J Chromatogr B Analyt Technol Biomed Life Sci
December 2018
Modern analytical ion-exchange chromatography is one of the conventional tools used for assessment of product-related quality attributes in bio-therapeutic monoclonal antibodies (mAbs). Here, we present an approach to resolve, identify, and quantify product-related substances of therapeutic mAb at its intact molecular level by cation exchange (CIEX) HPLC coupled directly to electrospray ionization - quadrupole time of flight mass spectrometry (ESI-QTOF-MS). This method utilizes pH gradient elution mode comprised of ammonium formate buffer components, and a weak cation exchange column as stationary phase.
View Article and Find Full Text PDFCD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis.
View Article and Find Full Text PDFWe present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample.
View Article and Find Full Text PDFNumerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP.
View Article and Find Full Text PDFAquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.
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