Protein Precipitation for the Purification of Therapeutic Protein

This thesis documents the application of precipitant scouting and analytical tools for the development of a precipitation process for the purification of therapeutic proteins in biopharmaceuticals. Precipitation has the potential to bypass the bottlenecks in productivity experienced with packed bed chromatographic separations, offering a fast, robust first purification step with volume reduction at low cost. In order to evaluate a large number of precipitation candidates, microscale investigations aided by automated liquid handling robotics were chosen, which conferred precision, speed, and complex experimental design with microliter material requirements and in-line analytics. A precipitation screening methodology was successfully built onto a Tecan liquid handling platform including product recovery and techniques for measuring soluble protein, liquid volumes, and recovered protein precipitate. The protocol was supplemented with a custombuild Excel VBA driven Tecan control tool. This accelerated progress by freeing up the potential of the liquid handling arm, which was curtailed by system software. These techniques were then used to characterise effective precipitants for the purification of monoclonal antibodies. Optimal conditions were identified on pure protein models, which were the bridged to process relevant cell culture fluid. Process performance, notably recovery yield and product quality were the investigated on the precipitant conditions brought forward. Process integration aspects were explored, by linking precipitation with an anion exchange chromatography step. This led to discussion on future work and process scale up considerations

The Effect of Fluid Periodization on Athletic Performance Outcomes in American Football Players

For decades strength and conditioning professionals have been seeking optimal training volumes and intensities to yield maximum performance outcomes without the onset of injury. Unfortunately, current studies apply experimental training techniques without considering the individuals’ response to the imposed training load. Due to the vast genetic variability and extraneous environmental factors that affect one’s ability to recover, results from such studies are controversial and inconclusive. Athlete monitoring systems offer an objective assessment that is purported to evaluate an individual’s physiological readiness to adapt to an overload stimulus and thus allow for daily manipulations in training loads (i.e., fluid periodization). However, little is known about the efficacy of this technology to enhance training outcomes. Therefore, the purpose of this study was to examine the effect of fluid periodization on performance outcomes in American football players. Sixty-one Division 1 collegiate American football players (Age: 19.7 ± 0.9 yr; Height: 1.88 ± 0.3 m; Mass: 107.3 ± 11.1 kg) participated in this study and were stratified into experimental (n=33) and control (n=28) groups. Performance outcomes were measured prior to and following the summer training program. Physiological readiness parameters (heart rate variability and direct current brain wave potential outcomes) were measured daily in the experimental group only with Omegawave technology prior to training sessions and adjustments in training volumes or intensity were made based upon physiological readiness outcomes. The control group trained according to the daily prescribed workout. The findings from this study indicate that the experimental group significantly improved in vertical jump, vertical power, aerobic efficiency and broad jump (P \u3c 0.01) compared to the control group. Additionally, significant improvements and effect sizes between groups were noted for fat-free mass (relative improvement: 54%, effect size: 0.30), vertical jump (relative improvement: 157%, effect size: 1.02), vertical power (relative improvement: 94%, effect size: 0.86), broad jump (relative improvement: effect size: 592%, 0.81), triple broad jump (relative improvement: 338%, effect size: 0.63), aerobic efficiency (relative improvement: 154%, effect size: 1.02), and medicine ball overhead throw (relative improvement: 50%, effect size: 0.26). In addition, the experimental group achieved these improvements with less core (-9.5%) and accessory (-13.2%) training volume (P \u3c 0.01). In conclusion, fluid periodization produced greater improvements in performance outcomes at a reduced training load compared to a similar unmodified periodization scheme. These findings highlight the importance of modifying training parameters based upon the daily physiological state of the athlete

Uranium (VI) Adsorbate Structures on Portlandite [Ca(OH)2] Type Surfaces Determined by Computational Modelling and X-ray Absorption Spectroscopy

Portlandite [Ca(OH)2] is a potentially dominant solid phase in the high pH fluids expected within the cementitious engineered barriers of Geological Disposal Facilities (GDF). This study combined X-ray Absorption Spectroscopy with computational modelling in order to provide atomic-scale data which improves our understanding of how a critically important radionuclide (U) will be adsorbed onto this phase under conditions relevant to a GDF environment. Such data are fundamental for predicting radionuclide mass transfer. Surface coordination chemistry and speciation of uranium with portlandite [Ca(OH)2] under alkaline groundwater conditions (ca. pH 12) were determined by both in situ and ex situ grazing incidence extended X-ray absorption fine structure analysis (EXAFS) and by computational modelling at the atomic level. Free energies of sorption of aqueous uranyl hydroxides, [UO2(OH)n]2–n (n = 0–5) with the (001), (100) and (203) or (101) surfaces of portlandite are predicted from the potential of mean force using classical molecular umbrella sampling simulation methods and the structural interactions are further explored using fully periodic density functional theory computations. Although uranyl is predicted to only weakly adsorb to the (001) and (100) clean surfaces, there should be significantly stronger interactions with the (203/101) surface or at hydroxyl vacancies, both prevalent under groundwater conditions. The uranyl surface complex is typically found to include four equatorially coordinated hydroxyl ligands, forming an inner-sphere sorbate by direct interaction of a uranyl oxygen with surface calcium ions in both the (001) and (203/101) cases. In contrast, on the (100) surface, uranyl is sorbed with its axis more parallel to the surface plane. The EXAFS data are largely consistent with a surface structural layer or film similar to calcium uranate, but also show distinct uranyl characteristics, with the uranyl ion exhibiting the classic dioxygenyl oxygens at 1.8 Å and between four and five equatorial oxygen atoms at distances between 2.28 and 2.35 Å from the central U absorber. These experimental data are wholly consistent with the adsorbate configuration predicted by the computational models. These findings suggest that, under the strongly alkaline conditions of a cementitious backfill engineered barrier, there would be significant uptake of uranyl by portlandite to inhibit the mobility of U(VI) from the near field of a geological disposal facility

The genome of Romanomermis culicivorax:revealing fundamental changes in the core developmental genetic toolkit in Nematoda

Background: The genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored. Results: We generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination. Conclusions: Despite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model

790-2 Baseline Electrocardiogram Predicts 30-day Mortality Among 32,812 Patients with Acute Myocardial Infarction Treated with Thrombolysis

To determine the initial electrocardiographic variables predictive of survival among patients with acute myocardial infarction, we analyzed the baseline 12-lead ECGs in 32,812 patients enrolled into the GUSTO trial. All patients had≥0.1mV of ST segment elevation in at least one lead and received thrombolytic therapy. Those with LBBB or ventricular rhythm were excluded from analysis. Clinical follow-up was > 99.5% complete. 2218 (6.8%) patients died within 30 days of the initial ECG. Death within 30 days was more common in patients with RBBB (17%), LAFB (14%), and LPFB (17%), than in those with a normal conduction pattern (6%). Patients with ECG evidence of previous MI in a location distinct from the acute MI had a higher risk of death (9.8% vs. 5.9%) than those without prior infarction (p<0.0001). The variable having the greatest univariate predictive power for 30-day survival was the sum of the absolute ST-segment deviation in each lead (x2=341), as shown in the following mortality curve.Other ST segment variables that predicted 30-day survival were the sum of ST-segment elevation in each lead (x2=287). the maximum ST elevation in anyone lead (X2=257), and the number of leads with ST elevation (x2=250). When multivariate modeling was performed the sum of the absolute ST deviations, number of leads with ST elevation, prior ECG MI, RBBB, and LAFB each added independent prognostic information.We conclude that an ECG at the time of presentation contains substantial prognostic information which can be used to help stratify risk among thrombelytic-treated patients with acute myocardial infarction

Ritonavir blocks AKT signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy).</p> <p>Results</p> <p>Here we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 μM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.</p> <p>Conclusion</p> <p>Our results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.</p