Development of a validated novel bead extraction method for the detection of anti-PEG antibodies in human serum

Abstract

Aims: Polyethylene glycol (PEG) is used in many applications including drug development. Due to exposure to environmental products, there is a high prevalence of preexisting anti-PEG antibodies in the global human population. The presence of anti-PEG antibodies is a concern for potentially reducing the efficacy of therapeutics after administration and represents a risk of safety events after exposure to PEGylated drug products. We developed and validated a creative and sensitive method for the detection of anti-PEG antibodies in human serum to support clinical programs for PEGylated drugs.

Methods: In this method, biotin-PEG streptavidin beads were used to extract anti-PEG antibodies from human serum for analysis in an anti-PEG ELISA assay. The same serum sample was analyzed in an anti-drug antibody assay.

Results: The anti-PEG antibody assay was validated with a screening cut point of 1.41 normalized signal, confirmatory cut point of 32.2% inhibition, sensitivity of 7.81 ng/mL and sufficient reproducibility, selectivity, and drug tolerance in accordance with the FDA 2019 Immunogenicity guidance.

Conclusion: This method of removal of anti-PEG antibodies enables the use of a single sample to detect anti-drug and anti-PEG antibodies to support drug development programs.

Keywords: ADA; BEAD; PEG; anti-PEG antibodies; immunogenicity.

Figure 1.

The BEAD extraction method. Magnetic streptavidin beads were labeled with mPEG Biotin and incubated with serum samples in a polypropylene plate overnight. After overnight incubation, the plate was placed on a plate magnet and the supernatant containing non-peg antibodies removed to be run in the core ADA assay. Acetic acid was added to the beads to elute the anti-peg antibodies and neutralized into a separate polypropylene and used in the anti-peg ELISA method.

Figure 2.

Removal of anti-peg antibodies from samples. (a) Samples were untreated (blue bar) or treated with 0.1 mg/mL (red bar), 1 mg/mL (green bar), 5 mg/mL (purple bar), and 10 mg/mL (orange bar) capture to remove anti-peg antibodies from sample. The samples were analyzed for anti-peg binding activity. (b) Human serum samples containing high (aPEG HPC) and low (aPEG LPC) levels of anti-peg antibodies, samples prepared with positive control spiked at high (aDrug HPC), medium (aDrug MPC) and low concentration (aDrug LPC) in human serum and negative serum pool samples (NC) were untreated (blue circles) or treated with 0.1 mg/mL (red squares) or 10 mg/mL (green triangles) capture beads, then acid eluted and assayed in the anti-peg ELISA assay.

Figure 3.

Comparison of anti-drug antibody levels with sample pretreatment. (a) Human serum samples containing high (aPEG HPC) and low (aPEG LPC) levels of anti-peg antibodies, samples prepared with positive control spiked at high (aDrug HPC), medium (aDrug MPC) and low concentration (aDrug LPC) in human serum and negative serum pool samples (NC) were untreated (blue circles) or subjected to an ACE (affinity capture elution) method with an acid dissociation step with 300 mm (red squares) or 600 mm (green triangles) acetic acid prior to analyzing in the core ADA assay. (b) The same samples were subjected to no treatment (blue circles) or treated with 0.1 mg/mL (red squares) or 10 mg/mL (green triangle) anti-peg bead capture and analyzed in the core ADA assay.

Figure 4.

Drug tolerance in core ADA assay after sample pretreatment. (a) This was assessed with positive control samples that were prepared by spiked positive control in human serum at varying concentrations at high (HPC; blue circles), mid (MPC; red squares), and low (LPC; green triangles) and incubated with increasing concentrations of drug (0 to 2.5 mg/mL). Samples were assayed in the conventional ACE ADA core method. (b) The same positive control samples were treated with the anti-peg depletion method and analyzed for drug tolerance in the core ADA method.

Figure 5.

Preliminary anti-peg screening and confirmatory cut point determination. (a) The screening cut point was determined by testing 56 individual serum samples in run 1 (blue circles) and run 2 (red squares) performed by two analysts. The response of each sample was normalized to the mean of the NC. (b) The confirmatory cut point was evaluated with the same 56 individual serum samples with excess PEG at 50 µg/mL (blue circles) or 400 µg/mL (red squares) and analyzed in the anti-peg assay.