Biotechnology Progress最新文献

The urgent need to replace the European-prohibited Triton X-100 in biomanufacturing has been hindered by insufficient data on alternative detergents' minimum effective concentrations (MECs) and process robustness in viral inactivation. This study makes systematic research including: (1) Establishment of MECs for novel Triton X-100 substitutes (TXR-1/VIS/13-S9/C16) achieving effective inactivation of Xenotropic murine leukemia virus and Pseudorabies virus (log₁₀ reduction factor >4) across diverse CHO harvest fluids; (2) Demonstration of broad-spectrum efficacy against various viruses, with TXR-1/VIS/13-S9 maintaining effective inactivation for Bovine viral diarrhea virus, Vesicular stomatitis virus, Baculovirus, and Herpes simplex virus type 1; (3) Identification of PS20's material-dependent inactivation dynamics, establishing standalone parameters (4 h at 37°C) that achieve equivalent viral inactivation to traditional tri(n-butyl)phosphate -combined methods without requiring lipase activity-a paradigm shift in detergent application. Crucially, process optimization revealed that extending exposure time (1-4 h) enhanced PS20/PS80 efficacy more effectively than two fold concentration increases, providing cost-effective solutions. These findings deliver broader design spaces for implementing eco-friendly detergents while ensuring compliance with EMA/ICH viral safety standards.

Hydroxylysine (Hyl) is a post-translational hydroxyl modification of lysine that is not commonly observed at very high levels and thus is not usually considered a product quality attribute (PQA). Post-translation modifications (PTMs) are considered potential PQAs when elevated levels are observed – requiring monitoring and investigation. In a recent monoclonal antibody expression using Media A, Hyl levels were observed at ~20%–35%. At such elevated percentage levels, Hyl was considered a PQA – triggering a root-cause investigation in the upstream activities like cell culture conditions and media components. Initial detection of the Hyl modification originated from non-quantitative, intact mass analysis with confirmation of site-location determined by peptide mapping. Through the root-cause investigation, it was determined that levels of Hyl were underestimated by ~10-fold using tryptic peptide mapping analysis without inclusion of miscleaved peptides. The analytical procedure was revised from trypsin-digestion to IdeS-digestion, a reduced mass analysis, to accurately and rapidly quantify Hyl levels of investigational samples. Proprietary Media B was utilized to reduce the Hyl level by 2-fold to ~10%–15%. Further investigation into the media and feed components determined that increasing concentration of Fe(III) content decreased Hyl levels. Supplementation of Fe(III) served as a robust mitigation strategy of Hyl reduction in upstream process. Media B was used to scale up to a 500 L bioreactor while maintaining the lower Hyl level. The analytical and cell culture methods developed in this study can be leveraged to detect and tune Hyl levels.

Elastin-like polypeptides (ELPs) are recombinant protein-like polymers whose macromolecular structure can be precisely controlled through genetic manipulation of their sequence and length. Their lower critical solution temperature (LCST) phase behavior facilitates purification via chromatography-free techniques and can be explored for self-assembly. As a result, ELPs are extensively investigated for diverse biological, biomedical, and biotechnological applications. So far, ELPs have mostly been isolated from bacteria grown in flasks or fermenters containing complex media that only yield limited amounts of biomass. We herein explored the use of the semi-defined ECPM1 medium, known to limit the accumulation of toxic metabolites and rich in glycerol as a low energy carbon source, to produce ELPs of different chain lengths and containing oxidation-sensitive methionine residues. We report the optimized bioproduction using ECPM1 of ELP[M₁V₃-n] with n = 20, 40, 80 in a fermenter in good yields and confirm their intact protein sequence using various chemical characterization techniques.

In order to explore the separation and purification methods of uricase and alkaline protease crude enzyme extracts, a combinatorial approach of Three Phase Partitioning (TPP) and Ion-Exchange Chromatography (IEC) was studied. TPP alone was able to separate and purify uricase and alkaline protease enzymes by 5 fold and 2.7 fold, respectively. Further application of ion-exchange chromatography purified enzymes with 99.99% purity in the form of single peaks on a chromatogram. The optimum TPP parameters for simultaneous separation and purification were 50% ammonium sulfate concentration, crude extract to solvent ratio of 1:1, and pH of 8.5. Ion exchange chromatography was performed on a fully automated AKTA start system equipped with a conductivity detector, UV detector, fraction collector, and buffer reservoirs used for isocratic or gradient elution profiles of the proteins under study. Successful purification of enzymes, including their molecular weight, was confirmed with SDS PAGE analysis. Furthermore, the uricase sequence from Bacillus licheniformis was also corroborated to be 87.6% homologous to uricase of B. subtilis using the bioinformatics tool BLASTp (Basic Local Alignment Search Tool for Protein), wherein it compares sequence similarity based on protein or nucleotide sequence. It should be emphasized that this study is the first report for tandem enzyme purification of two enzymes using an automated IEC - AKTA start system where partial purification of enzymes was carried out using TPP.

Digital twins (DT) are sophisticated mathematical models representing real-world physical processes, equipped with predictive capabilities that adapt alongside the physical system. The successful implementation of DT in bioprocessing offers numerous advantages, including enhanced understanding of processes, accelerated overall development timelines, and effective monitoring of critical process parameters (CPPs). A comprehensive end-to-end DT can facilitate informed control decisions and forecast how disturbances within the process may affect the final output, accelerating the overall development timelines while optimizing process efficiency and productivity. Tangential flow filtration (TFF) is a standard methodology in bioprocessing, commonly employed to concentrate and exchange buffers for bioproducts. The advancement of continuous process technologies has led to the emergence of alternative TFF methods, notably single-pass tangential flow filtration (SPTFF), which streamlines the process by eliminating the need for stream recirculation. Here, we present the development of a live DT of the SPTFF concentration step within the downstream continuous manufacturing line for a monoclonal antibody (mAb) process. A live DT, equipped with a state estimation tool, was implemented via the Siemens' gPROMS Digital Applications (gDAP) platform. The DT demonstrated the ability to monitor changes in membrane resistance, a typical process parameter that is not directly measured. This parameter is crucial for SPTFF control, as it allows for the constant setting of the concentration factor (CF) by adjusting the retentate flow rate based on the measured resistance and calculated transmembrane pressure (TMP). This achievement illustrates the potential of DT as effective tools for accurately tracking the complete state of the bioprocess.

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.

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 MgCl₂. 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.

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.

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 R² > 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.

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.