Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

Table 2

Affinity solid-phase PEGylation of some important proteins.

Protein	Affinity support	Used PEG reagent	Mono-PEGylated yield (%)	Formation of multi-PEGylated	Relative bioactivity^a (%)	Reference
L-Asparaginase	L-Asparagine Sepharose CL-4B	5 kDa cyanuric chloride mPEG	—	No	50.2^b	[43]
Catalase	Procion Red Sepharose CL-4B	6 kDa cyanuric chloride mPEG	—	No	46.5^c	[43]
Recombinant fibroblast growth factor 2 (FGF-2)	Heparin Sepharose	20 kDa mPEG butyraldehyde	58.3	No	105.4^d	[44]
Recombinant human keratynocite growth factor 1 (RhKGF-1)	Heparin Sepharose	20 kDa mPEG butyraldehyde	40	No	89.6^d	[45]
His-tagged recombinant staphylokinase (trSak)	Ni^2+-IDA Sepharose 4FF	Succinimidyl carbonate mPEG (5,10, 20 kDa)	5 kDa > 40; 10 kDa > 30; 20 kDa > 28	Yes (di-PEG) in PEGylation with 5 and 10 kDa	5 kDa > 83^e; 10 kDa > 81 20 kDa > 75	[42]

^aKept bioactivity after PEGylation respect to bioactivity of the unmodified protein. Bioactivity was measured in a different way for each protein. ^bMeasured absorbance at 500 nm of ammonia with Nessler's method. ^cSpectrophotometrical measure of the decrease in absorbance of H₂O₂ at 240 nm. ^dMitogenic activity measured by NIH3T3 cell proliferation assay. ^eIn vitro bioactivity was measured as the diameter of the halo around the well in Petri dishes with the fibrinogen powder.