A high throughput capillary electrophoresis method to obtain pharmacokinetics and quality attributes of a therapeutic molecule in circulation.

Therapeutic proteins circulating in blood are in a highly crowded, redox environment at high temperatures of ~37°C. These molecules circulate in the presence of enzymes and other serum proteins making it difficult to predict from in vitro studies the stability, aggregation or pharmacokinetics of a therapeutic protein in vivo. Here, we describe use of a high throughput capillary electrophoresis based microfluidic device (LabChip GXII) to obtain pharmacokinetics (PK) of a fluorescently labeled human mAb directly from serum. The non-labeled and labeled mAbs were evaluated in single dose rat PK studies using a traditional ELISA method or LabChip GXII, respectively. The fluorescent dye did not significantly alter clearance of this particular mAb, and PK parameters were comparable for labeled and unlabeled molecules. Further, from the CE profile we concluded that the mAb was resistant to fragmentation or aggregation during circulation. In a follow-up experiment, dimers were generated from the mAb using photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the same fluorophore. The extent of dimerization was incomplete and some monomer and higher molecular weight species were found in the preparation. In rat PK studies, the serum concentration-time profile of the three entities present in the dimer preparation could be followed simultaneously with the GXII technology. While further studies are warranted, we believe this method could be adapted to obtain PK of different forms of antibodies (oxidized, deamidated or various glycosylated species) and other proteins.

Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine.

Monoclonal antibodies in liquid formulation undergo nonenzymatic hydrolysis when stored at 5 degrees C for extended periods. This hydrolysis is enhanced at extreme pH and high temperature. In this study we discover that iron in the presence of histidine also enhanced cleavage of human immunoglobulin gamma (IgG) molecules containing a lambda light chain when incubated at 40 degrees C. The level of cleavage was concentration dependent on both iron and histidine levels. Enhanced cleavage with iron and histidine was not observed on IgG molecules containing a kappa light chain. Using CE-SDS to quantify levels of Fab+Fc, the Fab arm, and free light chain (LC) and heavy chain (HC) fragments, we show that cleavage resulted in elevated levels of free light and heavy chain fragments. Using MS we find elevated scission between residues E/C on the LC resulting in LC fragment 1-215. We also observed enhanced cleavage between S/C residues of the HC resulting in HC fragment 1-217. The corresponding Fab+Fc fragment beginning with cys-218 was not found. Instead, we find elevation of a Fab+Fc fragment that began with aspartic acid (cleavage between C/D). Further studies to understand how iron and histidine enhance cleavage of lambda light chain IgG molecules are ongoing.

Organic nanostructures on silicon, created with semitransparent polystyrene spheres and 248 nm laser pulses.

Arrays of nanostructures are made starting with a template of close-packed, polystyrene spheres on a silicon surface. The spheres are either 1.091 or 2.99 µm in diameter (d) and are of polystyrene (PS). They are irradiated with a pulse of either 308 or 248 nm light to which they are transparent and semitransparent, respectively. A transparent sphere with d = 1.091 µm diameter concentrates incident light onto a small substrate area. As has been previously reported, that creates silicon nanobumps that rise from circular craters. At 248 nm and d = 2.99 µm, the light energy is mainly absorbed, destroys the sphere, and leaves a shrunken mass (typically about 500 nm wide and 100 nm high) of organic material that is probably polystyrene and its thermal degradation products. At 248 nm and d = 1.091 µm, the residual organic structures are on the order of 300 nm wide and 100 nm high. A distinctive feature is that these organic structures are connected by filaments that are on the order of 50 nm wide and 10 nm high. Filaments form because the close-packed PS spheres expand into each other during the early part of the laser pulse, and then, as the main structures shrink, their viscoelasticity leads to threads between them. Our results with 248 nm and d = 1.091 µm differ from those described by Huang et al with 248 nm and d = 1.0 µm. Future studies might include the further effect of wavelength and fluence upon the process as well the use of other materials and the replacement of nanospheres by other focusing shapes, such as ellipsoids or rods.