 1-(Perfluorobutyl)undecanoic acid shows some similarity to the compression isotherm of tetradecanoic acid while lacking the phase transition of pentadecanoic acid. The isotherm for 1-(perfluorohexyl)undecanoic acid shows a temperature-dependent phase transition similar to the liquid expanded-liquid condensed transition of pentadecanoic acid. The π-A isotherm of 1-(perfluorooctyl)undecanoic acid resembles the highly condensed monolayers of hepta- and nonadecanoic acid. Thus, the partially fluorinated acids seem to exhibit isotherm characteristics similar to hydrocarbon acids with shorter chain lengths. The three partially fluorinated acids have a higher limiting area compared to hydrocarbon and perfluorocarbon acids, which may be atributed to the strong dipole moment of the CF 2 CH 2  linkage. Within this series of partially fluorinated carboxylic acids, the limiting area decreases with chain lengthening and a higher degree of fluorination. The collapse pressures of the fluorinated acids are smaller compared to their respective hydrocarbon analog.

Kinetics of paclitaxel 2′-N-methylpyridinium mesylate decomposition

This study was designed to examine the kinetics of decomposition of paclitaxel 2′-N-methylpyridinium mesylate (PNMM), a derivative of paclitaxel. Further, the potential for PNMM to act as a prodrug of paclitaxel was assessed in vitro. Stability studies of PNMM were conducted over a pH range of 4.0 to 8.0 at 25°C. The critical micelle concentration (CMC) of PNMM was determined by pulsating bubble surfactometry. Studies of the conversion of PNMM to paclitaxel were conducted in vitro in human plasma. Decomposition of PNMM followed apparent zero-order kinetics. The pH-rate profile exhibited no evidence of acid catalysis down to pH 4.0, while the rate was accelerated under base conditions. Surface tension studies suggested that PNMM formed micelles with a CMC of approximately 34 μg/mL. Conversion studies in phosphate buffer showed that no more than 5% of PNMM converted to paclitaxel, while in human plasma the conversion was about 25%. The degradation of PNMM was via apparent zero-order kinetics and was dependent upon pH. The observed apparent zero-order kinetics of decomposition of PNMM was consistent with the formation of micelles in phosphate buffer. In buffered aqueous media alone or in human plasma, PNMM did not convert quantitatively to paclitaxel. Thus, the limiting factor in the application of PNMM as a prodrug would appear to be the poor potential to convert to paclitaxel.

An FTRI study of the structure of human serum albumin adsorbed to polysulfone

Internal reflection infrared spectroscopy was employed in a study of the structural changes in human serum albumin that result from adsorption to cast films of polysulfone. Adsorption experiments were carried out in a flow-through cell at 30°C in the presence of either a H2O-based or D2O-based saline buffer. Spectral features show adsorption results in Amide I bandwidth broadening and diminished Amide II band intensities relative to the Amide I band. Deconvolution and band-fitting of spectra obtained in the presence of D20 show appearance of beta and aperiodic conformations at the expense of helical structures. The results suggest considerable change in the secondary structure of human serum albumin upon adsorption to polysulfone.

Histidine tagged protein recovery from tobacco extract by foam fractionation

Tobacco plants have the potential to be used for the production of proteins for pharmaceutical applications. This work describes a novel protein recovery strategy where the protein of interest is “tagged” with a histidine sequence, which forms a complex with cobalt ions and surfactant possessing a chelating functionality, such that the protein is recovered in the foamate of a foam fractionation step. His‐gus, a histidine‐tagged enzyme, was chosen as a model protein to study the feasibility of this strategy. The His‐gus is recovered from spiked prefoamed tobacco extract by foam fractionation in the presence of surfactant and cobalt ions with an enrichment of 1.29 and a recovery of 21.5% in terms of an adjusted activity.

Foam fractionation of binary mixtures of lysozyme and albumin

A nitrogen gas-based foam fractionation method was employed to separate model proteins, bovine serum albumin (BSA) and hen egg white lysozyme, from each other. Fractionation was characterized by the separation ratio and by recovery of proteins in the retentate as a function of the nominal pore size of the gas dispersion frit and solution conditions (pH and ionic strength). For binary mixtures of the proteins at pH 7.4, and ionic strength (mu) of 0.18 M, the recovery of lysozyme and the separation ratio were both dependent on the frit size employed to generate the foam. At low ionic strength (mu = 0.01 M), separation was only somewhat greater with the small pore size frits, although at values significantly lower than those found for high ionic strength. The diminished separations appear to be due to the only slight changes in recoveries observed for BSA and lysozyme.%Separation ratios of lysozyme from BSA in solutions either of high or low ionic strength were maximal at pH values equal to or less than the isoelectric point (pI) of BSA. Separation ratios were lower when foaming was carried out under low compared with high ionic strength. The recovery of lysozyme was enhanced by foaming from solutions of low pH and high ionic strength. Recoveries of BSA were greatest when the molecule was negatively charged. Electrical interactions between the positively charged lysozyme and negatively charged BSA may explain the diminished separation ratios and enhanced recoveries. Enzyme activity studies of lysozyme remaining in the retentate showed no change from prefoam activity.

The Influence of Water Content of Triglyceride Oils on the Solubility of Steroids

Purpose.To determine if hydration of long- and medium-chain triglyceride oils (long = soybean and olive, medium = Miglyol 812) has a significant effect on the ability to solubize the model hydrophobic compounds progesterone, estradiol, and testosterone.Methods.Soybean, olive, and Miglyol 812 oils were treated in one of two ways: hydrated or desiccated (hydrated, then dried). Solubility of 3H-labeled progesterone, estradiol, and testosterone in the triglycerides was measured by liquid scintillation counting.Results.Both hydration state and chain length of the triglycerides were shown to have a significant influence on the solubility of steroids. Solubility of estradiol hemihydrate and testosterone monohydrate in hydrated triglycerides is decreased by about 30%–40% compared with desiccated oils. The solubility of anhydrous testosterone was decreased by hydration of the oils due to conversion to the monohydrate crystalline form. In contrast, the solubility of progesterone was insensitive to the state of hydration of all oils.Conclusions.Hydration of triglyceride oils caused a significant decrease in the solubility of steroids, which may form hydrates or hemihydrates. Results suggest the need for knowledge of the hydration state of triglyceride oils to be used as pharmaceutical excipients.

Chemical Stability of Esters of Nicotinic Acid Intended for Pulmonary Administration by Liquid Ventilation

AbstractPurpose. It has been suggested that fluorocarbon liquid may be a unique vehicle for the delivery of drugs directly to the acutely injured lung. A prodrug approach was used as a means of enhancing the solubility of a model drug (nicotinic acid) in the fluorocarbon. The solubility, the chemical stability of the putative prodrugs, and the sensitivity to enzymatic hydrolysis was investigated. Methods. The solubility of each nicotinic acid ester was determined in buffer as a function of pH and in perflubron. The octanol/buffer partition coefficient was determined at pH 7.4. The chemical stability of the putative prodrugs was determined as a function of pH, temperature, buffer content, and ionic strength. In addition, sensitivity of the esters to enzymatic degradation was evaluated. Results. Compared with nicotinic acid, the solubility in perflubron of the esters was significantly enhanced. In aqueous buffers, the esters exhibited pseudo-first order degradation kinetics, with both acid and base catalyzed loss. Studies of the fluorobutyl ester indicate quantitative loss of the putative prodrug and release of the parent nicotinic acid. Porcine esterase accelerated the loss of fluorobutyl ester by a factor of over 200 compared with chemical hydrolysis at pH 7.4. Conclusions. The properties of the fluorinated esters suggest that they may be suitable candidates for further testing as possible prodrugs of nicotinic acid based upon higher solubility in perflubron, rapid release of the parent drug after simple hydrolysis, and sensitivity to the presence of a model esterase enzyme.

Mechanistic model and analysis of doxorubicin release from liposomal formulations.

Reliable and predictive models of drug release kinetics in vitro and in vivo are still lacking for liposomal formulations. Developing robust, predictive release models requires systematic, quantitative characterization of these complex drug delivery systems with respect to the physicochemical properties governing the driving force for release. These models must also incorporate changes in release due to the dissolution media and methods employed to monitor release. This paper demonstrates the successful development and application of a mathematical mechanistic model capable of predicting doxorubicin (DXR) release kinetics from liposomal formulations resembling the FDA-approved nanoformulation DOXIL® using dynamic dialysis. The model accounts for DXR equilibria (e.g. self-association, precipitation, ionization), the change in intravesicular pH due to ammonia release, and dialysis membrane transport of DXR. The model was tested using a Box-Behnken experimental design in which release conditions including extravesicular pH, ammonia concentration in the release medium, and the dilution of the formulation (i.e. suspension concentration) were varied. Mechanistic model predictions agreed with observed DXR release up to 19h. The predictions were similar to a computer fit of the release data using an empirical model often employed for analyzing data generated from this type of experimental design. Unlike the empirical model, the mechanistic model was also able to provide reasonable predictions of release outside the tested design space. These results illustrate the usefulness of mechanistic modeling to predict drug release from liposomal formulations in vitro and its potential for future development of in vitro - in vivo correlations for complex nanoformulations.

Perinatal sidestream cigarette smoke exposure and the developing pulmonary surfactant system in rats

1 Epidemiological studies have strongly implicated passive smoking with increased incidence of various respiratory diseases in children. Our earlier studies have shown that chronic exposure to tobacco smoke significantly changes the composition and the surface activity of the pulmonary surfactant in adult rats. The aim of the present study was to determine if perinatal exposure to sidestream cigarette smoke influences the composition and function of pulmonary surfactant system in developing rat pups. 2 Pregnant Sprague Dawley rats were exposed to sidestream cigarette smoke in a whole body exposure chamber for a total of 6 h each day and the pups born to these dams were further exposed to sidestream smoke for 2 h/day during postnatal period. Exposure of animals to smoke was ascertained by measuring their plasma cotinine. Surfactant analysis was performed on bronchoalveolar lavage fluids (BALF) collected from pups on postnatal day 1, 3, 7, 14, 21 and 35. The phospholipid (PL) content, percentage disaturated phosphatidylcholine (DSPC) and surfactant proteins (SP-A and SP-B) in BALF surfactant preparations from sham and smoke-exposed pups were compared. 3 Significant differences between the two groups were observed at two exposure points: a reduced level of SPA on day 1, and, a higher level of SP-A and PLs on day 21, in smoke-exposed pups. Surface activity analysis of the surfactant films by pulsating bubble surfactometer did not show any significant differences between the sham and smoke-exposed groups at any exposure point. 4 The results indicate that perinatal exposure to sidestream smoke is capable of producing biochemical changes in the composition of pulmonary surfactant at different stages of development but these changes do not influence surface tension reducing properties of the surfactant.