Overcoming challenges in co-formulation of proteins with contradicting stability profiles: EPO plus G-CSF

The co-formulation of drugs is widely used for small molecules, e.g. fixed-dose-combinations of synergistic medicines in the treatment of infections, diabetes or neurodegenerative diseases. For protein drugs, only a few studies have been published to elucidate the challenges of stabilizing two proteins in one formulation. Here, we show a systematic study with the model proteins EPO and G-CSF, which differ significantly in their physicochemical properties. We apply several analytical methods to investigate the stability of the co-formulated proteins in the liquid and solid state. Forced degradation studies at elevated temperature indicate poor stability of the liquid co-formulations. Therefore, we use lyophilization as a stabilization strategy. Finally, we adopt an elegant approach, in which the proteins are lyophilized at pH 4.0 and reconstituted with buffer at pH 7.0 to obtain high monomer recovery and to preserve the protein structure of both EPO and G-CSF. After reconstitution, both proteins in co-formulation remain stable for the timespan until eventual application in patients. With this case study, we demonstrate how to overcome some challenges during the co-formulation of therapeutic proteins

Structure-based Discovery of a new Protein-Aggregation Breaking Excipient

Reducing the aggregation of proteins is of utmost interest to the pharmaceutical industry. Aggregated proteins are often less active and can cause severe immune reactions in the patient upon administration. At the same time the biopharmaceutical market is pushing for high concentration formulations and products that do not require refrigerated storage conditions. For a given protein, the liquid formulation developer’s toolbox is limited to achieve these goals: pH, ionic strength and concentration of a very limited number of excipients are the only solution parameters to be varied. In this work, we present a structure-based approach to discover new molecules that successfully reduce the aggregation of proteins and apply it to the model protein Interferon-alpha-2a

SLN and NLC for topical, dermal, and transdermal drug delivery

Introduction: From a biopharmaceutical standpoint, the skin is recognized as an interesting route for drug delivery. In general, small molecules are able to penetrate the stratum corneum, the outermost layer of the skin. In contrast, the delivery of larger molecules, such as peptides and proteins, remains a challenge. Nanoparticles have been exploited not only to enhance skin penetration of drugs but also to expand the range of molecules to be clinically used.Areas covered: This review focus on Solid lipid nanoparticles (SLN) and Nanostructured lipid carriers (NLC) for skin administration. We discuss the selection criteria for lipids, surfactants, and surface modifiers commonly in use in SLN/NLC, their production techniques, and the range of drugs loaded in these lipid nanoparticles for the treatment of skin disorders.Expert opinion: Depending on the lipid and surfactant composition, different nanoparticle morphologies can be generated. Both SLN and NLC are composed of lipids that resemble those of the skin and sebum, which contribute to their enhanced biocompatibility, with limited toxicological risk. SLN and NLC can be loaded with very chemically different drugs, may provide a tunable release profile, can be produced in a sterilized environment, and be scaled-up without the need for organic solvents.The authors acknowledge CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support and for the fellowship of the second author (88887.368385/2019-00). Authors also acknowledge the support received from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, and co-financed by FEDER, under the Partnership Agreement PT2020, for the projects M-ERA-NET-0004/2015-PAIRED and UIDB/04469/2020 (strategic fund).info:eu-repo/semantics/publishedVersio

Analytical tools and evaluation strategies for nanostructured lipid carrier-based topical delivery systems

Introduction The inception of nanostructured lipid carriers (NLCs) proved to be a revolutionary step toward the treatment of dermatological disorders. To uncover its true potential, it is imperative that the system be characterized and evaluated comprehensively. Areas covered The present review has been written to furnish an in-depth account of analytical tools and evaluation procedures under one roof. Besides discussing the challenges of topical delivery and benefits of NLCs, the paper elaborates on their physicochemical characterization. Further, in vitro evaluation of NLCs for dermatological benefits, followed by their evaluation in a hydrogel/cream base is covered. Lastly, disease-specific evaluation of NLC-based formulations is presented. Expert opinion The research endeavors for NLCs have largely focused on the fabrication of NLCs for different bioactives. However, scientific efforts should be aimed toward the lesser explored realm of NLCs, i.e. exploitation of analytical techniques, such as Parelectric spectroscopy, Electron Spin Resonance, and Nuclear Magnetic Resonance spectroscopy. NLCs have been proven for their potential to foster the therapeutic modalities applicable to cutaneous disorders. More attention needs to be devoted to their evaluation for disease-specific parameters. The futuristic steps must involve clinical studies, to lay the path for their commercialization.info:eu-repo/semantics/publishedVersio

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions

Chemometrics in Protein Formulation:Stability Governed by Repulsion and Protein Unfolding

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor