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Supercritical fluids processing of recombinant human Growth Hormone
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2005 (English)In: Drug Development and Industrial Pharmacy, ISSN 0363-9045, E-ISSN 1520-5762, Vol. 31, no 2, 135-149 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the study was to investigate the feasibility of precipitating recombinant human growth hormone (hGH) from aqueous solutions using conventional and modified techniques of solution-enhanced dispersion (SEDS) by supercritical fluids. The study investigated the effect on hGH stability of adding isopropanol either as a cosolvent with the original aqueous protein solution (conventional process) or to the supercritical carbon dioxide before mixing with the aqueous protein solution (modified process). The influence of the addition of sucrose (with or without isopropanol) on the precipitation behavior and stability of the protein was also studied. Experiments were performed under various processing conditions (pressure 100-200 bars and temperature 40-50 degrees C), and with various flow rates and solution compositions (CO2/isopropanol and protein solution). Bioanalytical characterization of the resulting powders involved spectrophotometry, sodium dodecyl sulfate-polycrylamide gel electrophoresis, reverse-phase high performance liquid chromatography (RP-HPLC), and size exclusion chromatography. Solid-state characterization was performed using differential scanning calorimetry, X-ray powder diffraction, scanning electron microscopy, and Karl Fischer techniques. Results showed that with both conventional and modified methods, under optimum processing conditions, the presence of sucrose in the solution decreased the destabilizing effects of the solvent and/or process on the structure of hGH. More hGH was dissolved from the precipitated powders containing sucrose than from those containing only isopropanol. Reverse-phase HPLC indicated that about 94% of the hGH was recovered in its native form. The proportion of dimers and oligomers was reduced in the presence of sucrose; about 92% of the soluble protein was present in monomer form under optimal conditions. The remaining undissolved protein was in monomeric form. The precipitated powders were amorphous, containing particulate aggregates in the size range 1-6 microm with 5-10% residual moisture content. In conclusion, hGH was successfully precipitated from aqueous solution using SEDS technology. The presence of sucrose in the protein solution promoted the precipitation of hGH and reduced aggregation and improved dissolution.

Place, publisher, year, edition, pages
2005. Vol. 31, no 2, 135-149 p.
Keyword [en]
Chemistry; Pharmaceutical, Chromatography; Gel, Chromatography; High Pressure Liquid, Chromatography; Supercritical Fluid, Drug Stability, Human Growth Hormone/*chemistry, Precipitation, Recombinant Proteins, Solvents
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-91391DOI: 10.1081/DDC-200047368PubMedID: 15773281OAI: oai:DiVA.org:uu-91391DiVA: diva2:164105
Available from: 2004-02-18 Created: 2004-02-18 Last updated: 2013-05-16Bibliographically approved
In thesis
1. Preparation of Pharmaceutical Powders using Supercritical Fluid Technology: Pharmaceutical Applications and Physicochemical Characterisation of Powders
Open this publication in new window or tab >>Preparation of Pharmaceutical Powders using Supercritical Fluid Technology: Pharmaceutical Applications and Physicochemical Characterisation of Powders
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders.

The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO2) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive.

The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein.

In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 50 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 308
Pharmaceutics, Supercritical fluid, Gas Anti-Solvent, SEDS, Crystallisation, Particle design, Polymorphs, Dry powder inhalation, Solid-state behaviour, Therapeutic proteins, Precipitation, Stability, Recombinant human growth hormone (hGH), Galenisk farmaci
National Category
Pharmaceutical Sciences
urn:nbn:se:uu:diva-4006 (URN)91-554-5880-7 (ISBN)
Public defence
2004-03-12, B41, BMC, Husargatan 3, Uppsala, 10:15
Available from: 2004-02-18 Created: 2004-02-18Bibliographically approved

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