Design and development of TT30, a novel C3d-targeted C3/C5 convertase inhibitor for treatment of human complement alternative pathway-mediated diseases.

To selectively modulate human complement alternative pathway (CAP) activity implicated in a wide range of acute and chronic inflammatory conditions and to provide local cell surface and tissue-based inhibition of complement-induced damage, we developed TT30, a novel therapeutic fusion protein linking the human complement receptor type 2 (CR2/CD21) C3 fragment (C3frag = iC3b, C3dg, C3d)-binding domain with the CAP inhibitory domain of human factor H (fH). TT30 efficiently blocks ex vivo CAP-dependent C3frag accumulation on activated surfaces, membrane attack complex (MAC) formation and hemolysis of RBCs in a CR2-dependent manner, and with a ∼ 150-fold potency gain over fH, without interference of C3 activation or MAC formation through the classic and lectin pathways. TT30 protects RBCs from hemolysis and remains bound and detectable for at least 24 hours. TT30 selectively inhibits CAP in cynomolgus monkeys and is bioavailable after subcutaneous injection. Using a unique combination of targeting and effector domains, TT30 controls cell surface CAP activation and has substantial potential utility for the treatment of human CAP-mediated diseases.

Quantitative determination of lipase activity by liquid chromatography-mass spectrometry.

We developed a novel in vitro lipase assay based on the quantitation of fatty acids by liquid chromatography-mass spectrometry. Oleic acids enzymatically released from triolein substrates were isolated from the reaction mixture by reverse-phase chromatography, ionized in negative mode electrospray mass spectrometry and quantitated with the aid of [(13)C]-oleic acid internal standard. The enzymatic activity was measured by monitoring oleic acid productions at multiple time points. This method overcomes the substrate and pH limitations of conventional techniques and thus serves as a generic lipase activity assay.

Matrix-assisted laser desorption/ionization mass spectrometry for the evaluation of the C-terminal lysine distribution of a recombinant monoclonal antibody.

Recombinant monoclonal antibodies produced using mammalian cell lines contain multiple chemical modifications. One specific modification resides on the C-terminus of the heavy chain. Enzymes inside the cell can cleave the C-terminal lysine from the heavy-chain molecules, and variants with and without C-terminal lysine can be produced. In order to fully characterize the protein, there is a need for analytical methods that are able to account for the different product variants. Conventional analytical methods used for the measurement of the distribution of the two different variants are based on chemical or enzymatic degradation of the protein followed by chromatographic separation of the degradation products. Chromatographic separations with gradient elution have long run times, and analyses of multiple samples are time-consuming. This paper reports development of a novel method for the determination of the relative amounts of the two C-terminal heavy-chain variants based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) measurements of the cyanogen bromide degraded recombinant monoclonal antibody products. The distribution of the variants is determined from the MALDI-TOF mass spectra by measuring the peak areas of the two C-terminal peptides. The assay was used for the assessment of the C-terminal lysine distribution in different development lots. The method was able to differentiate between the products obtained using the same cell line as well as between products obtained from different cell lines.