Browsing by Author "Darkwah, Joseph"

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    Characterisation of freeze dried amino acids and gelatin based rapidly Disintegrating tablets
    (De Montfort University, 2011) Darkwah, Joseph
    Recent research has shown the feasibility of using individual or a combination of amino acids as a replacement component for sugars in RDT formulations. What has emerged from this work is the notion of an optimal concentration of amino acid, i.e. one that is sufficiently high to provide the desired mechanical strength but not too high to impact disintegration time. In this study, the degree of amino acid crystallinity in gelatin/amino acid based RDTs was investigated using terahertz pulsed spectroscopy. Three amino acids were investigated: alanine (89 g mol-1), serine (105 g mol-1), and proline (115 g mol-1). The three amino acids were studied by terahertz pulsed spectroscopy (in the frequency band 0.1 to 3 THz; 3 to 100 cm-1), both in the pure crystalline form (as received from the manufacturer) and in the form of a co-freeze-dried matrix with gelatin (in weight fractions of 10:90, 30:70, 50:50, 70:30). Each pure crystalline form of amino acid displayed one or two resonance peaks at characteristics wave numbers, which were in general agreement with the literature (with alanine at 75 cm-1 and 85 cm-1; proline at 48 cm-1 and 66 cm-1 and serine centred on 67 cm-1). Irrespective of the amino acid in question (viz. alanine, proline, or serine) all freeze-dried formulations containing 10% amino acid and 90% gelatin were found to have no crystallinity with respect to the amino acid component. On increasing the amino acid to 30%, only those formulations manufactured from serine showed evidence of crystallisation behaviour. Only on increasing the concentration of amino acid to 50% did the spectra of alanine display the distinct absorption bands of their crystalline reference counterparts. The degree of crystallinity in serine and alanine was estimated using calibration model built from partial least square regression (PLSR). The increased concentration of alanine to 50%w/w and 70% w/w in freeze dried gelatin matrix showed an estimated degree of crystallinity to be ~62% and ~72% (± 16%) respectively. Similarly the degree of crystallinity in 30%w/w and 50% w/w serine/gelatin freeze dried matrix were estimated to be ~55% and ~97% (± 10%) respectively. This work has shown a rank order of solubility within the freeze-dried gelatin matrix of proline > alanine > serine. Unsurprisingly, the same rank order exists for the aqueous solubility, with serine being the least soluble (~5 g ml-1) and proline being the most soluble (~162 g ml-1). The impact of hydrophobic interactions between the amino acid and gelatin are therefore less dominant in defining the crystallinity of the amino acid within the freeze-dried material.
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    Effect of acidic and basic pH on Thioflavin T absorbance and fluorescence
    (Springer, 2015-03-22) Hackl, E. V.; Darkwah, Joseph; Smith, Geoff; Ermolina, I.
    Thioflavin T (ThT) is a fluorescent dye able to enhance significantly its fluorescence quantum yield upon binding to protein amyloids. ThT assay is widely used to detect and quantify amyloids in a variety of conditions, including solutions with different pH levels. In the present work, the effect of acidic and basic pH on the conformation of the ThT molecule and its absorption and fluorescence properties was studied. The results show that both acidic and basic pH decrease significantly the intensity of ThT absorption in the visible region and fluorescence emission intensity. Low pHs induce an immediate “all-or-nothing” decrease in the ThT signal, while in alkaline solutions the ThT signal decreases gradually over time. pH-induced signal quenching is less in the presence of glycerol or protein aggregates. Two different mechanisms are responsible for the ThT signal quenching—the ThT hydroxylation at basic pH and protonation of the nitrogen atom of the dimethylamino group at acidic pH. ThT assays should be carefully carried out at basic or acidic pH as strong pH dependence of ThT could be responsible for misinterpretation and false positive/negative experimental results. The potential unsuitability of ThT as a probe in solutions with high pH (>9) has been shown. © 2015, European Biophysical Societies' Association.
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    Effect of arginine on the aggregation of protein in freeze-dried formulations containing sugars and polyol. 1. Formulation development
    (Springer, 2017-10-18) Hackl, E. V.; Darkwah, Joseph; Smith, Geoff; Ermolina, I.
    L-arginine was introduced into protein-based freeze-dried formulations to study the ability of arginine to reduce / prevent from protein aggregation during manufacturing, storage and reconstitution of lyophilized protein-based pharmaceuticals. As L-arginine is known to be very hygroscopic, additional excipients which could provide a moisture buffering capacity need to be introduced into the formulation. In the first part of our study – excipient formulation development – the screening of a number of sugars/polyols has been done in order to select the best combination of excipients that, in a complex with L-arginine, can i) produce freeze-dried cakes with elegant appearance, adequate mechanical properties and reconstitution times, and ii) resist/minimize the moisture sorption. Various freeze-dried cakes containing L-arginine in combination with mannitol, trehalose, lactose, and sucrose were produced and analyzed by TGA, DSC, texture analysis, moisture sorption, cake shrinkage, TVIM and SEM. The non-linear dependencies of the physicochemical properties of the freeze-dried cakes on the sugar-to-mannitol ratios were found. The best combinations of excipients (L-arginine, mannitol and trehalose) were selected to be used in the second part of this work, in which the impact of each selected formulation will be studied in relation to the aggregation of a protein.
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    Effect of Arginine on the Aggregation of Protein in Freeze-Dried Formulations Containing Sugars and Polyol: II. BSA Reconstitution and Aggregation
    (Springer International Publishing, 2018-07-06) Hackl, E. V.; Darkwah, Joseph; Smith, Geoff; Ermolina, I.
    The current paper continues our study on the ability of L-arginine to prevent/reduce the aggregation of proteins that results from the various stresses during the lyophilisation and/or storage of lyophilized protein-based products. The first part of our study, i.e. formulation development, was devoted to the rational design and optimization of an L-arginine containing lyophilized formulation which can resist the natural tendency of L-arginine to absorb atmosphere moisture. Mannitol and trehalose were chosen among other excipients to be included in the protein-based formulation, as mannitol in a combination with L-arginine has been shown to reduce moisture sorption while trehalose provides a degree of lyoprotection. In the present study, a number of formulations, which comprised bovine serum albumin (BSA) with and without L-arginine, and with five different ratios of trehalose-to-mannitol (from 30:70 to 80:20) were lyophilised and assessed. The internal structures and the moisture sorption/retention of the lyophilized formulations were characterised. To study the effect of L-arginine on BSA solid-phase stability, the lyophilized powder was exposed to accelerated storage conditions (high moisture (75% RH) and temperature (22 or 45 °C)) for up to 24 h. The lyophilized BSA formulations were then reconstituted and solution-state protein aggregation assessed by turbidimetry at 360 nm and fluorescence spectroscopy using the thioflavin T assay. It was demonstrated that L-arginine can be used in protein-based freeze-dried formulations to significantly reduce the aggregation of protein during the manufacturing, storage and subsequent reconstitution. The results also revealed the importance of a sufficient amount of mannitol in the arginine-containing formulations.
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    Protein stability: Impact of formulation excipients and manufacturing processes in protein-based pharmaceuticals
    (De Montfort University, 2017-08) Darkwah, Joseph
    Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.
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    Terahertz pulsed spectroscopy study of amino acids and gelatin.
    (Wiley-Blackwell, 2010) Darkwah, Joseph; Smith, Geoff; Ermolina, I.
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