Control of Mammalian Cell Behaviour Through Mimicry of the Extracellular Matrix Environment

This chapter looks at the control of mammalian cell behaviour through mimicry of the extracellular matrix enviromen

Discovering Novel Small Molecule Compound for Prevention of Monoclonal Antibody Self-Association

Designing an antibody with the desired affinity to the antigen is challenging, often achieved by lengthening the hydrophobic CDRs, which can lead to aggregation and cause major hindrance to the development of successful biopharmaceutical products. Aggregation can cause immunogenicity, viscosity and stability issues affecting both the safety and quality of the product. As the hydrophobic residues on the CDR are required for direct binding to antigens, it is not always possible to substitute these residues for aggregation-reduction purposes. Therefore, discovery of specific excipients to prevent aggregation is highly desirable for formulation development. Here, we used a combination of in silico screening methods to identify aggregation-prone regions on an aggregation-prone therapeutic antibody. The most aggregation-prone region on the antibody was selected to conduct virtual screening of compounds that can bind to such regions and act as an aggregation breaker. The most promising excipient candidate was further studied alongside plain buffer formulations and formulations with trehalose using coarse-grained molecular dynamics (CGMD) simulations with MARTINI force field. Mean interaction value between two antibody molecules in each formulation was calculated based on 1024 replicates of 512 ns of such CGMD simulations. Corresponding formulations with an excipient:antibody ratio of 1:5 were compared experimentally by measuring the diffusion interaction parameter kD and accelerated stability studies. Although the compound with the highest affinity score did not show any additional protective effects compared with trehalose, this study proved using a combination of in silico tools can aid excipient design and formulation development

Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range.

Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1–10 μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates’ generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development

Insights into the influence of the cooling profile on the reconstitution times of amorphous lyophilized protein formulations

Lyophilized protein formulations must be reconstituted back into solution prior to patient administration and in this regard long reconstitution times are not ideal. The factors that govern reconstitution time remain poorly understood. The aim of this research was to understand the influence of the lyophilization cooling profile (including annealing) on the resulting cake structure and reconstitution time. Three protein formulations (BSA 50 mg/ml, BSA 200 mg/ml and IgG1 40 mg/ml, all in 7% w/v sucrose) were investigated after cooling at either 0.5 °C/min, or quench cooling with liquid nitrogen with/without annealing. Significantly longer reconstitution times were observed for the lower protein concentration formulations following quench cool. Porosity measurements found concomitant increases in the surface area of the porous cake structure but a reduction in total pore volume. We propose that slow reconstitution results from either closed pores or small pores impeding the penetration of water into the lyophilized cake

Engineering biodegradable polyester particles with specific drug targeting and drug release properties

Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres and nanoparticles remain the focus of intensive research effort directed to the controlled release and in vivo localization of drugs. In recent years engineering approaches have been devised to create novel micro- and nano-particles which provide greater control over the drug release profile and present opportunities for drug targeting at the tissue and cellular levels. This has been possible with better understanding and manipulation of the fabrication and degradation processes, particularly emulsion-solvent extraction, and conjugation of polyesters with ligands or other polymers before or after particle formation. As a result, particle surface and internal porosity have been designed to meet criteria-facilitating passive targeting (e.g., for pulmonary delivery), modification of the drug release profile (e.g., attenuation of the burst release) and active targeting via ligand binding to specific cell receptors. It is now possible to envisage adventurous applications for polyester microparticles beyond their inherent role as biodegradable, controlled drug delivery vehicles. These may include drug delivery vehicles for the treatment of cerebral disease and tumor targeting, and codelivery of drugs in a pulsatile and/or time-delayed fashio

Modulation of the intestinal tight junctions using bacterial enterotoxins

This chapter discusses the modulation of the intestinal tight junctions using bacterial enterotoxins. Epithelial cell sheets limit the movement of solutes through the intercellular space by forming tight junctions (tjs) between adjacent epithelial cells, and therefore act as the major barrier between the internal and external environment of the body. There are three transepithelial pathways for molecules to pass from the intestinal lumen to the bloodstream: the passive transcellular pathway, the carrier-mediated transcellular pathway, and the paracellular pathway. Physicochemical properties, such as hydrophobicity, allow a molecule to passively partition from the intestinal lumen through the lipid bilayer into the cell. Some hydrophilic molecules, such as sugars and amino acids, are absorbed by specifically interacting with active transport systems on the cell membrane. Drug delivery via the paracellular pathway is less dependent on the physicochemical properties of the drug, and does not require a specific interaction with a transport system, and so is suitable for a large variety of molecules including peptides and proteins. Since opening of the tjs can cause the influx of other foreign substances, absorption enhancers acting via the paracellular pathway are required to modify the tight junctional structure reversibly. Early development of paracellular absorption enhancers was limited due to the lack of knowledge regarding the composition of the tjs and their regulatory role, resulting in unacceptable side-effects. Study of canonical enterotoxins (a class of exotoxin that acts on the intestinal epithelium) such as those released by Clostridium perfringens and Vibrio cholerae has increased our understanding to the point where one can exploit their mechanisms to facilitate peptide and protein delivery via the paracellular pathway

Engineering silica particles as oral drug delivery vehicles

Porous silica particles are emerging as complementary systems to polyester microspheres for the encapsulation and controlled delivery of small-organic drugs. Their recent application in pharmaceutics is strengthened by well-established characterization and synthetic routes from the chemical engineering sciences. Silica is an interesting scaffold material for the encapsulation of organic molecules. It can be formed into hierarchical structures over a wide range of length scales and interconnectivities. Encapsulation can therefore be tailored not only to the drug but the desired release properties. In addition to surfactant-templating of hierarchical silica structures, polypeptides from marine organisms may offer biological routes to novel silica materials. Silica sol-gels have also been evaluated as delivery vehicles, particularly with regard to generating hybrid systems with mesoporous silica or composite xerogels. This review will first focus on the detailed characterisation of pore size and structure of mesoporous silica with regards water penetration and drug diffusion. We then describe the pharmaceutical applications of silica materials with regard to improving oral bioavailability, multiparticulate systems for gastroretention or sustained release, composite xerogels and in vivo biocompatibility