PEGylated Proteins

Protein drugs have become a kind of important modern pharmaceutical products because of their high biological activity and good specificity. Polyethylene glycol (PEG) is a kind of non-toxic, high hydrophilic macromolecule polymer, combined with protein inactive necessary groups, increasing the relative molecular mass of the protein drugs, some antigenic determinant is covered on the surface of the charged property will occur some changes, these changes can often increase the stability of protein drugs and half-life in the body, lower immunogenicity1. The application of PEGylated technology in protein drugs has been widely concerned at home and abroad. A variety of PEGylated drugs on the market have also fully demonstrated their medicinal value and commercial value.

Main advantages of PEGylated protein2

Fig.1 Main advantages of PEGylated protein2 (2. Veronese, FM.; Pasut, G. 2005)

Physicochemical properties of PEG

PEG commonly used for modifying proteins and other polymers has a relative molecular weight of 200 ~ 20 000 and is a colorless transparent liquid. PEG with a relative molecular weight of more than 1 000 is white or beige paste or solid at room temperature, with a slight odor. PEG is soluble in water and most polar solvents, but insoluble in non-polar solvents such as aliphatic hydrocarbons, benzene and mineral oils. PEG has strong hygroscopicity, which decreases rapidly with the increase of molecular weight. It also has weak surface activity, and the surface tension decreases with the increase of its aqueous solution concentration. The viscosity of PEG solution with lower molecular weight was not high and increased with the increase of molecular weight.

PEGylated protein drugs technology

  • PEG activation

In order to bind PEG to the target molecule, one or both sides of the PEG molecule should be activated, and the functional groups required for activation should be selected according to the characteristics of the molecule to be connected. For proteins, the first to be activated in -OH terminal, you can use PEG activation reagent for activation (eg. cyanuric acid chloride). The common activation forms of mPEG are: SS-PEG, SC-PEG, T-PEG and so on.

  • PEGylated protein synthesis

Activated mPEG can theoretically react with the major amino acids in proteins, such as lysine, cysteine, histidine, aspartic acid, glutamate, serine, threonine, and so on, either at the N-terminal or C-terminal. However, the reaction with lysine, cysteine and N-terminal groups is the most common.  The purified PEGylated protein can be synthesized at pH8-9.5 for 30 min at room temperature.

Effects of PEG modification on protein drugs

  • Decreased immunogenicity
  • Increased thermal stability
  • Prolonged half-life in vivo3
  • Increased solubility
  • Changes in tissue distribution
  • Toxicity reducing
  • The ability to resist inactivation factor was improved
  • The optimal pH value changes

Bioanalysis of PEGylated protein drugs

  • Pharmacokinetics

PEGylated proteins

The development and implementation of an effective and safe PEG-protein conjugation requires the analysis of PEG-protein conjugation 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 curve of PEG-protein conjugation in serum with sensitivity, specificity, repeatability, and accuracy that meet acceptance criteria, a universal assay is needed for the detection of conjugation in complex biological samples. The bioanalytical scientist must evaluate which method is best for the object to be measured through the validation of the correct method.

Since PEGylated drugs release free drugs under the action of metabolic enzymes or acids and bases in vivo, monitoring the real-time changes of PEGylated drugs, free drugs and free PEG concentrations in vivo has very important practical significance for the pharmacodynamics and toxicology of PEGylated drugs4.

Colorimetric determination of PEGylated drugs requires high technical requirements and requires frequent cleaning of colorimetric dishes and collection of the precipitation formed by pegylated drugs and heteropoly acids, which is time-consuming.

Radionuclide labeling: the application of radionuclide labeling is limited due to its difficulty and high cost.

NMR has poor sensitivity and is not suitable for complex biological substrates, and it detects the concentration of total PEG and cannot distinguish free from conjugated PEG.

ELISA: The Fab of anti-PEG antibody was expressed on the surface of BALB/3T3 cells to capture pegylated molecules. This method is not suitable for the analysis of PEGylated drugs in complex biological matrix.

LC-MS/MS: Bioanalysis of PEGylated proteins should also take into account potential interfering components from endogenous proteins5.

PEGylated proteins

  • Immunogenicity analysis

Since 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 manage the risk of adverse reactions after patient exposure to PEGylated drugs.

Hemagglutination test was the earliest detection method used. The method is quick and simple, but has low sensitivity. In order to improve the sensitivity, Western blot, acoustic membrane microparticle technology (AMMP), enzyme-linked immunosorbent assay (ELISA) and flow cytometry can be used to amplify the signal by enzyme reaction or fluorescence. However, these techniques are usually not absolutely quantitative and the detection limits depend on experimental conditions.

Surface plasmon resonance (SPR) has the advantages of being super-sensitive, quantitative and fast, but it is not commonly used because of the need for special and expensive instruments and reagents.

Due to its high sensitivity and semi-quantitative antibody level, ELISA is the most widely used anti-PEG antibody detection technology. In direct ELISA, PEG-specific antibodies in serum or plasma are recognized by enzyme-coupled host IgG or IgM specific antibodies, which bind to PEG-coated surfaces to produce color reactions. In bridging ELISA, PEG-specific binding anti-PEG antibodies are detected by binding antigens rather than anti IgG or IgM6.


  1. Lawrence, PB.; Price, JL. How PEGylation influences protein conformational stability. Curr Opin Chem Biol. 2016 Oct; 34:88-94.
  2. Veronese, FM.; Pasut, G. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005 Nov 1;10(21):1451-8.
  3. Yang, Z.; et al. PEGylation confers greatly extended half-life and attenuated immunogenicity to recombinant methioninase in primates. Cancer Res. 2004 Sep 15;64(18):6673-8.
  4. Markman, J L.; et al. Nanomedicine therapeutic approaches to overcome cancer drug resistance.[J]. Advanced Drug Delivery Reviews. 2013, 65(13-14):1866-1879.
  5. Jiachang, Gong.; et al. Quantitative Analysis of Polyethylene Glycol (PEG) and PEGylated Proteins in Animal Tissues by LC-MS/MS Coupled with In-Source CID[J]. Analytical Chemistry. 2014, 86, 7642−7649.
  6. Liu, Y.; et al. A double antigen bridging immunogenicity ELISA for the detection of antibodies to polyethylene glycol polymers. J Pharmacol Toxicol Methods. 2011 Nov-Dec;64(3):238-45.

Reagents Solutions

Creative Diagnostics provides a set of specific anti-PEG monoclonal antibodies that have been validated to work in various types of immunoassays for sensitive measurement of PEGylated compounds, including PEG-proteins, PEG-liposomes and PEG nanoparticles.


  • Evaluate the PEGylation efficiency and PK/PD of a PEGylated drug candidate
  • Validated epitopes: bind to methoxy PEG (mPEG) or PEG backbone
  • Human antibodies available as standards for ADA assays
  • IgG & IgM isotypes from various species

Anti-PEG Antibodies List

Cat# Product Name Description
Rabbtit anti-PEG mAb (IgG, IgM)
DMABT-Z60095 Rabbit Anti-PEG Mab, IgG, Biotin Detects both linear and branched PEG.
DMABT-Z60369 Rabbit Anti-PEG Mab, IgM Binds to the PEG backbone.
Mouse anti-PEG mAb (IgG1, IgG2b, IgM)
CABT-L3126 Mouse Anti-PEG Mab, IgG1 Detects repeating subunits present in PEG.
CABT-L3127 Mouse Anti-PEG Mab, IgG1 Binds to the PEG backbone. Detects PEGylated compounds.
CABT-L3133 Mouse Anti-PEG Mab, IgG1, Biotin
CABT-L3128 Mouse Anti-PEG Mab, IgG1 Detects backbone of PEG molecule. Detects PEGylated compounds.
CABT-L3134 Mouse Anti-PEG Mab, IgG1, Biotin
CABT-L3129 Mouse Anti-mPEG Mab, IgG2b Detects the terminal methoxy group of PEG. Suitable for detection of PEGylated liposomes and nanoparticles.
CABT-L3136 Mouse Anti-mPEG Mab, IgG2b, Biotin
CABT-L3131 Mouse Anti-PEG Mab, IgM Binds to the PEG backbone, detects PEGylated compounds.
CABT-L3137 Mouse Anti-PEG Mab, IgM, Biotin
CABT-L3132 Mouse Anti-PEG Mab, IgM Recognizes PEG backbone. Binds to PEGylated nanoparticles or long-chain PEGylated molecules.
Rat anti-PEG mAb (IgG2a, IgM)
DMABT-Z60567 Rat Anti-PEG Mab, IgM Displays high affinity to both short and long PEG molecules.
CABT-L3139 Rat Anti-PEG Mab, IgM, Biotin
CABT-L3130 Rat Anti-PEG Mab, IgG2a Detects PEGylated compounds. Higher affinity, Wider binding range than CABT-L3128.
CABT-L3135 Rat Anti-PEG Mab, IgG2a, Biotin
Human Anti-PEG mAb Standards (IgG1, IgM)
CABT-L3140 Chimeric Human Anti-PEG Mab, IgG1 Fusion protein of mouse CABT-L3127 VH and VL with human IgG1.
CABT-L3141 Chimeric Human Anti-PEG Mab, IgM Fusion protein of mouse CABT-L3132 VH and VL with human IgM.
CABT-L3142 Humanized Anti-PEG Mab, IgG1 Recombinant antibody composed of humanized CABT-L3128 VH and VL with human IgG1 constant regions.


Creative Diagnostics provides ELISA kits for measurement of free and conjugated PEG as well as ELISA kits for detection of anti-PEG IgM and IgG in mouse, rat, monkey, and human serum or plasma. These assay kits are useful in clinical safety studies to determine the immunogenicity of PEGylated biotherapeutics.

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