Date Published: March 26, 2019
Publisher: Springer International Publishing
Author(s): Jinjiang Li, Mary E. Krause, Xiaodong Chen, Yuan Cheng, Weiguo Dai, John J. Hill, Min Huang, Susan Jordan, Daniel LaCasse, Linda Narhi, Evgenyi Shalaev, Ian C. Shieh, Justin C. Thomas, Raymond Tu, Songyan Zheng, Lily Zhu.
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.
Protein therapeutics 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.
Aggregation of therapeutic proteins can be induced by stresses encountered at vapor–liquid, solid–liquid, and liquid–liquid interfaces (Fig. 1) (1). Aggregation typically involves some degree of protein conformational change, relative to the folded monomer, that allows two or more proteins to form interprotein bonds that possess similar or greater stability than the intraprotein bonds of the native folded structure. The corresponding growth of aggregates typically occurs through addition of monomers and/or through combining aggregates to form soluble high molecular weight (HMW) aggregates. The mechanism by which the stresses encountered at interfaces promote protein aggregation relative to that in the bulk of solution is less well established. Whether the proteins distort/unfold and self-assemble into aggregates at the interface, and/or whether they release from the interface and further aggregate while in the bulk solution (or even disassemble and refold) is not definitively settled. In principle, each type of interfacial species can reversibly exchange with the corresponding bulk species through convective and/or diffusive mass transport between the bulk and interfaces. The adsorption and desorption processes may be at equilibrium or under mass-transfer control. If under equilibrium control, gentle sample mixing processes are not expected to affect the concentration of adsorbed species; however, if under mass transfer control, then mixing should enhance the adsorption and/or desorption rates via convective mass transfer.Fig. 1Protein interfacial behavior. Proteins from the bulk solution can adsorb to the interface leading to an adsorbed network of proteins. Surfactants can mitigate this adsorption. Modified from Morris et al. (1)
Biologics are developed and manufactured in two major stages. Biologic drug substance is the purified bulk material that has been concentrated to the target protein concentration, partially (or fully) formulated, and typically frozen until initiation of drug product manufacturing. The drug product is the fully formulated protein solution contained in a vial, syringe, or other delivery device that is ready for patient administration. Both protein drug substance and drug product process development and manufacturing workflows can expose proteins to various interfacial stress conditions (Fig. 2). During drug substance manufacturing, the protein is taken through a series of unit operations, including harvest, centrifugation or filtration for removal of cell debris, purification via column chromatography, filtration, virus reduction, concentration, and formulation/storage. In particular, the drug substance filtration, freezing/thawing, as well as unit operations that combine mechanical and interfacial stresses are particularly impactful and will be detailed here.Fig. 2Process flow diagram that indicates types of interfacial stress that can occur during unit operations for drug substance and drug product manufacture, as well as upon transportation and storage
During drug product manufacturing, a drug substance may be mixed with additional excipients, often after thawing, before being filled into vials, syringes, and other devices. The thawing process exposes the protein to the ice/liquid interface, then the mixing step exposes the protein to the air/water interface while being sheared via stirring. The effects of these stresses on biologics were described above. However, the unit operation that likely results in the most interfacial stress is the filling process. Once filled into vials, opportunities for exposure to interfacial stress emerge during storage, transport, and clinical administration.
The integrity of biological drugs for administration in clinical settings is very important because it affects not only the efficacy of the drugs but also the safety of patients. Understanding postproduction handling risks is therefore critical to the development of a robust product (76). The purpose of in-use stability testing is to establish a period of time for the preparation and administration of biological drugs used in the clinical settings while retaining quality within accepted specification. At this time, biological drugs are delivered via intravenous injection of either neat drug product or product that has been diluted with vehicle to achieve specific dosages in a clinical setting, or subcutaneously by various devices that are often self-administered by the patient. For IV administration, the commercially available fluids 0.9% sodium chloride injection, USP (normal saline), or 5% dextrose in water (D5W) are widely used as diluents. The infusion solutions for administration encounter various materials throughout the preparation and administration period, including syringes, tubes, IV bags, and in-line filters. In certain cases, it has been reported that the IV bag materials have an impact on aggregate formation (4). Additionally, the diluted solution may contain particles due to the lower concentration of surfactant after dilution (77); however, infusion solutions containing certain levels of protein aggregates or particles are not suitable for IV administration because they may reduce potency or increase immunogenicity (78). In-line filters are therefore often used to prevent any formed particulate matter from reaching the patient; however, protein adsorption to this filter or to other surfaces encountered throughout administration can lead to the patient receiving less than the intended dose, especially for very low dose therapies. Thus, in-use stability should be evaluated as a part of formulation development to avoid unforeseen problems.
Biologics subjected to interfacial stress can generate a diverse assortment of aggregated species ranging in size from dimers and other soluble aggregates, to subvisible or micrometer-sized particles, to particles in the hundreds of micrometers that are visible to the unaided eye. Due to this size diversity, no single analytical technique is capable of providing a comprehensive assessment of the variety of aggregates that can be generated by interfacial stress. The subsequent section presents a brief overview of the analytical tools useful for characterizing aggregated species, including techniques that assess secondary/tertiary/higher order structure, size, and morphology. An analytical and characterization strategy needs to be developed that utilizes techniques that provide insight into the composition, morphology, mechanism of formation, and quantitation (e.g., mass, particle count) of the aggregated species. It is beyond the scope of this paper to delve into the intricacies and limitations of each technology, as such information has been thoroughly reviewed elsewhere (79–82). Instead, the reader is directed to these reviews and the various citations contained throughout to achieve the appropriate level of understanding.
Biologic molecules encounter multiple types of interfaces during development, manufacturing, and throughout the lifetime of the product. Here, we have highlighted many of the interfacial stress conditions to which proteins are exposed, as well as the effect of these stresses on the stability of the drug substance and drug product, and methods used to characterize. While some mitigation strategies, such as the addition of surfactant, are relatively common, other risk assessment tools and strategies around evaluation of interfacial sensitivity of a given protein should be considered. Each protein has its own unique sensitivities, and in order to develop a robust drug product, it is important to understand the different risks for each molecule. While some recommendations have been made within each section, here we provide a summary of recommendations during different stages of biologics product development.
Exposure of proteins to interfaces during development, manufacturing, and storage is inevitable. Each protein has its own unique sensitivities, and in order to develop a robust drug product, it is important to understand the different risks for each molecule. While some mitigation strategies, such as the addition of surfactant, are relatively common, other risk assessment tools and strategies around the evaluation of interfacial sensitivity of a given protein should be considered. In this commentary, we have described the interfacial conditions to which proteins are exposed, the effects interfacial exposure have on the stability of the protein, potential stability studies and risk assessment tools to understand the impact on each individual molecule, as well as new analytical techniques that are available to explore the behavior of different molecules at the air/liquid and solid/liquid interface. Understanding the particular sensitivities of a molecule throughout the development process is key to development of a robust drug product.