Biological Insights into Therapeutic Protein Modifications throughout Trafficking and Their Biopharmaceutical Applications

Over the lifespan of therapeutic proteins, from the point of biosynthesis to the complete clearance from tested subjects, they undergo various biological modifications. Therapeutic influences and molecular mechanisms of these modifications have been well appreciated for some while remained less understood for many. This paper has classified these modifications into multiple categories, according to their processing locations and enzymatic involvement during the trafficking events. It also focuses on the underlying mechanisms and structural-functional relationship between modifications and therapeutic properties. In addition, recent advances in protein engineering, cell line engineering, and process engineering, by exploring these complex cellular processes, are discussed and summarized, for improving functional characteristics and attributes of protein-based biopharmaceutical products. 1. Introduction Nature has evolved complex biological processes that enable mammalian cells to conduct sophisticated physiological activities for living and adaptation. Primary structure of a protein obtained from genome projects serves as a nice starting point for the understanding of biological complexity but not sufficient to explain various functions and regulations. Majority of proteins from eukaryotic cells are subjected to certain kind of covalent modifications either during or after their ribosomal synthesis. An increasing appreciation of these cellular homeostatic modifications, which have been shown to initiate various biological functions and regulation mechanisms, should contribute to unfolding detailed knowledge about biological networks and living systems. One of the earliest pieces of evidences for the existence of posttranslational modifications can be traced back to the finding of protein phosphorylation published in 1883 [1, 2], in which protein casein was found containing stoichiometric amount of phosphate. One of the earliest modified proteins to be studied, the first glycoprotein, is likely the glycogen of liver which the French physiologist Claude Bernard famously identified as “glycogenous matter” in 1855 [3]. During the biosynthesis of all proteins, the building blocks are 19 standard amino acids (plus selenocysteine for a few selenoproteins) and one imino acid. Nonetheless upon hydrolysis, close to 200 different amino acids have been identified, indicating a significant posttranslational modification of the originally encoded sequences [4]. Over the years, more than 200,000 reported modification events have been unveiled by a PubMed analysis [5].