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Monoclonal antibodies (mAbs) are currently leading products in the global biopharmaceutical market. Multiple mAbs are in clinical development and novel biotherapeutic protein scaffolds, based on the canonical immunoglobulin G (IgG) fold, are emerging as treatment options for various medical conditions. However, fast approvals for biotherapeutics are challenging to achieve, because of difficult scientific development procedures and complex regulatory processes. Selecting molecular entities with superior physicochemical properties that proceed into clinical trials and the identification of stable formulations are crucial developability aspects. It is widely accepted that the solution pH has critical influences on both the protein's colloidal stability and its crystallization behavior. Furthermore, proteins usually crystallize best at solution conditions that enable high protein solubility, purity, stability, and monodispersity. Therefore, we hypothesize that the solution pH value is a central parameter that is linking together protein formulation, protein crystallization, and thermal protein stability. In order to experimentally test this hypothesis, we have investigated the effect of the solution pH on the thermal stabilities and crystallizabilities for three different mAbs. Combining biophysical measurements with high throughput protein (HTP) crystallization trials we observed a correlation in the buffer pH values for eminent mAb stability and successful crystallization. Specifically, differential scanning fluorimetry (DSF) was used to determine pH values that exert highest thermal mAb stabilities and additionally led to the identification of unfolding temperatures of individual mAb domains. Independently performed crystallization trials with the same mAbs resulted in their successful crystallization at pH values that displayed highest thermal stabilities. In summary, the presented results suggest a strategy how protein crystallization could be used as a screening method for the development of biotherapeutic protein formulations with improved in vitro stabilities.

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.

Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LTβR) binding Fv domain of an anti-LTβR/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley-Liss, Inc.