Purpose: Pegylation is a well-documented modification used to diminish the proteins’ immunogenicity and has been widely used as PK enhancer for biotherapeutics. However, in contrast to the generally accepted assumption that polyethylene glycol (PEG) is non-immunogenic and non-antigenic, the immune responses to the PEG itself have been reported to cause loss of product efficacy and adverse safety consequences Thus, screening and monitoring the anti-PEG antibodies are critical in understanding the safety and efficacy of the pegylated biotherapeutics. FDA and other global regulatory agencies require that for pegylated therapeutic protein products, the ADA assay should be able to detect both the anti-therapeutic protein antibodies and antibodies against the PEG moiety. For this purpose, we developed and validated a direct ELISA method for the determination of anti-PEG antibodies in human serum.
Methods: Serum anti-PEG antibodies are detected using a direct ELISA. Diluted serum samples were added to a microplate, which is coated with streptavidin followed by the binding with biotinylated PEG to streptavidin. The wells were washed to remove any unbound sample material and enzyme-labeled antibodies were added. Unbound enzyme-labeled antibodies were removed and a chromogenic substrate was added. The development of the colored reaction product was directly proportional to the amount of anti-PEG antibodies present in the sample. The microplate was then analyzed using a colorimetric plate reader. The method was thoroughly validated by determining the screening cut points and confirmatory cut points using 50 drug naïve normal human sera, calculating the assay sensitivity, and evaluating the matrix effect and free drug interference.
Results: The anti-PEG antibodies detection immunoassay was successfully developed and validated. Assay screening cut points and confirmation cut points were determined using robust statistical methods. The assay displayed acceptable precision. The sensitivity of the screening assay and the confirmatory assay were 44.5 and 49.6 ng/mL, respectively. Selectivity in normal human serum found to be acceptable with 8 out of 10 spiked samples (at 128 ng/mL) showed response greater than rCP in screening assay and 9 of the same spiked samples showed %inhibition greater than iCP, while 1 out of 10 un-spiked samples detected as positive, suggesting the presence of pre-exiting anti-PEG antibodies. The drug tolerance data shows that anti-PEG antibodies at 128 ng/mL could be detected in the presence of 12.8 ng/mL of 20 kDa PEG. No hook effect was observed for up to 10 µg/mL of positive control.
Conclusion: A direct ELISA method was successfully developed and validated to detect anti-PEG antibodies in human serum. The assay shows acceptable sensitivity, precision, selectivity and drug tolerance. This assay can be used for detecting the antibodies against the PEG moiety of the pegylated biotherapeutics.