Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Optimizing clarification processes in biopharmaceutical manufacturing through quality by design: Strategies, implications, and future prospects.
Yeop KW, Nam HJ, Shim CJ … +5 more , Yang SM, Kim HW, Park CI, Banerjee S, Mok Y
Biotechnol Prog · 2025 · PMID 40810245 · Full text
Biopharmaceutical manufacturing processes in which the product of interest is extracellularly expressed typically employ a clarification step following cell culture or fermentation. During clarification, crude cell cultu...
Biopharmaceutical manufacturing processes in which the product of interest is extracellularly expressed typically employ a clarification step following cell culture or fermentation. During clarification, crude cell culture fluid or fermentation broth is processed to remove insoluble solids, cells, debris, and other particulates, with the extracellular product of interest retained in the filtrate. Soluble impurities, such as host cell proteins (HCPs), may also be partially removed. Historically, the clarification process has been considered a limited contributor to Critical Quality Attributes (CQA). As part of upstream harvest, many biopharmaceutical companies have not fully developed quality control strategies from process development to manufacturing, complicating the application of Quality by Design (QbD) principles to this step. However, advancements in upstream and downstream processing (DSP) technologies, alongside increasing cell counts and titers, necessitate reevaluating clarification as a critical process contributing to drug product quality. Conducting controlled studies to define the process and establish parameters using QbD principles can improve control over process impurities and facilitate a logical quality control strategy, integrating quality into the process. This article describes a systematic approach to QbD for a harvest clarification process where the product of interest is extracellular and impurities are removed in the filtrate post-clarification. It highlights methods for optimizing the clarification unit operation using QbD principles, ensuring better process efficiency, and product quality.
Enrichment of full AAV capsids by preparative strong anion exchange chromatography.
Subramanian S, Dembek M, Auchus N … +5 more , Hines A, Devine PWA, Mcwhirter ÅH, Maloisel JL, Linke T
Biotechnol Prog · 2025 · PMID 40808509 · Publisher ↗
Recombinant adeno-associated virus (rAAV) vectors are the leading in vivo gene delivery platform for the treatment of various human diseases. Scalable manufacturing of rAAV has been successfully demonstrated; however, th...
Recombinant adeno-associated virus (rAAV) vectors are the leading in vivo gene delivery platform for the treatment of various human diseases. Scalable manufacturing of rAAV has been successfully demonstrated; however, the presence of non-genome containing empty AAV capsids still remains a significant downstream bottleneck. Separation of empty and full rAAV vectors with linear gradient anion exchange chromatography is challenging to implement at large scale and often achieves only a low recovery of full rAAV capsids. Here we present a workflow to separate empty from full rAAV capsids using Capto Q™ resin with isocratic elution as an alternative. The workflow is based on a preliminary conductivity screening that identifies an optimal empty capsid removal salt concentration, followed by an isocratic two-step elution method. This approach was successfully demonstrated with rAAV serotypes 8 and 9. Approximately 65% of full rAAV8 and rAAV9 capsids were recovered with an enrichment to greater than 80% and 90% full capsids, respectively. Process development using the same approach for rAAV6.2 proved to be more challenging and required a switch in elution salt and an increased concentration of MgCl. The optimized two-step purification protocol for AAV6.2 achieved the recovery of 68% of full capsids with a purity of greater than 80% full capsids.
Evaluation of triply periodic minimal surface geometries in 3D-printed PLA scaffolds for chondrogenic differentiation.
Kılıç MA, Akyürek M, Abidnejad R … +1 more , Karakoç A
Biotechnol Prog · 2025 · PMID 40787904 · Full text
Triply periodic minimal surface (TPMS) scaffolds are gaining attention in tissue engineering due to their continuous and interconnected porous architecture. In this study, three TPMS geometries-Gyroid, Diamond, and I-WP-...
Triply periodic minimal surface (TPMS) scaffolds are gaining attention in tissue engineering due to their continuous and interconnected porous architecture. In this study, three TPMS geometries-Gyroid, Diamond, and I-WP-were fabricated from polylactic acid (PLA) using fused deposition modeling (FDM), with all scaffolds designed to maintain the same overall porosity. Scaffold characterization included scanning electron microscopy (SEM), microcomputed tomography (micro-CT), compressive mechanical testing, and surface wettability analysis. Although porosity was constant, differences in Equivalent Circular Diameter (ECD) values were observed among the geometries, reflecting variations in pore morphology. Adipose-derived stem cells (ADSCs) were seeded onto the scaffolds and cultured under chondrogenic differentiation conditions for 21 days. Cell viability, gene expression (Col2, Col10, Sox9), and protein levels were assessed using RT-PCR and Western blot. All scaffold geometries supported cell attachment and chondrogenic differentiation to varying degrees. The Diamond geometry showed the highest chondrogenic marker expression at the mRNA level, while the Gyroid geometry promoted more stable protein expression with reduced hypertrophic signaling. These findings demonstrate that scaffold geometry, even under identical material and porosity conditions, can influence stem cell behavior. The results offer valuable insights for optimizing TPMS-based scaffold designs in cartilage tissue engineering applications.
Near-infrared spectroscopy coupled with convolutional neural network as a checkpoint tool for cell culture bioprocess media characterization.
Gangwar N, Balraj K, Rathore AS
Biotechnol Prog · 2025 · PMID 40787701 · Publisher ↗
As per the quality by design (QbD) paradigm, manufacturers are expected to identify critical raw materials that can contribute to variability in process performance and product quality. Further, manufacturers should be a...
As per the quality by design (QbD) paradigm, manufacturers are expected to identify critical raw materials that can contribute to variability in process performance and product quality. Further, manufacturers should be able to characterize and monitor the quality of these critical raw materials. Cell culture medium is universally accepted to be one such critical raw material for monoclonal antibody production. It is complex and comprises hundreds of components in varying proportions that are known to impact a multitude of critical quality attributes of a biotherapeutic product, particularly the post-translational modifications. In this study, a near-infrared (NIR) spectroscopy-based quantification method has been developed for media additives that are known to be potential glycan modulators. A one-dimensional convolution neural network (1D-CNN)-based chemometric model has been developed for estimating galactose and uridine concentrations in the various media formulations. Employing the advantage of data augmentation, the proposed 1D-CNN model delivers excellent prediction statistics (test R > 0.9) for predicting both analytes in real time. Further, this model has been used in combination with DoE-based experimental design for prediction of glycosylation using concentrations of media additives as input. In summary, predicted glycosylation distributions were in accordance with actual distribution without significant differences (p > 0.9) in the investigated media formulation. The proposed method and tool can play a critical role in facilitating real-time characterization and control of mammalian cell culture raw materials.
Enhancing rapid Protein A performance in monoclonal antibody processing: Anion exchange chromatographic clarification.
Vail A, Chau D, Heitkamp J … +1 more , Voloshin A
Biotechnol Prog · 2025 · PMID 40772735 · Full text
Clarification fidelity, including reduction of insoluble and soluble contaminants, has been demonstrated to significantly affect the performance and robustness of the Protein A capture chromatography step during the puri...
Clarification fidelity, including reduction of insoluble and soluble contaminants, has been demonstrated to significantly affect the performance and robustness of the Protein A capture chromatography step during the purification of monoclonal antibodies (mAb) and their derivatives expressed in CHO cell cultures. While the vast majority of previous studies have focused on the evaluation of these effects on conventional Protein A resins, in this study, we evaluated such effects on the new membrane- and fiber-based Protein A technologies. Both depth filtration and chromatographic clarification using charged functional fiber approaches have been studied, and we evaluated the effects of these methods on convective Protein A technology cycling robustness, as well as the purity of the product in the elution pool with respect to process-related contaminants. We found that clarification of CHO cell culture using anion exchange (AEX) fiber significantly increases the purity of the mAb in the elution pool with respect to host cell protein (at least 50% less) and DNA (>2 log less) as well as enables a higher number of Protein A cycles (at least 2X increase in fiber-based Protein A cycling lifetime) compared to CHO cell culture fluid clarified with conventional depth filtration. It is likely that this is due to superior DNA and sub-500 nm particle reduction during the chromatographic fiber clarification. This work elucidates the importance of a holistic process strategy when designing a biopharmaceutical purification process.
Methylcellulose has synergistic growth benefits with poloxamer in suspension CHO culture.
Katz JS, Jordan S, Flannery H … +8 more , Rigdon C, Serrano S, Burgett K, Van Bruggen S, Peacock J, Joshi A, Uplekar S, Wolfe L
Biotechnol Prog · 2025 · PMID 40767396 · Publisher ↗
Chemically defined cell culture media used in the growth of mammalian cells for biopharmaceutical applications is a complex mixture of various agents to promote cell growth and function. Poloxamer 188 (P188) is a well-kn...
Chemically defined cell culture media used in the growth of mammalian cells for biopharmaceutical applications is a complex mixture of various agents to promote cell growth and function. Poloxamer 188 (P188) is a well-known shear protectant added to media for use in CHO suspension culture but is not without drawbacks. This work explores the use of methylcellulose (MC), a well-known pharmaceutical polymer, in CHO media as an alternative and/or complementary additive to P188. IgG-producing DG44 CHO cell lines were cultured in a variety of suspension systems, up to 3 L reactors, to which MC and/or P188 were added. MC was an effective shear protectant in relatively lower shear systems but is less effective on its own in higher shear cultures. Across the range of conditions studied, MC and P188 were found to have a synergistic benefit with each other, where the combination of both additives produced cultures with higher viable cell densities than cultures containing either additive alone. These results indicate that MC is a viable option for use in media optimization studies as part of ongoing process intensification and optimization for CHO manufacturing.
Hands-free from inoculation to harvest: Microbial fermentation with multivariate model to automate induction of recombinant protein expression.
Reid J, Szto A, Chen A … +4 more , Gomes P, Kearse C, Ni J, Yuan T
Biotechnol Prog · 2025 · PMID 40765106 · Full text
Industrial fermentation continually improves biological process control for a wide range of microorganisms used in multi-billion-dollar industries including industrial enzymes, pharmaceuticals, foods, beverages, commodit...
Industrial fermentation continually improves biological process control for a wide range of microorganisms used in multi-billion-dollar industries including industrial enzymes, pharmaceuticals, foods, beverages, commodity chemicals, and bioenergy. In the case of recombinant protein production, batch and fed-batch phases of fermentation are usually followed by an induction phase, where chemical or thermal induction initiates the expression of a target protein. Fed-batch processes are usually automated, whereas "out-of-the-box" distributed control systems (DCS) are often unable to define the threshold for induction and respond accordingly. The present study demonstrates the integration of optical density (OD) process analytical technology (PAT) and Lucullus®, a process information management system (PIMS), to enable end-to-end automated fermentation at bench and pilot scale. Data aggregated from tens of fermenter runs and hundreds of offline training measurements enabled the development of an accurate multivariate model to predict OD in real-time. This eliminated the requirement to generate offline correlation models for each OD probe, allowed for model transfer, and incorporated additional predictor terms such as antifoam usage. Automating the induction phase enabled end-to-end fermentation, reducing labor and operational costs while increasing yield through higher reactor utilization within the same time period.
Harvesting AAV by tangential flow filtration using reverse asymmetric membranes.
Hao X, Horax R, Qian X … +3 more , Wheeler A, Shirataki H, Wickramasinghe SR
Biotechnol Prog · 2025 · PMID 40757753 · Full text
Efficient bioreactor clarification for harvesting virus particles is often challenging. Tangential flow filtration is attractive as it can be easily adapted for batch and perfusion operations. Here the feasibility of usi...
Efficient bioreactor clarification for harvesting virus particles is often challenging. Tangential flow filtration is attractive as it can be easily adapted for batch and perfusion operations. Here the feasibility of using reverse asymmetric hollow fiber membranes, where the more open support structure faces the feed stream, has been investigated for harvesting adeno associated virus serotype 2. The open support structure of these membranes stabilizes a secondary membrane consisting of rejected particulate matter. It is essential that the stabilized secondary membrane remains highly permeable. Flux stepping experiments were conducted in total recycle mode in order to determine the critical flux. The critical flux is the maximum stable flux. Higher fluxes lead to a rapid increase in transmembrane pressure under constant flux operation. The critical flux is shown to increase with increasing wall shear rate (feed flow rate). The reduction in turbidity of the permeate relative to the feed decreases with increasing wall shear rate. Harvesting adeno associated virus was conducted at a wall shear rate of 2000 s. The permeate flux was set at 15 Lm h. The feed was concentrated till the transmembrane pressure reached 3.5 kPa. Diafiltration then commenced using 3 diavolumes. While commencing diafiltration with a smaller feed volume will reduce diluent usage and dilution of the product, it is essential that the transmembrane pressure is not too high to create a compacted low permeability secondary membrane. Here the transmembrane pressure was almost constant at 3.5 kPa during diafiltration. Virus recovery was 94%.
Innovations in Dental Implants Integration: Optimizing dental implants performance utilizing stem cells and coatings.
Tsamesidis I, Christodoulou A, Stalika E … +2 more , Pouroutzidou GK, Kontonasaki E
Biotechnol Prog · 2025 · PMID 40750567 · Full text
The last two decades, between 2000 and 2024, significant steps were achieved regarding the interaction between various stem cells and titanium implant surfaces to improve dental implant integration. This literature revie...
The last two decades, between 2000 and 2024, significant steps were achieved regarding the interaction between various stem cells and titanium implant surfaces to improve dental implant integration. This literature review focuses on the potential effects of (i) bone marrow mesenchymal stem cells (BMSCs), (ii) periodontal ligament stem cells (PDLSCs), and (iii) dental follicle stem cells (DFSCs) in promoting osseointegration and tissue regeneration. Studies have shown that combining these stem cells with Ti implants enhances bone formation, accelerates implant osseointegration, and improves long-term implant stability. Additionally, animal models and bioreactors have been employed to evaluate the effects of stem cells on dental implant performance, with some studies showing promising results, although certain models have also yielded inconsistent outcomes. The interaction between stem cells and surface-modified Ti implants has emerged as a key area of research, with results indicating improved healing times and reduced failure rates. This article provides an overview of these findings, highlighting the role of stem cells in not only replacing lost teeth but also actively regenerating the surrounding biological structures for a more integrated and natural outcome.
Accelerating AAV purification process development using high-throughput resin tip module.
Li Q, Hadidi M, Benner S … +1 more , Ma J
Biotechnol Prog · 2025 · PMID 40678841 · Full text
Recombinant adeno-associated viruses (AAVs) with precise genome editing and cell-virus interaction have become a promising delivery tool for gene therapy. A robust AAV purification process is crucial for ensuring therape...
Recombinant adeno-associated viruses (AAVs) with precise genome editing and cell-virus interaction have become a promising delivery tool for gene therapy. A robust AAV purification process is crucial for ensuring therapeutic efficacy. The challenges of AAV purification process development encompass limited material availability during early-stage development, high cost-of-goods compared to traditional biologics, and short development timelines for the critical first-in-human stages. The key to overcoming these challenges is to leverage high throughput (HTP) methods. In this article, an integrated end-to-end HTP workflow is proposed, utilizing a resin tip as the purification module and incorporating an HTP analytical toolkit on one platform. Purification parameters, including binding capacity, resin selection, and buffer composition screening for AAV full/partial/empty capsids separation, are efficiently determined using a 25 μL resin tip and HTP analytical tools with only micro-volume sample requirements. The process parameters determined from the HTP workflow predict the trends of full capsid enrichment and partial capsid removal for the bench-scale purification. This HTP workflow is also applied for the assessment of the AAV quality attributes to accelerate early-stage cell line and cell culture development. Comparable AAV quality attributes are demonstrated to Robocolumn as the benchmark HTP purification method. By leveraging HTP analytical tools to instantly interpret the purification data, this integrated HTP workflow effectively accelerates AAV purification process development, with a 2% material volume requirement compared to the benchmark method, 96-well format screening, short turnaround time for analytical assays, and significant cost-of-goods savings for downstream process development.
PEGylation of polymerized albumin retains colloid osmotic pressure: Towards an enhanced potential plasma substitute.
Abdalbaqi A, Yahya A, Govender K … +5 more , Muñoz C, Van Moer GS, Lucas D, Cabrales P, Palmer AF
Biotechnol Prog · 2025 · PMID 40660704 · Full text
Plasma expanders (PEs) are commonly used to replace lost blood volume for septic shock patients with increased vascular permeability. Human serum albumin (HSA) is the preferred PE, due to its innate ability to restore bl...
Plasma expanders (PEs) are commonly used to replace lost blood volume for septic shock patients with increased vascular permeability. Human serum albumin (HSA) is the preferred PE, due to its innate ability to restore blood colloid osmotic pressure (COP). However, HSA is susceptible to protein extravasation under endothelial dysfunction leading to edema and exposing tissue to toxic HSA-bound metabolites. To prevent extravasation, the molecular diameter of HSA has been previously increased through chemical polymerization to yield polymerized HSA (PHSA). In this study, we further optimize PHSA size and COP via polyethylene glycol (PEG) surface conjugation. Previously synthesized PHSA that was size fractionated via tangential flow filtration (TFF) into two brackets (bracket A [500 kDa-0.2 μm] and bracket B [50-500 kDa]) served as precursors for subsequent PEGylation. Each PHSA bracket was thiolated with 2-iminothiolane hydrochloride (IT) and PEGylated with monofunctional 5 kDa maleimide PEG to yield PEGylated PHSA (PPHSA). All PPHSA solutions exhibited increased molecular size, zeta potential, and osmolality compared to their non-PEGylated precursor PHSA. At the same total protein concentration, PPHSA viscosity decreased compared to the precursor PHSA, while the COP remained consistent with HSA, indicating their potential to serve as PEs.
RETRACTION: A Novel pH-Responsive Nanoniosomal Emulsion for Sustained Release of Curcumin from a Chitosan-Based Nanocarrier: Emphasis on the Concurrent Improvement of Loading, Sustained Release, and Apoptosis Induction.
Biotechnol Prog · 2025 · PMID 40557742 · Publisher ↗
S. Haseli , M. Pourmadadi , A. Samadi , F. Yazdian , M. Abdouss , H. Rashedi , and M. Navaei-Nigjeh , "A Novel pH-Responsive Nanoniosomal Emulsion for Sustained Release of Curcumin from a Chitosan-Based Nanocarrier: Emph...
S. Haseli , M. Pourmadadi , A. Samadi , F. Yazdian , M. Abdouss , H. Rashedi , and M. Navaei-Nigjeh , "A Novel pH-Responsive Nanoniosomal Emulsion for Sustained Release of Curcumin from a Chitosan-Based Nanocarrier: Emphasis on the Concurrent Improvement of Loading, Sustained Release, and Apoptosis Induction," Biotechnology Progress 38, no. 5 (2022): e3280, https://doi.org/10.1002/btpr.3280. The above article, published online on 30 June 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, John A. Morgan; the American Institute of Chemical Engineers; the Society for Biological Engineering; and Wiley Periodicals LLC. The retraction has been agreed upon following an investigation into concerns raised by a third party, which revealed inappropriate duplication of image panels between this (Figure 4) and another article published by an overlapping group of authors, depicting a different experimental condition. The partial raw data provided by the authors could not address the original concerns, showed inconsistencies with the published results, and ultimately raised additional doubts about the study's overall reliability. Consequently, the editors have lost confidence in the presented data and decided to retract the paper. The authors' institute has been informed of the allegations and the decision to retract but remained unresponsive. The authors disagree with the retraction.
A digital shadow of CAR T cell expansion in a perfusion bioreactor: Informing optimal harvest times for autologous cell therapy.
Egan JR, Marí-Buyé N, Benítez-Cano EV … +9 more , Costa M, Wanika L, Chappell MJ, Schultz U, Ochs J, Effenberger M, Horna D, Rafiq Q, Goldrick S
Biotechnol Prog · 2025 · PMID 40546047 · Full text
Chimeric antigen receptor (CAR) T cell therapy has tremendous potential for the treatment of cancer and other diseases. To manufacture cells of the desired quantity and quality, it is important to expand the CAR T cells...
Chimeric antigen receptor (CAR) T cell therapy has tremendous potential for the treatment of cancer and other diseases. To manufacture cells of the desired quantity and quality, it is important to expand the CAR T cells ex vivo for an optimal duration. However, identifying the optimal harvest time requires knowledge of the cell concentration during the expansion period. To address this challenge, we have developed a digital shadow of CAR T cell expansion that provides a soft sensor of cell concentration in real-time. Specifically, a novel mechanistic mathematical model of cell growth within a proportional-integral-derivative (PID) controlled perfusion bioreactor has been developed using nonlinear ordinary differential equations. The model is fitted to data generated via bioreactor runs of the Aglaris FACER, in which both donor and patient cells have been expanded in two different media. Off-line data includes the initial and final cell concentrations, and online data includes the glucose and lactate concentrations as well as the perfusion rate. Training the digital shadow utilizes all the off-line and online data for each run. In contrast, real-time testing utilizes only the initial cell concentration and the available online data at the time of model fitting. Real-time testing shows that with at least 2.5 days of online data, the final cell concentration up to 2.5 days later is predicted with a mean relative error of 13% (standard deviation ≈ 6%). Informative real-time predictions of cell concentration via the digital shadow can guide decisions regarding the optimal harvest time of CAR T cells.
Innovating cell culture process development with deep learning-powered robotic experimentation using the first Industrial Smart Lab Framework.
Xu S, Huang Y, Shen X … +9 more , Mao R, Song Y, Ye W, Wang L, Tong X, Cao Y, Sun R, Zhou H, Zhou W
Biotechnol Prog · 2025 · PMID 40542657 · Publisher ↗
Traditional biologics process development, including antibody and recombinant protein production, typically relies on labor-intensive, iterative cell culture optimization to determine optimal process parameters. To addre...
Traditional biologics process development, including antibody and recombinant protein production, typically relies on labor-intensive, iterative cell culture optimization to determine optimal process parameters. To address this inefficiency, we introduced the Industrial Smart Lab Framework for Cell Culture (ISLFCC), an autonomous laboratory that combines deep learning and robotic experimentation to enhance cell culture processes. In this system, robotic arms sample various bioreactors for analysis, and the IoT system transmits these analysis results to decoder-only transformer deep learning models. Based on these analysis results, these models predict future cell states and recommend optimal actions, which are then executed by automation devices through the IoT system, such as adjusting nutrient feeds and temperature shifts. In a comparative case study, our AI-driven process development for three different cell clones resulted in an average titer increase of 26.8% and maintained lactate levels below 1 g/L without rebound in the late phase within just a single batch, surpassing traditional three-stage empirical process development methods. Moreover, our approach has greatly automated cell culture to ensure enhanced reproducibility, data accuracy, adaptability to various cell lines, and seamless scalability across production scales, marking the first implementation of high-throughput automated cell culture in 3 and 15 L bioreactors. By merging AI with robotic execution, ISLFCC provides a transformative framework that accelerates biologics development, representing a paradigm shift towards autonomous, data-driven biomanufacturing.
Impact of bioreactor process parameters and yeast biomass on Raman spectra.
Klaverdijk M, Nemati M, Ottens M … +1 more , Klijn ME
Biotechnol Prog · 2025 · PMID 40519078 · Full text
In-line Raman spectroscopy combined with chemometric modeling is a valuable process analytical technology (PAT) providing real-time quantitative information on cell culture compounds. Considering that compound quantifica...
In-line Raman spectroscopy combined with chemometric modeling is a valuable process analytical technology (PAT) providing real-time quantitative information on cell culture compounds. Considering that compound quantification through chemometric models depends on pre-processing to maintain consistent changes in intensity at certain wavenumbers, all causes of signal distortion should be well understood to prevent quantification inaccuracies. This work investigated spectral distortion caused by the changing bioreactor parameters temperature, bubble quantity, and medium viscosity. In addition, the isolated spectral contribution of Saccharomyces cerevisiae cells in suspension was also determined. A temperature range from 20 to 40°C resulted in peak shifts up to 0.8 cm to lower wavenumbers, bubbles generated under standard bioreactor operation conditions led to signal attenuation of up to 7.93% reduction in peak intensity, and changes in liquid viscosity resulted in complex peak shift behavior. Isolated biomass concentrations reaching 5 g/L caused up to 44.6% reduction in distinct peak intensity, which was similar to spectra from batch process fermentations. Correcting for the attenuation revealed spectral features of biomass associated with proteins and lipids in the 1000-1500 cm region. However, the spectral contribution of yeast biomass is dominated by signal extinction, which attenuates Raman spectra in a non-linear manner as biomass accumulates. The obtained knowledge on different sources of spectral distortion aids in the development of robust pre-processing and modeling strategies to obtain chemometric models applicable across experimental setups.
Optimization of multi-column chromatography for capture and polishing at high protein load.
Silva TC, Isaksson M, Nilsson B … +2 more , Eppink M, Ottens M
Biotechnol Prog · 2025 · PMID 40485304 · Full text
Integrated Continuous Biomanufacturing reduces manufacturing costs while maintaining product quality. A key contributor to high biopharmaceutical costs, specifically monoclonal antibodies (mAbs), is chromatography. Prote...
Integrated Continuous Biomanufacturing reduces manufacturing costs while maintaining product quality. A key contributor to high biopharmaceutical costs, specifically monoclonal antibodies (mAbs), is chromatography. Protein A ligands are usually preferred but still expensive in the manufacturing context, and batch chromatography under-utilizes the columns' capacity, compromising productivity to maintain high yields. Continuous chromatography increases columns' Capacity Utilization (CU) without sacrificing yield or productivity. This work presents the in-silico optimization of a 3 Column Periodic Counter-current Chromatography (3C-PCC) of a capture and polishing step for mAbs from a high titer harvest (c = 5 g/L). The 3C-PCC was modeled and Pareto-fronts for continuous and batch modes were used to optimize the 3C-PCC steps varying the flow rate and percentage of breakthrough achieved in the interconnected loading, maximizing Productivity and CU, for varying concentrations of mAb (batch mode concentration of 5 g/L and continuous mode concentration of 2.5, 5, 7.5, and 10 g/L). The shape of the breakthrough curve significantly impacts the optimization of 3C-PCC. The model output was validated for three different protein A ligands using a pure mAb solution. MAb Select SuRe pcc was selected to continuously capture mAb from a high-titer clarified cell culture supernatant (harvest). The product eluates were pooled and used for continuous polishing using a Cation-Exchange resin (CaptoS ImpAct). Experimental results validated model predictions (<7% deviation in the worst case) and a process with two 3C-PCC in sequence was proposed, with a productivity of approximately 100 mg/mL res/h.
A recurrent neural network for soft sensor development using CHO stable pools in fed-batch process for SARS-CoV-2 spike protein production as a vaccine antigen.
Reyes SJ, Voyer R, Durocher Y … +2 more , Henry O, Pham PL
Biotechnol Prog · 2025 · PMID 40454837 · Full text
Fed-batch recombinant therapeutic protein (RTP) production processes utilizing Chinese Hamster Ovary (CHO) cells can take a long period of time (>10 days). Within this period, not all critical features may be measured ro...
Fed-batch recombinant therapeutic protein (RTP) production processes utilizing Chinese Hamster Ovary (CHO) cells can take a long period of time (>10 days). Within this period, not all critical features may be measured routinely, and in fact, some are only measured once the process is terminated, complicating decision making. As a consequence, utilizing routine current day bioreactor online data to aid in next day predictions is a promising strategy for model predictive control-based feeding strategies. The article details the development of a proposed soft sensor that merges current day bioreactor online data and offline historical sampling data to generate predictions about the next day of the production process. This approach demonstrated the ability to track product titer, cell growth, key metabolites, and cumulative glucose consumption across the 17-day process with low normalized root mean squared error (nRMSE = 0.24) and low normalized mean absolute error (nMAE = 0.18) as well as high linearity with respect to ground data (average R = 0.97). It was also demonstrated that the same model architecture could effectively soft sense product titer and metabolic profiles (glucose, lactate, ammonia) without having sampling day's offline data as inputs to the model. This suggests that the proposed model could act as a true soft sensor of hard-to-determine variables such as the trimeric SARS-CoV-2 spike protein that relies on end-of-process measurements to acquire the data (labor-intensive semi-quantitative SDS-PAGE gels or ELISA assay). Instantaneous specific glucose consumption rates were also predicted and showed good agreement with experimental measurements, further offering opportunities for online glucose control.
A simple electrochemical genosensor based on polythiophene acetic acid film for detection of Schistosoma mansoni.
Oliveira MSML, Lucena RPS, Silva-Júnior AG … +5 more , Melo FL, Silva BM, Gomes ECS, Andrade CAS, Oliveira MDL
Biotechnol Prog · 2025 · PMID 40454539 · Publisher ↗
Schistosoma mansoni infection and other neglected diseases pose significant challenges in diagnosis and treatment, particularly in resource-constrained regions. Despite being useful, traditional techniques lack sensitivi...
Schistosoma mansoni infection and other neglected diseases pose significant challenges in diagnosis and treatment, particularly in resource-constrained regions. Despite being useful, traditional techniques lack sensitivity, offering frequent false-positive results, highlighting the emergence of innovative tools such as genosensors as a promising solution to this dilemma. In this work, we developed a simple electrochemical biosensor platform based on electropolymerized films of polythiophene acetic acid (PTAA) and a specific DNA probe for the detection of S. mansoni. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and atomic force microscopy (AFM) were used to assess the assembly process of the genosensor, as well as to evaluate biodetection assays. The developed biosensor was found to be effective in detecting the target analyte in pure and complex samples such as cerebrospinal fluid, urine, and plasma from infected patients at different concentrations. CV and EIS were extremely useful in the evaluation of the detection process based on the electron kinetics and charge transfer resistance (R) in the interface of the biosensor, where the hybridization with the target single-stranded S. mansoni DNA resulted in the variation of these parameters. The genosensor exhibited high sensitivity and selectivity, with a limit of detection of 0.451 pg.μL. As genosensors continue to evolve, they promise to revolutionize the field of neglected disease management, providing hope for improved healthcare outcomes worldwide.
3D hepatic spheroids and liver-organ on chip models displayed maintenance of hepatic functions and maturation profile in a long-term culture of the humanized HepaSH cells, a human cell population harvested from chimeric mice.
Wang H, Meschini C, Poulain S … +7 more , Kim SH, Arakawa H, Kato Y, Legallais C, Pereira U, Sakai Y, Leclerc E
Biotechnol Prog · 2025 · PMID 40448434 · Publisher ↗
Long-term functional hepatocyte and reproducible cultures are required in pharmaceutical industries to model chronic liver disorders and to perform associated drug testing. In this frame, we have investigated the behavio...
Long-term functional hepatocyte and reproducible cultures are required in pharmaceutical industries to model chronic liver disorders and to perform associated drug testing. In this frame, we have investigated the behavior of the HepaSH cells when cultivated in liver Biochips, in 3D Spheroids, and in Petri for 20 days. HepaSH is a newly developed humanized hepatocyte harvested from chimeric mice. After the cells' harvesting and inoculation, the HepaSH were successfully maintained in cultures in Petri dishes, spheroids, and Biochips for 20 days. The immunostaining confirmed the expressions of albumin, CYP1A2, and CYP3A4 in all conditions. Furthermore, the CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities were successfully detected in all conditions after 20 days of cultures. Continuous production of albumin and biliary acids was detected in Biochips, Spheroids, and Petri, among which Biochip culture showed the highest albumin secretion level. The RNA sequencing analysis revealed that Biochips and Spheroids cultures enriched hepatic maturation, xenobiotic, lipid, small molecule, steroid, and alcohol metabolisms compared to Petri cultures. Overall, our data demonstrated the feasibility of cultivating the HepaSH cells in Petri, Biochips, and Spheroids for 20 days in the presented protocol, while keeping important liver functions. Biochip and Spheroids cultures show advantages in hepatic maturation, drug metabolism-related gene expression, and albumin secretion (in biochips) compared with conventional Petri culture.
Effects of LEDs, macronutrients and culture conditions on biomass and artemisinin production using Artemisia annua L. suspension cultures.
Lim WH, Khaw ML, Yungeree O … +5 more , Hew WH, Parab AR, Chew BL, Wahyuni DK, Subramaniam S
Biotechnol Prog · 2025 · PMID 40410819 · Publisher ↗
Artemisinin is a sesquiterpene lactone extracted from the medicinal plant Artemisia annua L. (sweet wormwood). It has traditionally been utilized in artemisinin-based combination therapies (ACTs) for the malarial parasit...
Artemisinin is a sesquiterpene lactone extracted from the medicinal plant Artemisia annua L. (sweet wormwood). It has traditionally been utilized in artemisinin-based combination therapies (ACTs) for the malarial parasite, including drug-resistant strains. Natural artemisinin extraction is costly with low yields. Due to its effectiveness, there is a significant rise in the demand for artemisinin production. In vitro cell suspension culture offers a cost-effective and viable technique for artemisinin production. Therefore, this study aimed to optimize a protocol for cell suspension culture of A. annua L. to enhance biomass and artemisinin production. A successful cell suspension culture was initiated from induced callus. The highest cell biomass was obtained in suspension cultures grown with an initial inoculum size of 0.1 g of mixed type cell aggregates, in media with a pH of 6.2 and a rotation speed of 90 rpm. Macronutrient concentrations influenced both biomass and artemisinin content, with optimal biomass achieved at 19 mM KNO and 1.56 mM KHPO. The absence of these nutrients resulted in the highest artemisinin levels. Different LED wavelengths also significantly influenced biomass and artemisinin production. Red + blue LED increased cell biomass, while the highest artemisinin content was observed under red LED. The upscaling of the culture indicated a variation in biomass yield pattern, but the highest growth index was achieved in the 500 mL Erlenmeyer flask. This study successfully established a cell suspension culture for A. annua L., demonstrating the influence of macronutrients and red LED on biomass and artemisinin production, providing insights for potential large-scale production.
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