Expression and characterization of recombinant antibodies against H7 subtype avian influenza virus and their diagnostic potential.

Introduction: Monoclonal antibodies (mAbs) play a pivotal role in disease diagnosis as well as immunotherapy interventions. Traditional monoclonal antibody generation relies on animal immunization procedures predominantly involving mice; however, recent advances in in-vitro expression methodologies have enabled large-scale production suitable for both industrial applications as well as scientific investigations. Methods: In this study, two mAbs against H7 subtype avian influenza viruses (AIV) were sequenced and analyzed, and the DNA sequences encoding heavy chain (HC) and light chain (LC) were obtained and cloned into pCHO-1.0 expression vector. Then, the HC and LC expression plasmids were transfected into CHO-S cells to establish stable cell lines expressing these mAbs using a two-phase selection scheme with different concentrations of methotrexate and puromycin. Recombinant antibodies were purified from the cell culture medium, and their potential applications were evaluated using hemagglutination inhibition (HI), western blotting (WB), confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that the obtained recombinant antibodies exhibited biological activity similar to that of the parent antibodies derived from ascites and could be used as a replacement for animal-derived mAbs. A kinetic analysis of the two antibodies to the AIV HA protein, conducted using surface plasmon resonance (SPR), showed concordance between the recombinant and parental antibodies. Discussion: The data presented in this study suggest that the described antibody production protocol could avoid the use of experimental animals and better conform to animal welfare regulations, and provides a basis for further research and development of mAbs-based diagnostic products.

A novel approach for perfusion process design based on a "Grey-Box" kinetic model.

Perfusion cell-culture mode has caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, perfusion-culture processes can support higher cell densities, higher productivities and longer process times. However, due to the inherent operational complexity and high running costs, the development and design of perfusion-culture processes remain challenging. Here, we present a model-based approach to design optimized perfusion cultures of Chinese Hamster Ovary cells. Initially, four batches of bench-top reactor continuous-perfusion-culture data were used to fit the model parameters. Then, we proposed the model-based process design approach, aiming to quickly find out the "theoretically optimal" operational parameters combinations (perfusion rate and the proportion of feed medium in perfusion medium) which could achieve the target steady-state VCD while minimizing both medium cost and perfusion rate during steady state. Meanwhile, we proposed a model-based dynamic operational parameters-adjustment strategy to address the issue of cell-growth inhibition due to the high osmolality of concentrated perfusion medium. In addition, we employed a dynamic feedback control method to aid this strategy in preventing potential nutrient depletion scenarios. Finally, we test the feasibility of the model-based process design approach in both shake flask semi-perfusion culture (targeted at 5 × 107 cells/ml) and bench-top reactor continuous perfusion culture (targeted at 1.1 × 108 cells/ml). This approach significantly reduces the number of experiments needed for process design and development, thereby accelerating the advancement of perfusion-mode cell-culture processes.

Novel and effective screening system for recombinant protein production in CHO cells.

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.

Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells.

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.

Recent Advances and Insights in Designing ZnxCd1-xS-Based Photocatalysts for Hydrogen Production and Synergistic Selective Oxidation to Value-Added Chemical Production.

Combining the hydrogen (H2) extraction process and organic oxidation synthesis in photooxidation-reduction reactions mediated by semiconductors is a desirable strategy because rich chemicals are evolved as byproducts along with hydrogen in trifling conditions upon irradiation, which is the only effort. The bifunctional photocatalytic strategy facilitates the feasible formation of a C═O/C─C bond from a large number of compounds containing a X-H (X = C, O) bond; therefore, the production of H2 can be easily realized without support from third agents like chemical substances, thus providing an eco-friendly and appealing organic synthesis strategy. Among the widely studied semiconductor nanomaterials, ZnxCd1-xS has been continuously studied and explored by researchers over the years, and it has attracted much consideration owing to its unique advantages such as adjustable band edge position, rich elemental composition, excellent photoelectric properties, and ability to respond to visible light. Therefore, nanostructures based on ZnxCd1-xS have been widely studied as a feasible way to efficiently prepare hydrogen energy and selectively oxidize it into high-value fine chemicals. In this Review, first, the crystal and energy band structures of ZnxCd1-xS, the model of twin nanocrystals, the photogenerated charge separation mechanism of the ZB-WZ-ZB homojunction with crisscross bands, and the Volmer-Weber growth mechanism of ZnxCd1-xS are described. Second, the morphology, structure, modification, synthesis, and vacancy engineering of ZnxCd1-xS are surveyed, summarized, and discussed. Then, the research progress in ZnxCd1-xS-based photocatalysis in photocatalytic hydrogen extraction (PHE) technology, the mechanism of PHE, organic substance (benzyl alcohol, methanol, etc.) dehydrogenation, the factors affecting the efficiency of photocatalytic discerning oxidation of organic derivatives, and selective C-H activation and C-C coupling for synergistic efficient dehydrogenation of photocatalysts are described. Conclusively, the challenges in the applicability of ZnxCd1-xS-based photocatalysts are addressed for further research development along this line.

Enhancing protein productivities in CHO cells through adenosine uptake modulation - Novel insights into cellular growth and productivity regulation.

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.

Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

CHO cell engineering via targeted integration of circular miR-21 decoy using CRISPR/RMCE hybrid system.

Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibition of miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.

The Chinese hamster as an excellent experimental animal model.

Hamsters are valuable rodent models that are distinct from mice and rats. Currently, the main hamster species used for experimental research are the Syrian golden hamster and Chinese hamster, in addition to hamster species from other countries. Chinese hamsters are small, easy to run and feed, and inexpensive. They are prominent species found only in China and are part of the experimental animal resources of Chinese specialty. Chinese hamsters are distinguished by a black stripe on their back, short tail, pair of easily retractable cheek pouches, and pair of large drooping testes in males with 22 chromosomes. Due to their unique anatomical structure and biological features, Chinese hamsters have been used as a model in biomedical research. Moreover, the breeding and use of Chinese hamsters was comprehensively studied in 1958, with significant breakthroughs. We present a thorough review of the current developments and applications of Chinese hamsters and support the use of this species as a suitable and innovative experimental research model. With the success of Chinese hamster transgenic technology, this species will become more commonly employed in biological and medical research in the future.

Chemical characterization and influencing factors of gaseous and particulate components in cooking oil fume (COF) from traditional Chinese dishes: Insights from high-resolution mass spectrometry.

Cooking oil fumes (COF) are known to emit a wide range of organic compounds with significant impacts on human health and urban air quality. This study used HPLC-QToF-MS and Vocus PTR-TOF to explore the chemical constituents and influencing factors of the COF generated from eight typical Chinese dishes representing different areas in a laboratory kitchen. The results revealed that both CHO and CHON compounds exhibited strong reducibility and saturability, with CHO compounds being the dominant and CHON compounds showing greater diversity. 24 among 168 CHO compounds were identical with those generated from heating soybean oil, representing 72.4%-92.3% in abundance and 22.2%-29.2% in quantity. That was 5 among 113 CHON compounds, accounting for 7.8%-10% in abundance and 4.7%-6.7% in quantity. These findings suggest that the major CHO compounds from heating soybean oil continued to dominate the abundances in dishes. The diversity of CHO compounds and the presence of CHON compounds were influenced by the food ingredients. The VOC analysis indicated that oxygen-containing organics were the major components. 6 identical VOC species between cooking dishes and heating soybean oil were identified, comprising 36.02%-67.84% of the total VOCs mass. Notably, poor ventilation could result in even higher COF concentrations in the connected room compared to the kitchen itself.

Dialysis rolled scaffold bioreactor allows extended production of monoclonal antibody with reduced media use.

Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production.

New Species and Cytotoxicity Mechanism of Halohydroxybenzonitrile Disinfection Byproducts in Drinking Water.

Recently, seven dihalohydroxybenzonitriles (diHHBNs) have been determined as concerning nitrogenous aromatic disinfection byproducts (DBPs) in drinking water. Herein, eight new monohalohydroxybenzonitriles (monoHHBNs), including 3-chloro-2-hydroxybenzonitrile, 5-chloro-2-hydroxybenzonitrile, 3-chloro-4-hydroxybenzonitrile, 3-bromo-2-hydroxybenzonitrile, 5-bromo-2-hydroxybenzonitrile, 3-bromo-4-hydroxybenzonitrile, 5-iodo-2-hydroxybenzonitrile, and 3-iodo-4-hydroxybenzonitrile, were detected and identified in drinking water for the first time. Thereafter, the relative concentration-cytotoxicity contribution of each HHBN was calculated based on the acquired occurrence level and cytotoxicity data in this study, the genome-scale cytotoxicity mechanism was explored, and a quantitative structure-activity relationship (QSAR) model was developed. Results indicated that new monoHHBNs were present in drinking water at concentrations of 0.04-1.83 ng/L and exhibited higher cytotoxicity than some other monohalogenated aromatic DBPs. Notably, monoHHBNs showed concentration-cytotoxicity contribution comparable to diHHBNs, which have been previously identified as potential toxicity drivers in drinking water. Transcriptomic analysis revealed immunotoxicity and genotoxicity as dominant cytotoxicity mechanisms for HHBNs in Chinese hamster ovary (CHO-K1) cells, with potential carcinogenic effects. The QSAR model suggested oxidative stress and cellular uptake efficiency as important factors for their cytotoxicity, highlighting the importance of potential iodinated HHBNs in drinking water, such as 3,5-diiodo-2-hydroxybenzonitrile, for future studies. These findings are meaningful for better understanding the health risk and toxicological significance of HHBNs in drinking water.

Hydrogen Bond Strengthens Acceptor Group: The Curious Case of the C-H···O=C Bond.

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C-H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s-character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond's polarity is observed upon the formation of the C-H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

Multi-omic characterization of antibody-producing CHO cell lines elucidates metabolic reprogramming and nutrient uptake bottlenecks.

Characterizing the phenotypic diversity and metabolic capabilities of industrially relevant manufacturing cell lines is critical to bioprocess optimization and cell line development. Metabolic capabilities of production hosts limit nutrient and resource channeling into desired cellular processes and can have a profound impact on productivity. These limitations cannot be directly inferred from measured data such as spent media concentrations or transcriptomics. Here, we present an integrated multi-omic analysis pipeline combining exo-metabolomics, transcriptomics, and genome-scale metabolic network analysis and apply it to three antibody-producing Chinese Hamster Ovary cell lines to identify reprogramming features associated with high-producing clones and metabolic bottlenecks limiting product formation in an industrial bioprocess. Analysis of individual datatypes revealed a decreased nitrogenous byproduct secretion in high-producing clones and the topological changes in peripheral metabolic pathway expression associated with phase shifts. An integrated omics analysis in the context of the genome-scale metabolic model elucidated the differences in central metabolism and identified amino acid utilization bottlenecks limiting cell growth and antibody production that were not evident from exo-metabolomics or transcriptomics alone. Thus, we demonstrate the utility of a multi-omics characterization in providing an in-depth understanding of cellular metabolism, which is critical to efforts in cell engineering and bioprocess optimization.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) ß-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG ß-subunit CTP region (amino acids 115-149) was inserted between the ß-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG ß-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Mammalian cell expressed recombinant trimeric spike protein is a potent vaccine antigen and confers near-complete protection against SARS-CoV-2 infection in Hamster.

Numerous vaccine candidates have emerged in the fight against SARS-CoV-2, yet the challenges posed by viral evolution and the evasion of vaccine-induced immunity persist. The development of broadly protective vaccines is essential in countering the threat posed by variants of concern (VoC) capable of eluding existing vaccine defenses. Among the diverse SARS-CoV-2 vaccine candidates, detailed characterization of those based on the expression of the entire spike protein in mammalian cells have been limited. In our study, we engineered a recombinant prefusion-stabilized trimeric spike protein antigen, IMT-CVAX, encoded by the IMT-C20 gene. This antigen was expressed utilizing a suspension mammalian cell line (CHO-S). The establishment of a stable cell line expressing IMT-CVAX involved the integration of the gene into the CHO genome, followed by the expression, purification, and characterization of the protein. To gauge the vaccine potential of adjuvanted IMT-CVAX, we conducted assessments in small animals. Analyses of blood collected from immunized animals included measurements of anti-spike IgG, SARS-CoV-2 neutralization, and responses from GC-B and Tfh cells. Furthermore, the protective efficacy of IMT-CVAX was evaluated using a Hamster challenge model. Our findings indicate that adjuvanted IMT-CVAX elicits an excellent immune response in both mice and hamsters. Notably, sera from animals immunized with IMT-CVAX effectively neutralize a diverse range of SARS-CoV-2 variants. Moreover, IMT-CVAX immunization conferred complete protection to hamsters against SARS-CoV-2 infection. In hACE2 transgenic mice, IMT-CVAX vaccination induced a robust response from GC-B and Tfh cells. Based on our preclinical model assessments, adjuvanted IMT-CVAX emerges as a highly efficacious vaccine candidate. This protein-subunit-based vaccine exhibits promise for clinical development, offering an affordable solution for both primary and heterologous immunization against SARS-CoV-2 variants.