Chinese Hamster Ovary Cell Line Research Articles

Super7 passaging method to improve Chinese hamster ovary cell fed-batch performance.

Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process. Cultures passaged using this method had lower net growth rates and were more oxidative throughout 6 weeks of passaging. Fed-batch cultures inoculated by cells passaged using this method had increased net growth rates, oxidative metabolism, and volumetric productivity compared to cells passaged using a conventional strategy. Cells from unstable cell lines passaged by this new method produced 80%-160% more mAbs per unit volume than cells passaged by a conventional method. This new method, named Super7, provides the ability to mitigate the impact of production instability in CHO-K1 cell lines without a need for further cell line creation, genetic engineering, or medium development.

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system.

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.

Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration

High-yield production of therapeutic proteins using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

DirectedCHO: A new miniaturized directed evolution process for phenotype stability trial test of CHO cells before bioreactor scale-up

Most of the biopharmaceuticals that are currently on the market are expressed using the Chinese Hamster Ovary (CHO) cell lines. However, the production yield of these biopharmaceuticals is affected due to CHO cellular heterogeneity and challenges in adaptability during the bioreactor scale-up stage. In this communication, we report the protocol for the miniaturized directed evolution process for CHO cells. The results of the directed evolution process would guide adapting the CHO cell line before bioreactor scale-up. With our approach, we have established the protocol that can be used to streamline superior CHO cell lines for biopharmaceutical production which would be the first of its kind in Africa. Our directed evolution protocol includes a method for a low-cost multiplex directed evolution process that can be used on CHO cells using 20 stressors in 8 concentrations and provides stable trial results for the scale-up process. Using our process, we can provide a simple consumable kit that manufacturers can use for the CHO cell phenotype stability test before the scale-up process. With our approach, we would further develop a platform that can streamline superior CHO cell lines for biopharmaceutical production. This approach would be the first of its kind in South Africa/ Africa.

Fed-batch performance profiles for mAb production using different intensified N - 1 seed strategies are CHO cell-line dependent.

Recent optimizations of cell culture processes have focused on the final seed scale-up step (N - 1 stage) used to inoculate the production bioreactor (N-stage bioreactor) to enable higher inoculation cell densities (2-20 × 106 cells/mL), which could shorten the production culture duration and/or increase the volumetric productivity. N - 1 seed process intensification can be achieved by either non-perfusion (enriched-batch or fed-batch) or perfusion culture to reach those higher final N - 1 viable cell densities (VCD). In this study, we evaluated how different N - 1 intensification strategies, specifically enriched-batch (EB) N - 1 versus perfusion N - 1, affect cell growth profiles and monoclonal antibody (mAb) productivity in the final N-stage production bioreactor operated in fed-batch mode. Three representative Chinese Hamster Ovary (CHO) cell lines producing different mAbs were cultured using either EB or perfusion N - 1 seeds and found that the N-stage cell growth and mAb productivities were comparable between EB N - 1 and perfusion N - 1 conditions for two of the cell lines but were very different for the third. In addition, within the two similar cell growth cell lines, differences in cell-specific productivity were observed. This suggests that the impact of the N - 1 intensification process on production was cell-line dependent. This study revealed that the N - 1 intensification strategy and the state of seeds from the different N - 1 conditions may affect the outcome of the N production stage, and thus, the choice of N - 1 intensification strategy could be a new target for future upstream optimization of mAb production.

Duplication of a chromosome 2 segment in production CHO cell lines correlates with age-related growth improvement.

Monitoring the stability of recombinant Chinese Hamster Ovary (CHO) cell lines is essential to ensure the selection of production cell lines suitable for biomanufacturing. It has been frequently observed that recombinant CHO cell lines develop phenotypic changes upon aging, such as accelerated cell growth in late generation cultures. However, the mechanism responsible for age-correlated changes is poorly understood. In this study, we investigated the molecular mechanisms underlying the age-correlated cell growth improvement in Pfizer's platform fed-batch production process, by examining multiple cell lines derived from different CHO expression systems, expressing a variety of monoclonal antibodies (mAbs). Comprehensive whole-genome resequencing analysis revealed duplication of a continuous 50.2 Mbp segment in chromosome 2 (Chr2) specific to clones that showed age-correlated growth change as compared to clones that did not exhibit age-correlated growth change. Moreover, such age- and growth-related Chr2 duplication was independent of the presence or type of recombinant monoclonal antibody expression. When we compared transcriptome profiles from low-growth and high-growth cell lines, we found that>95% of the genes overexpressed in high-growth cell lines were in the duplicated Chr2 segment. To the best of our knowledge, this is the first report of large genomic duplication, specific to Chr2, being associated with age-correlated growth change. Investigation of the cause-and-effect relationship between the genes identified in the duplicated regions and age-correlated growth change is underway. We are confident that this effort will lead to improved cell line screening and targeted rational cell line engineering efforts to develop cell lines with improved stability performance.

Amino acid degradation pathway inhibitory by-products trigger apoptosis in CHO cells.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

The Chinese Hamster Ovary Cell-Based H9 HA Subunit Avian Influenza Vaccine Provides Complete Protection against the H9N2 Virus Challenge in Chickens.

(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.

A novel missense mutation (FGG c.1168G > T) in the gamma chain of fibrinogen causing congenital hypodysfibrinogenemia with bleeding phenotype

BackgroundFibrinogen plays pivotal roles in multiple biological processes. Genetic mutation of the fibrinogen coding genes can result in congenital fibrinogen disorders (CFDs). We identified a novel heterozygous missense mutation, FGG c.1168G > T (NCBI NM_000509.6), and conducted expression studies and functional analyses to explore the influence on fibrinogen synthesis, secretion, and polymerization.MethodsCoagulation tests were performed on the patients to detect the fibrinogen concentration. Whole-exome sequencing (WES) and Sanger sequencing were employed to detect the novel mutation. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cell lines were built to examine the recombinant fibrinogen synthesis and secretion by western blotting and enzyme-linked immunosorbent assay (ELISA). The functional analysis of fibrinogen was performed by thrombin-catalyzed fibrin polymerization assay. In silico molecular analyses were carried out to elucidate the potential molecular mechanisms.ResultsThe clinical manifestations, medical history, and laboratory tests indicated the diagnosis of hypodysfibrinogenemia with bleeding phenotype in two patients. The WES and Sanger sequencing revealed that they shared the same heterozygous missense mutation, FGG c.1168G > T. In the expression studies and functional analysis, the missense mutation impaired the recombinant fibrinogen's synthesis, secretion, and polymerization. Furthermore, the in silico analyses indicated novel mutation led to the hydrogen bond substitution.ConclusionThe study highlighted that the novel heterozygous missense mutation, FGG c.1168G > T, would change the protein secondary structure, impair the “A: a” interaction, and consequently deteriorate the fibrinogen synthesis, secretion, and polymerization.

Development of a novel tyrosine-based selection system for generation of recombinant Chinese hamster ovary cells

Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of phenylalanine hydroxylase (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key enzymes in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used PCBD1 as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium.

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems.

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Prediction of CHO cell line stability using expression of DNA repair genes.

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

In-house CHO HCPs platform: A promising approach for HCPs ELISA monitoring

A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities.Residual host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme‐linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts.This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.

CRISPR-Cas9 Mediated Stable Expression of Exogenous Proteins in the CHO Cell Line through Site-Specific Integration

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression.

Effect of adenosine and cordycepin on recombinant antibody production yields in two different Chinease hamster ovary cell lines.

In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO-DHFR- and GS-CHO knockout (GS-KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO-DHFR- cells but extended culture duration and significantly increased antibody titers in GS-KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS-KO CHO cells in medium- to large-scale batches. In case of cordycepin, a derivative of adenosine, CHO-DHFR- cells required higher concentration of cordycepin than GS-KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO-DHFR- cell cultures without glucose feeding.

3D Printed, Single-Use Bioreactor with Integrated Inline Sensors for Microbial and Mammalian Cell Cultivation—A Case Study

The development of upstream bioprocesses necessitates small, instrumented bioreactors for investigating and optimizing production processes in a cost-effective manner. Due to advances in both the equipment and the materials used in additive manufacturing, 3D printing of customized bioreactors is now in the realm of possibilities. In this study, a small-scale 3D printed bioreactor suitable for mammalian and microbial cultivations was developed, featuring a working volume of 90 mL, inline pH and dissolved oxygen probes and a levitating magnetic stirrer. Aeration channels and a sampling port were printed directly into the vessel walls. Additionally, the vessel was equipped with a 3D printed customizable optical biomass-sensor. The bioreactor’s performance was evaluated through technical characterization and proof of concept cultivations, demonstrating that mixing time and oxygen mass transfer were sufficient for cultivating mammalian as well as microbial cells at high cell densities. Specifically, an Escherichia coli fed-batch cultivation achieved a maximum OD600 of 204. Furthermore, a fed-batch cultivation of an IgG antibody-producing Chinese hamster ovary cell line reached a peak viable cell density of 10.2 × 106 cells mL−1 and a maximum product titer of 2.75 g L−1. Using a three-parameter fit, the inline biomass signal could be correlated to the corresponding offline values with satisfactory accuracy, making it possible to monitor cell growth in real-time.

Strategy for Developing a Stable CHO Cell Line that Produces Large Titers of Trastuzumab Antibody.

Trastuzumab (Herceptin®) is currently the main treatment option for breast cancer patients that overexpress the human epidermal growth factor receptor 2 (HER2). This antibody binds specifically to HER2, blocks cancer cell growth, and promotes effective cell death. In the present study, we sought to develop a robust and efficient process for the development of a stable Chinese hamster ovary (CHO) cell line with high trastuzumab expression and production. We adapted a process that combines transposon system-based vector construction, suspension cell culture, and a high selection process. The latter, involved enhanced green fluorescent protein (eGFP) expression, fluorescence-activated cell sorting (FACS), and semi-solid methylcellulose media. The construction of trastuzumab as a humanized monoclonal antibody was achieved by subcloning the synthesized light and heavy chain sequences into a suitable piggyBac expression vector. The optimized piggyBac vector used for the expression of trastuzumab in CHO cells resulted in the production of trastuzumab and reached 4.24 g/L in the T1A7 clone after a 7-day batch culture. The T1A7 clone was selected after screening over 1500 clones. The current simple workflow ensures strict monoclonality and relatively high production of trastuzumab. This workflow could potentially be implemented in Research and Development (R&D) laboratories, including in developing countries for the production of recombinant monoclonal antibodies in a cost-effective manner.

Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins.

The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids' large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.