Biotechnol. Prog. [JOURNAL]
Sun
200 papers
A PVA/chitosan/gelatin scaffold incorporating salicylic acid-loaded TiO₂ nanoparticles enhances collagen I expression and accelerates cutaneous wound repair in mice.
Safaiefar P, Reiisi S, Haghi M … +1 more , Shirian S
Biotechnol Prog · 2026 May · PMID 42087362 · Publisher ↗
Wound healing is a complex biological process requiring coordinated cellular and molecular responses, motivating the development of bioactive scaffolds capable of modulating the wound microenvironment. In this study, a n...
Wound healing is a complex biological process requiring coordinated cellular and molecular responses, motivating the development of bioactive scaffolds capable of modulating the wound microenvironment. In this study, a nanocomposite scaffold composed of chitosan, polyvinyl alcohol (PVA), and gelatin incorporating salicylic acid-loaded titanium dioxide nanoparticles (TiO₂ NPs) was fabricated and evaluated for wound-healing applications. TiO₂ nanoparticles synthesized via a co-precipitation method exhibited nanoscale dimensions, high crystallinity, a negative surface charge (-19.4 mV), and a drug-loading efficiency exceeding 90%. The scaffold showed good structural integrity under physiological conditions, excellent hemocompatibility (<5% hemolysis), and a swelling ratio of approximately 50%. Sustained, pH-independent release of salicylic acid was observed. In vitro studies demonstrated that the nanocomposite increased fibroblast migration by approximately 90% compared with untreated controls and significantly upregulated COL1 gene expression by 2.5-fold (p < 0.05), while maintaining high cell viability. In a murine excisional wound model, the nanocomposite-treated group achieved 85% wound closure by day 10, compared with 30% in untreated wounds and 50% in the drug-free scaffold group (p < 0.05), along with improved epidermal regeneration. Overall, these results indicate that controlled delivery of salicylic acid from a TiO₂-containing polymeric scaffold supports fibroblast activity, collagen-related responses, and wound closure, providing a rational basis for further optimization of multifunctional wound dressings.
On-deck pH measurement with integrated feedback control on a micro-scale platform for monoclonal antibody low pH viral inactivation.
Sharma P, von den Eichen N, Schweisgut S … +3 more , Robbel L, Schmitt M, Bracewell DG
Biotechnol Prog · 2026 May · PMID 42084290 · Publisher ↗
The increasing demand for efficient monoclonal antibody manufacturing has accelerated the adoption of high throughput process development (HTPD) platforms, which enable rapid, automated screening of downstream operations...
The increasing demand for efficient monoclonal antibody manufacturing has accelerated the adoption of high throughput process development (HTPD) platforms, which enable rapid, automated screening of downstream operations. However, the integration of non-chromatographic steps such as low pH viral inactivation (VI) within automated workflows remains limited, largely due to the absence of a micro-scale method for accurate, on-deck pH measurement and control. This study presents the development and implementation of an automated pH measurement and feedback control system using optical pH sensors immobilized within 96-well microplates. The approach enables non-invasive, real-time monitoring of pH across the acidic range required for VI and is fully compatible with standard liquid-handling platforms. Integration of a feedback control algorithm allowed autonomous acid and base addition to achieve precise target pH values during both acidification and neutralization phases. The method achieved strong agreement between measured and expected pH values following optimization of measurement conditions, including ionic strength adjustment. The system was further integrated with Sartobind® Q and cation exchange chromatography steps to demonstrate an end-to-end automated workflow. Systematic assessment of cation exchange chromatography performance under controlled loading conditions enabled direct visualization of separation behavior and early identification of sub-optimal operating regions, demonstrating the platform's capability to expand experimental space and accelerate mechanistic process understanding. This work establishes a micro-scale, fully automated downstream platform with pH control, bridging a critical technological gap and advancing the vision of an end-to-end HTPD system for biopharmaceutical purification.
Data-driven design of deterministic lateral displacement microfluidic devices for CTC separation.
Sarowar T, Mim SA, Chen X
Biotechnol Prog · 2026 Apr · PMID 42057373 · Publisher ↗
Circulating tumor cells (CTCs) are established biomarkers for cancer diagnosis and therapeutic monitoring, yet their extreme rarity in peripheral blood and size overlaps with leukocytes present substantial technical barr...
Circulating tumor cells (CTCs) are established biomarkers for cancer diagnosis and therapeutic monitoring, yet their extreme rarity in peripheral blood and size overlaps with leukocytes present substantial technical barriers to clinical deployment. Deterministic lateral displacement (DLD) is a promising microfluidic technique for label-free, continuous CTC separation. However, due to the extreme heterogeneity of cancer cells, rational device design requires computationally expensive iterative simulation across multi-dimensional geometric parameter spaces. Here we present a data-driven design framework that integrates validated computational fluid dynamics (CFD) simulations with supervised machine learning to enable rapid prediction of particle separation behavior and critical diameter in DLD arrays. A dataset of approximately 8.5 million trajectory data points was generated across 1160 unique combinations of array period number (N = 3-48) and particle diameter (1-20 μm) using COMSOL-based CFD. Four regression algorithms, Gradient Boosting, Random Forest, k-Nearest Neighbors, and Multi-Layer Perceptron, were trained and evaluated on trajectory prediction and critical diameter estimation. Random Forest achieved the highest accuracy, with a test-set R of 0.994 and a mean absolute error of 0.31 μm in critical diameter prediction. A clinical case study targeting colorectal CTC separation from blood demonstrated that the ML-guided design workflow converged on an optimal device configuration (N = 15, D = 10-12 μm) in under 2.3 s, representing a speedup exceeding four orders of magnitude compared to equivalent CFD iteration. The framework enables direct translation of CTC size distributions into DLD device configurations and provides a scalable foundation for reproducible microfluidic process development in biomedical applications.
Protein hydrolysates in cell culture: Toward multi-omics characterization.
Combe M, Wrage BM, Varela-Rohena A … +1 more , Sokolenko S
Biotechnol Prog · 2026 Apr · PMID 42050359 · Publisher ↗
The high cost of conventional cell culture media has driven demand for sustainable, cost-effective serum-free formulations in biopharmaceutical manufacturing and cultivated meat production. Although plant- and yeast-deri...
The high cost of conventional cell culture media has driven demand for sustainable, cost-effective serum-free formulations in biopharmaceutical manufacturing and cultivated meat production. Although plant- and yeast-derived hydrolysates are generally considered to be promising additives, significant variability in raw materials and processing conditions results in substantial compositional and batch-to-batch differences between products, making it challenging to predict effectiveness in cell culture a priori. This review examines hydrolysate media additives through the specific lens of comprehensive multi-omic characterization as a means of addressing this uncertainty. While typical characterization is often limited to metrics such as degree of hydrolysis or color, detailed metabolomic and peptidomic profiling remains uncommon. We identify a gap in systematic, high-resolution characterization and argue that integrating compositional and bioactivity analyses is essential to transition hydrolysates from undefined inputs toward semi-defined media components.
Addressing challenging impurities in fusion proteins: A comprehensive review and cost-effective strategy for HCP and low molecular weight species control.
Das A, Paranandi MR, Subbiramani K … +1 more , Reddy R
Biotechnol Prog · 2026 Apr · PMID 42026977 · Publisher ↗
Fusion proteins represent a rapidly expanding class of biotherapeutics engineered by combining functional domains from different proteins to enhance therapeutic efficacy, stability, and pharmacokinetics. Their unique des...
Fusion proteins represent a rapidly expanding class of biotherapeutics engineered by combining functional domains from different proteins to enhance therapeutic efficacy, stability, and pharmacokinetics. Their unique design enables extended half-life, reduced dosing frequency, and multi-targeting capabilities, making them highly versatile for diverse therapeutic indications. However, the manufacturing of fusion proteins presents significant challenges, particularly in managing complex product- and process-related impurities such as host cell proteins (HCPs), fragments, and low-molecular-weight (LMW) species. These impurities are difficult to eliminate due to their structural and physicochemical similarity to the target protein, complicating purification and scale-up. Part I of this paper provides a comprehensive review of published studies addressing impurity characterization and control strategies in fusion protein manufacturing. The review highlights critical impurity types, analytical tools for detection and quantification, and process strategies for effective removal while maintaining product integrity and regulatory compliance. Part II presents a case study from Kemwell Biopharma, illustrating a cost-effective platform purification strategy for complex fusion proteins. The approach is applicable to non-tagged, Fc-based, and multispecific fusion protein formats and demonstrates efficient removal of HCPs and LMW impurities while achieving high yield and purity. Together, the review and case study provide both a broad scientific understanding and a practical demonstration of scalable impurity control approaches, offering valuable insights for the development of robust and economically viable manufacturing processes for next-generation fusion protein therapeutics.
Strategies for removing two "problematic" host cell proteins, using an "end-to-end" approach.
Evangelist G, Kwiatkowski C, Malu K … +4 more , Liu S, Yi L, Khattak S, Stanley B
Biotechnol Prog · 2026 Apr · PMID 42026710 · Publisher ↗
Host cell proteins (HCPs) are critical process-related impurities of recombinant protein biopharmaceuticals that have the potential to impact product safety and efficacy. In this study, two residual HCPs, heat shock prot...
Host cell proteins (HCPs) are critical process-related impurities of recombinant protein biopharmaceuticals that have the potential to impact product safety and efficacy. In this study, two residual HCPs, heat shock protein 90 beta and perilipin-4-like, produced from a CHO cell line, were identified during the development of a late-stage platform Immunoglobulin G subclass 1 (IgG1) monoclonal antibody process. A risk assessment was performed that included identification of HCPs, homology with their human protein counterparts, and prior non-clinical and clinical experience. The outcome deemed these two species as "problematic" and target levels were established to guide approaches for removal. Given the high productivity of the upstream and downstream platform processes, the goal was to explore conditions that minimize deviations from the platform. An end-to-end approach was performed that evaluated downstream levers, including Protein A washes, polishing chromatography operational parameters, and exploration of depth filter media. Upstream levers were also explored, evaluating effects of temperature shift and modulation of iron and citrate to help control levels of both HCP species. The results presented in this study demonstrated the upstream and downstream conditions achieved effective removal of the two HCP species to meet drug substance targets.
Evaluating the impact of ionic strength on retention of minute virus of mice during small virus-retentive filtration.
Murphy C, Slocum A, Tierson M … +4 more , Cacace B, Genest P, Remington KM, Hale J
Biotechnol Prog · 2026 Apr · PMID 42010327 · Publisher ↗
Small virus-retentive filtration (VRF) is a critical unit operation utilized in the viral clearance package to ensure the viral safety of biotherapeutics. This step is relied upon to provide effective parvovirus retentio...
Small virus-retentive filtration (VRF) is a critical unit operation utilized in the viral clearance package to ensure the viral safety of biotherapeutics. This step is relied upon to provide effective parvovirus retention of ≥4 log reduction value (LRV) (e.g., minute virus of mice; MVM), and robust retention of larger viruses. Biomolecules, including multispecific antibodies and recombinant proteins, present purification challenges that require multiple chromatography columns to achieve final purity specifications. These additional polishing steps may utilize high ionic strength solution conditions to meet purity targets, and it is critical to assess if those process conditions impact the viral retention performance of the subsequent VRF operations. In one instance, retention performance was lower than expected (≤4 LRV) and was attributed to the use of a high ionic strength buffer. Two possible mechanisms were investigated, that is, reduction in the effective size of the MVM virus (~25 nm) and/or an increase in the membrane filter pore size, as observed with increase in the virus filter permeability. Based on modeling and experimental data presented here, at high ionic strength solution conditions the hydrodynamic size of MVM is reduced while virus-retentive membrane effective pore size is larger, as compared to low ionic strength conditions. Additionally, MVM LRV and membrane permeability of multiple commercial-grade virus filters were impacted by high ionic strength and process interruptions. These results demonstrate that high ionic strength solution conditions could impact parvovirus retention performance, and these findings may guide process development for biotherapeutics operating under similar atypical solution conditions.
Rapid development of a transferable Raman model using high-throughput cell culture for monitoring monoclonal antibody titer.
Umprecht A, Uth N, Kim H … +2 more , Spadiut O, Yang Y
Biotechnol Prog · 2026 · PMID 41937236 · Full text
Monoclonal antibody (mAb) titer monitoring is a key capability during process development and optimization, enabling timely decision making and increasing the speed of development. Raman spectroscopy is a prominent proce...
Monoclonal antibody (mAb) titer monitoring is a key capability during process development and optimization, enabling timely decision making and increasing the speed of development. Raman spectroscopy is a prominent process analytical technology (PAT), but resource-efficient calibration strategies for the development of transferable models are limited. This work demonstrates the development and successful transfer of a calibration model for monoclonal antibody concentration between two different cell lines with varied metabolic profiles expressing different antibodies. The root mean square error of prediction (RMSEP) for titer in the source cell line (0.266 g L) was comparable to that of the target cell line (0.325 g L). The transferable model was achieved by conducting a spiking study in a high-throughput parallel bioreactor system. Different experimental approaches with models trained on spiked versus native samples were compared. This analysis revealed that model transferability was influenced by the degree of correlation of lactate with antibody titer in the source process, emphasizing the importance of process knowledge in the development of Raman calibration models. Overall, the study presents evidence of the feasibility of transferable titer models, marking a significant advancement in process monitoring capabilities for high-throughput cell culture as well as introducing a generic methodology for calibration of transferable models in the context of upstream bioprocessing.
Disulfide bond reduction of antibodies during clarification: A bench-scale assay and a mitigation tool.
Tjandra H, Surendar D, Yoon I … +5 more , Rusly F, Su Y, Thai V, Liu Y, Hesslein A
Biotechnol Prog · 2026 Apr · PMID 41928694 · Publisher ↗
We investigated side-by-side clarification with a two-stage depth filter system against a single-stage charge chromatographic clarifier on a number of Chinese hamster ovary (CHO) cell cultures. We found that the one-stag...
We investigated side-by-side clarification with a two-stage depth filter system against a single-stage charge chromatographic clarifier on a number of Chinese hamster ovary (CHO) cell cultures. We found that the one-stage fibrous anion exchange (AEX) clarification met or exceeded the performance of the conventional two-stage depth filtration systems in terms of product recovery, removal of process-related impurities, and throughput. The one-stage anion clarification approach had a smaller footprint, a shorter set-up time, an easier setup, consumed less buffer flush volume, and operated at a lower differential pressure relative to those of conventional systems. In a comparability study at 200 L scale, antibodies in cell culture fluid (CCF) clarified by depth filtration system exhibited product quality degradation due to disulfide bond reduction (DSBR), whereas antibodies clarified by AEX fiber method were not. We surmised that the lower operating differential pressure and electrostatic adsorption mechanism of the anion clarification system minimized the potential of product to undergo DSBR. We developed a bench-scale process model that allowed us to assay DSBR and predict the process results. We then utilized it to demonstrate that the antibody was reduced when clarified with a two-stage 3 M™ Zeta Plus™ Depth Filter system but not with the anionic clarification system. This bench-scale DSBR assay allowed us to rank the relative disulfide bond reduction potentials of various monoclonal antibodies (mAbs) and demonstrate the reduction-mitigating impacts of the fibrous clarification approach on a reduction-prone antibody culture at 2000 L processing scale.
An efficient and robust strategy for developing cell lines through targeted integration.
McVey D, Garg C, Wang C … +11 more , Awan A, Zheng R, Zhang J, Sturner S, Langsdorf E, Yu L, Shankarling K, Rizzi G, Agarwal K, Tremml G, Khetan A
Biotechnol Prog · 2026 Apr · PMID 41927489 · Publisher ↗
Reducing the time needed and enhancing predictability of outcomes for cell line development is an ongoing focus of improvement. Targeted integration (TI) where the expression cassettes for a biologic are inserted at a pr...
Reducing the time needed and enhancing predictability of outcomes for cell line development is an ongoing focus of improvement. Targeted integration (TI) where the expression cassettes for a biologic are inserted at a predefined locus, a landing pad, is one of the strategies that is used for this purpose. To be commercially viable the landing pad host cell must support high titers and the transgenes in the landing pad must be highly transcribed. To make such a host TI cell line takes a great deal of time and effort with little guarantee of success. Here we describe a strategy where we converted a stable high-expressing mAb cell line and generated multiple TI host cell lines that are commercially relevant. We routinely generate 6 g/L mAb clones using our platform fit process and 8 to >10 g/L with process optimization that are phenotypically and genetically stable. We also created a duo-landing pad where both landing pads reside in the same locus. This gives the opportunity to simplify the construction of differentially expressed genes since both landing pads are expected to perform equally.
Design of experiments-guided membrane capture facilitates integration of continuous virus inactivation using Phi6 as a surrogate virus in an intensified downstream process.
Grünberg M, Lipski L, Fisicaro G … +6 more , Duvignau T, Meyer-Heinrichs K, Mocsy DC, Filz TJ, Quaas B, Stützer A
Biotechnol Prog · 2026 · PMID 41877579 · Full text
The growing demand for cost-efficient and flexible biomanufacturing has increased interest in process-intensified downstream platforms. This study evaluates an intensified monoclonal antibody (mAb) purification sequence,...
The growing demand for cost-efficient and flexible biomanufacturing has increased interest in process-intensified downstream platforms. This study evaluates an intensified monoclonal antibody (mAb) purification sequence, where two unit operations traditionally performed in batch mode-Protein A capture and low pH virus inactivation (VI)-are redesigned to enhance productivity and minimize resource usage. Rapid-cycling Protein A membrane chromatography was optimized using a design-of-experiments approach to address inherent membrane challenges such as buffer consumption. Wash step volumes were systematically reduced without compromising host cell protein or host cell DNA clearance, yielding a 70% reduction in total wash buffer consumption. At manufacturing scale, membrane adsorbers achieved critical quality attributes comparable to a benchmark resin, while increasing productivity ~20-fold and lowering capture-step costs. Protein A eluates were processed in a novel continuous virus inactivation (cVI) system using bacteriophage Phi6 as a surrogate for enveloped viruses. At residence times of 35 and 70 min, the cVI system achieved a ≥5-log reduction, equaling conventional batch performance without compromising mAb quality. The study demonstrates that membrane-based Protein A capture and continuous VI can be seamlessly integrated into an intensified DSP framework. This approach effectively maintains product quality while significantly reducing buffer usage and cost, thus supporting modular intensification strategies for clinical-scale mAb manufacturing.
A practical framework for clone selection, media-feed screening, and upstream process parameter optimization for a mAb CHO cell culture process.
Shah S, Kumar D, Dhamdhere A … +5 more , Devi S, Patekar S, Mahalingam V, Adhikary L, Chopda V
Biotechnol Prog · 2026 · PMID 41814514 · Publisher ↗
This study presents a data-driven workflow for upstream process development in biologics manufacturing, aimed at improving consistency, efficiency, and decision-making using established statistical and machine learning t...
This study presents a data-driven workflow for upstream process development in biologics manufacturing, aimed at improving consistency, efficiency, and decision-making using established statistical and machine learning tools. Rather than relying on subjective interpretation, the framework integrates multiple analytical methods in a structured manner to support key process development decisions. For clone selection, K-means clustering was applied and benchmarked with hierarchical clustering to identify top-performing candidates from a large clone pool. By incorporating multiple product quality attributes into the evaluation, the approach improves selection consistency and reduces subjectivity. In the media and feed screening stage, principal component analysis (PCA) was used to explore how specific combinations influence glycosylation patterns, enabling rapid identification of promising conditions for further optimization while reducing experimental burden. For upstream process parameter refinement, a sequential learning strategy based on multi-objective Bayesian optimization (MOBO) was applied to adaptively explore trade-offs among competing quality attributes and titer. This approach enabled more efficient identification of improved operating conditions compared with static experimental designs while minimizing experimental runs. While the methods used are well-established, their structured integration into a cohesive workflow demonstrates practical utility for industrial applications. Aligned with Bioprocessing 4.0 principles, this approach supports improved process understanding and informed decision-making in upstream bioprocess development.
Single-cell RNA sequencing reveals population-based shifts in gene expression heterogeneity in an unstable, clonally derived CHO cell line.
Peters TW, Leinas JT, O'Brien S … +5 more , Ochoa-Moreno H, Lachance D, Younan P, Szeto G, Clarke H
Biotechnol Prog · 2026 · PMID 41803018 · Publisher ↗
The genomic plasticity of Chinese Hamster Ovary cells allows them to be genetically engineered to produce foreign proteins with high specific productivities while placing a significant burden on their ability to retain t...
The genomic plasticity of Chinese Hamster Ovary cells allows them to be genetically engineered to produce foreign proteins with high specific productivities while placing a significant burden on their ability to retain this expression capacity over time. Many cell lines lose expression over time and need to be screened for expression consistency. Stability has been studied in clonally derived cell cultures by evaluating phenotypic heterogeneity in subclones derived from stable or unstable clones, but not well characterized within the primary, clonally derived cell culture. To address this, we employed single cell RNA sequencing in stable and unstable cell lines expressing the same monoclonal antibody, at early and late timepoints in their manufacturing lifespan. We observed higher expression heterogeneity and a more drastic redistribution of expression profile "clusters" over serial passaging in the unstable cell line. Differentially expressed genes in those clusters making up the unstable cell line suggest distinct changes in metabolic pathway activity in this cell line over time. Using molecular methods, changing gene expression profiles were confirmed and activation of molecular pathways consistent with a challenged protein homeostasis were shown to be enriched in cells over time in the unstable cell line. This report more broadly implicates the role of heterogeneity within a clonal population as a contributing factor to cell line instability, providing a biological context for further investigation.
Characterization of apoptosis in fed-batch cultures highlights the impact of timely process intervention on improving recombinant protein productivity.
Patel M, Madabhushi SR
Biotechnol Prog · 2026 · PMID 41738360 · Publisher ↗
Fed-batch processes using Chinese hamster ovary (CHO) cells are primarily employed to manufacture biologics. In these cultures, cells transition from exponential growth to the stationary phase, often resulting in a viabi...
Fed-batch processes using Chinese hamster ovary (CHO) cells are primarily employed to manufacture biologics. In these cultures, cells transition from exponential growth to the stationary phase, often resulting in a viability decline. This decline along, with similar reductions in cell growth and productivity is attributed to changes in cellular metabolism, the cell cycle, and apoptosis, potentially caused by nutrient imbalance and the accumulation of inhibitory byproducts. To enhance productivity in fed-batch processes, understanding the interactions among these factors is crucial for identifying intervention strategies to sustain cell culture performance. In this study, we evaluated the dynamics of the cell cycle and apoptosis in a mAb-producing cell line during a fed-batch process. Results showed that the cells transitioned to the G0/G1 phase during the stationary phase. Although viability remained above 80% throughout the culture, early signs of apoptosis were observed as early as Day 5, coinciding with product accumulation. Our investigation revealed that early apoptotic cells were not productive and that removing apoptotic stimuli or adding fresh nutrients did not rescue these cells. This underscores the necessity for early process interventions. Media exchange after Day 7, when cells are in stationary phase, resulted in higher viability, fewer early apoptotic cells, and approximately a 26% increase in productivity compared to conditions with no or earlier media exchange. This study highlights the significance of timely interventions to remove the inhibitory byproducts and replenish the culture with fresh media, based on detailed process characterization.
Sequential, chromosome-specific glutamine synthetase double knockout with Cas-CLOVER establishes enhanced CHO platforms for cell line development.
Limia CG, Steffey V, Cheng HC … +5 more , Machado D, Hart T, McHargue MC, Brizzee C, Crawford J
Biotechnol Prog · 2026 · PMID 41724722 · Full text
Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CH...
Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.
Scalable desolvation synthesis, storage stability, and comprehensive biophysical characterization of human hemoglobin nanoparticles as a potential hemoglobin-based oxygen carrier.
Greenfield A, Palmer AF
Biotechnol Prog · 2026 · PMID 41689261 · Full text
Hemoglobin (Hb)-based oxygen carriers (HBOCs) are materials that exploit Hb's native oxygen transport while avoiding the adverse side-effects associated with the exposure of the cell-free, unmodified Hb tetramer (i.e. va...
Hemoglobin (Hb)-based oxygen carriers (HBOCs) are materials that exploit Hb's native oxygen transport while avoiding the adverse side-effects associated with the exposure of the cell-free, unmodified Hb tetramer (i.e. vasoconstriction, systemic hypertension, and oxidative tissue injury) in circulation. There are many synthetic routes to generate HBOCs including chemical polymerization, polymer surface conjugation, and protein encapsulation that meet the material and O demands of different biomedical applications (e.g. emergency transfusion medicine, ex vivo organ perfusion, tumor oxygenation, etc.) especially when red blood cells may not be immediately available or desirable. There is currently no FDA approved HBOC in the US, therefore leaving room in the field for further HBOC design and optimization. An ideal HBOC should effectively bind and release O, have a scalable synthesis to meet material demand, and limit batch-to-batch variability to yield consistent, uniform material. This work demonstrates the successful synthesis of human Hb nanoparticles using a desolvation technique (hHb-dNPs) as a new HBOC candidate. hHb-dNPs demonstrated a monodisperse size distribution between 130 and 150 nm with spherical morphology, a negative surface charge, and hemocompatibility. hHb-dNPs exhibited high O affinity with decreased cooperativity similar to that of relaxed-state polymerized Hb. The methodology demonstrated scalability from 5 to 500 mL starting volume, producing products with similar biophysical properties regardless of the scale. hHb-dNPs demonstrated structural and biophysical stability for a month at 4°C and -80°C with various cryoprotectants, with the optimal combination being hHb-dNPs co-stored at -80°C with HSA. Considering these results, hHb-dNPs are a promising potential next generation HBOC.
Innovative methodology for flexible monitoring of various bioprocesses by using pre-existing Raman data coupled with automated transfer learning technique.
Schini A, El Radi H, Cailletaud J … +5 more , Sanchez C, Gay N, Ebel B, Guedon E, Jourdainne L
Biotechnol Prog · 2026 · PMID 41668216 · Full text
This study introduces an innovative approach for the flexible monitoring of bioprocesses using Raman spectroscopy coupled with automated transfer learning. Traditional Raman spectroscopy requires extensive process-specif...
This study introduces an innovative approach for the flexible monitoring of bioprocesses using Raman spectroscopy coupled with automated transfer learning. Traditional Raman spectroscopy requires extensive process-specific calibration, limiting its transferability across different conditions. To address this, we developed an automated method that utilizes the dynamic orthogonal projection (DOP) algorithm to use pre-existing chemometric models built from one process ("Input Process") to monitor a distinct process ("Target Process") which lacked its own Raman models. These new processes conditions varied in terms of culture mode, cell line, media, analyzer, and acquisition settings. This method used spectral data from Target Process to modify the existing spectral data from Input Process, aligning them with the new conditions. The approach was validated on Chinese Hamster Ovary cell cultures, targeting critical metabolic parameters such as glucose, lactate, glutamine, and viable cell density. The results showed that with only one batch used for the transfer, the average relative errors compared to offline values were around 10% for glucose and lactate but remained high for VCD and glutamine. After a second batch to perform the transfer on two batches, the relative errors were further reduced below 10% for most parameters. By effectively transferring models across different processes, this approach minimizes the need for extensive recalibrations, enhancing the efficiency and applicability of Raman spectroscopy in diverse bioprocess environments.
Evaluation of gene editing in CHO cells using the Cas-CLOVER system.
McLamarrah T, Aral E, Hoffman M … +11 more , Tedstone J, King T, Vitko J, Sebastião MJ, Escandell JM, Dias MM, McCall I, Machado D, Cairns V, DeMaria C, Scarcelli JJ
Biotechnol Prog · 2026 · PMID 41656178 · Full text
Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study,...
Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.
Enhancing mRNA vaccine production: Optimization of in vitro transcription for improved yield and purity.
Zhao K, Raffaele J, Holland DA … +4 more , Feibelman K, Lengel K, Shanter J, Wen E
Biotechnol Prog · 2026 · PMID 41612912 · Publisher ↗
In vitro transcription (IVT) is a powerful method to generate RNA which not only facilitates RNA research but also plays a key role in the development and manufacture of RNA-based vaccines. mRNA is produced via the IVT p...
In vitro transcription (IVT) is a powerful method to generate RNA which not only facilitates RNA research but also plays a key role in the development and manufacture of RNA-based vaccines. mRNA is produced via the IVT process with a DNA template that contains information for the target antigen. However, as many disease-causing viruses mutate quickly and the cost of raw materials is high for the IVT reaction, there is a need for a system to develop a cost-effective and efficient IVT process platform. In this paper, we showed how total nucleoside-5'-triphosphates (NTPs) input, Mg concentration and NTP preparation methods can influence IVT reaction yield and purity level of the final RNA constructs of different lengths and sequences. We propose an IVT design that will result in high RNA yield, high RNA integrity and low double-stranded RNA (dsRNA) concentrations for multiple RNA sequences. The approach presented here could significantly contribute to the development of a cost-effective, easy-to-adopt IVT process platform for RNA manufacturing.
CRISPR-Cas9 lipid nanoparticles for targeting cyclin-dependent kinases in the tumor microenvironment.
Mashreghi M, Rezazade Bazaz M, Jaafari MR
Biotechnol Prog · 2026 · PMID 41606801 · Publisher ↗
Due to the significant roles of cyclin-dependent kinases 4 and 6 (CDK4/6) in cancer progression, this study aimed to introduce clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein...
Due to the significant roles of cyclin-dependent kinases 4 and 6 (CDK4/6) in cancer progression, this study aimed to introduce clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) plasmid DNA (pDNA) encapsulated in lipid nanoparticles (LNPs) as a novel CDK4/6 inhibitor using a gene knock-out strategy for treating cancer. pDNA-LNP was prepared and characterized using a microfluidic system. The results indicated the hydrodynamic diameter of the pDNA-LNP was 90.0 ± 0.1 nm with the PDI of 0.1 and a negative zeta-potential. The cytotoxicity results demonstrated statistically significant differences at doses of 0.250, 500, and 1 μg of pDNA with the capabilities of pDNA-LNP in the induction of apoptosis, as depicted by the Annexin-V-FITC-PI method. Real-time quantitative PCR (qPCR) also indicated a significant reduction in the expression levels of both CDK4 and 6 in the cells that were treated with pDNA-LNP. The in vivo anti-tumor activities of pDNA-LNP have demonstrated that the formulation has the potential to decrease tumor size and improve survival parameters, including median survival time (MST), which was increased from 31 days for the PBS group to 51 days for the pDNA-LNP group at 0.5 μg. On the other hand, the dose of 1 μg had shown signs of toxicity, indicating the need to optimize dosing in future studies. In summary, these findings indicate that CRISPR-Cas9 encapsulated in the LNP can suppress tumor growth and offer a promising strategy for future cancer treatment approaches.
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