Enhancing CHO cell recombinant protein production using a perfusion-directed host evolution approach.

Today, more than 30 products using a manufacturing process based on mammalian cell culture have been approved for human therapy. Most of these products are currently supplied with stirred tank bioreactors operated in batch or fed-batch mode. However, the bioprocess industry needs to constantly increase the productivity of its cell culture processes in order to supply more material with minimal investments in additional equipments. Therefore, alternative production techniques must also be considered, such as the perfusion culture of mammalian cells immobilized in packed-bed bioreactors (PBRs). PBRs can reach very high cell density and hence very high volumetric productivity; so their potential for bioprocess applications must be further evaluated. A review of current state-of-the-art in PBRs development (Chapter 2) showed that the latest generation of PBRs used for bioprocess applications have achieved very high cell densities (i.e. 107-108 cells per milliliter) leading to outstandingly high volumetric productivity. However, the major bottleneck for bioprocess PBRs is their relatively small volume due to the impossibility to avoid nutrient concentration gradients in PBRs of large volume. The current maximal volume seems to be in the range 10-30 liters, and more than 10-fold scale-up would still be required make the PBRs a competitive production technique. Beside their use for bioprocessing applications, PBRs have proven to be an excellent tool to fulfill the requirements of compact bioartificial organs in biomedical applications: they can reach the cell density and volume of an organ. However, as observed during the development of bioartificial livers, a decrease of metabolic activity is frequently observed after 1-2 weeks of culture. Therefore, the main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro, and that maintain a stable phenotype for a minimum of 1-2 months to make them applicable to the PBR technology and to fulfill the clinical demand. PBRs are characterized by high cell density levels, thus by high metabolic rates, and accurate control of the cultures is required. Furthermore cell number cannot be determined directly in PBRs so an indirect method is needed. An indirect method based on the Glucose Consumption Rate (GCR) was developed to monitor a PBR process using recombinant Chinese Hamster Ovarian (CHO) cells cultured on Fibra-Cel® disk carriers (Chapter 3). A key step in this process was the switch from the cell growth phase to the production phase triggered by a reduction of the temperature. In this system, a GCR-based criterion was defined for the switch, and this control strategy proved to be robust, very simple, and was applied successfuly in routine operations for the monitoring and control of an industrial process at pilot-scale. The process operated with this GCR-based strategy yielded consistent, reproducible process performance across numerous bioreactor runs performed on multiple production sites. Another consequence of the high cell density reached in PBRs is the need for an intensive medium perfusion rate (feed and harvest) that should be used in order to keep the cells viable and productive. It appears that the perfusion rate is one of the central parameters of such a process: it drives the volumetric protein productivity, the protein product quality and has a very strong impact on the overall economics of the process. Therefore at industrial scale the optimal stationary packed-bed bioreactor process should operate with a perfusion rate as low as possible without compromising on quantity and quality of the product. In Chapter 4, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel® disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate (2.6 vvd), in order to rapidly produce material for pre-clinical and early clinical trials. A reduction of the medium perfusion rate by –25% and –50% was investigated. With a –25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a 30% loss of productivity was obtained when the medium perfusion rate was further reduced to –50% of its original level. The protein quality under reduced perfusion rate conditions was analysed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of –25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. In Chapter 5, another limitation found in high-density PBR cultures was studied: oxygen supply. A model describing the oxygenation in a PBR of Fibra-Cel® disk carriers was developed. With the help of this model, it was possible to identify that the initial PBR system operated in sub-optimal conditions. Using the model two options were proposed, which could improve the performance of the initial system by about 2-fold: by increasing the density of immobilized cells per volume of carrier from 6.1·107 to 1.2·108 cell·mL-1, or by increasing the packed-bed height from 0.2 to 0.4 meters. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result it would be possible to achieve a substantial improvement of about 2-fold higher productivity as compared with the basal conditions. Finally, this work compared the performance of two different CHO cell culture processes (PBR and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale (Chapter 6). The 1st-generation process was based on the Fibra-Cel® PBR technology. However, it appeared during the development of the candidate drug that high therapeutic doses were required (>100 milligrams per dose), and that future market demand would be in excess of 100 kilograms per year. This exceeded by far the production capacity of the 1st-generation process, and triggered a change of technology from a PBR process with limited scale-up capabilities to a fed-batch process with scale-up potential to several thousand litres. The volumetric productivity (in Product·m-3·year-1) reached with the fed-batch process was comparable to productivitiy of the PBR process. However, since the PBR system was limited in scale (0.6 m3 max.) compared to the volumes reached in suspension cultures (15 m3), the fed-batch was selected as the 2nd-generation process. In fact, the total product output per year (in Product·year-1) was about 18-fold higher for the fed-batch compared to the PBR. Data from perfusion and fed-batch harvest samples indicated that comparable product quality (relative abundance of monomers, dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. This illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development timelines.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells.

Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development.

Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. KEY POINTS: • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.

Background CHO (Chinese Hamster Ovary) cells are the cell line of choice for therapeutic protein production. Although the achieved volumetric titers have increased significantly over the past two decades, the establishment of wellproducing CHO cell lines is still difficult and not always successful [1]. Factors influencing productivity are the chosen host cell line, the genetic vectors, applied media, the cultivation strategy as well as the product itself. Several CHO host strains are available for recombinant protein production, however, they are often quite diverse in terms of growth rate, maximal achieved cell concentrations and specific productivities. Specific productivity is also related to the locus of integration of the transgenes due to positional effects caused by the chromatin environment. Previously it was described that Bacterial Artificial Chromosomes (BACs) carrying the Rosa26 locus are advantageous for the recombinant protein production in CHO cells, enhancing the specific productivity compared to plasmid derived recombinant CHO cells [2-4]. In this project we aim to identify factors influencing volumetric productivity using different CHO hosts, Rosa 26 BACs as genetic constructs and suitable cell culture media. First, different commonly used CHO host cell lines were analyzed in various cell culture media to identify which host strain performs best. Secondly, we generated a recombinant cell line, producing the highly glycosylated HIV envelope protein gp140 as an example for a difficult to express model protein. Gp140 expression was compared to an already existing gp140 cell line generated by a plasmid vector as expression system. Methods Cell culture: CHO-DUKX-B11 (ATCC-CRL-9096) and CHO-DG44 (life technologies) were serum-free cultivated in spinner flasks. CHO-K1 (ATCC-CCL-61) and CHO-S (life technologies) were serum-free cultivated in in shaker flasks. BAC Recombineering: E.coli carrying the Rosa 26 BAC (~220 kbp) were transformed with a plasmid coding for a recombinase. Consecutively, a plasmid carrying the gp140 (CN54) gene flanked by homologous regions to the BAC was used for the transformation of the recombinase positive E.coli cells. BAC positive colonies were selected and the BAC DNA was purified (NucleoBond Xtra BAC, Macherey Nagel). Transfection and selection: CHO-S host cells were transfected with linearized, lipid complexed (Lipofectin) CN54 Rosa26 BAC DNA. Recombinant clone selection was performed in 96-well plates using 0.5 mg/mL G418. BAC transfected CHO cells are able to express the transgene as well as a Neomycin resistance gene within the Rosa26 locus.

Chinese hamster ovary (CHO) cells is a widely used host cell line and can mass-produce recombinant proteins by using various amplifiable selectable marker gene and selection drugs. However, the establishment of highly productive cell line is a laborious and time-consuming work. In the present study, we developed a novel efficient method. We transfected CHO cells with plasmid expressing a gene of interest along with that expressing cell surface marker protein, and we selected recombinant CHO cells by their expression of cell surface marker protein and seed them into 96-well plates by single cell sorting. This method enabled a rapid and efficient establishment of recombinant CHO cells without drug selection. Furthermore, by using ras-amplified CHO cell line (CHO-hp1) as host cells, recombinant protein hyper-producing CHO cells can be easily obtained within a relatively short period. Actually, larger number of recombinant clones were obtained by using CHO-hp1 cells as host cells (188 clones/1,000 seeded wells), while clones cannot be obtained by using conventional CHO cells as host cells (0 clones/1,000 seeded wells). Furthermore, these recombinant cells obtained from CHO-hp1 cells demonstrated higher productivity (45 mg/106 cells/day). These results demonstrate that this method enables a rapid and efficient establishment of recombinant protein hyper-producing cell lines, and can be applicable to production of various recombinant proteins.

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200pF/cm (where 1pF/cm is equivalent to 1×106viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37°C to 29°C was studied during 13 days at cell densities above 100pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31°C was applied at cell density 100pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.

MicroRNAs (miRs) are a class of non-coding RNA that function to regulate global mammalian gene expression by mediating the translational repression of mRNAs harbouring a complementary target region sequence within their 3'UTR. The RNA-RNA binding event between the miR seed region and mRNA target region disrupts mRNA translation and hence repression of protein synthesis from multiple mRNA targets encompassing a variety of cellular processes and pathways. In addition to this multiplicity, miRs also exhibit a high level of promiscuity as a single miR may silence many mRNA targets and a single mRNA transcript may be under the regulation of multiple miRs. miR activity may therefore be manipulated to favour/inhibit cellular processes/pathways of interest to yield desirable cell phenotypes such as enhanced secretion, metabolism or growth. The engineering of miR activity has been applied to enhancing the production of recombinant proteins in the industrially-relevant Chinese hamster ovary (CHO) cell line. In the industrial setting, the utilization of CHO cells has become the dominant mammalian system for manufacturing biotherapeutic recombinant proteins due to their aptitude for accurate protein folding, assembly and performing 'human like' post-translational modifications. A historical Lonza microarray conducted on a range of GS-CHOK1SV IgG-producing cell lines identified changes in miR abundance throughout culture that correlated with growth or recombinant IgG productivity. From this screen, three miRs in particular (miR-15b, -16-1 and -34c) were selected for further study with regard to the impact of targeted miR knockdown and engineered pri-miR over-expression in recombinant and host CHO cell lines and transfectants with respect to establishing whether changes in the amounts of these miRs impacted upon CHO cell growth and productivity. The studies undertaken here have shown that the engineered over-expression of pri-miRs can improve the longevity (e.g. when over-expressing pri-miR-16-1-34c and -15b-34c-16-1) and maximum viable cell concentration (e.g. when over-expressing pri-miR-15b, -16-1 and -16-1-34c) of a CHOK1SV-GSKO host cell line whilst the expression of miR-sponges for targeted miR knockdown can enhance the cell specific productivity (e.g. when expressing miR-sponge-S6) in a variety of GS-CHOK1SV IgG-producing cell lines. In particular, miR-sponges derived from the 3'UTR of the SRPR? mRNA, which should be targeted by miR-34c, reduced miR-mediated repression of SRPR? expression and hence an increase in SRPR? mRNA and protein expression was observed. Specifically, the application of a SRPR?-derived miR-sponge construct constituting 6 miR-binding site motifs was shown to be both functional in relieving endogenous SRPR? from miR-mediated translational repression as well as potentially enabling an increase in cell specific productivity in selected recombinant CHO cell lines, suggesting that secretory capacity was limited by the availability of SRPR?. In conclusion, the studies presented here have demonstrated that the exploitation of miR activity can be an effective tool for CHO cell engineering for the tuning of recombinant protein production without placing any additional translational burdens on the cell.

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.

Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume—almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.