Challenges in the Quantification of Free TFPI in Human Plasma Following Marstacimab Treatment
TL;DR
The accurate measurement of free TFPI from marstacimab-treated human plasma was not achievable with the approaches tested, in part because marstacimab binds TFPI with a low-nanomolar affinity.
Abstract
Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. Marstacimab (PF‐06741086), a human immunoglobulin G1 monoclonal antibody, binds the TFPI Kunitz-2 domain to improve hemostatic control in individuals with hemophilia. Quantifying the free form of TFPI, i.e., not bound to marstacimab or endogenous binding proteins, is the most direct assessment of target coverage and modulation following treatment. This investigation describes efforts to develop a quantitative assay to measure free TFPI in human plasma to support the marstacimab drug development program. Three protein enrichment methods (competitive antibody capture, binding partner capture, and drug depletion) were assessed in combination with liquid chromatography–tandem mass spectrometry to develop a bioanalytical free target assay. Surface plasmon resonance (SPR) was used to determine the binding affinities of marstacimab and competitive antibody clones to TFPI. SPR analysis demonstrated marstacimab binds TFPI with a nanomolar affinity (equilibrium dissociation constant [KD], 3.7 nM), whereas antibody clones had picomolar binding affinities. All assay formats over-recovered TFPI, indicating disruption of the marstacimab:TFPI complex during sample handling and release of bound TFPI, under the experimental conditions reported here. As a result, accurate measurement of free TFPI was not feasible. These findings demonstrate that the sample manipulation required for analysis altered the binding equilibrium of the marstacimab:TFPI complex with release of bound TFPI that compromised assay specificity. In conclusion, the accurate measurement of free TFPI from marstacimab-treated human plasma was not achievable with the approaches tested, in part because marstacimab binds TFPI with a low-nanomolar affinity.