Gerard W. Hofland

Stabilization of Proteins in Dry Powder Formulations Using Supercritical Fluid Technology

Therapeutic proteins have become essential in the treatment of many diseases. Their formulation in dry form is often required to improve their stability. Traditional freeze-drying or spray-drying processes are often harmful to labile proteins and could be replaced by supercritical fluid (SCF) drying to produce particles with defined physicochemical characteristics in a mild single step. A survey of the current SCF drying processes for proteins is presented to give insight into the effect of SCF drying on protein stability and to identify issues that need further investigation. Methods used for drying aqueous and organic protein solutions are described. In particular, effects of process and formulation parameters on particle formation and protein stability are discussed. Although SCF methodology for drying proteins is still in its infancy, it can provide a serious alternative to existing drying methods for stabilizing proteins.

Fractionation of soybean proteins with pressurized carbon dioxide as a volatile electrolyte

Fractionation of specific proteins from plant material is a complex and involved science, yet pure protein extracts are in high demand by a wide range of food and pharmaceutical industries. In this study carbon dioxide has been used as a volatile electrolyte to isoelectrically precipitate two major protein constituents of soybean. Carbon dioxide was shown to be effective in purifying glycinin and beta-conglycinin in a three-step process as 95% and 80% concentrated fractions with precipitation yields of 28% and 21%, respectively. Recycling of the mixed precipitate of the intermediary step enables complete separation into the concentrated fractions. Fractionation acidity was precisely controlled by a simple modification of pressure. In addition, the occurrence of a pH overshoot was prevented at any point in the fractionation vessel, as the pH minimum was defined by its equilibrium relationship with carbon dioxide operating pressure. The removal of the glycinin precipitate was an important factor in the purification procedure. The yield of the individual concentrated glycinin and beta-conglycinin precipitate fractions was a function of carbon dioxide pressure, extract concentration and, to a much lesser extent, temperature.

Lipase-Catalyzed Ethanolysis of Milk Fat with a Focus on Short-Chain Fatty Acid Selectivity

Mixtures of fatty acid ethyl esters were produced by lipase-catalyzed ethanolysis of milk fat triglycerides. Three commercial immobilized lipases (Lipozyme TL, Lipozyme RM, and Novozym 435) were tested in different reaction conditions with the aim of maximizing the conversion of the short-chain fatty acid fraction of milk fat to flavor ethyl esters. The influence of the reactants molar ratio was investigated, as well as three different reaction media, that is, hexane, CO(2)-expanded liquid (GXL), and the solvent-free mixture. Novozym 435 showed the highest activity in all conditions. This lipase also exhibited selectivity for short-chain fatty acids, which, at short reaction times, resulted in a product mixture richer in short-chain fatty acids than the original milk fat. The highest selectivities were obtained in hexane and in CO(2)-expanded liquid fat, at low ethanol to fat ratios. Using dense CO(2) as the reaction cosolvent is attractive because it results in the largest short-chain fatty acid enrichment in the product mixture, while leaving no residues in the product.

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 approximately 1.5, approximately 5 or approximately 25 microns 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.

Carbon Dioxide Induced Soybean Protein Precipitation: Protein Fractionation, Particle Aggregation, and Continuous Operation

A novel protein fractionation technique using a volatile electrolyte has been developed. Carbon dioxide was used to isoelectrically precipitate 80% and 95% pure glycinin and β‐conglycinin fractions from soybean isolate. The protein fractions precipitated as primary particles 0.2−0.3 μm in diameter, which under optimum conditions may be recovered as aggregates up to 500 μm in diameter. The dependency of protein fractionation efficiency on aggregate settling rates has been demonstrated. The isoelectric points of the two main soybean fractions, glycinin and β‐conglycinin, were calculated to be pH 5.2 and 4.95, respectively. Solution pH was accurately controlled by pressure in the isoelectric pH range of the different soybean protein fractions, and a pH “overshoot” was eliminated. Volatile electrolyte technology was also applied to a continuous process in order to eliminate the particle recovery concerns associated with batch precipitation and to demonstrate the potential for scale‐up. Glycinin was effectively recovered on‐line (94% glycinin recovery) with a purity approaching that of the batch process (95%).

Dynamics of precipitation of casein with carbon dioxide

Abstract Carbon dioxide is a green alternative for mineral acids (e.g., sulphuric acid) in protein precipitation. The precipitation using a gaseous precipitant differs from the conventional precipitation in the way and the rate at which the precipitant can be applied to the aqueous solution. In this paper, the consequences of using carbon dioxide were investigated for the precipitation of the milk protein casein. Product properties, such as particle size, solids content and calcium release were investigated in a batch system at various mixing conditions and gas addition rates. In addition, mass transfer coefficients were determined from pH response data. The experiments revealed a strong influence of stirring rate and gas flow rate on the particle size. The main effect of varying the gas flow was through the change of acidification rate, much alike precipitation with sulphuric acid. At high acidification rates, the particle size of casein precipitated with carbon dioxide was smaller than with sulphuric acid.