One of the major challenges associated with the development of proteinbased
biotherapeutics lies in achieving persistent concentrations of the active molecule
in circulation. Most human proteins are rapidly cleared from circulation, mainly by
renal filtration, and so increasing their in vivo residence time to reach appropriate
therapeutic doses has been a matter of extensive investigation. The majority of
therapeutic proteins exhibit post-translational modifications (PTMs). Among them, Nand
O-glycosylation are the most abundant and complex modifications that proteins
can undergo, affecting diverse biological properties, including solubility, protease and
thermal stability, antigenicity, immunogenicity, bioactivity and pharmacokinetics.
Thus, glycosylation represents one of the most relevant attributes of many therapeutic
proteins, defining their potency and effectiveness. Also, both size and charge of
proteins are completely modified by the presence of glycans, so that manipulation of
this PTM represents a valuable tool to alter the pharmacokinetics and
pharmacodynamics of biotherapeutics. This chapter deals with different
glycoengineering strategies developed with the aim of increasing the plasma half-life of
proteins, as well as other properties. Specifically, engineering of proteins through the
addition of new glycosyl moieties is addressed. A thorough description of the
properties conferred to proteins by glycans is first presented, followed by a description
of the strategies developed for the rational manipulation of glycosylation parameters to
improve such properties. Different approaches to incorporate new N- and O-glycans into proteins are described and exemplified. Finally, the application of N- and Ohyperglycosylation
engineering to an emblematic protein such as recombinant human
IFN-α2b is presented.
Keywords: Antigenicity, Bioactivity, Biotherapeutics, CTP, Efficacy,
Glycoengineering, Glycosylation prediction, hCG, hEPO, hFSH, hIFN-α2b,
Immunogenicity, N-glycosylation, O-glycosylation, Peptide fusion,
Pharmacokinetics, Plasma clearance, Site-directed mutagenesis, Solubility,
Stability.
