The disinfection of drinking water is a major public health achievement; however, an unintended consequence of disinfection is the generation of disinfection by-products（DBPs）. Many of the identified ...DBPs exhibit in vitro and in vivo toxicity, generate a diversity of adverse biological effects, and may be hazards to the public health and the environment.Only a few DBPs are regulated by several national and international agencies and it is not clear if these regulated DBPs are the forcing agents that drive the observed toxicity and their associated health effects. In this study, we combine analytical chemical and biological data to resolve the forcing agents associated with mammalian cell cytotoxicity of drinking water samples from three cities. These data suggest that the trihalomethanes（THMs） and haloacetic acids may be a small component of the overall cytotoxicity of the organic material isolated from disinfected drinking water. Chemical classes of nitrogen-containing DBPs, such as the haloacetonitriles and haloacetamides, appear to be the major forcing agents of toxicity in these samples. These findings may have important implications for the design of epidemiological studies that primarily rely on the levels of THMs to define DBP exposure among populations. The TIC-Tox approach constitutes a beginning step in the process of identifying the forcing agents of toxicity in disinfected water.
Background
Real time process data facilitates timely decisions, enables better process control, and can increase quality assurance. Biological drugs (mol. Wt. ≥ 40 kDa) are manufactured using ...mammalian cells such as Chinese hamster ovary (CHO) cells in bioreactors and have significant risks of contamination during processing. In such processes, in‐line monitoring of biomass can provide real‐time cell growth profiles and indications of bioreactor health.
Methods
An in‐line conductivity/capacitance probe (Aber Instruments, Aberystwyth, UK) for monitoring CHO cell growth during fed batch cultures for producing an IgG1 monoclonal antibody was employed. Cell growth was measured in real‐time using the capacitance probe (pF cm−1) while being compared with off‐line measurements using a metabolic analyzer (Nova Biomedical, Waltham, MA, USA). Conductivity measurements (mS cm−1) detected variations in the solute concentrations in the bioreactor due to nutrient feed, bicarbonate buffer, and cellular metabolism by‐products.
Results and conclusion
Abnormal increases in conductivity were found to consistently correspond to bacterial contamination, which was confirmed by orthogonal methods. The contaminated bioreactor runs exhibited sharp increases in conductivity rates hours before dissolved oxygen levels precipitously decreased due to bacterial growth. It is proposed that in‐line measurement of conductivity could be employed for early detection of bacterial contaminations. The probe may be adopted in pharmaceutical aseptic aqueous liquid handling processes.
Graphical and Lay Summary
Single in‐line probe detects Chinese hamster ovary cell growth by capacitance (pF cm−1), and bacterial contamination by conductivity (mS cm−1) in bioreactors. An abnormal increase in conductivity not attributable to feeding nutrients or base addition and flat or deteriorating capacitance indicate bacterial contamination earlier than pH or dissolved oxygen (DO) responses. Precipitous drop is seen in DO and pH values when bacterial load overwhelms the bioreactor process controls at later time points.
Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger ...numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 μg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/μg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.
•Difficult-to-express proteins expressed in CHO cells using STEP® vector technology.•STEP® technology enables production of DTE proteins with high product homogeneity.•STEP® vectors combine speed of transient expression with reliable stable expression.•Rapid production of any type of DTE protein for large-scale GMP manufacturing.
Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the ...mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.
Background
Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can ...prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression.
Methods and results
In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line.
Conclusion
Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development.
(1) Background: Avian influenza has attracted widespread attention because of its severe effect on the poultry industry and potential threat to human health. The H9N2 subtype of avian influenza ...viruses was the most prevalent in chickens, and there are several commercial vaccines available for the prevention of the H9N2 subtype of avian influenza viruses. However, due to the prompt antigenic drift and antigenic shift of influenza viruses, outbreaks of H9N2 viruses still continuously occur, so surveillance and vaccine updates for H9N2 subtype avian influenza viruses are particularly important. (2) Methods: In this study, we constructed a stable Chinese hamster ovary cell line (CHO) to express the H9 hemagglutinin (HA) protein of the major prevalent H9N2 strain A/chicken/Daye/DY0602/2017 with genetic engineering technology, and then a subunit H9 avian influenza vaccine was prepared using the purified HA protein with a water-in-oil adjuvant. (3) Results: The results showed that the HI antibodies significantly increased after vaccination with the H9 subunit vaccine in specific-pathogen-free (SPF) chickens with a dose-dependent potency of the immunized HA protein, and the 50 μg or more per dose HA protein could provide complete protection against the H9N2 virus challenge. (4) Conclusions: These results indicate that the CHO expression system could be a platform used to develop the subunit vaccine against H9 influenza viruses in chickens.
As of early 2022, the coronavirus disease 2019 (COVID-19) pandemic remains a substantial global health concern. Different treatments for COVID-19, such as anti-COVID-19 neutralizing monoclonal ...antibodies (mAbs), have been developed under tight timelines. Not only mAb product and clinical development but also chemistry, manufacturing, and controls (CMC) process development at pandemic speed are required to address this highly unmet patient need. CMC development consists of early- and late-stage process development to ensure sufficient mAb manufacturing yield and consistent product quality for patient safety and efficacy. Here, we report a case study of late-stage cell culture process development at pandemic speed for mAb1 and mAb2 production as a combination therapy for a highly unmet patient treatment. We completed late-stage cell culture process characterization (PC) within approximately 4 months from the cell culture process definition to the initiation of the manufacturing process performance qualification (PPQ) campaign for mAb1 and mAb2, in comparison to a standard one-year PC timeline. Different strategies were presented in detail at different PC steps, i.e., pre-PC risk assessment, scale-down model development and qualification, formal PC experiments, and in-process control strategy development for a successful PPQ campaign that did not sacrifice quality. The strategies we present may be applied to accelerate late-stage process development for other biologics to reduce timelines.
The Yangtze River basin covers one-fifth of China’s land area and serves as a water source for one-third of China’s population. During long-distance water transport from upstream to downstream, ...various sources of dissolved organic matter (DOM) lead to considerable variation in DOM properties, significantly impacting water treatability and disinfection byproduct (DBP) formation after chlorination. Using size-exclusion chromatography and fluorescence spectroscopy, the spatial variation in DOM characteristics was comprehensively investigated on a basin scale. The formation of 36 DBPs and speciated total organic halogen in chlorinated samples was determined. Overall, the Yangtze River waters featured a high proportion of terrestrially derived humic substances that served as important precursors for trihalomethanes and haloacetic acids, which was responsible for the increase in total DBP formation along the Yangtze River. The downstream waters were characterized by high levels of microbially derived protein-like biopolymers, which significantly contributed to the formation of haloacetaldehydes and haloacetonitriles that dominated DBP-associated mammalian cell cytotoxicity. Moreover, the precursors of haloacetaldehydes and haloacetonitriles in downstream waters were highly hydrophilic, posing a challenge for water treatment. This study presents an extensive basin-scale study, providing insights into DOM variations along the Yangtze River, illustrating the impact of DOM properties on drinking water from a DBP perspective.
Psoralen and isopsoralen are the major components responsible for Psoraleae Fructus-induced hepatotoxicity. This study explored the role of metabolic activation by cytochrome P450 (CYP) enzymes in ...psoralen- and isopsoralen-induced cytotoxicity and its potential mechanisms. Inhibitors of CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 were used to screen specific CYP enzymes responsible for the metabolic activation of psoralen and isopsoralen in mouse primary hepatocytes, which was verified using the corresponding transfected cell lines. Network toxicology and transcriptome analyses were performed to explore the mechanisms underlying toxicity. Psoralen and isopsoralen decreased the viability of mouse primary hepatocytes, whereas the inhibition of CYP2C9, 2C19, 2D6, and 2E1 significantly increased their viability. Psoralen-induced cytotoxicity was significantly enhanced by the overexpression of CYP2C19 in Chinese hamster ovary cells, whereas the overexpression of the above CYP enzymes did not affect the cytotoxicity of isopsoralen. Psoralen- and isopsoralen-induced cytotoxic effects were associated with putative core targets (i.e., Fn1, Thbs1, and Tlr2) and multiple signaling pathways (e.g., PI3K-Akt, MAPK, and TNF pathways). Our results demonstrate that the metabolic activation of psoralen and isopsoralen is mediated by CYP enzymes, thereby regulating multiple core targets and signaling pathways and resulting in cytotoxicity.
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•Psoralen and isopsoralen metabolized by CYPs exhibit clear cytotoxicity.•CYP2C19 is the primary CYP enzyme involved in the metabolic activation of psoralen.•Specific CYPs that mediated cytotoxicity of isopsoralen further need more studies.•Psoralen and isopsoralen metabolized by CYPs may act on targets (Fn1, Thbs1, etc.).
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