Investigation of cell line specific responses to pH inhomogeneity and consequences for process design.

With increasing bioreactor volumes, the mixing time of the reactor increases as well, which creates an inhomogeneous environment for the cells. This can result in impaired process performance in large-scale production reactors. Particularly the addition of base through the reactor headspace can be problematic, since it creates an area, where cells are repeatedly exposed to an increased pH. The aim of this study is to simulate this large-scale phenomenon at lab-scale and investigate its impact. Two different cell lines were exposed to pH amplitudes of a maximal magnitude of 0.05 units (pH of 6.95). Both cell lines showed similar responses, like decreased viable cell counts, but unaffected lactate levels. However, cell line B showed an initially increased specific productivity in response to the introduced amplitudes, whereas cell line A showed a consistently lower specific productivity. Furthermore, the time point at which base addition is started influences the impact, which pH amplitudes have on process performance. When pH control was started earlier in the process, maximal viable cell counts decreased and the lactate metabolic shift was less pronounced. These results show that the potential negative impact of pH amplitudes can be minimized by strategic process design.

Development, characterization, and application of a 2-Compartment system to investigate the impact of pH inhomogeneities in large-scale CHO-based processes.

Large-scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2-Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large-scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N-glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large-scale bioreactors.

The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes.

A divalent human leukocyte antigen-B7 fusion-protein up-regulates CD25 and CD69 in alloreactive CD8+ T cells bypassing CD28 costimulation.

BACKGROUND: T cells recognize major histocompatibility complex (MHC) molecules and their cryptic antigenic peptides on antigen-presenting cells and are generally triggered to proliferate, and when sufficient, co-stimulation is available. In soluble form, monomeric MHC molecules can induce apoptosis, anergy, or decreases of the T-cell receptor (TCR). METHODS: A dimeric fusion protein of the human leukocyte antigens (HLA)-B7 was molecularly engineered and expressed in a B-cell line to allow secretion. Alloreactive T cells were generated according to the standard protocol. RESULTS: A dimer of approximately 160 kD was obtained, affinity purified, and used to study T-cell interaction. In immobilized form, this protein efficiently stimulated alloreactive T cells to proliferate and produce interleukin (IL)-2 and interferon (IFN)-gamma in a concentration-dependent manner, up-regulating CD25 and CD69 expression. In contrast, the soluble fusion protein induced T-cell apoptosis. CONCLUSIONS: The dichotomy in T-cell regulation by a divalent MHC fusion protein warrants the use of MHC multimers as custom-designed immune-regulatory molecules both in transplantation and autoimmune disease.

Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.