Browsing by Subject "formulation"

Now showing 1 - 3 of 3
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    Effect of the Thermodynamic and Physical State of the Freeze-Concentrate on Protein Stability
    (2017-12) Jena, Sampreeti
    In this dissertation research, specific interactions (excipient-excipient, excipient/protein-ice, protein-excipient) governing protein conformational stability and crystallization behavior of excipients in the freeze concentrate, were explored. Furthermore, the effects of formulation composition (type and mole fractions of excipients in the formulation) on afore-mentioned interactions, during freeze-thaw and freeze-drying of protein formulations, was investigated. Concentration dependent effects of excipients including the bulking agent, lyo/cryo-protectant and surfactant on the nucleation and growth of crystalline phases in the freeze concentrate were characterized and quantified. Changes in the secondary and tertiary conformations of model proteins (such as Bovine Serum Albumin and Immunoglobulin) due to crystallization of excipients, were determined as a function of formulation composition during freeze-thaw and freeze-drying. Infrared (IR) Spectroscopy was used to detect onset of crystallization the bulking agent and lyo/cryo-protectant. X-Ray Diffractometry (XRD) was used to characterize the polymorphic form of crystalline phases. Far UV circular Dichroism (CD) was used to characterize secondary conformation of protein in thawed and reconstituted (freeze-dried) formulations. IR Spectroscopy was used to characterize secondary conformation of protein in frozen and freeze-dried formulations. A bulking agent – lyo/cryo-protectant – protein system, a typical freeze-drying formulation, was chosen for characterization of frozen and freeze-dried formulations. It was observed that high concentrations of non-crystallizing components such as the protein and lyo/cryo-protectant (usually a disaccharide such as trehalose) inhibited crystallization of the (otherwise readily crystallizing) bulking agent (such as mannitol) and vice versa. At low concentrations, surfactants such as Polysorbate 20, prevented growth of crystalline phases due to amphiphilic interface coverage, but when their concentrations exceeded the critical micelle concentration (CMC), they enhanced degree of crystallinity in the formulation. Structural unfolding of the protein was detected upon crystallization of the lyo/cryo-protectant and micelle formation (when surfactant concentration exceeded CMC). Detection of protein aggregates in reconstituted solutions, confirmed that unfolding induced during freezing, thawing and drying processes, did not reverse upon reconstitution. Presence of ice surfaces and other crystalline interfaces (such as those introduced by the bulking agent) significantly contributed to protein degradation. In our model system, thawing induced stresses such as recrystallization were found to be more detrimental than the stresses induced by freezing and desiccation and hence, freeze-drying yielded better structural recovery of the protein than freeze-thaw in our model system. Secondary relaxations arising from the flexible polar groups on the protein surface (millisecond time scales) and dynamic ring flips of the monosaccharide units about the glycosidic linkage (microsecond time scales) of disaccharides (indicating flexibility of glycosidic linkage) were detected in our model freeze-dried system using Frequency Domain Dielectric Spectroscopy. In the presence of protein, flexibility of the glycosidic linkage was decreased and likewise, presence of disaccharides slowed down the dynamics of flexible protein groups, up to a critical protein to disaccharide mass ratio (= 0.5). Surfactant and higher protein to disaccharide mass ratios (≥ 0.5) produced the opposite effect. These secondary relaxations govern conformational stability of the protein and propensity of the disaccharide to crystallize during storage below the Tg. In the final part of the thesis, effects of slow freezing on lyo/cryo-protectant-protein formulations during cryo-vitrification was investigated. Chemical toxicity of cell penetrating lyo/cryo -protectants such as Dimethyl Sulfoxide (DMSO), frequently used for cryo-vitrification of organs and tissues, was shown to be dictated by their hydrogen bonding behavior (characterized by IR Spectroscopy). At temperatures where hydrogen bonding interactions between lyo/cryo-protectant and water were unfavorable, the lyo/cryo-protectant directly partitioned in the hydration shell of the protein and caused unfolding of the protein, potentially due to hydrophobic interactions. It was also ascertained that when the freeze concentrate is vitrified during freezing, rapid thawing is a necessity to minimize ice recrystallization during devitrification to minimize the damage to the proteins. This dissertation research has enhanced an overall understanding of interactions between the excipients, protein and crystalline interfaces (of ice and crystalline excipients such as bulking agent) as well as protein dynamics in the freeze-concentrate. This information is needed to identify stresses arising in the protein micro-environment that lead to conformational destabilization (and loss of activity) during preservation of protein formulations and is currently absent in literature.
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    Effects of Freezing and Freeze-drying on Protein Conjugated Nanoparticles
    (2018-10) Su, Yafan
    Biodegradable poly(D,L-lactide-co-glycolide) PLGA nanoparticles (NPs) have been extensively investigated for drug delivery. Protein conjugated NPs provide the advantage of active targeting. To overcome the instability associated with storage of aqueous dispersions, NPs are usually lyophilized. However, the freezing and freeze-drying stress can lead to nanoparticle aggregation and protein denaturation. As such, sucrose is widely used as a cryoprotectant and minimal ‘cryoprotectant to particle ratio’ is required. In this study, we used trypsin as a model protein to conjugate on the PEGlyated NPs. The effects of the freezing and freeze-drying process on trypsin conjugated NPs was studied by comparing the size of the NPs, the morphology of the NPs under fluorescent images and the trypsin activity after of the fresh made, freeze-thawed and lyophilized NPs. A minimum sucrose to NPs ratio was founded to provide the complete protection of the trypsin conjugated NPs.
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    UNDERSTANDING THE STABILITY OF SALTS AND COCRYSTALS IN A DRUG PRODUCT ENVIRONMENT
    (2021-06) Kaur, Navpreet
    Transition of a drug substance to drug product necessitates the use of excipients and often includes several unit operations.1 A risk associated with processing pharmaceutical solids is their propensity to undergo solid form transformations such as polymorphism and amorphization. Changes in the physical form during drug product manufacture or storage can have an influence on their chemical stability and product performance. The central goal of this thesis work is to mechanistically understand the influence of processing and formulation composition on the stability of pharmaceutical salts and cocrystals.Processing induced lattice disorder was investigated for caffeine-oxalic acid cocrystals. The unmilled cocrystals were stable in presence of excipient and water. However, very short milling times induced sufficient lattice disorder to induce cocrystal dissociation. Quantification of disorder was performed using X-ray diffractometry (XRD). The lattice disorder was proposed to be predominant on the particle surface experiencing shear and hence served to explain the disproportionate influence that low levels of disorder had on the stability of the cocrystals. Cocrystal dissociation was observed to be a water mediated reaction and was influenced by the pH of the microenvironment. Very low levels of lattice disorder, which cannot be characterized using bulk characterization tools such as XRD and thermal analysis, can induce chemical instability and lead to product failure. Disorder induced during processing was also imaging using atomic force microscopy. The second part of the thesis focused on understanding the challenges associated with the formulation development of levothyroxine sodium pentahydrate (LSP). The influence of pharmaceutical processing on the hydration state of LSP was investigated using single crystal and synchrotron X-ray diffractometry, and a novel crystal form of the drug was reported when it undergoes partial dehydration to form levothyroxine sodium monohydrate (LSM). LSM has a higher chemical reactivity than the pentahydrate form. The influence of excipients on the physical and chemical stability of LSP was investigated using synchrotron XRD and high performance liquid chromatography (HPLC). Hygroscopic and acidic excipients can induce dehydration and salt disproportionation of LSP, respectively. Microenvironment pH and excipient hygroscopicity were critical determinants of LSP stability.