Protein stability: Impact of formulation excipients and manufacturing processes in protein-based pharmaceuticals
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.
Protein stability, bovine serum albumin, L-arginine, L-lysine, freeze-drying, mannitol, moisture uptake, sucrose, trehalose, dynamic light scattering, protein agitation, mechanical agitation, low shear agitation, high shear agitation, Thioflavin T assay
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