1. PEGYLATION: AN EFFECTIVE TOOL FOR BIOMASKING
José A. Ramón, Vivian Saez, Carlos Peniche, Eugenio Hardy
Centro de Biomateriales (BIOMAT)
Universidad de La Habana
Cuba

2. Foreign substances, currently used, in drugs and medical devices:
Metals and their alloys, polymers, ceramics and composites of them used in various devices such as prostheses, stents, heart valves, etc.
Components of drug delivery systems (DDS): (i) phospholipids for the obtaining of liposomes and (ii) various polymers used in biodegradable micro/nano-particles.
Proteins and Peptides
Others

4. Main cause of these problems: AN UNDESIRED INTERACTION BETWEEN FOREIGN MATERIALS AND BODY

5. A solution: BIOMASKING Biomasking by conjugation
Technology`s name
Polymer…
PEGylation
Polyethyleneglycol (PEG)
HSAylation
Human serum albumin
Acylation
Lipids
HESylation
Hydroxyethyl starch
PASylation
Poly-Pro-Ala-Ser
XTENylation
A polypeptide

6. From Shah T. Bioconjugates: The adaptable challenge
From Shah T. Bioconjugates: The adaptable challenge. International BioPharm, Jan

7. From Shah T. Bioconjugates: The adaptable challenge
From Shah T. Bioconjugates: The adaptable challenge. International BioPharm, Jan

8. PEGylation of biomaterials and nanoparticles has been utilized as a strategy to evade immune recognition, improve solubility, and increase in vivo half-life.
Molino et al., Biomacromolecules April 9; 13(4): 974–981.

9. Challenges and Opportunities of current status of PEGylation

13. How develop a PEG-protein candidate?
Reaction
Purification
Characterization
Formulation

14. Reaction Protein PEG reagent Reaction conditions
PEG + prot. ---- PEG-prot
PEG-prot + PEG ---- (PEG)2-prot
(PEG)2-prot + nPEG ---- (PEG)n+2-prot
Protein
PEG reagent
Reaction conditions
Protein and PEG concentration pH
Buffer
Co-solvents

15. An easy way to select PEG reagent

16. Purification Chromatography Type Size-exclusion
It has very poor though put at production scale and also fails to separate other PEGylation variants that might be formed during the PEGylation reaction.
Hydrophobic interaction and reversed-phase
They have been used with some success, but the separation depends on the hydrophobicity of the protein relative to the PEG.
Immunoafinity
Very expensive.
Cation-exchange
It has been used to purify proteins that are PEGylated at amine groups, where the PEGylated proteins contain one less positive charge per PEG molecule attached.

17. Characterization Analytical Method Stoichiometry
MW determination (MALDI-TOF, GPC, SDS-PAGE)
Ratio protein/PEG by colorimetric analysis
Heterogeneity
IE-HPLC + peptides mapping
Purity
SDS-PAGE
Pharmacokinetic profiles
ELISA

18. From Ramon et al. PEGylated Interferon-α2b: A Branched 40K Polyethylene Glycol Derivative. Pharm Res 2005 (22):
It can be concluded that ELISA is still a useful way to obtain data for constructing PK profiles. Anti-PEG ELISAs have been described, but a better solution for detecting PEGylated protein would be a sandwich ELISA, identifying both the protein part and the PEG part of the conjugate. Two different antibodies are necessary; anti-protein antibody and anti-PEG antibody. Such a format would also eliminate errors due to the presence of endogenous proteins.

19. Formulation Liquid Formulation Lyophilizated Formulation
Easy manipulation and administration
Stable at room temperature
Cheaper manufacturing

20. Concluding…
From Ramon et al. PEGylated Interferon-α2b: A Branched 40K Polyethylene Glycol Derivative. Pharm Res 2005 (22):

21. Concluding…