Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. ...Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50 mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44 % compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.
•Medium composition and design space defined for perfusion cultures of Rhodovulum sulfidophilum.•Process scaled up to a 2 L bioreactor with ATF and effect of perfusion rate investigated.•Stable perfusion cultures for >20 days, with cell densities lager than in fed-batch.•Oligonucleotides continuously harvested, with titers 2 orders of magnitudes larger than literature.•First report on continuous biomanufacturing of oligonucleotides.
Cultivating Chinese hamster ovary (CHO) cells in microtiter plates (MTPs) with time‐resolved monitoring of the oxygen transfer rate (OTR) is highly desirable to provide process insights at increased ...throughput. However, monitoring of the OTR in MTPs has not been demonstrated for CHO cells, yet. Hence, a CHO cultivation process was transferred from shake flasks to MTPs to enable monitoring of the OTR in each individual well of a 48‐well MTP. For this, the cultivation of an industrially relevant, antibody‐producing cell line was transferred from shake flask to MTP based on the volumetric oxygen mass transfer coefficient (kLa). Culture behavior was well comparable (deviation of the final IgG titer less than 10%). Monitoring of the OTR in 48‐well MTPs was then used to derive the cytotoxicity of dimethyl sulfoxide (DMSO) based on a dose–response curve in a single experiment using a second CHO cell line. Logistic fitting of the dose–response curve determined after 100 h was used to determine the DMSO concentration that resulted in a cytotoxicity of 50% (IC50). A DMSO concentration of 2.70% ± 0.25% was determined, which agrees with the IC50 previously determined in shake flasks (2.39% ± 0.1%). Non‐invasive, parallelized, and time‐resolved monitoring of the OTR of CHO cells in MTPs was demonstrated and offers excellent potential to speed up process development and assess cytotoxicity.
Graphical and Lay Summary
Parallelized, small‐scale cultivation of mammalian cells combined with online‐monitoring is desirable to increase experimental throughput and reduce costs. In this study, the authors demonstrate a scale‐down from shake flasks to 48‐well microtiter plates (MTPs) and apply monitoring of the oxygen transfer rate (OTR) in the MTP to determine the cytotoxicity of DMSO. This work potentially opens up a variety of applications for mammalian cell culture characterization and process development based on OTR measurement in MTPs.
Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral ...envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale‐down technologies. The impact of shear during membrane filtration in a tangential flow filtration‐mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems.
Bioreactors are commonly used in the development and manufacture of a variety of therapeutics, including cell therapies, gene therapies, monoclonal antibodies and antibody drug conjugates. Adherent ...cells typically cultured on planar surfaces can also be cultivated in bioreactors by growing them on microcarrier beads. Separation of the final cell product from microcarrier beads during harvest presents its own challenges. Incorporation of the traditional separation method and centrifugation into an all-in-one system is very costly. Separation of microcarriers by gravity alone can lead to reduced yields and carryover of microcarriers beads.
Size exclusion-based separation of microcarrier beads allows for efficient collection of the final harvest, with little to no downstream microcarrier bead carryover. In the present study, we demonstrate that a small-size mesh disc can be incorporated into a bioreactor workflow for separation of microcarrier beads from the final cell product. We expanded bone-marrow derived mesenchymal stem cells on Sartorius SoloHill Star Plus or Corning CellBIND microcarrier beads in a Corning spinner flask bioreactor. Cell growth was monitored daily by nuclei counts and cells were allowed to expand until they reached greater than three population doublings. Upon harvest, the cells were enzymatically detached from the microcarrier beads in conical tubes using TrypLE Select and viable cell counts were acquired. For separation, the resulting cells and microcarrier suspension was subjected to a low shear straining using a mesh-disk scale down tool. Cell yield and viability was assessed after microcarrier separation. Cell counts and viability before and after microbead separation were recorded and microcarrier carryover was assessed in the flow-through.
This mesh disc is available in a range of standard mesh sizes (including 20, 40, 70, and 200 microns) and can be adapted into modular closed-system bioreactors through a range of commercial aseptic connection methods. The use of a mesh disc to separate adherent cells from microcarriers during the harvest step presents an aseptic, reduced shear-stress, and lower implementation and capital cost alternative to conventional separation methods like centrifugation.
Concentration gradients that occur in large industrial‐scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the ...response of strains to such heterogeneities to reduce the risk of failure during process scale‐up. Although there are various scale‐down techniques to study these effects, scale‐down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small‐scale parallel cultivation devices.
In this study, we combine mechanistic growth models with a parallel mini‐bioreactor system to create a high‐throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled‐down approach, a model‐based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large‐scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale‐down scheme, compared with the reference cultivations.
In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale‐down studies to select the most robust strains for bioprocess scale‐up.
Concentration gradients that occur in large industrial‐scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale‐up.
During the scale‐up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, ...tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed‐batch cultivations in the single multicompartment bioreactor (SMCB), a new scale‐down reactor system that offers new access to study large‐scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4–1.5 W m−3, large‐scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set‐up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large‐scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale‐up behavior and to predict cell line‐specific drawbacks at an early stage of process development.
Scaling up industrial microbial processes for commercial production is a high-stakes endeavor, requiring time and investment often exceeding that for laboratory microbe and process development. ...Omissions, oversights and errors can be costly, even fatal to the program. Approached properly, scale-up can be executed successfully. Three guiding principles are provided as a basis: begin with the end in mind; be diligent in the details; prepare for the unexpected. A detailed roadmap builds on these principles. There is a special emphasis on the fermentation step, which is usually the costliest and also impacts downstream processing. Examples of common scale-up mistakes and the recommended approaches are given. It is advised that engineering resources skilled in integrated process development and scale-up be engaged from the very beginning of microbe and process development to guide ongoing R&D, thus ensuring a smooth and profitable path to the large-scale commercial end.
A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL ...working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex‐centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N‐glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred‐tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N‐glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 106 and 120 × 106 cells/mL and a low cell‐specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.
In large‐scale quantitative mass spectrometry (MS)‐based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high‐pH reversed‐phase peptide chromatographic ...fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT‐based fractionation strategies against single‐shot label‐free quantification with spectral library‐free data independent acquisition (LFQ‐DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT‐fractionation approaches in a phosphoproteomics workflow, we compared two different high‐pH reversed‐phase fractionation strategies, microflow (MF) and stage‐tip fractionation (STF), while scaling the peptide input amount down from 12.5 to 1 μg per sample. Our results indicate that, for input amounts higher than 5 μg per sample, TMT labeling, followed by microflow fractionation (MF) and phospho‐enrichment, achieves the deepest phosphoproteome coverage, even compared to single shot direct‐DIA analysis. Conversely, STF of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 μg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of sample amount.
In large‐scale bioreactors, gradients in cultivation parameters such as oxygen, substrate, and pH result in fluctuating cell environments. pH fluctuations were identified as a critical parameter for ...bioprocess performance. Traditionally, scale‐down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strains and thus process performance. Here, we demonstrate the application of dynamic microfluidic single‐cell cultivation (dMSCC) as a novel scale‐down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model stress factor. A detailed comparison between two‐compartment reactor (two‐CR) scale‐down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 (~8 min) and disturbed pH 6 (~2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two‐CR 27%) compared to undisturbed cultivation at pH 7. Afterward, systematic experiments at symmetric and asymmetric pH oscillations, between pH ranges of 4–6 and 8–11 and different intervals from 1 to 20 min, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions of a putative large‐scale bioprocess, which is difficult to conduct using two‐CRs.
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