Bernardo Perez-Ramirez

Lyophilized Silk Fibroin Hydrogels for the Sustained Local Delivery of Therapeutic Monoclonal Antibodies

The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering β-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications.

Viscosity of concentrated therapeutic protein compositions

The use of monoclonal antibodies as therapeutic agents has been increasing steadily over the last decade for the treatment of various conditions. There is often a need to deliver a large dose of the protein, so there is a trend toward developing commercially viable liquid formulations of highly concentrated antibodies. Such concentrated solutions are associated with a number of challenges, including optimization of production processes, plus chemical and physical stability of the final product where solution viscosity becomes a critical quality attribute. Assessment of the rheological characteristics of concentrated compositions is essential as are development strategies to reduce the viscosity. This review covers the state-of-the-art rheology measurement techniques, focusing particularly on concentrated protein solutions. Current understanding of the mechanisms leading to high viscosity and control by formulation parameters is discussed.

Guide to collagen characterization for biomaterial studies.

The structure and remodeling of collagen in vivo is critical to the pathology and healing of many human diseases, as well as to normal tissue development and regeneration. In addition, collagen matrices in the form of fibers, coatings, and films are used extensively in biomaterial and biomedical applications. The specific properties of these matrices, both in terms of physical and chemical characteristics, have a direct impact on cellular adhesion, spreading, and proliferation rates, and ultimately on the rate and extent of new extracellular matrix formation in vitro or in vivo. In recent studies, it has also been shown that collagen matrix structure has a major impact on cell and tissue outcomes related to cellular aging and differentiation potential. Collagen structure is complex because of both diversity of source materials, chemistry, and structural hierarchy. With such significant impact of collagen features on biological outcomes, it becomes essential to consider an appropriate set of analytical tools, or guide, so that collagens attained from commercial vendors are characterized in a comparative manner as an integral part of studies focused on biological parameters. The analysis should include as a starting point: (a) structural detail-mainly focused on molecular mass, purity, helical content, and bulk thermal properties, (b) chemical features-mainly focused on surface elemental analysis and hydrophobicity, and (c) morphological features at different length scales. The application of these analytical techniques to the characterization of collagen biomaterial matrices is critical in order to appropriately correlate biological responses from different studies with experimental outcomes in vitro or in vivo. As a case study, the analytical tools employed for collagen biomaterial studies are reviewed in the context of collagen remodeling by fibroblasts. The goal is to highlight the necessity of understanding collagen biophysical and chemical features as a prerequisite to (a) studies with cells and tissue formation, and (b) suggest modes to establish comparative outcomes for studies conducted in different laboratories.

Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field.

Subvisible (2–100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application.

Mechanistic complexity of subvisible particle formation: Links to protein aggregation are highly specific

There is little knowledge available on the mechanistic features of the protein aggregation pathway, which lead to subvisible particles (SVPs) (0.1-100 µm in size). Additionally, the relationship between soluble aggregates (SAs) (those that are less than 0.1 µm in size) and SVP formation is largely unknown. To better understand these relationships and the mechanism of SVP formation, we conducted agitation experiments on three different classes of proteins; two antibodies [an immunoglobulin G (IgG) 1 and an IgG4] and a glycoprotein. A quantification of SVPs, using the Brightwell Microfluidics Instrument, and levels of SAs by size-exclusion chromatography were determined as a function of agitation time. Not surprisingly, the propensity to aggregate and particulate was different for each protein. However, integrated mass analysis in these studies showed that the relationship between SA and SVP formation is also protein and formulation dependent, and can vary greatly between molecules. Morphological and statistical analysis of SVPs in agitated and nonagitated samples revealed that changes in both the shape and the size distribution of the SVPs population are also protein dependent and highly defined. Collectively, these results suggest/illustrate the complexity of elucidating an aggregation mechanism that encompasses both SAs and SVPs.

Subvisible (2–100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Approaches for Early Developability Assessment of Proteins to Guide Quality by Design of Liquid Formulations

A systematic work process has been defined that strengthens the critical interface between discovery research and formulation development for the early identification of suitable protein candidates to be moved into formulation development and assess their developmentability/manufacturability potential. This predevelopment risk-based approach is essential for (i) understanding the solution behavior of therapeutic proteins, (ii) identifying preliminary critical quality attributes, (iii) optimizing liquid formulations earlier, and (iv) aligning with the quality by design requirements directed by industry guidelines, namely, ICH Q8(R2), Q9, and Q10.