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Gaithersburg, MD, United States

Li C.H.,Amgen Inc. | Narhi L.O.,Amgen Inc. | Wen J.,Amgen Inc. | Dimitrova M.,Formulation science | And 6 more authors.
Biochemistry | Year: 2012

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and CH2 domain unfolding first, followed by changes in the secondary structure and CH3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated CH2 domain melts at a temperature 4-5 C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ∼0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the CH2 domain, where the glycosylation of the Fc resides. © 2012 American Chemical Society.

Gourbatsi E.,University of Kent | Povey J.,University of Kent | Uddin S.,Formulation science | Smales C.M.,University of Kent
Biotechnology Letters | Year: 2016

Objectives: The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Results: Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Conclusions: Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins. © 2015, The Author(s).

Gerhardt A.,University of Colorado at Boulder | McGraw N.R.,University of Colorado at Boulder | Schwartz D.K.,University of Colorado at Boulder | Bee J.S.,Formulation science | And 2 more authors.
Journal of Pharmaceutical Sciences | Year: 2014

The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil-water interfaces and air-water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil-water interfaces (siliconized syringe walls), air-water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil-water-air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil-water interfaces, air-water interfaces, and agitation in the generation of particles in protein formulations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Zhou Q.,University of Minnesota | Zhou Q.,Huazhong University of Science and Technology | Shi L.,University of Minnesota | Marinaro W.,Formulation science | And 2 more authors.
Powder Technology | Year: 2013

Using a representative powder blend containing ibuprofen, the applicability of coating with silica nanoparticles using a dry comilling process to effectively enhance flowability of formulated pharmaceutical powders was investigated. Using a shear cell, we systematically studied effects of process parameters, including the total number of comilling cycles, silica loading level, type of screen (mesh size), impeller speed, and impeller type, on the efficiency of the nanocoating process. Impact of silica coating on powder tabletability was also assessed using a compaction simulator. Results confirm that coating with silica nano particles significantly improves flowability of the formulated ibuprofen blend. The two most prominent factors that lead to flowability improvement are 1) repeated comilling cycles and 2) higher silica loading. In addition, we have observed that simple blending is effective in coating silica nanoparticles to improve flowability of the formulated ibuprofen powders, which is not very cohesive. This suggests the possibility of improving flowability of a sub-optimum formulation by prolonging the blending process in presence of colloidal silica. Moreover, silica coated ibuprofen blend exhibits improved tabletability and compactibility without significantly impacting compressibility. The simultaneous improvement in powder tabletability and flowability shows the potential of the nanocoating strategy in improving tablet manufacturability of pharmaceutical powders. © 2013 Elsevier B.V.

Mason L.M.,University of Nottingham | Campinez M.D.,University of Seville | Pygall S.R.,Merck And Co. | Burley J.C.,University of Nottingham | And 4 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2015

Abstract Percolation theory has been used for several years in the design of HPMC hydrophilic matrices. This theory predicts that a minimum threshold content of polymer is required to provide extended release of drug, and that matrices with a lower polymer content will exhibit more rapid drug release as a result of percolation pathways facilitating the faster penetration of the aqueous medium. At present, percolation thresholds in HPMC matrices have been estimated solely through the mathematical modelling of dissolution data. This paper examines whether they can be also identified in a novel way: through the use of confocal laser scanning fluorescence microscopy (CLSM) to observe the morphology of the emerging gel layer during the initial period of polymer hydration and early gel formation at the matrix surface. In this study, matrices have been prepared with a polymer content of 5-30% w/w HPMC 2208 (Methocel K4M), with a mix of other excipients (a soluble drug (caffeine), lactose, microcrystalline cellulose and magnesium stearate) to provide a typical industrially realistic formulation. Dissolution studies, undertaken in water using USP apparatus 2 (paddle) at 50 rpm, provided data for the calculation of the percolation threshold through relating dissolution kinetic parameters to the excipient volumetric fraction of the dry matrix. The HPMC percolation threshold estimated this way was found to be 12.8% v/v, which was equivalent to a matrix polymer content of 11.5% w/w. The pattern of polymer hydration and gel layer growth during early gel layer formation was examined by confocal laser scanning fluorescence microscopy (CLSM). Clear differences in gel layer formation were observed. At polymer contents above the estimated threshold a continuous gel layer was formed within 15 min, whereas matrices with polymer contents below the threshold were characterised by irregular gel layer formation with little evidence of HPMC particle coalescence. According to percolation theory, this implies that a continuous cluster of HPMC particles was not formed. The images provide the first direct evidence of how the percolation threshold may be related to the success or failure of early gel layer development in HPMC matrices. It also shows how extended release characteristics are founded on the successful coalescence of hydrated polymer particles to form a continuous coherent diffusion barrier, which can then inhibit further percolation of the hydration medium. The correlation between percolation thresholds estimated from dissolution and imaging techniques suggests that confocal imaging may provide a more rapid method for estimating the percolation thresholds, facilitating the rational design of HPMC extended release matrices at lower polymer contents with minimal risk of dose dumping. © 2015 Elsevier B.V.

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