The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Improved precision of proteomic measurements in immunoprecipitation based purifications using relative quantitation.

Mass spectrometry coupled immunoprecipitation (MS-IP) studies are useful in identifying and quantitating potential binding partners of a target protein. However, they are often conducted without appropriate loading controls. Western blots are often used to analyze loading controls, yet there are limitations to their usefulness as analytical tools. One remedy for this is the use of selected reaction monitoring (SRM), where the areas under the curve (AUCs) of peptides from a protein of interest can be normalized to those from the constant regions of the immunoglobulins used for the IP. Using this normalization method, significant changes in relative peptide abundance were observed between samples when there appeared to be an unequal load based on immunoglobulin peptide abundance.