Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis

Abstract

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell–surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell–surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors

“…In previous benchmarking studies, highly validated sets of antibodies derived from published sequences have been used to generate data associated with their biophysical and chemical stability properties. [4][5][6][7][8][9] We chose to do something similar for this SPR-focused study, and selected antibodies from the patent literature that target PD-1, an important checkpoint protein in immuno-oncology that helps the immune system to regulate and eliminate tumors. The effort led us to 35 mAbs that we produced as isotype IgG4, being the most common format in fully documented cases.…”

Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

“…Therefore, use of simple predictive tools that can detect at early stage different developability issues is of high importance. Many approaches have been developed for in vitro or in silico evaluation of the various physicochemical features of Abs that can allow recognition of those with developability problems (6,(8)(9)(10)(11)(12)(13)(14)(15)(16). From these studies it is evident that only the simultaneous use of complementary analytical techniques would allow comprehensive interrogation of all possible developability issues of a particular Ab.…”

Pharmacokinetic prediction of an antibody in mice based on an in vitro cell-based approach using target receptor-expressing cells

“…Ultimately, given animal welfare considerations, all PK prediction studies should be performed in vitro. In vitro assays are frequently used to evaluate nonspecific binding by baculovirus particle-based ELISA 6 , extracellular matrix binding assays 8 , and heparin binding assays 7 . While none of these assays are able to quantitatively predict PK, they can be helpful for designing novel biologics to improve PK.…”

“…However, this approach involves the sacrifice of many experimental animals. A wide variety of in vitro assays have been used to evaluate nonspecific binding and antibody uptake, but none of these assays are able to quantitatively predict PK profiles [6][7][8] . In addition, cell-based assays such as FcRn-mediated transcytosis assays have been developed to predict the half-life of an antibody, but they can only be used to rank candidate antibodies by half-life [9][10][11][12] .…”