Flow cytometry screening strategy for the enrichment of high-producing Chinese Hamster ovary cells for monoclonal antibody manufacturing

Establishment of Chinese hamster ovary (CHO) cell lines for the production of monoclonal antibodies (mAbs) is generally conducted through transfection and pooling procedures with a laborious and time-consuming screening. It could be one of the most rate-limiting steps in the process development of mAb manufacturing. Therefore, an improvement to screening efficiency will give a positive impact to shortening the period of process development. We here propose an enrichment strategy for screening for high-producing cells using flow cytometry (FCM). A stable pool prepared by mAb-expression vector transfection was stained with a fluorescent-labeled antibody that binds to an mAb presented on the cell surface and was applied to FCM analysis and cells were separated and grouped based on their fluoresence. The volumetric productivity in a fed-batch culture of grouped cells, which showed the brightest of fluorescein isothiocyanate (FITC)-positive cells, was only 1.2 to 1.3 times higher than that of ungrouped cells, despite our expectation. Therefore, we tried to set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and selected the brightest 5% of FITC-positive cells from each group by FCM. The volumetric productivity of cells gated by FSC and SSC was 3.4- to 4.7-fold higher than ungrouped cells. Surprisingly, the selection with the highest volumetric productivity indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all selected cells. Our results showed that our new screening strategy by FCM to group cells with the smallest values of FSC and SSC based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells

Laser spectroscopic studies of the pure rotational U_0(0) and W_0(0) transitions of solid parahydrogen

High resolution spectrum of multipole-induced transitions of solid parahydrogen was recorded using diode and difference frequency laser spectroscopy. The J=4<--0 pure rotational U_0(0) transition observed in the diode spectrum agrees well in frequency with the value reported by Balasubramanian et al. [Phys. Rev. Lett. 47, 1277 (1981)] but we observed a spectral width smaller by about a factor of 4. The J=6<--0 W_0(0) transition was observed to be exceedingly sharp, with a width of ~70 MHz, using a difference frequency spectrometer with tone-burst modulation. This transition is composed of three components with varying relative intensity depending upon the direction of polarization of laser radiation. These components were interpreted as the splitting of the M levels in the J=6 state due to crystal field interactions. In addition, a new broad feature was found at 2452.4 cm^(−1) in the low resolution Fourier-transform infrared (FTIR) spectrum of solid hydrogen and was assigned to be the phonon branch W_R(0) transition of the W_0(0) line. The selection rules, crystal field splitting of J=4 and J=6 rotons, and the measured linewidth based on these observations are discussed

Effects of verapamil and lidocaine on two components of the re-entry circuit of verapamil-sensitive idiopathic left ventricular tachycardia

AbstractOBJECTIVESWe characterized pharmacologically the slow conduction zone of verapamil-sensitive idiopathic left ventricular tachycardia (ILVT) with regard to the late diastolic potential (LDP).BACKGROUNDWe showed that the slow conduction zone of ILVT could be divided into two components by LDP; that is, the distal component with a tachycardia-dependent conduction delay property and the proximal one without it.METHODSElectrophysiologic studies were performed in eight consecutive patients. The LDP was recorded during left ventricular (LV) mapping during ILVT. Entrainment was performed from the right ventricular outflow tract while recording LDP. The effects of lidocaine (1 mg/kg body weight) and verapamil (0.5 or 1.0 mg) were examined during entrainment.RESULTSThe LDPs preceding the Purkinje potential (PP) were serially recorded from the upper third to the middle of the LV septum along the narrow longitudinal line. The ventricular tachycardia (VT) cycle length increased after lidocaine (p < 0.05), and further after verapamil (p < 0.05). The increments in the VT cycle length after administration of the drugs strongly correlated with those in LDP-PP (r > 0.9 for both drugs). The interval from the ventricular potential to LDP was unchanged after administration of the drugs. In one patient, verapamil terminated VT by local conduction block between LDP and PP. The LDP-PP measured during entrainment increased after lidocaine, and further after verapamil, whereas the interval from the stimulus to LDP remained unchanged.CONCLUSIONSThe component distal to LDP is mainly calcium channel-dependent and partly depressed sodium channel-dependent. The proximal component is considered to be sodium channel-dependent (normal)

Automated screening procedure for the phenotypes of congenital fibrinogen disorders using novel parameters, |min1|c and Ac/|min1|c, obtained from clot waveform analysis using the Clauss method

ArticleClinica chimica acta. 521: 170-176(2021)journal articl

HETEROTACTIC POLY(N-ISOPROPYLACRYLAMIDE)

Radical polymerization of N-isopropylacrylamide (NIPAAm) in toluene at low temperatures, in the presence of fluorinated alcohols, produced heterotactic polymer comprising an alternating sequence of meso and racemo dyads. The heterotacticity reached 70% in triads when polymerization was carried out at –40°C using nonafluoro-tert-butanol as the added alcohol. NMR analysis revealed that formation of a 1:1 complex of NIPAAm and fluorinated alcohol through C=O•••H-O hydrogen bonding induces the heterotactic specificity. A mechanism for the heterotactic-specific polymerization is proposed. Examination of the phase transition behavior of aqueous solutions of heterotactic poly(NIPAAm) revealed that the hysteresis of the phase transition between the heating and cooling cycles depended on the average length of meso dyads in poly(NIPAAm)

High-Resolution Infrared Spectroscopy of Solid Hydrogen: The Tetrahexacontapole-Induced ΔJ=6 Transitions

Weak new features in the absorption spectrum of solid hydrogen were observed by high-resolution infrared spectroscopy, and assigned as transitions with total change in angular momentum of ΔJ=6. The tetrahexacontapole (64-pole)-induced rotational transition W_0(0) was studied with a difference frequency laser spectrometer and found to be very narrow, with a linewidth of 0.003 cm^-1 HWHM in 99.8% parahydrogen. Frequencies, linewidths, and integrated absorption strengths were measured for several pure rotational and rotation-vibration transitions from 1800 to 7000 cm^-1, and the tetrahexacontapole moment was determined