Controlling monoclonal antibody aggregation at the upstream stage itself can significantly reduce the burden on downstream processing and can improve the process yield. Hence, we have investigated ...the use of sugar osmolytes (glucose, mannose, sucrose and maltose) and formulation excipients (mannitol, polysorbate 20 and polysorbate 80) as medium additives to reduce protein aggregation during cell culture. Aggregate content in cell culture samples was estimated using a high-resolution size-exclusion chromatography technique, which efficiently resolved the antibody monomer and aggregates in the cell culture matrix i.e., without purification. Glucose, mannose, maltose and the polysorbates effectively reduced the mean aggregate content over the course of the culture. Sugar-based additives exhibited a higher degree of variation during aggregate quantitation as compared to polysorbate additives, rendering the latter a preferred additive. Therefore, this study demonstrated the potential of sugar osmolytes and formulation excipients as media additives during cell culture to reduce aggregate formation, without negatively impacting cell growth and antibody production, facilitated by the monitoring of aggregate content in cell culture samples without purification.
•Aggregation during cell culture increases with batch duration thus requiring control.•Glucose and mannose supplementation reduced the mean final aggregate content by half.•High variability in aggregate quantities was observed upon sugar supplementation.•Polysorbates may be preferred over sugars to control aggregation.•SE-HPLC is an effective tool for monitoring aggregation during cell culture.
Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product ...biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)‐producing CHO cells using transcriptome analysis by RNA‐seq, and mechanistically dissected AAT function using a variety of transporter‐specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.
Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro.
Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits ...of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not cross-react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ϕC31 and Wβ integrases, and show that Wβ has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.
A compendium of stable hotspots in the CHO genome Hilliard, William; Lee, Kelvin H.
Biotechnology and bioengineering,
August 2023, 2023-Aug, 2023-08-00, 20230801, Letnik:
120, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The use of targeted integration for industrial CHO cell line development currently requires significant upfront effort to identify genomic loci capable of supporting multigram per liter therapeutic ...protein production from a limited number of transgene copies. To address this barrier to widespread adoption, we characterized transgene expression from thousands of stable hotspots in the CHO genome using the Thousands of Reporters Integrated in Parallel high‐throughput screening method. This genome‐scale data set was used to define a limited set of epigenetic properties of hotspot regions with sizes on the order of 10 kb. Cell lines with landing pad integrations at eight retargeted hotspot candidates consistently exhibited higher transgene mRNA expression than a commercially viable hotspot in equivalent culture conditions. Initial benchmarking of NISTmAb and trastuzumab productivity from one of these hotspots yielded mAb productivities of approximately 0.7–2 g/L (qP range: 2.9–8.2 pg/cell/day) in small‐scale fed‐batches. These findings indicate the list of hotspot candidates identified here will be a valuable resource for targeted integration platform development within the CHO community.
Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and ...therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering.
We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs.
Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins.
These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.
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•SINEUP noncoding RNAs can be manipulated to improve the translation of secreted proteins.•Serum-free growing CHO-S cells are prone to SINEUP activity.•A novel natural SINEUP, antisense to a secreted protein (elastin), has been discovered.•The successful implementation of SINEUP molecules to cell factories will prove useful to increase protein manufacturing to be used for therapeutic.
The physiological significance of metabotropic acetylcholine receptors in parasitic nematodes remains largely unexplored. Here, three different Trichinella spiralis G protein-coupled acetylcholine ...receptors (TsGAR-1, -2, and -3) were identified in the genome of T. spiralis. The phylogenetic analyses showed that TsGAR-1 and -2 receptors belong to a distinct clade specific to invertebrates, while TsGAR-3 is closest to the cluster of mammalian-type muscarinic acetylcholine receptors (mAChR). The mRNA of TsGAR-1, -2, and -3 was detected in muscle larvae, newborn larvae, and adults. The functional aequorin-based assay in Chinese hamster ovary cells revealed that all three types of T. spiralis GARs trigger the G
pathway upon activation of the receptor with the acetylcholine ligand. TsGAR-1 and TsGAR-2 showed atypical affinity with classical muscarinic agonists, while TsGAR-3 was sensitive to all muscarinic agonists tested. High concentrations of propiverine antagonist blocked the activities of all three TsGARs, while atropine and scopolamine antagonists effectively inhibited only TsGAR-3. Our data indicate that the distinct pharmacological profile of TsGAR-1 and -2 receptors, as well as the phylogenetic distance between them and their mammalian orthologs, place them as attractive targets for the development of selective anthelmintic drugs interfering with nematodes' cholinergic system.
Gene editing with all its own advantages in molecular biology applications has made easy manipulation of various production hosts with the discovery and implementation of modern gene editing tools ...such as Crispr (Clustered regularly interspaced short palindromic repeats), TALENs (Transcription activator-like effector nucleases) and ZFNs (Zinc finger nucleases). With the advent of these modern tools, it is now possible to manipulate the genome of industrial production hosts such as yeast and mammalian cells which allows developing a potential and cost effective recombinant therapeutic protein. These tools also allow single editing to multiple genes for knocking-in or knocking-out of a host genome quickly in an efficient manner. A recent study on "multiplexed" gene editing revolutionized the knock-out and knock-in events of yeast and CHO, mammalian cells genome for metabolic engineering as well as high, stable, and consistent expression of a transgene encoding complex therapeutic protein such as monoclonal antibody. The gene of interest can either be integrated or deleted at single or multiple loci depending on the strategy and production requirement. This review will give a gist of all the modern tools with a brief description and advances in genetic manipulation using three major tools being implemented for the modification of such hosts with the emphasis on the use of Crispr-Cas9 for the "multiplexing gene-editing approach" for genetic manipulation of yeast and CHO mammalian hosts that ultimately leads to a fast track product development with consistent, improved product yield, quality, and thus affordability for a population at large.
Controlling the process of CHO cell fed‐batch culture is critical for biologics quality control. However, the biological complexity of cells has hampered the reliable process understanding for ...industrial manufacturing. In this study, a workflow was developed for the consistency monitoring and biochemical marker identification of the commercial‐scale CHO cell culture process through 1H NMR assisted with multivariate data analysis (MVDA). Firstly, a total of 63 metabolites were identified in this study object in 1H NMR spectra of the CHO cell‐free supernatants. Secondly, multivariate statistical process control (MSPC) charts were used to evaluate process consistency. According to MSPC charts, the batch‐to‐batch quality consistency was high, indicating the CHO cell culture process at the commercial scale was well‐controlled and stable. Then, the biochemical marker identification was provided through orthogonal partial least square discriminant analysis (OPLS‐DA) based S‐line plots during the cell logarithmic expansion, stable growth, and decline phases. Identified biochemical markers of the three cell growth phases were as follows: L‐glutamine, pyroglutamic acid, 4‐hydroxyproline, choline, glucose, lactate, alanine, and proline were of the logarithmic growth phase; isoleucine, leucine, valine, acetate, and alanine were of the stable growth phase; acetate, glycine, glycerin, and gluconic acid were of the cell decline phase. Additional potential metabolic pathways that might influence the cell culture phase transitions were demonstrated. The workflow proposed in this study demonstrates that the combination of MVDA tools and 1H NMR technology is highly appealing to the research of the biomanufacturing process, and applies well to provide guidance in future work on consistency evaluation and biochemical marker monitoring of the production of other biologics.
Graphical and Lay Summary
For the quality control of biologics, the process control of cell‐fed‐batch culture is crucial. Enhancing the cell culture procedure has advantages for improving protein expression, sustainability, and other product quality characteristics. In this study, a workflow was developed using 1H NMR technology, assisted by multivariate data analysis tools, for consistency monitoring and biochemical marker identification of the entire commercial‐scale CHO cell culture process. The results were verified using multi‐tier analysis of product quality attributes and metabolic pathways. The study's proposed approach can be used to provide valuable guidance for subsequent work on consistency evaluation, biochemical marker monitoring, and production control of mAb and other biologics.
