Development of releasable PEG for transient protein PEGylation

PEGylation is a widely used strategy in the pharmaceutical industry to overcome hurdles in delivery of protein therapeutics. It involves linking PEG chains to bioactive molecules such as proteins, peptides, and oligonucleotides to increase molecular weight, reduce renal excretion, improve solubility, prolong retention time in blood, and decrease immunogenicity. Various strategies exist, including random and site-specific conjugation. Simple random conjugation can reduce receptor interaction due to steric entanglement while site-specific conjugation offers better preservation of receptor binding and homogeneity. The interaction between PEG and proteins is typically covalent, which can hinder protein-receptor interactions or catalytic sites. The use of smaller PEG molecules can minimize this constraint but reduces protection in the bloodstream and prolongation of half-life. Releasable PEGs release native proteins slowly, which prevents the efficacy loss observed in stable covalent PEGylation or random conjugation. This work was committed to develop new releasable PEGs useful for the random PEGylation to ε-amino groups, belonging to the ubiquitous lysine of a protein, of human Growth Hormone (hGH). A 40 kDa linear PEG containing an acetal linker chemically modified to be hydrolyzed in the physiological environment was evaluated and compared to a similar non-releasable PEG used as control. To minimize early hydrolysis reactions of the conjugates, it was important to work only with basic pH buffers throughout the synthesis. Once the optimal reaction condition was identified, the pure monoPEGylated protein was purified from the other reaction products. Ion exchange chromatography allowed for the separation of all species present in the reaction mixture. Due to overlapping of the biconjugate and monoconjugates elution peaks a size exclusion chromatography was used to separate the two components. After this, the purified monoPEGylated hGH underwent several characterizations. Mass spectrometry was used to ensure that the protein of interest remained intact and did not degrade in the reaction environment, as well as after the various purification and lyophilization steps. The conjugation was confirmed by using matrix-assisted laser desorption/ionization (MALDI) spectrometry. Furthermore, the preservation of the protein’s secondary structure was assessed by circular dichroism spectroscopy. To assess the ability of these monoconjugates to release the protein of interest, hGH, an in-vitro release study was conducted. The conjugates were exposed to three different environments to confirm the effect of the pH level on the release. Specifically, the conjugates were exposed to buffer solution with pH 5, pH 7.4 and pH 9. To monitor this study reverse phase chromatography was used and by integrating the intensity variation of the monoconjugate peak overtime it was possible to compare the release rate in the different conditions. The study confirmed that the conjugate was rapidly hydrolyzed in an acidic environment, with 50% of it being released after only 20 hours. At physiological pH and temperature, the release was more controlled, with approximately 50% of the conjugate being hydrolyzed after 72 hours. In contrast, at basic pH, only 15% of the conjugate released the protein after 72 hours. The analysis also included non-releasable PEGylated proteins, serving as a negative control, confirming the stability of these conjugates by not releasing hGH. SDS-PAGE was performed for a qualitative analysis of the release. The released hGH from condition with pH 5 and pH 7.4 was collected during the chromatographic analyzes to be characterized by mass spectrometry. The spectra were compared to that of standard hGH to prove the final aim of the project, namely that the released protein was identical to the native one, thus avoiding possible activity limitations due to covalent PEGylation.