Poly(ethylene glycol) (PEG) is a widely used polymer in drug delivery systems and is often directly conjugated to a drug therapeutic. PEG is a water-soluble, biologically inert, non-immunogenic synthetic polymer consisted of ethylene glycol (-CH2-CH2-O-) in repeating units (Figure 1). PEG is produced by anionic polymerization of ethylene oxide initiated by a nucleophilic attack of a methoxide ion on the epoxide ring (Figure 2). PEGylation of a protein conjugate is the process by which PEG is either non-covalently or covalently attached to a potential protein, peptide or antibody of interest to produce alterations in the physiochemical properties of the macromolecule in areas like conformation, electrostatic binding and hydrophobicity. PEGylation is used as a strategy to overcome challenges such as instability, inadequate circulation half-life and immunogenicity of a drug candidate intended for a therapeutic use.

Considerations Prior to PEGylation

The structure and size of a PEG polymer is an important parameter that is considered prior to protein PEGylation. The mass of bound PEG was found important for determination of the time it remains in blood. Modern improvements of PEG’s properties involve the synthesis of a less polydispersed PEG product, such as the branched form. Initially, the only PEG polymer used for several years was 5kDa in size that is “end-capped” at the part of the chain with a methoxyl group and terminated at the hydroxyl group. However, there were pitfalls associated to conjugating proteins with these polymers. On one hand this produced the presence of a significant amount of diol impurities in samples which led to the formation of covalent protein aggregates. On the other hand, the presence of different lengths of side chains within the PEG polymer (high polydispersity) which led to conjugate products with different masses. Both produced heterogeneity in the final PEGylated protein product. To overcome this, in the 1990s, low polydisperse, activated, high mass branched PEG polymers (30-40kDa in size) came to market. Branched PEG (PEG2) are characterized by two chains of PEG polymers attached to one activated moiety. This polymer form allows for higher masking of the protein surface by allowing for linking a double mass of polymer by the modification of one amino acid residue in the protein, leaving the protein more biologically active. Given there is a high chance of masking the catalytic or recognition sites across a protein, it becomes necessary to optimize the number of linking chains and mass (the degree of modification) such that it maintains the most biological activity and bioavailability possible. The hydrophobicity of the PEG end-group is a consideration as a result. Sherman et al., ‘s study using anti-PEG antibodies to examine the immunogenicity of three different PEG analogs, t-butoxy-PEG (tBu-PEG) methoxy-PEG (mPEG) and hydroxyl-PEG (OH-PEG) found the bulkier (higher molecular weight), more hydrophobic attachment at the “end-cap”, the greater the non-immunogenic character. OH-PEGs were suggested to be better for PEGylated therapeutics in comparison as the bulkier side-chains may result in increased shielding.

Another important parameter prior to PEGylation of a protein is targeted conjugation within the protein sequence. Unfortunately, there are only a few strategies for site-specific conjugation such as conjugation to an amino terminal group of a protein at a low pH. PEG-polymer conjugation at an amino group occurs by alkylation or acylation. Alkylation maintains a positively charged amine, while acylation yields neutral amides which may change the surface character of the protein which can reduce bioavailability and activity of the protein conjugate. However, it has been found that at epsilon-amino groups of lysine, which have a higher acid dissociation constant (pKA) than the protein a-amino terminal, makes way for more preferential site-specific conjugation because at a low pH, the a-amino group is only reactive.

Advantages of Conjugating Proteins with PEG

The key advantages of attaching PEG to a biomacromolecule are to pass along PEG’s 1) high solubility in aqueous and organic solutions, 2) its hydrophilic character and 3) its non-immunogenic properties. Repeating side chain units of PEG are attached to a protein such that they can sterically shield antigenic epitopes from immune system recognition as PEG is a non-immunogenic polymer. This can help a therapeutic conjugate avoid degradation by phagocytic responses from the reticuloendothelial system or proteolytic enzymes that look to remove foreign particles or soluble substances in circulation and tissues. Conjugation of a protein to PEG can increase hydrophilicity through the hydrogen bonding provided by the PEG side chain to water molecules that increases the hydrodynamic volume and produces a stable hydration shell surrounding the polymer that can increase the bound macromolecules’s solubility by result. This increase in hydrodynamic size contributes towards producing a resistance to renal filtration in the kidneys causing clearance of the drug conjugate to occur more slowly.

Enzo’s sensitive PEGylated Protein ELISA kit allows for quantitative detection of a diverse set of PEGylated molecules in plasma, serum, tissue and other biological samples using a competitive ELISA format. The kit is applicable for drug development and pharmaceutical manufacturing applications including drug formulations, pharmacokinetics analysis, drug comparison, lead candidate identification, lot release criteria and in-process QC studies. The kit has also been validated for quantification of PEGylated target molecules in complex biological matrices, extending its utility for monitoring drug levels or its accumulation in tissue. The assay measures <1ng/ml of PEGylated molecules and is validated for a wide range of molecular weight linear and branched PEGs, both in free form and as protein conjugates (Figure 3). With the ability to analyze up to 35 samples in duplicate in just 2 hours, the kit is fit for rapid, high throughput analysis. Intra-assay precision was determined by assaying 16 replicates of two buffer controls containing PEG-BSA in a single assay. Figure 4 shows that in development this ELISA reported a range of low intra-assay %CVs between 3.4 – 4.2%. On the other hand, inter-assay precision was determined by back-calculating three standards off 16 standard curves produced in multiple assays over several days. Figure 5 shows that in product development, this ELISA reported a range of low inter-assay %CVs between 1.3 – 5.7%. The PEGylated ELISA kit is a highly precision kit with a high standard of reproducibility.

Disadvantages of Conjugating Proteins with PEG

PEG is the most used polymer and is utilized as a gold standard for polymer-based drug delivery systems that conjugate PEG to a drug candidates. PEGylation of potential chemical leads that are drug candidates have been found to be desirable because it increases the retention time, reduces the immunogenicity and increases the stability of the drug to be metabolized by enzymes. Possible side effects and complications have been found from the accumulation of metabolized PEGylated therapeutics in various tissues so examination of the influence of PEGylated products has been a focus to characterize hypersensitivity, unexpected changes in pharmacokinetic behavior, toxicity and antagonism from degradation by mechanical stress from them. Assaying for concentrations of PEGylated substituents can be helpful tool for drug development applications such as pharmacokinetics analysis, drug comparison, lead candidate identification, lot release criteria and in-process QC studies.

In addition to the high reproducibility afforded by Enzo’s PEGylated ELISA kit, it also offers a highly sensitive immunoassay with a limit of detection less than 1 ng/ml with a dynamic range from 1.75 ng/ml to 225ng/ml. Sensitivity of the assay was dependent on the molecular weight of PEG tested and quantity of PEG molecules conjugated to target molecule and evaluated prepared conjugates using 10 kDa linear PEG offered at 1, 3, 5, 10 and 15-fold molar excess to BSA (Figure 6).

Enzo Life Sciences offers a comprehensive bioprocess portfolio with thoroughly validated ELISAs for contamination monitoring of Protein A and Host Cell Protein (HCP) residuals for process optimization in your biologics preparations. We also have our widely cited PROTEOSTAT® assays for monitoring protein aggregation and protein stability under systematic thermal stress conditions to enhance your manufacturing workflows. Please check out our Bioprocess Optimization platform and our PEGylation Application Note. For more information or contact our Technical Support Team for further assistance.

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