Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim.

The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product.

Ca2+-Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1.

Most chemical neurotransmission occurs through Ca(2+)-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca(2+) sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca(2+) dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca(2+) for the assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H(+)-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca(2+)-Calmodulin (CaM)-dependent manner. Ca(2+)-CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca(2+)-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca(2+)-CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes.

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.

Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet.

It is widely accepted that glycoprotein (GP) Ib contains one Ibalpha and one Ibbeta subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibalpha, C484 or C485, forms the disulfide bond with Ibbeta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibbeta. In the context of isolated peptides containing the Ibalpha or Ibbeta transmembrane domain and nearby Cys residue, C484 and C485 in the Ibalpha peptide were both capable of forming a disulfide bond with the Ibbeta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibbeta subunits but only 1 Ibalpha and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibalpha, not the number of Ibbeta subunits therein. Overall, these results demonstrate that Ibalpha is covalently connected to 2 Ibbeta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alphabeta2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.