Clot formation and fibrinolysis assays reveal functional differences among hemostatic agents in hemophilia A plasma.

Background: Measuring the activity of hemostatic agents used to treat hemophilia A often requires drug-specific assays. In vitro assays show hemophilic clots have abnormal characteristics, including prolonged clotting time and decreased resistance to fibrinolysis. The ability of certain agents to correct these parameters in vitro is associated with hemostatic efficacy in vivo. Objectives: To compare effects of established and emerging hemostatic agents on clot formation and fibrinolysis in hemophilia A plasma. Methods: Pooled and individual hemophilia A platelet-poor plasmas were spiked with replacement (recombinant factor VIII [rFVIII], PEGylated rFVIII, polysialylated rFVIII, and porcine rFVIII) or bypassing (emicizumab, rFVIIa, and activated prothrombin complex concentrate) products. Effects on tissue factor-initiated clot formation and fibrinolysis were measured by turbidity. Results: Compared to normal pooled plasma, hemophilia-pooled plasma showed reduced clot formation and increased fibrinolysis, and all replacement agents improved these characteristics. rFVIII and PEGylated rFVIII produced similar effects at similar concentrations, whereas polysialylated rFVIII produced slightly higher and porcine rFVIII slightly lower effects at these concentrations. Bypassing agents enhanced clot formation and stability, but patterns differed from replacement agents. The clotting rate showed a concentration-response relationship for all agents. High concentrations of all products produced effects that exceeded the normal range in at least some parameters. Responses of individual donors varied, but all agents improved clot formation and stability in all donors tested. Conclusion: Clotting and fibrinolysis assays reveal hemostatic effects of replacement and bypassing therapies at clinically relevant concentrations. These assays may help characterize hemostatic agents and optimize dosing.

Adsorbed protein film on pump surfaces leads to particle formation during fill-finish manufacturing.

During fill-finish manufacturing, therapeutic proteins may aggregate or form subvisible particles in response to the physical stresses encountered within filling pumps. Understanding and quantitating this risk is important since filling may be the last unit operation before the patient receives their dose. We studied particle formation from lab-scale to manufacturing-scale using sensitive and robust protein formulations. Filling experiments with a ceramic rotary piston pump were integrated with a rinse-stripping method to investigate the relationship between protein adsorption and particle formation. For a sensitive protein, multilayer film formation on the piston surface correlated with high levels of subvisible particles in solution. For a robust protein formulation, adsorption and subvisible particle formation were minimal. These results support an aggregation mechanism that is initiated by adsorption to pump surfaces and propagated by mechanical and/or hydrodynamic disruption of the film. The elemental analysis confirmed that ceramic wear debris remained at trace levels and did not contribute appreciably to protein aggregation.

Embryonic exposures to mono-2-ethylhexyl phthalate induce larval steatosis in zebrafish independent of Nrf2a signaling.

Mono-2-ethylhexyl phthalate (MEHP) is the primary metabolite of the ubiquitous plasticizer and toxicant, di-2-ethylhexyl phthalate. MEHP exposure has been linked to abnormal development, increased oxidative stress, and metabolic syndrome in vertebrates. Nuclear factor, Erythroid 2 Like 2 (Nrf2), is a transcription factor that regulates gene expression in response to oxidative stress. We investigated the role of Nrf2a in larval steatosis following embryonic exposure to MEHP. Wild-type and nrf2a mutant (m) zebrafish embryos were exposed to 0 or 200 µg/l MEHP from 6 to either 96 (histology) or 120 hours post fertilization (hpf). At 120 hpf, exposures were ceased and fish were maintained in clean conditions until 15 days post fertilization (dpf). At 15 dpf, fish lengths and lipid content were examined, and the expression of genes involved in the antioxidant response and lipid processing was quantified. At 96 hpf, a subset of animals treated with MEHP had vacuolization in the liver. At 15 dpf, deficient Nrf2a signaling attenuated fish length by 7.7%. MEHP exposure increased hepatic steatosis and increased expression of peroxisome proliferator-activated receptor alpha target fabp1a1. Cumulatively, these data indicate that developmental exposure alone to MEHP may increase risk for hepatic steatosis and that Nrf2a does not play a major role in this phenotype.