Biotechnology Progress最新文献

Scaled production of cultivated meat (CM) will co-produce large volumes of spent media. Recycling of abundant metabolites such as lactic acid in spent media offers an opportunity for valorization and reduction of the carbon footprint of CM production; however, the feasibility has yet to be examined. We modeled a conceptual five-step lactic acid recovery process integrated into a previously modeled CM facility and analyzed the corresponding cost and environmental impacts of recovering an 88% lactic acid solution. At an anticipated lactic acid concentration in spent media of 3 g/L, we found the net cost of recovery would be $0.71/kg lactic acid, with a 7.5-year simple payback period. Sales of lactic acid as a co-product could offset $0.06/kg of the cost of CM production. Depending on allocation scenarios, the environmental impact of CM production with an integrated recovery process had a -1.0 to +0.2 kg CO₂ eq effect on the carbon footprint and a -22 to +3 MJ effect on cumulative energy demand per kg of CM. Recovery of lactic acid from spent media also had a 25% lower carbon footprint than conventional fermentation processes. These model results suggest that recovery of lactic acid may be an economically viable and environmentally beneficial practice if validated in future CM production facilities. This original study provides crucial guidance for lactic acid valorization and other media recycling strategies that can be broadly applied to animal cell biomanufacturing industries.

Perfusion technologies play a growing role in the implementation of continuous processes for biotherapeutics production in mammalian-based manufacturing. However, their application to alternative production hosts is limited. Cell retention systems are of key importance for the efficiency of perfusion bioreactors. In this study, we investigate two cell retention technologies for the development of lab-scale Komagataella phaffii continuous processes. An acoustic-based process (AP) and a membrane-based process (MP) were developed using an acoustic cell separator (ACS) and a vibrating membrane filtration (VMF) device, respectively. Both systems allowed for continuous cell recycle and production of scFv13R4 antibody fragment for 8 days (AP) and 9 days (MP), without loss in productivity, while maintaining high viability (greater than 90%). Higher volumetric and specific productivities were achieved during the AP process, namely 50.63 ± 1.63 mg L^-1 day^-1 and 1.09 ± 0.07 mg g^-1 day^-1, against the 32.29 ± 1.21 mg L^-1 day^-1 and 0.44 ± 0.02 mg g^-1 day^-1 afforded by the MP process. The VMF device provided 100% separation efficiency with biomass accumulating up to concentrations of 74.1 ± 0.1 g L^-1 dry cell weight (DCW), whereas the acoustic device reached 55.1 ± 0.47 g L^-1 DCW at 98% separation efficiency. The acoustic device showed selectivity towards larger and more complex cells in the yeast population, which might be linked to the observed higher productivities for the AP process. This study discusses the advantages and drawbacks of both cell retention technologies and provides an outlook towards their future investigation in K. phaffii perfusion processes.

Chinese hamster ovary (CHO) cells have emerged as the predominant mammalian host for the production of therapeutic recombinant proteins, including monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and fusion proteins. To meet the growing demand for biologics and reduce manufacturing costs, the exploitation of efficient cell line development platforms is essential. Over the past decades, various selection markers, such as dihydrofolate reductase (DHFR), glutamine synthetase (GS), and antibiotic resistance genes, have been widely utilized in the development of production cell lines. In this study, we introduce the proline selection system, an alternative metabolic selection strategy, as an efficient approach to optimize our CHO cell line development platform. By employing yeast PRO1 and PRO2 genes as selection markers, proline selection effectively complements GS selection to establish high-producing cell lines for both mAbs and bsAbs. In particular, the integration of PRO1 and PRO2 genes into a single plasmid, in conjunction with the GS gene, significantly enhances productivity for asymmetric molecules. Optimized chain configuration across proline and GS selection plasmids can further boost protein yield. Additionally, the overexpression of regulator proteins can be leveraged with proline selection to enhance antibody production or fine-tune product quality. Taken together, the incorporation of proline selection into CHO cell line development, particularly when combined with GS selection, provides a consistent and streamlined strategy to meet the growing demand for high-quality biologics in the pharmaceutical industry.

Measurement and imaging of intra-matrix protein therapeutics diffusion is important due to the emergence of injectable biologics currently in various stages of research and clinical testing. These therapeutics are developed for delivery to hyaluronic acid (HA)-rich anatomical sites such as subcutaneous tissue, the vitreous humor, and knee joints, depending on the target tissue. Understanding their diffusion behavior is essential for optimizing drug delivery strategies. Our work presents an image analysis method suited for tracking IgG diffusion in low viscosity HA matrices representative of the vitreous humor, where diffusion occurs more rapidly unlike a previously reported analysis method for higher viscosity matrices where protein diffusion is significantly slower. The current method utilizes scanner images at 6.3 MP resolution, and an algorithm that removes background and calculates protein mass and concentration measured directly within matrices formulated to represent HA in an intravitreal environment. We report and demonstrate a robust method for predicting protein diffusion coefficient from images of label-free protein diffusing in a low viscosity HA matrix.

Artificial intelligence and automation are no longer just buzzwords in the biopharmaceutical industry. The manufacturing of a class of biologics, comprising monoclonal antibodies, cell therapies, and gene therapies, is far more complex than that of traditional small molecule drugs. Therefore, applications based on artificial intelligence are essential for successfully manufacturing this new class of biologics more quickly and more economically. Some biologics manufacturers, academic researchers, and young entrepreneurs have already begun implementing artificial intelligence-based applications to increase operational efficiency, enhance process understanding, improve process monitoring, and achieve better regulatory compliance. Regulatory guidance from health agencies on the use of artificial intelligence and machine learning is acting as a catalyst in the adoption process of these new technologies by the biopharmaceutical industry. Research in artificial intelligence and machine learning has also advanced significantly in the last decade. At the same time, new cloud technologies have made the development and deployment of machine learning applications much easier. Several examples of artificial intelligence and machine learning applications in monoclonal antibodies manufacturing already exist. Cell and gene therapy, which present the future of medicine, will also benefit from this new technology. Overall, advancements in this domain will essentially help better serve patients' needs.

RETRACTION: H. Faraji, M. Ramezani, B. Mashkani, H. R. Sadeghnia, H. M. Benhangi, S. H. Teshnizi, and F. Soltani, “ Comparison of Expression Optimization of New Derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK,” Biotechnology Progress 35, no. 4 (2019): e2819. 10.1002/btpr.2819.

The above article, published online on 11 April 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between journal Editor-in-Chief, John A. Morgan; American Institute of Chemical Engineers, and Wiley Periodicals, LLC. A third party reported that Figure 7 contained several repeated image elements and that a number of these elements were copied from a previous publication by some of the same authors (Faraji et al. 2017 [https://doi.org/10.1080/10826068.2016.1252924]). An investigation by the publisher confirmed these concerns and also found that the protein marker in Figure 9 had been copied from another publication (Pednekar et al 2016 [https://doi.org/10.3389/fimmu.2016.00567]) and that elements in Figure 11B had been duplicated and manipulated.

The authors did not respond to an inquiry and request for original data by the publisher. The retraction has been agreed to because the evidence of image manipulation fundamentally compromises the editors’ confidence in the results presented.

Nanoemulsions, with droplet sizes between 20 and 200 nm, have emerged as a promising vaccine adjuvant and drug delivery system, enhancing the solubility of hydrophobic drugs for diverse applications. Sterile filtration of nanoemulsions is particularly challenging due to the similar size between the nanodroplets and the 0.2 μm nominal pore size rating of sterile filters. One approach to reducing membrane fouling, and enhancing filtration capacity and yield, is to employ an appropriate prefilter, but there are currently no clear guidelines on how to select the prefilter pore size, chemistry, or morphology for sterile filtration of nanoemulsions. This study examined the performance of a range of prefilters with varying pore morphologies and surface chemistries. Sessile drop contact angles were used to evaluate the prefilter hydrophobicity, and bubble point and mercury intrusion porosimetry were used to evaluate the pore characteristics of the different prefilters. The best performance was achieved using a relatively hydrophobic 0.45 μm prefilter made of polyvinylidene fluoride but modified with a somewhat hydrophilic (oxygen-containing) coating. This prefilter reduced the surface tension of the nanoemulsion and provided more than a 2-fold increase in capacity for a variety of sterile filters. These results provide critical insights into the factors influencing nanoemulsion filtration and offer a framework for selection of appropriate prefilters in biopharmaceutical manufacturing.

Real-time detection of foodborne pathogens such as Staphylococcus aureus (S. aureus) is essential for ensuring food safety. In this study, we evaluate the performance of an electrochemical aptasensor developed from gold nanoparticles (AuNPs)-immobilized screen-printed carbon electrode for the detection of low concentrations of S. aureus in chicken extract media. Using cyclic voltammetry (CV), the dynamic interaction between the aptamer-modified electrode and S. aureus was monitored across four bacterial concentrations of 1, 5, 10, and 20 colony-forming units per milliliter (CFU/mL) at 35-min intervals over 350 min. The aptasensor demonstrated a concentration-dependent response with increasingly lower maximum CV signals and faster time to equilibrium as CFU increased. Real-time kinetic and equilibrium parameters were extracted to understand the binding behavior of the pathogen to the electrode surface. Critical parameters such as the kinetic rate constant (k) of 0.0274 min^-1 and equilibrium dissociation constants ( $K_d$ ) of 7.35 CFU/mL, were derived from the CV signals. Langmuir isotherm modeling yielded a maximum binding capacity ( $B_{\max }$ ) of 33.55 μA. In addition, a Hill coefficient (n_H) of 0.65 was obtained, which indicates a slightly negative cooperativity. These findings demonstrate the capability of the aptasensor for real-time detection of S. aureus, offering a robust framework for field-deployable pathogen monitoring in food matrices.

Various technologies, including precipitation, flocculation, depth filtration, microfiltration, and centrifugation, have been developed to clarify mammalian cell culture fluids. For processing volumes between 2000 and 5000 L, continuous centrifugation followed by depth filtration is the preferred method. This process starts with the removal of cells and large debris through continuous centrifugation, followed by the filtration of small debris and some impurities. The newly introduced single-use centrifuge, designed to prevent cross-contamination and mimic traditional continuous centrifuges, was evaluated for its performance, particularly focusing on its impact on cell lysis and subsequent filtration and purification processes. The single-use centrifuge showed better performance in reducing turbidity and lactate dehydrogenase levels (LDH) in the supernatant, indicating less cell lysis compared to the conventional centrifuge. A separation load factor range of 0.91-2.73 was identified as optimal for balancing centrifugation throughput and product quality. Both centrifuge types had a comparable impact on the performance of subsequent depth filtration, supporting a load capacity of at least 100 L/m². No significant differences in product quality, including SE-HPLC, NR/R CE-SDS, icIEF, HCP, and rDNA, were observed between the conventional and single-use centrifuges. These harvest strategies did not affect the subsequent purification steps. For volumes up to 5000 L, both centrifuge types are viable; however, for larger volumes, the conventional centrifuge is necessary due to the scale limitations of the single-use centrifuge.

Castellano BM, Tang D, Marsters S, Lam C, Liu P, Rose CM, Sandoval W, Ashkenazi A, Snedecor B, Misaghi S. Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling. Biotechnol Prog. 2023 Sep-Oct;39(5):e3354. doi: 10.1002/btpr.3354.

We have noticed that in Fig. 4D of the article an immunoblot image representing the actin control was inadvertently depicted again in the BiP panel. We have now updated this figure with the appropriate BiP immunoblot image and have hence corrected the figure accordingly. All the article's conclusions remain unchanged as the article sections relating to the Fig. 4D were originally written based on the corrected figure. Please find the corrected Figure 4D below. This correction notice belongs to Figure 4 legends, section (d), page 7 of the published article:

We apologize for this error.