Analytical Approaches for Assessing Aggregation of Protein Biopharmaceuticals

Abstract

Production of protein-based pharmaceuticals is a major issue in conventional pharmacology, biomedicine and nanomedicine. Being mostly obtained by genetic engineering, the quality and activity of protein drugs is a steady matter of concern. Although the physiology of the host recombinant cells, mostly mammalian and microbial, is progressively understood, the complexity of the cellular quality control systems escapes rational protein and process engineering, and recombinant proteins are often unstable, aggregate and/or do not reach the fully native conformation compatible with proper biological activity. In this review, we summarize the main biological aspects of protein folding and misfolding, mainly focusing in microbial cells, the newest insights in the biological control of protein quality and the main and analytical approaches that are suitable for the fast evaluation of the conformational quality and aggregation of recombinant drugs, even if showing apparent solubility.

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

“…The IB formation and its maintenance involve a complex network of intracellular responses related to culture conditions, leading to complex and stable structures sometimes showing bioactivity [13,22]. Due to their different physicochemical properties, IBs have been proposed for various uses, such as catalysts, support materials, drug delivery agents, cell therapy, and immunogens, and their recent application has become an important new topic in biology, medicine and biotechnology [11,[23][24][25][26][27][28][29][30]. However, the study of their physicochemical properties is a recent area, and few reports have been published about that [18,[31][32][33].…”

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

“…The main objective of the current study was to explore the capability of K. marxianus to secrete various heterologous proteins at different temperatures. In fact, for both eukaryotic and prokaryotic hosts, it has been reported that growth temperature affects the yield of properly folded recombinant proteins [13,14]. In E. coli a common strategy to express the target protein in a soluble state consists in evaluating different growth temperatures.…”

Post-production protein stability: trouble beyond the cell factory

“…The high concentration at which proteins drugs are stored and administered [50] specifically favors aggregation [51]. In this context, diverse analytical procedures have been developed and specifically adapted to the detection of therapeutic protein aggregation [52-54]. Importantly, aggregation does not only render drug inactivation during storage, and fast clearance, reduction of activity, limited bioavailability and proteolytic digestion upon administration, but it also stimulates undesired immunogenicity [55].…”