Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

Improvement of cerebral metabolism mediated by Ro5-4864 is associated with relief of intracranial pressure and mitochondrial protective effect in experimental brain injury.

PURPOSE: To investigate the possible impact of reduction of mitochondrial membrane permeabilization by modulation of the 18 kDa translocator protein mediated by Ro5-4864 over post-traumatic cerebral edema and metabolic crisis. METHODS: Cerebral microdialysis and intracranial pressure (ICP) monitoring were performed in Sprague-Dawley rats treated by intraperitoneal injection of either dimethylsulfoxide (vehicle) or Ro5-4864 following cortical contusion and further correlated with quantitative assessment of mitochondrial damage, water content in the injured tissue, modified neurological severity score, and lesion size. RESULTS: Ro5-4864 resulted in a profound decrease in ICP that correlated with improved cerebral metabolism characterized by significantly higher glucose and pyruvate and lower lactate concentrations in the pericontusional area in comparison with vehicle-treated animals. Reduced ICP correlated with reduced water content in the injured tissue; improved metabolism was associated with reduced mitochondrial damage evidenced by electron microscopy. Both effects were associated with a profound and significant reduction in glycerol release and lesion size, and correlated with improved neurological recovery. CONCLUSIONS: The present study shows that Ro5-4864 has a favorable effect on the fate of injured brain, presumably mediated by improvement of metabolism. It further suggests that improvement of metabolism may contribute to ICP relief.

TVP1022 protects neonatal rat ventricular myocytes against doxorubicin-induced functional derangements.

Our recent studies demonstrated that propargylamine derivatives such as rasagiline (Azilect, Food and Drug Administration-approved anti-Parkinson drug) and its S-isomer TVP1022 protect cardiac and neuronal cell cultures against apoptotic-inducing stimuli. Studies on structure-activity relationship revealed that their neuroprotective effect is associated with the propargylamine moiety, which protects mitochondrial viability and prevents apoptosis by activating Bcl-2 and protein kinase C-epsilon and by down-regulating the proapoptotic protein Bax. Based on the established cytoprotective and neuroprotective efficacies of propargylamine derivatives, as well as on our recent study showing that TVP1022 attenuates serum starvation-induced and doxorubicin-induced apoptosis in neonatal rat ventricular myocytes (NRVMs), we tested the hypothesis that TVP1022 will also provide protection against doxorubicin-induced NRVM functional derangements. The present study demonstrates that pretreatment of NRVMs with TVP1022 (1 microM, 24 h) prevented doxorubicin (0.5 microM, 24 h)-induced elevation of diastolic [Ca(2+)](i), the slowing of [Ca(2+)](i) relaxation kinetics, and the decrease in the rates of myocyte contraction and relaxation. Furthermore, pretreatment with TVP1022 attenuated the doxorubicin-induced reduction in the protein expression of sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase, Na(+)/Ca(2+) exchanger 1, and total connexin 43. Finally, TVP1022 diminished the inhibitory effect of doxorubicin on gap junctional intercellular coupling (measured by means of Lucifer yellow transfer) and on conduction velocity, the amplitude of the activation phase, and the maximal rate of activation (dv/dt(max)) measured by the Micro-Electrode-Array system. In summary, our results indicate that TVP1022 acts as a novel cardioprotective agent against anthracycline cardiotoxicity, and therefore potentially can be coadmhence, theinistered with doxorubicin in the treatment of malignancies in humans.

Hyperbaric oxygen treatment improves GFR in rats with ischaemia/reperfusion renal injury: a possible role for the antioxidant/oxidant balance in the ischaemic kidney.

BACKGROUND: Ischaemic kidney injury continues to play a dominant role in the pathogenesis of acute renal failure (ARF) in many surgical and medical settings. A major event in the induction of renal injury is related to the generation of oxygen-free radicals. Hyperbaric oxygen therapy (HBO) is indicated for treatment of many ischaemic events but not for ARF. Therefore, the present study examined the effects of HBO on kidney function and renal haemodynamics in rats with ischaemic ARF. METHODS: Renal ischaemia was induced by unilateral renal artery clamping (45 min) in rats. Within 24 h following ischaemia, rats were treated twice with HBO of 100% O(2) at 2.5 absolute atmospheres for 90 min each (+HBO). Untreated rats (-HBO) served as a control. Forty-eight hours later, GFR, RBF and endothelial-dependent vasorelaxation were measured. In addition, the immunoreactive staining of 4-hydroxy-2-noneal (4-HNE), a major product of endogenous lipid peroxidation, and superoxide dismutase (SOD) were assessed. RESULTS: In the -HBO group, GFR was reduced by 94% compared with the untouched normal kidney (ischaemic: 0.06 +/- 0.03 ml/min, normal: 1.02 +/- 0.13 ml). In contrast, in the +HBO group, GFR of the ischaemic kidney (0.36 +/- 0.07 ml/min) was reduced only by 68% compared with the contralateral normal kidney (1.12 +/- 0.12 ml/min). In line with these findings, HBO improved the vasodilatory response to ACh as expressed in enhancement of both total and regional renal blood flow. In addition, HBO reduced the formation of 4-HNE by 33% and 76% and increased SOD by 30% and 70% in the cortex and outer stripe region of the medulla of the ischaemic kidney, respectively. CONCLUSION: HBO attenuates the decline in GFR following renal ischaemia, and improves endothelial-dependent vasorelaxation, suggesting that treatment with HBO may be beneficial in the setting of ischaemic ARF.

Neuroprotective effect of hyperbaric oxygen therapy in brain injury is mediated by preservation of mitochondrial membrane properties.

Recent experimental data have shown that hyperbaric oxygen therapy (HBOT) was associated increased Bcl-2 expression at the injury site that correlated with reduced apoptosis. We hypothesized that HBOT mediated enhancement of Bcl-2 expression and increased intracellular oxygen bio-availability may both contribute to preserve mitochondrial integrity and reduce the activation of the mitochondrial pathway of apoptosis. For this purpose, a cortical lesion was created in the parietal cortex of Sprague-Dawley rats by dynamic cortical deformation (DCD) and outcome measures in non-treated animals were compared with that of HBOT treated rats. Morphological analysis showed a profound reduction in neuronal counts in the perilesional area and a marked rarefaction of the density of the axonal-dendritic network. In treated animals, however, there was a significant attenuation of the impact of DCD over perilesional neurons, characterized by significantly higher cell counts and denser axonal network. In mitochondria isolated from injured brain tissue, there was a profound loss of mitochondrial transmembrane potential (Deltapsi(M)) that proved to be substantially reversed by HBOT. This finding correlated with a significant reduction of caspases 3 and 9 activation in HBOT treated animals but not of caspase 8, indicating a selective effect over the intrinsic pathway of apoptosis. All together, our results indicate that the neuroprotective effect of HBOT may represent the consequence of preserved mitochondrial integrity and subsequent inhibition of the mPTP and reduction of the mitochondrial pathway of apoptosis.

The 1,4,5-inositol trisphosphate pathway is a key component in Fas-mediated hypertrophy in neonatal rat ventricular myocytes.

OBJECTIVE: Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas-->phospholipase C (PLC)-->1,4,5-inositol trisphosphate (1,4,5-IP3)-->sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy. METHODS: The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence. RESULTS: Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122, xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002. CONCLUSIONS: Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3beta pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.