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SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Targeting specific cells for sequencing is important for applications in cancer, microbiology, and infectious disease. Nucleic acid cytometry (NAC) is a powerful approach for accomplishing this because it allows specific cells to be isolated based on sequence biomarkers that are otherwise impossible to detect. However, existing methods require specialized microfluidic devices, limiting adoption. Here, a modified workflow is described that uses particle-templated emulsification (PTE) and flow cytometry to conduct the essential steps of cell detection and sorting normally accomplished by microfluidics. Our microfluidic-free workflow allows facile isolation and sequencing of cells, viruses, and nucleic acids and thus provides a powerful enrichment approach for targeted sequencing applications.

Integration of a large-sized DNA fragment into a chromosome is an important strategy for characterization of cellular functions in microorganisms. Magnetotactic bacteria synthesize intracellular organelles comprising membrane-bound single crystalline magnetite, also referred to as magnetosomes. Magnetosomes have gained interest in both scientific and engineering sectors as they can be utilized as a material for biomedical and nanotechnological applications. Although genetic engineering of magnetosome biosynthesis mechanism has been investigated, the current method requires cumbersome gene preparation processes. Here, the chromosomal integration of a plasmid containing ≈27 magnetosome genes (≈26 kbp region) in a non-magnetic mutant of Magnetospirillum magneticum AMB-1 using a broad-host-range plasmid is shown. The genome sequencing of gene-complemented strains reveals the chromosomal integration of the plasmid with magnetosome genes at a specific site, most likely by catalysis of an endogenous transposase. Magnetosome production is successfully enhanced by integrating a variation of magnetosome gene operons in the chromosome. This chromosomal integration mechanism will allow the design of functional magnetosomes de novo and M. magneticum AMB-1 may be used as a chassis for the designed magnetosome production.

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree-positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid [3HP]) into an evolved PDC-negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (μmax ) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux ( > 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid-producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering.

As a late colonizer of herbivore dung, Podospora anserina has evolved an enzymatic machinery to degrade the more recalcitrant fraction of plant biomass, suggesting a great potential for biotechnology applications. The authors investigated its transcriptome during growth on two industrial feedstocks, soybean hulls (SBH) and corn stover (CS). Initially, CS and SBH results in the expression of hemicellulolytic and amylolytic genes, respectively, while at later time points a more diverse gene set is induced, especially for SBH. Substrate adaptation is also observed for carbon catabolism. Overall, SBH resulted in a larger diversity of expressed genes, confirming previous proteomics studies. The results not only provide an in depth view on the transcriptomic adaptation of P. anserina to substrate composition, but also point out strategies to improve saccharification of plant biomass at the industrial level.

Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

Research on renewable biofuels produced by microorganisms has enjoyed considerable advances in academic and industrial settings. As the renewable ethanol market approaches maturity, the demand is rising for the commercialization of more energy-dense fuel targets. Many strategies implemented in recent years have considerably increased the diversity and number of fuel targets that can be produced by microorganisms. Moreover, strain optimization for some of these fuel targets has ultimately led to their production at industrial scale. In this review, the recent metabolic engineering approaches for augmenting biofuel production derived from alcohols, isoprenoids, and fatty acids in several microorganisms are discussed. In addition, the successful commercialization ventures for each class of biofuel targets are discussed.

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community-acquired methicillin-resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG-DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short-chain fatty acids including acetic, butyric and propionic acids with anti-USA300 activities are produced by PEG-DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis-laden PEG-DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG-DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.

To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications. Stable isotope tracing and targeted metabolomics identifies specific, deleterious impacts of enzyme-based passaging methods on the metabolic behavior of human embryonic stem cells (hESCs). Enzymatic passaging of hESCs decreases glucose catabolism, fatty acid synthesis, and glycan abundance. These results provide insights into potential selective pressures placed on hESCs during expansion and subculture.

The blood-brain barrier (BBB) represents an obstacle in targeting and delivering therapeutics to the central nervous system. In order to discover new BBB-targeting molecules, we panned a phage-displayed nonimmune human single-chain antibody fragment (scFv) library against a representative BBB model comprised of hydrocortisone-treated primary rat brain endothelial cells. Parallel screens were performed with or without pre-subtraction against primary rat heart and lung endothelial cells in an effort to identify antibodies that may have binding selectivity toward brain endothelial cells. After three rounds of screening, three unique scFvs, scFv15, scFv38, and scFv29, were identified that maintained binding to primary rat brain endothelial cells, both in phage and soluble scFv format. While scFv29 and to a lesser extent, scFv15, exhibited some brain endothelial cell specificity in tissue culture, scFv29 did not appear to bind a BBB antigen in vivo. In contrast, both scFv15 and scFv38 were capable of immunolabeling rat brain vessels in vivo and displayed brain vascular selectivity with respect to all peripheral organs tested other than heart. Taken together, scFv15 and scFv38 represent two new antibodies that are capable of binding antigens that are expressed at the BBB in vivo.

Interaction between biomaterials and cells is a critical aspect for successful application of tissue engineering research. Technological advances within the past decade have enabled a number of studies to investigate how the spatial organization of cell-adhesive ligands impacts complex and rich cell behaviors ranging from adhesion to differentiation. Cells in their native environment are surrounded by chemical and physical factors spanning a range of length scales from nanometers to hundreds of microns. Furthermore, signals in the form of cell-adhesive ligands presented from this environment in different size scales and/or geometrical arrangements can change how a cell senses and responds to its surroundings. Biology can thus convey information not only in the concentration of a ligand but through its ability to change the spatial organization of these cues, raising questions both on the mechanisms by which it patterns such information and on the means by which a cell interprets it. This review discusses major findings associated with various systems developed to study cell-adhesive ligand presentation as well as an overview of the important material systems used in these studies. Promising material systems to further investigations in this field are also examined. Future directions will likely include determining how cells sense local and global ligand concentrations, understanding underlying mechanisms that regulate cell behaviors, and investigating the function of more complex cell types and diverse ligands.

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues.

Monitoring the stability of recombinant Chinese Hamster Ovary (CHO) cell lines is essential to ensure the selection of production cell lines suitable for biomanufacturing. It has been frequently observed that recombinant CHO cell lines develop phenotypic changes upon aging, such as accelerated cell growth in late generation cultures. However, the mechanism responsible for age-correlated changes is poorly understood. In this study, we investigated the molecular mechanisms underlying the age-correlated cell growth improvement in Pfizer's platform fed-batch production process, by examining multiple cell lines derived from different CHO expression systems, expressing a variety of monoclonal antibodies (mAbs). Comprehensive whole-genome resequencing analysis revealed duplication of a continuous 50.2 Mbp segment in chromosome 2 (Chr2) specific to clones that showed age-correlated growth change as compared to clones that did not exhibit age-correlated growth change. Moreover, such age- and growth-related Chr2 duplication was independent of the presence or type of recombinant monoclonal antibody expression. When we compared transcriptome profiles from low-growth and high-growth cell lines, we found that >95% of the genes overexpressed in high-growth cell lines were in the duplicated Chr2 segment. To the best of our knowledge, this is the first report of large genomic duplication, specific to Chr2, being associated with age-correlated growth change. Investigation of the cause-and-effect relationship between the genes identified in the duplicated regions and age-correlated growth change is underway. We are confident that this effort will lead to improved cell line screening and targeted rational cell line engineering efforts to develop cell lines with improved stability performance., (© 2024 Wiley-VCH GmbH.)

The biopharmaceutical industry is under increased pressure to maximize efficiency, enhance quality compliance, and reduce the cost of drug substance manufacturing. Ways to reduce costs associated with manufacturing of complex biological molecules include maximizing efficiency of chromatography purification steps. For example, process analytical technology (PAT) tools can be employed to improve column resin life, prevent column operating failures, and decrease the time it takes to solve investigations of process deviations. We developed a robust method to probe the shape of the chromatogram for indications of column failure or detrimental changes in the process. The approach herein utilizes raw data obtained from manufacturing followed by a pre-processing routine to align chromatograms and patch together the different chromatogram phases in preparation for multivariate analysis. A principal component analysis (PCA) was performed on the standardized chromatograms to compare different batches, and resulted in the identification specific process change that affected the profile. In addition, changes in the chromatogram peaks were used to create predictive models for impurity clearance. This approach has the potential for early detection of column processing issues, improving timely resolution in large-scale chromatographic operations., (© 2023 Wiley-VCH GmbH.)

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The fast-growing interest in cell and gene therapy (C&GT) products has led to a growing demand for the production of plasmid DNA (pDNA) and viral vectors for clinical and commercial use. Manufacturers, regulators, and suppliers need to develop strategies for establishing robust and agile supply chains in the otherwise empirical field of C&GT. A model-based methodology that has great potential to support the wider adoption of C&GT is presented, by ensuring efficient timelines, scalability, and cost-effectiveness in the production of key raw materials. Specifically, key process and economic parameters are identified for (1) the production of pDNA for the forward-looking scenario of non-viral-based Chimeric Antigen Receptor (CAR) T-cell therapies from clinical (200 doses) to commercial (40,000 doses) scale and (2) the commercial (40,000 doses) production of pDNA and lentiviral vectors for the current state-of-the-art viral vector-based CAR T-cell therapies. By applying a systematic global sensitivity analysis, we quantify uncertainty in the manufacturing process and apportion it to key process and economic parameters, highlighting cost drivers and limitations that steer decision-making. The results underline the cost-efficiency and operational flexibility of non-viral-based therapies in the overall C&GT supply chain, as well as the importance of economies-of-scale in the production of pDNA., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Background/aims: Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls., Methods and Results: Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range and offering similar spectral shape across all probes was first constructed, improving prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales in the model, reducing prediction bias. External parameter orthogonalization, a preprocessing method, also mitigated the interference from feeding, which further improved model performance. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R 2 of 92.4%., Conclusion: The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. Additionally, the incorporation of at-line spectra following orthogonal design into the calibration set was shown to generate calibration models that are more robust than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectral collection is easier, faster, and more material-sparing than in-line spectra collection., (© 2022 Wiley-VCH GmbH.)

The chemical industry has made a contribution to modern society by providing cost-competitive products for our daily use. However, it now faces a serious challenge regarding environmental pollutions and greenhouse gas emission. With the rapid development of molecular biology, biochemistry, and synthetic biology, industrial biotechnology has evolved to become more efficient for production of chemicals and materials. However, in contrast to chemical industries, current industrial biotechnology (CIB) is still not competitive for production of chemicals, materials, and biofuels due to their low efficiency and complicated sterilization processes as well as high-energy consumption. It must be further developed into "next-generation industrial biotechnology" (NGIB), which is low-cost mixed substrates based on less freshwater consumption, energy-saving, and long-lasting open continuous intelligent processing, overcoming the shortcomings of CIB and transforming the CIB into competitive processes. Contamination-resistant microorganism as chassis is the key to a successful NGIB, which requires resistance to microbial or phage contaminations, and available tools and methods for metabolic or synthetic biology engineering. This review proposes a list of contamination-resistant bacteria and takes Halomonas spp. as an example for the production of a variety of products, including polyhydroxyalkanoates under open- and continuous-processing conditions proposed for NGIB., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Whole-cell biocatalysis for C-H oxyfunctionalization depends on and is often limited by O 2 mass transfer. In contrast to oxygenases, molybdenum hydroxylases use water instead of O 2 as an oxygen donor and thus have the potential to relieve O 2 mass transfer limitations. Molybdenum hydroxylases may even allow anaerobic oxyfunctionalization when coupled to anaerobic respiration. To evaluate this option, the coupling of quinoline hydroxylation to denitrification is tested under anaerobic conditions employing Pseudomonas putida (P. putida) 86, capable of aerobic growth on quinoline. P. putida 86 reduces both nitrate and nitrite, but at low rates, which does not enable significant growth and quinoline hydroxylation. Introduction of the nitrate reductase from Pseudomonas aeruginosa enables considerable specific quinoline hydroxylation activity (6.9 U g CDW -1 ) under anaerobic conditions with nitrate as an electron acceptor and 2-hydroxyquinoline as the sole product (further metabolization depends on O 2 ). Hydroxylation-derived electrons are efficiently directed to nitrate, accounting for 38% of the respiratory activity. This study shows that molybdenum hydroxylase-based whole-cell biocatalysts enable completely anaerobic carbon oxyfunctionalization when coupled to alternative respiration schemes such as nitrate respiration., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches ≥200 × 10 6  cells mL -1 . The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production., (© 2018The Authors. Biotechnology Journal Published by Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim.)

The reported impact of shear stress on protein aggregation has been contradictory. At high shear rates, the occurrence of cavitation or entrapment of air is reasonable and their effects possibly misattributed to shear stress. Nine different proteins (α-lactalbumin, two antibodies, fibroblast growth factor 2, granulocyte colony stimulating factor [GCSF], green fluorescence protein [GFP], hemoglobin, human serum albumin, and lysozyme) are tested for their aggregation behavior on vapor/liquid interfaces generated by cavitation and compared it to the isolated effects of high shear stress and air/liquid interfaces generated by foaming. Cavitation induced the aggregation of GCSF by +68.9%, hemoglobin +4%, and human serum albumin +2.9%, compared to a control, whereas the other proteins do not aggregate. The protein aggregation behaviors of the different proteins at air/liquid interfaces are similar to cavitation, but the effect is more pronounced. Air-liquid interface induced the aggregation of GCSF by +94.5%, hemoglobin +35.5%, and human serum albumin (HSA) +31.1%. The results indicate that the sensitivity of a certain protein toward cavitation is very similar to air/liquid-induced aggregation. Hence, hydroxyl radicals cannot be seen as the driving force for protein aggregation when cavitation occurs. Further, high shear rates of up to 10 8  s -1 do not affect any of the tested proteins. Therefore, also within this study generated extremely high isolated shear rates cannot be considered to harm structural integrity when processing proteins., (© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), is one of the most valuable biopolymers because of its flexible mechanical properties. In this study, the goal is to establish a scaled-up process of low cost P(3HB-co-4HB) from a 7.5-L fermentor to 1- and 5-m 3 industrial bioreactors, respectively, using Halomonas bluephagenesis TD40 grown on glucose, γ-butyrolactone, and waste corn steep liquor (CSL) as substrates, under open non-sterile and fed-batch or continuous conditions. The non-sterile process enables the energy reduction for less steam consumption. Moreover, waste gluconate is successfully utilized to replace glucose as a carbon source for cell growth and PHA accumulation in 7.5-L fermentor, which opens the possibility of 60% of raw material cost reduction for recycling the waste resources. A mathematical model and rational calculation is established to help guide the feeding strategy and scale-up, respectively, leading to 100 g L -1 cell dry weight (CDW) containing 60.4% P(3HB-co-mol 13.5% 4HB) after 36 h of growth in the 5 m 3 vessel. An even higher P(3HB-co-4HB) content of 74% is achieved by decreasing the use of waste CSL. A stable and continuous open process for efficient low-cost production of P(3HB-co-4HB) is successfully developed coupling fermentation with the downstream extraction processing., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The growing market of biopharmaceuticals and the constant developments in upstream fermentation have generated a strong demand for new downstream purification methods. Magnetic separation in combination with functional magnetic particles has been known for many years as a promising candidate for a direct capturing tool in protein purification but the lack of suitable GMP-compliant purification equipment has prevented the launch of this technology in large scale bioprocessing. To tackle this bottle-neck, the principle of a "rotor-stator" high-gradient magnetic separator is fully redesigned to meet the rigorous requirements of modern cGMP biotechnology purification processes. In order to fulfill regulatory requirements, the separation chamber is reengineered to allow effective cleaning and sterilization in place while maintaining excellent separation capacities and efficiencies. Two kinds of commercially available magnetic particles are used to validate key performance data and determine system related parameters in order to calculate process performance figures for process optimization of the new magnetic separation device. With separation capacities of over 400 g of magnetic particles per liter of separation chamber volume and separation efficiencies as well as recovery rates over 99%, the system is able to process more than 200 l crude feedstock per day and capture more than 1.6 kg target compounds., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)