Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Evaluation of single-use disk stack continuous centrifuge to harvest monoclonal antibody from cell culture fluid.
Xu A, Gong L, Deng C … +8 more , Sheng W, Hua C, Lu B, Li C, Ma J, Zhou J, Zhang X, Cui Y
Biotechnol Prog · 2026 · PMID 41131805 · Publisher ↗
Various technologies, including precipitation, flocculation, depth filtration, microfiltration, and centrifugation, have been developed to clarify mammalian cell culture fluids. For processing volumes between 2000 and 50...
Various technologies, including precipitation, flocculation, depth filtration, microfiltration, and centrifugation, have been developed to clarify mammalian cell culture fluids. For processing volumes between 2000 and 5000 L, continuous centrifugation followed by depth filtration is the preferred method. This process starts with the removal of cells and large debris through continuous centrifugation, followed by the filtration of small debris and some impurities. The newly introduced single-use centrifuge, designed to prevent cross-contamination and mimic traditional continuous centrifuges, was evaluated for its performance, particularly focusing on its impact on cell lysis and subsequent filtration and purification processes. The single-use centrifuge showed better performance in reducing turbidity and lactate dehydrogenase levels (LDH) in the supernatant, indicating less cell lysis compared to the conventional centrifuge. A separation load factor range of 0.91-2.73 was identified as optimal for balancing centrifugation throughput and product quality. Both centrifuge types had a comparable impact on the performance of subsequent depth filtration, supporting a load capacity of at least 100 L/m. No significant differences in product quality, including SE-HPLC, NR/R CE-SDS, icIEF, HCP, and rDNA, were observed between the conventional and single-use centrifuges. These harvest strategies did not affect the subsequent purification steps. For volumes up to 5000 L, both centrifuge types are viable; however, for larger volumes, the conventional centrifuge is necessary due to the scale limitations of the single-use centrifuge.
Correction to "Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling".
Biotechnol Prog · 2025 · PMID 41131719 · Publisher ↗
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Optimizing monoclonal antibody biosimilar production via transfer and active learning for targeted quality profiles.
Kumar J, Sultana R, Saripalla D … +3 more , Chopda V, Mahalingam V, Adhikary L
Biotechnol Prog · 2026 · PMID 41131713 · Publisher ↗
Biosimilar development of monoclonal antibodies (mAbs) is gaining significant momentum as numerous blockbuster biologics approach their patent expiry in the current decade. A critical challenge in biosimilar development...
Biosimilar development of monoclonal antibodies (mAbs) is gaining significant momentum as numerous blockbuster biologics approach their patent expiry in the current decade. A critical challenge in biosimilar development lies in achieving product quality attributes(PQAs) comparable to the innovator product. PQAs in upstream processing are influenced by multiple factors, including cell line selection, media composition, feeding strategy, supplements, and bioreactor process parameters, with physical parameter optimization playing a pivotal role in enhancing both product titer and modulating PQAs. In this study, we systematically evaluated the impact of physical process parameters-pH and temperature along with initial seeding density (ISD)-on N-glycan profiles and charge variants across four biosimilar development projects (Projects 1-4). Stepwise regression models were developed between process parameters and product quality attributes using JMP software to establish parameter-attribute relationships. Our results demonstrated that lowering culture pH reduced %acidic variants and %galactosylation while increasing %basic variants and %afucosylation (AF). Increased culture temperature resulted in an increase in %acidic variants and a decrease in %AF. This parameter-attribute relationships knowledge base was directly applied in experimental design to expedite the development of a fifth mAb biosimilar development (Project 5), substantially reducing experimental iterations and development timelines, exemplifying the practical implementation of Bioprocessing 4.0 principles.
Dynamic pH profiles drive higher cell-specific and volumetric productivity.
Klaubert SR, Chitwood DG, Peng D … +4 more , Redman E, Anderson JYL, Sandoval NR, Harcum SW
Biotechnol Prog · 2026 · PMID 41115826 · Full text
Mammalian cell cultures in bioreactors rely heavily on critical process parameter control to ensure optimal growth, productivity, and reproducibility to produce recombinant therapeutic proteins. Culture pH has been shown...
Mammalian cell cultures in bioreactors rely heavily on critical process parameter control to ensure optimal growth, productivity, and reproducibility to produce recombinant therapeutic proteins. Culture pH has been shown to be a critical parameter that influences growth, productivity, and critical quality attributes. Typically, pH is either controlled to a set-point throughout the culture or uses a single pH shift to achieve higher productivity and more desirable charge variant profiles. The pH is usually maintained by CO and base additions. For CO controlled cultures, using a set-point can result in an accumulation of CO, which has detrimental effects on mammalian cell growth and protein production. In this study, a dynamic pH profile was implemented that allowed the pH control in the bioreactor to mimic the natural uncontrolled pH profile observed in shake flask cultures. This dynamic pH profile employs multiple pH shifts during the exponential phase of a single IgG producing CHO-K1 cell line. The results show that a dynamic pH profile was able to successfully alleviate CO accumulation and increase the cell-specific, as well as overall culture productivity. Impacts of the dynamic pH profile on product quality attributes, including glycosylation and charge variants, were also evaluated, showing mixed impacts on the glycosylation pattern and a positive impact on charge variants. Since the ideal glycosylation pattern is highly dependent on the intended function of the recombinant antibody, impacts on product quality should be evaluated on a "per process" basis.
Optimization of amino acid composition in CHO cell perfusion medium using definitive screening design and H NMR-based consumption profiling.
Xu S, Wan Y, Shi Y … +5 more , Jiao J, Gao D, Chen Z, Wang H, Qu H
Biotechnol Prog · 2026 · PMID 41099189 · Publisher ↗
Perfusion culture is acknowledged as a promising platform for sustained high-density cell production, while concurrently necessitating stringent control over medium nutrient composition. A multi-component medium optimiza...
Perfusion culture is acknowledged as a promising platform for sustained high-density cell production, while concurrently necessitating stringent control over medium nutrient composition. A multi-component medium optimization strategy has been developed in this study, integrating the targeted feeding approach (TAFE), H nuclear magnetic resonance (H NMR) analysis, and definitive screening design (DSD). Nine pivotal amino acids were selected through quantitative profiling of cellular uptake kinetics and literature evidence. Their concentrations were optimized using a DSD within only 24 experimental runs. The optimized formulation was demonstrated to maintain stable cell density, high viability (>97%), and excellent monoclonal antibody production in both shake flask semi-perfusion (38.63 pg/cell/day) and 3L bioreactor systems (45-61.5 pg/cell/day), while significantly reducing the accumulation of lactate and ammonium. These results suggest that the proposed strategy can effectively enhance both productivity and metabolic stability, offering excellent scalability and engineering applicability. This work provides a novel and efficient pathway for the development of perfusion culture media in biopharmaceutical manufacturing.
Enzymatic properties of a bacterial microcystinase A produced in Saccharomyces cerevisiae.
Silva FG, Greiner R, Lopes DD … +3 more , Hector RE, Hashimoto EH, Hirooka EY
Biotechnol Prog · 2026 · PMID 41099154 · Publisher ↗
Microcystins (MCs) are toxins produced by cyanobacteria, posing a significant emerging threat to human and public health. Therefore, control strategies combining frequent toxin monitoring with removal techniques are urge...
Microcystins (MCs) are toxins produced by cyanobacteria, posing a significant emerging threat to human and public health. Therefore, control strategies combining frequent toxin monitoring with removal techniques are urgently needed. In this context, microcystin degradation using the bacterial enzyme microcystinase A, originally derived from Sphingosinicella microcystinivorans B9, has been identified as a sustainable and effective approach. To facilitate access to the enzyme, the gene encoding microcystinase A was successfully expressed in the Saccharomyces cerevisiae PE-2 strain. The recombinant microcystinase A was produced as an intracellular enzyme and applied in MC degradation assays. Optimal conditions for enzymatic activity were identified at 42.2°C and pH 6.3. The maximum degradation rate of microcystin was determined to be 3.09 mg/L/h, and a K of 2.81 μM was obtained when assays were performed at 37°C and pH 7.4. The recombinant microcystinase A remained fully active for 2 h at 20°C. Exposure to 50°C for 1 h resulted in 60% residual activity, while 30 min at 65°C led to complete inactivation. The enzyme was also denatured when exposed to alkaline pH conditions. Therefore, this study provides key data on recombinant microcystinase A, supporting further investigations into its potential applications for MC degradation, particularly under mildly acidic conditions and temperatures up to 45°C.
Identification of the differential and synergic lipotoxic patterns of oleic acid, palmitic acid, and their mixture in 3D HepG2/C3A tissue using liver-on-chip technology.
Morisseau L, Pawlowski V, Plaisance V … +6 more , Lucas M, Legallais C, Sakai Y, Abderrahmani A, Jellali R, Leclerc E
Biotechnol Prog · 2026 · PMID 41078319 · Publisher ↗
The metabolic dysfunction-associated steatotic liver disease (MASLD, previously formerly known as non-alcoholic fatty liver disease, NAFLD) is rapidly expanding worldwide in parrallel with the obesity pandemic. Dietary f...
The metabolic dysfunction-associated steatotic liver disease (MASLD, previously formerly known as non-alcoholic fatty liver disease, NAFLD) is rapidly expanding worldwide in parrallel with the obesity pandemic. Dietary fatty acids including oleic (OA) and palmitic acids (PA) contribute to the hepatic intracellular triglyceride accumulation, and are therefore thought to play key roles in disease development and progression. Taking advantage of the cutting-edge organ-on-chip technology that mimics the 3D organ dynamic environment, we aimed at investigating the role of OA, PA and a 2:1 OA/PA mixture on the growth and function of the HepG2/C3A, a liver cell line model, over 2 and 7 days. OA supported sustained cell growth, leading to dense 3D tissues, whereas PA and OA exposure did not affect cell proliferation. PA treatment downregulated the GLUT2, INSRA, SREBP1, FASN, mRNA levels indicating a lipid metabolism perturbation in our model. The cell dysfunction caused by OA, PA, and OA/PA was associated with an increase in reactive oxygen species (ROS) production over time. Intracellular lipid monitored by oil red O was higher in cells exposed to OA than in the control ones and cells cultured with PA. Our data confirm the role of fatty acids on the growth and dysfunction of HepG2/C3A cells, and highlight distinct mechanisms through which OA and PA exert their effects.
Enhanced deoxyviolacein bioproduction via enzyme condensation with a yeast glycolytic enzyme-derived peptide tag.
Ito R, Ito-Harashima S, Segawa N … +2 more , Miura N, Kataoka M
Biotechnol Prog · 2026 · PMID 41075295 · Full text
In microbial production, regulating endogenous and exogenous synthetic pathways is essential. Techniques to induce intracellular enzyme condensation have attracted attention as a means to increase apparent enzyme activit...
In microbial production, regulating endogenous and exogenous synthetic pathways is essential. Techniques to induce intracellular enzyme condensation have attracted attention as a means to increase apparent enzyme activity with minimal transcriptional and translational burden on the cell. Artificial enzyme condensation can be induced by tagging enzymes with specific proteins or peptides. In our previous study, we identified novel peptide tags derived from condensate-forming Saccharomyces cerevisiae glycolytic enzymes and their potential use in controlling intracellular metabolism by inducing artificial condensates in S. cerevisiae cells. Herein, we evaluated the condensate formation of two enzymes using peptide tags and the branched violacein biosynthetic pathway from Pseudoalteromonas luteoviolacea in S. cerevisiae, and tested the effects of modulating biosynthesis to increase the production of deoxyviolacein, a byproduct with antibacterial and anticancer activities. We used several protein and peptide tags with a simplified expression system, and all the tags successfully induced artificial condensate formation in the cell. Additionally, introducing a short peptide tag successfully increased deoxyviolacein production by approximately twofold, displaying a higher efficacy compared to FUS, a previously reported N-terminal 213 amino acid region with an intrinsically disordered property. These results demonstrate the potential use of peptide tags to enhance bioproduction through the regulation of endogenous and exogenous synthetic pathways. The methods contribute to the development of novel strategies for microorganisms to be used for bioproduction through the controlled condensation of metabolic enzymes in cells.
Automation of an integrated micro-scale platform for monoclonal antibody process development by incorporation of a depth filter mimic.
Sharma P, Sebastian P, Robbel L … +2 more , Schmitt M, Bracewell DG
Biotechnol Prog · 2026 · PMID 41041729 · Full text
High throughput process development (HTPD) has been widely adopted for efficient development and optimization of chromatographic operations in monoclonal antibody (mAb) purification. However, the integration of non-chrom...
High throughput process development (HTPD) has been widely adopted for efficient development and optimization of chromatographic operations in monoclonal antibody (mAb) purification. However, the integration of non-chromatographic unit operations, particularly depth filtration following protein A chromatography, which is essential for the removal of process- and product-related impurities prior to the ion exchange chromatography (IEX) operations, remains a challenge due to the absence of commercially available micro-scale depth filtration tools. This limits the integration of this unit operation within the purification sequence, restricting the analysis of process interactions and overall process understanding. In this study, a micro-scale HTPD platform was designed and evaluated to enable integration of a depth filtration mimic, Sartobind® Q anion exchange adsorber, within a mAb purification sequence. This was achieved by translating laboratory-scale protocols to the micro-scale using workflow design tools and executed on an automated liquid handling system. Step yields and impurity clearance were assessed to confirm the equivalence of scale-down. The Sartobind® Q membrane achieved effective removal of host cell DNA (hcDNA), while subsequent IEX operations removed host cell proteins (HCPs) and high molecular weight components (HMWC), meeting target product quality specifications. The platform demonstrated robustness across varying impurity profiles, supporting its applicability for diverse process intermediates. Comparative analysis with laboratory-scale operations confirmed the performance and scalability of the micro-scale system, reducing the total run time by greater than 50%. The integrated HTPD platform offers a resource-efficient, scalable approach for comprehensive mAb purification process development and is suitable for developability assessments during early-stage development.
Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity.
Mazzei A, Martewicz S, Amiri R … +3 more , Cui M, Elvassore N, Luni C
Biotechnol Prog · 2026 · PMID 41035413 · Full text
Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization,...
Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell-specific and context-specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. We show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.
Scale-up of a monoclonal antibody CHO fed-batch production in stirred tank bioreactors: Effect of hydrodynamic conditions and feeding regimen.
Lemire L, Reyes SJ, Durocher Y … +3 more , Voyer R, Henry O, Pham PL
Biotechnol Prog · 2026 · PMID 41020541 · Full text
Key hydrodynamic-related parameters such as volumetric power input (P/V), impeller configuration, aeration strategy, and maximum gas sparge rate, as well as an appropriate feeding strategy, must be carefully selected to...
Key hydrodynamic-related parameters such as volumetric power input (P/V), impeller configuration, aeration strategy, and maximum gas sparge rate, as well as an appropriate feeding strategy, must be carefully selected to improve production yields in bioreactor. In this study, the feeding regimen was found to have an important impact on cell growth and productivity of a cumate-inducible CHO fed-batch cell culture. A low-volume feeding regimen avoided a rapid increase in osmolality, allowing for prolonged cell viability and a 33% increase in volumetric titer compared to the high-volume feeding regimen. Both sparged air and oxygen were used for dissolved oxygen (DO) control, utilizing three levels of airflow rates. An optimum airflow rate of 0.0031 vvm was found to improve cell growth, longevity, and thus final titer. A larger air cap required increased gas flow rates, which led to an earlier cell mortality. Scale-up from 1-L to 10-L bioreactor using constant P/V and air cap volumetric gas flow rate (vvm) allowed for comparable cell growth and productivity. Further investigation of the effect of mixing and aeration was done by maintaining P/V and vvm constant throughout the cell culture, which further improved product titers at 11 days after induction. Our study also demonstrates that keeping a constant volume by removing a culture amount equal to the feed volume added at each sampling event can significantly improve the final volumetric titer. This finding shows the benefit of developing a concentrated feed to reduce the volume increase, which in turn could greatly ease the scale-up task.
Quantitative variation in the camptothecin produced by diverse endophytic microorganisms of Ophiorrhiza mungos L.
Theresa M, Pradeep AN, Ravi A … +9 more , Mathew M, Premnath M, Aravindakumar CT, Appukuttan R, Nair IC, Mathew L, Bender SF, van der Heijden MGA, Krishnankutty RE
Biotechnol Prog · 2026 · PMID 41014254 · Publisher ↗
Endophytic microorganisms (EMs) residing in medicinal plants form a promising resource of anticancer compounds such as camptothecin (CPT). Given the increasing therapeutic demand for CPT, its sustainable production is of...
Endophytic microorganisms (EMs) residing in medicinal plants form a promising resource of anticancer compounds such as camptothecin (CPT). Given the increasing therapeutic demand for CPT, its sustainable production is of high significance. This study has investigated the EMs isolated from different parts of Ophiorrhiza mungos for the CPT biosynthetic potential. Preliminary screening of EMs for the CPT synthesis was carried out by HPLC analysis of culture extracts, and the HPLC-positive extracts were further confirmed via LC-MS/MS. From a total of 175 EMs screened in the study, 17 strains (14 bacterial and 3 fungal) were found to be CPT producing, with most of them being sourced from the root tissues. Among the bacterial strains, Alcaligenes faecalis subsp. phenolicus S18 exhibited the highest CPT yield (1294.52 μg/L) followed by Bacillus tequilensis (309.02 μg/L). From the fungal strains, Aspergillus sp., S109, S42, and S111 yielded CPT of 22.07, 18.98, and 13.26 μg/L, respectively. Overall, CPT yield among the bacterial producers ranged from 1294.52 to 5.16 μg/L, predominantly from the Bacillus, Acinetobacter, Alcaligenes, and Pseudomonas genera. This study provides the first report on the CPT production by A. faecalis and Aspergillus sp. isolated from O. mungos, and also the first documentation of CPT synthesis in Stenotrophomonas, Fictibacillus, Acinetobacter, and Pseudomonas genera. These findings highlight the potential of novel microbial sources as high-yielding, reliable, and cost-effective alternatives to support commercial CPT production.
Secondary feed filtration and storage conditions influence trace element availability and process performance at 2000 L scale.
Jain AR, Reyes JS, Yuan C … +5 more , Zhao Q, Sun R, Zhou H, Sisodiya V, Rajendra Y
Biotechnol Prog · 2026 · PMID 40996342 · Publisher ↗
Achieving consistent CHO cell culture performance during process scale-up is critical but often challenged by subtle changes in operational parameters. This study investigates how differences in feed media filtration and...
Achieving consistent CHO cell culture performance during process scale-up is critical but often challenged by subtle changes in operational parameters. This study investigates how differences in feed media filtration and storage during scale-up can impact CHO cell culture performance. A 70% reduction in titer and a 25% drop in peak viable cell density (VCD) were observed at 2000 L scale. Root cause analysis revealed that the secondary filtration of feed media was likely a contributing factor. Trace element analysis confirmed significant copper(II) ions (Cu) loss in feed media at 2000 L, likely due to precipitation during storage and subsequent removal by secondary sterile filtration. This resulted in continued lactate accumulation and reduced titer. Feed storage conditions had an impact on Cu stability, with room temperature storage accelerating Cu loss when compared to storage at 2 to 8°C. By eliminating the secondary filtration step and optimizing feed media storage conditions, process performance was successfully restored at 2000 L scale, matching smaller scale performance. This study highlights how feed filtration and storage critically affect micronutrient stability and availability during scale-up. While secondary filtration may be used for additional microbial control, it can inadvertently alter feed composition, affecting cell metabolism and productivity. Thorough evaluation of feed stability, filtration, and storage strategies is therefore key to ensuring consistent bioreactor performance across scales.
An economically cost-effective production medium optimization of Bacillus subtilis Ö-4-68: A potential probiotic for aquaculture.
Demirhan-Yazıcı S, Karaca K, Nalbantsoy A … +1 more , Eltem R
Biotechnol Prog · 2026 · PMID 40990158 · Publisher ↗
Probiotic use has become more important in aquaculture for healthy and sustainable output. In particular, Bacillus spp. have emerged as effective probiotic agents, improving gut health, enhancing the immune system, promo...
Probiotic use has become more important in aquaculture for healthy and sustainable output. In particular, Bacillus spp. have emerged as effective probiotic agents, improving gut health, enhancing the immune system, promoting growth, and providing protection against pathogens in fish. Therefore, the application of Bacillus in aquaculture offers a strategic approach to increasing productivity while reducing the reliance on antibiotics. In this study, the antibacterial activities of Bacillus isolates, whose probiotic properties will be determined, against test bacteria that are fish pathogens such as Aeromonas hydrophila, Vibrio anguillarum, Lactococcus garvieae, and Yersinia ruckeri were determined by using cross-streak method and agar well diffusion methods. Then, antibiotic resistances of 75 isolates determined to have antibacterial activity were screened against 9 different antibiotics by the agar disc diffusion method. Gastric juice (pH 2.5) tolerance of 55 isolates determined to be sensitive to antibiotics was examined, and the tolerance of 13 isolates to gastric juice was determined. Optimum growth characteristics at acidic pH, surface hydrophobicity, bile tolerance, and protease, amylase, lipase, and cellulase activities, hemolytic activities, coagulase activities, bacterial adhesion abilities, and biofilm production properties of these isolates were determined. As a result, Bacillus subtilis Ö-4-68, with the best probiotic properties, was selected from the examined isolates, and production medium optimization was carried out with laboratory scale statistical experiment design (Response Surface Methodology, RSM) for high amount of biomass production. As a result of the trials, an economical cost-effective production medium content with high biomass production was determined.
Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells.
Khan MA, Beyer GJ, Goosby N … +2 more , Ortiz L, Palmer AF
Biotechnol Prog · 2026 · PMID 40970691 · Full text
Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC-encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidat...
Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC-encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto-oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled-up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol-chloroform precipitation and multiple biophysical analyses (SDS-PAGE, SEC-HPLC, MALDI-TOF, and LC-MS/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.
Exploring the design space for Triton X-100 substitutes in viral inactivation applications.
Du Y, Wu S
Biotechnol Prog · 2026 · PMID 40898976 · Publisher ↗
The urgent need to replace the European-prohibited Triton X-100 in biomanufacturing has been hindered by insufficient data on alternative detergents' minimum effective concentrations (MECs) and process robustness in vira...
The urgent need to replace the European-prohibited Triton X-100 in biomanufacturing has been hindered by insufficient data on alternative detergents' minimum effective concentrations (MECs) and process robustness in viral inactivation. This study makes systematic research including: (1) Establishment of MECs for novel Triton X-100 substitutes (TXR-1/VIS/13-S9/C16) achieving effective inactivation of Xenotropic murine leukemia virus and Pseudorabies virus (log reduction factor >4) across diverse CHO harvest fluids; (2) Demonstration of broad-spectrum efficacy against various viruses, with TXR-1/VIS/13-S9 maintaining effective inactivation for Bovine viral diarrhea virus, Vesicular stomatitis virus, Baculovirus, and Herpes simplex virus type 1; (3) Identification of PS20's material-dependent inactivation dynamics, establishing standalone parameters (4 h at 37°C) that achieve equivalent viral inactivation to traditional tri(n-butyl)phosphate -combined methods without requiring lipase activity-a paradigm shift in detergent application. Crucially, process optimization revealed that extending exposure time (1-4 h) enhanced PS20/PS80 efficacy more effectively than two fold concentration increases, providing cost-effective solutions. These findings deliver broader design spaces for implementing eco-friendly detergents while ensuring compliance with EMA/ICH viral safety standards.
Elucidation of cell culture impacts on hydroxylysine levels in monoclonal antibodies using high-throughput analytical quantification and media components.
Kargupta R, Rivera S, Kochert B … +10 more , Devenney K, Donelly D, Atieh T, Li F, Pan J, Patel D, Tayi V, Chauhan G, Chmielowski R, Abbondanzo SJ
Biotechnol Prog · 2025 · PMID 40874408 · Publisher ↗
Hydroxylysine (Hyl) is a post-translational hydroxyl modification of lysine that is not commonly observed at very high levels and thus is not usually considered a product quality attribute (PQA). Post-translation modific...
Hydroxylysine (Hyl) is a post-translational hydroxyl modification of lysine that is not commonly observed at very high levels and thus is not usually considered a product quality attribute (PQA). Post-translation modifications (PTMs) are considered potential PQAs when elevated levels are observed - requiring monitoring and investigation. In a recent monoclonal antibody expression using Media A, Hyl levels were observed at ~20%-35%. At such elevated percentage levels, Hyl was considered a PQA - triggering a root-cause investigation in the upstream activities like cell culture conditions and media components. Initial detection of the Hyl modification originated from non-quantitative, intact mass analysis with confirmation of site-location determined by peptide mapping. Through the root-cause investigation, it was determined that levels of Hyl were underestimated by ~10-fold using tryptic peptide mapping analysis without inclusion of miscleaved peptides. The analytical procedure was revised from trypsin-digestion to IdeS-digestion, a reduced mass analysis, to accurately and rapidly quantify Hyl levels of investigational samples. Proprietary Media B was utilized to reduce the Hyl level by 2-fold to ~10%-15%. Further investigation into the media and feed components determined that increasing concentration of Fe(III) content decreased Hyl levels. Supplementation of Fe(III) served as a robust mitigation strategy of Hyl reduction in upstream process. Media B was used to scale up to a 500 L bioreactor while maintaining the lower Hyl level. The analytical and cell culture methods developed in this study can be leveraged to detect and tune Hyl levels.
Production of recombinant methionine-containing elastin-like polypeptides in a fermenter using ECPM1 medium.
Delhaes A, Bataille L, Médéric M … +2 more , Lecommandoux S, Garanger E
Biotechnol Prog · 2025 · PMID 40842372 · Full text
Elastin-like polypeptides (ELPs) are recombinant protein-like polymers whose macromolecular structure can be precisely controlled through genetic manipulation of their sequence and length. Their lower critical solution t...
Elastin-like polypeptides (ELPs) are recombinant protein-like polymers whose macromolecular structure can be precisely controlled through genetic manipulation of their sequence and length. Their lower critical solution temperature (LCST) phase behavior facilitates purification via chromatography-free techniques and can be explored for self-assembly. As a result, ELPs are extensively investigated for diverse biological, biomedical, and biotechnological applications. So far, ELPs have mostly been isolated from bacteria grown in flasks or fermenters containing complex media that only yield limited amounts of biomass. We herein explored the use of the semi-defined ECPM1 medium, known to limit the accumulation of toxic metabolites and rich in glycerol as a low energy carbon source, to produce ELPs of different chain lengths and containing oxidation-sensitive methionine residues. We report the optimized bioproduction using ECPM1 of ELP[MV-n] with n = 20, 40, 80 in a fermenter in good yields and confirm their intact protein sequence using various chemical characterization techniques.
Integrated approach for purification of uricase and protease from Bacillus licheniformis by TPP and IEC.
Pawar S, Rathod V
Biotechnol Prog · 2025 · PMID 40842303 · Publisher ↗
In order to explore the separation and purification methods of uricase and alkaline protease crude enzyme extracts, a combinatorial approach of Three Phase Partitioning (TPP) and Ion-Exchange Chromatography (IEC) was stu...
In order to explore the separation and purification methods of uricase and alkaline protease crude enzyme extracts, a combinatorial approach of Three Phase Partitioning (TPP) and Ion-Exchange Chromatography (IEC) was studied. TPP alone was able to separate and purify uricase and alkaline protease enzymes by 5 fold and 2.7 fold, respectively. Further application of ion-exchange chromatography purified enzymes with 99.99% purity in the form of single peaks on a chromatogram. The optimum TPP parameters for simultaneous separation and purification were 50% ammonium sulfate concentration, crude extract to solvent ratio of 1:1, and pH of 8.5. Ion exchange chromatography was performed on a fully automated AKTA start system equipped with a conductivity detector, UV detector, fraction collector, and buffer reservoirs used for isocratic or gradient elution profiles of the proteins under study. Successful purification of enzymes, including their molecular weight, was confirmed with SDS PAGE analysis. Furthermore, the uricase sequence from Bacillus licheniformis was also corroborated to be 87.6% homologous to uricase of B. subtilis using the bioinformatics tool BLASTp (Basic Local Alignment Search Tool for Protein), wherein it compares sequence similarity based on protein or nucleotide sequence. It should be emphasized that this study is the first report for tandem enzyme purification of two enzymes using an automated IEC - AKTA start system where partial purification of enzymes was carried out using TPP.
Use of live digital twin (shadow) soft sensor to monitor membrane degradation in continuous manufacturing single pass tangential flow filtration.
Taylor R, Mandur J, Amira U … +10 more , Al-Inati N, Marin-Celis J, Hatch S, Cerezo LF, Pinto N, Metsi-Guckel E, Matos T, Brower M, Mehta K, Sarkar A
Biotechnol Prog · 2025 · PMID 40813261 · Publisher ↗
Digital twins (DT) are sophisticated mathematical models representing real-world physical processes, equipped with predictive capabilities that adapt alongside the physical system. The successful implementation of DT in...
Digital twins (DT) are sophisticated mathematical models representing real-world physical processes, equipped with predictive capabilities that adapt alongside the physical system. The successful implementation of DT in bioprocessing offers numerous advantages, including enhanced understanding of processes, accelerated overall development timelines, and effective monitoring of critical process parameters (CPPs). A comprehensive end-to-end DT can facilitate informed control decisions and forecast how disturbances within the process may affect the final output, accelerating the overall development timelines while optimizing process efficiency and productivity. Tangential flow filtration (TFF) is a standard methodology in bioprocessing, commonly employed to concentrate and exchange buffers for bioproducts. The advancement of continuous process technologies has led to the emergence of alternative TFF methods, notably single-pass tangential flow filtration (SPTFF), which streamlines the process by eliminating the need for stream recirculation. Here, we present the development of a live DT of the SPTFF concentration step within the downstream continuous manufacturing line for a monoclonal antibody (mAb) process. A live DT, equipped with a state estimation tool, was implemented via the Siemens' gPROMS Digital Applications (gDAP) platform. The DT demonstrated the ability to monitor changes in membrane resistance, a typical process parameter that is not directly measured. This parameter is crucial for SPTFF control, as it allows for the constant setting of the concentration factor (CF) by adjusting the retentate flow rate based on the measured resistance and calculated transmembrane pressure (TMP). This achievement illustrates the potential of DT as effective tools for accurately tracking the complete state of the bioprocess.
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