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Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor–liquid, solid–liquid, and liquid–liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.

Chemically linked Double network (DN) hydrogels display extraordinary mechanical attributes but mostly suffer from poor self-healing property and unsatisfactory biocompatibility due to the irreversible breaks in their chemical-linked networks and the use of toxic chemical crosslinking agents. To address these limitations, we developed a novel κcarrageenan/polyacrylamide (KC/PAM) DN hydrogel through a dual physical-crosslinking strategy, with the ductile, hydrophobically associated PAM being the first network, and the rigid potassium ion (K +) cross-linked KC being the second network. The dual physically cross-linked DN (DPC-DN) hydrogels with optimized KC concentration exhibit excellent fracture tensile stress (1320 ± 46 kPa) and toughness (fracture energy: 6900 ± 280 kJ/m 3), comparable to those fully chemically linked DN hydrogels and physically-chemically cross-linked hybrid DN hydrogels. Moreover, owing to their unique dual physical-crosslinking structures, the KC/PAM hydrogels also demonstrated rapid self-recovery, remarkable notch-insensitivity, self-healing capability, as well as excellent cytocompatibility towards stem cells. Accordingly, this work presents a new strategy towards fabricating self-repairing DPC-DN hydrogels with outstanding mechanical behaviors and biocompatibility. The new type of DN hydrogels demonstrates strong potentiality in many challenging biomedical applications such as artificial diaphragm, tendon, and cartilage.

Aggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.

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