Biotechnology Progress最新文献

Chemically defined cell culture media used in the growth of mammalian cells for biopharmaceutical applications is a complex mixture of various agents to promote cell growth and function. Poloxamer 188 (P188) is a well-known shear protectant added to media for use in CHO suspension culture but is not without drawbacks. This work explores the use of methylcellulose (MC), a well-known pharmaceutical polymer, in CHO media as an alternative and/or complementary additive to P188. IgG-producing DG44 CHO cell lines were cultured in a variety of suspension systems, up to 3 L reactors, to which MC and/or P188 were added. MC was an effective shear protectant in relatively lower shear systems but is less effective on its own in higher shear cultures. Across the range of conditions studied, MC and P188 were found to have a synergistic benefit with each other, where the combination of both additives produced cultures with higher viable cell densities than cultures containing either additive alone. These results indicate that MC is a viable option for use in media optimization studies as part of ongoing process intensification and optimization for CHO manufacturing.

Industrial fermentation continually improves biological process control for a wide range of microorganisms used in multi-billion-dollar industries including industrial enzymes, pharmaceuticals, foods, beverages, commodity chemicals, and bioenergy. In the case of recombinant protein production, batch and fed-batch phases of fermentation are usually followed by an induction phase, where chemical or thermal induction initiates the expression of a target protein. Fed-batch processes are usually automated, whereas “out-of-the-box” distributed control systems (DCS) are often unable to define the threshold for induction and respond accordingly. The present study demonstrates the integration of optical density (OD) process analytical technology (PAT) and Lucullus®, a process information management system (PIMS), to enable end-to-end automated fermentation at bench and pilot scale. Data aggregated from tens of fermenter runs and hundreds of offline training measurements enabled the development of an accurate multivariate model to predict OD in real-time. This eliminated the requirement to generate offline correlation models for each OD probe, allowed for model transfer, and incorporated additional predictor terms such as antifoam usage. Automating the induction phase enabled end-to-end fermentation, reducing labor and operational costs while increasing yield through higher reactor utilization within the same time period.

Efficient bioreactor clarification for harvesting virus particles is often challenging. Tangential flow filtration is attractive as it can be easily adapted for batch and perfusion operations. Here the feasibility of using reverse asymmetric hollow fiber membranes, where the more open support structure faces the feed stream, has been investigated for harvesting adeno associated virus serotype 2. The open support structure of these membranes stabilizes a secondary membrane consisting of rejected particulate matter. It is essential that the stabilized secondary membrane remains highly permeable. Flux stepping experiments were conducted in total recycle mode in order to determine the critical flux. The critical flux is the maximum stable flux. Higher fluxes lead to a rapid increase in transmembrane pressure under constant flux operation. The critical flux is shown to increase with increasing wall shear rate (feed flow rate). The reduction in turbidity of the permeate relative to the feed decreases with increasing wall shear rate. Harvesting adeno associated virus was conducted at a wall shear rate of 2000 s⁻¹. The permeate flux was set at 15 Lm⁻² h⁻¹. The feed was concentrated till the transmembrane pressure reached 3.5 kPa. Diafiltration then commenced using 3 diavolumes. While commencing diafiltration with a smaller feed volume will reduce diluent usage and dilution of the product, it is essential that the transmembrane pressure is not too high to create a compacted low permeability secondary membrane. Here the transmembrane pressure was almost constant at 3.5 kPa during diafiltration. Virus recovery was 94%.

The last two decades, between 2000 and 2024, significant steps were achieved regarding the interaction between various stem cells and titanium implant surfaces to improve dental implant integration. This literature review focuses on the potential effects of (i) bone marrow mesenchymal stem cells (BMSCs), (ii) periodontal ligament stem cells (PDLSCs), and (iii) dental follicle stem cells (DFSCs) in promoting osseointegration and tissue regeneration. Studies have shown that combining these stem cells with Ti implants enhances bone formation, accelerates implant osseointegration, and improves long-term implant stability. Additionally, animal models and bioreactors have been employed to evaluate the effects of stem cells on dental implant performance, with some studies showing promising results, although certain models have also yielded inconsistent outcomes. The interaction between stem cells and surface-modified Ti implants has emerged as a key area of research, with results indicating improved healing times and reduced failure rates. This article provides an overview of these findings, highlighting the role of stem cells in not only replacing lost teeth but also actively regenerating the surrounding biological structures for a more integrated and natural outcome.

Recombinant adeno-associated viruses (AAVs) with precise genome editing and cell-virus interaction have become a promising delivery tool for gene therapy. A robust AAV purification process is crucial for ensuring therapeutic efficacy. The challenges of AAV purification process development encompass limited material availability during early-stage development, high cost-of-goods compared to traditional biologics, and short development timelines for the critical first-in-human stages. The key to overcoming these challenges is to leverage high throughput (HTP) methods. In this article, an integrated end-to-end HTP workflow is proposed, utilizing a resin tip as the purification module and incorporating an HTP analytical toolkit on one platform. Purification parameters, including binding capacity, resin selection, and buffer composition screening for AAV full/partial/empty capsids separation, are efficiently determined using a 25 μL resin tip and HTP analytical tools with only micro-volume sample requirements. The process parameters determined from the HTP workflow predict the trends of full capsid enrichment and partial capsid removal for the bench-scale purification. This HTP workflow is also applied for the assessment of the AAV quality attributes to accelerate early-stage cell line and cell culture development. Comparable AAV quality attributes are demonstrated to Robocolumn as the benchmark HTP purification method. By leveraging HTP analytical tools to instantly interpret the purification data, this integrated HTP workflow effectively accelerates AAV purification process development, with a 2% material volume requirement compared to the benchmark method, 96-well format screening, short turnaround time for analytical assays, and significant cost-of-goods savings for downstream process development.

Plasma expanders (PEs) are commonly used to replace lost blood volume for septic shock patients with increased vascular permeability. Human serum albumin (HSA) is the preferred PE, due to its innate ability to restore blood colloid osmotic pressure (COP). However, HSA is susceptible to protein extravasation under endothelial dysfunction leading to edema and exposing tissue to toxic HSA-bound metabolites. To prevent extravasation, the molecular diameter of HSA has been previously increased through chemical polymerization to yield polymerized HSA (PHSA). In this study, we further optimize PHSA size and COP via polyethylene glycol (PEG) surface conjugation. Previously synthesized PHSA that was size fractionated via tangential flow filtration (TFF) into two brackets (bracket A [500 kDa–0.2 μm] and bracket B [50–500 kDa]) served as precursors for subsequent PEGylation. Each PHSA bracket was thiolated with 2-iminothiolane hydrochloride (IT) and PEGylated with monofunctional 5 kDa maleimide PEG to yield PEGylated PHSA (PPHSA). All PPHSA solutions exhibited increased molecular size, zeta potential, and osmolality compared to their non-PEGylated precursor PHSA. At the same total protein concentration, PPHSA viscosity decreased compared to the precursor PHSA, while the COP remained consistent with HSA, indicating their potential to serve as PEs.

RETRACTION: S. Haseli, M. Pourmadadi, A. Samadi, F. Yazdian, M. Abdouss, H. Rashedi, and M. Navaei-Nigjeh, “ A Novel pH-Responsive Nanoniosomal Emulsion for Sustained Release of Curcumin from a Chitosan-Based Nanocarrier: Emphasis on the Concurrent Improvement of Loading, Sustained Release, and Apoptosis Induction,” Biotechnology Progress 38, no. 5 (2022): e3280, https://doi.org/10.1002/btpr.3280.

The above article, published online on 30 June 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, John A. Morgan; the American Institute of Chemical Engineers; the Society for Biological Engineering; and Wiley Periodicals LLC.

The retraction has been agreed upon following an investigation into concerns raised by a third party, which revealed inappropriate duplication of image panels between this (Figure 4) and another article published by an overlapping group of authors, depicting a different experimental condition.

The partial raw data provided by the authors could not address the original concerns, showed inconsistencies with the published results, and ultimately raised additional doubts about the study's overall reliability. Consequently, the editors have lost confidence in the presented data and decided to retract the paper. The authors’ institute has been informed of the allegations and the decision to retract but remained unresponsive. The authors disagree with the retraction.

Chimeric antigen receptor (CAR) T cell therapy has tremendous potential for the treatment of cancer and other diseases. To manufacture cells of the desired quantity and quality, it is important to expand the CAR T cells ex vivo for an optimal duration. However, identifying the optimal harvest time requires knowledge of the cell concentration during the expansion period. To address this challenge, we have developed a digital shadow of CAR T cell expansion that provides a soft sensor of cell concentration in real-time. Specifically, a novel mechanistic mathematical model of cell growth within a proportional-integral-derivative (PID) controlled perfusion bioreactor has been developed using nonlinear ordinary differential equations. The model is fitted to data generated via bioreactor runs of the Aglaris FACER, in which both donor and patient cells have been expanded in two different media. Off-line data includes the initial and final cell concentrations, and online data includes the glucose and lactate concentrations as well as the perfusion rate. Training the digital shadow utilizes all the off-line and online data for each run. In contrast, real-time testing utilizes only the initial cell concentration and the available online data at the time of model fitting. Real-time testing shows that with at least 2.5 days of online data, the final cell concentration up to 2.5 days later is predicted with a mean relative error of 13% (standard deviation ≈ 6%). Informative real-time predictions of cell concentration via the digital shadow can guide decisions regarding the optimal harvest time of CAR T cells.

Traditional biologics process development, including antibody and recombinant protein production, typically relies on labor-intensive, iterative cell culture optimization to determine optimal process parameters. To address this inefficiency, we introduced the Industrial Smart Lab Framework for Cell Culture (ISLFCC), an autonomous laboratory that combines deep learning and robotic experimentation to enhance cell culture processes. In this system, robotic arms sample various bioreactors for analysis, and the IoT system transmits these analysis results to decoder-only transformer deep learning models. Based on these analysis results, these models predict future cell states and recommend optimal actions, which are then executed by automation devices through the IoT system, such as adjusting nutrient feeds and temperature shifts. In a comparative case study, our AI-driven process development for three different cell clones resulted in an average titer increase of 26.8% and maintained lactate levels below 1 g/L without rebound in the late phase within just a single batch, surpassing traditional three-stage empirical process development methods. Moreover, our approach has greatly automated cell culture to ensure enhanced reproducibility, data accuracy, adaptability to various cell lines, and seamless scalability across production scales, marking the first implementation of high-throughput automated cell culture in 3 and 15 L bioreactors. By merging AI with robotic execution, ISLFCC provides a transformative framework that accelerates biologics development, representing a paradigm shift towards autonomous, data-driven biomanufacturing.

In-line Raman spectroscopy combined with chemometric modeling is a valuable process analytical technology (PAT) providing real-time quantitative information on cell culture compounds. Considering that compound quantification through chemometric models depends on pre-processing to maintain consistent changes in intensity at certain wavenumbers, all causes of signal distortion should be well understood to prevent quantification inaccuracies. This work investigated spectral distortion caused by the changing bioreactor parameters temperature, bubble quantity, and medium viscosity. In addition, the isolated spectral contribution of Saccharomyces cerevisiae cells in suspension was also determined. A temperature range from 20 to 40°C resulted in peak shifts up to 0.8 cm⁻¹ to lower wavenumbers, bubbles generated under standard bioreactor operation conditions led to signal attenuation of up to 7.93% reduction in peak intensity, and changes in liquid viscosity resulted in complex peak shift behavior. Isolated biomass concentrations reaching 5 g/L caused up to 44.6% reduction in distinct peak intensity, which was similar to spectra from batch process fermentations. Correcting for the attenuation revealed spectral features of biomass associated with proteins and lipids in the 1000–1500 cm⁻¹ region. However, the spectral contribution of yeast biomass is dominated by signal extinction, which attenuates Raman spectra in a non-linear manner as biomass accumulates. The obtained knowledge on different sources of spectral distortion aids in the development of robust pre-processing and modeling strategies to obtain chemometric models applicable across experimental setups.