Development of a new control strategy based on prior knowledge that enables rapid and streamlined process characterization

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Investigation into the Usability of Micromechanical Models to Predict the Behavior of a Nanocomposite Polymer

The purpose of this thesis was to determine the acceptability of a set of existing nanocomposite test specimens for tensile testing and then to determine the Young\u27s and shear modulus of these test specimens. If the test specimens were found to be acceptable, the accuracy of three micromechanical models was to be evaluated by comparing their predictions to the mechanical properties determined from testing the specimens. The set of existing nanocomposite test specimens had a distinct concave shape on two surfaces that were believed to be intended as flat. In order to determine if they adhered to the Type I geometry for reinforced composites as listed in the Standard Test Method for Tensile Properties of Plastics a program was written using a coordinate measuring machine to measure the cross sectional area of the test specimens. The geometry was found to meet the requirements of the standard and then the tensile testing procedure from the standard was followed. During process verification of the testing procedure, the specimens were found to behave in an unexpected way for a material that was supposed to be homogeneous and isotropic. The test specimens were found to consistently break outside of the narrow test section. An investigation into the behavior of the test specimens using dissection, impact testing, and hardness testing found that a core had formed inside the test specimens during fabrication and therefore the specimens were not homogeneous and isotropic. Since the three micromechanical models under investigation for this thesis had the assumption that the material is homogeneous and isotropic it was determined that the three micromechanical models should not be used to predict the mechanical properties of the set of test specimens

Online control of cell culture redox potential prevents antibody reduction

The phenomenon of monoclonal antibody (mAb) interchain disulfide bond reduction during manufacturing processes has been reported widely across the biotechnology industry. Reduction results in loss of product, requires more complex purification processes, and leads to reduced stability of the final drug product. Here, we show the development of a control system to prevent mAb reduction in the bioreactor based on the cell culture redox potential. Cell culture redox potential indicates the reducing/oxidizing potential of the extracellular environment. This work describes the process development for an IgG2 mAb where a high level of mAb reduction was observed at harvest. Further analysis revealed reduced mAb in the bioreactor prior to harvest. To understand the impact of reducing/oxidizing environment on mAb reduction, the process was run with several different conditions including increased concentrations of metal ions, 2-mercaptoethanol, glutathione, cystine, and an increased DO set-point. Samples were taken from these bioreactors on days 12 and 14 and the amounts of intact mAb in the cell culture supernatants were quantified. Many of these bioreactors were run with redox probes that allowed monitoring of the cell culture redox potential. Analysis of the data revealed a clear correlation between the cell culture redox potential and mAb reduction. Based on this information, we identified a threshold redox potential above which the mAb remained intact and below which there was significant and highly variable amounts of reduced mAb. Using this knowledge, we developed three control schemes to mitigate mAb reduction. These control methodologies functioned by increasing the concentrations of dissolved oxygen (DO), Cu, or both DO and Cu to maintain the redox potential above the threshold value. The redox control strategies based on the addition of Cu or Cu and DO maintained the cell culture redox potential above the threshold value and prevented mAb reduction, whereas the control strategy based on DO control alone was insufficient to maintain the redox potential above the threshold value and had high levels of reduced mAb (Figure 1). Importantly, the redox control strategies did not significantly impact the cell growth, viability, mAb production, or product aggregates. This method of using on-line cell culture redox potential can be used to predict the likelihood of reduction occurring in the bioreactor and evaluate the effectiveness of new mitigation/control strategies; it can also be extended to prevent mAb reduction from occurring during or after the harvest. Finally, the methods described in this work to control mAb reduction would ensure simpler purification processes, improved product quality, and prolonged drug product stability compared to processes with uncontrolled reduction Please click Additional Files below to see the full abstract

Two-Allergen Model Reveals Complex Relationship between IgE Crosslinking and Degranulation

SummaryAllergy is an immune response to complex mixtures of multiple allergens, yet current models use a single synthetic allergen. Multiple allergens were modeled using two well-defined tetravalent allergens, each specific for a distinct IgE, thus enabling a systematic approach to evaluate the effect of each allergen and percentage of allergen-specific IgE on mast cell degranulation. We found the overall degranulation response caused by two allergens is additive for low allergen concentrations or low percent specific IgE, does not change for moderate allergen concentrations with moderate to high percent specific IgE, and is reduced for high allergen concentrations with moderate to high percent specific IgE. These results provide further evidence that supraoptimal IgE crosslinking decreases the degranulation response and establishes the two-allergen model as a relevant experimental system to elucidate mast cell degranulation mechanisms

Inhibition of weak-affinity epitope-IgE interactions prevents mast cell degranulation

Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design

Role of System Gly in glycine transport in monolayer cultures of liver cells

The high-affinity component of glycine uptake by the hepatoma cell line HTC and by the ordinary rat hepatocyte corresponds to System Gly, the agency serving for glycine uptake by pigeon red blood cells and rabbit reticulocytes, and at most to only a minor extent to System ASC. This component was identified in HTC by its sensitivity to inhibition by sarcosine but scarcely by 2-(methylamino) isobutyric acid, by its insensitivity to lowering of the pH, and by the unique relation of its rate to the square of the Na+ concentration. The identity of the low-affinity component with System A was confirmed by opposite properties, and by its stimulation by insulin or amino acid starvation. Both components differed sharply from the System ASC uptake as measured with threonine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24480/1/0000755.pd

A heterobivalent ligand inhibits mast cell degranulation via selective inhibition of allergen-IgE interactions in vivo

Current treatments for allergies include epinephrine and antihistamines, which treat the symptoms after an allergic response has taken place; steroids, which result in local and systemic immune suppression; and IgE-depleting therapies, which can be used only for a narrow range of clinical IgE titers. The limitations of current treatments motivated the design of a heterobivalent inhibitor (HBI) of IgE-mediated allergic responses that selectively inhibits allergen-IgE interactions, thereby preventing IgE clustering and mast cell degranulation. The HBI was designed to simultaneously target the allergen binding site and the adjacent conserved nucleotide binding site (NBS) found on the Fab of IgE Abs. The bivalent targeting was accomplished by linking a hapten to an NBS ligand with an ethylene glycol linker. The hapten moiety of HBI enables selective targeting of a specific IgE, whereas the NBS ligand enhances avidity for the IgE. Simultaneous bivalent binding to both sites provided HBI with 120-fold enhancement in avidity for the target IgE compared with the monovalent hapten. The increased avidity for IgE made HBI a potent inhibitor of mast cell degranulation in the rat basophilic leukemia mast cell model, in the passive cutaneous anaphylaxis mouse model of allergy, and in mice sensitized to the model allergen. In addition, HBI did not have any observable systemic toxic effects even at elevated doses. Taken together, these results establish the HBI design as a broadly applicable platform with therapeutic potential for the targeted and selective inhibition of IgE-mediated allergic responses, including food, environmental, and drug allergies

Features of amino acid structure enhancing or obstructing cosubstrate reactivity of Na+ in transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32883/1/0000261.pd