Bioanalysis of PEGylated Drugs



The development and deployment of an effective and safe PEG-protein conjugate requires the analysis of PEG-protein conjugates and PEGs 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 acceptable sensitivity, specificity, reproducibility, and accuracy, a general analytical method for the detection of conjugates in complex biological samples is required. Bioanalytical scientists must evaluate which method is best for the analyte through proper method validation.

In view of the fact that PEGylated drugs will release free drugs under the action of metabolic enzymes or acid-base in vivo, and monitor the real-time changes in the concentrations of PEG-conjugated drugs, free drugs, and free PEG in vivo. Pharmacodynamics and toxicology studies of PEGylated drugs has very important practical significance.

PEGylated drugs can be studied by colorimetry, enzyme-linked immunosorbent assay (ELISA), radionuclide labeling, NMR, liquid chromatography, liquid chromatography (such as LC-MS/MS, Q-TOF, etc.) .

The colorimetric determination of PEGylated drugs has high technical requirements, and requires frequent cleaning of the cuvettes and collection of the precipitates formed by PEGylated drugs and heteropolyacids, which is time-consuming. The radionuclide labeling method limits its application due to the difficulty of labeling and the high price. NMR has poor sensitivity and is not suitable for complex biological matrices, and NMR detects the concentration of the total PEG and cannot distinguish between free and bound forms. ELISA provides a fast and convenient technique for the analysis of PEGylated drugs that, in some cases, is difficult to achieve with other methods. However, PEGylated drugs will undergo a series of degradation and metabolism processes in the body. ELISA method can only detect active drugs (binding to targets), and cannot effectively distinguish PEGylated drugs, free drug molecules, PEG groups and metabolites. Suitable for analysis of PEGylated drugs in complex biological matrices. While LC/MS can detect total circulating drugs, it provides a possible solution for PEGylated drug analysis, and is widely used for its advantages of sensitivity and resolution. However, potential interfering components from endogenous proteins should also be considered when bioanalyzing PEGylated proteins using LC/MS.


ELISA is an immunochemical analysis technique used to quantitatively or qualitatively detect the presence of specific proteins. Instruments for analysis based on ELISA are microplate reader, MSD and Simoa.

Antigen-binding fragments (Fabs) of anti-PEG antibodies were expressed on the surface of BALB/3T3 cells for capture of 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: 58.6ng/mL for PEG2000, 14.6ng/mL for PEG5000, and 3.7ng/mL for PEG10kDa and PEG20kDa.

2. LC/MS

LC/MS is a rapidly evolving technique that has taken bioanalysis to the next level. LC/MS can achieve similar sensitivity to immunoassays with reduced method development time. Due to its high sensitivity and selectivity, this technique has been applied in the quantitative analysis of PEG and PEGylated drugs in recent years.

LC-MS/MS-based multiple reaction monitoring (MRM) is a sensitive and selective scanning method commonly used for LC-MS/MS bioanalysis of small molecule drugs. In MRM mode, the precursor ion of the analyte is first selected in Q1; the selected ion will thus be fragmented by collision-induced dissociation (CID); one or more specific fragment ions are selected again in Q2 and delivered to Q3 detection.

LC-MS/MS is a common analytical technique for small molecule drugs, and great progress has been made in the bioanalysis of macromolecular drugs in recent years. However, undissociated PEGylated proteins are generally beyond the detection range. Solving this problem can digest the target PEGylated protein into low molecular weight peptides. A highly specific peptide fragment was chosen as a surrogate analyte for whole PEG-protein conjugate quantification.

There are also studies using collision-induced dissociation (CID) technology to establish a CIDIn-Quadrupole LC-Q/Q/TOF tandem mass spectrometry method based on collision energy CE, using MSAll and SWATH (SequentialWindowedAcquisitionofAllTheoreticalFragmentIonMassSpectra) scanning modes, by monitoring PEG specificity and drug-specific fragment ions to complete the analysis of PEGylated drugs and free PEG. This method has good reproducibility, high sensitivity, and high-resolution fragment ion selection can effectively eliminate the influence of interfering ions. It is suitable for PEG in complex biological matrices. And the analysis of PEGylated drugs provides a powerful analytical tool for the design and safety and efficacy evaluation of PEGylated drugs.