Biotechnology Progress

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ISSN / EISSN : 8756-7938 / 1520-6033
Published by: Wiley (10.1002)
Total articles ≅ 5,694
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Jonathan Diep, Huong Le, , Ewelina Zasadzinska, Jasmine Tat, Pheng Yam, Ryan Zastrow, ,
Published: 28 July 2021
Biotechnology Progress; doi:10.1002/btpr.3192

Abstract:
Cell line development (CLD) represents a critical, yet time-consuming step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence activated cell sorting. Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform process. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared to standard process-derived clones over 50 population doubling levels. Taken together, the data support early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturing profiles.
Zahra Malekpour, Vajihe Akbari, Jaleh Varshosaz,
Published: 22 July 2021
Biotechnology Progress; doi:10.1002/btpr.3195

Abstract:
Periodontal diseases can lead to soft tissue defects. Tissue engineering can provide functional replacements for damaged tissues. Recently, electrospun nanofibers have attracted great interest for tissue engineering and drug delivery applications. This has been revealed that statins exhibit positive impacts on the proliferation and regeneration of periodontal tissues. Electrospun simvastatin loaded poly (lactic-co-glycolic acid) (SIM-PLGA-NF) were prepared using electrospinning technique. Optimal conditions for preparation of SIM-PLGA-NF (PLGA concentration of 30 wt%, voltage of 15 kV, and flow rate of 1.5 ml h-1) were identified using a 23 factorial design. The optimized SIM-PLGA-NFs (diameter of 640.2±32.5 nm and simvastatin entrapment efficacy of 99.6±1.5%) were surface modified with 1% w/v hyaluronic acid solution (1%HA- SIM-PLGA-NF) to improve their compatibility with fibroblasts and potential application as a periodontal tissue engineering scaffold. HA-SIM-PLGA NFs were analyzed using SEM, FTIR and XRD. 1%HA- SIM-PLGA-NF had uniform, bead-free and interwoven morphology, which is similar to the ECM. The mechanical performance of SIM-PLGA-NFs and release profile of simvastatin from these nanofibers have been also greatly improved after coating with HA. In vitro cellular tests showed that the proliferation, adhesion and differentiation of fibroblast cells positively enhanced on the surface of 1%HA- SIM-PLGA-NF. These results demonstrate the potential application of 1% HA-SIM-PLGA-NFs as a scaffold for periodontal tissue engineering.
Kazem Mashayekhi, Mojtaba Sankian, Saeed Mohammadian Haftcheshmeh, Ramezan Ali Taheri, Kazem Hassanpour,
Published: 4 July 2021
Biotechnology Progress; doi:10.1002/btpr.3191

Abstract:
To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1–T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1–T4 aptamers with 5′-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (Kd) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1–T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1–T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of Kd showed that trimerization could effectively enhance Kd of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.
Sergio D'Ambrosio, Michela Ventrone, Alberto Alfano, Chiara Schiraldi,
Published: 27 June 2021
Biotechnology Progress; doi:10.1002/btpr.3184

Abstract:
Microscale fermentation systems are important high throughput tools in clone selection, and bioprocess set up and optimization, since they provide several parallel experiments in controlled conditions of pH, temperature, agitation, and gas flow rate. In this work we evaluated the performance of biotechnologically relevant strains with different respiratory requirements in the micro-Matrix microbioreactor. In particular Escherichia coli K4 requires well aerated fermentation conditions to improve its native production of chondroitin-like capsular polysaccharide, a biomedically attractive polymer. Results from batch and fed-batch experiments demonstrated high reproducibility with those obtained on 2 L reactors, although highlighting a pronounced volume loss for longer-term experiments. Basfia succiniciproducens and Actinobacillus succinogenes need CO2 addition for the production of succinic acid, a building block with several industrial applications. Different CO2 supply modes were tested for the two strains in 24 h batch experiments and results well compared with those obtained on lab-scale bioreactors. Overall, it was demonstrated that the micro-Matrix is a useful scale-down tool that is suitable for growing metabolically different strains in simple batch process, however, a series of issues should still be addressed in order to fully exploit its potential.
, Anders Jensen, Claus Felby, Lisbeth Garbrecht Thygesen
Published: 26 June 2021
Biotechnology Progress; doi:10.1002/btpr.3189

Abstract:
Understanding of how the plant cell walls of different plant species respond to pretreatment can help improve saccharification in bioconversion processes. Here, we studied the chemical and structural modifications in lignin and hemicellulose in hydrothermally pretreated poplar and wheat straw using wet chemistry and 2D heteronuclear single quantum coherence nuclear magnetic resonance (NMR) and their effects on cellulose conversion. Increased pretreatment severity reduced the levels of β─O─4 linkages with concomitant relatively increased levels of β─5 and β─β structures in the NMR spectra. β─5 structures appeared at medium and high severities for wheat straw while only β─β structures were observed at all pretreatment severities for poplar. These structural differences accounted for the differences in cellulose conversion for these biomasses at different severities. Changes in the hemicellulose component include a complete removal of arabinosyl and 4-O-methyl glucuronosyl substituents at low and medium pretreatment severities while acetyl groups were found to be relatively resistant toward hydrothermal pretreatment. This illustrates the importance of these groups, rather than xylan content, in the detrimental role of xylan in cellulose saccharification and helps explain the higher poplar recalcitrance compared to wheat straw. The results point toward the need for both enzyme preparation development and pretreatment technologies to target specific plant species.
Emily A. Berckman,
Published: 25 June 2021
Biotechnology Progress; doi:10.1002/btpr.3190

Abstract:
The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.
Published: 24 June 2021
Biotechnology Progress; doi:10.1002/btpr.3188

Abstract:
Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography-mass spectrometry and inductively coupled plasma-optical emission spectroscopy (ICP-OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP-OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.
, Bhumit A. Patel, Adrian Gospodarek, Jayesh Desai, Alejandro de Janon Gutiérrez, Edita Botonjic‐Sehic, Mark Brower, Nuno D. S. Pinto
Published: 23 June 2021
Biotechnology Progress; doi:10.1002/btpr.3187

Abstract:
Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.
Zheng Zhang, Ji Chen, JungHao Wang, Qiao Gao, Zhujun Ma, Shurong Xu, Li Zhang, Jill Cai,
Published: 20 June 2021
Biotechnology Progress; doi:10.1002/btpr.3186

Abstract:
The global pandemic outbreak COVID-19 (SARS-COV-2), has prompted many pharmaceutical companies to develop vaccines and therapeutic biologics for its prevention and treatment. Most of the therapeutic biologics are common human IgG antibodies, which were identified by next-generation sequencing with the B cells from the convalescent patients. To fight against pandemic outbreaks like COVID-19, biologics development strategies need to be optimized to speed up the timeline. Since the advent of therapeutic biologics, strategies of transfection and cell line selection have been continuously improved for greater productivity and efficiency. Next-generation sequencing (NGS) has also been implemented for accelerated cell bank testing. These recent advances enable us to rethink and reshape the chemistry, manufacturing and controls (CMC) strategy in order to start supplying GMP materials for clinical trials as soon as possible. We elucidated an accelerated CMC workflow for biologics, including using GMP-compliant pool materials for Phase I clinical trials, selecting the final clone with product quality similar to that of Phase I materials for late-stage development and commercial production.
Steven C. Huhn, Yang Ou, Xiaoyan Tang, Bo Jiang, Ren Liu, Henry Lin,
Published: 18 June 2021
Biotechnology Progress; doi:10.1002/btpr.3185

Abstract:
Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.
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