Biological Medicines Prepared Using Vibration Processing Are Able to Influence Their Targets Without Direct Contact With Them.

Recently, there has been a radical change in understanding of the nature of drugs based on highly diluted solutions. It has been established that their activity does not depend on the content of the original substance in dilutions, but is a consequence of the technological processing (TP) of dilutions with vibration, which accompanies each dilution during the preparation of solutions and, among others, leads to the formation of nanoparticles with certain properties. Repeated vibration treatment leads to the appearance of modifying activity that is absent in the original substance, and these effects of TP solutions can be exerted without direct contact with their targets, which clearly indicates the physical nature of the TP solution's activity. In the framework of this article, a statistically significant effect of TP antibodies to the insulin receptor on glucose consumption by CHO cells both with and without contact exposure, as compared with control (P < 0.05) was shown in the vast majority of the experiments. The obtained results shed light on a possible source of activity of drugs based on TP antibodies, which should be associated with the applied vibration effect and can manifest itself both with contact exposure and without it.

Modulating antibody N-glycosylation through feed additives using a multi-tiered approach.

Glycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product.

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.

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.

Evaluation of NAD+ precursors for improved metabolism and productivity of antibody-producing CHO cell.

In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.

SiMPl-GS: Advancing Cell Line Development via Synthetic Selection Marker for Next-Generation Biopharmaceutical Production.

Rapid and efficient cell line development (CLD) process is essential to expedite therapeutic protein development. However, the performance of widely used glutamine-based selection systems is limited by low selection efficiency, stringency, and the inability to select multiple genes. Therefore, an AND-gate synthetic selection system is rationally designed using split intein-mediated protein ligation of glutamine synthetase (GS) (SiMPl-GS). Split sites of the GS are selected using a computational approach and validated with GS-knockout Chinese hamster ovary cells for their potential to enable cell survival in a glutamine-free medium. In CLD, SiMPl-GS outperforms the wild-type GS by selectively enriching high producers. Unlike wild-type GS, SiMPl-GS results in cell pools in which most cells produce high levels of therapeutic proteins. Harnessing orthogonal split intein pairs further enables the selection of four plasmids with a single selection, streamlining multispecific antibody-producing CLD. Taken together, SiMPl-GS is a simple yet effective means to expedite CLD for therapeutic protein production.

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.

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism.

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO-K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO-K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Matrix attachment regions enhance transgene expression by manipulating position-dependent effects in stably transfected CHO-K1 cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluorescent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recombinant protein production in CHO-K1 cells. This insight underscores the importance of strategic MAR incorporation in vector design for optimized recombinant protein expression.

Enhancing recombinant antibody yield in Chinese hamster ovary cells.

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.

The new frontier in CHO cell line development: From random to targeted transgene integration technologies.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells.

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.

Sialyllactose supplementation enhances sialylation of Fc-fusion glycoprotein in recombinant Chinese hamster ovary cell culture.

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.

Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target.

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

Ectoine enhances recombinant antibody production in Chinese hamster ovary cells by promoting cell cycle arrest.

Chinese hamster ovary (CHO) cells represent the most preferential host cell system for therapeutic monoclonal antibody (mAb) production. Enhancing mAb production in CHO cells can be achieved by adding chemical compounds that regulate the cell cycle and cell survival pathways. This study investigated the impact of ectoine supplementation on mAb production in CHO cells. The results showed that adding ectoine at a concentration of 100 mM on the 3rd day of cultivation improved mAb production by improving cell viability and extending the culture duration. RNA sequencing analysis revealed differentially expressed genes associated with cell cycle regulation, cell proliferation, and cellular homeostasis, in particular promotion of cell cycle arrest, which was then confirmed by flow cytometry analysis. Ectoine-treated CHO cells exhibited an increase in the number of cells in the G0/G1 phase. In addition, the cell diameter was also increased. These findings support the hypothesis that ectoine enhances mAb production in CHO cells through mechanisms involving cell cycle arrest and cellular homeostasis. Overall, this study highlights the potential of ectoine as a promising supplementation strategy to enhance mAb production not only in CHO cells but also in other cell lines.

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.

Modeling of cell cultivation for monoclonal antibody production processes considering lactate metabolic shifts.

Demand for monoclonal antibodies (mAbs) is rapidly increasing. To achieve higher productivity, there have been improvements to cell lines, operating modes, media, and cultivation conditions. Representative mathematical models are needed to narrow down the growing number of process alternatives. Previous studies have proposed mechanistic models to depict cell metabolic shifts (e.g., lactate production to consumption). However, the impacts of variations of some operating conditions have not yet been fully incorporated in such models. This paper offers a new mechanistic model considering variations in dissolved oxygen and glutamine depletion on cell metabolism applied to a novel Chinese hamster ovary (CHO) cell line. Expressions for the specific rates of lactate production, glutamine consumption, and mAb production were formulated for stirred and shaken-tank reactors. A deeper understanding of lactate metabolic shifts was obtained under different combinations of experimental conditions. Lactate consumption was more pronounced in conditions with higher DO and low glutamine concentrations. The model offers mechanistic insights that are useful for designing advanced operation strategies. It can be used in design space generation and process optimization for better productivity and product quality.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells.

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody-producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation. KEY POINTS: • Proteome profiling of recombinant CHO cells under mild TM treatment. • Identified protein clusters are associated with the unfolded protein response (UPR). • The compared cell lines revealed noticeable disparities in protein expression levels.

CRISPR Deletion of miR-27 Impacts Recombinant Protein Production in CHO Cells.

MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.