Search Results (116)

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells. Full article

Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing Fc-related effector functions. N-linked glycan distribution of therapeutic IgG1s affects Fc-related effector functions such as CDC (complement-dependent cytotoxicity) and ADCC (antibody dependent cell-mediated cytotoxicity) biological activities and efficacy in vivo. Hence, as a critical quality attribute (CQA), the glycosylation profile of therapeutic IgG1s must be consistently preserved, which is primarily influenced by manufacturing process factors. In the era of biosimilars, it is challenging for biopharmaceutical manufacturers to not only obtain the desired glycan distribution consistently but also to meet the innovator molecule specifications as per the regulatory agencies. Methods: This study investigates the CHO fed-batch process parameters that affect the titer and terminal galactosylation of the TNF-α blocker-IgG1. It was hypothesized that galactose supplementation would enhance the galactosylation of TNF-α blocker-IgG1. Results: It was observed that such in-cultivation process shift does not affect cell culture parameters yet significantly enhances the galactosylation of TNF-α blocker-IgG1. Interestingly, the results indicate that supplementing D-galactose from the exponential phase of the CHO fed-batch process had the greatest effect on Fc galactosylation, increasing the amount of total galactosylated TNF-α blocker-IgG1 from 7.7% to 15.8%. Conclusions: Our results demonstrate a relatively easy and viable technique for cell culture engineering that is more appropriate for industrial production than costly in vitro glycoengineering. Full article

Follicle stimulating hormone (FSH) stands as one of the most prevalently used reproductive hormones in the field of animal-assisted reproduction. Conventionally, pituitary FSH is sourced from the heterologous pituitary glands of pigs and sheep procured from slaughterhouses, and it typically exists in the form of crude FSH. The specific challenges inherent in FSH-based assisted reproduction drugs has significantly spurred the interest in exploring novel alternatives, aiming to reduce the reliance on these traditional sources in relevant production processes. In this study, the α- and β-FSH genes were retrieved from pituitary cDNA libraries. These genes were selected to construct a recombinant protein—the novel cow recombinant FSH (CrFSH)—through the application of the homologous recombination method. Notably, the β-subunit was extended by a carboxy-terminal peptide (CTP). After successfully integrating the two genes into Chinese hamster ovary (CHO) cells, the recombinant protein (approximately 33 kDa) in the culture supernatant was detected using Western blotting (WB). The results of the GCs proliferation experiment indicated that both 1.2 µg/mL pFSH and 20–20,000 ng/mL CrFSH could significantly promote the proliferation of GCs in vitro. Remarkably, on the 4th day after treatment, 20 ng/mL of CrFSH had a higher GCs proliferation rate than 1.2 μg/mL of pFSH (p < 0.001). Additionally, cyclic adenosine monophosphate (cAMP) induction assay in GCs unequivocally confirmed that CrFSH possesses superior activity compared to pFSH. These findings underscore that this recombinant protein holds great potential as a promising candidate for FSH production in assisted reproduction approaches for dairy herds. Full article

Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes—glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells. Full article

The metabolism of drugs and foreign substances in humans typically involves multiple enzymatic steps, particularly in phase-1 biotransformation in the liver, where various cytochrome P450 monooxygenases (CYPs) play crucial roles. This complexity can lead to a wide range of metabolites. Understanding the contributions of individual CYPs and their interactions within these intricate enzyme cascades can be challenging. We recently developed an in vitro biotransformation platform employing various Chinese Hamster Ovarian (CHO) cell clones. These clones express human cytochrome P450 oxidoreductase (CPR), and each is defined by a specific human CYP enzyme expression, thus exhibiting no detectable endogenous CYP enzyme activity (mono-CYP CHO platform). In this study, we investigated whether the mono-CYP CHO platform is a suitable tool for modeling complex drug metabolization reactions in vitro. Tamoxifen (TAM) was selected as a model substance due to its role as a prodrug widely used in breast cancer therapy, where its main active metabolite, endoxifen, arises from a two-step metabolism primarily involving the CYP system. Specifically, the combined activity of CYP3A4 and CYP2D6 is believed to be essential for efficient endoxifen production. However, the physiological metabolization pathway of TAM is more complex and interconnected, and the reasons for TAM’s therapeutic success and variability among patients are not yet fully understood. Analogous to our recently introduced mono-CYP3A4 CHO cells, we generated a CHO cell line expressing human CPR and CYP2D6, including analysis of CYP2D6 expression and specific activity. Comparative studies on the metabolization of TAM were performed with both mono-CYP CHO models individually and in co-culture with intact cells as well as with isolated microsomes. Supernatants were analyzed by HPLC to calculate individual CYP activity for each metabolite. All the picked mono-CYP2D6 clones expressed similar CYP2D6 protein amounts but showed different enzyme activities. Mono-CYP2D6 clone 18 was selected as the most suitable for TAM metabolization based on microsomal activity assays. TAM conversion with mono-CYP2D6 and -3A4 clones, as well as the combination of both, resulted in the formation of the expected main metabolites. Mono-CYP2D6 cells and microsomes produced the highest detected amounts of 4-hydroxytamoxifen and endoxifen, along with N-desmethyltamoxifen and small amounts of N,N-didesmethyltamoxifen. N-desmethyltamoxifen was the only TAM metabolite detected in notable quantities in mono-CYP3A4, while 4-hydroxytamoxifen and endoxifen were present only in trace amounts. In CYP2D6/3A4 co-culture and equal mixtures of both CYP microsomes, all metabolites were detected at concentrations around 50% of those in individual clones, indicating no significant synergistic effects. In conclusion, our mono-CYP CHO model confirmed the essential role of CYP2D6 in synthesizing the active TAM metabolite endoxifen and indicated that CYP2D6 is also involved in producing the by-metabolite N,N-didesmethyltamoxifen. The differences in metabolite spectra between the two mono-CYP models highlight the CYP specificity and sensitivity of our in vitro system. Full article

Cell-based therapy is a promising direction for the treatment of various diseases. However, it is associated with several problems, primarily related to reproducibility and standardization. In this context, the development of new methods for the production of cell-based preparations is of particular relevance. Recently, a novel technology named ‘crossing’ has been developed. It comprises the multi-stage vibrational processing of two closely spaced test tubes containing the initial substance and a neutral carrier (water or lactose). As a result, the neutral carrier acquires some properties of the initial substance, and artificial products, vibrational iterations, are obtained. Some vibrational iterations are also capable of exerting a modifying effect on the initial substance (or its target in the body), changing its physico-chemical/biological properties. Earlier, we demonstrated the possibility of obtaining vibrational iterations from biological molecules (antibodies). In this study, we evaluated the biological effects of vibrational iterations obtained by the crossing technology using cells grown in culture. This work shows that vibrational iterations obtained from CHO-S cell culture affect the ability of CHO-S cells to utilize glucose in the presence of insulin. The data demonstrate the prospect of developing fundamentally new biological drugs based on vibrational iterations, including for the treatment of diabetes mellitus. Full article

Background: Chinese hamster ovary (CHO) cell metabolism is complex, influenced by nutrients like glucose and glutamine and metabolites such as lactate. Real-time monitoring is necessary for optimizing culture conditions and ensuring consistent product quality. Raman spectroscopy has emerged as a robust process analytical technology (PAT) tool due to its non-invasive, in situ capabilities. This study evaluates Raman spectroscopy for monitoring key metabolic parameters and IgG titer in CHO cell cultures. Methods: Raman spectroscopy was applied to five 10 L-scale CHO cell cultures. Partial least squares (PLS) regression models were developed from four batches, including one with induced cell death, to enhance robustness. The models were validated against blind test sets. Results: PLS models exhibited high predictive accuracy (R² > 0.9). Glucose and IgG titer predictions were reliable (RMSEP = 0.51 g/L and 0.12 g/L, respectively), while glutamine and lactate had higher RMSEP due to lower concentrations. Specific Raman bands contributed to the specificity of glucose, lactate, and IgG models. Predictions for viable (VCD) and total cell density (TCD) were less accurate due to the absence of direct Raman signals. Conclusions: This study confirms Raman spectroscopy’s potential for real-time, in situ bioprocess monitoring without manual sampling. Chemometric analysis enhances model robustness, supporting automated control systems. Raman data could enable continuous feedback regulation of critical nutrients like glucose, ensuring consistent critical quality attributes (CQAs) in biopharmaceutical production. Full article

Cyanide (CN) is a potent, fast-acting toxicant that impacts endogenous biomolecules in the nervous system, including acid-sensing ion channels (ASICs), which play a vital role in various neurological and psychological conditions. Here, we demonstrate that CN rapidly potentiates ASIC currents in cultured mouse cortical neurons in a dose-dependent manner while causing a leftward shift in the pH dose–response curve. Notably, this potentiation was unaffected by a 30-min CN treatment or the presence of ATP in the recording pipette. Further investigations into the role of zinc revealed that TPEN, a high-affinity zinc chelator, did not enhance ASIC currents following CN pretreatment, nor did CN influence the potentiation of ASIC currents induced by TPEN. Low-affinity zinc blocked the potentiation of ASIC currents by CN. CN potentiated ASIC currents in cortical neurons from ASIC2 but not from ASIC1a knockout mice. In experiments with CHO cells expressing homomeric ASIC1a and heteromeric ASIC1a/2, CN potentiated ASIC1a currents but had no effect on homomeric ASIC1b, ASIC2a, or ASIC3 channels. Mutating lysine 133 (K133) to arginine (R) in the extracellular domain of ASIC1a abolished CN’s effect, suggesting that CN potentiates ASIC1a currents primarily via high-affinity zinc binding, with K133 being critical for this modulation. Full article

This study examines the differences in performance between orbitally shaken bioreactors (OSBs) and stirred tank bioreactors (STBs) in Chinese Hamster Ovary (CHO) cell perfusion culture in response to the growing market demand for monoclonal antibodies (mAbs). Although OSBs demonstrated higher cell densities, a notable reduction in specific antibody production rates was observed during the mid-to-late phases of the culture compared with STBs. To elucidate the underlying mechanisms, the rheological behaviour of high-density cell suspensions in both reactor types was initially characterised, confirming their adherence to the Sisko fluid model. Computational Fluid Dynamics (CFD) analysis revealed the influence of these rheological properties on the shear stress distribution and mass transfer. This analysis identified the key limiting factors for achieving higher cell densities: mass transfer efficiency in OSBs and shear stress in STBs. Using an Euler–Lagrangian cell-tracking methodology to analyse cellular “lifelines”, it was determined that OSBs exhibited approximately twice the number and frequency of shear stress peak occurrences compared to STBs. This persistent mechanical stimulation likely contributes to the reduced specific antibody production rates observed. This comprehensive investigation not only clarifies the comparative advantages and limitations of different bioreactor types in perfusion culture but also provides a robust theoretical basis and technical guidance for informed reactor selection, optimisation, and scale-up in industrial production environments. Full article

Chimeric orthoflaviviruses derived from the insect-specific Binjari virus (BinJV) offer a promising basis for safe orthoflavivirus vaccines. However, these vaccines have so far only been produced using adherent C6/36 Aedes albopictus mosquito cell cultures grown in serum-supplemented media, limiting their scalable manufacture. To address this, we adapted C6/36 cells for serum-free suspension culture using Sf900-III medium, achieving high peak cell densities (up to 2.5 × 10⁷ cells/mL). Higher agitation rates reduced cell aggregation, and cryopreservation and direct-to-suspension revival were successful, confirming the adapted line’s stability for research and industrial applications. Despite this, BinJV-based chimeric orthoflaviviruses, including BinJV/WNV_KUN, a candidate vaccine for West Nile virus, and similar vaccines (BinJV/DENV2 and BinJV/JEV_NSW22) for dengue 2 virus and Japanese encephalitis virus, respectively, exhibited substantially reduced titres in C6/36 cultures infected in Sf900-III, a phenomenon attributed to the medium’s acidic pH. Switching to the more alkaline, serum-free CD-FortiCHO medium enhanced the replication of these chimeric viruses to peak titres between 1.7 × 10⁷ and 7.6 × 10⁹ infectious units per mL whilst preserving viral integrity. These findings suggest that suspension-adapted C6/36 cultures in CD-FortiCHO medium can support high-yield vaccine production for various orthoflaviviruses and highlight the important role of cell culture media pH for orthoflavivirus bioprocessing. This scalable mosquito cell-based system could reduce production costs and improve vaccine accessibility, supporting efforts to combat arbovirus-related public health challenges. Full article

Chinese hamster ovary (CHO) cells are commonly used to produce recombinant therapeutic proteins (RTPs). The yield of RTPs in CHO cells has been greatly improved through cell editing and optimization of culture media, cell culture processes, and expression vectors. However, the heterogeneity of cell clones and product aggregation considerably affect the yield and quality of RTPs. Recently, novel technologies such as semi-targeted and site-specific transgene integration, endoplasmic reticulum-residents, and cell culture process optimization have been used to address these issues. In this review, novel developments in the field of CHO cell expression system heterogeneity are summarized. Moreover, the advantages and limitations of the new strategies are discussed, and important methods for the control of RTP quality are outlined. Full article

Recently, using a panel of recombinant CHO cell lines, we identified the coxsackie and adenovirus receptor (CAR) and histo-blood group antigens (HBGAs) or sialic acid as the minimum requirement for susceptibility to rhesus enteric calicivirus (ReCV) infections. While ReCVs cause lytic infection in LLC-MK2 cells, recombinant CHO (rCHO) cell lines did not exhibit any morphological changes upon infection. To monitor infectious virus production, rCHO cell cultures had to be freeze–thawed and titrated on LLC-MK2 monolayers. This raised the question of whether ReCV infection in rCHO cells is persistent and whether non-enveloped progeny virions are released from the infected cells. Here, we used the rCHO-CAR+ cell line and a CAR and sialic acid-dependent recovirus strain (FT7) and found that these cells were persistently infected, and infectious virus was continuously produced and released into the culture without showing any visible cell damage. Viral capsid protein and replication intermediate double-stranded RNA (dsRNA) were detectable in almost all cells for at least 12 passages. We suspect a fully exosomal viral exit mechanism without a lytic cycle in these cells. rCHO cell may provide a valuable system for ReCV production (producer cell line) and serve as a model for investigating enteric calicivirus non-lytic viral exit mechanisms and the properties of the released, most likely membrane-cloaked, infectious progeny virions. Full article

Ustekinumab is a fully human IgG1k monoclonal antibody that binds with high affinity and specificity to the p40 subunit of interleukins (IL-) 12 and 23, inhibiting their activity by preventing binding to their receptors. The European extension of the patent (Supplementary Protection Certificate) of ustekinumab expired on 20 July 2024. Biosimilar alternatives to ustekinumab are now an additional option for treating patients. The efficacy data for this drug in moderate-to-severe psoriasis obtained both from clinical trials and indirect comparisons through meta-analyses, are superior to those of etanercept and adalimumab, and its safety profile is more favorable than that of tumor necrosis factor (TNF) inhibitors. Several ustekinumab biosimilars have already been approved by regulatory agencies: between October 2023 and October 2024, Wezlana^® (Amgen ABP 654), Uzpruvo^® (Alvotech AVT04) and Pyzchiva^® (Samsung/Bioepis SB17) have been approved by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). SteQeyma^® (Celltrion Healthcare CT-P43) was approved by the EMA in August 2024. Otulfi^® (Fresenius Kabi/Formycon) was approved by the FDA in October 2024. Several other potential biosimilar candidates are under development, including BAT2206 (Bio-Thera), DMB-3115 (Dong-A ST), QX001S (Qyuns Therapeutic), BFI-751 (BioFactura), NeuLara (Neuclone), ONS3040 (Oncobiologics), and BOW090 (Epirus Biopharmaceuticals). In most cases, these monoclonal antibodies are expressed in cell lines (e.g., Chinese Hamster Ovary, CHO) different from those used for the originator (Sp2/0 spleen cell murine myeloma); of note, the cell line of origin is not a requirement for biosimilarity in the totality-of-evidence comparison exercise and may facilitate the production and reduce the immunogenicity of biosimilars originated in CHO cultures. This narrative review summarizes the available data on characteristics of the full comparability exercises and comparative clinical trials of these drugs. Full article

Research has shown that Raman spectroscopy could be applied to monitor various components in mammalian cell culture in real time. In the process of application, it is necessary to ensure the performance of the Raman-based model. The variable selection strategy is an effective method that significantly influences the model performance and simplification. In this study, different variable selection strategies were evaluated, and the optimal variable selection strategy was determined for monitoring the CHO cell culture process. Firstly, a wide variety of spectral regions involving the Raman fingerprinting region and the C-H stretching region were investigated. Secondly, six different variable selection algorithms were meticulously assessed. Thirdly, the combination of different variable selection algorithms was used to improve model performance and simplify the model. Finally, the monitoring of cell culture processes was implemented. The findings underscored that commonly used spectral regions could improve the model performance but could not simplify the model well. Moving-window partial least square (MWPLS), genetic algorithm (GA), and random frog (RF) are more suitable for Raman modeling of the cell culture process, but they must be used after the spectral region selection. The combination of three variable selection algorithms (MWPLS-GA-RF) improved the model’s performance by 16–70% by selecting 30–60 variables, effectively simplifying the model. For glucose, lactate, viable cell density, and ammonium ion, real-time monitoring was performed well. This study will be helpful for researchers to select suitable variable selection strategies for building models for the real-time monitoring of cell culture. Full article

Background/Objectives: There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental live virus VEE vaccines in the plasmid-transfected cell culture. Methods: Here, we used the high-fidelity polymerase chain reaction (PCR) to prepare synthetic, transcriptionally active PCR (TAP) fragments encoding the V4020 genome. Results: TAP fragment initiated the replication of the V4020 live virus vaccine in TAP fragment-transfected cells. A transfection of less than 1 ug of TAP fragment resulted in the replication of the V4020 vaccine virus in CHO cells. Conclusion: We conclude that not only plasmid DNA but also synthetic PCR-generated DNA fragments can be used for the manufacturing of live vaccines for VEEV and, potentially, other viruses. Full article