Protein PEGylation for the design of biobetters: from reaction to purification processes

Abstract

The covalent attachment of polyethylene glycol (PEG) to therapeutical proteins is an important route to develop biobetters for biomedical, biotech and pharmaceutical industries. PEG conjugation can shield antigenic epitopes of the protein, reduce degradation by proteolytic enzymes, enhance long-term stability and maintain or even improve pharmacokinetic and pharmacodynamics characteristics of the protein drug. Nonetheless, correct information in terms of the PEGylation process from reaction to downstream processing is of paramount importance for the industrial application and processing scale-up. In this review we present and discuss the main steps in protein PEGylation, namely: PEGylation reaction, separation of the products and final characterization of structure and activity of the resulting species. These steps are not trivial tasks, reason why bioprocessing operations based on PEGylated proteins relies on the use of analytical tools according to the specific pharmaceutical conjugate that is being developed. Therefore, the appropriate selection of the technical and analytical methods may ensure success in implementing a feasible industrial process.

Both IgM and IgG Antibodies against Polyethylene Glycol Can Alter the Biological Activity of Methoxy Polyethylene Glycol-Epoetin Beta in Mice

“…In contrast to foreign proteins modified with multiple, short PEG molecules, many pegylated biopharmaceutics are derived from human proteins and are conjugated with one long linear or branched PEG chain. [2] PEG-EPO is a good example of such a drug: it is derived from human erythropoietin and is modified with a single linear 30 kDa PEG chain. Until now, there is no evidence that the infusion of PEG-EPO induces the formation of new anti-PEG antibodies in human patients.…”

“…At least twenty pegylated drugs have been approved by the FDA ( Table 1) and dozens of pegylated medicines are in different stages of clinical development [1]. Pegylation ranges from the random attachment of multiple short PEG chains in drugs such as Adagen (11~17 5 kDa PEG molecules) and Oncaspar (69~82 5 kDa PEG molecules) to the site-specific introduction of a single long PEG chain in drugs such as Plegridy (20 kDa PEG) and Jivi (60 kDa branched PEG) [2][3][4][5]. The attachment of a single long linear or branched PEG chain has become the standard for recent pegylated medicines [1].…”

Improvement of Certolizumab Fab′ properties by PASylation technology

“…To overcome this problem, the C-terminal part of the Fab′ fragment has been covalently fused to a single 40 kDa PEG molecule. PEG is a biocompatible polymer used to enhance the pharmacokinetics of small therapeutic proteins 11 , 12 . The PEG polymer, as a conjugate, confers some advantages to proteins such as increasing half-life, hydrodynamic volume and solubility, while PEGylation harbors disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity due to the shielding effect and increased viscosity of the sample (reviewed by Zhang et al . )…”

Production of a novel N‐terminal PEGylated crisantaspase

“…It has advantage that usually protein N‐terminal groups have lower p Ka values (∼7.5–8.0) than lysine residues (∼10.5). Thus, the reaction is carried out under pH conditions in which the lysine residues are protonated, whereas the N‐terminal amino group is predominantly deprotonated and available for conjugation with the polymer . NHS ester of succinate of mPEG and NHS ester of carbonate of mPEG are the main agents to N‐terminal PEGylated proteins production …”