Search Results (23)

Electrospinning can be used to prepare nanofiber mats from diverse polymers and polymer blends. A large area of research is the application of nanofibrous membranes for tissue engineering. Typically, cell adhesion and proliferation as well as the viability of mammalian cells are tested by seeding the cells on substrates, cultivating them for a defined time and finally dyeing them to enable differentiation between cells and substrates under a white light or fluorescence microscope. While this procedure works well for cells cultivated in well plates or petri dishes, other substrates may undesirably also be colored by the dye. Here we show investigations of the optical contrast between dyed CHO DP-12 (Chinese hamster ovary) cells and different electrospun nanofiber mats, dyed with haematoxylin-eosin (H&E), PromoFluor 488 premium, 4,6-diamidino-2-phenylindole (DAPI) or Hoechst 33342, and give the optimum dyeing parameters for maximum optical contrast between cells and nanofibrous substrates. Full article

The evaluation of the analytical capabilities of a novel disposable flow cell for spectroscopic bioprocess monitoring is presented. The flow cell is presterilized and can be connected to any kind of bioreactor by weldable tube connections. It is clamped into a reusable holder, which is equipped with SMA-terminated optical fibers or an integrated light source and detection unit. This modular construction enables spectroscopic techniques like UV-Vis spectroscopy or turbidity measurements by scattered light for modern disposable bioreactors. A NIR scattering module was used for biomass monitoring in different cultivations. A high-cell-density fed-batch cultivation with Komagataella phaffii and a continuous perfusion cultivation with a CHO DG44 cell line were conducted. A high correlation between the sensor signal and biomass or viable cell count was observed. Furthermore, the sensor shows high sensitivity during low turbidity states, as well as a high dynamic range to monitor high turbidity values without saturation effects. In addition to upstream processing, the sensor system was used to monitor the purification process of a monoclonal antibody. The absorption module enables simple and cost-efficient monitoring of downstream processing and quality control measurements. Recorded absorption spectra can be used for antibody aggregate detection, due to an increase in overall optical density. Full article

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115–149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000–5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34–40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39–46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC₅₀ value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system. Full article

Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism. Full article

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) are amenable for use in a clinical setting for treatment of osteoarthritis (OA), which remains one of the major illnesses worldwide. Aside from iPSC-derived iMSCs, chondrocytes (iCHO) and extracellular vesicles (EV) are also promising candidates for treatment of OA. Manufacturing and quality control of iPSC-derived therapies is mainly manual and thus highly time consuming and susceptible to human error. A major challenge in translating iPSC-based treatments more widely is the lack of sufficiently scaled production technologies from seeding to fill-and-finish. Formerly, the Autostem platform was developed for the expansion of tissue-derived MSCs at scale in stirred tank bioreactors and subsequent fill-and-finish. Additionally, the StemCellDiscovery platform was developed to handle plate-based cultivation of adherent cells including their microscopic analysis. By combining the existing automation technology of both platforms, all required procedures can be integrated in the AutoCRAT system, designed to handle iPSC expansion, differentiation to iMSCs and iCHOs, pilot scale expansion, and formulation of iMSCs as well as extracellular vesicles and their purification. Furthermore, the platform is equipped with several in-line and at-line assays to determine product quality, purity, and safety. This paper highlights the need for adaptable and modular automation concepts. It also stresses the importance of ensuring safety of generated therapies by incorporating automated release testing and cleaning solutions in automated systems. The adapted platform concepts presented here will help translate these technologies for clinical production at the necessary scale. Full article

Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293F^TM and ExpiCHO-S^TM with the transfection reagents ExpiFectamine^TM and polyethylenimine. We discovered that there are significant differences between Expi293F^TM and ExpiCHO-S^TM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization. Full article

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Full article

Chemometric models for on-line process monitoring have become well established in pharmaceutical bioprocesses. The main drawback is the required calibration effort and the inflexibility regarding system or process changes. So, a recalibration is necessary whenever the process or the setup changes even slightly. With a large and diverse Raman dataset, however, it was possible to generate generic partial least squares regression models to reliably predict the concentrations of important metabolic compounds, such as glucose-, lactate-, and glutamine-indifferent CHO cell cultivations. The data for calibration were collected from various cell cultures from different sites in different companies using different Raman spectrophotometers. In testing, the developed “generic” models were capable of predicting the concentrations of said compounds from a dilution series in FMX-8 mod medium, as well as from an independent CHO cell culture. These spectra were taken with a completely different setup and with different Raman spectrometers, demonstrating the model flexibility. The prediction errors for the tests were mostly in an acceptable range (<10% relative error). This demonstrates that, under the right circumstances and by choosing the calibration data carefully, it is possible to create generic and reliable chemometric models that are transferrable from one process to another without recalibration. Full article

Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM) is applied in view of early detection of cellular responses to the cytostatic drug doxorubicin. Therefore, we determined cell-substrate topology of cultivated CHO cells transfected with a membrane-associated Green Fluorescent Protein (GFP) in the nanometer range prior to and subsequent to the application of doxorubicin. Cell-substrate distances increased up to a factor of 2 after 24 h of application. A reduction of these distances by again a factor 2 was observed upon cell aging, and an influence of the cultivation time is presently discussed. Applicability of VA-TIRFM was supported by measurements of MCF-7 breast cancer cells after membrane staining and incubation with doxorubicin, when cell-substrate distances increased again by a factor ≥ 2. So far, our method needs well-defined cell ages and staining of cell membranes or transfection with GFP or related molecules. Use of intrinsic fluorescence or even light-scattering methods to various cancer cell lines could make this method more universal in the future, e.g., in the context of early detection of apoptosis. Full article

Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added to replenish nutrients and regulate pH values. Some effects of hyperosmolality, such as increased cell size and growth inhibition, have been elucidated by previous research, but the impact of hyperosmolality and the specific effects of the added osmotic-active reagents have rarely been disentangled. In this study, CHO cells were exposed to four osmotic conditions between 300 mOsm/kg (physiologic condition) and 530 mOsm/kg (extreme hyperosmolality) caused by the addition of either high-glucose-supplemented industrial feed or mannitol as an osmotic control. We present novel single-cell cultivation data revealing heterogeneity in mass gain and cell division in response to these treatments. Exposure to extreme mannitol-induced hyperosmolality and to high-glucose-oversupplemented feed causes cell cycle termination, mtDNA damage, and mitochondrial membrane depolarization, which hints at the onset of premature stress-induced senescence. Thus, this study shows that both mannitol-induced hyperosmolality (530 mOsm/kg) and glucose overfeeding induce severe negative effects on cell growth and mitochondrial activity; therefore, they need to be considered during process development for commercial production. Full article

A current focus of biopharmaceutical research and production is seed train process intensification. This allows for intermediate cultivation steps to be avoided or even for the direct inoculation of a production bioreactor with cells from cryovials or cryobags. Based on preliminary investigations regarding the suitability of high cell densities for cryopreservation and the suitability of cells from perfusion cultivations as inoculum for further cultivations, an ultra-high cell density working cell bank (UHCD-WCB) was established for an immunoglobulin G (IgG)-producing Chinese hamster ovary (CHO) cell line. The cells were previously expanded in a wave-mixed bioreactor with internal filter-based perfusion and a 1 L working volume. This procedure allows for cryovial freezing at 260 × 10⁶ cells mL⁻¹ for the first time. The cryovials are suitable for the direct inoculation of N−1 bioreactors in the perfusion mode. These in turn can be used to inoculate subsequent IgG productions in the fed-batch mode (low-seed fed-batch or high-seed fed-batch) or the continuous mode. A comparison with the standard approach shows that cell growth and antibody production are comparable, but time savings of greater than 35% are possible for inoculum production. Full article

The link between hydrodynamics and biological process behavior of antibody-producing mammalian cell cultures is still not fully understood. Common methods to describe dependencies refer mostly to averaged hydrodynamic parameters obtained for individual cultivation systems. In this study, cellular effects and locally resolved hydrodynamics were investigated for impellers with different spatial hydrodynamics. Therefore, the hydrodynamics, mainly flow velocity, shear rate and power input, in a single- and a three-impeller bioreactor setup were analyzed by means of CFD simulations, and cultivation experiments with antibody-producing Chinese hamster ovary (CHO) cells were performed at various agitation rates in both reactor setups. Within the three-impeller bioreactor setup, cells could be cultivated successfully at much higher agitation rates as in the single-impeller bioreactor, probably due to a more uniform flow pattern. It could be shown that this different behavior cannot be linked to parameters commonly used to describe shear effects on cells such as the mean energy dissipation rate or the Kolmogorov length scale, even if this concept is extended by locally resolved hydrodynamic parameters. Alternatively, the hydrodynamic heterogeneity was statistically quantified by means of variance coefficients of the hydrodynamic parameters fluid velocity, shear rate, and energy dissipation rate. The calculated variance coefficients of all hydrodynamic parameters were higher in the setup with three impellers than in the single impeller setup, which might explain the rather stable process behavior in multiple impeller systems due to the reduced hydrodynamic heterogeneity. Such comprehensive insights lead to a deeper understanding of the bioprocess. Full article

During process development, bioprocess data need to be converted into applicable knowledge. Therefore, it is crucial to evaluate the obtained data under the usage of transparent and reliable data reduction and correlation techniques. Within this contribution, we show a generic Monte Carlo error propagation and regression approach applied to two different, industrially relevant cultivation processes. Based on measurement uncertainties, errors for cell-specific growth, uptake, and production rates were determined across an evaluation chain, with interlinked inputs and outputs. These uncertainties were subsequently included in regression analysis to derive the covariance of the regression coefficients and the confidence bounds for prediction. The usefulness of the approach is shown within two case studies, based on the relations across biomass-specific rate control limits to guarantee high productivities in E. coli, and low lactate formation in a CHO cell fed-batch could be established. Besides the possibility to determine realistic errors on the evaluated process data, the presented approach helps to differentiate between reliable and unreliable correlations and prevents the wrong interpretations of relations based on uncertain data. Full article

The development of continuous bioprocesses—which require cell retention systems in order to enable longer cultivation durations—is a primary focus in the field of modern process development. The flow environment of microfluidic systems enables the granular manipulation of particles (to allow for greater focusing in specific channel regions), which in turn facilitates the development of small continuous cell separation systems. However, previously published systems did not allow for separation control. Additionally, the focusing effect of these systems requires constant, pulsation-free flow for optimal operation, which cannot be achieved using ordinary peristaltic pumps. As described in this paper, a 3D printed cell separation spiral for CHO-K1 (Chinese hamster ovary) cells was developed and evaluated optically and with cell experiments. It demonstrated a high separation efficiency of over 95% at up to 20 × 10⁶ cells mL^?1. Control over inlet and outlet flow rates allowed the operator to adjust the separation efficiency of the device while in use—thereby enabling fine control over cell concentration in the attached bioreactors. In addition, miniaturized 3D printed buffer devices were developed that can be easily attached directly to the separation unit for usage with peristaltic pumps while simultaneously almost eradicating pump pulsations. These custom pulsation dampeners were closely integrated with the separator spiral lowering the overall dead volume of the system. The entire device can be flexibly connected directly to bioreactors, allowing continuous, pulsation-free cell retention and process operation. Full article

There is a profound need in bioprocess manufacturing for low-cost single-use sensors that allow timely monitoring of critical product and production attributes. One such opportunity is screen-printed enzyme-based electrochemical sensors, which have the potential to enable low-cost online and/or off-line monitoring of specific parameters in bioprocesses. In this study, such a single-use electrochemical biosensor for lactate monitoring is designed and evaluated. Several aspects of its fabrication and use are addressed, including enzyme immobilization, stability, shelf-life and reproducibility. Applicability of the biosensor to off-line monitoring of bioprocesses was shown by testing in two common industrial bioprocesses in which lactate is a critical quality attribute (Corynebacterium fermentation and mammalian Chinese hamster ovary (CHO) cell cultivation). The specific response to lactate of the screen-printed biosensor was characterized by amperometric measurements. The usability of the sensor at typical industrial culture conditions was favorably evaluated and benchmarked with commonly used standard methods (HPLC and enzymatic kits). The single-use biosensor allowed fast and accurate detection of lactate in prediluted culture media used in industrial practice. The design and fabrication of the biosensor could most likely be adapted to several other critical bioprocess analytes using other specific enzymes. This makes this single-use screen-printed biosensor concept a potentially interesting and versatile tool for further applications in bioprocess monitoring. Full article