Development of a strategy for transient protein PEGylation with releasable PEGs: a case study with human growth hormone

The use of protein therapeutics is, to date, one of the preferred strategies for the treatment of numerous diseases. This is due to the many advantages that these new drugs possess over traditional “small molecules”. However, therapeutic proteins also have quite a few issues: immunogenicity, degradation by enzymes, and above all, rapid renal clearance. The most widely used strategy in the pharmaceutical industry to overcome these challenges is PEGylation. PEGylation involves the attachment of polyethylene glycol (PEG) chains to bioactive molecules such as proteins, peptides, oligonucleotides, and others. This strategy makes it possible to overcome the limitations presented by the use of therapeutic proteins and also ensures the achievement of other advantages: increase in protein size resulting in reduced renal clearance, reduction in enzymatic degradation, increased solubility in aqueous media, and reduced immunogenicity. With the evolution of this technique, different PEGylation strategies have been developed. This work is based precisely on a new PEGylation strategy called "transient PEGylation". Indeed, it consists of the development of new releasable PEGs to achieve a “transient PEGylation” at the level of ε-amino groups of lysines of a protein, namely human growth hormone (hGH). For this purpose, two releasable PEGs of about 40 kDa containing a linker with an acetal modified that can hydrolyze in the physiological medium, releasing the protein in its native form, were used. The release of these polymers was compared with that of a non-releasable PEG, used as control. As a first step, it is necessary to set up the conjugation reaction between the PEGs and hGH. In this regard, the first step is to exchange the hGH with the reaction buffer, which is 0.2 M borate pH 9.5. The hGH thus exchanged is then reacted overnight with the PEG solubilized in ACN. The second step is the purification of the desired product, the monoconjugate, from the other species present in the reaction. This is accomplished through two purification steps: the first step involves the use of ion-exchange chromatography to separate the free PEG from the monoconjugate and biconjugate; the second step employed size-exclusion chromatography to obtain the monoconjugate in pure form. The purity of the monoconjugate is confirmed by reversed-phase chromatography analysis. The pure monoconjugates asre subsequently subjected to a series of characterizations and studies to test their ability to release hGH in its native form. At the end of the project, all three monoconjugates were subjected to in vivo pharmacokinetic studies in rats. The preliminary data obtained revealed an increase in the half-life of hGH when conjugated to these PEGs, confirming that this strategy was effective in reducing the frequency of protein administrations and ensuring a better in vivo activity of the protein.

The use of protein therapeutics is, to date, one of the preferred strategies for the treatment of numerous diseases. This is due to the many advantages that these new drugs possess over traditional “small molecules”. However, therapeutic proteins also have quite a few issues: immunogenicity, degradation by enzymes, and above all, rapid renal clearance. The most widely used strategy in the pharmaceutical industry to overcome these challenges is PEGylation. PEGylation involves the attachment of polyethylene glycol (PEG) chains to bioactive molecules such as proteins, peptides, oligonucleotides, and others. This strategy makes it possible to overcome the limitations presented by the use of therapeutic proteins and also ensures the achievement of other advantages: increase in protein size resulting in reduced renal clearance, reduction in enzymatic degradation, increased solubility in aqueous media, and reduced immunogenicity. With the evolution of this technique, different PEGylation strategies have been developed. This work is based precisely on a new PEGylation strategy called "transient PEGylation". Indeed, it consists of the development of new releasable PEGs to achieve a “transient PEGylation” at the level of ε-amino groups of lysines of a protein, namely human growth hormone (hGH). For this purpose, two releasable PEGs of about 40 kDa containing a linker with an acetal modified that can hydrolyze in the physiological medium, releasing the protein in its native form, were used. The release of these polymers was compared with that of a non-releasable PEG, used as control. As a first step, it is necessary to set up the conjugation reaction between the PEGs and hGH. In this regard, the first step is to exchange the hGH with the reaction buffer, which is 0.2 M borate pH 9.5. The hGH thus exchanged is then reacted overnight with the PEG solubilized in ACN. The second step is the purification of the desired product, the monoconjugate, from the other species present in the reaction. This is accomplished through two purification steps: the first step involves the use of ion-exchange chromatography to separate the free PEG from the monoconjugate and biconjugate; the second step employed size-exclusion chromatography to obtain the monoconjugate in pure form. The purity of the monoconjugate is confirmed by reversed-phase chromatography analysis. The pure monoconjugates are subsequently subjected to a series of characterizations and studies to test their ability to release hGH in its native form. At the end of the project, all three monoconjugates were subjected to in vivo pharmacokinetic studies in rats. The preliminary data obtained revealed an increase in the half-life of hGH when conjugated to these PEGs, confirming that this strategy was effective in reducing the frequency of protein administrations and ensuring a better in vivo activity of the protein.