ABSTRACT
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.
Subject(s)
Immunoglobulins/analysis , Immunoprecipitation , Peptides/analysis , Proteomics , Animals , Aorta/chemistry , Aorta/cytology , Cell Line , Endothelial Cells/chemistry , Endothelial Cells/cytology , Mass Spectrometry , SwineABSTRACT
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.
Subject(s)
Biological Variation, Individual , Filgrastim/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Animals , Cell Line, Tumor , Cell Proliferation , Enzyme-Linked Immunosorbent Assay/standards , Humans , Mice , STAT3 Transcription Factor/metabolismABSTRACT
Monoclonal antibody therapeutics are a heterogeneous mixture of glycoforms. Multiple methods exist for defining the glycan composition and relative abundance of species present. In the current report, two MS-based methods were compared for their ability to both identify glycans and monitor differences in the glycoprofile. Gross changes in the glycoprofile can be identified either by visual inspection of fluorescence profiles and correlated to glycan identities when coupled with online MS/MS (LC-F-MS/MS) or through extracted ion chromatograms using LC-MS. In the present study, both an LC-F-MS/MS method and an automated LC-MS label free approach were able to identify minor differences in low abundance glycoforms, and data indicate a disparity in glycosylation between the analyzed batches of US and foreign-sourced mAb X. Thus, either method may be useful in characterizing monoclonal antibody therapeutics products and could serve as a potential screening test for understanding process, comparability, similarity, and possibly detecting counterfeit agents.