Fed-batch Research Articles

Deep-learning-assisted medium optimization improves hyaluronic acid production by Streptococcus zooepidemicus.

Deep-learning-assisted medium optimization improves hyaluronic acid production by Streptococcus zooepidemicus.

Production of multi-subunit proteins in CHO cells by transposase-mediated integration of subunit-splitting vectors

The Chinese hamster ovary (CHO) cell line is a widely employed system for producing therapeutic proteins. In our previous study, we established an efficient method for generating stable cell lines by utilizing the Tol2 transposon system, combined with cycloheximide (CHX) resistance as a selection marker. This DNA-based transposon allows the integration of the gene of interest into various genomic loci within the host cell genome. To further develop this system, we performed gene transfer using single vectors carrying each subunit separately, rather than using dual vector linking subunits in tandem, to express monoclonal antibodies. The resulting cell lines exhibited stable protein production for an extended period of up to 12 weeks, as well as high productivity in fed-batch cultures. We found that the copy numbers of vector construct integrated in the genome varied for different mAbs, suggesting these cell lines maintained the vector constructs at copy numbers for effective gene expression. This study highlights the potential usefulness of the Tol2 transposon system in producing multi-subunit proteins, such as bispecific antibodies and Fc-fusion proteins, thereby promoting advancements in biopharmaceutical production.

Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.

Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.

Establishment of a high-throughput scale-down clone screening platform for intensified fed-batch culture of CHO cells.

To develop a scale-down clone screening platform for the intensified fed-batch (IFB) process to allow efficient identification of high expressing clones fitting the IFB culture strategy in bioreactor. Three monoclonal antibodies (mAbs) were used in the development and validation of the IFB specific clone screening platform for CHO cells. The IFB platform significantly improved titer levels, achieving an average titer of 8g/L and the highest titer of 9.6g/L. With similar cell viability, lactate profile and titer levels, both the spin tube model and the AMBR250@ bioreactor system were effective in screening clones suitable for IFB process. The addition of aurintricarboxylic acid (ATA) and uridine in the process optimization led to a further increase in expression levels in both systems, achieving the highest titer of 12.2g/L. This IFB-process specific clone screening serves as an alternative platform for industry application that can increase the effectiveness and efficiency of screening high-expressing CHO cell lines for IFB production.

Enhanced cell-specific productivity through delayed supplementation of antioxidants in intensified processes.

Antioxidant supplementation to serum-free culture media is a common strategy to enhance productivity through oxidative stress alleviation. In this study, it was hypothesized that certain antioxidants can improve the specific productivity of a CHO-GS cell line expressing a bi-specific antibody. A fed-batch (FB) screening study investigated several antioxidants and revealed rosmarinic acid (RoA) and retinyl acetate (RAc), to a lesser extent, improved cell productivity. Contrary to the previous literature reports, the addition of RoA and/or RAc resulted in slower cell growth and reduced peak viable cell density, counteracting the enhanced specific productivity. We hypothesized that supplementing RoA/RAc after the exponential growth phase would increase titer through enhanced specific productivity without substantially impeding cell growth. This hypothesis was tested in three different ways: (1) supplementing RoA/RAc to the feed, rather than the basal media, in the FB process; (2) implementing the intensified fed-batch (iFB) process mode which started with high seeding VCD, bypassing the exponential cell growth phase; (3) supplementing RoA/RAc to the production phase perfusion media, rather than the growth phase perfusion media, in the perfusion-based continuous manufacturing (CM) process. All three methods were proven effective in titer improvement, which supported the hypothesis. Additionally, RoA/RAc significantly impacted product quality, with variations depending on the process mode and components. Overall, their supplementation led to decreased N-glycan mannose percentage and increased product fragmentation and aggregation. These changes do not fully align with the previous reports, highlighting that the supplementation strategy needs to be evaluated carefully based on cell line and expressed molecule type.

Engineering an Integrated Bioprocess to Produce Human Dental Pulp Stem Cell-Alginate-Based Bone Organoids.

Bone tissue engineering (BTE) emerged as a practical approach to tackle prosthetic industry limitations. We merge aspects from developmental biology, engineering and medicine with the aim to produce fully functional bone tissue. Mesenchymal stem cells have the capability of self-renewal and specific lineage differentiation. Herein lies their potential for BTE. Among MSCs, human dental pulp stem cells have a higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs under specific conditions. In addition, these cells are readily accessible and can be extracted through a subtle extraction procedure. Thus, they garner fewer moral concerns than most MSCs available and embody a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their true value in BTE, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Here, we study the osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel perfusion bioreactor. The system is compared with traditional 3D static and fed-batch culture methodologies. The novel system performed better, producing higher alkaline phosphatase activity, and more homogeneous, dense and functional bone constructs. Additionally, cell constructs produced by the in-house-designed system were richer in mature osteoblast-like and mineralizing osteocyte-like cells. In conclusion, this study reports the development of a novel bioprocess able to produce hDPSC-based bone-like constructs, providing new insights into hDPSCs' therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities.

Recombinant production of a diamine oxidase in Komagataella phaffii and in vitro degradation of histamine in simulated intestinal fluid.

Recombinant production of a diamine oxidase in Komagataella phaffii and in vitro degradation of histamine in simulated intestinal fluid.

Development and Characterization of the NISTCHO Reference Cell Line.

Well-characterized reference materials enable successful collaborations within the scientific community by establishing common reagents for benchmarking studies and reducing the barriers to sharing materials and information. Here, we report the development of NISTCHO, a recombinant Chinese hamster ovary cell line expressing a nonoriginator version of the NISTmAb IgG1. We evaluated candidate clonal cell lines in a fed-batch cell culture model to assess growth and productivity of the cell lines and protein quality attributes of the recombinant IgG produced, which demonstrated suitability of multiple candidates. Selection of a preferred candidate was accomplished through sequencing-based analysis of the transgene integration sites, and a base-pair resolution map of the transgene integration site was developed and verified using PCR-based methods. Lastly, a validation study performed by an independent laboratory confirmed the robustness of the preferred candidate, which has been selected for further development as the NISTCHO reference cell line. Together, these results describe the origin of this new reference material and will serve as the foundation for future interlaboratory studies using the NISTCHO cell line.

Dual-purpose Bacillus subtilis fermentation: enhanced nattokinase production via oxygen-enriched fed-batch cultivation and natto starter preparation from harvested biomass.

Nattokinase (NK) is one of the most important functional components in natto, but its content is low. In this study, the fermentation conditions using Bacillus subtilis JZ08-02 for high-yield NK production were investigated, and the residual bacterial pellets were used to prepare a natto starter. Batch fermentation of NK was conducted using a 5L fermenter, and soybean milk and glucose were used as the substrates. When the stirring speed was increased from 450 to 650 rpm with air supply at 1.0vvm, NK was increased from 4859 ± 142 to 12,294 ± 226IU/mL. When pure oxygen was supplied, 15,013 ± 550IU/mL of NK was obtained. When fed-batch fermentation was conducted, the titer was further elevated to 18,014 ± 112IU/mL, which was increased by about 76% compared with the previous result. The experimental findings revealed that aeration control and nutrient feeding regimens exerted pronounced effects on NK productivity during submerged fermentation. The crude enzyme supernatant was obtained by centrifugation and the precipitate was collected. With optimized protectant, the bacterial pellets were freeze-dried with 90.1% cell survival rate. Using economical and edible feedstocks, this study achieved a significant enhancement in NK fermentation yield via oxygen-enriched fed-batch cultivation. At the same time, a natto starter was prepared as a by-product using the residual cell waste.

Engineering Komagataella phaffii for citric acid production through carbon-conserving supply of acetyl-CoA.

The oxidative formation of AcCoA limits the glycolytic pathway yield (YPGLY) for citric acid due to the NADH overflow and carbon loss as CO2. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid production, utilizing the non-native citric acid producer Komagataella phaffii as an orthogonal test system. The combination of different metabolic engineering strategies enabled K. phaffii to acquire the ability to produce extracellular citric acid. By constructing the GATHCYC in the cytosol and peroxisomes, the intracellular concentration of AcCoA increased. Overexpression of the genes encoding pyruvate carboxylase (PYC2), citrate synthase (CIT2) and citrate exporter protein (cexA) in the peroxisomal AcCoA strains boosted the citric acid production. Thus, the best producer strain reached a citric acid titer of 51.3±0.9gL-1 and a yield of 0.59±0.01gg-1 after 76h of glucose-limited fed-batch cultivation. Our results highlight the potential of using GATHCYC to provide an efficient supply of acetyl-CoA to enhance citric acid production. This approach could be exploited for the production of other AcCoA-derived compounds of industrial relevance in different cell factories.

Efficient production of recombinant human FVII in CHO cells using the piggyBac transposon system.

Efficient production of recombinant human FVII in CHO cells using the piggyBac transposon system.

Temporal Galactose-Manganese Feeding in Fed-Batch and Perfusion Bioreactors Modulates UDP-Galactose Pools for Enhanced mAb Glycosylation Homogeneity.

Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N-linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N-glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N-glycosylation and how mAb glycoform composition responds to real-time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N-glycosylation pathway to modulate N-glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N-GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real-time for bench-scale bioprocesses operated in both fed-batch and perfusion modes to build an understanding of how temporal changes in mAb N-glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose-occupied glycoforms over time under glucose-starved fed-batch cultures. Comparable galactosylation profiles were also observed between fed-batch (nutrient-limited) and perfusion (non-nutrient-limited) bioprocess conditions. In summary, our results demonstrate the utility of real-time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed-batch and perfusion bioprocessing conditions to produce higher-quality biologics.

Flux Sampling Suggests Metabolic Signatures of High Antibody-Producing CHO Cells.

Chinese hamster ovary (CHO) cells remain the industry standard for producing numerous therapeutic proteins, particularly monoclonal antibodies (mAbs). However, achieving higher recombinant protein titers remains an ongoing challenge and a fundamental understanding of the cellular mechanism driving improved bioprocess performance remains elusive. To directly address these challenges and achieve substantial improvements, a more in-depth understanding of cellular function within a bioprocess environment may be required. Over the past decade, significant advancements have been made in the building of genome-scale metabolic models (GEMs) for CHO cells, bridging the gap between high information content 'omics data and the ability to perform in silico phenotypic predictions. Here, time-course transcriptomics has been employed to constrain culture phase-specific GEMs, representing the early exponential, late exponential, and stationary/death phases of CHO cell fed-batch bioreactor culture. Temporal bioprocess data, including metabolite uptake and secretion rates, as well as growth and productivity, has been used to validate flux sampling results. Additionally, high mAb-producing solutions have been identified and the metabolic signatures associated with improved mAb production have been hypothesized. Finally, constraint-based modeling has been utilized to infer specific amino acids, cysteine, histidine, leucine, isoleucine, asparagine, and serine, which could drive increased mAb production and guide optimal media and feed formulations.

Expression and characterization of cold-adapted xylanase Xyl-L in Pichia pastoris for xylooligosaccharide (XOS) preparation

Background Xylan, the second most abundant polysaccharide in plant biomass, requires endoxylanases for its hydrolysis into xylooligosaccharides (XOS). Xylanases have been widely used in industries such as animal feed, bakery, juice production, and paper pulp. Recently, XOS have gained attention for their health benefits, including improved digestion, reduced cholesterol, and antioxidant effects. The cold-adapted GH10 xylanase of Antarctic origin Xyl-L was previously expressed in Escherichia coli, showing promising low-temperature activity. However, Pichia pastoris is currently a preferred host for industrial xylanase production due to its ability to express complex proteins and secrete them into the culture medium. This study explored the expression of Xyl-L in P. pastoris and evaluated its potential for XOS production using common flours as substrates, aiming for applications in the food and nutraceutical industry. Results Comparison between AOX1 (PAOX1) and GAP (PGAP) promoters for recombinant Xyl-L production in P. pastoris showed that the PAOX1 promoter resulted in higher activity per wet-cell weight. Co-transforming PAOX1-Xyl strains with plasmids encoding genes aiding in protein folding (HAC1 or PDI1) did not enhance Xyl-L catalytic activity compared to the parental PAOX1 strain. Thus, PAOX1-Xyl was cultivated in 3 L bioreactors in fed-batch cultures; it is presumed that the enzyme is produced with glycosylations within its structure, given its migration within the SDS-PAGE gels. The produced Xyl-L was purified from the culture supernatant, resulting in peak xylanase activity after 90 h, with specific activity of 5.10 ± 0.21 U/mg, at pH 7.5 and 25∘C, using beechwood xylan. It also showed a Km of 3.5 mg/mL and a kcat of 9.16 s-1. Xyl-L maintained over 80% of relative activity between pH 5.6-8.6 and 37-44∘ C, and was activated by CaCl2 and MgCl2, but inhibited by MnCl2. Xyl-L was tested using several flours (whole wheat, rye, oatmeal and all-purpose) as substrates, where XOS with a polymerization degree (DP) of 2 were obtained from each substrate, whole wheat flour generated XOS with DP 3, and XOS with DP 2, 3 and 4 were produced when beechwood xylan was used as substrate.Conclusions The xylanase Xyl-L was successfully expressed in P. pastoris and proved to be able to degrade various flour substrates, producing XOS with DP ranging from 2 to 4, indicating its potential applications in the nutraceutical and food industries. Further studies must be performed to optimize its production in bioreactors.

N-acetyl-D-mannosamine, a novel additive, effectively reducing high mannose glycosylation of monoclonal antibody without affecting other quality attributes.

N-linked glycosylation stands as a pivotal quality attribute for monoclonal antibodies (mAbs), particularly the high mannose (Man5) variant, which significantly influences the pharmacokinetics of mAbs. Traditional approaches to modulate Man5 have frequently resulted in suboptimal outcomes. In this investigation, we introduced a novel additive, N-acetyl-d-mannosamine (ManNAc), which selectively targeted and reduced Man5 without compromising other product quality attributes (PQAs). The study further examined optimal concentrations and timing for the incorporation of ManNAc in the mAbs expression process utilizing CHO-K1 cells within a fed-batch shaker flask culture mode. In the ManNAc titration experiments, we established groups at concentrations of 5, 10, 15, 20, 40, 60, 80, and 100 mM. The findings revealed a concentration-dependent decrease in Man5, with reductions reaching as low as 2.9% from an initial 8.9%. Importantly, cellular growth, metabolism, and other PQAs remained unaffected. Regarding the timing of ManNAc addition, groups were set for days N-1, 0, 5, and 11. The results indicated that ManNAc addition on Day 11 did not affect Man5 levels, whereas earlier additions proved effective. A full factorial design was employed to assess the interplay between ManNAc concentration and addition timing, revealing no significant interaction. Consequently, it is recommended to administer 20-40 mM ManNAc prior to Day 4. The strategy of introducing 20 mM ManNAc on Day 0 has been successfully implemented across 12 clones, achieving an average Man5 reduction of 46%. Collectively, these findings delineate a novel and efficacious strategy for the Man5 modulation, promising enhanced control over this critical quality attribute in mAbs production.

Secretory and metabolic engineering of squalene in Yarrowia lipolytica.

Secretory and metabolic engineering of squalene in Yarrowia lipolytica.

Screening of Non-Conventional Yeasts on Low-Cost Carbon Sources and Valorization of Mizithra Secondary Cheese Whey for Metabolite Production.

The production of microbial metabolites such as (exo)polysaccharides, lipids, or mannitol through the cultivation of microorganisms on sustainable, low-cost carbon sources is of high interest within the framework of a circular economy. In the current study, two non-extensively studied, non-conventional yeast strains, namely, Cutaneotrichosporon curvatus NRRL YB-775 and Papiliotrema laurentii NRRL Y-3594, were evaluated for their capability to grow on semi-defined lactose-, glycerol-, or glucose-based substrates and produce value-added metabolites. Three different nitrogen-to-carbon ratios (i.e., 20, 80, 160 mol/mol) were tested in shake-flask batch experiments. Pretreated secondary cheese whey (SCW) was used for fed-batch bioreactor cultivation of P. laurentii NRRL Y-3594, under nitrogen limitation. Based on the screening results, both strains can grow on low-cost substrates, yielding high concentrations of microbial biomass (>20 g/L) under nitrogen-excess conditions, with polysaccharides comprising the predominant component (>40%, w/w, of dry biomass). Glucose- and glycerol-based cultures of C. curvatus promote the secretion of mannitol (13.0 g/L in the case of glucose, under nitrogen-limited conditions). The lipids (maximum 2.2 g/L) produced by both strains were rich in oleic acid (≥40%, w/w) and could potentially be utilized to produce second-generation biodiesel. SCW was nutritionally sufficient to grow P. laurentii strain, resulting in exopolysaccharides secretion (25.6 g/L), along with dry biomass (37.9 g/L) and lipid (4.6 g/L) production.

Metabolic engineering of Escherichia coli for enhanced production of p-coumaric acid via L-phenylalanine biosynthesis pathway.

p-Coumaric acid (p-CA), an invaluable phytochemical, has novel bioactivities, including antiproliferative, anxiolytic, and neuroprotective effects, and is the main precursor of various flavonoids, such as caffeic acid, naringenin, and resveratrol. Herein, we report the engineering of Escherichia coli for de novo production of p-CA via the PAL-C4H pathway. As the base strain, we used the E. coli H-02 strain, which was previously engineered for sufficient supplementation of L-phenylalanine, the main precursor of p-CA. For the bioconversion of L-Phe to p-CA, we constructed and optimized an expression system for phenylalanine ammonia lyase (SmPAL), codon-optimized cinnamate 4-hydroxylase (AtC4H), and its redox partner, cytochrome P450 reductase (AtCPR1). We confirmed that the engineered cell showed higher production of p-CA at 30°C and the addition of 0.5mM 5-aminolevulinic acid could increase the production titer further. Subsequently, the main pathways of acetic acid (poxB and pta-ackA) were eliminated to reduce its accumulation and restore cell growth. Next, to increase the available pool of cofactor (NADPH), the co-expression system of the zwf gene in the pentose phosphate pathway (PPP) was integrated into genome and the expression level was optimized with synthetic promoters. Finally, by optimizing fed-batch culture in a 5 L-scale bioreactor, the engineered strain achieved 1.5g/L p-CA with a productivity of 31.8mg/L/h.

Dual Antibiotic-Free Plasmid Systems Enable High-Efficiency l-Fucose Biosynthesis.

l-Fucose, a functional monosaccharide with significant commercial potential in the pharmaceutical, nutraceutical, and cosmetic industries, faces challenges in microbial production due to antibiotic-dependent plasmid maintenance systems. This study presents a dual antibiotic-free plasmid strategy in engineered Escherichia coli BL21(DE3) to achieve high-efficiency l-fucose biosynthesis. By integration of the hok/sok toxin-antitoxin system and a cysC-based auxotrophic selection into two plasmids, genetic stability and plasmid retention were ensured without antibiotics. Metabolic pathway optimization involved enhancing GDP-l-fucose supply via promoter replacements, genomic integration of key enzymes (α1,2-fucosyltransferase and α-l-fucosidase), and blocking l-fucose degradation. The engineered strain demonstrated robust performance, producing 7.99 g/L of l-fucose in shake-flask fermentation and 61.91 g/L via fed-batch cultivation─both antibiotic-free. This titer represents the highest reported l-fucose yield to date, highlighting the effectiveness of combining toxin-antitoxin and auxotrophic systems for sustainable, high-productivity microbial manufacturing.

Upcycling depolymerized PET waste into polyhydroxyalkanoates and triacylglycerols by a newly isolated Rhodococcus sp. strain

The use of post-consumer polyethylene terephthalate (PET) wastes, which often contain various additives and contaminants such as metals and pigments that make mechanical recycling and reusability difficult, as feedstocks for microbial synthesis of value-added bio-based products is an emerging sustainable strategy for managing such wastes. This study evaluated the ability of a strain isolated from a plastic-contaminated site, Rhodococcus sp. isolate Ave7, to use terephthalic acid (TPA) obtained by chemically depolymerizing PET waste, as sole feedstock for cell growth and production of polyhydroxyalkanoates (PHAs) and triacylglycerols (TAGs) as intracellular storage compounds. The fed-batch bioreactor cultivation resulted in a cell dry weight production of 3.85 g/L, with PHA and TAG contents of 15.0 wt.% and 15.4 wt.%, respectively. Overall, the culture consumed 16.5 g/L TPA over a period of 73 h. The produced PHA was mainly composed of 3-hydroxyvalerate (3HV) monomers (> 90 wt.%). The accumulated TAGs presented a fatty acids profile rich in octadecenoic acid (C18:1; 52 wt.%), hexadecanoic acid (C16:0; 32 wt.%) and octadecanoic acid (C18:0; 12 wt.%). Overall, the strain Rhodococcus sp. Ave7 demonstrated a high capacity for TPA removal, converting it into cell biomass, PHA and TAGs, thus rendering this bioprocess a promising solution to reduce the plastic waste burden, in a circular and sustainable approach.