Protein based drugs and the use of polymers

By: Negeen Kargar

Protein based formulations are an important class of medicines. However, proteins are sensitive molecules that generally clear rapidly from the systemic circulation. Much thought needs to be put into the delivery issues of a protein in order to maximize their bioavailability. In spite of this, there are still many examples of such medicine that have successfully reached the market or which are currently in clinical trials [1]. Numbers of different approaches are now being used to prolong the circulation half-life of protein-based medicines. This is the addition of poly ethylene glycol (PEG) to proteins PEGylation. The neutrality and size of the PEG moiety helps the protein to avoid immune response and the reticular endothelial system response that would clear protein[1].

Polyethylene glycol (PEG) is formed by ring opening polymerisation of polyethylene oxide.

Figure 1: PEG and m PEG.

The step-growth polymerization technique relies on the condensation of hydroxy-acids or of mixtures of di acids and diols. The major drawbacks of this poly condensation mechanism are the high temperatures and long reaction times generally required that favour the side reactions, together with the deleterious effect on the molecular weight of any short deviation from the reaction stoichiometry. Such reactions are also limited to equilibrium. Water must consequently be removed from the polymerization medium to increase the conversion and the molecular weight [2].

Water solubility is the important property of PEG used for the crystallization of proteins, as it increases the effective concentration of protein in suspension. Subsequent hydration of PEG can increase its molecular size. Different coupling groups have been added to the PEG for instance, Methoxy-PEG. The stability of the linker, the number of PEG per molecule of protein, and the geometry of PEG all affect the chemistry of conjugation [3].

PEGs that are conjugated to proteins, immunoglobulins or enzymes can have different polydispersities. The terminal –OH group of PEG can also be substituted with a (-CH₃) group. High molecular weight PEG is also sometimes branched. PEG is widely used as an excipient for a large variety of drugs. PEG is removed by oxidation of the –OH group to a -COOH group immediately alcohol by alcohol dehydrogenase [3].

A major challenge of PEG biologics is the viability of radio labeling. The dose of PEGyated drug is very low, and human beings are routinely exposed to PEG products. Metabolic products of PEG are the same regardless of route of administration. PEG biologics do not present any toxicity. This is due to the low toxicological profile of PEG [4].

PEGs with different molecular weights and geometries are used widely in pharmaceutics. PEG is an inert molecule therefore needs a linker to couple it with a protein [4].

Different PEGylation reagents are available including amine reactive PEGs, site-specific N-terminus active PEGs and site-specific thiol reactive PEG reagents. These were categorized as first, second or third generation of PEGylation technology. PEGylation reagents of the first, second and third generation are classified as acylating, anrylating and alkyating reagents respectively [5].

PEGylation is a technology that involves covalent attachment of the polyethylene glycol to a drug in order to improve several aspects of its pharmaceutics. For instance, prolonged half-life, water solubility, immunogenicity, antigenecity, target specificity and higher stability to enzymatic degradation therefore therapeutic efficacy [2].

Figure 2: Small drug -polymer conjugation. A polymeric backbone stabilizes the drug to be delivered to the specific site.

When using PEGylated drugs, the molecular half-life of the therapeutic molecule is increased 5 to 10 fold [6]. PEGylation enhances the efficacy of the drug. Therefore, proteins and antibodies are the important targets of such therapeutic agents [7] .

Pre definition of the stoichiometric ratio of protein to PEG is essential due to the effect of molecular weight during bio-conjugation. The degree of PEGylation (mono, bi, tri) and location of the PEGlyation sites can have dramatic impact on the desired activity of the PEGylated protein. When PEG causes a loss of activity the mechanism by which the activity is lost may vary for enzyme, allosteric regulator or receptor directed ligands. The PEGylation process may also contribute to loss of biological activity caused by adverse coupling conditions for example extreme pH, temperature, or the presence of an oxidizing or reducing agent [8].

Figure 3: Protein surface shielding effect offered by conjugated polymer chains [9].

Protein surface shielding effect offered by conjugated polymer chains. The conjugation of protein to polymer can preserve many biological functions while increasing the molecular size, reducing renal filtration and reduction in degradation by protolytic enzymes [10].

Site-specific N-terminus addition to the PEG is the reaction of PEG to amine residue of the amino acids on proteins such as Lysine (εamine pKa 9.3-9.5), the guanidinyl group of arginine (pKa >12) , the imidazolyl nitrogen group of histidine (pKa 6.7-7.1) , and the alpha amine of the N terminus [11]. In site specific N-terminus these amino acids have all a pKa in the range of 6-12. However, the ideal pH for the amine reactivity is about pH 8, but often this reactivity occurs at the natural pH [11]. The conjugation of the PEG with amine residues of the amino acids is limited by the reactivity of the hydroxyl group[11] .

First generation of the PEGylation method is limited due to the diol contamination rapid degradation of conjugate [11]. In first generation of PEG the reaction of PEG hydroxyl group leads to oxidation and attaching of PEG to any amine residue of the amino acid [11].

Second generation derivatives of PEG reagents are stable [12], and at random site form stable amide bonds via nucleophilic addition [12]. However, it is attached to proteins at the random sites the main issue of conjugation using second- PEG generation methological is the positional isomers. These positional isomers cause reduction in antigenic properties of protein therefore the ratio of the molecular weight of protein to PEG is very important[13].

Occasionally PEG has to be in excess in order for the PEG to bind with nucleophilic groups on the protein. Some protein requires PEG to be in excess, as the PEG has to bind to all the exposed amino acids. This often leads to the reduction of the innate immunogenic properties of the protein. PEGylation can be optimized by the stoichiometry of the reagent and purification of deactivated species, but requires additional effort and cost [14].

Numerous other strategies have been examined for the site-specific PEGylation of proteins [15]. They include reaction to specific sites like arginine using diketone derivatives of PEG-phynylglioxale, PEG hydrazide and hydroxyl groups using PEG isocyanate [2]. Many of these PEG derivatives could be prepared directly from monomethoxy poly (ethylene glycol) (figure1) [16]. Although designed for site-specificity, only PEG hydrazide displays site-specific conjugation, whereas the PEG diketone and PEG isocyanate could react to any nucleophilic site [16]. Although site-specificity can be achieved, the presence of one or more carboxylic acids present in the protein would yield multiple sites of PEG attachment [16].

Third generation of PEGylation describes the site specifically binding of PEG to amine or cysteine residues. Site-specific binding of PEG is homogenous and has particular In Vitro biological activities e.g. cell proliferation assays. [14]. Any based protein amine group could be used for site-specific binding by reductive alkylation. For example, Neulasta® PEG-GCSF is an amine site-specific PEG active derivative of recombinant human methionyl granulocyte-colony stimulating factor an FDA approved cytokine [17] by being selective with site specific bonding the purification and modification steps can be reduced and protein can keep their native structure [18]. However, Neulasta® can form few positional isomers, this is due to the toxic reagents used for the preparation also this product work at controlled temperature and pH [11].

Site-specific PEGylation may also be used to reduce the formation positional isomers, reduce instability and increase the selectivity. Isolation of individual positional isomers is not a cost effective process and is dependent n the structure and molecular weight of PEG. Improvements in the chemistry of PEG, such as the modification the cysteine residue can lead to a more site-specific approach. The reagent for cysteine can site-specific modification be synthesized. This reagent would be capable to increase the bioavailability, blood circulation time of the drug, optimize pharmacokinetics, decrease the immunogenicity, and decrease the frequency of administration of the PEG protein conjugated drug [11].

After over three decades of study, PEGylation technology can be regarded as a tried and tested method for the drug delivery of biopharmaceuticals, especially proteins. By now, biopharmaceuticals are a multibillion-dollar market and it is expected to grow rapidly within the next decade. In other words, there is still much more to be learned about PEGylation. Clinically it is proven which is a key towards further understanding of this subject and has taken over twenty years of research.

The sales of several PEGylated drugs such as PEG-INTRON have reached 3 billion US dollars, comparable to the sales of conventional synthetic blockbuster drugs.