Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics.

Lactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.

Dye-free determination of the focalization position for the hollow fiber flow field flow fractionation (HF5) of proteins.

Proteins are separated in field flow fractionation (FFF) according to a well-established mechanism described as the "Normal or Brownian" mode. In the case of the sub-technique using a hollow fiber, the focalization/relaxation position can be observed visually only with a transparent holder and using dyes as samples. Whatever the choice of instrumentation, a dye-free method is proposed to determine the center of the zone from experimental fractograms by means of only two sample elutions. It is also possible to determine and model the kinematics of the sample toward the equilibrium focalization/relaxation position as well as the real dimensions of the fiber during the separation process.

Instrumentation of hollow fiber flow field flow fractionation for selective cell elution.

Hollow fiber flow field flow fractionation (HF5) columns can be built with minimized cost and instrumental skills by incorporation of commercial hollow fibers into holders made of classical chromatographic tubing and connectors. The proposed design leads to differential elution of human cells of different origin. Suspensions of red blood cells (RBC) and adherent human colorectal cancer (CRC) cell lines were used. These CRC nucleated population have been linked to cell-to-cell and cell to instrument interactions that are limiting factors in terms of recovery and viability. These interactions can be limited depending on injection/elution conditions. By using RBC we observed that the focalization/relaxation step played a major role in the elution process. However, HF5 opens a large potential, which completes the diversity shown by SdFFF in cell sorting methodologies and technologies.