Production Of Recombinant Therapeutic Proteins Research Articles

Overexpression of YTHDF3 increases the specific productivity of the recombinant protein in CHO cells by promoting the translation process.

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

Screening Strategies for High-Yield Chinese Hamster Ovary Cell Clones.

Chinese hamster ovary (CHO) cells are by far the most commonly used mammalian expression system for recombinant expression of therapeutic proteins in the pharmaceutical industry. The development of high-yield stable cell lines requires processes of transfection, selection, screening and adaptation, among which the screening process requires tremendous time and determines the level of forming highly productive monoclonal cell lines. Therefore, how to achieve productive cell lines is a major question prior to industrial manufacturing. Cell line development (CLD) is one of the most critical steps in the production of recombinant therapeutic proteins. Generation of high-yield cell clones is mainly based on the time-consuming, laborious process of selection and screening. With the increase in recombinant therapeutic proteins expressed by CHO cells, CLD has become a major bottleneck in obtaining cell lines for manufacturing. The basic principles for CLD include preliminary screening for high-yield cell pool, single-cell isolation and improvement of productivity, clonality and stability. With the development of modern analysis and testing technologies, various screening methods have been used for CLD to enhance the selection efficiency of high-yield clonal cells. This review provides a comprehensive overview on preliminary screening methods for high-yield cell pool based on drug selective pressure. Moreover, we focus on high throughput methods for isolating high-yield cell clones and increasing the productivity and stability, as well as new screening strategies used for the biopharmaceutical industry.

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells.

In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.

Transfer of continuous manufacturing process principles for mAb production in a GMP environment: A step in the transition from batch to continuous.

Implementation of continuous in lieu of batch upstream processing (USP) and downstream process (DSP) for the production of recombinant therapeutic protein is a significant paradigm change. The present report describes how the first kilograms of monoclonal antibody were produced with equipment originally designed for batch operations while using continuous manufacturing processes and principles. Project timelines for the delivery of clinical material have driven this ambition and helped the transition. Nevertheless, because of equipment availability, a tradeoff between the envisaged continuous downstream process (cDSP) operations and the ones described in this article had to be taken. A total of 2.1 kg of monoclonal antibody were produced in two GMP runs for clinical trials. For USP, a 200-L single-use pilot scale bioreactor was upgraded to enable perfusion operation. DSP steps were designed to be easily transferable to cDSP for later clinical or commercial productions. An in-line conditioning buffer preparation strategy was tested in a discontinuous way to prove its efficiency and the purification cascade was structured in parallel to the continuous collection of antibody-containing cell culture supernatant. This strategy will avoid any process change when later moving to the continuous equipment that is currently under qualification. Alignment between small-scale references runs and the GMP runs in terms of productivity and quality confirmed that the presented approach was valid. Thus, we demonstrate that existing fed-batch infrastructure can be adapted to continuous manufacturing without significant additional investments. Such approach is useful to evaluate next-generation manufacturing processes before making large investments.

Combination of sodium butyrate and decitabine promotes transgene expression in CHO cells via apoptosis inhibition

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for production of recombinant therapeutic proteins (RTPs). Small-molecule additives related to cell cycle apoptosis and autophagy regulation have been used to promote RTP production. By combining two small-molecule additives, positive synergistic effects on transgene expression were observed in CHO cells. In the present study, six small-molecule additives were used, including hydrocinnamic acid (HCA), sodium butyrate (NaB), lithium acetate (LiAc), sodium succinate dibasic hexahydrate (SDH), decitabine (DAC), and sodium propionate (SP). Experiments to test the effects of their pairwise combinations on two different recombinant CHO cell lines (rCHO) were designed using Design-Expert 12.0. Different effects of various pairs of small molecules on apoptosis- and autophagy-related protein expression were observed in the rCHOs. The results showed that compared to the control culture, NaB alone increased the volumetric yield and specific productivity (Qp) by 166% and 143%, respectively. The volumetric yield and Qp of NaB combined with DAC (Cg1)-treated cells increased by 178% and 212%, respectively. Cg1 selectively blocked the cells in the G0/G1 cell cycle stage. The relative expression levels of B-cell lymphoma 2 (Bcl-2), Beclin 1, and microtubule-associated protein light chain 3 (LC3B) in Cg1-treated CHO cells were significantly increased, while relative levels of cleaved caspase-3 expression were significantly decreased. In conclusion, Cg1 had the most obvious effect on RTP production and Qp in CHO cells, suggesting the Cg1 combination of small molecules may be used to improve the expression of recombinant protein in CHO cells.

Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

Improvement of the efficiency and quality in developing a new CHO host cell line.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.

Application of machine learning methods to pathogen safety evaluation in biological manufacturing processes.

The production of recombinant therapeutic proteins from animal or human cell lines entails the risk of endogenous viral contamination from cell substrates and adventitious agents from raw materials and environment. One of the approaches to control such potential viral contamination is to ensure the manufacturing process can adequately clear the potential viral contaminants. Viral clearance for production of human monoclonal antibodies is achieved by dedicated unit operations, such as low pH inactivation, viral filtration, and chromatographic separation. The process development of each viral clearance step for a new antibody production requires significant effort and resources invested in wet laboratory experiments for process characterization studies. Machine learning methods have the potential to help streamline the development and optimization of viral clearance unit operations for new therapeutic antibodies. The current work focuses on evaluating the usefulness of machine learning methods for process understanding and predictive modeling for viral clearance via a case study on low pH viral inactivation.

A Lab-Scale Fermentation Course with an Emphasis on Recombinant Protein Production

Fermentation is responsible for the production of myriad products across a variety of industrial sectors. In particular, the biomanufacturing industry requires a labor force proficient in fermentation and its associated technologies to drive the production of recombinant protein therapeutics. This paper describes the development of a course that introduces students to fermentation technologies for protein production at the laboratory scale. The course aims to familiarize students with the theory underlying the operation of bioreactors, the growth of microorganisms, and the expression and characterization of recombinant proteins. The eight-week course consists of a weekly lecture covering topics including properties of proteins, expression systems, microbial growth, gas-liquid mass transfer, sterilization, and process controls. In addition to lectures, students participate in weekly lab sessions involving the evaluation of growth kinetics, protein production, expression system optimization, and oxygenation and substrate feeding strategies. Student learning is assessed through bi-weekly quizzes and team lab reports, as well as a final group presentation on a technology relevant to the production of recombinant therapeutic proteins. Analysis of student responses on pre- and post-course surveys informed efficacy of course delivery. Overall, student responses on pre- and post-course surveys indicate achievement of learning objectives.

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.

Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.

Expression, Purification, and Evaluation of Anti-IL-2Rα Antibody Secreted by Leishmania tarentolae

Producing a functional anti-IL-2Rα antibody in Leishmania tarentolae, a trypanosomatid protozoan non-pathogenic to human, is a cost-effective and safe alternative for the production of therapeutic recombinant proteins. Expression cassettes encoding heavy and light chains of the anti-IL-2Rα antibody were cloned into two separate pLEXSY vectors. The plasmids were then used to transfect L. tarentolae laboratory strain p10 and stable expression of a recombinant humanized anti-IL-2Rα antibody was obtained. The heavy and light chains successfully assembled to produce a tetrameric 150 kDa antibody. The antibody was glycosylated and functional as assessed by enzymatic deglycosylation and cell proliferation assays respectively. L. tarentolae can be used as an efficient, cost and labor-effective expression system for the production of therapeutic recombinant proteins.

Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems.

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae.

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

Therapeutic recombinant protein production in plants: Challenges and opportunities

Societal Impact StatementTherapeutic protein production in plants is an area of great potential for increasing and improving the production of proteins for the treatment or prevention of disease in humans and other animals. There are a number of key benefits of this technique for scientists and society, as well as regulatory challenges that need to be overcome by policymakers. Increased public understanding of the costs and benefits of therapeutic protein production in plants will be instrumental in increasing the acceptance, and thus the medical and veterinary impact, of this approach.SummaryTherapeutic recombinant proteins are a powerful tool for combating many diseases which have previously been hard to treat. The most utilized expression systems are Chinese Hamster Ovary cells and Escherichia coli, but all available expression systems have strengths and weaknesses regarding development time, cost, protein size, yield, growth conditions, posttranslational modifications and regulatory approval. The plant industry is well established and growing and harvesting crops is easy and affordable using current infrastructure. Growth conditions are generally simple: sunlight, water, and the addition of cheap, available fertilizers. There are multiple options for plant expression systems, including species, genetic constructs and protein targeting, each best suited to a particular type of therapeutic protein production. Transient expression systems provide a mechanism to rapidly transfect plants and produce therapeutic protein in a matter of weeks, rather than the months it can take for many competing expression systems, while proteins targeted to cereal seeds can be harvested, stored and potentially purified much more easily than in competing systems. Current challenges for plant expression systems include a lack of regulatory approval, environmental containment concerns and nonhuman glycosylation, which may limit the scope of the type of therapeutic proteins that can be manufactured in plants. The specific strengths of plant expression systems could facilitate the production of certain therapeutic proteins quickly and cheaply in the near future.

Selective removal of closely related clipped protein impurities using poly(ethylenimine)- grafted anion-exchange chromatography resin

Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k′) and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins.

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.

HEK293 Cells Overexpressing Nuclear Factor E2-Related Factor-2 Improve Expression of Recombinant Coagulation Factor VII.

The mammalian expression system plays a key central role in the production of therapeutic recombinant proteins. Conspicuously, any improvements in the expression system which lead to a higher expression level would have an impact especially in bio-pharmaceutical industries. In the current study, to take steps toward the improvement of expression of recombinant protein, first, we established a stable HEK293 cell line to overexpress a well-known cytoprotective and antioxidant gene, Nrf2. Next, we transiently expressed human recombinant coagulation factor VII, as an example of human recombinant protein, in the engineered-HEK293 cell line. Our results revealed that the established cells had a higher growth rate and were able to endure to UV-induced oxidative stress. Furthermore, within our expectation, our results revealed that the expression level of recombinant FVII in Nrf2-engineered HEK293 cells (315ng/ml) was higher than the HEK293 (198ng/ml) cells and it was functional in a coagulation test assay. Moreover, our new cell line could be a suitable cell to express other recombinant proteins especially for large-scale production of recombinant protein under other culture condition such as lower serum and suspension culture that imposed advantages especially in terms of cost benefits in bio-pharmaceutical industries.

Increased growth rate and productivity following stable depletion of miR-7 in a mAb producing CHO cell line causes an increase in proteins associated with the Akt pathway and ribosome biogenesis

Cell line engineering using microRNAs represents a desirable route for improving the efficiency of recombinant protein production by CHO cells. In this study we generated stable CHO DP12 cells expressing a miR-7 sponge transcript which sequesters miR-7 from its endogenous targets. Depletion of miR-7 results in a 65% increase in cell growth and >3-fold increase in yield of secreted IgG protein. Quantitative labelfree LC-MS/MS proteomic profiling was carried out to identify the targets of miR-7 and understand the functional drivers of the improved CHO cell phenotypes. Subcellular enrichment and total proteome analysis identified more than 3000 proteins per fraction resulting in over 5000 unique proteins identified per timepoint analysed. Early stage culture analysis identified 117 proteins overexpressed in miR-7 depleted cells. A subset of these proteins are involved in the Akt pathway which could be the underlying route for cell density improvement and may be exploited more specifically in the future. Late stage culture identified 160 proteins overexpressed in miR-7 depleted cells with some of these involved in ribosome biogenesis which may be causing the increased productivity through improved translational efficiency. This is the first in-depth proteomic profiling of the IgG producing CHO DP12 cell line stably depleted of miR-7. SignificanceChinese hamster ovary (CHO) cells are the mammalian cell expression system of choice for production of recombinant therapeutic proteins. There is much research ongoing to characterise CHO cell factories through the application of systems biology approaches that will enable a fundamental understanding of CHO cell physiology, and as a result, a better knowledge and understanding of recombinant protein production. This study profiles the proteomic effects of microRNA-7 depletion on the IgG producing CHO DP12 cell line. This is one of the very few studies that attempts to identify the functioning proteins driving improved CHO cell phenotypes resulting from microRNA manipulation. Using subcellular enrichment and total proteome analysis we identified over 5000 unique proteins in miR-7 depleted CHO cells. This work has identified a cohort of proteins involved in the Akt pathway and ribosome biogenesis. These proteins may drive improved CHO cell phenotypes and are of great interest for future work.