Pharmacokinetics and Bioanalysis of PEGylated Drugs

In recent years, macromolecule conjugation to polyethylene glycol (PEG) has become an effective strategy to alter the pharmacokinetics (PK) of various drugs, thereby improving their therapeutic potential. However, PEG modification leads to the loss of binding affinity due to steric hindrance that interferes with drug-target binding interactions. The pharmacodynamic (PD) properties of a drug are measured at the molecular level through parameters such as receptor binding affinity or enzymatic activity. This loss of potency is offset by the longer circulating half-life of the drug, so the role of PEG is to alter the balance between pharmacodynamic and pharmacokinetic properties, compensating for the reduced binding affinity with increased systemic exposure. The resulting changes in PK-PD have in some cases enabled the development of drugs that would otherwise have been undeveloped, and in other cases led to the improvement of existing drugs.

Pharmacokinetics of PEGylated Drugs

Key characteristics of PEG polymers include PEG molecular weight (MW), branching and end group chemistry. Factors such as the modification site of PEG, the size of PEG, and the connection between peptide and PEG will all affect biological activity. The size and hydrophobicity of the end groups are key to determining binding affinity. The dynamics of proteins attached to polymers are essentially affected by the dynamics of the polymer itself. Therefore, it is necessary to analyze the plasma kinetics and tissue distribution of PEG and PEG protein conjugates separately before evaluating specific PEG-protein conjugates.

Fig. 1. In vivo pharmacokinetic characteristics of PEG polymers (Expert Opin Drug Metab Toxicol. 2014, 10(12): 1691-702).

Absorption

PEG is an amphiphilic substance with long chains that can bind multiple water molecules. It is larger in size and may not easily enter the blood circulation from the gastrointestinal environment, so it is less absorbed. In topical administration experiments, the transdermal absorption rate of PEG also depends on their molecular weight. Low molecular weight PEG can enter the body through intact skin to a low extent, while PEG with a molecular weight higher than 4000 Da can only be absorbed by the body when the protective barrier of the skin is damaged. PEG with a molecular weight of 2000 is considered a critical value for uptake by epithelial cell membranes via paracellular transport or endocytosis.

At the injection site, PEG-50 remained at the injection site longer than PEG-6 after intramuscular and subcutaneous injections, indicating that the absorption of PEG from intramuscular and subcutaneous injection sites is molecular weight dependent. However, for intraperitoneal injection, the in vivo absorption of PEG of different molecular weights is very similar. The difference in drug-time curves of PEG with different molecular weights is mainly related to the pore size of the renal vascular bed.

Distribution

PEGylation may alter the tissue distribution of the drug due to the physicochemical changes it causes in modifying the parent drug. The relative preferential distribution of PEGylated drugs in certain tissue sites can be regarded as the basis for drug targeting, and the molecular weight of PEG is an important factor in determining targeting properties. Macromolecules with prolonged circulation can accumulate in tumor tissue. For PEG molecules of 10 KDa or larger, the relative uptake in tumor tissue is higher than in normal tissue. The drug-PEG-liposome combination can significantly change the biodistribution of the parent drug and specifically bind to targeted tumor cells in the body. Studies have shown that PEG-coated doxorubicin-loaded liposomes significantly increase the distribution in tumor sites. In addition, compared with the non-PEGylated control group, the distribution of PEGylated nanospheres used for regional lymph node imaging diagnosis has been significantly changed, and the localization of the target site has been enhanced.

Metabolism

PEG is generally considered a non-biodegradable polymer, but reports clearly indicate that PEG can be oxidatively degraded by various enzymes, such as alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome p450-dependent oxidase. Phase I metabolism of PEG is mainly mediated by alcohol dehydrogenase and aldehyde dehydrogenase. Molecular weight has an important influence on the phase I metabolism of PEG. Approximately 25% of the dose of PEG 400 is metabolized in the body, but metabolism decreases with increasing MW. PEG with a molecular weight less than 400 can be converted into toxic metabolites in the body by alcohol dehydrogenase, while PEG used for drug or preparation modification has a larger molecular weight and is rarely degraded by enzymes.

Fig. 2. Different linkages between PEG and drugs (Expert Opin Drug Metab Toxicol. 2014, 10(12): 1691-702).

PEGylation affects the metabolism of attached drugs through two mechanisms: shielding plasma enzymes through steric hindrance effects and reducing RES phagocytosis. Therefore, after the drug is modified with PEG, PEG polymers with larger particle sizes can also be metabolized by enzymes, but the rate of biotransformation is significantly slower than systemic elimination. In addition, the linking bonds between PEG and the drug also play a role in the metabolism of the parent drug, because they determine the release rate of the parent drug.

Excretion

PEGylation may lead to physicochemical changes in the parent drug compound, which may result in a less efficient drug clearance process. In general, therapeutic proteins have a very limited lifetime in circulation due to efficient elimination mechanisms in the body, such as proteolysis, specific cell-mediated protein degradation pathways, and capture by RES. The circulating half-life (t1/2) of PEG increases with increasing molecular weight. For example, when the molecular weight increases from 6 kDa to 50 kDa, t1/2 increases from 18 minutes to 16.5 hours. PEG with a molecular weight of 40 to 50 kDa can delay glomerular filtration of small molecules. For example, the systemic clearance rate after intravenous injection of IFN-α reaches 6.6-29.2 lit/hr. When coupled to 5 kDa linear PEG, the systemic clearance rate is significantly reduced to 2.5-5 lit/hr.

Protein clearance depends on the protein's net ionic charge at physiological pH, molecular weight, and the presence of protein-specific receptors in the cell responsible for protein uptake. PEG molecules and PEG protein conjugates with a molecular weight less than 20 kDa can be cleared through urine, while PEG protein conjugates with large molecular weights are cleared slowly through urine and feces. As the mass of PEG molecules adhered to the drug increases, the main elimination pathway shifts from the renal pathway to the hepatic pathway.

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Bioanalysis of PEGylated Drugs

PK Studies of PEGylated Drugs

Developing and developing an effective and safe PEG-protein conjugate requires analysis of the PEG-protein conjugate and PEG at different stages. The quality of bioanalytical data from preclinical and clinical studies is entirely dependent on analytical methods that are selective, sensitive, and reproducible. In order to determine the drug-time profile of PEG-protein conjugates in serum with sensitivity, specificity, repeatability, and accuracy that meet acceptance criteria, an analytical method is needed that is versatile and capable of detecting conjugates in complex biological samples. Bioanalytical scientists must evaluate which method is best for the analyte through proper method validation. In view of the fact that PEGylated drugs release free drugs under the action of metabolic enzymes or acid-base in the body, it is of great practical significance to monitor the real-time changes in the concentration of PEG-bound drugs, free drugs and free PEG in the body for the pharmacodynamic and toxicological studies of PEGylated drugs.

PEGylated drugs can be studied using colorimetry, enzyme-linked immunoassay (ELISA), radionuclide labeling, NMR, liquid chromatography, and liquid mass spectrometry (such as LC-MS/MS, Q-TOF, etc.). Colorimetric determination of PEGylated drugs has high technical requirements and requires frequent cleaning of the cuvette and collection of the precipitates formed between PEGylated drugs and heteropoly acids, which is time-consuming. The radionuclide labeling method limits its application due to reasons such as difficulty in labeling and high price. NMR has poor sensitivity and is not suitable for complex biological matrices. Moreover, NMR detects the overall concentration of PEG and cannot distinguish between free and bound forms. Therefore, ELISA and liquid mass spectrometry are currently used more frequently.

• Enzyme-linked immunoassay (ELISA)

ELISA is an immunochemical analysis technique used to quantitatively or qualitatively detect the presence of specific proteins. Instruments for analysis based on enzyme-linked immunoassay include microplate reader, MSD and Simoa. The ELISA method provides a fast and convenient technology for the analysis of PEGylated drugs. The antigen-binding fragment (Fab) of the anti-PEG antibody was expressed on the surface of BALB/3T3 cells and used to capture PEGylated molecules. Captured PEG and PEGylated molecules were quantified by subsequent addition of biotinylated AGP3 antibody, streptavidin-conjugated horseradish peroxidase (streptavidin-HRP), and ABTS substrate. The detection limits of this method for PEG and PEG-conjugated drugs are: 58.6 ng/mL for PEG 2000, 14.6 ng/mL for PEG 5000, and 3.7 ng/mL for PEG 10 kDa and PEG 20 kDa.

• LC/MS

LC-MS is a rapidly evolving technology that has taken bioanalysis to a new level. LC-MS can achieve similar sensitivity to immunoassays and shorten method development time. Due to its high sensitivity and selectivity, this technology has been applied in the quantitative analysis of PEG and PEGylated drugs in recent years. However, undissociated PEGylated proteins are generally beyond the detection range. This problem can be solved by digesting the target PEGylated protein into low molecular weight peptides. A highly specific peptide fragment was selected as a surrogate analyte for quantification of the entire PEG-protein conjugate.

Immunogenicity Analysis of PEGylated Drugs

• PEG immunogenicity generation mechanism

For PEGylated proteins and peptides, close attention should be paid to their potential immunogenicity and antigenicity, which may be caused by the variable molecular weight of native protein fragments and conjugates. Due to the introduction of partial protein fragments or foreign species, PEGylation may lead to the formation of new epitopes. Among these anti-drug antibodies, anti-PEG IgG and IgM have been shown to cause accelerated drug blood clearance (ABC phenomenon) and hypersensitivity reactions (HSRs) leading to severe allergic symptoms and even fatal allergic reactions. Correct detection of anti-PEG antibodies (APA) may predict adverse immune reactions to PEGylated drugs, thereby improving their effectiveness and safety.

Fig. 3. Immunogenicity of polyethylene glycol (ACS Nano. 2021, 15(9): 14022-14048).

• Factors affecting PEG immunogenicity

The immunogenicity of PEGylated drugs depends on the characteristics of the polymer and its modified drugs, such as PEG chain structure and length, terminal groups, degree of PEGylation, linker, characteristics of the parent drug, and patient factors.

PEG chain structure and length: Anti-PEG antibodies are specific for either the backbone or the terminal groups of PEG, which have different binding properties. Binding affinity to the backbone is low and the number of antibodies bound is limited by the length of the PEG.

Terminal group: Antibodies induced by PEG-OH coupled proteins have similar affinities for methoxy-PEG and PEG-OH, while antibodies induced by methoxy-PEG coupled proteins have stronger recognition ability for methoxy-PEG than PEG-OH. Among the commonly used PEG end groups, the binding affinity for antibody formation increases in the following order: hydroxyl (–OH) < amino (–NH³⁺) < methoxy (–O-CH₃) < butoxy (–O-( CH₂)₃)-CH₃) < tert-butoxy(–O-(CH₃)₃).

Degree of PEGylation: The degree of PEGylation of different proteins or liposomes is a key factor in immunogenicity. Proteins usually have no more than three PEG molecules attached to them, while liposomes have more PEG binding sites. Some studies have explored potential factors affecting immunogenicity by adjusting the molecular weight and coupling degree of PEG. In the presence of Freund's adjuvant, free PEG has no or only very weak immunogenicity, while PEG conjugated to macromolecules induces significant anti-PEG antibodies in immunized animals.

Linker: The linker between PEG and protein/carrier may affect the immunogenicity of PEG. After injection of PEG-asparaginase, both the amide bond and the succinate bond between PEG-asparaginase can induce anti-PEG antibodies to a similar extent. In addition to anti-PEG antibodies, anti-succinate linker antibodies have been reported in patients with PEG-asparaginase hyperreactivity.

Characteristics of the parent drug: If the PEG-modified drug is a protein, its immunogenicity is mainly through the T cell-dependent (TD) pathway, and the inherent immunogenicity of the protein synergistically promotes the secretion of PEG-specific antibodies. For example, Takeda's Omontys®, the modified protein itself is immunogenic, but its PEGylated formulation induces a much stronger anti-PEG immune response than non-immunogenic protein-bound PEG, which is why the drug was recalled.

• PEG immunogenicity analysis method

Because the production of anti-PEG antibodies may reduce the efficacy and safety of PEGylated drugs, clinically relevant anti-PEG antibody titers need to be determined to control the risk of adverse effects in patients exposed to PEGylated drugs. The earliest applied detection method was the coagulation test. This method is fast and simple, but has low sensitivity. In order to improve sensitivity, methods such as Western blot, acoustic membrane microparticle technology (AMMP), ELISA, and flow cytometry can be used to amplify the signal through enzymatic reaction or fluorescence. However, these techniques are often not absolutely quantitative, and detection limits depend on experimental conditions. Surface plasmon resonance technology (SPR) has the advantages of ultra-sensitivity, quantification, and rapidity, but this method is not commonly used because it requires special and expensive instruments and reagents.

ELISA is currently the most widely used anti-PEG antibody detection technology due to its high sensitivity and ability to semi-quantitate antibody levels. In the direct anti-PEG ELISA method, PEG-specific antibodies in serum or plasma are recognized by enzyme-conjugated host IgG or IgM-specific antibodies and bind to the PEG-coated surface to produce a color reaction. In the bridged immunogenicity ELISA method, PEG-specific binding to anti-PEG antibodies is detected by binding to the antigen rather than anti-host IgG or IgM. Due to the bivalent or multivalent nature of anti-PEG antibodies, these antibodies are sandwiched between two layers of PEG antigen. The first antigen is coated on the surface for capture, the second, usually a biotinylated antigen, is preincubated with the sample and finally detected by a streptavidin-enzyme conjugate.

In Conclusion

PEG is increasingly used as a bioconjugated polymer in drug therapy. PEGylated drugs have made great progress in recent years, and as the field of biotherapeutics expands, the emergence of PEGylated drugs is expected to continue to accelerate. At present, PEGylation modification has been widely used to improve the pharmacokinetic properties of therapeutic drugs. It is believed that in the near future, with the continuous improvement of modification technology, more and more excellent PEGylated drugs will appear, providing patients with more and better treatment options. A comprehensive understanding of the pharmacokinetics and bioanalysis of PEG-modified drugs is of great significance for designing more efficient and better-targeted PEGylated drugs and better controlling adverse reactions.

References

1. Zhang, X. et al. Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns. Expert Opin Drug Metab Toxicol. 2014, 10(12): 1691-702.
2. Chen, B.M. et al. Polyethylene Glycol Immunogenicity: Theoretical, Clinical, and Practical Aspects of Anti-Polyethylene Glycol Antibodies. ACS Nano. 2021, 15(9): 14022-14048.