In high concentration systems (>50 mg/ml), the changes in protein particle size and light scattering may behave in nonlinear fashions [69-71]. to stimulate new dialogs that would bridge the interface between physical characterizations of protein aggregates in biotherapeutics and the functional attributes of the immune system. Keywords:monoclonal antibodies, high-concentration protein formulations, anti-drug antibodies, T cell epitopes, bioprocessing == I. Introduction == The rapid expansion of biologic drugs in recent years has led to a pressing need for early and precise control of product quality, in which protein aggregation remains a key challenge because of its implications in potency and safety [1]. Understanding the molecular mechanisms of protein aggregation is critical for developing mitigation strategies. Proteins aggregate through three main mechanisms, broadly defined by the seeding entity: native monomers, denatured proteins, and pre-existing aggregates [2,3]. Monomers of native proteins can self-associate into oligomers through complementarity of charge-charge interactions, or through covalent linkages formed between hydrophilic and hydrophobic residues around the protein exterior. Low molecular weights, non-covalent oligomers can revert to their native says, but as the oligomers increase in size over time, the associations become irreversible. It is assumed that in any given protein product there exists a denatured fraction [4], Prazosin HCl which has a propensity to undergo Prazosin HCl irreversible aggregation. Because protein conformation is usually a dynamic phenomenon, partially unfolded proteins may refold under certain conditions. However, the free energy and kinetics generally favor aggregation rather than refolding [5]. Native protein monomers can also aggregate by adhering to pre-existing protein oligomers, contaminants, or vessel surface, rapidly expand via nucleation. Aggregates can be classified as soluble and insoluble [6]. Soluble aggregates have low molecular mass and may be reversible. A small amount of soluble aggregates, between 5 to 10%, for example, may be acceptable in biologic products, because the belief is usually that it is generally impractical to eliminate aggregates below these levels [7]. When protein aggregation exceeds the solution solubility limit, aggregates become irreversible and precipitate out of solution. The tolerable quantity of insoluble aggregates appears to correlate with the size of particulates detected in the protein product upon reconstitution. Particles as low as 150 m in diameter in injectable products may be detected visually [8]. Perhaps paradoxically, particles that below 150 m are more likely to elicit immune reactions [9-11]. Particles with a hydrodynamic radius of 50-100m are generally considered subvisible [6], with those that are 10 m occlude blood flow [12]. Described in the U.S. Pharmacopeia (USP) chapter <788> is usually a subvisible particle counting method, Rabbit Polyclonal to TSPO which sets the acceptable limit of particulate matter in a container of 100 mL to be 6000 particles 10 m and 600 particles 25 m. Based on the standards, 10 m is usually most often invoked as a limit in analytical regulatory guidance. Because many recently developed protein products are administered subcutaneously or intramuscularly, limiting aggregates based on the 10 m threshold to avoid blood vessel occlusion has become less relevant. Furthermore, aggregates 10 m possess a greater risk in product stability and immunogenicity [9-11,13,14]. Therefore, the U.S. Food and Drug Administration (FDA) has tighten the regulatory scrutiny of aggregates below 10 m in biologic products [9]. Beyond the classifications based on the aggregation mechanisms, solubility, and size, protein aggregation can be delineated as intrinsic and extrinsic. Intrinsic protein aggregation arises within the protein formulation during the synthesis and purification actions. Extrinsic aggregation, in contrast, results from the contacts of protein with external sources during processing, such as glass surfaces inside containers, stainless steel of bioprocessing gear, or silicon oil Prazosin HCl droplets inside pre-filled syringes. Because these two categories intertwine and collectively contribute to protein aggregates detected downstream, specific attribution to each is usually often not done. In this review, we will focus on intrinsic protein aggregation, in which prediction and mitigation approaches can be applied as early as in the drug development process. Summarized inFigure 1are the.