SEC Based Method for Size Determination of Immune Complexes of Therapeutic Antibodies in Animal Matrix

Abstract

Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).

Generation, Characterization, and Quantitative Bioanalysis of Drug/Anti-drug Antibody Immune Complexes to Facilitate Dedicated In Vivo Studies

“…Different research groups developed technologies and methods for the analysis and quantification of ICs in buffer and sera (6,24–26). Boysen et al developed a SEC-based method to visualize ICs in sera by adding Fab fragments with a fluorescence-tag to preformed ICs (27). Although a fast and generic method, it has different drawbacks: with a limit of detection of 10 μg/ml, only high concentrated samples can be studied.…”

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

“…An SEC method to determine the ratio of free therapeutic mAbs and antidrug antibody complexed mAb in the serum of animals was described by Boysen et al . .…”

“…Whereas the retention time of the intact protein and heavy chain fragment coincided in the RP-LC methods, baseline resolution could only be achieved between intact antibody, the heavy chain, and light chain fragments with SEC. An SEC method to determine the ratio of free therapeutic mAbs and antidrug antibody complexed mAb in the serum of animals was described by Boysen et al [46].…”

Biomarkers for nonclinical infusion reactions in marketed biotherapeutics and considerations for study design

“…TK data can be used to demonstrate either increased test article (TA) clearance or sustained exposure to the TA if an ADA assay is not available, and complement activation can help elucidate the mechanism behind the IR. Another possible peripheral blood biomarker for immunogenicity is the measurement of circulating immune complexes (CICs), which can be measured by size-exclusion chromatography – high-pressure liquid chromatography SEC-HPLC (ADA bound to the biotherapeutic), SEC-ELISA, or by ELISA in Raji cells (complement bound to either ADA or TA) 48 , 49 . Even if an ADA assay is available, other biomarkers beyond ADA or TK are critical.…”

“…(mL) Sample collection & handling considerations Expected modulation Timing a Pharmacokinetic/Pharmacodynamic TK serum or plasma 0.5–1.5 Per protocol b Increased clearance/decreased TA 2-4 h after IR PD/Target 0.5–1 Target with decreased or no engagement a Immunogenicity ADA serum or plasma 0.5–1 Per protocol. For small species with TK cohorts, sample from TK, Main study, and Recovery groups Positive 2-4 h after IR or prior to termination in all animals CIC serum or plasma 0.5–1 Method dependent 48 , 49 Positive/Increased Complement ∗∗2 , ∗∗5 , ∗6 , 18 , 19 , ∗∗51 CH50 serum 0.5–1.8 Process and freeze immediately. Minimize freeze/thaw cycles Decreased 2-4 h after IR C3a serum or plasma 0.5–1.8 Increased C5a Increased Bb Increased Sc5b-9 Increased Cytokines ∗∗2 , ∗∗5 , 10 , 12 , 19 , ∗50 , ∗∗51 Multi-plexed assay (cytokines most commonly observed listed) IL-6, TNF-α, IFNγ, IL1-b, IL-2 serum or plasma 0.5–1.0 Process and freeze immediately.…”