Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Just passing through: Deploying aquaporins in microbial cell factories.
Jenkins Sánchez LR, Sips LM, Van Bogaert INA
Biotechnol Prog · 2024 · PMID 39051848 · Publisher ↗
As microbial membranes are naturally impermeable to even the smallest biomolecules, transporter proteins are physiologically essential for normal cell functioning. This makes transporters a key target area for engineerin...
As microbial membranes are naturally impermeable to even the smallest biomolecules, transporter proteins are physiologically essential for normal cell functioning. This makes transporters a key target area for engineering enhanced cell factories. As part of the wider cellular transportome, aquaporins (AQPs) are responsible for transporting small polar solutes, encompassing many compounds which are of great interest for industrial biotechnology, including cell feedstocks, numerous commercially relevant polyols and even weak organic acids. In this review, examples of cell factory engineering by targeting AQPs are presented. These AQP modifications aid in redirecting carbon fluxes and boosting bioconversions either by enhanced feedstock uptake, improved intermediate retention, increasing product export into the media or superior cell viability against stressors with applications in both bacterial and yeast production platforms. Additionally, the future potential for AQP deployment and targeting is discussed, showcasing hurdles and considerations of this strategy as well as recent advances and future directions in the field. By leveraging the natural diversity of AQPs and breakthroughs in channel protein engineering, these transporters are poised to be promising tools capable of enhancing a wide variety of biotechnological processes.
Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches.
Lenser M, Ngo HG, Sarrafha L … +1 more , Rajendra Y
Biotechnol Prog · 2025 · PMID 39016635 · Publisher ↗
Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ov...
Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.
Proteomics-based method to comprehensively model the removal of host cell protein impurities.
Disela R, Keulen D, Fotou E … +5 more , Neijenhuis T, Le Bussy O, Geldhof G, Pabst M, Ottens M
Biotechnol Prog · 2024 · PMID 39016609 · Full text
Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell prote...
Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.
A comparison of different intensified upstream processes highlighting the advantage of WuXi Biologics' Ultra-high Productivity platform (WuXiUP) in improved product quality and purification yield.
Zheng X, Fang M, Zou Y … +3 more , Wang S, Zhou W, Zhou H
Biotechnol Prog · 2024 · PMID 38980213 · Publisher ↗
WuXiUP, WuXi Biologics' Ultra-high Productivity platform, is an intensified and integrated continuous bioprocess platform developed for production of various biologics including monoclonal antibodies, fusion proteins, an...
WuXiUP, WuXi Biologics' Ultra-high Productivity platform, is an intensified and integrated continuous bioprocess platform developed for production of various biologics including monoclonal antibodies, fusion proteins, and bispecific antibodies. This process technology platform has manifested its remarkable capability in boosting the volumetric productivity of various biologics and has been implemented for large-scale clinical material productions. In this paper, case studies of the production of different pharmaceutical proteins using two high-producing and intensified culture modes of WuXiUP and the concentrated fed-batch (CFB), as well as the traditional fed-batch (TFB) are discussed from the perspectives of cell growth, productivity, and protein quality. Both WuXiUP and CFB outperformed TFB regarding volumetric productivity. Additionally, distinctive advantages in product quality profiles in the WuXiUP process, such as reduced acidic charge variants and fragmentation, are revealed. Therefore, a simplified downstream purification process with only two chromatographic steps can be developed to deliver the target product at a satisfactory purity and an extremely-high yield.
Rapid total sialic acid monitoring during cell culture process using a machine learning model based soft sensor.
Behdani AM, Zhao Y, Yao G … +7 more , Wasalathanthri D, Hodgman E, Borys M, Li G, Khetan A, Wijesinghe D, Leone A
Biotechnol Prog · 2024 · PMID 38953182 · Publisher ↗
Total sialic acid content (TSA) in biotherapeutic proteins is often a critical quality attribute as it impacts the drug efficacy. Traditional wet chemical assays to quantify TSA in biotherapeutic proteins during cell cul...
Total sialic acid content (TSA) in biotherapeutic proteins is often a critical quality attribute as it impacts the drug efficacy. Traditional wet chemical assays to quantify TSA in biotherapeutic proteins during cell culture typically takes several hours or longer due to the complexity of the assay which involves isolation of sialic acid from the protein of interest, followed by sample preparation and chromatographic based separation for analysis. Here, we developed a machine learning model-based technology to rapidly predict TSA during cell culture by using typically measured process parameters. The technology features a user interface, where the users only have to upload cell culture process parameters as input variables and TSA values are instantly displayed on a dashboard platform based on the model predictions. In this study, multiple machine learning algorithms were assessed on our dataset, with the Random Forest model being identified as the most promising model. Feature importance analysis from the Random Forest model revealed that attributes like viable cell density (VCD), glutamate, ammonium, phosphate, and basal medium type are critical for predictions. Notably, while the model demonstrated strong predictability by Day 14 of observation, challenges remain in forecasting TSA values at the edges of the calibration range. This research not only emphasizes the transformative power of machine learning and soft sensors in bioprocessing but also introduces a rapid and efficient tool for sialic acid prediction, signaling significant advancements in bioprocessing. Future endeavors may focus on data augmentation to further enhance model precision and exploration of process control capabilities.
Biotechnological potential of yeast cell wall: An overview.
Jofre FM, Queiroz SS, Sanchez DA … +3 more , Arruda PV, Santos JCD, Felipe MDGA
Biotechnol Prog · 2024 · PMID 38934212 · Publisher ↗
The yeast cell wall is a complex structure whose main function is to protect the cell from physical and chemical damage, providing it with rigidity. It is composed of a matrix of covalently linked polysaccharides and pro...
The yeast cell wall is a complex structure whose main function is to protect the cell from physical and chemical damage, providing it with rigidity. It is composed of a matrix of covalently linked polysaccharides and proteins, including β-glucans, mannoproteins, and chitin, whose proportion can vary according to the yeast species and environmental conditions. The main components of the yeast cell wall have relevant properties that expand the possibilities of use in different industrial sectors, such as pharmaceutical, food, medical, veterinary, and cosmetic. Some applications include bioremediation, enzyme immobilization, animal feed, wine production, and hydrogel production. In the literature it is the description of the cell wall composition of model species like Saccharomyces cerevisiae and Candida albicans, however, it is important to know that this composition can vary according to the species or the culture medium conditions. Thus, understanding the structural composition of different species holds promise as an alternative to expanding the utilization of residual yeast from different bioprocesses. In the context of a circular economy, the conversion of residual yeast into valuable products is an attractive prospect for researchers aiming to develop sustainable technologies. This review provides an overview of yeast cell wall composition and its significance in biotechnological applications, considering prospects to increase the diversification of these compounds in industry.
Modeling of cell cultivation for monoclonal antibody production processes considering lactate metabolic shifts.
Okamura K, Badr S, Ichida Y … +2 more , Yamada A, Sugiyama H
Biotechnol Prog · 2024 · PMID 38924316 · Full text
Demand for monoclonal antibodies (mAbs) is rapidly increasing. To achieve higher productivity, there have been improvements to cell lines, operating modes, media, and cultivation conditions. Representative mathematical m...
Demand for monoclonal antibodies (mAbs) is rapidly increasing. To achieve higher productivity, there have been improvements to cell lines, operating modes, media, and cultivation conditions. Representative mathematical models are needed to narrow down the growing number of process alternatives. Previous studies have proposed mechanistic models to depict cell metabolic shifts (e.g., lactate production to consumption). However, the impacts of variations of some operating conditions have not yet been fully incorporated in such models. This paper offers a new mechanistic model considering variations in dissolved oxygen and glutamine depletion on cell metabolism applied to a novel Chinese hamster ovary (CHO) cell line. Expressions for the specific rates of lactate production, glutamine consumption, and mAb production were formulated for stirred and shaken-tank reactors. A deeper understanding of lactate metabolic shifts was obtained under different combinations of experimental conditions. Lactate consumption was more pronounced in conditions with higher DO and low glutamine concentrations. The model offers mechanistic insights that are useful for designing advanced operation strategies. It can be used in design space generation and process optimization for better productivity and product quality.
Primary clarification of mammalian cell culture fluid using enhanced sedimentation on inclined surfaces inside the single-use disposable Sudhin BioSettler150.
Glascock CB, Kompala DS
Biotechnol Prog · 2024 · PMID 38898736 · Publisher ↗
The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and...
The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h.
Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.
Kanis FC, Broude CN, Hellwarth EB … +5 more , Gibbons WJ, Sen AK, Adams AM, Wang X, Jones JA
Biotechnol Prog · 2024 · PMID 38888046 · Full text
N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for...
N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.
The development of an automated microscope image tracking and analysis system.
McAfee L, Heath Z, Anderson W … +3 more , Hozi M, Orr JW, Kang YA
Biotechnol Prog · 2024 · PMID 38888043 · Publisher ↗
Microscopy image analysis plays a crucial role in understanding cellular behavior and uncovering important insights in various biological and medical research domains. Tracking cells within the time-lapse microscopy imag...
Microscopy image analysis plays a crucial role in understanding cellular behavior and uncovering important insights in various biological and medical research domains. Tracking cells within the time-lapse microscopy images is a fundamental technique that enables the study of cell dynamics, interactions, and migration. While manual cell tracking is possible, it is time-consuming and prone to subjective biases that impact results. In order to solve this issue, we sought to create an automated software solution, named cell analyzer, which is able to track cells within microscopy images with minimal input required from the user. The program of cell analyzer was written in Python utilizing the open source computer vision (OpenCV) library and featured a graphical user interface that makes it easy for users to access. The functions of all codes were verified through closeness, area, centroid, contrast, variance, and cell tracking test. Cell analyzer primarily utilizes image preprocessing and edge detection techniques to isolate cell boundaries for detection and analysis. It uniquely recorded the area, displacement, speed, size, and direction of detected cell objects and visualized the data collected automatically for fast analysis. Our cell analyzer provides an easy-to-use tool through a graphical user interface for tracking cell motion and analyzing quantitative cell images.
Metagenomics analysis yields assembled genomes from prokaryotic anaerobes with polymer-degrading potential.
Blair EM, Brown JL, Li D … +2 more , Holden PA, O'Malley MA
Biotechnol Prog · 2024 · PMID 38881311 · Full text
Anaerobic microbial communities are often highly degradative, such as those found in the herbivore rumen and large-scale anaerobic digesters. Since the microbial communities in these systems degrade recalcitrant organic...
Anaerobic microbial communities are often highly degradative, such as those found in the herbivore rumen and large-scale anaerobic digesters. Since the microbial communities in these systems degrade recalcitrant organic polymers, we hypothesize that some microbes in anaerobic environments may be involved in man-made plastic association, deformation, or even breakdown. While efforts have been put toward characterizing microbial communities, many microbes remain unidentified until they can be sufficiently cultivated to generate enough genetic material to assemble high-quality metagenome assemblies and reference genomes. In this study, microbial consortia from goat fecal pellets and anaerobic digester sludge were cultivated for over 6 weeks to assemble metagenomes from novel anaerobic taxa with potential degradative activity. To select for microbes with potential plastic-degrading abilities, plastic strips were included in culture, though the presence of plastic did not appear to enrich for particularly degradative consortia, yet it did select for novel species that otherwise may not have been characterized. Whole-genome shotgun sequencing enabled assembly of 72 prokaryotic metagenome-assembled genomes (MAGs) with >90% completion, <5% contamination, and an N50 >10,000 bp; 17 of these MAGs are classified as novel species given their lack of similarity to publicly available genomes and MAGs. These 72 MAGs vary in predicted carbohydrate-degrading abilities, with genes predicted to encode fewer than 10 or up to nearly 400 carbohydrate-active enzymes. Overall, this enrichment strategy enables characterization of less abundant MAGs in a community, and the MAGs identified here can be further mined to advance understanding of degradative anaerobic microbial consortia.
High-throughput in silico workflow for optimization and characterization of multimodal chromatographic processes.
Altern SH, Lyall JY, Welsh JP … +5 more , Burgess S, Kumar V, Williams C, Lenhoff AM, Cramer SM
Biotechnol Prog · 2024 · PMID 38856182 · Publisher ↗
While high-throughput (HT) experimentation and mechanistic modeling have long been employed in chromatographic process development, it remains unclear how these techniques should be used in concert within development wor...
While high-throughput (HT) experimentation and mechanistic modeling have long been employed in chromatographic process development, it remains unclear how these techniques should be used in concert within development workflows. In this work, a process development workflow based on HT experiments and mechanistic modeling was constructed. The integration of HT and modeling approaches offers improved workflow efficiency and speed. This high-throughput in silico (HT-IS) workflow was employed to develop a Capto MMC polishing step for mAb aggregate removal. High-throughput batch isotherm data was first generated over a range of mobile phase conditions and a suite of analytics were employed. Parameters for the extended steric mass action (SMA) isotherm were regressed for the multicomponent system. Model validation was performed using the extended SMA isotherm in concert with the general rate model of chromatography using the CADET modeling software. Here, step elution profiles were predicted for eight RoboColumn runs across a range of ionic strength, pH, and load density. Optimized processes were generated through minimization of a complex objective function based on key process metrics. Processes were evaluated at lab-scale using two feedstocks, differing in composition. The results confirmed that both processes obtained high monomer yield (>85%) and removed of aggregate species. Column simulations were then carried out to determine sensitivity to a wide range of process inputs. Elution buffer pH was found to be the most critical process parameter, followed by resin ionic capacity. Overall, this study demonstrated the utility of the HT-IS workflow for rapid process development and characterization.
Correction to "Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide".
Biotechnol Prog · 2024 · PMID 38801176 · Publisher ↗
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Buffer effects on protein sieving losses in ultrafiltration and their relationship to biophysical properties.
Mohammadzadehmarandi A, Zydney AL
Biotechnol Prog · 2024 · PMID 38780204 · Full text
The design of effective ultrafiltration/diafiltration processes for protein formulation requires the use of membranes with very high protein retention. The objective of this study was to examine the effects of specific b...
The design of effective ultrafiltration/diafiltration processes for protein formulation requires the use of membranes with very high protein retention. The objective of this study was to examine the effects of specific buffers on the retention of a model protein (bovine serum albumin) during ultrafiltration. Albumin retention at pH 4.8 was significantly reduced in phosphate buffer compared with that in acetate, citrate, and histidine. This behavior was consistent with a small change in the effective albumin hydrodynamic diameter as determined by dynamic light scattering. The underlying conformational changes leading to this change in diameter were explored using circular dichroism spectroscopy and differential scanning calorimetry. These results provide important insights into the factors controlling protein retention during ultrafiltration and diafiltration.
Effect of culture hydrodynamics on Arthrospira platensis production using a single-use photobioreactor system through a CFD supported approach.
Demirden SF, Erdogan B, Öncel DŞ … +1 more , Oncel SS
Biotechnol Prog · 2024 · PMID 38766884 · Publisher ↗
Laboratory scale conventional single-use bioreactor was used to investigate the effect of different stirrer speeds on the Arthrospira platensis (Spirulina platensis) culture. Experiments were handled in two steps. First...
Laboratory scale conventional single-use bioreactor was used to investigate the effect of different stirrer speeds on the Arthrospira platensis (Spirulina platensis) culture. Experiments were handled in two steps. First step was the selection of the stirring speeds, which was simulated via using CFD, and the second was the long term cultivation with the selected speed. During 10 days of batches as the first step, under identical culture conditions, stirrer speed of 230 rpm gave higher results, compared to 130 and 70 rpm, with respect to dry biomass weight, absorbance value (AB) and chlorophyll-a concentration. Volumetric productivity during the growth phase of the cultures were calculated as 0.39 ± 0.03, 0.28 ± 0.01, and 0.19 ± 0.02 g L d, from the fast to the slower speeds. According to the results a 17 day batch was handled with 230 rpm in order to monitor the effects on the culture. The culture reached a volumetric productivity of 0.33 ± 0.04 g L d. Statistical analysis showed the significance of the parameters related with the stirring speed.
A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.
Wäneskog M, Rasmussen TB, Jensen ED
Biotechnol Prog · 2024 · PMID 38757558 · Full text
Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins...
Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.
Targeted delivery of interleukin-12 plasmid into HepG2 cells through folic acid conjugated graphene oxide nanocarrier.
Safari F, Bardania H, Dehshahri A … +8 more , Hallaj-Nezhadi S, Asfaram A, Mohammadi V, Baneshi M, Bahramianpour S, Akrami N, Khalvati B, Mirzaei A
Biotechnol Prog · 2024 · PMID 38757348 · Publisher ↗
Successful gene therapy relies on carriers to transfer genetic materials with high efficiency and low toxicity in a targeted manner. To enhance targeted cell binding and uptake, we developed and synthesized a new gene de...
Successful gene therapy relies on carriers to transfer genetic materials with high efficiency and low toxicity in a targeted manner. To enhance targeted cell binding and uptake, we developed and synthesized a new gene delivery vector based on graphene oxide (GO) modified by branched polyethyleneimine (BPEI) and folic acid (FA). The GO-PEI-FA nanocarriers exhibit lower toxicity compared to unmodified PEI, as well as having the potential to efficiently condense and protect pDNA. Interestingly, increasing the polymer content in the polyplex formulation improved plasmid transfer ability. Substituting graphene oxide for PEI at an N/P ratio of 10 in the HepG2 and THP1 cell lines improved hIL-12 expression by up to approximately eightfold compared to simple PEI, which is twice as high as GO-PEI-FA in Hek293 at the same N/P ratio. Therefore, the GO-PEI-FA described in this study may serve as a targeting nanocarrier for the delivery of the hIL-12 plasmid into cells overexpressing folic acid receptors, such as those found in hepatocellular carcinoma.
SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.
Ganapathy K, Lam C, Tsukuda J … +6 more , Sargon A, Nava A, Harms P, Shen A, Barnard G, Misaghi S
Biotechnol Prog · 2024 · PMID 38716635 · Publisher ↗
Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalab...
Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.
Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media.
Ou J, Cui W, Zhao Y … +7 more , Tang Y, Williams A, Wasalathanthri D, Xu J, Lee J, Borys MC, Khetan A
Biotechnol Prog · 2024 · PMID 38699906 · Publisher ↗
Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO...
Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40-80°C) and pH (7.6-10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.
Developing tardigrade-inspired material: Track membranes functionalized with Dsup protein for cell-free DNA isolation.
Zarubin M, Andreev E, Kravchenko E … +3 more , Pinaeva U, Nechaev A, Apel P
Biotechnol Prog · 2024 · PMID 38699905 · Publisher ↗
When developing functionalized biomaterials, the proteins from extremophilic organisms, in particular unique tardigrade disordered proteins, are of great value. The damage suppressor protein (Dsup), initially discovered...
When developing functionalized biomaterials, the proteins from extremophilic organisms, in particular unique tardigrade disordered proteins, are of great value. The damage suppressor protein (Dsup), initially discovered in the tardigrade Ramazzottius varieornatus and found to be an efficient DNA protector under oxidative and irradiation stress, has been hypothesized to possess a good potential for the development of the material, which can isolate cell-free DNA. With this in mind, DNA-nonadsorbing polyethylene terephthalate track membranes have been functionalized using the Dsup protein via covalent bonding with glutaraldehyde. The filtration experiments have verified the ability of track membranes with the immobilized Dsup protein to adsorb cell-free DNA, with an accumulation capacity of 70 ± 19 mg m. The resulting track membrane-based biomaterial might be used in various devices for filtration and separation of cell-free DNA molecules from biological solutions and environmental samples, and also for their accumulation, storage, and further manipulation.
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