Stabilization of α‐chymotrypsin upon PEGylation correlates with reduced structural dynamics

Abstract

Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein α-chymotrypsin (α-CT) was covalently modified with PEGs with molecular weights (MW) of 700, 2000 and 5000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (Tm) of α-CT by up to 6°C and the free energy of unfolding (ΔGU (25°C)) by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG MW and it leveled off after an average of four PEG molecules were bound to α-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG MW. Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, poly(ethylene glycol) thermodynamically stabilizes α-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.

Investigation of PEG Crystallization in Frozen and Freeze‐Dried PEGylated Recombinant Human Growth Hormone–Sucrose Systems: Implications on Storage Stability

“…PEGylation does not always result in an altered protein structure and dynamics. 11,12,14 We cannot, therefore, generalize our observations of PEG crystallization from frozen PEG-rhGH solutions to other proteins.…”

“…[4][5][6][7][8][9][10][11] Although PEGylation does not cause significant changes in the secondary structure of proteins, changes in the tertiary and quaternary structure have been reported. 12 Svergun et al 13 recently showed that PEGylation elongates the protein hemoglobin, wherein a part of the PEG interacts with the protein and a major portion of the PEG protrudes away from the protein.…”

“…An important consequence of PEGylation appears to be a more compact protein structure and reduced structural dynamics after conjugation. 11,12 However, there are contrasting examples where PEGylation did not alter the internal dynamics in a protein. 14 Currently, 10 approved products containing PEGylated peptides, proteins, and small molecules are available for therapeutic use, 8,15,16 of which three are manufactured by freeze-drying.…”

Effect of site-specific PEGylation on the fibrinolytic activity, immunogenicity, and pharmacokinetics of staphylokinase

“…Modification of the Sak molecule with PEG might influence protein thermostability [22,24]. After PEGylation, the thermal stability of Sak(E80C-PEG) slightly decreased when compared with r-Sak.…”

“…An important advantage of PEGylation is the reduction or elimination of protein immunogenicity [4,24,25]. The immunogenicity studies indicated that Sak(E80C-PEG) had greatly reduced immunogenicity in guinea pigs.…”