Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

Filamentous fungi cultivated as biopellets are well established in biotechnology industries. A distinctive feature of filamentous fungi is that hyphal growth and fungal morphology affect product titers and require tailored process conditions. Within the pellet, mass transfer, substrate consumption, and biomass formation are intricately linked to the local hyphal fraction and pellet size. This study combined oxygen concentration measurements with microelectrode profiling and three-dimensional X-ray microtomography measurements of the same fungal pellets for the first time. This allowed for the precise correlation of micromorphological information with local oxygen concentrations of two Aspergillus niger strains (hyperbranching and regular branching). The generated results showed that the identified oxygen-penetrated outer pellet regions exhibited a depth of 90-290 µm, strain-specific, with the active part percentage in the pellet ranging from 18% to 69%, without any difference between strains. Using a 1D continuum diffusion consumption model, the oxygen concentration in the pellets was computed depending on the local hyphal fraction. The best simulation results were achieved by individually estimating the oxygen-related biomass yield coefficient of the consumption term within each examined pellet, with an average estimated value of 1.95 (± 0.72) kg biomass per kg oxygen. The study lays the foundation for understanding oxygen supply in fungal pellets and optimizing processes and pellet morphologies accordingly.

Mathematical modeling and computer simulation are fundamental for optimizing biotechnological processes, enabling cost reduction and scalability, thereby driving advancements in the bioindustry. In this work, mathematical modeling and estimation of fermentative kinetic parameters were carried out to produce 1,3-propanediol (1,3-PDO) from residual glycerol and Klebsiella pneumoniae BLh-1. The Markov chain Monte Carlo method, using the Metropolis-Hastings algorithm, was applied to experimental data from a batch bioreactor under aerobic and anaerobic conditions. Sensitivity analysis and parameter evolution studies were conducted. The root-mean-square error (rRMSE) was chosen as the validation and calibration metric for the developed mathematical model. The results indicated that the average tolerance of glycerol was 174.68 and 44.85 g L, the inhibitory products was 150.95 g L for ethanol and 35.56 g L for 1,3-PDO, and the maximum specific rate of cell growth was 0.189 and 0.275 h, for aerobic and anaerobic cultures, respectively. The model presented excellent fits in both crops, with rRMSE values between 0.09 - 33.74% and 3.58 - 31.82%, for the aerobic and anaerobic environment, respectively. With this, it was possible to evaluate and extract relevant information for a better understanding and control of the bioprocess.

We describe a novel device to mimic the metastasis of cancer cells from the colon into the liver in a human model. The colon mimic is connected to the liver model by a gravity-driven recirculating unidirectional flow of a blood surrogate and can mimic the five steps of the metastatic cascade: invasion in the colon, intravasation into the bloodstream, systemic transportation, extravasation into the liver, and colonization in the liver. The colon mimic uses established normal colon epithelial organoid cells (NL) and human umbilical vein endothelial cells (HUVEC) plated on opposite sides of a membrane. To better mimic the colon structure the NL side of the membrane is exposed to air to establish an air-liquid interface. The liver mimic consists of human liver sinusoidal endothelial cells (HHSEC) and epithelial hepatic cells (HepG2 C3A) plated in Matrigel on opposite sides of a membrane. Labeled colorectal cancer cells/clusters (CA) from organoids are introduced into an established normal colon epithelial cell (NL) layer from the same patient before assembly of the system or alternatively NL organoids and fluorescently labeled CA organoids from the same patient were prepared as a ratio of 10:1 NL:CA and established together before assembly of the system. Cell viability is greater than 85% in this system. We demonstrate that over 5 days of operation that the five steps of the metastatic cascade are replicated. This novel device allows an in vitro estimate of metastatic capability (as measured by using percentages of the labeled areas per device through ImageJ) in response to selected variables. In this study, the metastatic capability depends on the source of cancer cells (e.g., the patient), the clumping of cancer cells, glucose concentration, and oxygen levels (hypoxia). For the first time, this new in vitro system mimics all five steps of the metastatic cascade in a single device and provides a new device to probe and observe the process of metastasis in a human-based model in only 5 days. The rapid observation is due to the use of a high concentration of cancer cells in the colon (e.g. 10%) and the absence of the immune system. Our device makes it possible to probe aspects of each step of metastasis and interactions between steps.

Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed-)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small-scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4-fold higher cell-specific functional virus yield and 2.8-fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set-up, resulting in a 1.4-fold higher cell-specific functional virus yield and 3.1-fold higher volumetric virus yield.

In modern bioprocessing, cell culture media is one of the most significant cost drivers, yet the nutrients and other critical factors in the media are often not fully utilized. With the renewed emphasis on reducing the cost of bioprocessing, there is much interest in reducing the overall use of cell culture media. In this work, we introduce a mesoscale microfluidic separation device based on the ion concentration polarization (ICP) process to regenerate the spent media for reuse by removing critical waste products from the cell culture that are known to inhibit the growth of the cells. We demonstrated that up to 75% of spent culture media can be regenerated and reused without affecting the cell viability. A detailed analysis of the materials consumed during antibody production indicated that one could improve the water process mass intensity by up to 33% by regenerating and recycling the media. Given that ICP separation systems have already been scaled up to support large-volume processing, it would be feasible to deploy this technology for manufacturing scale bioreactors (e.g., 50 L perfusion culture of CHO cells), reducing the overall operation cost and water use.

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals manufactured by well-established processes using Chinese Hamster Ovary (CHO) cells. Next-generation biomanufacturing using alternative hosts like Komagataella phaffii could improve the accessibility of these medicines, address broad societal goals for sustainability, and offer financial advantages for accelerated development of new products. Antibodies produced by K. phaffii, however, may manifest unique molecular quality attributes, like host-dependent, product-related variants, that could raise potential concerns for clinical use. We demonstrate here conservative modifications to the amino acid sequence of aglycosylated antibodies based on the human IgG1 isotype that minimize product-related variations when secreted by K. phaffii. A combination of 2-3 changes of amino acids reduced variations across six different aglycosylated versions of commercial mAbs. Expression of a modified sequence of NIST mAb in both K. phaffii and CHO cells showed comparable biophysical properties and molecular variations. These results suggest a path toward the production of high-quality mAbs that could be expressed interchangeably by either yeast or mammalian cells. Improving molecular designs of proteins to enable a range of manufacturing strategies for well-characterized biopharmaceuticals could accelerate global accessibility and innovations.

Harmonizing unit operations in the downstream process of monoclonal antibodies (mAbs) has a high potential to overcome throughput limitations and reduce manufacturing costs. This study proposes a streamlined clarification and capture (S-CC) process concept for the continuous processing of cell broth harvested from a connected bioreactor. The process was realized with a fluidized bed centrifuge connected to depth and sterile filters, a surge tank, and a multi-column chromatography (MCC) unit. The MCC unit was operated in the rapid cycling simulated moving bed (RC-BioSMB) mode with five convective diffusive membrane adsorbers (MAs). A control strategy and the surge tank were used to adjust the loading flow rate of the MCC unit. The mAb was recovered with a total process yield of 90%, with high removal of the process-related impurities HCP (2.1 LRV) and DNA (2.9 LRV). Moreover, the S-CC process productivity of 4.2 g h was up to 5.3 times higher than for comparable, hypothetical batch MA processes. In addition, the buffer consumption of the capture step could be reduced from 2.0 L g in batch mode to 1.2 L g in the RC-BioSMB mode. These results demonstrate the high potential of streamlined interconnected unit operations to improve the overall mAb downstream process performance.

Antibacterial resistance has emerged as a significant global concern, necessitating the urgent development of new antibacterial drugs. Antimicrobial peptides (AMPs) are naturally occurring peptides found in various organisms. Coupled with a wide range of antibacterial activity, AMPs are less likely to develop drug resistance and can act as potential agents for treating bacterial infections. However, their characteristics, such as low activity, instability, and toxicity, hinder their clinical application. Consequently, researchers are inclined towards artificial design and optimization based on natural AMPs. This review discusses the research advancements in the field of artificial designing and optimization of various AMPs. Moreover, it highlights various strategies for designing such peptides, aiming to provide valuable insights for developing novel AMPs.

Recombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 10 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV-1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV-1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.

Helicobacter pylori cells undergo chemotaxis toward several small molecules, called chemo-attractants, including urea produced by the epithelial cells of the stomach. The biophysical mechanisms of chemotaxis are not well understood in H. pylori. Here, we developed point sources of urea by encapsulating it in Poly(lactic-co-glycolic acid) or PLGA microbeads for H. pylori chemotaxis studies. Microscopy and Dynamic Light Scattering characterization indicated that the PLGA particles had an average diameter of < 0.8 μm. The particles were relatively stable and had a net negative surface charge. Absorbance measurements indicated that the beads released ~70% of the urea over a 2-week period, with most of the release occurring within the first 24-h period. Varying pH (2.0-7.0) had little effect on the rate of urea release. A diffusion model predicted that such beads could generate sufficient urea gradients to chemotactically attract H. pylori cells. Single-bead single-cell chemotaxis assays confirmed the predictions, revealing that H. pylori continued to be attracted to beads even after most of the urea had been released in the first 24 h. Our work highlights a novel use of PLGA microbeads as delivery vehicles for stimulating a chemotaxis response in H. pylori, with potential applications in bacterial eradication strategies.

Characterizing co-formulated monoclonal antibodies (mAbs) poses significant challenges in the pharmaceutical industry. Due to the high structural similarity of the mAbs, traditional analytical methods, compounded by the lengthy method development process, hinder product development and manufacturing efficiency. There is increasing critical need in the pharmaceutical industry to streamline analytical approaches, minimizing time and resources, ensuring a rapid clinical entry and cost-effective manufacturing. This study investigates the application of process analytical technologies (PAT) to address such challenges. Our investigation introduces two complementary technologies, on-line ultra-performance liquid chromatography (online UPLC) and multimode fluorescence spectroscopy (MMFS), as potential PAT tools tailored for characterizing critical quality attributes (CQA) in co-formulated mAb products. Specifically, the CQAs under evaluation include the total protein concentration of the mAbs within the co-formulation and the ratio of mAb A to mAb B. Online UPLC enables direct and automated measurement of the CQAs through physical separation, while MMFS determines them in a non-destructive and more swift manner based on chemometric modeling. We demonstrate these technologies' comparable performance to conventional methods, alongside substantial benefits such as reduced analytical turnaround time and decreased laboratory efforts. Ultimately, integrating them as innovative PAT tools expedites the delivery of therapeutic solutions to patients and enhances manufacturing efficiency, aligning with the imperative for swift translation of scientific discoveries into clinical benefits.

There are a number of new format antibody-inspired molecules with multiple antigen binding capabilities in development and clinical evaluation. Here, we describe the impact of the sequence and configuration of a unique bispecific antibody format (termed BYbe) using a panel of four BYbe's and the three IgG1s from which they were derived on their production in a Chinese hamster ovary (CHO) cell expression system. Following transfection and selection, one bispecific antibody format yielded fewer mini-pools in comparison to the other bispecific cell pools. When the top 12 expressing stable mini-pools of all BYbe configurations and sequences were evaluated, both the dsscFv sequence and antibody chain configuration or placement directly impacted productivity. The cell-specific productivity (qP, pg/cell/day) was lower in all BYbe cell pools compared to the IgG1 cell lines. However, when the actual molecules/cell/day produced were considered, three of the four bispecific cell pools outproduced the parental IgG1 cell pools. While gene copy number did not correlate to productivity, mRNA analysis showed that for specific BYbe formats there was a strong correlation with productivity. In summary, we describe how bispecific antibody format configuration impacts the cell line construction process and yield of product from CHO cells.

Large-scale fermentations (»100 m³) often encounter concentration gradients which may significantly affect microbial activities and production performance. Reliably investigating such scenarios in silico would allow to optimize bioproduction. But related simulations are very rare in particular for large bubble columns. Here, we pioneer the spatially resolved investigation of a 600 m³ bubble column operating for Escherichia coli based l-phenylalanine fed-batch production. Microbial kinetics are derived from experimental data. Advanced Euler-Lagrange (EL) computational fluid dynamics (CFD) simulations are applied to track individual bubble dynamics that result from a recently developed bubble breakage model. Thereon, the complex nonlinear characteristics of hydrodynamics, mass transfer, and microbial activities are simulated for large scale and compared with real data. As a key characteristic, zones for upriser, downcomer, and circulation cells were identified that dominate mixing and mass transfer. This results in complex gradients of glucose, dissolved oxygen, and microbial rates dividing the bioreactor into sections. Consequently, alternate feed designs are evaluated splitting real feed rates in two feeds at different locations. The opposite reversed installation of feed spots and spargers improved the product synthesis by 6.24% while alternate scenarios increased the growth rate by 11.05%. The results demonstrate how sophisticated, spatially resolved simulations of hydrodynamics, mass transfer, and microbial kinetics help to optimize bioreactors in silico.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pH). We then investigated how changes in lactate metabolism impacted pH, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pH rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pH decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pH which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.

Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application. Here, we demonstrated that continuous selective L-L ISPR provides the solution for maintaining the productivity of de novo produced 2PE at an industrial pilot scale. A unique bioreactor concept called "Fermentation Accelerated by Separation Technology" (FAST) utilizes hydrostatic pressure differences to separate aqueous- and extractant streams within one unit operation, where both production and product extraction take place - allowing for the control of the concentration of the inhibiting compound. Controlled aqueous 2PE levels (0.43 ± 0.02 g kg) and extended production times (>100 h) were obtained and co-inhibiting by-product formation was reduced, resulting in a twofold increase of the final product output of batch L-L ISPR approaches. This study establishes that continuous selective L-L ISPR, enabled by FAST, can be applied for more economically viable production of inhibiting products.

Cellular metabolism plays a role in the observed variability of a drug substance's Critical Quality Attributes (CQAs) made by biomanufacturing processes. Therefore, here we describe a new approach for monitoring biomanufacturing processes that measures a set of metabolic reaction rates (named Critical Metabolic Parameters (CMP) in addition to the macroscopic process conditions currently being used as Critical Process Parameters (CPP) for biomanufacturing. Constraint-based systems biology models like Flux Balance Analysis (FBA) are used to estimate metabolic reaction rates, and metabolic rates are used as inputs for multivariate Batch Evolution Models (BEM). Metabolic activity was reproducible among batches and could be monitored to detect a deliberately induced macroscopic process shift (i.e., temperature change). The CMP approach has the potential to enable "golden batches" in biomanufacturing processes to be defined from the internal metabolic activity and to aid in detecting process changes that may impact the quality of the product. Overall, the data suggested that monitoring of metabolic activity has promise for biomanufacturing process control.

Flavin-dependent halogenase (FDH) is highly prized in pharmaceutical and chemical industries for its exceptional capacity to produce halogenated aromatic compounds with precise regioselectivity. This study has devised a multi-enzyme self-assembly strategy to construct an effective and reliable in vitro coenzyme cycling system tailored for FDHs. Initially, tri-enzyme self-assembling nanoclusters (TESNCs) were developed, comprising glucose dehydrogenase (GDH), flavin reductase (FR) and FDH. The TESNCs exhibited enhanced thermal stability and conversion efficiency compared to free triple enzyme mixtures during the conversion of L-Trp to 6-Cl-L-Trp, resulting in a 2.1-fold increase in yield. Subsequently, an ordered co-immobilization of GDH, FR, and FDH was established, further amplifying the stability and catalytic efficiency of the FDH coenzyme cycle system. Compared to the free TESNCs, the immobilized TESNCs demonstrated a 4.2-fold increase in catalytic efficiency in a 5 mL reaction system. This research provides an effective strategy for developing a robust and efficient coenzyme recycling system for FDHs.

1,5-Pentanediol (1,5-PDO) is an important five-carbon alcohol, widely used in polymer and pharmaceutical industries. Considering the substantial energy (ATP and NADPH) requirements of previous pathways, an energy-conserving artificial pathway with a higher theoretical yield (0.75 mol/mol glucose) was designed and constructed in this study. In this pathway, lysine is converted into 1,5-PDO by decarboxylation, two transamination, and two reduction reactions. For the purpose of full pathway construction, 5-aminopetanal reductase and 5-amino-1-pentanol (5-APO) transaminase were identified and characterized. By implementing strategies such as modular optimization of gene expression, enhancing lysine biosynthesis and increasing NADPH supply, the engineered strains were able to produce 1502.8 mg/L 5-APO and 726.2 mg/L 1,5-PDO in shake flasks and 11.7 g/L 1,5-PDO in a 3 L bioreactor. This work provides a new and promising pathway for the efficient production of 5-APO and 1,5-PDO.