Establishing a small scale model with MULTIVARIATE and bayesian statistics

One of the primary challenges during process characterization is establishing the suitability of the small scale models used to generate the data. Monte Carlo simulations of small scale data are able to account for increased variability in process parameters seen at large scale. However, this approach requires an accurate estimate of the large-scale variability, which is complicated by the small size of typical large-scale data sets. Bayesian statistics offer an alternate approach in which scale effects are accounted for by directly incorporating scaling offsets into predictive models. In this context, Bayesian methods are advantageous because they explicitly account for uncertainty in datasets, which is essential when attempting to estimate scaling offsets based on the small manufacturing-scale datasets. A CHO cell line known to demonstrate scale differences in lactate production was used as an experimental system, where lab-scale bioreactors typically underestimated the lactate levels observed in large-scale bioreactors. To address this issue, multifactor DOE experiments were run on process conditions known in influence lactate, including glucose, pH, cell generational age, base control and cell bank. Conditions were established for high and low lactate at small scale and a multivariate model of lactate production was established. To address the issue of scale predictability, Monte Carlo simulations were performed using both frequentist and hierarchal Bayesian methods. Both models more accurately described the large-scale lactate levels, and gave a more realistic picture of the robustness of the bioreactor process at scale. Overall the Bayesian model tended to predict a higher percentage of runs that would result in high-lactate and therefore a more accurate picture of large scale. Pilot studies were completed to verify the ability of each model to predict high and low-lactate production at large-scale

iSKID: From integrated pilot scale runs to GMP implementation approach

One of the most compelling business reasons for integrated processing is the ability to de-risk capital investment due to a significantly more productive process that takes less space and fewer campaigns to generate clinical and commercial material. Boehringer Ingelheim and Pfizer developed the iSKID, a fully integrated and automated system that hydraulically links the perfusion bioreactor with several downstream unit operations (2xProtein A columns, continuous viral inactivation, AEX in flow through mode, and SPTFF). The Protein A elution cycles are discrete and separated by \u3e2hrs, allowing the ability to discard cycles that do not meet process specifications. The discreteness between product cycles and hydraulic linkage enables the sanitization between cycles for a robust bioburden control strategy. Each cycle is captured in a single use mixer (SUM), where the product is pooled in stable conditions until viral filtration, ultrafiltration/diafiltration and final filtration are performed in batch mode. Identical iSKID prototypes at 100L scale were used at three different sites to generate product quality, process, and bioburden data from three different molecules. The data has been used to understand implementation gaps in GMP facilities and process platforms (CMC1/CMC2). In addition, the team identified specific items to present to the FDA’s Emerging Technology Team (ETT). These items include our strategies for batch definition, microbial control, and process control. In this talk, we will use the data generated from the consistency runs to elaborate on the robustness of the process and touch upon the strategies to be presented to the ETT

The Electrooculogram and a New Blink Detection Algorithm

Accurate and efficient real-time cognitive workload assessment has many important applications, and physiological monitoring has proven quite helpful with this assessment. One such physiological signal, the electrooculogram (EOG), can provide blink rate and blink duration measures. In a recent study, we developed and validated a robust blink detection algorithm based on the vertical EOG (VEOG). This algorithm does not require baseline data and is adaptive in the sense that it works for a wide variety of individuals without any experimenter adjustments. The performance of the algorithm is quantified using truth data based on video recordings. The algorithm produced blink rate and blink duration data for participants in a simulated remotely piloted aircraft experiment. Although this paper focuses on the blink detection algorithm, some results from the study will be included. Specifically, it was found that participants blinked fewer times and with a shorter duration in the more difficult experimental conditions

Saccade Detection Using Polar Coordinates – a New Algorithm

Over the past few decades substantial research has been conducted regarding saccades (rapid eye movements). There are two components of this research. First there is the detection of the saccades, and second how to interpret the saccades features (amplitude, length, and velocity) to inform specific areas of research. This involves both experimental research and clinical applications. The detection of saccades is typically accomplished using two approaches, including cameras and the electrooculogram (EOG). Both of these approaches require algorithms to process the raw data, detect saccades, and calculate the saccade features. The current effort focuses on detecting saccades in the EOG using a new algorithm based on polar coordinates. The details of this algorithm will be presented, as will a calibration procedure and validation of the algorithm’s accuracy. This algorithm was used in a recent study in which operator workload was manipulated. The saccade features produced by the algorithm were analyzed with respect to the workload manipulations. These results will be discussed

Modeling Linear and Cyclic PKS Intermediates through Atom Replacement

[Image: see text] The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs

The Evolving Role of Chemical Synthesis in Antibacterial Drug Discovery

The discovery and implementation of antibiotics in the early twentieth century transformed human health and wellbeing. Chemical synthesis enabled the development of the first antibacterial substances, organoarsenicals and sulfa drugs, but these were soon outshone by a host of more powerful and vastly more complex antibiotics from nature: penicillin, streptomycin, tetracycline, and erythromycin, among others. These primary defences are now significantly less effective as an unavoidable consequence of rapid evolution of resistance within pathogenic bacteria, made worse by widespread misuse of antibiotics. For decades medicinal chemists replenished the arsenal of antibiotics by semisynthetic and to a lesser degree fully synthetic routes, but economic factors have led to a subsidence of this effort, which places society on the precipice of a disaster. We believe that the strategic application of modern chemical synthesis to antibacterial drug discovery must play a critical role if a crisis of global proportions is to be averted