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This book gives pharmaceutical scientists an up-to-date resource on protein aggregation and its consequences, and available methods to control or slow down the aggregation process. While significant progress has been made in the past decade, the current understanding of protein aggregation and its consequences is still immature. Prevention or even moderate inhibition of protein aggregation has been mostly experimental. The knowledge in this book can greatly help pharmaceutical scientists in the development of therapeutic proteins, and also instigate further scientific investigations in this area. This book fills such a need by providing an overview on the causes, consequences, characterization, and control of the aggregation of therapeutic proteins.
Aggregation of therapeutic proteins is currently one of the major challenges in the bio-pharmaceutical industry, because aggregates could induce immunogenic responses and compromise the quality of the product. Current scientific efforts, both in industry and academia, are focused on developing rational approaches to screen different drug candidates and predict their stability under different conditions. Moreover, aggregation is promoted in highly concentrated protein solutions, which are typically required for subcutaneous injection. In order to gain further understanding about the mechanisms that lead to aggregation, an approach that combined rheology, neutron scattering, and molecular simulations was undertaken. Two model systems were studied in this work: Bovine Serum Albumin in surfactant-free Phosphate Buffered Saline at pH = 7.4 at concentrations from 11 mg/mL up to ~519 mg/mL, and a monoclonal antibody in 20 mM Histidine/Histidine Hydrochloride at pH = 6.0 with 60 mg/mL trehalose and 0.2 mg/mL polysorbate-80 at concentrations from 53 mg/mL up to ~220 mg/mL. The antibody used here has three mutations in the CH2 domain, which result in lower stability upon incubation at 40 C with respect to the wild-type protein, based on size-exclusion chromatography assays. This temperature is below 49 C, where unfolding of the least stable, CH2 domain occurs. This dissertation focuses on identifying the role of aggregation on the viscosity of protein solutions. The protein solutions of this work show an increase in the low shear viscosity in the absence of surfactants, because proteins adsorb at the air/water interface forming a viscoelastic film that affects the measured rheology. Stable surfactant-laden protein solutions behave as simple Newtonian fluids. However, the surfactant-laden antibody solution also shows an increase in the low shear viscosity from bulk aggregation, after prolonged incubation at 40 C.Small-angle neutron scattering experiments were used to characterize the antibody aggregates responsible for this non-Newtonian response. From the neutron scattering data, a weak barrier leading to reversible aggregation is identified. Therefore, proteins aggregate weakly after colliding hydrodynamically, unless they find a favorable contact with high binding energy. Two types of antibody aggregates were identified: oligomers with average radius of gyration of ~10 nm, and fractal aggregates larger than ~ 0.1 [mu]m formed by a reaction-limited aggregation process. A characteristic upturn in the scattered intensity at low wavevector and a low shear viscosity increase are observed in aggregated protein solutions. These features are removed by filtering with a 0.2 [mu]m filter, which also eliminates the submicron fractal aggregates. Biophysical characterization supports the conclusions from the rheology and neutron scattering experiments. Finally, molecular dynamics simulations were used to understand the effects of disulfide bonds on the conformational stability of serum albumin. Changes in disulfide bonds in the native structure could lead to partial unfolding, and the formation of aggregates through inter-molecular disulfide bonds. Therefore, it is important to understand the role of each disulfide bond on the structure and dynamics of the protein. After removing disulfide bonds, changes occur in the dynamic correlations between different residues, the secondary and tertiary structure of albumin. However, not all disulfide bonds affect the conformation of the protein. Removal of all disulfide bonds using molecular dynamics is proposed as a practical prescreening tool to identify disulfide bonds that are important for the conformational stability. As a result, some disulfide bonds can be mutated without affecting the conformation of the protein.
Successful biofunctional surface engineering will determine the future of medical devices such as orthopedic implants, stents, catheters, vaccine scaffolds, wound dressings, and extracorporeal circulation devices. Moreover, the biosensor and diagnostic chip technology will evolve rapidly due to the growing medical need for personalized medicine. A
This book covers the physical side of colloidal science from the individual forces acting between particles smaller than a micrometer that are suspended in a liquid, through the resulting equilibrium and dynamic properties. A variety of internal forces both attractive and repulsive act in conjunction with Brownian motion and the balance between them all decides the phase behaviour. On top of this various external fields, such as gravity or electromagnetic fields, diffusion and non-Newtonian rheology produce complex effects, each of which is of important scientific and technological interest. The authors aim to impart a sound, quantitative understanding based on fundamental theory and experiments with well-characterised model systems. This broad grasp of the fundamentals lends insight and helps to develop the intuitive sense needed to isolate essential features of the technological problems and design critical experiments. The main prerequisites for understanding the book are basic fluid mechanics, statistical mechanics and electromagnetism, though self contained reviews of each subject are provided at appropriate points. Some facility with differential equations is also necessary. Exercises are included at the end of each chapter, making the work suitable as a textbook for graduate courses in chemical engineering or applied mathematics. It will also be useful as a reference for individuals in academia or industry undertaking research in colloid science.
This work covers advances in the interactions of proteins with their solvent environment and provides fundamental physical information useful for the application of proteins in biotechnology and industrial processes. It discusses in detail structure, dynamic and thermodynamic aspects of protein hydration, as well as proteins in aqueous and organic solvents as they relate to protein function, stability and folding.
Specific ion effects are important in numerous fields of science and technology. This book summarizes the main ideas that came up over the years. It presents the efforts of theoreticians and supports it by the experimental results stemming from various techniques.
Parenteral Medications is an authoritative, comprehensive reference work on the formulation and manufacturing of parenteral dosage forms, effectively balancing theoretical considerations with practical aspects of their development. Previously published as a three-volume set, all volumes have been combined into one comprehensive publication that addresses the plethora of changes in the science and considerable advances in the technology associated with these products and routes of administration. Key Features: Provides a comprehensive reference work on the formulation and manufacturing of parenteral dosage forms Addresses changes in the science and advances in the technology associated with parenteral medications and routes of administration Includes 13 new chapters and updated chapters throughout Contains the contributors of leading researchers in the field of parenteral medications Uses full color detailed illustrations, enhancing the learning process The fourth edition not only reflects enhanced content in all the chapters but also highlights the rapidly advancing formulation, processing, manufacturing parenteral technology including advanced delivery and cell therapies. The book is divided into seven sectionss: Section 1 - Parenteral Drug Administration and Delivery Devices; Section 2 - Formulation Design and Development; Section 3 - Specialized Drug Delivery Systems; Section 4 - Primary Packaging and Container Closure Integrity; Section 5 - Facility Design and Environmental Control; Section 6 - Sterilization and Pharmaceutical Processing; Section 7 - Quality Testing and Regulatory Requirements
The 3rd World Congress on Genetics, Geriatrics, and Neurodegenerative Disease Research (GeNeDis 2018), focuses on recent advances in genetics, geriatrics, and neurodegeneration, ranging from basic science to clinical and pharmaceutical developments. It also provides an international forum for the latest scientific discoveries, medical practices, and care initiatives. Advanced information technologies are discussed, including the basic research, implementation of medico-social policies, and the European and global issues in the funding of long-term care for elderly people.
This book is immensely useful for graduate students as well as researchers to understand the basics of molecular biology and Recombinant DNA Technology. It provides a comprehensive overview of different approaches for the synthesis of recombinant proteins from E. coli including their cloning, expression and purification. Recent advances in genomics, proteomics, and bioinformatics have facilitated the use of Recombinant DNA Technology for evaluating the biophysical and biochemical properties of various proteins. The book starts with an introductory chapter on gene cloning, protein expression and purification and its implication in current research and commercial applications. Each chapter provides a lucid set of principles, tools and techniques for both students and instructors. The protocols described have been aptly exemplified, and troubleshooting techniques have been included to aid better understanding. Moreover, the set of questions at the end of each chapter have been particularly formulated to help effective learning.