Biotechnol. Prog. [JOURNAL]
Sun
200 papers
Purification and concentration of model viruses using single-pass tangential flow filtration.
Mejía-Manzano LA, Manchester L, Wallis G … +4 more , Wang K, Lee BP, Betenbaugh M, Heldt CL
Biotechnol Prog · 2026 Jul · PMID 42400311 · Publisher ↗
The vaccine and viral vector industry is growing at an accelerated rate. To improve harvest and purification processes, the development of continuous membrane-based operations, such as normal flow filtration (NFF) and si...
The vaccine and viral vector industry is growing at an accelerated rate. To improve harvest and purification processes, the development of continuous membrane-based operations, such as normal flow filtration (NFF) and single pass tangential flow filtration (SPTFF) for concentration were explored. This work was conducted using two model viruses, non-enveloped porcine parvovirus (PPV) and enveloped Suid herpesvirus (SuHV). The viruses are in the same family as the gene therapy vectors adeno associated virus and herpes simplex virus, respectively. SPTFF design started with batch TFF for membrane selection. Hollow fiber membranes with a 100 and 300 kDa molecular weight cut off were defined for PPV and SuHV SPTFF operations, respectively. The SPTFF runs for PPV did not provide any concentration of the virus and low protein and DNA removal, unlike batch TFF. Two hollow fiber membranes run at 10 mL/min and 2 psi were the best condition for SuHV concentration, with approximately 100-fold titer concentration and protein and DNA removal of 37% ± 5% and 32% ± 8%, respectively. This concentration was superior to the batch TFF and indicated a strong dependence on flow rate and transmembrane pressure. For NFF, filters selection and performance tests were carried out for NFF of PPV and SuHV, as well as cleaning protocols for hollow fiber membranes. The ultimate goal is to integrate this work into the continuous purification of viral vectors produced in mammalian cell cultures to reduce costs and increase throughput.
Advanced glucose control strategies leveraging Raman spectroscopy for optimized mammalian cell culture manufacturing.
Banner M, Ray D, Ahanger I … +10 more , Zurlo F, Hitchcock N, Mistry R, Kozakowska-McDonnell K, Cheeks M, Turner R, Welsh J, Farid SS, Thomas M, Goldrick S
Biotechnol Prog · 2026 Jun · PMID 42374623 · Publisher ↗
Maintaining consistent quality in the manufacturing of biotherapeutic proteins in mammalian cell culture is challenging, with unplanned deviations causing inconsistencies and potential batch failure. Current methods for...
Maintaining consistent quality in the manufacturing of biotherapeutic proteins in mammalian cell culture is challenging, with unplanned deviations causing inconsistencies and potential batch failure. Current methods for monitoring and controlling critical process parameters (CPPs) rely on slow, labor-intensive offline analyses. This is particularly problematic in upstream manufacturing, where infrequent measurements hinder real-time CPP monitoring and result in suboptimal control. This study developed and implemented a robust, standardized framework that integrates Raman spectroscopy with real-time machine learning models and bioreactor control via OPC-UA, enabling seamless communication and effective control of mammalian cell culture CPPs. High-accuracy ML models (R >0.92) enabled real-time monitoring of glucose, lactate, viable cell density, and antibody titre. The glucose model was integrated into an automated process analytical technology (PAT) control strategy to maintain glucose at a predefined setpoint. The PAT strategy was compared to a manual bolus glucose control under high and low glucose feeding regimes. The PAT control approach observed an increase in biotherapeutic product titre by up to 35% and reduced glycation by up to 27%. These results indicated that both glucose setpoints and fluctuations impacted cell culture performance. In conclusion, this study highlighted the value of PAT tools in automating control loops, consequently improving process performance with enhanced productivity and quality.
Mechanistic deconvolution of BSA size variants by constrained Raman pseudo-Voigt hard modeling during anion-exchange chromatography.
Heyer-Müller J, Schiemer R, Robbel L … +2 more , Schmitt M, Hubbuch J
Biotechnol Prog · 2026 Jun · PMID 42357842 · Publisher ↗
In biopharmaceutical manufacturing, protein aggregation is a critical quality attribute, necessitating rapid and reliable analytical strategies during downstream processes like anion-exchange chromatography (AEX). While...
In biopharmaceutical manufacturing, protein aggregation is a critical quality attribute, necessitating rapid and reliable analytical strategies during downstream processes like anion-exchange chromatography (AEX). While Raman spectroscopy enables continuous monitoring of protein secondary structure, standard data-driven regression models struggle to decouple intrinsic structural changes from gradient-induced solvent and buffer drifts under dynamic chromatographic conditions. Addressing this methodological gap, this study establishes a constrained pseudo-Voigt hard modeling framework for the mechanistic deconvolution of bovine serum albumin (BSA) size variants during in-line Raman monitoring of AEX processes. By explicitly defining a parametric background model to capture salt-induced spectral drift, the methodology effectively isolates matrix variations from genuine protein-specific signals. The constrained hard model was applied to 285 in-line spectra across diverse chromatographic conditions, achieving reconstruction fidelity while maintaining stable, physically interpretable component identities. The mechanistically derived Amide I center of mass emerged as a robust, aggregation-sensitive descriptor that preserves structural information despite strong concentration dynamics. Furthermore, the extracted spectral features demonstrated strong predictive performance for monomer concentration and acceptable accuracy for high molecular weight components. Collectively, these results demonstrate that constrained spectral hard modeling provides a highly interpretable, robust, and calibration-light alternative to classical partial least squares approaches for the real-time monitoring of protein size variants.
Status and future of recombinant adeno-associated virus vector manufacturing.
Agbogbo F, Dismuke D
Biotechnol Prog · 2026 Jun · PMID 42339583 · Publisher ↗
Sixty years of adeno-associated virus (AAV) research illustrates a trajectory marked by basic science exploration, iterative innovation, persistent challenges, a number of clinical setbacks, as well as commercial therape...
Sixty years of adeno-associated virus (AAV) research illustrates a trajectory marked by basic science exploration, iterative innovation, persistent challenges, a number of clinical setbacks, as well as commercial therapeutic triumphs. This continual evolution has led to recombinant AAV (rAAV) becoming a cornerstone of modern gene therapy. Significant advancements in molecular design, process development, and manufacturing have been made over the past three decades; these improvements are expected to significantly improve the safety, efficacy, and economics of rAAV gene therapies. Beyond rare disease, rAAV vectors have the potential to be used in prevalent conditions such as arthritis, heart failure, diabetes, Alzheimer's disease, and Parkinson's disease. Meeting the vector demands of these disease treatments will require continued innovations in rAAV manufacturing, including further improvements in process optimization and molecular engineering. In addition, the adoption of process intensification and automation strategies, pioneered in other biologics such as monoclonal antibody manufacturing, should prove pivotal in advancing the scale, robustness, and efficiency of rAAV production.
Multifaceted algae as an ingredient in alternative meat formulations.
Phadnis G, Prakash G
Biotechnol Prog · 2026 Jun · PMID 42332354 · Publisher ↗
Rising global meat demand and nutritional awareness have fuelled interest in sustainable, ethical protein sources. Animal agriculture generates greenhouse gas emissions, land degradation, and water scarcity, creating a n...
Rising global meat demand and nutritional awareness have fuelled interest in sustainable, ethical protein sources. Animal agriculture generates greenhouse gas emissions, land degradation, and water scarcity, creating a need for plant-based meat alternatives. While plant sources face drawbacks such as incomplete amino acid profiles, anti-nutritional factors, and land requirements. Algae emerge as a superior option, delivering exceptionally high protein content (up to 70% dry weight), complete essential amino acids, omega-3 fatty acids, vitamins, polysaccharides, and potent antioxidants, surpassing plant sources in nutrient density, bioavailability, and environmental footprint. This review evaluates the nutritional, environmental, and technological potential of key algal species (microalgae and macroalgae) for meat substitute applications. Algal formulations excel over plant-based counterparts with superior protein quality (PDCAAS >0.9 vs. often <0.8 for plants), rapid biomass growth (10-50× faster than plants), and no arable land requirements, enabling scalable, low-water production. The review addresses challenges such as off-flavors, digestibility, and cost through solutions such as strain selection, biorefinery optimization, and hybrid cultivation systems. An overview of key market players highlights the growing role of algae in alternative meats. By integrating nutritional and industrial perspectives, this work reveals trends positioning algae at the forefront for health-conscious consumers, advocating a "best-of-everything" approach with diverse species to revolutionize sustainable food systems.
In-line Raman spectroscopy real-time glucose prediction method for commercial pneumococcal vaccine drug substance fermentation manufacturing process control.
Thomas GP, Marks BC, Alvarado-Hernandez BB … +5 more , Woodling M, O'Connor J, Ortiz R, Higgins J, Shanter J
Biotechnol Prog · 2026 Jun · PMID 42324489 · Publisher ↗
Traditional off-line analysis methods to quantify residual metabolite concentrations in a drug substance fermentation process consume valuable time, require costly resources, and demand potentially hazardous manual opera...
Traditional off-line analysis methods to quantify residual metabolite concentrations in a drug substance fermentation process consume valuable time, require costly resources, and demand potentially hazardous manual operations when working with pathogenic biological organisms. This case study focuses on the successful method development and validation of Raman spectroscopy as an in-line process analytical technology (PAT) to replace the existing off-line assay for real-time glucose quantification. The value and feasibility of Raman spectroscopy as an in situ PAT method are recognized and demonstrated as an enabling technology suited to mitigate challenges fundamental to lab-scale process development and commercial manufacturing. Herein, application of chemometric techniques and multivariate analysis of Raman spectral data is described to develop a partial least squares model capable of predicting real-time and accurate glucose concentrations with a root mean square error of prediction of 1.2 g/L. Through this work, the predictive Raman PAT glucose quantification method was validated to make process decisions in a commercially licensed Pneumococcal vaccine drug substance manufacturing facility for the first time at our company. The method was integral to the process control strategy and applied to accurately and robustly indicate the glucose concentration target ± 2 g/L for triggering subsequent process operations. Several advantages were realized through this work including advanced process control, reduced operating costs, and improved safety posture.
Prolonged autophagy induction correlates with host cell protein reduction in CHO cell culture.
Sahoo A, Tsukidate T, Pendyala G … +3 more , Yang RS, Li X, Madabhushi SR
Biotechnol Prog · 2026 Jun · PMID 42322119 · Publisher ↗
Autophagy, a cellular recycling process regulated by the CLEAR signaling pathway, plays a pivotal role in maintaining cellular homeostasis. We hypothesize that this process may regulate and reduce high-risk host cell pro...
Autophagy, a cellular recycling process regulated by the CLEAR signaling pathway, plays a pivotal role in maintaining cellular homeostasis. We hypothesize that this process may regulate and reduce high-risk host cell proteins (HCPs) levels by targeting intracellular proteins and organelles for degradation. This study investigates the relationship between autophagy induction and the reduction of HCPs, including polysorbate-degrading enzymes (PSDEs), to enhance the stability of therapeutic biologics such as monoclonal antibodies (mAbs). Using clonal analysis, we identified upregulation of the CLEAR pathway in one clone, correlating with a significant reduction in lipase activity and PSDE abundance. Furthermore, autophagy modulators, such as 3-methyladenine (3-MA), selectively decreased PSDE levels in both glucose-supplemented batch culture and fed-batch cultures. This resulted in a 62% reduction in lipase activity that corresponded to a 22% improvement in polysorbate-80 stability. Additionally, 3-MA treatment increased mAb specific productivity and altered glycosylation profiles, increasing afucosylation and galactosylation levels. These findings highlight autophagy induction as a promising strategy to modulate product quality profiles and reduce high-risk HCPs in biologics production.
Viscosity of concentrated mRNA solutions: Implications for downstream processing.
Javidanbardan A, Najafi F, Wang Z … +1 more , Zydney AL
Biotechnol Prog · 2026 Jun · PMID 42295144 · Publisher ↗
Messenger RNA (mRNA) therapeutics can be used to protect against infectious disease and for the treatment of cancers and genetic disorders. In order to obtain the desired therapeutic dosage, it is necessary to concentrat...
Messenger RNA (mRNA) therapeutics can be used to protect against infectious disease and for the treatment of cancers and genetic disorders. In order to obtain the desired therapeutic dosage, it is necessary to concentrate the mRNA to levels where the viscosity of the mRNA solutions can become significant. However, there is currently no quantitative data on the viscosity of these concentrated mRNA solutions. Experiments were performed with two model mRNA constructs: a firefly luciferase (Fluc) mRNA (2.117 kb) and a self-amplifying RNA (9.442 kb) over a wide range of concentrations. The viscosity of the larger saRNA was more than 140 mPa·s at a concentration of 28 mg/mL, which is more than 10× larger than that for the Fluc mRNA under the same conditions. The viscosity was a weak function of shear rate (from 10 to 100 s), with some shear-thinning behavior. The viscosity data were analyzed using several available models from the literature, providing additional insights into the underlying physical behavior. The potential implications of the mRNA viscosity in the downstream processing of mRNA therapeutics are also discussed.
Characterization and immunogenicity of nanoparticle vaccines displaying embecovirus spike proteins.
Halfmann PJ, Duffy A, Wee A … +4 more , Anderson N, Terrell J, Kawaoka Y, Kane RS
Biotechnol Prog · 2026 Jun · PMID 42290307 · Publisher ↗
Endemic human coronaviruses OC43 and HKU1 cause widespread respiratory infections and can be associated with severe illness in immunocompromised and elderly individuals. Frequent adaptive evolution in the spike proteins...
Endemic human coronaviruses OC43 and HKU1 cause widespread respiratory infections and can be associated with severe illness in immunocompromised and elderly individuals. Frequent adaptive evolution in the spike proteins of these embecoviruses and the potential for zoonotic transmission from a large animal reservoir necessitates the characterization of the immunogenic landscape of the spike proteins of embecoviruses. Here, we constructed nanoparticle vaccines displaying the spike antigens from OC43, HKU1 A, or HKU1 B, as well as a bivalent formulation incorporating spike antigens from OC43 and HKU1 A. Immunization of mice elicited spike-specific IgG antibody responses, with endpoint titers demonstrating cross-reactivity among clade-matched viruses. Notably, the bivalent formulation elicited antibody responses comparable to those of monovalent vaccines against matched antigens. These findings inform future design of vaccines against human-infecting embecoviruses and could serve as an important step toward a universal vaccine against common cold causing coronaviruses.
A straightforward approach for converting existing batch production to an integrated continuous manufacturing suite by adopting membrane-based technology.
Fang M, Shi C, Zhou A … +4 more , Zhu H, Zhou H, Gu S, Zhou W
Biotechnol Prog · 2026 Jun · PMID 42272058 · Publisher ↗
The rapidly developing antibody market poses challenges to the production cost and efficiency, which can be addressed through continuous process. However, a hurdle of high capital investment remains a concern especially...
The rapidly developing antibody market poses challenges to the production cost and efficiency, which can be addressed through continuous process. However, a hurdle of high capital investment remains a concern especially for the company with existing fed-batch capacity. This study proposes a straightforward approach to convert existing batch production suites into continuous ones using membrane-based technology. A conceptual validation was conducted at a 100 L pilot scale, and a head-to-head comparison was carried out. The results demonstrated that Protein A membrane chromatography could achieve a productivity 6.3 times higher than that of periodic counter-current chromatography (PCC), without requiring the purchase of any dedicated equipment. Using Q membrane, compared with traditional column anion exchange chromatography, can achieve a 16-fold increase in load capacity and a 25-fold increase in productivity. Finally, VF-UF/DF (Virus Filtration-Ultrafiltration/diafiltration) linkage process was designed, resulting in a 20% reduction in process time. During the 23-day upstream perfusion and 15-day downstream processing, the cumulative titer reached 51.0 g/L, with an 83.1% yield in downstream processing. With downstream process time shortening from 4 to 1.5 days, the process capacity could be improved by 8 times using the existing batch production suites and the same facility area. PATs, including ultra-performance liquid chromatography, Raman spectroscopy, FlowVPX, and an inline turbidity meter, were integrated to monitor process CQAs (Critical Quality Attributes): monomer purity, protein concentration, and turbidity, to enable efficient process control in the future.
Endotoxin removal by virus filters.
Wang C, Wang S, Peng S … +1 more , Fei L
Biotechnol Prog · 2026 Jun · PMID 42252523 · Publisher ↗
Endotoxins pose a significant risk to drug product safety due to their potent pyrogenic activity. However, developing a universal decontamination method for protein solutions remains challenging, as current technologies...
Endotoxins pose a significant risk to drug product safety due to their potent pyrogenic activity. However, developing a universal decontamination method for protein solutions remains challenging, as current technologies struggle to reconcile efficient endotoxin removal with high product recovery and operational simplicity. This study explores the novel application of commercial virus filters for endotoxin clearance. We systematically evaluated filters from multiple manufacturers across a range of initial endotoxin concentrations and buffer conditions. The results demonstrate that Planova BioEX and Valpha PES virus filters reduced endotoxin levels by 88%-99% at initial endotoxin concentrations below 5 EU/mg of a therapeutic monoclonal antibody, while achieving near-complete protein recovery (>95%). A comparative study revealed that under low pH conditions (pH 5.5), virus filters outperformed Mustang E and Mustang Q membranes in removing low levels of endotoxin. Under high pH conditions (pH 7.5), their performance was comparable. This difference likely stems from distinct removal mechanisms: Mustang membranes rely on electrostatic interactions, whereas virus filters (~20 nm pore size) operate via size exclusion. Dynamic light scattering results indicated the majority of the mAb-endotoxin complexes exceeded 20 nm, supporting the size-based removal mechanism of virus filters. These findings demonstrate that virus filtration has the potential to serve as a viable alternative for endotoxin removal in preclinical protein processing, particularly for high-pH sensitive proteins and acidic proteins. It also represents a valuable addition to the endotoxin clearance toolbox for clinical manufacturing.
A mechanistic model for recombinant adeno-associated virus production dynamics in a helper virus-assisted producer cell line.
Sha S, Hossler P, Abrahamyan A … +1 more , Warren J
Biotechnol Prog · 2026 Jun · PMID 42244226 · Publisher ↗
Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo gene therapy, with over 200 clinical trials currently underway worldwide. Achieving a manufacturing process that is sufficiently...
Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo gene therapy, with over 200 clinical trials currently underway worldwide. Achieving a manufacturing process that is sufficiently productive with the requisite product quality is critically important for commercialization success. Mammalian producer cell lines (PCLs) are stably transfected to integrate AAV structural and non-structural genes and are frequently combined with infection by wild-type adenovirus (Ad5) to provide helper elements. Within the PCL, both Ad5 amplification and rAAV production occur. During AAV manufacturing with PCLs, the upstream process targets the generation of high rAAV titers and full-capsid percentages. This is followed by a downstream process designed to effectively clear Ad5 and other process-related impurities, while further enriching full rAAV capsids. To explore potential improvements at the cellular level, we developed a mechanistic model based on empirical cell culture data, capturing intracellular dynamics of rAAV assembly and Ad5 amplification. Sensitivity analysis using this model identified cellular processes associated with rAAV yield and full-particle percentage. Notably, the analysis confirmed the critical influence of Ad5 amplification on both rAAV yield and quality. Further analysis, including nonlinear optimization, identified additional targets for cell line and helper virus engineering to evaluate process performance.
Continuous concentration and diafiltration tangential flow filtration with scalable dual membrane technology.
Helling A, Borujeni EE, Leuthold M … +3 more , Tindal S, Fernandez-Cerezo L, Brower M
Biotechnol Prog · 2026 Jun · PMID 42231136 · Publisher ↗
Intensified, integrated, and continuous bioprocessing are key trends in current biologics biomanufacturing. They model more efficient and economical production in upstream and downstream processes. Continuous or single-p...
Intensified, integrated, and continuous bioprocessing are key trends in current biologics biomanufacturing. They model more efficient and economical production in upstream and downstream processes. Continuous or single-pass (SP) tangential flow filtration (TFF) and particularly diafiltration (buffer exchange) are key applications needed in downstream processing. This work presents a novel flat sheet membrane cassette for TFF diafiltration with optional simultaneous ultrafiltration (concentration). It enables effective buffer exchange of feed streams in a single-pass, without dilution and with reduced shear stress to the valuable product. Contaminants such as salts, media components, or any small molecules smaller than the respective membrane cut-off are continuously removed while the needed buffer volume is comparable to conventional diafiltration processes with recirculation. The flat-sheet membrane cassette design resembles classic TFF cassettes and can therefore be scaled up and down and handled analogously. In addition, the design allows for a closed configuration, suitable for aseptic or bioburden-controlled conditions. To introduce the buffer, only one additional external connection port is required. This allows continuous diafiltration without significant increase in operator or process control complexity. This work presents ultrafiltration and diafiltration application data using bovine serum albumin (BSA) as a model protein and real antibodies processed with an automated lab-scale process system for reproducible data collection. The process performance was evaluated based on buffer exchange efficiency, transmembrane pressure (TMP) and permeate flux rates under varying process parameters, namely feed protein concentration, feed flow rate and diafiltration volumes. Buffer consumptions similar to conventional recirculation diafiltration have been observed with even reduced buffer consumptions at diafiltration volumes below 3. Buffer exchange values ranged over a broad range depending on the process parameters while relevant settings achieved a ~97 to >99% buffer exchange at, for example, 3, 5 or 7 diafiltration volumes at relatively low TMP between 50 and 350 mbar using both BSA and mAb. Typical permeability values and feed flow rates ranged from 10 to 60 L/(mh) and 1 to 7 L/(mh), respectively, using BSA and mAb protein concentrations from ~3 to 80 g/L and were significantly depending on the set process parameters. The data demonstrates how this new SPTFF concept can contribute to intensified and continuous biomanufacturing.
Generation of high-expressing transposon-based stable pools to produce (1 + 1) and (2 + 1) bispecific common light chain antibodies.
Ramos S, Karunakaran S, Monney T … +7 more , Bornert O, Reimann A, Boldog F, Dyson M, Srivastava A, Minshull J, Caro LN
Biotechnol Prog · 2026 Jun · PMID 42230311 · Publisher ↗
Bi- and multispecific antibodies can deliver effective protein-based therapeutics by enabling novel biology. Transient expression of these complex asymmetric antibody formats may become a bottleneck in providing sufficie...
Bi- and multispecific antibodies can deliver effective protein-based therapeutics by enabling novel biology. Transient expression of these complex asymmetric antibody formats may become a bottleneck in providing sufficient amounts of high-quality material for preclinical discovery and developability assessment to support candidate selection activities. In contrast, transposase-based stable cell line generation is being recognized as a robust and rapid path to enable the evaluation of product quality from stable pool derived material. Transposase-based systems feature a semi-targeted integration of multiple copies of the gene(s) of interest in the host cell's genome resulting in the rapid generation of homogeneous high-expressing (up to g/L range) stable pools after 1-3 weeks of selection phase. Here, we evaluated the Leap-In® transposase technology for the expression of IGI's 1 + 1 and 2 + 1 bispecific, common light chain antibodies, based on IGI's proprietary BEAT® (Bispecific Engagement of Antibodies based on T cell Receptor) platform. To generate stable CHO pools expressing IGI's bispecific molecules, transposon-based constructs expressing the common light chain and the two heavy chain genes were co-transfected with the Leap-In® transposase mRNA. Based on the proportion of the different chain combinations detected in the harvests, their expression levels were further optimized through vector engineering to facilitate heterodimer assembly and maximize productivity. The best pool displayed a titer of 2.33 g/L in 14-day fed-batch cultures, and heterodimer represented 93.8% of the expressed species. The purified antibody displayed 97.4% purity, as determined by SE-HPLC and binding to targets within range, as determined by SPR and cell-based affinity.
Binding of bacteria to magnetic nanoparticles in suspension: Experiments and kinetic models.
Houser BJ, Bryner CA, Rappleyea AD … +6 more , Forstrom BL, Camacho AN, Wood JB, Harrison RG, Chesnel K, Pitt WG
Biotechnol Prog · 2026 May · PMID 42174394 · Publisher ↗
With the rise of antimicrobial-resistant and multi-drug-resistant bacteria, the use of magnetic nanoparticles (MNPs) has become a promising tool for separation processes in conjunction with in vitro diagnostics for the r...
With the rise of antimicrobial-resistant and multi-drug-resistant bacteria, the use of magnetic nanoparticles (MNPs) has become a promising tool for separation processes in conjunction with in vitro diagnostics for the rapid identification of bacterial species in various medical and biological fluids. However, there are few studies on the time required for MNPs to successfully capture and remove bacteria from suspensions. This study presents two mathematical models that describe the kinetics of bacterial capture using polydopamine-coated MNPs (pDA-MNPs) in phosphate-buffered saline. The model parameters are derived from data from the capture of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans, which exhibit high adherence to pDA-MNPs. Results show that during the first 3 min, bacterial capture and removal by magnetic particles show second-order kinetics overall, and first order each with respect to bacterial concentration and to magnetic particle concentration. At longer times, the data are better fit by a model of fast and slow parallel capture mechanisms. pDA-MNPs capture S. epidermidis faster than S. aureus, and capture S. mutans more slowly than the other bacterial species. Rate constants derived at very high bacterial concentrations (~10 CFU/mL) were also found to fit experimental data collected at very low concentrations (~750 CFU/mL), showing applicability of the model spanning several orders of magnitude in bacterial concentration. Analysis of the kinetic parameters provides insights into optimizing the time and MNP concentrations necessary to achieve a desired bacterial capture efficiency in medical or environmental diagnostic applications.
Machine learning-assisted clone selection for intensified cell culture processes.
Wolnick N, Schmitt J, Siltanen C … +4 more , Armstrong J, Hendrick S, Atchley A, Downey B
Biotechnol Prog · 2026 May · PMID 42169499 · Publisher ↗
Intensified fed-batch processes are becoming increasingly prevalent among biomanufacturers due to their superior space-time yields relative to traditional, non-intensified fed-batch processes. However, the shift towards...
Intensified fed-batch processes are becoming increasingly prevalent among biomanufacturers due to their superior space-time yields relative to traditional, non-intensified fed-batch processes. However, the shift towards intensified manufacturing has unexpectedly made optimal clone selection more challenging. Clone selection, traditionally an empirical screening of candidates on the basis of productivity, has been complicated by the observation that clonal productivity rankings can vary substantially between intensified and non-intensified processes. In the absence of a small-scale intensified empirical screen, clones destined for intensified manufacturing are selected based on their performance in the non-intensified process. This mismatch risks selection of suboptimal clones, representing a missed opportunity to generate additional product at no extra cost. This study presents a machine learning approach to improve clone selection for intensified processes by predicting and ranking clonal productivity. Models were trained on non-intensified clone performance data routinely collected during cell line development to facilitate seamless integration into existing capabilities. To evaluate generalizability and simulate real-world application, independent clone panels expressing two distinct monoclonal antibodies were withheld from model training and used in comparative testing against the legacy clone selection method. The ranking model identified the most productive clone of those expressing the first mAb, representing a 68.5% higher titer than the clone selected by the legacy method. For the second mAb, the ranking model identified the second most productive clone, resulting in a marginally lower titer. These results demonstrate the potential of machine learning models as practical tools for improving the selection of high productivity clones in intensified bioprocessing.
β-1,4-galactosyltransferase 1 inhibitors modulate cellular N-glycan profiles.
Kranjc J, Pajk S, Anderluh M … +2 more , Pišlar A, Brinc M
Biotechnol Prog · 2026 May · PMID 42165174 · Publisher ↗
N-glycosylation is a key post-translational modification of proteins influencing their physicochemical properties as well as biological activity. This study investigates the in vivo activity of four β-1,4-galactosyltrans...
N-glycosylation is a key post-translational modification of proteins influencing their physicochemical properties as well as biological activity. This study investigates the in vivo activity of four β-1,4-galactosyltransferase 1 inhibitors in cell cultures producing recombinant IgG antibodies. Inhibitors, previously identified as potent inhibitors in on-target assay, were evaluated in fed-batch bioprocesses. Dose-dependent effects on cell culture performance were assessed following compound addition. N-glycan profiles were analyzed using InstantPC labeling and UHPLC-FLD, while intracellular compound concentrations were quantified via LC-MS/MS following time-resolved sampling of treated cell lysates. Bioprocess parameters, including cell growth, viability, and productivity, were monitored throughout cultivation. The study revealed divergent behaviors between on-target potency and in vivo activity, underscoring the importance of cellular permeability, metabolic stability, and off-target effects of potential inhibitors used in cell culture. These findings provide new insights into the challenges of glycosylation pathway modulation and inform strategies for future glycoengineering tool development.
Multivariate phase-dependent optimization of bioprocesses boosts performance and quality-Why timing (of exposure) matters.
Kienzle S, Junghans L, Wittmann A … +5 more , Wieschalka S, Takors R, Radde NE, Presser B, Nold V
Biotechnol Prog · 2026 May · PMID 42144897 · Publisher ↗
Applying a single parameter set to describe complex mammalian kinetics often is too simplistic, as it fails to capture sensitive cell-to-environment interactions that may be exploited to optimize production performance....
Applying a single parameter set to describe complex mammalian kinetics often is too simplistic, as it fails to capture sensitive cell-to-environment interactions that may be exploited to optimize production performance. To resolve this time dependency, intra-experimental parameter shifts as part of design of dynamic experiments (DoDE) can be performed to study mammalian growth and production kinetics in fed-batch processes. This enables growth phase-dependent optimization, aligned with cellular requirements. Here, we provide a comprehensive, head-to-head comparison of our phase-dependent optimization approach with intra-experimental shifts of process parameters to a static optimization that retains parameter settings through the entire bioprocess. Showcasing a monoclonal antibody production process development scenario, the study examines growth phase-dependent effects of temperature (T) and dissolved oxygen (DO) together with time-invariant parameters for feed and seeding cell density. While the static optimization suggests settings near the center of the design space, phase-dependent optimization finds an optimum by shifting T and DO between the exponential growth, transition, and production phases. Overall, the phase-wise optimized process gives an experimentally validated ~30% increase in product titer while maintaining comparable product quality. Furthermore, the approach breaks the correlation between product titer and acidic charged variants: both depend on T but at different timeframes. Additionally, DoDE uncovers a crucial interaction between T and DO, with low T and high DO during the exponential growth phase, leading to strong lactate accumulation. The data demonstrate the advantages of phase-dependent optimization enabled by DoDE. The results may serve as a good practice example for follow-up research.
NS0 and CHO cell lines generate distinct quality profiles for non-originator NISTmAbs.
Bush X, Azer N, Kim J … +2 more , Powers DN, Fratz-Berilla EJ
Biotechnol Prog · 2026 May · PMID 42129584 · Publisher ↗
The selection of host cell lines and culture media plays a central role in influencing the quality attributes of monoclonal antibodies, particularly in biosimilar development and process comparability. This study used st...
The selection of host cell lines and culture media plays a central role in influencing the quality attributes of monoclonal antibodies, particularly in biosimilar development and process comparability. This study used standardized upstream conditions to evaluate the impact of host cell line selection on product quality attributes of a non-originator version of the NISTmAb by comparing results from antibodies made in murine myeloma NS0 or Chinese Hamster Ovary (CHO) cells. NISTCHO and NS0 clones achieved comparable peak density and productivity when cultured identically, demonstrating the critical role of media formulation for NISTCHO performance. Distinct cell line-specific differences were noted in aggregation, fragmentation, charge variant distribution, and glycosylation profiles. Non-originator NISTmAb derived from NS0 cells exhibited glycoforms that were nearly 100% fucosylated with higher relative levels of sialylation and galactosylation. The NS0-derived material also displayed elevated levels of acidic charge variant species, decreased main charge variant species, and increased aggregation. Conversely, non-originator NISTmAb derived from NISTCHO exhibited an average of 15-20% relative afucosylation, resulting in an increased binding affinity to FcγRIIIa, which can be of critical importance to quality due to potential enhancement of ADCC potency. These results highlight the intrinsic biology of the host cell line and culture media composition as influential factors for microheterogeneity in post-translational modifications and structural characteristics. As of December 2025, 14 (17.3%) of FDA-approved biosimilars are manufactured using a different cell type than that of the originator product, necessitating comparisons among cell substrates and their effect on product quality of interest for comparative analytical assessment.
Improved methodology for direct monitoring of large-scale manufacturing cell culture using Raman spectroscopy data from process development.
El Radi H, Chen H, Su X … +6 more , Hu W, Zhang P, Jin L, Cao X, Wang H, Lu J
Biotechnol Prog · 2026 · PMID 42125845 · Publisher ↗
Process analytical technology (PAT) is a framework that encourages the implementation of analytical tools for real-time monitoring of critical batch components during the production of pharmaceuticals. In mammalian cell...
Process analytical technology (PAT) is a framework that encourages the implementation of analytical tools for real-time monitoring of critical batch components during the production of pharmaceuticals. In mammalian cell cultures, Raman spectroscopy as a PAT tool has demonstrated its value for accurate in-line monitoring of various process parameters as well as quality attributes. However, as process development cultures are transferred to manufacturing scales, the small-scale models are often adjusted either with new calibration data or with transfer learning algorithms. This model adjustment step makes the overall process transfer to manufacturing less straightforward from an analytical standpoint, requiring additional resources and specific data treatment expertise to fine tune the models. In this work, we compare a specific spectra processing approach against existing approaches reported in the literature for processing Raman spectra in the context of scale-up challenges. The proposed novelty is a preprocessing strategy which computes spectra normalization based on the 3100-3700 cm band. Data generated at 3 L scale were used to monitor a 3000 L bioreactor without model recalibration or transfer learning with equivalent accuracy as a same scale 3 L bioreactor monitoring. While broader validation case would be warranted, these results demonstrate a direct scale-up model implementation in large scale manufacturing under the studied conditions.
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