Chinese Hamster Ovary Research Articles

Exploring the Potentiality of a Plant Platform for Monoclonal Antibody Production in Veterinary Medicine.

Canine atopic dermatitis (CAD) is an allergic, inflammatory, and pruritic skin disease associated with the production of IgE antibodies against environmental allergens and mainly house dust mite allergens. This complex dermatological pathology involves Interleukin 31 (IL-31) as a central itch mediator. One of the most effective CAD treatments is a caninized monoclonal antibody (mAb) called Lokivetmab. It is produced in CHO cells and targets specifically canine IL-31 (cIL-31) and blocks its cellular messaging. This treatment has undoubtedly contributed to a breakthrough in dermatitis-related pruritus. However, its production in mammalian cells requires time-consuming procedures, high production costs, and investment. Plants are considered an emerging protein production platform for recombinant biopharmaceuticals due to their cost-effectiveness and rapidity for production. Here, we use transient expression in Nicotiana benthamiana plants to produce recombinant canine Interleukin 31 (cIL-31) and an anti-IL-31 monoclonal antibody (M1). First, we describe the production and characterization of M1 and then its activity on an IL-31-induced pruritic model in dogs compared to its commercial homolog. Dogs treated with the plant-made M1 mAb have shown similar improvements to Lokivetmab-treated ones after different challenges using canine IL-31. Furthermore, M1 injections were not associated with any side effects. These results demonstrate the safety and efficacy of this plant-made Lokivetmab biosimilar to control dogs' pruritus in a well-established model. Finally, this study shows that the plant-production platform can be utilized to produce rapidly functional mAbs and bring hope to the immunotherapy field of veterinary medicine.

Combining descriptive and predictive modeling to systematically design depth filtration-based harvest processes for biologics.

Advances in upstream production of biologics-particularly intensified fed-batch processes beyond 10% cell solids-have severely strained harvest operations, especially depth filtration. Bioreactors containing high amounts of cell debris (more than 40% particles <10 µm in diameter) are increasingly common and drive the need for more robust depth filtration processes, while accelerated timelines emphasize the need for predictive tools to accelerate development. Both needs are constrained by the current limited mechanistic understanding of the harvest filter-feedstream system. Historically, process development relied on screening scale-down depth filter devices and conditions to define throughput before fouling, indicated by increasing differential pressure and/or particle breakthrough (measured via turbidity). This approach is straightforward, but resource-intensive, and its results are inherently limited by the variability of the feedstream. Semi-empirical models have been developed from first principles to describe various mechanisms of filter fouling, that is, pore constriction, pore blocking, and/or surface deposit. Fitting these models to experimental data can assist in identifying the dominant fouling mechanism. Still, this approach sees limited application to guide process development, as it is descriptive, not predictive. To address this gap, we developed a hybrid modeling approach. Leveraging historical bench scale filtration process data, we built a partial least squares regression model to predict particle breakthrough from filter and feedstream attributes, and leveraged the model to demonstrate prediction of filter performance a priori. The fouling models are used to interpret and provide physical meaning to these computational models. This hybrid approach-combining the mechanistic insights of fouling models and the predictive capability of computational models-was used to establish a robust platform strategy for depth filtration of Chinese hamster ovary cell cultures. As new data continues to teach the computational models, in silico tools will become an essential part of harvest process development by enabling prospective experimental design, reducing total experimental load, and accelerating development under strict timelines.

Multi-cell-line learning for the data-driven construction of mechanistic metabolic models.

Mammalian cells are commonly used as hosts in cell culture for biologics production in the pharmaceutical industry. Structured mechanistic models of metabolism have been used to capture complex cellular mechanisms that contribute to varying metabolic shifts in different cell lines. However, little research has focused on the impact of temporal changes in enzyme abundance and activity on the modeling of cell metabolism. In this work, we present a framework for constructing mechanistic models of metabolism that integrate growth-signaling control of enzyme activity and transcript dynamics. The proposed approach is applied to build models for three Chinese hamster ovary (CHO) cell lines using fed-batch culture data and time-series transcript profiles. Leveraging information from the transcriptome data, we develop a parameter estimation approach based on multi-cell-line (MCL) learning, which combines data sets from different cell lines and trains the individual cell-line models jointly to improve model accuracy. The computational results demonstrate the important role of growth signaling and transcript variability in metabolic models as well as the virtue of the MCL approach for constructing cell-line models with a limited amount of data. The resulting models exhibit a high level of accuracy in predicting distinct metabolic behaviors in the different cell lines; these models can potentially be used to accelerate the process and cell-line development for the biomanufacturing of new protein therapeutics.

Antibody glycan quality predicted from CHO cell culture media markers and machine learning

N-glycosylation can have a profound effect on the quality of mAb therapeutics. In biomanufacturing, one of the ways to influence N-glycosylation patterns is by altering the media used to grow mAb cell expression systems. Here, we explore the potential of machine learning (ML) to forecast the abundances of N-glycan types based on variables related to the growth media. The ML models exploit a dataset consisting of detailed glycomic characterisation of Anti-HER fed-batch bioreactor cell cultures measured daily under 12 different culture conditions, such as changes in levels of dissolved oxygen, pH, temperature, and the use of two different commercially available media. By performing spent media quantitation and subsequent calculation of pseudo cell consumption rates (termed media markers) as inputs to the ML model, we were able to demonstrate a small subset of media markers (18 selected out of 167 mass spectrometry peaks) in a Chinese Hamster Ovary (CHO) cell cultures are important to model N-glycan relative abundances (Regression - correlations between 0.80–0.92; Classification - AUC between 75.0–97.2). The performances suggest the ML models can infer N-glycan critical quality attributes from extracellular media as a proxy. Given its accuracy, we envisage its potential applications in biomaufactucuring, especially in areas of process development, downstream and upstream bioprocessing.

Dynamic optimization of an integrated cultivation-aggregation model for mAb production.

Due to their proteinaceous structure, monoclonal antibodies (mAbs) are susceptible to irreversible aggregation, with harmful consequences on drug efficacy and patient safety. To mitigate this risk in modern biopharmaceutical processes, it is critical to comply with current good manufacturing practices (cGMP) and pursue operating strategies minimizing irreversible aggregation whilst also maximizing mAb throughput. These conflicting objectives are targeted in this study by formulating and analyzing an integrated dynamic model accounting for both cultivation and aggregation of mAbs from a Chinese Hamster Ovary (CHO) cell line. Two manipulated dynamic variables are considered here in simulation studies: firstly temperature manipulation within a batch reactor, and secondly feed flow manipulation within a series of isothermal fed-batch reactors. Following this, dynamic optimization investigations have been conducted, firstly with the single objective of maximizing mAb throughput and secondly with multiple (two) objectives of maximizing mAb throughput while also minimizing irreversible aggregate content, simultaneously. The study provides key insight into tradeoffs of how simultaneous temperature and feed flowrate manipulation affects mAb throughput and aggregation inside bioreactors.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

A phase I clinical study to evaluate the safety, tolerability, immunogenicity, pharmacokinetics and preliminary efficacy of SCB-219M in patients with chemotherapy-induced thrombocytopenia (CIT): An interim analysis.

e15015 Background: CIT is a serious, chemotherapy-associated complication observed in a wide range of cancer patients with various types of cancer. Incidence of CIT can occur in about 15-25% of patients undergoing standard chemotherapy regiments with platinum, taxane and/or gemcitabine-based regimen. CIT can lead to chemotherapy dose delay or dose reduction, and potentially bleeding events. Currently, there is no FDA-approved therapy for CIT. SCB-219M, a thrombopoietin receptor agonist (TPO-RA) mimetic bispecific Fc-fusion protein produced from CHO cells can efficiently raise PLT count under normal and pathological conditions in laboratory animals. Methods: This is a single-arm, multi-center, open-label, dose escalation Phase Ia clinical study to evaluate the safety, tolerability, and immunogenicity, PK profile and preliminary efficacy of a single ascending dose of SCB-219M administered one day after a chemotherapy in prior CIT patients with malignant solid tumors and lymphoma. The study design of accelerated titration combined with "3 + 3" is used, and the number of subjects planned to enroll is 12 to be assigned to 4 cohorts with 2, 6, 10 and 15 mg/kg subcu administration of SCB-219M, respectively. Enrollment criteria include episode of CIT experienced (&lt;75 × 109/L in PLT count) in prior cycle of chemotherapies and PLT count has recovered to &gt;75 × 109/L prior to initiation of this study . The chemotherapy regimen allowed include either mono or combination chemotherapy (including targeted therapy and/or immunotherapy). The observation period of dose-limiting toxicities (DLTs) is 21 days. Chemotherapy is planned at each study site. There will be no change in the planned dose or frequency of chemotherapy in the study. Results: As of Dec 20, 2023, 9 prior CIT patients including 3 at 15 mg/kg were enrolled. SCB-219M was well tolerated. There were no SAEs or AESIs (Adverse Event of Special Interest) observed. DLT was not reached. A favorable PK profile, in particular in serum availability (AUC) and half-life (T1/2) was obtained. All 9 prior CIT patients in the study maintained PLT counts &gt;75 x 109/L from 1-week following chemotherapies with a single dose of SCB-219M, and durable responses experienced through at least 3 weeks. Conclusions: In this interim analysis, drug safety, PK profile and durable efficacy are expected to support SCB-219M dosing interval of ≥2-weeks or a single dosing regimen within a chemotherapy cycle. Accordingly, a Phase Ib trial evaluating repeated dosing of SCB-219M is planned to initiate in 2024. Clinical trial information: NCT05426369 .

Combination treatment effect of an immunotherapeutic agent with microtubule inhibitors.

e14619 Background: The microtubule (MT) is a polymer formed by self-assembly of αβ-tubulin heterodimers. MT inhibitors either stabilize or destabilize microtubule assembly and halt cell proliferation during the mitotic phase of the cell cycle. However, due to non-specificity, high resistance, and adverse reactions observed with MT-targeting drugs, various approaches to novel combination chemotherapies are being used in the clinic. The specific goal of this study is to investigate the ability of an immunotherapeutic agent to alter the activity of MT-targeting chemotherapeutics when used in a combination treatment approach. Methods: The combination treatment effect of the immunotherapeutic agent with microtubule inhibitors was investigated using two methods: 1) Tubulin polymerization studies using purified tubulin and microtubule inhibitors, paclitaxel (a microtubule stabilizing agent) and dolastatin 10 (a microtubule destabilizing agent), studied alone or in combination with the immunotherapeutic agent. (2) Co-culture system expressing the stably expressing Nuclear Factor of Activated T cells (NFAT)- Renilla luciferase (NFAT- Rluc) reporter gene to study anti-tumor immune response of MT inhibitors alone or in combination with the immunotherapeutic agent. Results: Spectrophotometric-based tubulin polymerization studies demonstrated that the immunotherapeutic agent (0.01 - 0.1 mg/mL) altered tubulin polymerization when studied in combination with either paclitaxel (10 µM) or dolastatin 10 (0.5 mM). Luminescence-based reporter gene expression studies in a co-culture system, consisting of CD3− antigen presenting Chinese hamster ovary and CD3+ effector Jurkat T cells, demonstrated the combination treatment effectively induced NFAT- Rluc activity when compared to the controls, the immunotherapeutic agent alone or to the MT-inhibitor alone. Statistical analysis was done using one-way analysis of variance (ANOVA). A significant difference ( p &lt; 0.05) in mean IC50 values (expressed as ng/mL ± SEM, N=4) was observed between the immunotherapeutic plus dolastatin 10 versus the immunotherapeutic agent alone, and a 6.5-fold decrease (85% reduction) in the IC50 value was observed for induction of NFAT- Rluc activity in the presence of immunotherapeutic plus dolastatin 10 versus the immunotherapeutic alone. There was a notable decrease (1.9-fold or 46% reduction) in the IC50 value observed for induction of NFAT- Rluc activity in the presence of the immunotherapeutic plus paclitaxel versus the immunotherapeutic alone. Conclusions: These results suggest a boost in T-cell stimulation by the immunotherapeutic agent in the presence of MT inhibitors. The implications of enhanced T-cell activation on antitumor immunity and modulation of tubulin polymerization by a combination treatment of the immunotherapeutic agent and microtubule inhibitors are currently under investigation.

Discarded CHO cells as a valuable source of bioactive peptides for sustainable biotechnological applications

Repurposing discarded cells stands as a groundbreaking paradigm shift in sustainable biotechnology, with profound implications across diverse industrial sectors. Our study proposes a transformative concept by harnessing histone proteins from discarded CHO cells to produce bioactive peptides. We systematically isolated and hydrolyzed histones using Trypsin and Neutrase enzymes, optimizing reaction conditions. Ultrafiltration yielded distinct peptide fractions (<3 kDa and 3–10 kDa), which we analyzed for DPP-IV inhibition, antioxidant potential, and other activities. Furthermore, LC-Q-TOF-MS analysis and in silico tools unveiled the structural composition of bioactive peptides within these fractions. Three peptide sequences with high bioactivity potential were identified: KLPFQR, VNRFLR, and LSSCAPVFL. Our findings demonstrated exceptional DPP-IV inhibition, potent antioxidant effects, and effective anti-lipid peroxidation activities, surpassing reference compounds. Hemolytic activity assessment indicated promising biocompatibility, enhancing therapeutic application prospects. Pioneering the strategic repurposing of discarded cells, this research addresses cost-efficiency in cell-based studies and promotes sustainable use of biological resources across sectors. This novel approach offers an efficient, eco-friendly method for bioactive molecule procurement and resource management, revolutionizing cell culture studies and biotechnological applications.

Interaction of Angiotensin-(1−7) with kinins in the kidney circulation: Role of B1 receptors

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1−7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1−7) (1.0–25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 μM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 μM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1−7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1−7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1−7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1−7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Ayurvedic herbal formulations Haridra Khanda and Manjisthadi Kwath (brihat) in the management of allergic rhinitis: A pharmacological study

This study aims to pharmacologically validate Haridra Khanda (HK) and Manjishthadi Kwatham (brihat) (MMK) in allergy management using invivo and invitro studies to rationalize the prescription of these two ayurvedic polyherbal drug formulations, which are currently used in Indian government hospitals. Experimental animals received HK and MMK orally from day 0 to day 14 and histamine (1 mg/kg b.w/i.v) and 1 % evans blue (EB) (0.1 mL) via tail vein on day 14. The compound 48/80 (intracutaneous) challenged mice model followed the same technique. The former mimicked acute anaphylaxis and the latter mast cell degranulation. For both models, EB dye leakage was quantified spectrophotometrically to determine vascular permeability. Plasma histamine was measured in Compound 48/80-induced animals using LC-ESI-MS/MS. The guineapig received HK and MMK p.o. and 0.6 % histamine sprayed in a histamine chamber to simulate allergic rhinitis. Blood eosinophil count and sneeze rate were measured in histamine-challenged guineapigs. Goat R.B.C. membrane stability assay (mammalian cell membrane toxicity) and intracellular histamine-induced cytosolic Ca2+ release assay in Chinese hamster ovary (CHO) cells were performed in vitro. For both histamine and Compound 48/80 challenged animals, HK (22.81 % and 14.58 %) and MMK (19.71 % and 22.40 %) significantly reduced EB dye leakage (p < 0.05). Both formulations, HK and MMK considerably (p < 0.05) decreased plasma histamine (29.62 % and 25.37 % respectively) in mice and eosinophilic count (11.56 % and 9.94 % respectively) and sneeze rate (42.58 % and 29.03 % respectively) in guinea pigs. In membrane stability experiment, HK and MMK reduced RBC lysis. Both HK and MMK raw/dialysate blocked CHO cell cytosolic Ca2+ release. HK and MMK activities mimic mast cell stabilization with possible H1 receptor inactivation seen by decreased Ca2+ efflux and thus indicate potential for allergic rhinitis management. The combination of activities is usually related with curative and prophylactic therapy and might lead future clinical trials and therapies.

Enhancing recombinant antibody yield in Chinese hamster ovary cells

ABSTRACT A range of recombinant monoclonal antibodies (rMAbs) have found application in treating diverse diseases, spanning various cancers and immune system disorders. Chinese hamster ovary (CHO) cells have emerged as the predominant choice for producing these rMAbs due to their robustness, ease of transfection, and capacity for posttranslational modifications akin to those in human cells. Transient transfection and/or stable expression could be conducted to express rMAbs in CHO cells. To bolster the yield of rMAbs in CHO cells, a multitude of approaches have been developed, encompassing vector optimization, medium formulation, cultivation parameters, and cell engineering. This review succinctly outlines these methodologies when also addressing challenges encountered in the production process, such as issues with aggregation and fucosylation.

Sodium valproate promotes low metabolism and high protein expression in CHO-engineered cell lines

Chinese hamster ovary cells are widely used in various biopharmaceutical industries as preferred host cells for the production of recombinant proteins. In an effort to increase yield and reduce production costs, the incorporation of small molecule additives into the culture medium has emerged as an effective approach to augment the expression of recombinant proteins, thus increasing overall efficiency. Here, we examined the impact of five small molecules on protein expression in CHO-engineered cell lines. These molecules included sodium valproate, alanyl-L-tyrosine, ascorbic acid, sodium citrate and ferrous sulphate. Their optimal concentrations were determined through univariate analysis aimed at enhancing cell growth and protein production. Our findings revealed that inoculating cells at a density of 1.0 × 106 cells/mL with the simultaneous addition of sodium valproate at a concentration of 2 mM for 0 h yielded the most favorable results. Notably, while valproate inhibited cell growth in a dose-dependent manner, it simultaneously extended cell culture longevity. Additionally, sodium valproate was observed to decrease glucose consumption, collectively increasing protein expression. In conclusion, our data suggest that sodium valproate is a potent chemical agent capable of increasing the production of recombinant protein in CHO cells. This enhancement is achieved by the modulation of metabolic pathways in CHO-engineered cell lines, thereby facilitating a state of low metabolism and high protein expression.

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.

Technoeconomic Analysis of Intensified PEGylated Biopharmaceutical Recombinant Protein Production: Alpha Antitrypsin as a Model Case

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by the insufficient production of the AAT protein. Due to availability limitations, not all AATD patients receive protein therapy treatment. In this study, the technoeconomic analysis of different processes (conventional and intensified) producing 200 kg/year of PEGylated recombinant AAT (PEG-AAT) using a Chinese hamster ovary cell line was investigated. All bioprocesses consist of upstream, downstream, and PEGylation sections. A base-case model (process A) of the conventional fed-batch production bioreactor was developed using SuperPro Designer software (Version 13) to evaluate the economic feasibility of the process. The cost of goods (COG) was estimated to be approximately USD 387.6/g. Furthermore, an intensified process (B) was modeled and evaluated to reduce the COG. Process intensification was implemented in the process (N-1 perfusion bioreactor). The specific operating COG for process B was found to be 10% less than that of process A. Scenario analysis was performed to assess the impact of process capacity (100–1000 kg/year) and cell-specific productivity (30–90 pg/cell/day). With an increase in process capacity, the specific operating COG was reduced for all processes. Increasing cell-specific productivity decreases the specific operating COG at different rates for each process, depending on the titer level. Future investigations into the PEGylation section are required since it has the highest COG of all the sections.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Boltzmann Model Predicts Glycan Structures from Lectin Binding.

Glycans are complex oligosaccharides that are involved in many diseases and biological processes. Unfortunately, current methods for determining glycan composition and structure (glycan sequencing) are laborious and require a high level of expertise. Here, we assess the feasibility of sequencing glycans based on their lectin binding fingerprints. By training a Boltzmann model on lectin binding data, we predict the approximate structures of 88 ± 7% of N-glycans and 87 ± 13% of O-glycans in our test set. We show that our model generalizes well to the pharmaceutically relevant case of Chinese hamster ovary (CHO) cell glycans. We also analyze the motif specificity of a wide array of lectins and identify the most and least predictive lectins and glycan features. These results could help streamline glycoprotein research and be of use to anyone using lectins for glycobiology.

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Conserved cysteines prevent C-mannosylation of mucin Cys domains.

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucinCysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.