Biotechnology Progress

The hollow fiber filter is the primary cell-retention device used in high-density perfusion cell culture and often used in an alternating tangential flow (ATF) configuration. The limited commercially available diaphragm pumps for ATF prevent utilization of vertical space when scaling beyond 500 L. Stacking hollow fiber filters coupled with viscous cell culture imposes vacuum pressure exceeding facility capabilities. Additionally, the longer filter assembly increases the hold-up volume and exceeds the diaphragm pump's fluid exchange capacity...

A critical measure of the quality of pharmaceutical proteins is the preservation of native conformations of the active pharmaceutical ingredients. Denaturation of the active proteins in any step before administration into patients could lead to loss of potency and/or aggregation, which is associated with an increased risk of immunogenicity of the products. Interfacial stress enhances protein instability as their adsorption to the air-liquid and liquid-solid interfaces are implicated in the formation of denatured proteins and aggregates...

Light management strategy is crucial for improving microalgal production in terms of higher biomass and economically valuable bioactive molecules. However, green light has received less attention in developing light managements for algae and higher plant due to its low absorption rate by chlorophyll. In this study, the effects of green light supplementation, in the combination with red and blue light were investigated in Haematococcus pluvialis. 10% and 20% of green light supplementations were applied in 3:2 ratios of red and blue LED light combinations as an expense of red-light...

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production...

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis...

Tumor spheroid models have garnered significant attention in recent years as they can efficiently mimic in vivo models, and in addition, they offer a more controlled and reproducible environment for evaluating the efficacy of cancer drugs. In this study, we present the design and fabrication of a micromold template to form multicellular spheroids in a high-throughput and controlled-sized fashion. Briefly, polydimethylsiloxane-based micromolds at varying sizes and geometry were fabricated via soft lithography using 3D-printed molds as negative templates...

Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their "developability" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude...

Cell line generation of mammalian cells is a time-consuming and labor-intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource-intensive and time-consuming due to necessary sampling and offline analysis effort...

Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties...

Alzheimer's disease and other tauopathies are characterized by the misfolding and aggregation of the tau protein into oligomeric and fibrillar structures. Antibodies against tau play an increasingly important role in studying these neurodegenerative diseases and the generation of tools to diagnose and treat them. The development of antibodies that recognize tau protein aggregates, however, is hindered by complex immunization and antibody selection strategies and limitations to antigen presentation. Here, we have taken a facile approach to identify single-domain antibodies, or nanobodies, that bind to many forms of tau by screening a synthetic yeast surface display nanobody library against monomeric tau and creating multivalent versions of our lead nanobody, MT3...

Lung cancer has a high incidence rate and a low cure rate, hence the urgent need for effective treatment methods. Current lung cancer drugs have several drawbacks, including low specificity, poor targeting, drug resistance, and irreversible damage to normal tissues. Therefore, there is a need to develop a safe and effective new drug that can target and kill tumor cells. In this study, we combined nanotechnology and biotechnology to develop a CD133 ligand-modified etoposide-liposome complex (Lipo@ETP-CD133) for targeted therapy of lung cancer...

Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role...

Biopharmaceutical manufacturing entails a series of highly regulated steps. The manufacturing of safe and efficacious drug product (DP) requires testing of critical quality attributes (CQAs) against specification limits. DP potency concentration, which measures the dosage strength of a particular DP, is a CQA of great interest. In order to minimize the DP potency out-of-specification (OOS) risk, sterile fill finish (SFF) process adjustments may be needed. Varying the potency targets can be one such process adjustment...

Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand-alone batch mode...

Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale-down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing...

Raman spectroscopy has been used to measure the concentration of a pharmaceutically relevant model amine intermediate for positive allosteric modulators of nicotinic acetylcholine receptor in a ω-transaminase-catalyzed conversion. A model based on a one-dimensional convolutional neural network was developed to translate raw data augmented Raman spectra directly into substrate concentrations, with which the conversion from ketone to amine by ω-transaminase could be determined over time. The model showed very good predictive capabilities, with R2 values higher than 0...

Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High-throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench-scale glass fermentors or small-scale steam-in-place (SIP) fermentors. However, such scale-down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small-scale models...

Biopharmaceutical manufacture is transitioning from batch to integrated and continuous biomanufacturing (ICB). The common framework for most ICB, potentially enables a global biomanufacturing ecosystem utilizing modular and multi-function manufacturing equipment. Integrating unit operation hardware and software from multiple suppliers, complex supply chains enabled by multiple customized single-use flow paths, and large volume buffer production/storage make this ICB vision difficult to achieve with commercially available manufacturing equipment...

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs...

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis...